Repression of β-galactosidase synthesis in Escherichia ...
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Portland State University Portland State University
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Dissertations and Theses Dissertations and Theses
1972
Repression of β-galactosidase synthesis in Repression of -galactosidase synthesis in
Escherichia coli by salicylates Escherichia coli by salicylates
Joan Carlyn Olson Portland State University
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All AB31RACT OP 1112 TIf3SIS OF Joan Carlyn Olson for the l-aster ot
SCience in 3io1oGi presented July 2S 1972 bull
Title Repression ot J3-0ilactosid~se ~nthesis in 3scnericnia ~
co11 by Salicy1ates bull
APROVJD 3Y 373 OF Tti TH35IS CmrTSS
( Jo~
~middotr 1-~ers Chaiman
Earl Fisher -
Gordon L Kilgour
Salicylic acid and to a lesser eytent aspirin ~ave been shoim
toreprecs f3-galactosidase ~nthesis in Zscherichia coli The represshy
~on is not due to decreased inducer uptake nor does it result from
eOr1petition idth inducer for repressor Dinitrophenol does not eert
~ ror- n d tmiddotSJIiII-ct cJ ccrs on )J-gt-scos~ ase syn ue~ls Cycli~ ~dcnosine 35 shy
- J1onophoshate partial1r relieves the repressionbull Zle eten t 01 the
relief by cyclic adenoJine Ionopilospnateseem to depend on the conshy
centratiol of s4~cz1ic acid This indicates there raay be direct
interaction betueen the ac tions ot CJclic admosine monogthosphate and
8ali~J1io acid in E ooli oellSe
RpoundPRs3Imi OF j3-GALtICrOSIDASE SYUiHESIS
IN SCIE1ITCHIA COLI BY SLIcLATES
by
JOAN CARLY OLSDrl
bull A thesis subnitted in partial tuliilL1cnt of the
requira~ents tor the degree opound
luSTER OF S8r DICE
in
BIOLOGY
Portland State Univelsity 1972
PORTlAND STATE UNIVERSITY UBliARl
TO THZ OFrICB OF GDUArS STUDI2S lJD RZ33A1CH
The re~~ers of the COr~~ttee a~rove the thesis of
~oan Carlyn Olson presented July 28 1972bull
John i llyers Chailan
Barl Fisher bull
1fiJ ~~~~~
GordonL Kilgour
AFPROE~1
Earl risher Head Dea=~ent of BioloGY
David r Clari Dean of Gr~dlace St1dies
July 28 1972
TABLE OFmiddot CONTENTS
PAGE
LI3r OF fABLES iv
LIST FIGURSS v
GI~PTEa
I INrODUCITON 1
II iiflTSRIALS ANTI HSTHODS
B~cterial strains h
Grouth Conditions 4
~-Gnlaco~oridase Assar
rEaterials
III RESULTS
The Effects of Salicr1ates on j3-G2Jactosidase Synthesis 6
The Zifcct of Cyclic AfP on Repression by Salicylic Acid 9
The Effect of Salicylatcs on Gonsti tutivo xzyme S~rnthesis 16
The Effect of 24-Dinitro~hcnol on j3-Gllactosidase Synthesis
IV DISCUSSION ~3
26BIBLIOGRAPHY
28ltIP lZmIX
LIST OF FIGURZS
FIGURE PAGE
1 Effect of Salicylic Acid on Differential Rate opound
--Galactosidase Synthesis 11
2 Effectof Cyciic tjp e1d Salicylic Acid on Differential
f3 -Gal~cto3idase Synthesis 13
3 ~ffect of Cyclic 1lmiddotiP and Salicrlic Acid on (3Galactoshy
sidase Synthesis 15
h Effect of cJclicMi cud Salicylic Acid on j3-Galactoshy
sidase Synthesis 17
5 Sffect of Salicylic Acid on DLfferelltial 13 -Galactosidase
~~~thesis in Constitutive Strain 3300 19
6 Effect of mJP on Differential f3 -Galactosidase Synthesis 22
LIST OF TABLES
TABLE PAGE
I
II
Effects of Asoirin on Differe~tial Rate o~ Synthesis
of 3-Galactosidase
Effect of Salicylic Acid on DifferenGial Rate of
Synthesis of fl-Galactosidase
7
8
INrrWDUCTION
Although aspirin is one of the EOSt commonLy us~d drugs its
actual nechnnism of action within the body still remains unclear
Acetylsalicylic acid belongs to the group of drugs referred to as salishy cylates all of which have SLllilar pharmacological properties rheir
activity is believed to be dependent on the drugs salicylic acid conshy
tent and substitutions on the carboxyl or hydroxyl groups alter only
their toxicity or potency (1)
Aspirin is most COnt~only recognized for its analgeSiC antipyreshy
tic and antiirulamTatory effects Recently Vane (2) demonstrated
antiinflan~atorJ acids such as aspirin salicylic acid mld indoshy
methaCin inlJibit enzymes which catalyze prostaglandin (PGE2 and PGF2~)
synthesis in cell free extracts of guinea pig lungs PG11 and PG~
when injected into the third ventricle of cats also proves to be a
potent inducer of fevers (2) Thus Vane postulated aspirin produces
its an~ipyretic and antiinf1aw~tory responses by inhibiting synthesis
of prostaglandins ~~though prostaglandins when administered intradershy
m2~ly in nan do not produce pain (2) some of aspirins ~~gesic activity
may result from reduction of inllt3ll111ation (3) Smith and lillis (4)
supryorted this evidence when they independently observed aspirin to
inhibit conversion of arachidonic acid into prostaglandins in h~~an
platelets
All abbreviations used in text are defined in appendix
2
Aspirin has many other diverse actions within the body Included
among these is its abllity to prevent collagen and epinepnrine induced
aggregation of platelets Platelet ageregation occurs in two phasesa
The first is characterized by a rapid change in the shape of platelets
the second or actual agflegation of cells follol1s the release of ADP
fram platelets (10) Aspirin blocks the second phase of agcregation
(7)(24) This again relates the actions of aspirin to prostaglandins
for POE and PG~ are also known to have effects on platelet aggregation1
Other actions of aspirin are its ability to lower blood sugar in
certain diabetic ~atients (1) to alter ion transport and membrane potenshy
tial in v8ried cell types (1)(16) to reduce lipogenosis and depress
free fatty acid release in muscle tissue (1) and tomiddot uncouple oxidative
phosphorylation (5)
lnother compound cyclic adenosine )5-mollophosphate (cUP) has
also been shmmiddotrn to have a broad spectrum of effects at sites similar to
those acted upon by salicylates These include 1) the mediation by
cM~ of prostaglandins i~1ibition of platelet agsregation (7)(10)
2) the mediatio~ by Ci~1P of insulins induction of glucose upt~~e in
hep~tic cells (11) ) its co~plex effects on ion transport and ma~brnne
potentials in various cell types (18)(25)(17) and 4) its stimulation of
lipolysis in ht~ and rat fat cells (27)
Because cyclic ffi in1 tictes the (lction alld release of many hormones
it has become knowIl as a ubiquitous second messengertl bull Adenyl cyclase
is activated when an external stimulus such as a hormone comes in con-
tact 111th the cell surface The inCleae in intracellular levels of
cyclic lIP activate other specific enzymes ACTIJI s stiI1ulation of
3
adenyl cyclase in the adrenal cortex follolled by the activation of enzymes
synthesizinEcortiaol exenpliiies this (12) Also thyroid hormone secretion
is induced middothen TSH activates thyroid adenyl cyclase (13)
lhere is some evidence cM may in a sirilar manner mediate
the synthesis ~f prostaglandins (10) EPinephrine and nore~inephrine both
known to stimulcte ade~yl cyclase in certai~ cells stimulate release of
prostaglandins from the dog sp~een (14) In addition it has been shown
prostaglandins stL~u1ate adenyl cyclase in human platelets (7)
The question arose -lhether soe of the effects of aspirin might
be mediated by its effects on the ~~1P systems of various target cells
In order to determine whether a relationship exists between the action
of aspirin ald cyclic ANP the effect of salicylates on j3-galactosidase
synthesis in the bacterial ce11 was obs~rved Synthesis of (3 -galactoshy
sidase in Jscherichia coli is known to require cyclic AImiddotrp (15) In
E COli c~middot~ acts at the transcriptional level of enz~ne synthesis by
promoting initiation of ~-galactosidase nu~fA synthesis The nucleotide
is believed to activate a protein called cMP receptor protein which
in turn allows the protein to bind to the promoter reGion of the lac
operon Its presence enhances the binding oflli1A pol~erase to the
proper initiation site of lac DNA (6) COllsequentlyincreased syntheshy
sis of B-galactosi(ase occurs with optimum concenlirations of cAllP and
repreSSion occurs ~hen the ct1middotp concentration is louered
This paplusmner reorts the effects salicylates have on j3-galactosishy
dase SJIlthesis in E coli and the relationship between cAllPs and salicylic
acid t s mechanism of ac tion within the bacterial cell
YJATERIALS AND NlTHODS
Bacterial strains
For the initial experiments Escherichia coli strain 3000 (lac i +)
was used To determine salicylates effect on inducer uptake a conshy
stitutive strain 3300 (lac i -) was used Both strains llere provided by
Dr John 11 Hyers
Grmmiddotrth Conditions
Bacteria liera grown in double strength Hinimal Broth Davis
medium (Bacto) supplelllented k1th 0002 vitamin B and either 02
glucose or 05 glycerol Double strength media lIas used since this
has essentially the same constituents as the minilal medium used in
earlier experiments observing effects of cyclic AmiddotU on E coli (7)
-Before each experiment fres11 stationary cultures were prepared
from bacteria1 slants StatioIlaty cultures were inoculatr-d into
Klett Flasks to obtain an initia1 ceU density of 10-8ml ald gr01ID on
a shaker at 370 C until log phase was reached To induce fl-galactoshy
sidase -syntheSis isopropyl j3-D-thiogalactopyranoside (rrrG) was added
to the cells in log phase producing a final concentration of 5xl0-4 11
FUrther a1ditions depending on the experiment liere made to log phase
cultures
All cell denSity measurements were taken on a K1ett-~~erson
Photoelectric Colorimeter (Model 80003) using a 660 mp filter
5 ~Galactosida3e Ass~
j3-galactosidase was assayed according to the procedure of Pardee
Jacob and Honod (6) with slight Iilodification At specific timeslt
09 ml of sample lUlS ldthdrakll fro-n the cultures and added ~o tubes
containing 2 drops of 025 sodiurn dodecyl sulfate (SOO) end 2 drops
chloroform (9) Both toluene treatment and SDS - chloroform treatshy
ment were pre~narily tested to deterwine the best method of releasshy
ing enzyme from the middotcell The latter did not alter enzyme stabilitY
and proved to increase the sensitivity of the assq
The amount of enzyme present was measured spectrophotomatr1cally
using a KJett-5UlllIllerson Photoelectric Colorimeter (lmiddotiodel 600-3) witil a
420 111)I1ilter One unit of enzyme is defined as the amount producing
1 II1j1 mole of o-nitrophenol per minute at 280 0 pH 70 The units of
enzyme in the sample can be calculated from the 1act 1 17fL mole per ml
of o-nitrophenol has a Klett reading of 045 under experirlental condishy
tions used here
To verify the assay procedure With E coJ strain 3000 enzyme
activity was confirmed to be linear in relation to time and concentrashy
tion
l-Iaterials
All chevicals were commercially obtained Cyclic adenosine
3 5-monophosphatie was obtained from Sigma and its purity 1-1as
confirmed chromatographically and spectrophotometrlcally Based on
readings vy a Cry Spectrophotometer the cyclic ffJ proved to be
99 pure b-leight Cyclic AHPs biolobical activity was confiIr4ed by
its ability to reverse glucose repression
RESULTS
Tho Effects of Salicylates on 13-GaJactosidase Synthesis
Various concentrations of aspirin were added to E coli strain
3000 cultures grmrlng on glycerol medium At a 5 x 10-3f concentration
ofacetylsa1i~lic aci~the ~fferential rate of j3-galactosidase synshy
thesis decreased (Table I) This 1-1aS accompanied by a slight decrease
in bacterial growth rate At 1 x 10-~ and 25 X 10-3M aspirin had
no significant effect on j3-galactos1dase synthesis
Sinceaspirin1s pharmacological action depends on its salicylic
acid content (1) the effect of salicylic acid on j3-galactosidase synshy
thesis ~1as detemined At all concentrations salicylic acid produced
a greater effect on the differential rate of enzyme synthesis than
aspirin (Table II) Salicylic acid also inhibited grorrth to a considershy
able extent but in all cases the effect on enzyme synthesis was
greater
Control experiments showed both aspirin and salicylic acid had
no effect on the enz~e ass~ system
Since the utjlization of glycerol requires the inducible enzoJlne
glycerol kinase kriown to be sensitive to catabolite repression (~)
it Has postulated the effect of salicylic ~cid on grouth rate resulted
from an inhibition of glYcerol kinase synthesis Such an inhibition
would have the effect of masking decreases in the differential rate
of ~-galactosidcse synthesis For this reason it was decided to use
a carbon source not subject to catabolite repreSSion and presumably
-7
TABLE I
EFFECTS OF ASPIRIN on
DrFFEIl3NTIAL RATE OF SYNTHESIS OF P-GALACTOSIDASE
Elcp 170
Aspirin (N)
Diff Rate En~ Synth (EUrilLKR)
at Control Rate
Grol-rth Rate (1gen time)
of Control Rate
1 1 -( cO3)1x10
25x10-3 5xl0-3
21 -( con3~)5x10shy
3 I -(con3- ) 5xl0shy
361 100 361 100 361 100 254 70
313 100 210 67
33bull3 100 259 78
72hr
63hr bull67hr 60br
100 87 93 825
65hr
54hr 100
83
67hr
58hr 100 87
E coli strain 3000 1-las grOl-m in glycerol raedium until log phase was obtained At -rms time the culture was divided and transferred to fiasks containing 5 x 10- H IPTa ard the indicated amount of dissolved aspirin The control flask contained water in place of the dissolved aspirin solution To insure dilutions would not 1irrdt the media flasks were also initi~ supplemented vnth 4x y~~ Broth Davis
At varioUs cell densities 09 ml samples were taken and assayed for j3-Galactosidase activit7
8
Exp No
1
2bull
3
TABLE II
EFFECT OF SALICYLIC ACID ON
DIFFER11ITIAL It4TE OF SYNTHESIS OF J3-GALACTOSID~sE
Salicylic Acid (N)
-(contro1) x 10shy
-(contro~23 387 x 10
-(contrpound)1 x 10
DtiL Rate ~~th (EUII1lJb Iffi) of
control rate
Growth Rate (ligen time) of
Control
370 62
100 n
60hr
33hr 100
283 65
100 23
71hr
44Ihr 100 62
315 206
bull100 6
60hr
2hr 100 87
Cells l-ere gr01ffl and sampled as in Table I ITith the exception that disshysolved salicylic acid was added to ce~s in log phase in place of aspirin
9
not subject to salicylic aci~ inhibition In all subsequent eXperiments
glucose lias used as carbon source
Upon the addition of 5 x lO-~ salicylic acid to cells grovID in
glucose media there gtmiddotas an ilnmecliate cessation of a-galactosidase synshy
thesis lasting over a generation of grotrth This l-laS followed by a
recovery of srnthesis but at a decreascdrate relative to the control
(fig 1) tt 1 x 10-~i salicylic acid deceased the differential rate
of enzY1e synthesis but tlere 1-TaS a)parently no lag in synthesis after
its addition
From this data it apears salicjlic acid more effectively represses
j3-galactosidase synthesis in glucose grovID cells than in cells grovm
in glycerol redia It should be noted hOHever the inhibitory effect
of salicylic acid on grot-lth rate is the srune vJhether the carbon source
is glucose or glycerol The reason for this inhibition ra1lains obscure
The strect or CYclic AlP on Heprcssion by Snlicylic Acid
If salicylic acid acts by decreasing cyclic PlW levels then cpJmiddot~
should revese the efects of se1icylates Figure 2 shms the resiuts
of rul experinent lihere 5 x lO-3M cyclic AHP and 25 x 10-31-1 salicylic
acid were added silulta1eously to glucose grotm cells It can be seen
that cPJP revJrses the repression caused by salicylic acid Hm1ever
the reversal is only partial since the dif~erential ra-ce is not restored
to the level of the control plus c~tmiddotp cells
Salicylic acid in these experi~ents also caused a decrease in
gromh rate and it lla3 further decreased when cAt-1P las added to the
cells Because of this the data from Figure 2 was replotted with time
units on the abscis3a in place of cell density (Fig 3)~ This allows
10
FIGURE 1
EFFECT OF SLICYLIC ACID ON THE DIFF3P3NrIAL RJTE OF [3-GALACTOSIDA3E SYNTHESIS
strain 3000 Was grovffi in glucose modiUI1 until log phase wst reached
Approrinate1y 1 hour follOldn~ induction rlith IPTG the culture was
separated and trs1sierred to flasks containing the indicted additions
Samples uere talten and assayed for j3-galactosidase as in Table I
Since diaBo1ved salicylic acid lias added a dilution in cell densitr
occurred The first point on the graph represents samples taken L~Jnedi-
ately c)fter the culture Was separatedand diluted
Grorth Rate Differential Enz Rate Flask A Control 75hr 308 EUllilAKR
B Salicylic Acid 68hr 180 EUlllJfoKR
(1 x 10-31)
o Sa1icylic Acid 35hr 95 EUralAlffi (5 x 10-31-1)
12
FIGlllE 2
EFF3Cr OF CYCLIC AmiddotlP 1jJ 3lLICYLIC ACID ON DIFF~JTIAL f3-01AClOSImE SYUTH33IS
IFTG was added to a log phase culture of strain 3000 grown in glucose
medium Approximately 45 minutes after induction the culture was divided
equally into flasks containing the indicated additions 32J1lplcs were
ta~en and ~-Galactosidase was assayed as previously indica~ed The
arroH at 30 Klett unts represents the time the culture ylaS divided
and trcmsferred
DifLerential Rate Growth tate Enzyme Synthesis
Flask A Control bull67hr 23 lJrrJ KIt
B Cyclic mp (5xlO-311) 57hr 315EUrr~6KR
c SalicyliQ acid 4lhr 135ZUmlKR(25xlO-)N)
D Salicy~c acid bull 35hr 25 ~1JrrJLKR 25xlO ~ 11 + CJllmiddotfP (5xlO-)~1)
+
0
t
o
oft
$+
~
Ft flJ rsi
m
m
mlt
l ~ ~
sect4( ~
m~ ~ e1
54 PI rd ISJ
aI ttl
14
FIGURE 3
EFF~T OF CYCLIC AHP UID SJLICYLIC ACID ON j)-GLACTO3IDASE SYNTIIESIS
This represents the smne experireental data as in Figure 1 In the
graph enz~e units ~re plotted in rel~tion to tL~e rather th~n in
relation to cell denitr rIle arrml indicates the time the culture
lras divided and all additions l-Tere i1ade
bull
bullbull Q
-Ld
bull
16 the effect of 321icylic acid ~o be noted indeendently of itsmiddot effect
on generlttion time Even on this graph cAli is shown to partially
relieve repression caused by salicyla~es
Previously Pastaa ~md PerL1an (21)(22) emphasized cAHP had no
effect on growth rate of E coli strain 300 rlhich 112S in contrast to
these results Their dltta homver referto using 1 x lO-3z cANP
Since 5 x 10- 3r crolic lp is believed to overcome both tranient and
catabolite reression caused by r~ucose in strain 3000 (23) the effect
of this concentration on salicylic acids repression was observed
Sirlilar results Nere obtcgtined in a second eXgerimcnt using
S x 10-31-1 salicylic acid and 5 x lO-3H Cl]~ (Fig 4) rhe degree of
reversal ias again only partial Because grorlth inhibition at this conshy
centration of salicylic acid tdth cAHP ias so great the curve is not
extended ver ffX
From these data it appears there is an interaction between the
effects of sali~Jlic acid and those of cMmiddotP Since repression proshy
duced by the higher concentration of salictlic acid seems to be less
effectively reversed by 5 x 10-3N cMP there i~ a possibility the
degree of interaction between the tlO may be concentration dependent
The 3ffect of Sdic-lates on Consti tutive Enzyme Synthesis
To eliminate the possibility salicylic acid may repress j3-galactoshy
sidase synthesis by preventing inducer upt~~e or by competing with
inducer for repressor an experiYlent vTaS carried out using strain 3300
constitutive for fo-galactosidase synthemiddotsis From Figure 5 it is evident
that 25 x 10-311 Jalicylic acid had the same effect on strain 3300 as it
had previously on strain 3000 Therefore salicylates act independently
17
B
uJ ~ l ~ l cJ J L o o- Ul t z 3 w r N Ia Z 312L fa m sa 1m m Em2 ru KLETI UNITS
EFFECT OF CYCLIC Almiddotp lm SLICYLIC ACID OU DIFFrRENfIAL j2gt-GALACTOSIDA3E SYNlHESIS
Figure 4 Strain 3000 culture las grOiID sampled and assayed for fl-g3~actosidase as in Figure 2 uitIl the exception a hig1er concentrC)tion of salic7l1ic acid i-as added after separshyation Since sliicylic acid 35 in dissolved form a dilution occurred upon its addition This uas compensated for in
J
-fA
other fiCsls by an equal dilution
flask Pt Control
Bbull Control + cAHP (5xlO-3H)
c Salicl~ic acid (5xlO H)
D salicl~c acid
Grmmiddotrth Rc1te 71hr
52hr
22hr
15hr (5xlO 3 I) + cMi (5xlO- 1middot1)
Diff Enz Rate 22 EUmlAKR
31 t1Jm1~KR
12 1UmlAIffi
16 EUmlAKR
18
FIGUR35
EFECr 01 SALICYLIC ACID OH DIIT2HE~rrAL fgt-GAL~C0SIDASE SYIJIl133I3 nr COnSrITlJlIV3TAIH 3300
strain 3300 iolaS gron in glucose medium Jllen the cells reached log
phase25 x lO-~1 snlicylic acid ~as added Cells were sa1pled ltmd
4asJared 80S in earlier experiments IPTG (5 x 10- 1-1) liaS added to
control flasks to note the degree of const~tutiveness of tile strain
The first points ngain represent sa~les trucen in~ediately poundter the
culture was divided and additions ware made
Growth Rate Diff Enz Synth Flask A Control bull89hr 65 EUrnlAKR
B Contro~ + IPTG bull89hr 65 EUmJ6KR (5xlO-~middotI)
o SalicYli~3acid bull54hr 46 ~iJ~KR (25x 10 H)
D Sa1icrli~ acid 5hhr 30 EUml~KR (25xlR--i-r) + IPTG (5xlO- M)
lSi JT1
bull 0
Fa-
I
a +
tsJ
yen
+
~
v
+
tsi IS1 m
m
tn
til sectll
m ~ ~ 2
bull
ef ~
~ ~ tsJ
uf
shy
20
of inducer uptake and of inducer-s derepression of lac mrrNA synthesis
The zrecl of 24-Di1ikophcnol ont30alactosidase Synthesis
Since salicylates are kno-Vffi to be uncouplers of oxidative phosshy
phorylation (5) it has been sJgccsted sOrle of their phaITlClcological
effects result from their uncoupling action Tis i-lould irply that
other uncouplers should preferentially inhibit ~-gilactosidase syntheshy
sis For this reason the effect of 2 4-dini trophenol (Dr~) on J-galacshy
tosidase synthesis Has observed Because DrIP is more effective in inhibishy
ting oxidative phosphorylation than salicylic acid and is consequently
more toxic a concentration of the drug i-laS found vhic uould inhibit
grOl-rth to ap~roximately the sa11e degree as 25 x 10-31gt1 salicylic acid
This was found to be 25 x 10-411 Figure 6 illustrates DNP does not
repress differential synthesis of j3-g1actosidase This implies that
althoueh DNP 1m-lers ATP levels there is no apparent effect of the drug
on lsvels of cAl-IP Also it appears salic-rlic acid is not exerting its
repression of enzyrre sJ~~hesis by inhibiting oxidative phosphorylation
21
FIGUllS 6
EFFECrl OF DJP ON DIFFif2rIlL ~GLCiO3D~S STImiddotrYliESIS
Jhen strain 3000 cells reacled log ph~se they tmiddotrere indlccd 1ith IPTG
AproxL~ately one hour later the culture was divide1 a~d tr~ferred
to flasks containing the indicated adlitions The cellsas in preVious
experirrents Tere sarlpled at various tLTIes and assayed for B-galactoshy
sidase activity Since 2h-dinitrophenol t-rag in solution its addition
resuted in dilution of cultures This dilution las equally conpensated
for in control flasks nle first points on the graph represent samples
taken innediately after sepration of the cuJture
GroYTth Rate Difierential inz Rate Flask A Control 75hr 22 L1JmlAKR
B Contro + cAll 64hr 50 EUmlAm (5y10- H) 4z
C DNP (25xlO- -I) 42hr 22 EOIraAKR
D DNP (25xl2~4zr) + 29hr 42 UmlAKR cAJP (5x10 -0
N
N
v
o
-shy-shy+
-shy
+
bull-
DISCU3SION
Salicylic acid ruld to a lesser extent aspirin cause repression
of f3-gclactosidase srnthesis in scherichia coli This effect is not
due to an inhibition of inducer uptake nor is it due ~o interference
l-rith inducers inactivation of lac repressor since strnins constitutive
for 3-gJactosidase 3J1lthesis are also repressed b~r sclicyli~ acid
In addition because dinitrophenol is liithout effect on differential
fl-galactosidase synthesis it appecrs salicrlates do not repress enzyme
s~mthesis by inhibiting oxidative phospllorylation
At these concentr2tions however salicylic acid does decrease
growth rate and direct or indirect effects of this on the observed
repression of enzyme synt~esis still ra~ain Since the differential
rate of enzyme synthesis i~ decreased and also since 25 x 10-411 DN
decreases grotrth rate aproximately the same degree middotuitl1cut having I
effects on differential synthesis salicylates inhibition of growth
is not the sole factor responsible for its effects on enZJcre synthesis
-11en 5 x 10-31 cyclic JUfP 1-ms added to cells treated Kith 25 x 10-3
and 5 x lO-3H srJicylic acid repression of differential enzye s~1lhesis
1ES pextially relieved This sugests salicllic acid represses J3-gJ~ac-
tosidase srnthesis br directly 1011erinti intIacellular levels of cyclic AMP
or by indirectly acting on asita which in turn releases cANP from the
cell Since cMP only partially relieves repression caused by wlicylic
acid and because there appears to be an inverse relationship between
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
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All AB31RACT OP 1112 TIf3SIS OF Joan Carlyn Olson for the l-aster ot
SCience in 3io1oGi presented July 2S 1972 bull
Title Repression ot J3-0ilactosid~se ~nthesis in 3scnericnia ~
co11 by Salicy1ates bull
APROVJD 3Y 373 OF Tti TH35IS CmrTSS
( Jo~
~middotr 1-~ers Chaiman
Earl Fisher -
Gordon L Kilgour
Salicylic acid and to a lesser eytent aspirin ~ave been shoim
toreprecs f3-galactosidase ~nthesis in Zscherichia coli The represshy
~on is not due to decreased inducer uptake nor does it result from
eOr1petition idth inducer for repressor Dinitrophenol does not eert
~ ror- n d tmiddotSJIiII-ct cJ ccrs on )J-gt-scos~ ase syn ue~ls Cycli~ ~dcnosine 35 shy
- J1onophoshate partial1r relieves the repressionbull Zle eten t 01 the
relief by cyclic adenoJine Ionopilospnateseem to depend on the conshy
centratiol of s4~cz1ic acid This indicates there raay be direct
interaction betueen the ac tions ot CJclic admosine monogthosphate and
8ali~J1io acid in E ooli oellSe
RpoundPRs3Imi OF j3-GALtICrOSIDASE SYUiHESIS
IN SCIE1ITCHIA COLI BY SLIcLATES
by
JOAN CARLY OLSDrl
bull A thesis subnitted in partial tuliilL1cnt of the
requira~ents tor the degree opound
luSTER OF S8r DICE
in
BIOLOGY
Portland State Univelsity 1972
PORTlAND STATE UNIVERSITY UBliARl
TO THZ OFrICB OF GDUArS STUDI2S lJD RZ33A1CH
The re~~ers of the COr~~ttee a~rove the thesis of
~oan Carlyn Olson presented July 28 1972bull
John i llyers Chailan
Barl Fisher bull
1fiJ ~~~~~
GordonL Kilgour
AFPROE~1
Earl risher Head Dea=~ent of BioloGY
David r Clari Dean of Gr~dlace St1dies
July 28 1972
TABLE OFmiddot CONTENTS
PAGE
LI3r OF fABLES iv
LIST FIGURSS v
GI~PTEa
I INrODUCITON 1
II iiflTSRIALS ANTI HSTHODS
B~cterial strains h
Grouth Conditions 4
~-Gnlaco~oridase Assar
rEaterials
III RESULTS
The Effects of Salicr1ates on j3-G2Jactosidase Synthesis 6
The Zifcct of Cyclic AfP on Repression by Salicylic Acid 9
The Effect of Salicylatcs on Gonsti tutivo xzyme S~rnthesis 16
The Effect of 24-Dinitro~hcnol on j3-Gllactosidase Synthesis
IV DISCUSSION ~3
26BIBLIOGRAPHY
28ltIP lZmIX
LIST OF FIGURZS
FIGURE PAGE
1 Effect of Salicylic Acid on Differential Rate opound
--Galactosidase Synthesis 11
2 Effectof Cyciic tjp e1d Salicylic Acid on Differential
f3 -Gal~cto3idase Synthesis 13
3 ~ffect of Cyclic 1lmiddotiP and Salicrlic Acid on (3Galactoshy
sidase Synthesis 15
h Effect of cJclicMi cud Salicylic Acid on j3-Galactoshy
sidase Synthesis 17
5 Sffect of Salicylic Acid on DLfferelltial 13 -Galactosidase
~~~thesis in Constitutive Strain 3300 19
6 Effect of mJP on Differential f3 -Galactosidase Synthesis 22
LIST OF TABLES
TABLE PAGE
I
II
Effects of Asoirin on Differe~tial Rate o~ Synthesis
of 3-Galactosidase
Effect of Salicylic Acid on DifferenGial Rate of
Synthesis of fl-Galactosidase
7
8
INrrWDUCTION
Although aspirin is one of the EOSt commonLy us~d drugs its
actual nechnnism of action within the body still remains unclear
Acetylsalicylic acid belongs to the group of drugs referred to as salishy cylates all of which have SLllilar pharmacological properties rheir
activity is believed to be dependent on the drugs salicylic acid conshy
tent and substitutions on the carboxyl or hydroxyl groups alter only
their toxicity or potency (1)
Aspirin is most COnt~only recognized for its analgeSiC antipyreshy
tic and antiirulamTatory effects Recently Vane (2) demonstrated
antiinflan~atorJ acids such as aspirin salicylic acid mld indoshy
methaCin inlJibit enzymes which catalyze prostaglandin (PGE2 and PGF2~)
synthesis in cell free extracts of guinea pig lungs PG11 and PG~
when injected into the third ventricle of cats also proves to be a
potent inducer of fevers (2) Thus Vane postulated aspirin produces
its an~ipyretic and antiinf1aw~tory responses by inhibiting synthesis
of prostaglandins ~~though prostaglandins when administered intradershy
m2~ly in nan do not produce pain (2) some of aspirins ~~gesic activity
may result from reduction of inllt3ll111ation (3) Smith and lillis (4)
supryorted this evidence when they independently observed aspirin to
inhibit conversion of arachidonic acid into prostaglandins in h~~an
platelets
All abbreviations used in text are defined in appendix
2
Aspirin has many other diverse actions within the body Included
among these is its abllity to prevent collagen and epinepnrine induced
aggregation of platelets Platelet ageregation occurs in two phasesa
The first is characterized by a rapid change in the shape of platelets
the second or actual agflegation of cells follol1s the release of ADP
fram platelets (10) Aspirin blocks the second phase of agcregation
(7)(24) This again relates the actions of aspirin to prostaglandins
for POE and PG~ are also known to have effects on platelet aggregation1
Other actions of aspirin are its ability to lower blood sugar in
certain diabetic ~atients (1) to alter ion transport and membrane potenshy
tial in v8ried cell types (1)(16) to reduce lipogenosis and depress
free fatty acid release in muscle tissue (1) and tomiddot uncouple oxidative
phosphorylation (5)
lnother compound cyclic adenosine )5-mollophosphate (cUP) has
also been shmmiddotrn to have a broad spectrum of effects at sites similar to
those acted upon by salicylates These include 1) the mediation by
cM~ of prostaglandins i~1ibition of platelet agsregation (7)(10)
2) the mediatio~ by Ci~1P of insulins induction of glucose upt~~e in
hep~tic cells (11) ) its co~plex effects on ion transport and ma~brnne
potentials in various cell types (18)(25)(17) and 4) its stimulation of
lipolysis in ht~ and rat fat cells (27)
Because cyclic ffi in1 tictes the (lction alld release of many hormones
it has become knowIl as a ubiquitous second messengertl bull Adenyl cyclase
is activated when an external stimulus such as a hormone comes in con-
tact 111th the cell surface The inCleae in intracellular levels of
cyclic lIP activate other specific enzymes ACTIJI s stiI1ulation of
3
adenyl cyclase in the adrenal cortex follolled by the activation of enzymes
synthesizinEcortiaol exenpliiies this (12) Also thyroid hormone secretion
is induced middothen TSH activates thyroid adenyl cyclase (13)
lhere is some evidence cM may in a sirilar manner mediate
the synthesis ~f prostaglandins (10) EPinephrine and nore~inephrine both
known to stimulcte ade~yl cyclase in certai~ cells stimulate release of
prostaglandins from the dog sp~een (14) In addition it has been shown
prostaglandins stL~u1ate adenyl cyclase in human platelets (7)
The question arose -lhether soe of the effects of aspirin might
be mediated by its effects on the ~~1P systems of various target cells
In order to determine whether a relationship exists between the action
of aspirin ald cyclic ANP the effect of salicylates on j3-galactosidase
synthesis in the bacterial ce11 was obs~rved Synthesis of (3 -galactoshy
sidase in Jscherichia coli is known to require cyclic AImiddotrp (15) In
E COli c~middot~ acts at the transcriptional level of enz~ne synthesis by
promoting initiation of ~-galactosidase nu~fA synthesis The nucleotide
is believed to activate a protein called cMP receptor protein which
in turn allows the protein to bind to the promoter reGion of the lac
operon Its presence enhances the binding oflli1A pol~erase to the
proper initiation site of lac DNA (6) COllsequentlyincreased syntheshy
sis of B-galactosi(ase occurs with optimum concenlirations of cAllP and
repreSSion occurs ~hen the ct1middotp concentration is louered
This paplusmner reorts the effects salicylates have on j3-galactosishy
dase SJIlthesis in E coli and the relationship between cAllPs and salicylic
acid t s mechanism of ac tion within the bacterial cell
YJATERIALS AND NlTHODS
Bacterial strains
For the initial experiments Escherichia coli strain 3000 (lac i +)
was used To determine salicylates effect on inducer uptake a conshy
stitutive strain 3300 (lac i -) was used Both strains llere provided by
Dr John 11 Hyers
Grmmiddotrth Conditions
Bacteria liera grown in double strength Hinimal Broth Davis
medium (Bacto) supplelllented k1th 0002 vitamin B and either 02
glucose or 05 glycerol Double strength media lIas used since this
has essentially the same constituents as the minilal medium used in
earlier experiments observing effects of cyclic AmiddotU on E coli (7)
-Before each experiment fres11 stationary cultures were prepared
from bacteria1 slants StatioIlaty cultures were inoculatr-d into
Klett Flasks to obtain an initia1 ceU density of 10-8ml ald gr01ID on
a shaker at 370 C until log phase was reached To induce fl-galactoshy
sidase -syntheSis isopropyl j3-D-thiogalactopyranoside (rrrG) was added
to the cells in log phase producing a final concentration of 5xl0-4 11
FUrther a1ditions depending on the experiment liere made to log phase
cultures
All cell denSity measurements were taken on a K1ett-~~erson
Photoelectric Colorimeter (Model 80003) using a 660 mp filter
5 ~Galactosida3e Ass~
j3-galactosidase was assayed according to the procedure of Pardee
Jacob and Honod (6) with slight Iilodification At specific timeslt
09 ml of sample lUlS ldthdrakll fro-n the cultures and added ~o tubes
containing 2 drops of 025 sodiurn dodecyl sulfate (SOO) end 2 drops
chloroform (9) Both toluene treatment and SDS - chloroform treatshy
ment were pre~narily tested to deterwine the best method of releasshy
ing enzyme from the middotcell The latter did not alter enzyme stabilitY
and proved to increase the sensitivity of the assq
The amount of enzyme present was measured spectrophotomatr1cally
using a KJett-5UlllIllerson Photoelectric Colorimeter (lmiddotiodel 600-3) witil a
420 111)I1ilter One unit of enzyme is defined as the amount producing
1 II1j1 mole of o-nitrophenol per minute at 280 0 pH 70 The units of
enzyme in the sample can be calculated from the 1act 1 17fL mole per ml
of o-nitrophenol has a Klett reading of 045 under experirlental condishy
tions used here
To verify the assay procedure With E coJ strain 3000 enzyme
activity was confirmed to be linear in relation to time and concentrashy
tion
l-Iaterials
All chevicals were commercially obtained Cyclic adenosine
3 5-monophosphatie was obtained from Sigma and its purity 1-1as
confirmed chromatographically and spectrophotometrlcally Based on
readings vy a Cry Spectrophotometer the cyclic ffJ proved to be
99 pure b-leight Cyclic AHPs biolobical activity was confiIr4ed by
its ability to reverse glucose repression
RESULTS
Tho Effects of Salicylates on 13-GaJactosidase Synthesis
Various concentrations of aspirin were added to E coli strain
3000 cultures grmrlng on glycerol medium At a 5 x 10-3f concentration
ofacetylsa1i~lic aci~the ~fferential rate of j3-galactosidase synshy
thesis decreased (Table I) This 1-1aS accompanied by a slight decrease
in bacterial growth rate At 1 x 10-~ and 25 X 10-3M aspirin had
no significant effect on j3-galactos1dase synthesis
Sinceaspirin1s pharmacological action depends on its salicylic
acid content (1) the effect of salicylic acid on j3-galactosidase synshy
thesis ~1as detemined At all concentrations salicylic acid produced
a greater effect on the differential rate of enzyme synthesis than
aspirin (Table II) Salicylic acid also inhibited grorrth to a considershy
able extent but in all cases the effect on enzyme synthesis was
greater
Control experiments showed both aspirin and salicylic acid had
no effect on the enz~e ass~ system
Since the utjlization of glycerol requires the inducible enzoJlne
glycerol kinase kriown to be sensitive to catabolite repression (~)
it Has postulated the effect of salicylic ~cid on grouth rate resulted
from an inhibition of glYcerol kinase synthesis Such an inhibition
would have the effect of masking decreases in the differential rate
of ~-galactosidcse synthesis For this reason it was decided to use
a carbon source not subject to catabolite repreSSion and presumably
-7
TABLE I
EFFECTS OF ASPIRIN on
DrFFEIl3NTIAL RATE OF SYNTHESIS OF P-GALACTOSIDASE
Elcp 170
Aspirin (N)
Diff Rate En~ Synth (EUrilLKR)
at Control Rate
Grol-rth Rate (1gen time)
of Control Rate
1 1 -( cO3)1x10
25x10-3 5xl0-3
21 -( con3~)5x10shy
3 I -(con3- ) 5xl0shy
361 100 361 100 361 100 254 70
313 100 210 67
33bull3 100 259 78
72hr
63hr bull67hr 60br
100 87 93 825
65hr
54hr 100
83
67hr
58hr 100 87
E coli strain 3000 1-las grOl-m in glycerol raedium until log phase was obtained At -rms time the culture was divided and transferred to fiasks containing 5 x 10- H IPTa ard the indicated amount of dissolved aspirin The control flask contained water in place of the dissolved aspirin solution To insure dilutions would not 1irrdt the media flasks were also initi~ supplemented vnth 4x y~~ Broth Davis
At varioUs cell densities 09 ml samples were taken and assayed for j3-Galactosidase activit7
8
Exp No
1
2bull
3
TABLE II
EFFECT OF SALICYLIC ACID ON
DIFFER11ITIAL It4TE OF SYNTHESIS OF J3-GALACTOSID~sE
Salicylic Acid (N)
-(contro1) x 10shy
-(contro~23 387 x 10
-(contrpound)1 x 10
DtiL Rate ~~th (EUII1lJb Iffi) of
control rate
Growth Rate (ligen time) of
Control
370 62
100 n
60hr
33hr 100
283 65
100 23
71hr
44Ihr 100 62
315 206
bull100 6
60hr
2hr 100 87
Cells l-ere gr01ffl and sampled as in Table I ITith the exception that disshysolved salicylic acid was added to ce~s in log phase in place of aspirin
9
not subject to salicylic aci~ inhibition In all subsequent eXperiments
glucose lias used as carbon source
Upon the addition of 5 x lO-~ salicylic acid to cells grovID in
glucose media there gtmiddotas an ilnmecliate cessation of a-galactosidase synshy
thesis lasting over a generation of grotrth This l-laS followed by a
recovery of srnthesis but at a decreascdrate relative to the control
(fig 1) tt 1 x 10-~i salicylic acid deceased the differential rate
of enzY1e synthesis but tlere 1-TaS a)parently no lag in synthesis after
its addition
From this data it apears salicjlic acid more effectively represses
j3-galactosidase synthesis in glucose grovID cells than in cells grovm
in glycerol redia It should be noted hOHever the inhibitory effect
of salicylic acid on grot-lth rate is the srune vJhether the carbon source
is glucose or glycerol The reason for this inhibition ra1lains obscure
The strect or CYclic AlP on Heprcssion by Snlicylic Acid
If salicylic acid acts by decreasing cyclic PlW levels then cpJmiddot~
should revese the efects of se1icylates Figure 2 shms the resiuts
of rul experinent lihere 5 x lO-3M cyclic AHP and 25 x 10-31-1 salicylic
acid were added silulta1eously to glucose grotm cells It can be seen
that cPJP revJrses the repression caused by salicylic acid Hm1ever
the reversal is only partial since the dif~erential ra-ce is not restored
to the level of the control plus c~tmiddotp cells
Salicylic acid in these experi~ents also caused a decrease in
gromh rate and it lla3 further decreased when cAt-1P las added to the
cells Because of this the data from Figure 2 was replotted with time
units on the abscis3a in place of cell density (Fig 3)~ This allows
10
FIGURE 1
EFFECT OF SLICYLIC ACID ON THE DIFF3P3NrIAL RJTE OF [3-GALACTOSIDA3E SYNTHESIS
strain 3000 Was grovffi in glucose modiUI1 until log phase wst reached
Approrinate1y 1 hour follOldn~ induction rlith IPTG the culture was
separated and trs1sierred to flasks containing the indicted additions
Samples uere talten and assayed for j3-galactosidase as in Table I
Since diaBo1ved salicylic acid lias added a dilution in cell densitr
occurred The first point on the graph represents samples taken L~Jnedi-
ately c)fter the culture Was separatedand diluted
Grorth Rate Differential Enz Rate Flask A Control 75hr 308 EUllilAKR
B Salicylic Acid 68hr 180 EUlllJfoKR
(1 x 10-31)
o Sa1icylic Acid 35hr 95 EUralAlffi (5 x 10-31-1)
12
FIGlllE 2
EFF3Cr OF CYCLIC AmiddotlP 1jJ 3lLICYLIC ACID ON DIFF~JTIAL f3-01AClOSImE SYUTH33IS
IFTG was added to a log phase culture of strain 3000 grown in glucose
medium Approximately 45 minutes after induction the culture was divided
equally into flasks containing the indicated additions 32J1lplcs were
ta~en and ~-Galactosidase was assayed as previously indica~ed The
arroH at 30 Klett unts represents the time the culture ylaS divided
and trcmsferred
DifLerential Rate Growth tate Enzyme Synthesis
Flask A Control bull67hr 23 lJrrJ KIt
B Cyclic mp (5xlO-311) 57hr 315EUrr~6KR
c SalicyliQ acid 4lhr 135ZUmlKR(25xlO-)N)
D Salicy~c acid bull 35hr 25 ~1JrrJLKR 25xlO ~ 11 + CJllmiddotfP (5xlO-)~1)
+
0
t
o
oft
$+
~
Ft flJ rsi
m
m
mlt
l ~ ~
sect4( ~
m~ ~ e1
54 PI rd ISJ
aI ttl
14
FIGURE 3
EFF~T OF CYCLIC AHP UID SJLICYLIC ACID ON j)-GLACTO3IDASE SYNTIIESIS
This represents the smne experireental data as in Figure 1 In the
graph enz~e units ~re plotted in rel~tion to tL~e rather th~n in
relation to cell denitr rIle arrml indicates the time the culture
lras divided and all additions l-Tere i1ade
bull
bullbull Q
-Ld
bull
16 the effect of 321icylic acid ~o be noted indeendently of itsmiddot effect
on generlttion time Even on this graph cAli is shown to partially
relieve repression caused by salicyla~es
Previously Pastaa ~md PerL1an (21)(22) emphasized cAHP had no
effect on growth rate of E coli strain 300 rlhich 112S in contrast to
these results Their dltta homver referto using 1 x lO-3z cANP
Since 5 x 10- 3r crolic lp is believed to overcome both tranient and
catabolite reression caused by r~ucose in strain 3000 (23) the effect
of this concentration on salicylic acids repression was observed
Sirlilar results Nere obtcgtined in a second eXgerimcnt using
S x 10-31-1 salicylic acid and 5 x lO-3H Cl]~ (Fig 4) rhe degree of
reversal ias again only partial Because grorlth inhibition at this conshy
centration of salicylic acid tdth cAHP ias so great the curve is not
extended ver ffX
From these data it appears there is an interaction between the
effects of sali~Jlic acid and those of cMmiddotP Since repression proshy
duced by the higher concentration of salictlic acid seems to be less
effectively reversed by 5 x 10-3N cMP there i~ a possibility the
degree of interaction between the tlO may be concentration dependent
The 3ffect of Sdic-lates on Consti tutive Enzyme Synthesis
To eliminate the possibility salicylic acid may repress j3-galactoshy
sidase synthesis by preventing inducer upt~~e or by competing with
inducer for repressor an experiYlent vTaS carried out using strain 3300
constitutive for fo-galactosidase synthemiddotsis From Figure 5 it is evident
that 25 x 10-311 Jalicylic acid had the same effect on strain 3300 as it
had previously on strain 3000 Therefore salicylates act independently
17
B
uJ ~ l ~ l cJ J L o o- Ul t z 3 w r N Ia Z 312L fa m sa 1m m Em2 ru KLETI UNITS
EFFECT OF CYCLIC Almiddotp lm SLICYLIC ACID OU DIFFrRENfIAL j2gt-GALACTOSIDA3E SYNlHESIS
Figure 4 Strain 3000 culture las grOiID sampled and assayed for fl-g3~actosidase as in Figure 2 uitIl the exception a hig1er concentrC)tion of salic7l1ic acid i-as added after separshyation Since sliicylic acid 35 in dissolved form a dilution occurred upon its addition This uas compensated for in
J
-fA
other fiCsls by an equal dilution
flask Pt Control
Bbull Control + cAHP (5xlO-3H)
c Salicl~ic acid (5xlO H)
D salicl~c acid
Grmmiddotrth Rc1te 71hr
52hr
22hr
15hr (5xlO 3 I) + cMi (5xlO- 1middot1)
Diff Enz Rate 22 EUmlAKR
31 t1Jm1~KR
12 1UmlAIffi
16 EUmlAKR
18
FIGUR35
EFECr 01 SALICYLIC ACID OH DIIT2HE~rrAL fgt-GAL~C0SIDASE SYIJIl133I3 nr COnSrITlJlIV3TAIH 3300
strain 3300 iolaS gron in glucose medium Jllen the cells reached log
phase25 x lO-~1 snlicylic acid ~as added Cells were sa1pled ltmd
4asJared 80S in earlier experiments IPTG (5 x 10- 1-1) liaS added to
control flasks to note the degree of const~tutiveness of tile strain
The first points ngain represent sa~les trucen in~ediately poundter the
culture was divided and additions ware made
Growth Rate Diff Enz Synth Flask A Control bull89hr 65 EUrnlAKR
B Contro~ + IPTG bull89hr 65 EUmJ6KR (5xlO-~middotI)
o SalicYli~3acid bull54hr 46 ~iJ~KR (25x 10 H)
D Sa1icrli~ acid 5hhr 30 EUml~KR (25xlR--i-r) + IPTG (5xlO- M)
lSi JT1
bull 0
Fa-
I
a +
tsJ
yen
+
~
v
+
tsi IS1 m
m
tn
til sectll
m ~ ~ 2
bull
ef ~
~ ~ tsJ
uf
shy
20
of inducer uptake and of inducer-s derepression of lac mrrNA synthesis
The zrecl of 24-Di1ikophcnol ont30alactosidase Synthesis
Since salicylates are kno-Vffi to be uncouplers of oxidative phosshy
phorylation (5) it has been sJgccsted sOrle of their phaITlClcological
effects result from their uncoupling action Tis i-lould irply that
other uncouplers should preferentially inhibit ~-gilactosidase syntheshy
sis For this reason the effect of 2 4-dini trophenol (Dr~) on J-galacshy
tosidase synthesis Has observed Because DrIP is more effective in inhibishy
ting oxidative phosphorylation than salicylic acid and is consequently
more toxic a concentration of the drug i-laS found vhic uould inhibit
grOl-rth to ap~roximately the sa11e degree as 25 x 10-31gt1 salicylic acid
This was found to be 25 x 10-411 Figure 6 illustrates DNP does not
repress differential synthesis of j3-g1actosidase This implies that
althoueh DNP 1m-lers ATP levels there is no apparent effect of the drug
on lsvels of cAl-IP Also it appears salic-rlic acid is not exerting its
repression of enzyrre sJ~~hesis by inhibiting oxidative phosphorylation
21
FIGUllS 6
EFFECrl OF DJP ON DIFFif2rIlL ~GLCiO3D~S STImiddotrYliESIS
Jhen strain 3000 cells reacled log ph~se they tmiddotrere indlccd 1ith IPTG
AproxL~ately one hour later the culture was divide1 a~d tr~ferred
to flasks containing the indicated adlitions The cellsas in preVious
experirrents Tere sarlpled at various tLTIes and assayed for B-galactoshy
sidase activity Since 2h-dinitrophenol t-rag in solution its addition
resuted in dilution of cultures This dilution las equally conpensated
for in control flasks nle first points on the graph represent samples
taken innediately after sepration of the cuJture
GroYTth Rate Difierential inz Rate Flask A Control 75hr 22 L1JmlAKR
B Contro + cAll 64hr 50 EUmlAm (5y10- H) 4z
C DNP (25xlO- -I) 42hr 22 EOIraAKR
D DNP (25xl2~4zr) + 29hr 42 UmlAKR cAJP (5x10 -0
N
N
v
o
-shy-shy+
-shy
+
bull-
DISCU3SION
Salicylic acid ruld to a lesser extent aspirin cause repression
of f3-gclactosidase srnthesis in scherichia coli This effect is not
due to an inhibition of inducer uptake nor is it due ~o interference
l-rith inducers inactivation of lac repressor since strnins constitutive
for 3-gJactosidase 3J1lthesis are also repressed b~r sclicyli~ acid
In addition because dinitrophenol is liithout effect on differential
fl-galactosidase synthesis it appecrs salicrlates do not repress enzyme
s~mthesis by inhibiting oxidative phospllorylation
At these concentr2tions however salicylic acid does decrease
growth rate and direct or indirect effects of this on the observed
repression of enzyme synt~esis still ra~ain Since the differential
rate of enzyme synthesis i~ decreased and also since 25 x 10-411 DN
decreases grotrth rate aproximately the same degree middotuitl1cut having I
effects on differential synthesis salicylates inhibition of growth
is not the sole factor responsible for its effects on enZJcre synthesis
-11en 5 x 10-31 cyclic JUfP 1-ms added to cells treated Kith 25 x 10-3
and 5 x lO-3H srJicylic acid repression of differential enzye s~1lhesis
1ES pextially relieved This sugests salicllic acid represses J3-gJ~ac-
tosidase srnthesis br directly 1011erinti intIacellular levels of cyclic AMP
or by indirectly acting on asita which in turn releases cANP from the
cell Since cMP only partially relieves repression caused by wlicylic
acid and because there appears to be an inverse relationship between
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
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RpoundPRs3Imi OF j3-GALtICrOSIDASE SYUiHESIS
IN SCIE1ITCHIA COLI BY SLIcLATES
by
JOAN CARLY OLSDrl
bull A thesis subnitted in partial tuliilL1cnt of the
requira~ents tor the degree opound
luSTER OF S8r DICE
in
BIOLOGY
Portland State Univelsity 1972
PORTlAND STATE UNIVERSITY UBliARl
TO THZ OFrICB OF GDUArS STUDI2S lJD RZ33A1CH
The re~~ers of the COr~~ttee a~rove the thesis of
~oan Carlyn Olson presented July 28 1972bull
John i llyers Chailan
Barl Fisher bull
1fiJ ~~~~~
GordonL Kilgour
AFPROE~1
Earl risher Head Dea=~ent of BioloGY
David r Clari Dean of Gr~dlace St1dies
July 28 1972
TABLE OFmiddot CONTENTS
PAGE
LI3r OF fABLES iv
LIST FIGURSS v
GI~PTEa
I INrODUCITON 1
II iiflTSRIALS ANTI HSTHODS
B~cterial strains h
Grouth Conditions 4
~-Gnlaco~oridase Assar
rEaterials
III RESULTS
The Effects of Salicr1ates on j3-G2Jactosidase Synthesis 6
The Zifcct of Cyclic AfP on Repression by Salicylic Acid 9
The Effect of Salicylatcs on Gonsti tutivo xzyme S~rnthesis 16
The Effect of 24-Dinitro~hcnol on j3-Gllactosidase Synthesis
IV DISCUSSION ~3
26BIBLIOGRAPHY
28ltIP lZmIX
LIST OF FIGURZS
FIGURE PAGE
1 Effect of Salicylic Acid on Differential Rate opound
--Galactosidase Synthesis 11
2 Effectof Cyciic tjp e1d Salicylic Acid on Differential
f3 -Gal~cto3idase Synthesis 13
3 ~ffect of Cyclic 1lmiddotiP and Salicrlic Acid on (3Galactoshy
sidase Synthesis 15
h Effect of cJclicMi cud Salicylic Acid on j3-Galactoshy
sidase Synthesis 17
5 Sffect of Salicylic Acid on DLfferelltial 13 -Galactosidase
~~~thesis in Constitutive Strain 3300 19
6 Effect of mJP on Differential f3 -Galactosidase Synthesis 22
LIST OF TABLES
TABLE PAGE
I
II
Effects of Asoirin on Differe~tial Rate o~ Synthesis
of 3-Galactosidase
Effect of Salicylic Acid on DifferenGial Rate of
Synthesis of fl-Galactosidase
7
8
INrrWDUCTION
Although aspirin is one of the EOSt commonLy us~d drugs its
actual nechnnism of action within the body still remains unclear
Acetylsalicylic acid belongs to the group of drugs referred to as salishy cylates all of which have SLllilar pharmacological properties rheir
activity is believed to be dependent on the drugs salicylic acid conshy
tent and substitutions on the carboxyl or hydroxyl groups alter only
their toxicity or potency (1)
Aspirin is most COnt~only recognized for its analgeSiC antipyreshy
tic and antiirulamTatory effects Recently Vane (2) demonstrated
antiinflan~atorJ acids such as aspirin salicylic acid mld indoshy
methaCin inlJibit enzymes which catalyze prostaglandin (PGE2 and PGF2~)
synthesis in cell free extracts of guinea pig lungs PG11 and PG~
when injected into the third ventricle of cats also proves to be a
potent inducer of fevers (2) Thus Vane postulated aspirin produces
its an~ipyretic and antiinf1aw~tory responses by inhibiting synthesis
of prostaglandins ~~though prostaglandins when administered intradershy
m2~ly in nan do not produce pain (2) some of aspirins ~~gesic activity
may result from reduction of inllt3ll111ation (3) Smith and lillis (4)
supryorted this evidence when they independently observed aspirin to
inhibit conversion of arachidonic acid into prostaglandins in h~~an
platelets
All abbreviations used in text are defined in appendix
2
Aspirin has many other diverse actions within the body Included
among these is its abllity to prevent collagen and epinepnrine induced
aggregation of platelets Platelet ageregation occurs in two phasesa
The first is characterized by a rapid change in the shape of platelets
the second or actual agflegation of cells follol1s the release of ADP
fram platelets (10) Aspirin blocks the second phase of agcregation
(7)(24) This again relates the actions of aspirin to prostaglandins
for POE and PG~ are also known to have effects on platelet aggregation1
Other actions of aspirin are its ability to lower blood sugar in
certain diabetic ~atients (1) to alter ion transport and membrane potenshy
tial in v8ried cell types (1)(16) to reduce lipogenosis and depress
free fatty acid release in muscle tissue (1) and tomiddot uncouple oxidative
phosphorylation (5)
lnother compound cyclic adenosine )5-mollophosphate (cUP) has
also been shmmiddotrn to have a broad spectrum of effects at sites similar to
those acted upon by salicylates These include 1) the mediation by
cM~ of prostaglandins i~1ibition of platelet agsregation (7)(10)
2) the mediatio~ by Ci~1P of insulins induction of glucose upt~~e in
hep~tic cells (11) ) its co~plex effects on ion transport and ma~brnne
potentials in various cell types (18)(25)(17) and 4) its stimulation of
lipolysis in ht~ and rat fat cells (27)
Because cyclic ffi in1 tictes the (lction alld release of many hormones
it has become knowIl as a ubiquitous second messengertl bull Adenyl cyclase
is activated when an external stimulus such as a hormone comes in con-
tact 111th the cell surface The inCleae in intracellular levels of
cyclic lIP activate other specific enzymes ACTIJI s stiI1ulation of
3
adenyl cyclase in the adrenal cortex follolled by the activation of enzymes
synthesizinEcortiaol exenpliiies this (12) Also thyroid hormone secretion
is induced middothen TSH activates thyroid adenyl cyclase (13)
lhere is some evidence cM may in a sirilar manner mediate
the synthesis ~f prostaglandins (10) EPinephrine and nore~inephrine both
known to stimulcte ade~yl cyclase in certai~ cells stimulate release of
prostaglandins from the dog sp~een (14) In addition it has been shown
prostaglandins stL~u1ate adenyl cyclase in human platelets (7)
The question arose -lhether soe of the effects of aspirin might
be mediated by its effects on the ~~1P systems of various target cells
In order to determine whether a relationship exists between the action
of aspirin ald cyclic ANP the effect of salicylates on j3-galactosidase
synthesis in the bacterial ce11 was obs~rved Synthesis of (3 -galactoshy
sidase in Jscherichia coli is known to require cyclic AImiddotrp (15) In
E COli c~middot~ acts at the transcriptional level of enz~ne synthesis by
promoting initiation of ~-galactosidase nu~fA synthesis The nucleotide
is believed to activate a protein called cMP receptor protein which
in turn allows the protein to bind to the promoter reGion of the lac
operon Its presence enhances the binding oflli1A pol~erase to the
proper initiation site of lac DNA (6) COllsequentlyincreased syntheshy
sis of B-galactosi(ase occurs with optimum concenlirations of cAllP and
repreSSion occurs ~hen the ct1middotp concentration is louered
This paplusmner reorts the effects salicylates have on j3-galactosishy
dase SJIlthesis in E coli and the relationship between cAllPs and salicylic
acid t s mechanism of ac tion within the bacterial cell
YJATERIALS AND NlTHODS
Bacterial strains
For the initial experiments Escherichia coli strain 3000 (lac i +)
was used To determine salicylates effect on inducer uptake a conshy
stitutive strain 3300 (lac i -) was used Both strains llere provided by
Dr John 11 Hyers
Grmmiddotrth Conditions
Bacteria liera grown in double strength Hinimal Broth Davis
medium (Bacto) supplelllented k1th 0002 vitamin B and either 02
glucose or 05 glycerol Double strength media lIas used since this
has essentially the same constituents as the minilal medium used in
earlier experiments observing effects of cyclic AmiddotU on E coli (7)
-Before each experiment fres11 stationary cultures were prepared
from bacteria1 slants StatioIlaty cultures were inoculatr-d into
Klett Flasks to obtain an initia1 ceU density of 10-8ml ald gr01ID on
a shaker at 370 C until log phase was reached To induce fl-galactoshy
sidase -syntheSis isopropyl j3-D-thiogalactopyranoside (rrrG) was added
to the cells in log phase producing a final concentration of 5xl0-4 11
FUrther a1ditions depending on the experiment liere made to log phase
cultures
All cell denSity measurements were taken on a K1ett-~~erson
Photoelectric Colorimeter (Model 80003) using a 660 mp filter
5 ~Galactosida3e Ass~
j3-galactosidase was assayed according to the procedure of Pardee
Jacob and Honod (6) with slight Iilodification At specific timeslt
09 ml of sample lUlS ldthdrakll fro-n the cultures and added ~o tubes
containing 2 drops of 025 sodiurn dodecyl sulfate (SOO) end 2 drops
chloroform (9) Both toluene treatment and SDS - chloroform treatshy
ment were pre~narily tested to deterwine the best method of releasshy
ing enzyme from the middotcell The latter did not alter enzyme stabilitY
and proved to increase the sensitivity of the assq
The amount of enzyme present was measured spectrophotomatr1cally
using a KJett-5UlllIllerson Photoelectric Colorimeter (lmiddotiodel 600-3) witil a
420 111)I1ilter One unit of enzyme is defined as the amount producing
1 II1j1 mole of o-nitrophenol per minute at 280 0 pH 70 The units of
enzyme in the sample can be calculated from the 1act 1 17fL mole per ml
of o-nitrophenol has a Klett reading of 045 under experirlental condishy
tions used here
To verify the assay procedure With E coJ strain 3000 enzyme
activity was confirmed to be linear in relation to time and concentrashy
tion
l-Iaterials
All chevicals were commercially obtained Cyclic adenosine
3 5-monophosphatie was obtained from Sigma and its purity 1-1as
confirmed chromatographically and spectrophotometrlcally Based on
readings vy a Cry Spectrophotometer the cyclic ffJ proved to be
99 pure b-leight Cyclic AHPs biolobical activity was confiIr4ed by
its ability to reverse glucose repression
RESULTS
Tho Effects of Salicylates on 13-GaJactosidase Synthesis
Various concentrations of aspirin were added to E coli strain
3000 cultures grmrlng on glycerol medium At a 5 x 10-3f concentration
ofacetylsa1i~lic aci~the ~fferential rate of j3-galactosidase synshy
thesis decreased (Table I) This 1-1aS accompanied by a slight decrease
in bacterial growth rate At 1 x 10-~ and 25 X 10-3M aspirin had
no significant effect on j3-galactos1dase synthesis
Sinceaspirin1s pharmacological action depends on its salicylic
acid content (1) the effect of salicylic acid on j3-galactosidase synshy
thesis ~1as detemined At all concentrations salicylic acid produced
a greater effect on the differential rate of enzyme synthesis than
aspirin (Table II) Salicylic acid also inhibited grorrth to a considershy
able extent but in all cases the effect on enzyme synthesis was
greater
Control experiments showed both aspirin and salicylic acid had
no effect on the enz~e ass~ system
Since the utjlization of glycerol requires the inducible enzoJlne
glycerol kinase kriown to be sensitive to catabolite repression (~)
it Has postulated the effect of salicylic ~cid on grouth rate resulted
from an inhibition of glYcerol kinase synthesis Such an inhibition
would have the effect of masking decreases in the differential rate
of ~-galactosidcse synthesis For this reason it was decided to use
a carbon source not subject to catabolite repreSSion and presumably
-7
TABLE I
EFFECTS OF ASPIRIN on
DrFFEIl3NTIAL RATE OF SYNTHESIS OF P-GALACTOSIDASE
Elcp 170
Aspirin (N)
Diff Rate En~ Synth (EUrilLKR)
at Control Rate
Grol-rth Rate (1gen time)
of Control Rate
1 1 -( cO3)1x10
25x10-3 5xl0-3
21 -( con3~)5x10shy
3 I -(con3- ) 5xl0shy
361 100 361 100 361 100 254 70
313 100 210 67
33bull3 100 259 78
72hr
63hr bull67hr 60br
100 87 93 825
65hr
54hr 100
83
67hr
58hr 100 87
E coli strain 3000 1-las grOl-m in glycerol raedium until log phase was obtained At -rms time the culture was divided and transferred to fiasks containing 5 x 10- H IPTa ard the indicated amount of dissolved aspirin The control flask contained water in place of the dissolved aspirin solution To insure dilutions would not 1irrdt the media flasks were also initi~ supplemented vnth 4x y~~ Broth Davis
At varioUs cell densities 09 ml samples were taken and assayed for j3-Galactosidase activit7
8
Exp No
1
2bull
3
TABLE II
EFFECT OF SALICYLIC ACID ON
DIFFER11ITIAL It4TE OF SYNTHESIS OF J3-GALACTOSID~sE
Salicylic Acid (N)
-(contro1) x 10shy
-(contro~23 387 x 10
-(contrpound)1 x 10
DtiL Rate ~~th (EUII1lJb Iffi) of
control rate
Growth Rate (ligen time) of
Control
370 62
100 n
60hr
33hr 100
283 65
100 23
71hr
44Ihr 100 62
315 206
bull100 6
60hr
2hr 100 87
Cells l-ere gr01ffl and sampled as in Table I ITith the exception that disshysolved salicylic acid was added to ce~s in log phase in place of aspirin
9
not subject to salicylic aci~ inhibition In all subsequent eXperiments
glucose lias used as carbon source
Upon the addition of 5 x lO-~ salicylic acid to cells grovID in
glucose media there gtmiddotas an ilnmecliate cessation of a-galactosidase synshy
thesis lasting over a generation of grotrth This l-laS followed by a
recovery of srnthesis but at a decreascdrate relative to the control
(fig 1) tt 1 x 10-~i salicylic acid deceased the differential rate
of enzY1e synthesis but tlere 1-TaS a)parently no lag in synthesis after
its addition
From this data it apears salicjlic acid more effectively represses
j3-galactosidase synthesis in glucose grovID cells than in cells grovm
in glycerol redia It should be noted hOHever the inhibitory effect
of salicylic acid on grot-lth rate is the srune vJhether the carbon source
is glucose or glycerol The reason for this inhibition ra1lains obscure
The strect or CYclic AlP on Heprcssion by Snlicylic Acid
If salicylic acid acts by decreasing cyclic PlW levels then cpJmiddot~
should revese the efects of se1icylates Figure 2 shms the resiuts
of rul experinent lihere 5 x lO-3M cyclic AHP and 25 x 10-31-1 salicylic
acid were added silulta1eously to glucose grotm cells It can be seen
that cPJP revJrses the repression caused by salicylic acid Hm1ever
the reversal is only partial since the dif~erential ra-ce is not restored
to the level of the control plus c~tmiddotp cells
Salicylic acid in these experi~ents also caused a decrease in
gromh rate and it lla3 further decreased when cAt-1P las added to the
cells Because of this the data from Figure 2 was replotted with time
units on the abscis3a in place of cell density (Fig 3)~ This allows
10
FIGURE 1
EFFECT OF SLICYLIC ACID ON THE DIFF3P3NrIAL RJTE OF [3-GALACTOSIDA3E SYNTHESIS
strain 3000 Was grovffi in glucose modiUI1 until log phase wst reached
Approrinate1y 1 hour follOldn~ induction rlith IPTG the culture was
separated and trs1sierred to flasks containing the indicted additions
Samples uere talten and assayed for j3-galactosidase as in Table I
Since diaBo1ved salicylic acid lias added a dilution in cell densitr
occurred The first point on the graph represents samples taken L~Jnedi-
ately c)fter the culture Was separatedand diluted
Grorth Rate Differential Enz Rate Flask A Control 75hr 308 EUllilAKR
B Salicylic Acid 68hr 180 EUlllJfoKR
(1 x 10-31)
o Sa1icylic Acid 35hr 95 EUralAlffi (5 x 10-31-1)
12
FIGlllE 2
EFF3Cr OF CYCLIC AmiddotlP 1jJ 3lLICYLIC ACID ON DIFF~JTIAL f3-01AClOSImE SYUTH33IS
IFTG was added to a log phase culture of strain 3000 grown in glucose
medium Approximately 45 minutes after induction the culture was divided
equally into flasks containing the indicated additions 32J1lplcs were
ta~en and ~-Galactosidase was assayed as previously indica~ed The
arroH at 30 Klett unts represents the time the culture ylaS divided
and trcmsferred
DifLerential Rate Growth tate Enzyme Synthesis
Flask A Control bull67hr 23 lJrrJ KIt
B Cyclic mp (5xlO-311) 57hr 315EUrr~6KR
c SalicyliQ acid 4lhr 135ZUmlKR(25xlO-)N)
D Salicy~c acid bull 35hr 25 ~1JrrJLKR 25xlO ~ 11 + CJllmiddotfP (5xlO-)~1)
+
0
t
o
oft
$+
~
Ft flJ rsi
m
m
mlt
l ~ ~
sect4( ~
m~ ~ e1
54 PI rd ISJ
aI ttl
14
FIGURE 3
EFF~T OF CYCLIC AHP UID SJLICYLIC ACID ON j)-GLACTO3IDASE SYNTIIESIS
This represents the smne experireental data as in Figure 1 In the
graph enz~e units ~re plotted in rel~tion to tL~e rather th~n in
relation to cell denitr rIle arrml indicates the time the culture
lras divided and all additions l-Tere i1ade
bull
bullbull Q
-Ld
bull
16 the effect of 321icylic acid ~o be noted indeendently of itsmiddot effect
on generlttion time Even on this graph cAli is shown to partially
relieve repression caused by salicyla~es
Previously Pastaa ~md PerL1an (21)(22) emphasized cAHP had no
effect on growth rate of E coli strain 300 rlhich 112S in contrast to
these results Their dltta homver referto using 1 x lO-3z cANP
Since 5 x 10- 3r crolic lp is believed to overcome both tranient and
catabolite reression caused by r~ucose in strain 3000 (23) the effect
of this concentration on salicylic acids repression was observed
Sirlilar results Nere obtcgtined in a second eXgerimcnt using
S x 10-31-1 salicylic acid and 5 x lO-3H Cl]~ (Fig 4) rhe degree of
reversal ias again only partial Because grorlth inhibition at this conshy
centration of salicylic acid tdth cAHP ias so great the curve is not
extended ver ffX
From these data it appears there is an interaction between the
effects of sali~Jlic acid and those of cMmiddotP Since repression proshy
duced by the higher concentration of salictlic acid seems to be less
effectively reversed by 5 x 10-3N cMP there i~ a possibility the
degree of interaction between the tlO may be concentration dependent
The 3ffect of Sdic-lates on Consti tutive Enzyme Synthesis
To eliminate the possibility salicylic acid may repress j3-galactoshy
sidase synthesis by preventing inducer upt~~e or by competing with
inducer for repressor an experiYlent vTaS carried out using strain 3300
constitutive for fo-galactosidase synthemiddotsis From Figure 5 it is evident
that 25 x 10-311 Jalicylic acid had the same effect on strain 3300 as it
had previously on strain 3000 Therefore salicylates act independently
17
B
uJ ~ l ~ l cJ J L o o- Ul t z 3 w r N Ia Z 312L fa m sa 1m m Em2 ru KLETI UNITS
EFFECT OF CYCLIC Almiddotp lm SLICYLIC ACID OU DIFFrRENfIAL j2gt-GALACTOSIDA3E SYNlHESIS
Figure 4 Strain 3000 culture las grOiID sampled and assayed for fl-g3~actosidase as in Figure 2 uitIl the exception a hig1er concentrC)tion of salic7l1ic acid i-as added after separshyation Since sliicylic acid 35 in dissolved form a dilution occurred upon its addition This uas compensated for in
J
-fA
other fiCsls by an equal dilution
flask Pt Control
Bbull Control + cAHP (5xlO-3H)
c Salicl~ic acid (5xlO H)
D salicl~c acid
Grmmiddotrth Rc1te 71hr
52hr
22hr
15hr (5xlO 3 I) + cMi (5xlO- 1middot1)
Diff Enz Rate 22 EUmlAKR
31 t1Jm1~KR
12 1UmlAIffi
16 EUmlAKR
18
FIGUR35
EFECr 01 SALICYLIC ACID OH DIIT2HE~rrAL fgt-GAL~C0SIDASE SYIJIl133I3 nr COnSrITlJlIV3TAIH 3300
strain 3300 iolaS gron in glucose medium Jllen the cells reached log
phase25 x lO-~1 snlicylic acid ~as added Cells were sa1pled ltmd
4asJared 80S in earlier experiments IPTG (5 x 10- 1-1) liaS added to
control flasks to note the degree of const~tutiveness of tile strain
The first points ngain represent sa~les trucen in~ediately poundter the
culture was divided and additions ware made
Growth Rate Diff Enz Synth Flask A Control bull89hr 65 EUrnlAKR
B Contro~ + IPTG bull89hr 65 EUmJ6KR (5xlO-~middotI)
o SalicYli~3acid bull54hr 46 ~iJ~KR (25x 10 H)
D Sa1icrli~ acid 5hhr 30 EUml~KR (25xlR--i-r) + IPTG (5xlO- M)
lSi JT1
bull 0
Fa-
I
a +
tsJ
yen
+
~
v
+
tsi IS1 m
m
tn
til sectll
m ~ ~ 2
bull
ef ~
~ ~ tsJ
uf
shy
20
of inducer uptake and of inducer-s derepression of lac mrrNA synthesis
The zrecl of 24-Di1ikophcnol ont30alactosidase Synthesis
Since salicylates are kno-Vffi to be uncouplers of oxidative phosshy
phorylation (5) it has been sJgccsted sOrle of their phaITlClcological
effects result from their uncoupling action Tis i-lould irply that
other uncouplers should preferentially inhibit ~-gilactosidase syntheshy
sis For this reason the effect of 2 4-dini trophenol (Dr~) on J-galacshy
tosidase synthesis Has observed Because DrIP is more effective in inhibishy
ting oxidative phosphorylation than salicylic acid and is consequently
more toxic a concentration of the drug i-laS found vhic uould inhibit
grOl-rth to ap~roximately the sa11e degree as 25 x 10-31gt1 salicylic acid
This was found to be 25 x 10-411 Figure 6 illustrates DNP does not
repress differential synthesis of j3-g1actosidase This implies that
althoueh DNP 1m-lers ATP levels there is no apparent effect of the drug
on lsvels of cAl-IP Also it appears salic-rlic acid is not exerting its
repression of enzyrre sJ~~hesis by inhibiting oxidative phosphorylation
21
FIGUllS 6
EFFECrl OF DJP ON DIFFif2rIlL ~GLCiO3D~S STImiddotrYliESIS
Jhen strain 3000 cells reacled log ph~se they tmiddotrere indlccd 1ith IPTG
AproxL~ately one hour later the culture was divide1 a~d tr~ferred
to flasks containing the indicated adlitions The cellsas in preVious
experirrents Tere sarlpled at various tLTIes and assayed for B-galactoshy
sidase activity Since 2h-dinitrophenol t-rag in solution its addition
resuted in dilution of cultures This dilution las equally conpensated
for in control flasks nle first points on the graph represent samples
taken innediately after sepration of the cuJture
GroYTth Rate Difierential inz Rate Flask A Control 75hr 22 L1JmlAKR
B Contro + cAll 64hr 50 EUmlAm (5y10- H) 4z
C DNP (25xlO- -I) 42hr 22 EOIraAKR
D DNP (25xl2~4zr) + 29hr 42 UmlAKR cAJP (5x10 -0
N
N
v
o
-shy-shy+
-shy
+
bull-
DISCU3SION
Salicylic acid ruld to a lesser extent aspirin cause repression
of f3-gclactosidase srnthesis in scherichia coli This effect is not
due to an inhibition of inducer uptake nor is it due ~o interference
l-rith inducers inactivation of lac repressor since strnins constitutive
for 3-gJactosidase 3J1lthesis are also repressed b~r sclicyli~ acid
In addition because dinitrophenol is liithout effect on differential
fl-galactosidase synthesis it appecrs salicrlates do not repress enzyme
s~mthesis by inhibiting oxidative phospllorylation
At these concentr2tions however salicylic acid does decrease
growth rate and direct or indirect effects of this on the observed
repression of enzyme synt~esis still ra~ain Since the differential
rate of enzyme synthesis i~ decreased and also since 25 x 10-411 DN
decreases grotrth rate aproximately the same degree middotuitl1cut having I
effects on differential synthesis salicylates inhibition of growth
is not the sole factor responsible for its effects on enZJcre synthesis
-11en 5 x 10-31 cyclic JUfP 1-ms added to cells treated Kith 25 x 10-3
and 5 x lO-3H srJicylic acid repression of differential enzye s~1lhesis
1ES pextially relieved This sugests salicllic acid represses J3-gJ~ac-
tosidase srnthesis br directly 1011erinti intIacellular levels of cyclic AMP
or by indirectly acting on asita which in turn releases cANP from the
cell Since cMP only partially relieves repression caused by wlicylic
acid and because there appears to be an inverse relationship between
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
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TO THZ OFrICB OF GDUArS STUDI2S lJD RZ33A1CH
The re~~ers of the COr~~ttee a~rove the thesis of
~oan Carlyn Olson presented July 28 1972bull
John i llyers Chailan
Barl Fisher bull
1fiJ ~~~~~
GordonL Kilgour
AFPROE~1
Earl risher Head Dea=~ent of BioloGY
David r Clari Dean of Gr~dlace St1dies
July 28 1972
TABLE OFmiddot CONTENTS
PAGE
LI3r OF fABLES iv
LIST FIGURSS v
GI~PTEa
I INrODUCITON 1
II iiflTSRIALS ANTI HSTHODS
B~cterial strains h
Grouth Conditions 4
~-Gnlaco~oridase Assar
rEaterials
III RESULTS
The Effects of Salicr1ates on j3-G2Jactosidase Synthesis 6
The Zifcct of Cyclic AfP on Repression by Salicylic Acid 9
The Effect of Salicylatcs on Gonsti tutivo xzyme S~rnthesis 16
The Effect of 24-Dinitro~hcnol on j3-Gllactosidase Synthesis
IV DISCUSSION ~3
26BIBLIOGRAPHY
28ltIP lZmIX
LIST OF FIGURZS
FIGURE PAGE
1 Effect of Salicylic Acid on Differential Rate opound
--Galactosidase Synthesis 11
2 Effectof Cyciic tjp e1d Salicylic Acid on Differential
f3 -Gal~cto3idase Synthesis 13
3 ~ffect of Cyclic 1lmiddotiP and Salicrlic Acid on (3Galactoshy
sidase Synthesis 15
h Effect of cJclicMi cud Salicylic Acid on j3-Galactoshy
sidase Synthesis 17
5 Sffect of Salicylic Acid on DLfferelltial 13 -Galactosidase
~~~thesis in Constitutive Strain 3300 19
6 Effect of mJP on Differential f3 -Galactosidase Synthesis 22
LIST OF TABLES
TABLE PAGE
I
II
Effects of Asoirin on Differe~tial Rate o~ Synthesis
of 3-Galactosidase
Effect of Salicylic Acid on DifferenGial Rate of
Synthesis of fl-Galactosidase
7
8
INrrWDUCTION
Although aspirin is one of the EOSt commonLy us~d drugs its
actual nechnnism of action within the body still remains unclear
Acetylsalicylic acid belongs to the group of drugs referred to as salishy cylates all of which have SLllilar pharmacological properties rheir
activity is believed to be dependent on the drugs salicylic acid conshy
tent and substitutions on the carboxyl or hydroxyl groups alter only
their toxicity or potency (1)
Aspirin is most COnt~only recognized for its analgeSiC antipyreshy
tic and antiirulamTatory effects Recently Vane (2) demonstrated
antiinflan~atorJ acids such as aspirin salicylic acid mld indoshy
methaCin inlJibit enzymes which catalyze prostaglandin (PGE2 and PGF2~)
synthesis in cell free extracts of guinea pig lungs PG11 and PG~
when injected into the third ventricle of cats also proves to be a
potent inducer of fevers (2) Thus Vane postulated aspirin produces
its an~ipyretic and antiinf1aw~tory responses by inhibiting synthesis
of prostaglandins ~~though prostaglandins when administered intradershy
m2~ly in nan do not produce pain (2) some of aspirins ~~gesic activity
may result from reduction of inllt3ll111ation (3) Smith and lillis (4)
supryorted this evidence when they independently observed aspirin to
inhibit conversion of arachidonic acid into prostaglandins in h~~an
platelets
All abbreviations used in text are defined in appendix
2
Aspirin has many other diverse actions within the body Included
among these is its abllity to prevent collagen and epinepnrine induced
aggregation of platelets Platelet ageregation occurs in two phasesa
The first is characterized by a rapid change in the shape of platelets
the second or actual agflegation of cells follol1s the release of ADP
fram platelets (10) Aspirin blocks the second phase of agcregation
(7)(24) This again relates the actions of aspirin to prostaglandins
for POE and PG~ are also known to have effects on platelet aggregation1
Other actions of aspirin are its ability to lower blood sugar in
certain diabetic ~atients (1) to alter ion transport and membrane potenshy
tial in v8ried cell types (1)(16) to reduce lipogenosis and depress
free fatty acid release in muscle tissue (1) and tomiddot uncouple oxidative
phosphorylation (5)
lnother compound cyclic adenosine )5-mollophosphate (cUP) has
also been shmmiddotrn to have a broad spectrum of effects at sites similar to
those acted upon by salicylates These include 1) the mediation by
cM~ of prostaglandins i~1ibition of platelet agsregation (7)(10)
2) the mediatio~ by Ci~1P of insulins induction of glucose upt~~e in
hep~tic cells (11) ) its co~plex effects on ion transport and ma~brnne
potentials in various cell types (18)(25)(17) and 4) its stimulation of
lipolysis in ht~ and rat fat cells (27)
Because cyclic ffi in1 tictes the (lction alld release of many hormones
it has become knowIl as a ubiquitous second messengertl bull Adenyl cyclase
is activated when an external stimulus such as a hormone comes in con-
tact 111th the cell surface The inCleae in intracellular levels of
cyclic lIP activate other specific enzymes ACTIJI s stiI1ulation of
3
adenyl cyclase in the adrenal cortex follolled by the activation of enzymes
synthesizinEcortiaol exenpliiies this (12) Also thyroid hormone secretion
is induced middothen TSH activates thyroid adenyl cyclase (13)
lhere is some evidence cM may in a sirilar manner mediate
the synthesis ~f prostaglandins (10) EPinephrine and nore~inephrine both
known to stimulcte ade~yl cyclase in certai~ cells stimulate release of
prostaglandins from the dog sp~een (14) In addition it has been shown
prostaglandins stL~u1ate adenyl cyclase in human platelets (7)
The question arose -lhether soe of the effects of aspirin might
be mediated by its effects on the ~~1P systems of various target cells
In order to determine whether a relationship exists between the action
of aspirin ald cyclic ANP the effect of salicylates on j3-galactosidase
synthesis in the bacterial ce11 was obs~rved Synthesis of (3 -galactoshy
sidase in Jscherichia coli is known to require cyclic AImiddotrp (15) In
E COli c~middot~ acts at the transcriptional level of enz~ne synthesis by
promoting initiation of ~-galactosidase nu~fA synthesis The nucleotide
is believed to activate a protein called cMP receptor protein which
in turn allows the protein to bind to the promoter reGion of the lac
operon Its presence enhances the binding oflli1A pol~erase to the
proper initiation site of lac DNA (6) COllsequentlyincreased syntheshy
sis of B-galactosi(ase occurs with optimum concenlirations of cAllP and
repreSSion occurs ~hen the ct1middotp concentration is louered
This paplusmner reorts the effects salicylates have on j3-galactosishy
dase SJIlthesis in E coli and the relationship between cAllPs and salicylic
acid t s mechanism of ac tion within the bacterial cell
YJATERIALS AND NlTHODS
Bacterial strains
For the initial experiments Escherichia coli strain 3000 (lac i +)
was used To determine salicylates effect on inducer uptake a conshy
stitutive strain 3300 (lac i -) was used Both strains llere provided by
Dr John 11 Hyers
Grmmiddotrth Conditions
Bacteria liera grown in double strength Hinimal Broth Davis
medium (Bacto) supplelllented k1th 0002 vitamin B and either 02
glucose or 05 glycerol Double strength media lIas used since this
has essentially the same constituents as the minilal medium used in
earlier experiments observing effects of cyclic AmiddotU on E coli (7)
-Before each experiment fres11 stationary cultures were prepared
from bacteria1 slants StatioIlaty cultures were inoculatr-d into
Klett Flasks to obtain an initia1 ceU density of 10-8ml ald gr01ID on
a shaker at 370 C until log phase was reached To induce fl-galactoshy
sidase -syntheSis isopropyl j3-D-thiogalactopyranoside (rrrG) was added
to the cells in log phase producing a final concentration of 5xl0-4 11
FUrther a1ditions depending on the experiment liere made to log phase
cultures
All cell denSity measurements were taken on a K1ett-~~erson
Photoelectric Colorimeter (Model 80003) using a 660 mp filter
5 ~Galactosida3e Ass~
j3-galactosidase was assayed according to the procedure of Pardee
Jacob and Honod (6) with slight Iilodification At specific timeslt
09 ml of sample lUlS ldthdrakll fro-n the cultures and added ~o tubes
containing 2 drops of 025 sodiurn dodecyl sulfate (SOO) end 2 drops
chloroform (9) Both toluene treatment and SDS - chloroform treatshy
ment were pre~narily tested to deterwine the best method of releasshy
ing enzyme from the middotcell The latter did not alter enzyme stabilitY
and proved to increase the sensitivity of the assq
The amount of enzyme present was measured spectrophotomatr1cally
using a KJett-5UlllIllerson Photoelectric Colorimeter (lmiddotiodel 600-3) witil a
420 111)I1ilter One unit of enzyme is defined as the amount producing
1 II1j1 mole of o-nitrophenol per minute at 280 0 pH 70 The units of
enzyme in the sample can be calculated from the 1act 1 17fL mole per ml
of o-nitrophenol has a Klett reading of 045 under experirlental condishy
tions used here
To verify the assay procedure With E coJ strain 3000 enzyme
activity was confirmed to be linear in relation to time and concentrashy
tion
l-Iaterials
All chevicals were commercially obtained Cyclic adenosine
3 5-monophosphatie was obtained from Sigma and its purity 1-1as
confirmed chromatographically and spectrophotometrlcally Based on
readings vy a Cry Spectrophotometer the cyclic ffJ proved to be
99 pure b-leight Cyclic AHPs biolobical activity was confiIr4ed by
its ability to reverse glucose repression
RESULTS
Tho Effects of Salicylates on 13-GaJactosidase Synthesis
Various concentrations of aspirin were added to E coli strain
3000 cultures grmrlng on glycerol medium At a 5 x 10-3f concentration
ofacetylsa1i~lic aci~the ~fferential rate of j3-galactosidase synshy
thesis decreased (Table I) This 1-1aS accompanied by a slight decrease
in bacterial growth rate At 1 x 10-~ and 25 X 10-3M aspirin had
no significant effect on j3-galactos1dase synthesis
Sinceaspirin1s pharmacological action depends on its salicylic
acid content (1) the effect of salicylic acid on j3-galactosidase synshy
thesis ~1as detemined At all concentrations salicylic acid produced
a greater effect on the differential rate of enzyme synthesis than
aspirin (Table II) Salicylic acid also inhibited grorrth to a considershy
able extent but in all cases the effect on enzyme synthesis was
greater
Control experiments showed both aspirin and salicylic acid had
no effect on the enz~e ass~ system
Since the utjlization of glycerol requires the inducible enzoJlne
glycerol kinase kriown to be sensitive to catabolite repression (~)
it Has postulated the effect of salicylic ~cid on grouth rate resulted
from an inhibition of glYcerol kinase synthesis Such an inhibition
would have the effect of masking decreases in the differential rate
of ~-galactosidcse synthesis For this reason it was decided to use
a carbon source not subject to catabolite repreSSion and presumably
-7
TABLE I
EFFECTS OF ASPIRIN on
DrFFEIl3NTIAL RATE OF SYNTHESIS OF P-GALACTOSIDASE
Elcp 170
Aspirin (N)
Diff Rate En~ Synth (EUrilLKR)
at Control Rate
Grol-rth Rate (1gen time)
of Control Rate
1 1 -( cO3)1x10
25x10-3 5xl0-3
21 -( con3~)5x10shy
3 I -(con3- ) 5xl0shy
361 100 361 100 361 100 254 70
313 100 210 67
33bull3 100 259 78
72hr
63hr bull67hr 60br
100 87 93 825
65hr
54hr 100
83
67hr
58hr 100 87
E coli strain 3000 1-las grOl-m in glycerol raedium until log phase was obtained At -rms time the culture was divided and transferred to fiasks containing 5 x 10- H IPTa ard the indicated amount of dissolved aspirin The control flask contained water in place of the dissolved aspirin solution To insure dilutions would not 1irrdt the media flasks were also initi~ supplemented vnth 4x y~~ Broth Davis
At varioUs cell densities 09 ml samples were taken and assayed for j3-Galactosidase activit7
8
Exp No
1
2bull
3
TABLE II
EFFECT OF SALICYLIC ACID ON
DIFFER11ITIAL It4TE OF SYNTHESIS OF J3-GALACTOSID~sE
Salicylic Acid (N)
-(contro1) x 10shy
-(contro~23 387 x 10
-(contrpound)1 x 10
DtiL Rate ~~th (EUII1lJb Iffi) of
control rate
Growth Rate (ligen time) of
Control
370 62
100 n
60hr
33hr 100
283 65
100 23
71hr
44Ihr 100 62
315 206
bull100 6
60hr
2hr 100 87
Cells l-ere gr01ffl and sampled as in Table I ITith the exception that disshysolved salicylic acid was added to ce~s in log phase in place of aspirin
9
not subject to salicylic aci~ inhibition In all subsequent eXperiments
glucose lias used as carbon source
Upon the addition of 5 x lO-~ salicylic acid to cells grovID in
glucose media there gtmiddotas an ilnmecliate cessation of a-galactosidase synshy
thesis lasting over a generation of grotrth This l-laS followed by a
recovery of srnthesis but at a decreascdrate relative to the control
(fig 1) tt 1 x 10-~i salicylic acid deceased the differential rate
of enzY1e synthesis but tlere 1-TaS a)parently no lag in synthesis after
its addition
From this data it apears salicjlic acid more effectively represses
j3-galactosidase synthesis in glucose grovID cells than in cells grovm
in glycerol redia It should be noted hOHever the inhibitory effect
of salicylic acid on grot-lth rate is the srune vJhether the carbon source
is glucose or glycerol The reason for this inhibition ra1lains obscure
The strect or CYclic AlP on Heprcssion by Snlicylic Acid
If salicylic acid acts by decreasing cyclic PlW levels then cpJmiddot~
should revese the efects of se1icylates Figure 2 shms the resiuts
of rul experinent lihere 5 x lO-3M cyclic AHP and 25 x 10-31-1 salicylic
acid were added silulta1eously to glucose grotm cells It can be seen
that cPJP revJrses the repression caused by salicylic acid Hm1ever
the reversal is only partial since the dif~erential ra-ce is not restored
to the level of the control plus c~tmiddotp cells
Salicylic acid in these experi~ents also caused a decrease in
gromh rate and it lla3 further decreased when cAt-1P las added to the
cells Because of this the data from Figure 2 was replotted with time
units on the abscis3a in place of cell density (Fig 3)~ This allows
10
FIGURE 1
EFFECT OF SLICYLIC ACID ON THE DIFF3P3NrIAL RJTE OF [3-GALACTOSIDA3E SYNTHESIS
strain 3000 Was grovffi in glucose modiUI1 until log phase wst reached
Approrinate1y 1 hour follOldn~ induction rlith IPTG the culture was
separated and trs1sierred to flasks containing the indicted additions
Samples uere talten and assayed for j3-galactosidase as in Table I
Since diaBo1ved salicylic acid lias added a dilution in cell densitr
occurred The first point on the graph represents samples taken L~Jnedi-
ately c)fter the culture Was separatedand diluted
Grorth Rate Differential Enz Rate Flask A Control 75hr 308 EUllilAKR
B Salicylic Acid 68hr 180 EUlllJfoKR
(1 x 10-31)
o Sa1icylic Acid 35hr 95 EUralAlffi (5 x 10-31-1)
12
FIGlllE 2
EFF3Cr OF CYCLIC AmiddotlP 1jJ 3lLICYLIC ACID ON DIFF~JTIAL f3-01AClOSImE SYUTH33IS
IFTG was added to a log phase culture of strain 3000 grown in glucose
medium Approximately 45 minutes after induction the culture was divided
equally into flasks containing the indicated additions 32J1lplcs were
ta~en and ~-Galactosidase was assayed as previously indica~ed The
arroH at 30 Klett unts represents the time the culture ylaS divided
and trcmsferred
DifLerential Rate Growth tate Enzyme Synthesis
Flask A Control bull67hr 23 lJrrJ KIt
B Cyclic mp (5xlO-311) 57hr 315EUrr~6KR
c SalicyliQ acid 4lhr 135ZUmlKR(25xlO-)N)
D Salicy~c acid bull 35hr 25 ~1JrrJLKR 25xlO ~ 11 + CJllmiddotfP (5xlO-)~1)
+
0
t
o
oft
$+
~
Ft flJ rsi
m
m
mlt
l ~ ~
sect4( ~
m~ ~ e1
54 PI rd ISJ
aI ttl
14
FIGURE 3
EFF~T OF CYCLIC AHP UID SJLICYLIC ACID ON j)-GLACTO3IDASE SYNTIIESIS
This represents the smne experireental data as in Figure 1 In the
graph enz~e units ~re plotted in rel~tion to tL~e rather th~n in
relation to cell denitr rIle arrml indicates the time the culture
lras divided and all additions l-Tere i1ade
bull
bullbull Q
-Ld
bull
16 the effect of 321icylic acid ~o be noted indeendently of itsmiddot effect
on generlttion time Even on this graph cAli is shown to partially
relieve repression caused by salicyla~es
Previously Pastaa ~md PerL1an (21)(22) emphasized cAHP had no
effect on growth rate of E coli strain 300 rlhich 112S in contrast to
these results Their dltta homver referto using 1 x lO-3z cANP
Since 5 x 10- 3r crolic lp is believed to overcome both tranient and
catabolite reression caused by r~ucose in strain 3000 (23) the effect
of this concentration on salicylic acids repression was observed
Sirlilar results Nere obtcgtined in a second eXgerimcnt using
S x 10-31-1 salicylic acid and 5 x lO-3H Cl]~ (Fig 4) rhe degree of
reversal ias again only partial Because grorlth inhibition at this conshy
centration of salicylic acid tdth cAHP ias so great the curve is not
extended ver ffX
From these data it appears there is an interaction between the
effects of sali~Jlic acid and those of cMmiddotP Since repression proshy
duced by the higher concentration of salictlic acid seems to be less
effectively reversed by 5 x 10-3N cMP there i~ a possibility the
degree of interaction between the tlO may be concentration dependent
The 3ffect of Sdic-lates on Consti tutive Enzyme Synthesis
To eliminate the possibility salicylic acid may repress j3-galactoshy
sidase synthesis by preventing inducer upt~~e or by competing with
inducer for repressor an experiYlent vTaS carried out using strain 3300
constitutive for fo-galactosidase synthemiddotsis From Figure 5 it is evident
that 25 x 10-311 Jalicylic acid had the same effect on strain 3300 as it
had previously on strain 3000 Therefore salicylates act independently
17
B
uJ ~ l ~ l cJ J L o o- Ul t z 3 w r N Ia Z 312L fa m sa 1m m Em2 ru KLETI UNITS
EFFECT OF CYCLIC Almiddotp lm SLICYLIC ACID OU DIFFrRENfIAL j2gt-GALACTOSIDA3E SYNlHESIS
Figure 4 Strain 3000 culture las grOiID sampled and assayed for fl-g3~actosidase as in Figure 2 uitIl the exception a hig1er concentrC)tion of salic7l1ic acid i-as added after separshyation Since sliicylic acid 35 in dissolved form a dilution occurred upon its addition This uas compensated for in
J
-fA
other fiCsls by an equal dilution
flask Pt Control
Bbull Control + cAHP (5xlO-3H)
c Salicl~ic acid (5xlO H)
D salicl~c acid
Grmmiddotrth Rc1te 71hr
52hr
22hr
15hr (5xlO 3 I) + cMi (5xlO- 1middot1)
Diff Enz Rate 22 EUmlAKR
31 t1Jm1~KR
12 1UmlAIffi
16 EUmlAKR
18
FIGUR35
EFECr 01 SALICYLIC ACID OH DIIT2HE~rrAL fgt-GAL~C0SIDASE SYIJIl133I3 nr COnSrITlJlIV3TAIH 3300
strain 3300 iolaS gron in glucose medium Jllen the cells reached log
phase25 x lO-~1 snlicylic acid ~as added Cells were sa1pled ltmd
4asJared 80S in earlier experiments IPTG (5 x 10- 1-1) liaS added to
control flasks to note the degree of const~tutiveness of tile strain
The first points ngain represent sa~les trucen in~ediately poundter the
culture was divided and additions ware made
Growth Rate Diff Enz Synth Flask A Control bull89hr 65 EUrnlAKR
B Contro~ + IPTG bull89hr 65 EUmJ6KR (5xlO-~middotI)
o SalicYli~3acid bull54hr 46 ~iJ~KR (25x 10 H)
D Sa1icrli~ acid 5hhr 30 EUml~KR (25xlR--i-r) + IPTG (5xlO- M)
lSi JT1
bull 0
Fa-
I
a +
tsJ
yen
+
~
v
+
tsi IS1 m
m
tn
til sectll
m ~ ~ 2
bull
ef ~
~ ~ tsJ
uf
shy
20
of inducer uptake and of inducer-s derepression of lac mrrNA synthesis
The zrecl of 24-Di1ikophcnol ont30alactosidase Synthesis
Since salicylates are kno-Vffi to be uncouplers of oxidative phosshy
phorylation (5) it has been sJgccsted sOrle of their phaITlClcological
effects result from their uncoupling action Tis i-lould irply that
other uncouplers should preferentially inhibit ~-gilactosidase syntheshy
sis For this reason the effect of 2 4-dini trophenol (Dr~) on J-galacshy
tosidase synthesis Has observed Because DrIP is more effective in inhibishy
ting oxidative phosphorylation than salicylic acid and is consequently
more toxic a concentration of the drug i-laS found vhic uould inhibit
grOl-rth to ap~roximately the sa11e degree as 25 x 10-31gt1 salicylic acid
This was found to be 25 x 10-411 Figure 6 illustrates DNP does not
repress differential synthesis of j3-g1actosidase This implies that
althoueh DNP 1m-lers ATP levels there is no apparent effect of the drug
on lsvels of cAl-IP Also it appears salic-rlic acid is not exerting its
repression of enzyrre sJ~~hesis by inhibiting oxidative phosphorylation
21
FIGUllS 6
EFFECrl OF DJP ON DIFFif2rIlL ~GLCiO3D~S STImiddotrYliESIS
Jhen strain 3000 cells reacled log ph~se they tmiddotrere indlccd 1ith IPTG
AproxL~ately one hour later the culture was divide1 a~d tr~ferred
to flasks containing the indicated adlitions The cellsas in preVious
experirrents Tere sarlpled at various tLTIes and assayed for B-galactoshy
sidase activity Since 2h-dinitrophenol t-rag in solution its addition
resuted in dilution of cultures This dilution las equally conpensated
for in control flasks nle first points on the graph represent samples
taken innediately after sepration of the cuJture
GroYTth Rate Difierential inz Rate Flask A Control 75hr 22 L1JmlAKR
B Contro + cAll 64hr 50 EUmlAm (5y10- H) 4z
C DNP (25xlO- -I) 42hr 22 EOIraAKR
D DNP (25xl2~4zr) + 29hr 42 UmlAKR cAJP (5x10 -0
N
N
v
o
-shy-shy+
-shy
+
bull-
DISCU3SION
Salicylic acid ruld to a lesser extent aspirin cause repression
of f3-gclactosidase srnthesis in scherichia coli This effect is not
due to an inhibition of inducer uptake nor is it due ~o interference
l-rith inducers inactivation of lac repressor since strnins constitutive
for 3-gJactosidase 3J1lthesis are also repressed b~r sclicyli~ acid
In addition because dinitrophenol is liithout effect on differential
fl-galactosidase synthesis it appecrs salicrlates do not repress enzyme
s~mthesis by inhibiting oxidative phospllorylation
At these concentr2tions however salicylic acid does decrease
growth rate and direct or indirect effects of this on the observed
repression of enzyme synt~esis still ra~ain Since the differential
rate of enzyme synthesis i~ decreased and also since 25 x 10-411 DN
decreases grotrth rate aproximately the same degree middotuitl1cut having I
effects on differential synthesis salicylates inhibition of growth
is not the sole factor responsible for its effects on enZJcre synthesis
-11en 5 x 10-31 cyclic JUfP 1-ms added to cells treated Kith 25 x 10-3
and 5 x lO-3H srJicylic acid repression of differential enzye s~1lhesis
1ES pextially relieved This sugests salicllic acid represses J3-gJ~ac-
tosidase srnthesis br directly 1011erinti intIacellular levels of cyclic AMP
or by indirectly acting on asita which in turn releases cANP from the
cell Since cMP only partially relieves repression caused by wlicylic
acid and because there appears to be an inverse relationship between
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
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TABLE OFmiddot CONTENTS
PAGE
LI3r OF fABLES iv
LIST FIGURSS v
GI~PTEa
I INrODUCITON 1
II iiflTSRIALS ANTI HSTHODS
B~cterial strains h
Grouth Conditions 4
~-Gnlaco~oridase Assar
rEaterials
III RESULTS
The Effects of Salicr1ates on j3-G2Jactosidase Synthesis 6
The Zifcct of Cyclic AfP on Repression by Salicylic Acid 9
The Effect of Salicylatcs on Gonsti tutivo xzyme S~rnthesis 16
The Effect of 24-Dinitro~hcnol on j3-Gllactosidase Synthesis
IV DISCUSSION ~3
26BIBLIOGRAPHY
28ltIP lZmIX
LIST OF FIGURZS
FIGURE PAGE
1 Effect of Salicylic Acid on Differential Rate opound
--Galactosidase Synthesis 11
2 Effectof Cyciic tjp e1d Salicylic Acid on Differential
f3 -Gal~cto3idase Synthesis 13
3 ~ffect of Cyclic 1lmiddotiP and Salicrlic Acid on (3Galactoshy
sidase Synthesis 15
h Effect of cJclicMi cud Salicylic Acid on j3-Galactoshy
sidase Synthesis 17
5 Sffect of Salicylic Acid on DLfferelltial 13 -Galactosidase
~~~thesis in Constitutive Strain 3300 19
6 Effect of mJP on Differential f3 -Galactosidase Synthesis 22
LIST OF TABLES
TABLE PAGE
I
II
Effects of Asoirin on Differe~tial Rate o~ Synthesis
of 3-Galactosidase
Effect of Salicylic Acid on DifferenGial Rate of
Synthesis of fl-Galactosidase
7
8
INrrWDUCTION
Although aspirin is one of the EOSt commonLy us~d drugs its
actual nechnnism of action within the body still remains unclear
Acetylsalicylic acid belongs to the group of drugs referred to as salishy cylates all of which have SLllilar pharmacological properties rheir
activity is believed to be dependent on the drugs salicylic acid conshy
tent and substitutions on the carboxyl or hydroxyl groups alter only
their toxicity or potency (1)
Aspirin is most COnt~only recognized for its analgeSiC antipyreshy
tic and antiirulamTatory effects Recently Vane (2) demonstrated
antiinflan~atorJ acids such as aspirin salicylic acid mld indoshy
methaCin inlJibit enzymes which catalyze prostaglandin (PGE2 and PGF2~)
synthesis in cell free extracts of guinea pig lungs PG11 and PG~
when injected into the third ventricle of cats also proves to be a
potent inducer of fevers (2) Thus Vane postulated aspirin produces
its an~ipyretic and antiinf1aw~tory responses by inhibiting synthesis
of prostaglandins ~~though prostaglandins when administered intradershy
m2~ly in nan do not produce pain (2) some of aspirins ~~gesic activity
may result from reduction of inllt3ll111ation (3) Smith and lillis (4)
supryorted this evidence when they independently observed aspirin to
inhibit conversion of arachidonic acid into prostaglandins in h~~an
platelets
All abbreviations used in text are defined in appendix
2
Aspirin has many other diverse actions within the body Included
among these is its abllity to prevent collagen and epinepnrine induced
aggregation of platelets Platelet ageregation occurs in two phasesa
The first is characterized by a rapid change in the shape of platelets
the second or actual agflegation of cells follol1s the release of ADP
fram platelets (10) Aspirin blocks the second phase of agcregation
(7)(24) This again relates the actions of aspirin to prostaglandins
for POE and PG~ are also known to have effects on platelet aggregation1
Other actions of aspirin are its ability to lower blood sugar in
certain diabetic ~atients (1) to alter ion transport and membrane potenshy
tial in v8ried cell types (1)(16) to reduce lipogenosis and depress
free fatty acid release in muscle tissue (1) and tomiddot uncouple oxidative
phosphorylation (5)
lnother compound cyclic adenosine )5-mollophosphate (cUP) has
also been shmmiddotrn to have a broad spectrum of effects at sites similar to
those acted upon by salicylates These include 1) the mediation by
cM~ of prostaglandins i~1ibition of platelet agsregation (7)(10)
2) the mediatio~ by Ci~1P of insulins induction of glucose upt~~e in
hep~tic cells (11) ) its co~plex effects on ion transport and ma~brnne
potentials in various cell types (18)(25)(17) and 4) its stimulation of
lipolysis in ht~ and rat fat cells (27)
Because cyclic ffi in1 tictes the (lction alld release of many hormones
it has become knowIl as a ubiquitous second messengertl bull Adenyl cyclase
is activated when an external stimulus such as a hormone comes in con-
tact 111th the cell surface The inCleae in intracellular levels of
cyclic lIP activate other specific enzymes ACTIJI s stiI1ulation of
3
adenyl cyclase in the adrenal cortex follolled by the activation of enzymes
synthesizinEcortiaol exenpliiies this (12) Also thyroid hormone secretion
is induced middothen TSH activates thyroid adenyl cyclase (13)
lhere is some evidence cM may in a sirilar manner mediate
the synthesis ~f prostaglandins (10) EPinephrine and nore~inephrine both
known to stimulcte ade~yl cyclase in certai~ cells stimulate release of
prostaglandins from the dog sp~een (14) In addition it has been shown
prostaglandins stL~u1ate adenyl cyclase in human platelets (7)
The question arose -lhether soe of the effects of aspirin might
be mediated by its effects on the ~~1P systems of various target cells
In order to determine whether a relationship exists between the action
of aspirin ald cyclic ANP the effect of salicylates on j3-galactosidase
synthesis in the bacterial ce11 was obs~rved Synthesis of (3 -galactoshy
sidase in Jscherichia coli is known to require cyclic AImiddotrp (15) In
E COli c~middot~ acts at the transcriptional level of enz~ne synthesis by
promoting initiation of ~-galactosidase nu~fA synthesis The nucleotide
is believed to activate a protein called cMP receptor protein which
in turn allows the protein to bind to the promoter reGion of the lac
operon Its presence enhances the binding oflli1A pol~erase to the
proper initiation site of lac DNA (6) COllsequentlyincreased syntheshy
sis of B-galactosi(ase occurs with optimum concenlirations of cAllP and
repreSSion occurs ~hen the ct1middotp concentration is louered
This paplusmner reorts the effects salicylates have on j3-galactosishy
dase SJIlthesis in E coli and the relationship between cAllPs and salicylic
acid t s mechanism of ac tion within the bacterial cell
YJATERIALS AND NlTHODS
Bacterial strains
For the initial experiments Escherichia coli strain 3000 (lac i +)
was used To determine salicylates effect on inducer uptake a conshy
stitutive strain 3300 (lac i -) was used Both strains llere provided by
Dr John 11 Hyers
Grmmiddotrth Conditions
Bacteria liera grown in double strength Hinimal Broth Davis
medium (Bacto) supplelllented k1th 0002 vitamin B and either 02
glucose or 05 glycerol Double strength media lIas used since this
has essentially the same constituents as the minilal medium used in
earlier experiments observing effects of cyclic AmiddotU on E coli (7)
-Before each experiment fres11 stationary cultures were prepared
from bacteria1 slants StatioIlaty cultures were inoculatr-d into
Klett Flasks to obtain an initia1 ceU density of 10-8ml ald gr01ID on
a shaker at 370 C until log phase was reached To induce fl-galactoshy
sidase -syntheSis isopropyl j3-D-thiogalactopyranoside (rrrG) was added
to the cells in log phase producing a final concentration of 5xl0-4 11
FUrther a1ditions depending on the experiment liere made to log phase
cultures
All cell denSity measurements were taken on a K1ett-~~erson
Photoelectric Colorimeter (Model 80003) using a 660 mp filter
5 ~Galactosida3e Ass~
j3-galactosidase was assayed according to the procedure of Pardee
Jacob and Honod (6) with slight Iilodification At specific timeslt
09 ml of sample lUlS ldthdrakll fro-n the cultures and added ~o tubes
containing 2 drops of 025 sodiurn dodecyl sulfate (SOO) end 2 drops
chloroform (9) Both toluene treatment and SDS - chloroform treatshy
ment were pre~narily tested to deterwine the best method of releasshy
ing enzyme from the middotcell The latter did not alter enzyme stabilitY
and proved to increase the sensitivity of the assq
The amount of enzyme present was measured spectrophotomatr1cally
using a KJett-5UlllIllerson Photoelectric Colorimeter (lmiddotiodel 600-3) witil a
420 111)I1ilter One unit of enzyme is defined as the amount producing
1 II1j1 mole of o-nitrophenol per minute at 280 0 pH 70 The units of
enzyme in the sample can be calculated from the 1act 1 17fL mole per ml
of o-nitrophenol has a Klett reading of 045 under experirlental condishy
tions used here
To verify the assay procedure With E coJ strain 3000 enzyme
activity was confirmed to be linear in relation to time and concentrashy
tion
l-Iaterials
All chevicals were commercially obtained Cyclic adenosine
3 5-monophosphatie was obtained from Sigma and its purity 1-1as
confirmed chromatographically and spectrophotometrlcally Based on
readings vy a Cry Spectrophotometer the cyclic ffJ proved to be
99 pure b-leight Cyclic AHPs biolobical activity was confiIr4ed by
its ability to reverse glucose repression
RESULTS
Tho Effects of Salicylates on 13-GaJactosidase Synthesis
Various concentrations of aspirin were added to E coli strain
3000 cultures grmrlng on glycerol medium At a 5 x 10-3f concentration
ofacetylsa1i~lic aci~the ~fferential rate of j3-galactosidase synshy
thesis decreased (Table I) This 1-1aS accompanied by a slight decrease
in bacterial growth rate At 1 x 10-~ and 25 X 10-3M aspirin had
no significant effect on j3-galactos1dase synthesis
Sinceaspirin1s pharmacological action depends on its salicylic
acid content (1) the effect of salicylic acid on j3-galactosidase synshy
thesis ~1as detemined At all concentrations salicylic acid produced
a greater effect on the differential rate of enzyme synthesis than
aspirin (Table II) Salicylic acid also inhibited grorrth to a considershy
able extent but in all cases the effect on enzyme synthesis was
greater
Control experiments showed both aspirin and salicylic acid had
no effect on the enz~e ass~ system
Since the utjlization of glycerol requires the inducible enzoJlne
glycerol kinase kriown to be sensitive to catabolite repression (~)
it Has postulated the effect of salicylic ~cid on grouth rate resulted
from an inhibition of glYcerol kinase synthesis Such an inhibition
would have the effect of masking decreases in the differential rate
of ~-galactosidcse synthesis For this reason it was decided to use
a carbon source not subject to catabolite repreSSion and presumably
-7
TABLE I
EFFECTS OF ASPIRIN on
DrFFEIl3NTIAL RATE OF SYNTHESIS OF P-GALACTOSIDASE
Elcp 170
Aspirin (N)
Diff Rate En~ Synth (EUrilLKR)
at Control Rate
Grol-rth Rate (1gen time)
of Control Rate
1 1 -( cO3)1x10
25x10-3 5xl0-3
21 -( con3~)5x10shy
3 I -(con3- ) 5xl0shy
361 100 361 100 361 100 254 70
313 100 210 67
33bull3 100 259 78
72hr
63hr bull67hr 60br
100 87 93 825
65hr
54hr 100
83
67hr
58hr 100 87
E coli strain 3000 1-las grOl-m in glycerol raedium until log phase was obtained At -rms time the culture was divided and transferred to fiasks containing 5 x 10- H IPTa ard the indicated amount of dissolved aspirin The control flask contained water in place of the dissolved aspirin solution To insure dilutions would not 1irrdt the media flasks were also initi~ supplemented vnth 4x y~~ Broth Davis
At varioUs cell densities 09 ml samples were taken and assayed for j3-Galactosidase activit7
8
Exp No
1
2bull
3
TABLE II
EFFECT OF SALICYLIC ACID ON
DIFFER11ITIAL It4TE OF SYNTHESIS OF J3-GALACTOSID~sE
Salicylic Acid (N)
-(contro1) x 10shy
-(contro~23 387 x 10
-(contrpound)1 x 10
DtiL Rate ~~th (EUII1lJb Iffi) of
control rate
Growth Rate (ligen time) of
Control
370 62
100 n
60hr
33hr 100
283 65
100 23
71hr
44Ihr 100 62
315 206
bull100 6
60hr
2hr 100 87
Cells l-ere gr01ffl and sampled as in Table I ITith the exception that disshysolved salicylic acid was added to ce~s in log phase in place of aspirin
9
not subject to salicylic aci~ inhibition In all subsequent eXperiments
glucose lias used as carbon source
Upon the addition of 5 x lO-~ salicylic acid to cells grovID in
glucose media there gtmiddotas an ilnmecliate cessation of a-galactosidase synshy
thesis lasting over a generation of grotrth This l-laS followed by a
recovery of srnthesis but at a decreascdrate relative to the control
(fig 1) tt 1 x 10-~i salicylic acid deceased the differential rate
of enzY1e synthesis but tlere 1-TaS a)parently no lag in synthesis after
its addition
From this data it apears salicjlic acid more effectively represses
j3-galactosidase synthesis in glucose grovID cells than in cells grovm
in glycerol redia It should be noted hOHever the inhibitory effect
of salicylic acid on grot-lth rate is the srune vJhether the carbon source
is glucose or glycerol The reason for this inhibition ra1lains obscure
The strect or CYclic AlP on Heprcssion by Snlicylic Acid
If salicylic acid acts by decreasing cyclic PlW levels then cpJmiddot~
should revese the efects of se1icylates Figure 2 shms the resiuts
of rul experinent lihere 5 x lO-3M cyclic AHP and 25 x 10-31-1 salicylic
acid were added silulta1eously to glucose grotm cells It can be seen
that cPJP revJrses the repression caused by salicylic acid Hm1ever
the reversal is only partial since the dif~erential ra-ce is not restored
to the level of the control plus c~tmiddotp cells
Salicylic acid in these experi~ents also caused a decrease in
gromh rate and it lla3 further decreased when cAt-1P las added to the
cells Because of this the data from Figure 2 was replotted with time
units on the abscis3a in place of cell density (Fig 3)~ This allows
10
FIGURE 1
EFFECT OF SLICYLIC ACID ON THE DIFF3P3NrIAL RJTE OF [3-GALACTOSIDA3E SYNTHESIS
strain 3000 Was grovffi in glucose modiUI1 until log phase wst reached
Approrinate1y 1 hour follOldn~ induction rlith IPTG the culture was
separated and trs1sierred to flasks containing the indicted additions
Samples uere talten and assayed for j3-galactosidase as in Table I
Since diaBo1ved salicylic acid lias added a dilution in cell densitr
occurred The first point on the graph represents samples taken L~Jnedi-
ately c)fter the culture Was separatedand diluted
Grorth Rate Differential Enz Rate Flask A Control 75hr 308 EUllilAKR
B Salicylic Acid 68hr 180 EUlllJfoKR
(1 x 10-31)
o Sa1icylic Acid 35hr 95 EUralAlffi (5 x 10-31-1)
12
FIGlllE 2
EFF3Cr OF CYCLIC AmiddotlP 1jJ 3lLICYLIC ACID ON DIFF~JTIAL f3-01AClOSImE SYUTH33IS
IFTG was added to a log phase culture of strain 3000 grown in glucose
medium Approximately 45 minutes after induction the culture was divided
equally into flasks containing the indicated additions 32J1lplcs were
ta~en and ~-Galactosidase was assayed as previously indica~ed The
arroH at 30 Klett unts represents the time the culture ylaS divided
and trcmsferred
DifLerential Rate Growth tate Enzyme Synthesis
Flask A Control bull67hr 23 lJrrJ KIt
B Cyclic mp (5xlO-311) 57hr 315EUrr~6KR
c SalicyliQ acid 4lhr 135ZUmlKR(25xlO-)N)
D Salicy~c acid bull 35hr 25 ~1JrrJLKR 25xlO ~ 11 + CJllmiddotfP (5xlO-)~1)
+
0
t
o
oft
$+
~
Ft flJ rsi
m
m
mlt
l ~ ~
sect4( ~
m~ ~ e1
54 PI rd ISJ
aI ttl
14
FIGURE 3
EFF~T OF CYCLIC AHP UID SJLICYLIC ACID ON j)-GLACTO3IDASE SYNTIIESIS
This represents the smne experireental data as in Figure 1 In the
graph enz~e units ~re plotted in rel~tion to tL~e rather th~n in
relation to cell denitr rIle arrml indicates the time the culture
lras divided and all additions l-Tere i1ade
bull
bullbull Q
-Ld
bull
16 the effect of 321icylic acid ~o be noted indeendently of itsmiddot effect
on generlttion time Even on this graph cAli is shown to partially
relieve repression caused by salicyla~es
Previously Pastaa ~md PerL1an (21)(22) emphasized cAHP had no
effect on growth rate of E coli strain 300 rlhich 112S in contrast to
these results Their dltta homver referto using 1 x lO-3z cANP
Since 5 x 10- 3r crolic lp is believed to overcome both tranient and
catabolite reression caused by r~ucose in strain 3000 (23) the effect
of this concentration on salicylic acids repression was observed
Sirlilar results Nere obtcgtined in a second eXgerimcnt using
S x 10-31-1 salicylic acid and 5 x lO-3H Cl]~ (Fig 4) rhe degree of
reversal ias again only partial Because grorlth inhibition at this conshy
centration of salicylic acid tdth cAHP ias so great the curve is not
extended ver ffX
From these data it appears there is an interaction between the
effects of sali~Jlic acid and those of cMmiddotP Since repression proshy
duced by the higher concentration of salictlic acid seems to be less
effectively reversed by 5 x 10-3N cMP there i~ a possibility the
degree of interaction between the tlO may be concentration dependent
The 3ffect of Sdic-lates on Consti tutive Enzyme Synthesis
To eliminate the possibility salicylic acid may repress j3-galactoshy
sidase synthesis by preventing inducer upt~~e or by competing with
inducer for repressor an experiYlent vTaS carried out using strain 3300
constitutive for fo-galactosidase synthemiddotsis From Figure 5 it is evident
that 25 x 10-311 Jalicylic acid had the same effect on strain 3300 as it
had previously on strain 3000 Therefore salicylates act independently
17
B
uJ ~ l ~ l cJ J L o o- Ul t z 3 w r N Ia Z 312L fa m sa 1m m Em2 ru KLETI UNITS
EFFECT OF CYCLIC Almiddotp lm SLICYLIC ACID OU DIFFrRENfIAL j2gt-GALACTOSIDA3E SYNlHESIS
Figure 4 Strain 3000 culture las grOiID sampled and assayed for fl-g3~actosidase as in Figure 2 uitIl the exception a hig1er concentrC)tion of salic7l1ic acid i-as added after separshyation Since sliicylic acid 35 in dissolved form a dilution occurred upon its addition This uas compensated for in
J
-fA
other fiCsls by an equal dilution
flask Pt Control
Bbull Control + cAHP (5xlO-3H)
c Salicl~ic acid (5xlO H)
D salicl~c acid
Grmmiddotrth Rc1te 71hr
52hr
22hr
15hr (5xlO 3 I) + cMi (5xlO- 1middot1)
Diff Enz Rate 22 EUmlAKR
31 t1Jm1~KR
12 1UmlAIffi
16 EUmlAKR
18
FIGUR35
EFECr 01 SALICYLIC ACID OH DIIT2HE~rrAL fgt-GAL~C0SIDASE SYIJIl133I3 nr COnSrITlJlIV3TAIH 3300
strain 3300 iolaS gron in glucose medium Jllen the cells reached log
phase25 x lO-~1 snlicylic acid ~as added Cells were sa1pled ltmd
4asJared 80S in earlier experiments IPTG (5 x 10- 1-1) liaS added to
control flasks to note the degree of const~tutiveness of tile strain
The first points ngain represent sa~les trucen in~ediately poundter the
culture was divided and additions ware made
Growth Rate Diff Enz Synth Flask A Control bull89hr 65 EUrnlAKR
B Contro~ + IPTG bull89hr 65 EUmJ6KR (5xlO-~middotI)
o SalicYli~3acid bull54hr 46 ~iJ~KR (25x 10 H)
D Sa1icrli~ acid 5hhr 30 EUml~KR (25xlR--i-r) + IPTG (5xlO- M)
lSi JT1
bull 0
Fa-
I
a +
tsJ
yen
+
~
v
+
tsi IS1 m
m
tn
til sectll
m ~ ~ 2
bull
ef ~
~ ~ tsJ
uf
shy
20
of inducer uptake and of inducer-s derepression of lac mrrNA synthesis
The zrecl of 24-Di1ikophcnol ont30alactosidase Synthesis
Since salicylates are kno-Vffi to be uncouplers of oxidative phosshy
phorylation (5) it has been sJgccsted sOrle of their phaITlClcological
effects result from their uncoupling action Tis i-lould irply that
other uncouplers should preferentially inhibit ~-gilactosidase syntheshy
sis For this reason the effect of 2 4-dini trophenol (Dr~) on J-galacshy
tosidase synthesis Has observed Because DrIP is more effective in inhibishy
ting oxidative phosphorylation than salicylic acid and is consequently
more toxic a concentration of the drug i-laS found vhic uould inhibit
grOl-rth to ap~roximately the sa11e degree as 25 x 10-31gt1 salicylic acid
This was found to be 25 x 10-411 Figure 6 illustrates DNP does not
repress differential synthesis of j3-g1actosidase This implies that
althoueh DNP 1m-lers ATP levels there is no apparent effect of the drug
on lsvels of cAl-IP Also it appears salic-rlic acid is not exerting its
repression of enzyrre sJ~~hesis by inhibiting oxidative phosphorylation
21
FIGUllS 6
EFFECrl OF DJP ON DIFFif2rIlL ~GLCiO3D~S STImiddotrYliESIS
Jhen strain 3000 cells reacled log ph~se they tmiddotrere indlccd 1ith IPTG
AproxL~ately one hour later the culture was divide1 a~d tr~ferred
to flasks containing the indicated adlitions The cellsas in preVious
experirrents Tere sarlpled at various tLTIes and assayed for B-galactoshy
sidase activity Since 2h-dinitrophenol t-rag in solution its addition
resuted in dilution of cultures This dilution las equally conpensated
for in control flasks nle first points on the graph represent samples
taken innediately after sepration of the cuJture
GroYTth Rate Difierential inz Rate Flask A Control 75hr 22 L1JmlAKR
B Contro + cAll 64hr 50 EUmlAm (5y10- H) 4z
C DNP (25xlO- -I) 42hr 22 EOIraAKR
D DNP (25xl2~4zr) + 29hr 42 UmlAKR cAJP (5x10 -0
N
N
v
o
-shy-shy+
-shy
+
bull-
DISCU3SION
Salicylic acid ruld to a lesser extent aspirin cause repression
of f3-gclactosidase srnthesis in scherichia coli This effect is not
due to an inhibition of inducer uptake nor is it due ~o interference
l-rith inducers inactivation of lac repressor since strnins constitutive
for 3-gJactosidase 3J1lthesis are also repressed b~r sclicyli~ acid
In addition because dinitrophenol is liithout effect on differential
fl-galactosidase synthesis it appecrs salicrlates do not repress enzyme
s~mthesis by inhibiting oxidative phospllorylation
At these concentr2tions however salicylic acid does decrease
growth rate and direct or indirect effects of this on the observed
repression of enzyme synt~esis still ra~ain Since the differential
rate of enzyme synthesis i~ decreased and also since 25 x 10-411 DN
decreases grotrth rate aproximately the same degree middotuitl1cut having I
effects on differential synthesis salicylates inhibition of growth
is not the sole factor responsible for its effects on enZJcre synthesis
-11en 5 x 10-31 cyclic JUfP 1-ms added to cells treated Kith 25 x 10-3
and 5 x lO-3H srJicylic acid repression of differential enzye s~1lhesis
1ES pextially relieved This sugests salicllic acid represses J3-gJ~ac-
tosidase srnthesis br directly 1011erinti intIacellular levels of cyclic AMP
or by indirectly acting on asita which in turn releases cANP from the
cell Since cMP only partially relieves repression caused by wlicylic
acid and because there appears to be an inverse relationship between
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
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LIST OF FIGURZS
FIGURE PAGE
1 Effect of Salicylic Acid on Differential Rate opound
--Galactosidase Synthesis 11
2 Effectof Cyciic tjp e1d Salicylic Acid on Differential
f3 -Gal~cto3idase Synthesis 13
3 ~ffect of Cyclic 1lmiddotiP and Salicrlic Acid on (3Galactoshy
sidase Synthesis 15
h Effect of cJclicMi cud Salicylic Acid on j3-Galactoshy
sidase Synthesis 17
5 Sffect of Salicylic Acid on DLfferelltial 13 -Galactosidase
~~~thesis in Constitutive Strain 3300 19
6 Effect of mJP on Differential f3 -Galactosidase Synthesis 22
LIST OF TABLES
TABLE PAGE
I
II
Effects of Asoirin on Differe~tial Rate o~ Synthesis
of 3-Galactosidase
Effect of Salicylic Acid on DifferenGial Rate of
Synthesis of fl-Galactosidase
7
8
INrrWDUCTION
Although aspirin is one of the EOSt commonLy us~d drugs its
actual nechnnism of action within the body still remains unclear
Acetylsalicylic acid belongs to the group of drugs referred to as salishy cylates all of which have SLllilar pharmacological properties rheir
activity is believed to be dependent on the drugs salicylic acid conshy
tent and substitutions on the carboxyl or hydroxyl groups alter only
their toxicity or potency (1)
Aspirin is most COnt~only recognized for its analgeSiC antipyreshy
tic and antiirulamTatory effects Recently Vane (2) demonstrated
antiinflan~atorJ acids such as aspirin salicylic acid mld indoshy
methaCin inlJibit enzymes which catalyze prostaglandin (PGE2 and PGF2~)
synthesis in cell free extracts of guinea pig lungs PG11 and PG~
when injected into the third ventricle of cats also proves to be a
potent inducer of fevers (2) Thus Vane postulated aspirin produces
its an~ipyretic and antiinf1aw~tory responses by inhibiting synthesis
of prostaglandins ~~though prostaglandins when administered intradershy
m2~ly in nan do not produce pain (2) some of aspirins ~~gesic activity
may result from reduction of inllt3ll111ation (3) Smith and lillis (4)
supryorted this evidence when they independently observed aspirin to
inhibit conversion of arachidonic acid into prostaglandins in h~~an
platelets
All abbreviations used in text are defined in appendix
2
Aspirin has many other diverse actions within the body Included
among these is its abllity to prevent collagen and epinepnrine induced
aggregation of platelets Platelet ageregation occurs in two phasesa
The first is characterized by a rapid change in the shape of platelets
the second or actual agflegation of cells follol1s the release of ADP
fram platelets (10) Aspirin blocks the second phase of agcregation
(7)(24) This again relates the actions of aspirin to prostaglandins
for POE and PG~ are also known to have effects on platelet aggregation1
Other actions of aspirin are its ability to lower blood sugar in
certain diabetic ~atients (1) to alter ion transport and membrane potenshy
tial in v8ried cell types (1)(16) to reduce lipogenosis and depress
free fatty acid release in muscle tissue (1) and tomiddot uncouple oxidative
phosphorylation (5)
lnother compound cyclic adenosine )5-mollophosphate (cUP) has
also been shmmiddotrn to have a broad spectrum of effects at sites similar to
those acted upon by salicylates These include 1) the mediation by
cM~ of prostaglandins i~1ibition of platelet agsregation (7)(10)
2) the mediatio~ by Ci~1P of insulins induction of glucose upt~~e in
hep~tic cells (11) ) its co~plex effects on ion transport and ma~brnne
potentials in various cell types (18)(25)(17) and 4) its stimulation of
lipolysis in ht~ and rat fat cells (27)
Because cyclic ffi in1 tictes the (lction alld release of many hormones
it has become knowIl as a ubiquitous second messengertl bull Adenyl cyclase
is activated when an external stimulus such as a hormone comes in con-
tact 111th the cell surface The inCleae in intracellular levels of
cyclic lIP activate other specific enzymes ACTIJI s stiI1ulation of
3
adenyl cyclase in the adrenal cortex follolled by the activation of enzymes
synthesizinEcortiaol exenpliiies this (12) Also thyroid hormone secretion
is induced middothen TSH activates thyroid adenyl cyclase (13)
lhere is some evidence cM may in a sirilar manner mediate
the synthesis ~f prostaglandins (10) EPinephrine and nore~inephrine both
known to stimulcte ade~yl cyclase in certai~ cells stimulate release of
prostaglandins from the dog sp~een (14) In addition it has been shown
prostaglandins stL~u1ate adenyl cyclase in human platelets (7)
The question arose -lhether soe of the effects of aspirin might
be mediated by its effects on the ~~1P systems of various target cells
In order to determine whether a relationship exists between the action
of aspirin ald cyclic ANP the effect of salicylates on j3-galactosidase
synthesis in the bacterial ce11 was obs~rved Synthesis of (3 -galactoshy
sidase in Jscherichia coli is known to require cyclic AImiddotrp (15) In
E COli c~middot~ acts at the transcriptional level of enz~ne synthesis by
promoting initiation of ~-galactosidase nu~fA synthesis The nucleotide
is believed to activate a protein called cMP receptor protein which
in turn allows the protein to bind to the promoter reGion of the lac
operon Its presence enhances the binding oflli1A pol~erase to the
proper initiation site of lac DNA (6) COllsequentlyincreased syntheshy
sis of B-galactosi(ase occurs with optimum concenlirations of cAllP and
repreSSion occurs ~hen the ct1middotp concentration is louered
This paplusmner reorts the effects salicylates have on j3-galactosishy
dase SJIlthesis in E coli and the relationship between cAllPs and salicylic
acid t s mechanism of ac tion within the bacterial cell
YJATERIALS AND NlTHODS
Bacterial strains
For the initial experiments Escherichia coli strain 3000 (lac i +)
was used To determine salicylates effect on inducer uptake a conshy
stitutive strain 3300 (lac i -) was used Both strains llere provided by
Dr John 11 Hyers
Grmmiddotrth Conditions
Bacteria liera grown in double strength Hinimal Broth Davis
medium (Bacto) supplelllented k1th 0002 vitamin B and either 02
glucose or 05 glycerol Double strength media lIas used since this
has essentially the same constituents as the minilal medium used in
earlier experiments observing effects of cyclic AmiddotU on E coli (7)
-Before each experiment fres11 stationary cultures were prepared
from bacteria1 slants StatioIlaty cultures were inoculatr-d into
Klett Flasks to obtain an initia1 ceU density of 10-8ml ald gr01ID on
a shaker at 370 C until log phase was reached To induce fl-galactoshy
sidase -syntheSis isopropyl j3-D-thiogalactopyranoside (rrrG) was added
to the cells in log phase producing a final concentration of 5xl0-4 11
FUrther a1ditions depending on the experiment liere made to log phase
cultures
All cell denSity measurements were taken on a K1ett-~~erson
Photoelectric Colorimeter (Model 80003) using a 660 mp filter
5 ~Galactosida3e Ass~
j3-galactosidase was assayed according to the procedure of Pardee
Jacob and Honod (6) with slight Iilodification At specific timeslt
09 ml of sample lUlS ldthdrakll fro-n the cultures and added ~o tubes
containing 2 drops of 025 sodiurn dodecyl sulfate (SOO) end 2 drops
chloroform (9) Both toluene treatment and SDS - chloroform treatshy
ment were pre~narily tested to deterwine the best method of releasshy
ing enzyme from the middotcell The latter did not alter enzyme stabilitY
and proved to increase the sensitivity of the assq
The amount of enzyme present was measured spectrophotomatr1cally
using a KJett-5UlllIllerson Photoelectric Colorimeter (lmiddotiodel 600-3) witil a
420 111)I1ilter One unit of enzyme is defined as the amount producing
1 II1j1 mole of o-nitrophenol per minute at 280 0 pH 70 The units of
enzyme in the sample can be calculated from the 1act 1 17fL mole per ml
of o-nitrophenol has a Klett reading of 045 under experirlental condishy
tions used here
To verify the assay procedure With E coJ strain 3000 enzyme
activity was confirmed to be linear in relation to time and concentrashy
tion
l-Iaterials
All chevicals were commercially obtained Cyclic adenosine
3 5-monophosphatie was obtained from Sigma and its purity 1-1as
confirmed chromatographically and spectrophotometrlcally Based on
readings vy a Cry Spectrophotometer the cyclic ffJ proved to be
99 pure b-leight Cyclic AHPs biolobical activity was confiIr4ed by
its ability to reverse glucose repression
RESULTS
Tho Effects of Salicylates on 13-GaJactosidase Synthesis
Various concentrations of aspirin were added to E coli strain
3000 cultures grmrlng on glycerol medium At a 5 x 10-3f concentration
ofacetylsa1i~lic aci~the ~fferential rate of j3-galactosidase synshy
thesis decreased (Table I) This 1-1aS accompanied by a slight decrease
in bacterial growth rate At 1 x 10-~ and 25 X 10-3M aspirin had
no significant effect on j3-galactos1dase synthesis
Sinceaspirin1s pharmacological action depends on its salicylic
acid content (1) the effect of salicylic acid on j3-galactosidase synshy
thesis ~1as detemined At all concentrations salicylic acid produced
a greater effect on the differential rate of enzyme synthesis than
aspirin (Table II) Salicylic acid also inhibited grorrth to a considershy
able extent but in all cases the effect on enzyme synthesis was
greater
Control experiments showed both aspirin and salicylic acid had
no effect on the enz~e ass~ system
Since the utjlization of glycerol requires the inducible enzoJlne
glycerol kinase kriown to be sensitive to catabolite repression (~)
it Has postulated the effect of salicylic ~cid on grouth rate resulted
from an inhibition of glYcerol kinase synthesis Such an inhibition
would have the effect of masking decreases in the differential rate
of ~-galactosidcse synthesis For this reason it was decided to use
a carbon source not subject to catabolite repreSSion and presumably
-7
TABLE I
EFFECTS OF ASPIRIN on
DrFFEIl3NTIAL RATE OF SYNTHESIS OF P-GALACTOSIDASE
Elcp 170
Aspirin (N)
Diff Rate En~ Synth (EUrilLKR)
at Control Rate
Grol-rth Rate (1gen time)
of Control Rate
1 1 -( cO3)1x10
25x10-3 5xl0-3
21 -( con3~)5x10shy
3 I -(con3- ) 5xl0shy
361 100 361 100 361 100 254 70
313 100 210 67
33bull3 100 259 78
72hr
63hr bull67hr 60br
100 87 93 825
65hr
54hr 100
83
67hr
58hr 100 87
E coli strain 3000 1-las grOl-m in glycerol raedium until log phase was obtained At -rms time the culture was divided and transferred to fiasks containing 5 x 10- H IPTa ard the indicated amount of dissolved aspirin The control flask contained water in place of the dissolved aspirin solution To insure dilutions would not 1irrdt the media flasks were also initi~ supplemented vnth 4x y~~ Broth Davis
At varioUs cell densities 09 ml samples were taken and assayed for j3-Galactosidase activit7
8
Exp No
1
2bull
3
TABLE II
EFFECT OF SALICYLIC ACID ON
DIFFER11ITIAL It4TE OF SYNTHESIS OF J3-GALACTOSID~sE
Salicylic Acid (N)
-(contro1) x 10shy
-(contro~23 387 x 10
-(contrpound)1 x 10
DtiL Rate ~~th (EUII1lJb Iffi) of
control rate
Growth Rate (ligen time) of
Control
370 62
100 n
60hr
33hr 100
283 65
100 23
71hr
44Ihr 100 62
315 206
bull100 6
60hr
2hr 100 87
Cells l-ere gr01ffl and sampled as in Table I ITith the exception that disshysolved salicylic acid was added to ce~s in log phase in place of aspirin
9
not subject to salicylic aci~ inhibition In all subsequent eXperiments
glucose lias used as carbon source
Upon the addition of 5 x lO-~ salicylic acid to cells grovID in
glucose media there gtmiddotas an ilnmecliate cessation of a-galactosidase synshy
thesis lasting over a generation of grotrth This l-laS followed by a
recovery of srnthesis but at a decreascdrate relative to the control
(fig 1) tt 1 x 10-~i salicylic acid deceased the differential rate
of enzY1e synthesis but tlere 1-TaS a)parently no lag in synthesis after
its addition
From this data it apears salicjlic acid more effectively represses
j3-galactosidase synthesis in glucose grovID cells than in cells grovm
in glycerol redia It should be noted hOHever the inhibitory effect
of salicylic acid on grot-lth rate is the srune vJhether the carbon source
is glucose or glycerol The reason for this inhibition ra1lains obscure
The strect or CYclic AlP on Heprcssion by Snlicylic Acid
If salicylic acid acts by decreasing cyclic PlW levels then cpJmiddot~
should revese the efects of se1icylates Figure 2 shms the resiuts
of rul experinent lihere 5 x lO-3M cyclic AHP and 25 x 10-31-1 salicylic
acid were added silulta1eously to glucose grotm cells It can be seen
that cPJP revJrses the repression caused by salicylic acid Hm1ever
the reversal is only partial since the dif~erential ra-ce is not restored
to the level of the control plus c~tmiddotp cells
Salicylic acid in these experi~ents also caused a decrease in
gromh rate and it lla3 further decreased when cAt-1P las added to the
cells Because of this the data from Figure 2 was replotted with time
units on the abscis3a in place of cell density (Fig 3)~ This allows
10
FIGURE 1
EFFECT OF SLICYLIC ACID ON THE DIFF3P3NrIAL RJTE OF [3-GALACTOSIDA3E SYNTHESIS
strain 3000 Was grovffi in glucose modiUI1 until log phase wst reached
Approrinate1y 1 hour follOldn~ induction rlith IPTG the culture was
separated and trs1sierred to flasks containing the indicted additions
Samples uere talten and assayed for j3-galactosidase as in Table I
Since diaBo1ved salicylic acid lias added a dilution in cell densitr
occurred The first point on the graph represents samples taken L~Jnedi-
ately c)fter the culture Was separatedand diluted
Grorth Rate Differential Enz Rate Flask A Control 75hr 308 EUllilAKR
B Salicylic Acid 68hr 180 EUlllJfoKR
(1 x 10-31)
o Sa1icylic Acid 35hr 95 EUralAlffi (5 x 10-31-1)
12
FIGlllE 2
EFF3Cr OF CYCLIC AmiddotlP 1jJ 3lLICYLIC ACID ON DIFF~JTIAL f3-01AClOSImE SYUTH33IS
IFTG was added to a log phase culture of strain 3000 grown in glucose
medium Approximately 45 minutes after induction the culture was divided
equally into flasks containing the indicated additions 32J1lplcs were
ta~en and ~-Galactosidase was assayed as previously indica~ed The
arroH at 30 Klett unts represents the time the culture ylaS divided
and trcmsferred
DifLerential Rate Growth tate Enzyme Synthesis
Flask A Control bull67hr 23 lJrrJ KIt
B Cyclic mp (5xlO-311) 57hr 315EUrr~6KR
c SalicyliQ acid 4lhr 135ZUmlKR(25xlO-)N)
D Salicy~c acid bull 35hr 25 ~1JrrJLKR 25xlO ~ 11 + CJllmiddotfP (5xlO-)~1)
+
0
t
o
oft
$+
~
Ft flJ rsi
m
m
mlt
l ~ ~
sect4( ~
m~ ~ e1
54 PI rd ISJ
aI ttl
14
FIGURE 3
EFF~T OF CYCLIC AHP UID SJLICYLIC ACID ON j)-GLACTO3IDASE SYNTIIESIS
This represents the smne experireental data as in Figure 1 In the
graph enz~e units ~re plotted in rel~tion to tL~e rather th~n in
relation to cell denitr rIle arrml indicates the time the culture
lras divided and all additions l-Tere i1ade
bull
bullbull Q
-Ld
bull
16 the effect of 321icylic acid ~o be noted indeendently of itsmiddot effect
on generlttion time Even on this graph cAli is shown to partially
relieve repression caused by salicyla~es
Previously Pastaa ~md PerL1an (21)(22) emphasized cAHP had no
effect on growth rate of E coli strain 300 rlhich 112S in contrast to
these results Their dltta homver referto using 1 x lO-3z cANP
Since 5 x 10- 3r crolic lp is believed to overcome both tranient and
catabolite reression caused by r~ucose in strain 3000 (23) the effect
of this concentration on salicylic acids repression was observed
Sirlilar results Nere obtcgtined in a second eXgerimcnt using
S x 10-31-1 salicylic acid and 5 x lO-3H Cl]~ (Fig 4) rhe degree of
reversal ias again only partial Because grorlth inhibition at this conshy
centration of salicylic acid tdth cAHP ias so great the curve is not
extended ver ffX
From these data it appears there is an interaction between the
effects of sali~Jlic acid and those of cMmiddotP Since repression proshy
duced by the higher concentration of salictlic acid seems to be less
effectively reversed by 5 x 10-3N cMP there i~ a possibility the
degree of interaction between the tlO may be concentration dependent
The 3ffect of Sdic-lates on Consti tutive Enzyme Synthesis
To eliminate the possibility salicylic acid may repress j3-galactoshy
sidase synthesis by preventing inducer upt~~e or by competing with
inducer for repressor an experiYlent vTaS carried out using strain 3300
constitutive for fo-galactosidase synthemiddotsis From Figure 5 it is evident
that 25 x 10-311 Jalicylic acid had the same effect on strain 3300 as it
had previously on strain 3000 Therefore salicylates act independently
17
B
uJ ~ l ~ l cJ J L o o- Ul t z 3 w r N Ia Z 312L fa m sa 1m m Em2 ru KLETI UNITS
EFFECT OF CYCLIC Almiddotp lm SLICYLIC ACID OU DIFFrRENfIAL j2gt-GALACTOSIDA3E SYNlHESIS
Figure 4 Strain 3000 culture las grOiID sampled and assayed for fl-g3~actosidase as in Figure 2 uitIl the exception a hig1er concentrC)tion of salic7l1ic acid i-as added after separshyation Since sliicylic acid 35 in dissolved form a dilution occurred upon its addition This uas compensated for in
J
-fA
other fiCsls by an equal dilution
flask Pt Control
Bbull Control + cAHP (5xlO-3H)
c Salicl~ic acid (5xlO H)
D salicl~c acid
Grmmiddotrth Rc1te 71hr
52hr
22hr
15hr (5xlO 3 I) + cMi (5xlO- 1middot1)
Diff Enz Rate 22 EUmlAKR
31 t1Jm1~KR
12 1UmlAIffi
16 EUmlAKR
18
FIGUR35
EFECr 01 SALICYLIC ACID OH DIIT2HE~rrAL fgt-GAL~C0SIDASE SYIJIl133I3 nr COnSrITlJlIV3TAIH 3300
strain 3300 iolaS gron in glucose medium Jllen the cells reached log
phase25 x lO-~1 snlicylic acid ~as added Cells were sa1pled ltmd
4asJared 80S in earlier experiments IPTG (5 x 10- 1-1) liaS added to
control flasks to note the degree of const~tutiveness of tile strain
The first points ngain represent sa~les trucen in~ediately poundter the
culture was divided and additions ware made
Growth Rate Diff Enz Synth Flask A Control bull89hr 65 EUrnlAKR
B Contro~ + IPTG bull89hr 65 EUmJ6KR (5xlO-~middotI)
o SalicYli~3acid bull54hr 46 ~iJ~KR (25x 10 H)
D Sa1icrli~ acid 5hhr 30 EUml~KR (25xlR--i-r) + IPTG (5xlO- M)
lSi JT1
bull 0
Fa-
I
a +
tsJ
yen
+
~
v
+
tsi IS1 m
m
tn
til sectll
m ~ ~ 2
bull
ef ~
~ ~ tsJ
uf
shy
20
of inducer uptake and of inducer-s derepression of lac mrrNA synthesis
The zrecl of 24-Di1ikophcnol ont30alactosidase Synthesis
Since salicylates are kno-Vffi to be uncouplers of oxidative phosshy
phorylation (5) it has been sJgccsted sOrle of their phaITlClcological
effects result from their uncoupling action Tis i-lould irply that
other uncouplers should preferentially inhibit ~-gilactosidase syntheshy
sis For this reason the effect of 2 4-dini trophenol (Dr~) on J-galacshy
tosidase synthesis Has observed Because DrIP is more effective in inhibishy
ting oxidative phosphorylation than salicylic acid and is consequently
more toxic a concentration of the drug i-laS found vhic uould inhibit
grOl-rth to ap~roximately the sa11e degree as 25 x 10-31gt1 salicylic acid
This was found to be 25 x 10-411 Figure 6 illustrates DNP does not
repress differential synthesis of j3-g1actosidase This implies that
althoueh DNP 1m-lers ATP levels there is no apparent effect of the drug
on lsvels of cAl-IP Also it appears salic-rlic acid is not exerting its
repression of enzyrre sJ~~hesis by inhibiting oxidative phosphorylation
21
FIGUllS 6
EFFECrl OF DJP ON DIFFif2rIlL ~GLCiO3D~S STImiddotrYliESIS
Jhen strain 3000 cells reacled log ph~se they tmiddotrere indlccd 1ith IPTG
AproxL~ately one hour later the culture was divide1 a~d tr~ferred
to flasks containing the indicated adlitions The cellsas in preVious
experirrents Tere sarlpled at various tLTIes and assayed for B-galactoshy
sidase activity Since 2h-dinitrophenol t-rag in solution its addition
resuted in dilution of cultures This dilution las equally conpensated
for in control flasks nle first points on the graph represent samples
taken innediately after sepration of the cuJture
GroYTth Rate Difierential inz Rate Flask A Control 75hr 22 L1JmlAKR
B Contro + cAll 64hr 50 EUmlAm (5y10- H) 4z
C DNP (25xlO- -I) 42hr 22 EOIraAKR
D DNP (25xl2~4zr) + 29hr 42 UmlAKR cAJP (5x10 -0
N
N
v
o
-shy-shy+
-shy
+
bull-
DISCU3SION
Salicylic acid ruld to a lesser extent aspirin cause repression
of f3-gclactosidase srnthesis in scherichia coli This effect is not
due to an inhibition of inducer uptake nor is it due ~o interference
l-rith inducers inactivation of lac repressor since strnins constitutive
for 3-gJactosidase 3J1lthesis are also repressed b~r sclicyli~ acid
In addition because dinitrophenol is liithout effect on differential
fl-galactosidase synthesis it appecrs salicrlates do not repress enzyme
s~mthesis by inhibiting oxidative phospllorylation
At these concentr2tions however salicylic acid does decrease
growth rate and direct or indirect effects of this on the observed
repression of enzyme synt~esis still ra~ain Since the differential
rate of enzyme synthesis i~ decreased and also since 25 x 10-411 DN
decreases grotrth rate aproximately the same degree middotuitl1cut having I
effects on differential synthesis salicylates inhibition of growth
is not the sole factor responsible for its effects on enZJcre synthesis
-11en 5 x 10-31 cyclic JUfP 1-ms added to cells treated Kith 25 x 10-3
and 5 x lO-3H srJicylic acid repression of differential enzye s~1lhesis
1ES pextially relieved This sugests salicllic acid represses J3-gJ~ac-
tosidase srnthesis br directly 1011erinti intIacellular levels of cyclic AMP
or by indirectly acting on asita which in turn releases cANP from the
cell Since cMP only partially relieves repression caused by wlicylic
acid and because there appears to be an inverse relationship between
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
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LIST OF TABLES
TABLE PAGE
I
II
Effects of Asoirin on Differe~tial Rate o~ Synthesis
of 3-Galactosidase
Effect of Salicylic Acid on DifferenGial Rate of
Synthesis of fl-Galactosidase
7
8
INrrWDUCTION
Although aspirin is one of the EOSt commonLy us~d drugs its
actual nechnnism of action within the body still remains unclear
Acetylsalicylic acid belongs to the group of drugs referred to as salishy cylates all of which have SLllilar pharmacological properties rheir
activity is believed to be dependent on the drugs salicylic acid conshy
tent and substitutions on the carboxyl or hydroxyl groups alter only
their toxicity or potency (1)
Aspirin is most COnt~only recognized for its analgeSiC antipyreshy
tic and antiirulamTatory effects Recently Vane (2) demonstrated
antiinflan~atorJ acids such as aspirin salicylic acid mld indoshy
methaCin inlJibit enzymes which catalyze prostaglandin (PGE2 and PGF2~)
synthesis in cell free extracts of guinea pig lungs PG11 and PG~
when injected into the third ventricle of cats also proves to be a
potent inducer of fevers (2) Thus Vane postulated aspirin produces
its an~ipyretic and antiinf1aw~tory responses by inhibiting synthesis
of prostaglandins ~~though prostaglandins when administered intradershy
m2~ly in nan do not produce pain (2) some of aspirins ~~gesic activity
may result from reduction of inllt3ll111ation (3) Smith and lillis (4)
supryorted this evidence when they independently observed aspirin to
inhibit conversion of arachidonic acid into prostaglandins in h~~an
platelets
All abbreviations used in text are defined in appendix
2
Aspirin has many other diverse actions within the body Included
among these is its abllity to prevent collagen and epinepnrine induced
aggregation of platelets Platelet ageregation occurs in two phasesa
The first is characterized by a rapid change in the shape of platelets
the second or actual agflegation of cells follol1s the release of ADP
fram platelets (10) Aspirin blocks the second phase of agcregation
(7)(24) This again relates the actions of aspirin to prostaglandins
for POE and PG~ are also known to have effects on platelet aggregation1
Other actions of aspirin are its ability to lower blood sugar in
certain diabetic ~atients (1) to alter ion transport and membrane potenshy
tial in v8ried cell types (1)(16) to reduce lipogenosis and depress
free fatty acid release in muscle tissue (1) and tomiddot uncouple oxidative
phosphorylation (5)
lnother compound cyclic adenosine )5-mollophosphate (cUP) has
also been shmmiddotrn to have a broad spectrum of effects at sites similar to
those acted upon by salicylates These include 1) the mediation by
cM~ of prostaglandins i~1ibition of platelet agsregation (7)(10)
2) the mediatio~ by Ci~1P of insulins induction of glucose upt~~e in
hep~tic cells (11) ) its co~plex effects on ion transport and ma~brnne
potentials in various cell types (18)(25)(17) and 4) its stimulation of
lipolysis in ht~ and rat fat cells (27)
Because cyclic ffi in1 tictes the (lction alld release of many hormones
it has become knowIl as a ubiquitous second messengertl bull Adenyl cyclase
is activated when an external stimulus such as a hormone comes in con-
tact 111th the cell surface The inCleae in intracellular levels of
cyclic lIP activate other specific enzymes ACTIJI s stiI1ulation of
3
adenyl cyclase in the adrenal cortex follolled by the activation of enzymes
synthesizinEcortiaol exenpliiies this (12) Also thyroid hormone secretion
is induced middothen TSH activates thyroid adenyl cyclase (13)
lhere is some evidence cM may in a sirilar manner mediate
the synthesis ~f prostaglandins (10) EPinephrine and nore~inephrine both
known to stimulcte ade~yl cyclase in certai~ cells stimulate release of
prostaglandins from the dog sp~een (14) In addition it has been shown
prostaglandins stL~u1ate adenyl cyclase in human platelets (7)
The question arose -lhether soe of the effects of aspirin might
be mediated by its effects on the ~~1P systems of various target cells
In order to determine whether a relationship exists between the action
of aspirin ald cyclic ANP the effect of salicylates on j3-galactosidase
synthesis in the bacterial ce11 was obs~rved Synthesis of (3 -galactoshy
sidase in Jscherichia coli is known to require cyclic AImiddotrp (15) In
E COli c~middot~ acts at the transcriptional level of enz~ne synthesis by
promoting initiation of ~-galactosidase nu~fA synthesis The nucleotide
is believed to activate a protein called cMP receptor protein which
in turn allows the protein to bind to the promoter reGion of the lac
operon Its presence enhances the binding oflli1A pol~erase to the
proper initiation site of lac DNA (6) COllsequentlyincreased syntheshy
sis of B-galactosi(ase occurs with optimum concenlirations of cAllP and
repreSSion occurs ~hen the ct1middotp concentration is louered
This paplusmner reorts the effects salicylates have on j3-galactosishy
dase SJIlthesis in E coli and the relationship between cAllPs and salicylic
acid t s mechanism of ac tion within the bacterial cell
YJATERIALS AND NlTHODS
Bacterial strains
For the initial experiments Escherichia coli strain 3000 (lac i +)
was used To determine salicylates effect on inducer uptake a conshy
stitutive strain 3300 (lac i -) was used Both strains llere provided by
Dr John 11 Hyers
Grmmiddotrth Conditions
Bacteria liera grown in double strength Hinimal Broth Davis
medium (Bacto) supplelllented k1th 0002 vitamin B and either 02
glucose or 05 glycerol Double strength media lIas used since this
has essentially the same constituents as the minilal medium used in
earlier experiments observing effects of cyclic AmiddotU on E coli (7)
-Before each experiment fres11 stationary cultures were prepared
from bacteria1 slants StatioIlaty cultures were inoculatr-d into
Klett Flasks to obtain an initia1 ceU density of 10-8ml ald gr01ID on
a shaker at 370 C until log phase was reached To induce fl-galactoshy
sidase -syntheSis isopropyl j3-D-thiogalactopyranoside (rrrG) was added
to the cells in log phase producing a final concentration of 5xl0-4 11
FUrther a1ditions depending on the experiment liere made to log phase
cultures
All cell denSity measurements were taken on a K1ett-~~erson
Photoelectric Colorimeter (Model 80003) using a 660 mp filter
5 ~Galactosida3e Ass~
j3-galactosidase was assayed according to the procedure of Pardee
Jacob and Honod (6) with slight Iilodification At specific timeslt
09 ml of sample lUlS ldthdrakll fro-n the cultures and added ~o tubes
containing 2 drops of 025 sodiurn dodecyl sulfate (SOO) end 2 drops
chloroform (9) Both toluene treatment and SDS - chloroform treatshy
ment were pre~narily tested to deterwine the best method of releasshy
ing enzyme from the middotcell The latter did not alter enzyme stabilitY
and proved to increase the sensitivity of the assq
The amount of enzyme present was measured spectrophotomatr1cally
using a KJett-5UlllIllerson Photoelectric Colorimeter (lmiddotiodel 600-3) witil a
420 111)I1ilter One unit of enzyme is defined as the amount producing
1 II1j1 mole of o-nitrophenol per minute at 280 0 pH 70 The units of
enzyme in the sample can be calculated from the 1act 1 17fL mole per ml
of o-nitrophenol has a Klett reading of 045 under experirlental condishy
tions used here
To verify the assay procedure With E coJ strain 3000 enzyme
activity was confirmed to be linear in relation to time and concentrashy
tion
l-Iaterials
All chevicals were commercially obtained Cyclic adenosine
3 5-monophosphatie was obtained from Sigma and its purity 1-1as
confirmed chromatographically and spectrophotometrlcally Based on
readings vy a Cry Spectrophotometer the cyclic ffJ proved to be
99 pure b-leight Cyclic AHPs biolobical activity was confiIr4ed by
its ability to reverse glucose repression
RESULTS
Tho Effects of Salicylates on 13-GaJactosidase Synthesis
Various concentrations of aspirin were added to E coli strain
3000 cultures grmrlng on glycerol medium At a 5 x 10-3f concentration
ofacetylsa1i~lic aci~the ~fferential rate of j3-galactosidase synshy
thesis decreased (Table I) This 1-1aS accompanied by a slight decrease
in bacterial growth rate At 1 x 10-~ and 25 X 10-3M aspirin had
no significant effect on j3-galactos1dase synthesis
Sinceaspirin1s pharmacological action depends on its salicylic
acid content (1) the effect of salicylic acid on j3-galactosidase synshy
thesis ~1as detemined At all concentrations salicylic acid produced
a greater effect on the differential rate of enzyme synthesis than
aspirin (Table II) Salicylic acid also inhibited grorrth to a considershy
able extent but in all cases the effect on enzyme synthesis was
greater
Control experiments showed both aspirin and salicylic acid had
no effect on the enz~e ass~ system
Since the utjlization of glycerol requires the inducible enzoJlne
glycerol kinase kriown to be sensitive to catabolite repression (~)
it Has postulated the effect of salicylic ~cid on grouth rate resulted
from an inhibition of glYcerol kinase synthesis Such an inhibition
would have the effect of masking decreases in the differential rate
of ~-galactosidcse synthesis For this reason it was decided to use
a carbon source not subject to catabolite repreSSion and presumably
-7
TABLE I
EFFECTS OF ASPIRIN on
DrFFEIl3NTIAL RATE OF SYNTHESIS OF P-GALACTOSIDASE
Elcp 170
Aspirin (N)
Diff Rate En~ Synth (EUrilLKR)
at Control Rate
Grol-rth Rate (1gen time)
of Control Rate
1 1 -( cO3)1x10
25x10-3 5xl0-3
21 -( con3~)5x10shy
3 I -(con3- ) 5xl0shy
361 100 361 100 361 100 254 70
313 100 210 67
33bull3 100 259 78
72hr
63hr bull67hr 60br
100 87 93 825
65hr
54hr 100
83
67hr
58hr 100 87
E coli strain 3000 1-las grOl-m in glycerol raedium until log phase was obtained At -rms time the culture was divided and transferred to fiasks containing 5 x 10- H IPTa ard the indicated amount of dissolved aspirin The control flask contained water in place of the dissolved aspirin solution To insure dilutions would not 1irrdt the media flasks were also initi~ supplemented vnth 4x y~~ Broth Davis
At varioUs cell densities 09 ml samples were taken and assayed for j3-Galactosidase activit7
8
Exp No
1
2bull
3
TABLE II
EFFECT OF SALICYLIC ACID ON
DIFFER11ITIAL It4TE OF SYNTHESIS OF J3-GALACTOSID~sE
Salicylic Acid (N)
-(contro1) x 10shy
-(contro~23 387 x 10
-(contrpound)1 x 10
DtiL Rate ~~th (EUII1lJb Iffi) of
control rate
Growth Rate (ligen time) of
Control
370 62
100 n
60hr
33hr 100
283 65
100 23
71hr
44Ihr 100 62
315 206
bull100 6
60hr
2hr 100 87
Cells l-ere gr01ffl and sampled as in Table I ITith the exception that disshysolved salicylic acid was added to ce~s in log phase in place of aspirin
9
not subject to salicylic aci~ inhibition In all subsequent eXperiments
glucose lias used as carbon source
Upon the addition of 5 x lO-~ salicylic acid to cells grovID in
glucose media there gtmiddotas an ilnmecliate cessation of a-galactosidase synshy
thesis lasting over a generation of grotrth This l-laS followed by a
recovery of srnthesis but at a decreascdrate relative to the control
(fig 1) tt 1 x 10-~i salicylic acid deceased the differential rate
of enzY1e synthesis but tlere 1-TaS a)parently no lag in synthesis after
its addition
From this data it apears salicjlic acid more effectively represses
j3-galactosidase synthesis in glucose grovID cells than in cells grovm
in glycerol redia It should be noted hOHever the inhibitory effect
of salicylic acid on grot-lth rate is the srune vJhether the carbon source
is glucose or glycerol The reason for this inhibition ra1lains obscure
The strect or CYclic AlP on Heprcssion by Snlicylic Acid
If salicylic acid acts by decreasing cyclic PlW levels then cpJmiddot~
should revese the efects of se1icylates Figure 2 shms the resiuts
of rul experinent lihere 5 x lO-3M cyclic AHP and 25 x 10-31-1 salicylic
acid were added silulta1eously to glucose grotm cells It can be seen
that cPJP revJrses the repression caused by salicylic acid Hm1ever
the reversal is only partial since the dif~erential ra-ce is not restored
to the level of the control plus c~tmiddotp cells
Salicylic acid in these experi~ents also caused a decrease in
gromh rate and it lla3 further decreased when cAt-1P las added to the
cells Because of this the data from Figure 2 was replotted with time
units on the abscis3a in place of cell density (Fig 3)~ This allows
10
FIGURE 1
EFFECT OF SLICYLIC ACID ON THE DIFF3P3NrIAL RJTE OF [3-GALACTOSIDA3E SYNTHESIS
strain 3000 Was grovffi in glucose modiUI1 until log phase wst reached
Approrinate1y 1 hour follOldn~ induction rlith IPTG the culture was
separated and trs1sierred to flasks containing the indicted additions
Samples uere talten and assayed for j3-galactosidase as in Table I
Since diaBo1ved salicylic acid lias added a dilution in cell densitr
occurred The first point on the graph represents samples taken L~Jnedi-
ately c)fter the culture Was separatedand diluted
Grorth Rate Differential Enz Rate Flask A Control 75hr 308 EUllilAKR
B Salicylic Acid 68hr 180 EUlllJfoKR
(1 x 10-31)
o Sa1icylic Acid 35hr 95 EUralAlffi (5 x 10-31-1)
12
FIGlllE 2
EFF3Cr OF CYCLIC AmiddotlP 1jJ 3lLICYLIC ACID ON DIFF~JTIAL f3-01AClOSImE SYUTH33IS
IFTG was added to a log phase culture of strain 3000 grown in glucose
medium Approximately 45 minutes after induction the culture was divided
equally into flasks containing the indicated additions 32J1lplcs were
ta~en and ~-Galactosidase was assayed as previously indica~ed The
arroH at 30 Klett unts represents the time the culture ylaS divided
and trcmsferred
DifLerential Rate Growth tate Enzyme Synthesis
Flask A Control bull67hr 23 lJrrJ KIt
B Cyclic mp (5xlO-311) 57hr 315EUrr~6KR
c SalicyliQ acid 4lhr 135ZUmlKR(25xlO-)N)
D Salicy~c acid bull 35hr 25 ~1JrrJLKR 25xlO ~ 11 + CJllmiddotfP (5xlO-)~1)
+
0
t
o
oft
$+
~
Ft flJ rsi
m
m
mlt
l ~ ~
sect4( ~
m~ ~ e1
54 PI rd ISJ
aI ttl
14
FIGURE 3
EFF~T OF CYCLIC AHP UID SJLICYLIC ACID ON j)-GLACTO3IDASE SYNTIIESIS
This represents the smne experireental data as in Figure 1 In the
graph enz~e units ~re plotted in rel~tion to tL~e rather th~n in
relation to cell denitr rIle arrml indicates the time the culture
lras divided and all additions l-Tere i1ade
bull
bullbull Q
-Ld
bull
16 the effect of 321icylic acid ~o be noted indeendently of itsmiddot effect
on generlttion time Even on this graph cAli is shown to partially
relieve repression caused by salicyla~es
Previously Pastaa ~md PerL1an (21)(22) emphasized cAHP had no
effect on growth rate of E coli strain 300 rlhich 112S in contrast to
these results Their dltta homver referto using 1 x lO-3z cANP
Since 5 x 10- 3r crolic lp is believed to overcome both tranient and
catabolite reression caused by r~ucose in strain 3000 (23) the effect
of this concentration on salicylic acids repression was observed
Sirlilar results Nere obtcgtined in a second eXgerimcnt using
S x 10-31-1 salicylic acid and 5 x lO-3H Cl]~ (Fig 4) rhe degree of
reversal ias again only partial Because grorlth inhibition at this conshy
centration of salicylic acid tdth cAHP ias so great the curve is not
extended ver ffX
From these data it appears there is an interaction between the
effects of sali~Jlic acid and those of cMmiddotP Since repression proshy
duced by the higher concentration of salictlic acid seems to be less
effectively reversed by 5 x 10-3N cMP there i~ a possibility the
degree of interaction between the tlO may be concentration dependent
The 3ffect of Sdic-lates on Consti tutive Enzyme Synthesis
To eliminate the possibility salicylic acid may repress j3-galactoshy
sidase synthesis by preventing inducer upt~~e or by competing with
inducer for repressor an experiYlent vTaS carried out using strain 3300
constitutive for fo-galactosidase synthemiddotsis From Figure 5 it is evident
that 25 x 10-311 Jalicylic acid had the same effect on strain 3300 as it
had previously on strain 3000 Therefore salicylates act independently
17
B
uJ ~ l ~ l cJ J L o o- Ul t z 3 w r N Ia Z 312L fa m sa 1m m Em2 ru KLETI UNITS
EFFECT OF CYCLIC Almiddotp lm SLICYLIC ACID OU DIFFrRENfIAL j2gt-GALACTOSIDA3E SYNlHESIS
Figure 4 Strain 3000 culture las grOiID sampled and assayed for fl-g3~actosidase as in Figure 2 uitIl the exception a hig1er concentrC)tion of salic7l1ic acid i-as added after separshyation Since sliicylic acid 35 in dissolved form a dilution occurred upon its addition This uas compensated for in
J
-fA
other fiCsls by an equal dilution
flask Pt Control
Bbull Control + cAHP (5xlO-3H)
c Salicl~ic acid (5xlO H)
D salicl~c acid
Grmmiddotrth Rc1te 71hr
52hr
22hr
15hr (5xlO 3 I) + cMi (5xlO- 1middot1)
Diff Enz Rate 22 EUmlAKR
31 t1Jm1~KR
12 1UmlAIffi
16 EUmlAKR
18
FIGUR35
EFECr 01 SALICYLIC ACID OH DIIT2HE~rrAL fgt-GAL~C0SIDASE SYIJIl133I3 nr COnSrITlJlIV3TAIH 3300
strain 3300 iolaS gron in glucose medium Jllen the cells reached log
phase25 x lO-~1 snlicylic acid ~as added Cells were sa1pled ltmd
4asJared 80S in earlier experiments IPTG (5 x 10- 1-1) liaS added to
control flasks to note the degree of const~tutiveness of tile strain
The first points ngain represent sa~les trucen in~ediately poundter the
culture was divided and additions ware made
Growth Rate Diff Enz Synth Flask A Control bull89hr 65 EUrnlAKR
B Contro~ + IPTG bull89hr 65 EUmJ6KR (5xlO-~middotI)
o SalicYli~3acid bull54hr 46 ~iJ~KR (25x 10 H)
D Sa1icrli~ acid 5hhr 30 EUml~KR (25xlR--i-r) + IPTG (5xlO- M)
lSi JT1
bull 0
Fa-
I
a +
tsJ
yen
+
~
v
+
tsi IS1 m
m
tn
til sectll
m ~ ~ 2
bull
ef ~
~ ~ tsJ
uf
shy
20
of inducer uptake and of inducer-s derepression of lac mrrNA synthesis
The zrecl of 24-Di1ikophcnol ont30alactosidase Synthesis
Since salicylates are kno-Vffi to be uncouplers of oxidative phosshy
phorylation (5) it has been sJgccsted sOrle of their phaITlClcological
effects result from their uncoupling action Tis i-lould irply that
other uncouplers should preferentially inhibit ~-gilactosidase syntheshy
sis For this reason the effect of 2 4-dini trophenol (Dr~) on J-galacshy
tosidase synthesis Has observed Because DrIP is more effective in inhibishy
ting oxidative phosphorylation than salicylic acid and is consequently
more toxic a concentration of the drug i-laS found vhic uould inhibit
grOl-rth to ap~roximately the sa11e degree as 25 x 10-31gt1 salicylic acid
This was found to be 25 x 10-411 Figure 6 illustrates DNP does not
repress differential synthesis of j3-g1actosidase This implies that
althoueh DNP 1m-lers ATP levels there is no apparent effect of the drug
on lsvels of cAl-IP Also it appears salic-rlic acid is not exerting its
repression of enzyrre sJ~~hesis by inhibiting oxidative phosphorylation
21
FIGUllS 6
EFFECrl OF DJP ON DIFFif2rIlL ~GLCiO3D~S STImiddotrYliESIS
Jhen strain 3000 cells reacled log ph~se they tmiddotrere indlccd 1ith IPTG
AproxL~ately one hour later the culture was divide1 a~d tr~ferred
to flasks containing the indicated adlitions The cellsas in preVious
experirrents Tere sarlpled at various tLTIes and assayed for B-galactoshy
sidase activity Since 2h-dinitrophenol t-rag in solution its addition
resuted in dilution of cultures This dilution las equally conpensated
for in control flasks nle first points on the graph represent samples
taken innediately after sepration of the cuJture
GroYTth Rate Difierential inz Rate Flask A Control 75hr 22 L1JmlAKR
B Contro + cAll 64hr 50 EUmlAm (5y10- H) 4z
C DNP (25xlO- -I) 42hr 22 EOIraAKR
D DNP (25xl2~4zr) + 29hr 42 UmlAKR cAJP (5x10 -0
N
N
v
o
-shy-shy+
-shy
+
bull-
DISCU3SION
Salicylic acid ruld to a lesser extent aspirin cause repression
of f3-gclactosidase srnthesis in scherichia coli This effect is not
due to an inhibition of inducer uptake nor is it due ~o interference
l-rith inducers inactivation of lac repressor since strnins constitutive
for 3-gJactosidase 3J1lthesis are also repressed b~r sclicyli~ acid
In addition because dinitrophenol is liithout effect on differential
fl-galactosidase synthesis it appecrs salicrlates do not repress enzyme
s~mthesis by inhibiting oxidative phospllorylation
At these concentr2tions however salicylic acid does decrease
growth rate and direct or indirect effects of this on the observed
repression of enzyme synt~esis still ra~ain Since the differential
rate of enzyme synthesis i~ decreased and also since 25 x 10-411 DN
decreases grotrth rate aproximately the same degree middotuitl1cut having I
effects on differential synthesis salicylates inhibition of growth
is not the sole factor responsible for its effects on enZJcre synthesis
-11en 5 x 10-31 cyclic JUfP 1-ms added to cells treated Kith 25 x 10-3
and 5 x lO-3H srJicylic acid repression of differential enzye s~1lhesis
1ES pextially relieved This sugests salicllic acid represses J3-gJ~ac-
tosidase srnthesis br directly 1011erinti intIacellular levels of cyclic AMP
or by indirectly acting on asita which in turn releases cANP from the
cell Since cMP only partially relieves repression caused by wlicylic
acid and because there appears to be an inverse relationship between
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
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INrrWDUCTION
Although aspirin is one of the EOSt commonLy us~d drugs its
actual nechnnism of action within the body still remains unclear
Acetylsalicylic acid belongs to the group of drugs referred to as salishy cylates all of which have SLllilar pharmacological properties rheir
activity is believed to be dependent on the drugs salicylic acid conshy
tent and substitutions on the carboxyl or hydroxyl groups alter only
their toxicity or potency (1)
Aspirin is most COnt~only recognized for its analgeSiC antipyreshy
tic and antiirulamTatory effects Recently Vane (2) demonstrated
antiinflan~atorJ acids such as aspirin salicylic acid mld indoshy
methaCin inlJibit enzymes which catalyze prostaglandin (PGE2 and PGF2~)
synthesis in cell free extracts of guinea pig lungs PG11 and PG~
when injected into the third ventricle of cats also proves to be a
potent inducer of fevers (2) Thus Vane postulated aspirin produces
its an~ipyretic and antiinf1aw~tory responses by inhibiting synthesis
of prostaglandins ~~though prostaglandins when administered intradershy
m2~ly in nan do not produce pain (2) some of aspirins ~~gesic activity
may result from reduction of inllt3ll111ation (3) Smith and lillis (4)
supryorted this evidence when they independently observed aspirin to
inhibit conversion of arachidonic acid into prostaglandins in h~~an
platelets
All abbreviations used in text are defined in appendix
2
Aspirin has many other diverse actions within the body Included
among these is its abllity to prevent collagen and epinepnrine induced
aggregation of platelets Platelet ageregation occurs in two phasesa
The first is characterized by a rapid change in the shape of platelets
the second or actual agflegation of cells follol1s the release of ADP
fram platelets (10) Aspirin blocks the second phase of agcregation
(7)(24) This again relates the actions of aspirin to prostaglandins
for POE and PG~ are also known to have effects on platelet aggregation1
Other actions of aspirin are its ability to lower blood sugar in
certain diabetic ~atients (1) to alter ion transport and membrane potenshy
tial in v8ried cell types (1)(16) to reduce lipogenosis and depress
free fatty acid release in muscle tissue (1) and tomiddot uncouple oxidative
phosphorylation (5)
lnother compound cyclic adenosine )5-mollophosphate (cUP) has
also been shmmiddotrn to have a broad spectrum of effects at sites similar to
those acted upon by salicylates These include 1) the mediation by
cM~ of prostaglandins i~1ibition of platelet agsregation (7)(10)
2) the mediatio~ by Ci~1P of insulins induction of glucose upt~~e in
hep~tic cells (11) ) its co~plex effects on ion transport and ma~brnne
potentials in various cell types (18)(25)(17) and 4) its stimulation of
lipolysis in ht~ and rat fat cells (27)
Because cyclic ffi in1 tictes the (lction alld release of many hormones
it has become knowIl as a ubiquitous second messengertl bull Adenyl cyclase
is activated when an external stimulus such as a hormone comes in con-
tact 111th the cell surface The inCleae in intracellular levels of
cyclic lIP activate other specific enzymes ACTIJI s stiI1ulation of
3
adenyl cyclase in the adrenal cortex follolled by the activation of enzymes
synthesizinEcortiaol exenpliiies this (12) Also thyroid hormone secretion
is induced middothen TSH activates thyroid adenyl cyclase (13)
lhere is some evidence cM may in a sirilar manner mediate
the synthesis ~f prostaglandins (10) EPinephrine and nore~inephrine both
known to stimulcte ade~yl cyclase in certai~ cells stimulate release of
prostaglandins from the dog sp~een (14) In addition it has been shown
prostaglandins stL~u1ate adenyl cyclase in human platelets (7)
The question arose -lhether soe of the effects of aspirin might
be mediated by its effects on the ~~1P systems of various target cells
In order to determine whether a relationship exists between the action
of aspirin ald cyclic ANP the effect of salicylates on j3-galactosidase
synthesis in the bacterial ce11 was obs~rved Synthesis of (3 -galactoshy
sidase in Jscherichia coli is known to require cyclic AImiddotrp (15) In
E COli c~middot~ acts at the transcriptional level of enz~ne synthesis by
promoting initiation of ~-galactosidase nu~fA synthesis The nucleotide
is believed to activate a protein called cMP receptor protein which
in turn allows the protein to bind to the promoter reGion of the lac
operon Its presence enhances the binding oflli1A pol~erase to the
proper initiation site of lac DNA (6) COllsequentlyincreased syntheshy
sis of B-galactosi(ase occurs with optimum concenlirations of cAllP and
repreSSion occurs ~hen the ct1middotp concentration is louered
This paplusmner reorts the effects salicylates have on j3-galactosishy
dase SJIlthesis in E coli and the relationship between cAllPs and salicylic
acid t s mechanism of ac tion within the bacterial cell
YJATERIALS AND NlTHODS
Bacterial strains
For the initial experiments Escherichia coli strain 3000 (lac i +)
was used To determine salicylates effect on inducer uptake a conshy
stitutive strain 3300 (lac i -) was used Both strains llere provided by
Dr John 11 Hyers
Grmmiddotrth Conditions
Bacteria liera grown in double strength Hinimal Broth Davis
medium (Bacto) supplelllented k1th 0002 vitamin B and either 02
glucose or 05 glycerol Double strength media lIas used since this
has essentially the same constituents as the minilal medium used in
earlier experiments observing effects of cyclic AmiddotU on E coli (7)
-Before each experiment fres11 stationary cultures were prepared
from bacteria1 slants StatioIlaty cultures were inoculatr-d into
Klett Flasks to obtain an initia1 ceU density of 10-8ml ald gr01ID on
a shaker at 370 C until log phase was reached To induce fl-galactoshy
sidase -syntheSis isopropyl j3-D-thiogalactopyranoside (rrrG) was added
to the cells in log phase producing a final concentration of 5xl0-4 11
FUrther a1ditions depending on the experiment liere made to log phase
cultures
All cell denSity measurements were taken on a K1ett-~~erson
Photoelectric Colorimeter (Model 80003) using a 660 mp filter
5 ~Galactosida3e Ass~
j3-galactosidase was assayed according to the procedure of Pardee
Jacob and Honod (6) with slight Iilodification At specific timeslt
09 ml of sample lUlS ldthdrakll fro-n the cultures and added ~o tubes
containing 2 drops of 025 sodiurn dodecyl sulfate (SOO) end 2 drops
chloroform (9) Both toluene treatment and SDS - chloroform treatshy
ment were pre~narily tested to deterwine the best method of releasshy
ing enzyme from the middotcell The latter did not alter enzyme stabilitY
and proved to increase the sensitivity of the assq
The amount of enzyme present was measured spectrophotomatr1cally
using a KJett-5UlllIllerson Photoelectric Colorimeter (lmiddotiodel 600-3) witil a
420 111)I1ilter One unit of enzyme is defined as the amount producing
1 II1j1 mole of o-nitrophenol per minute at 280 0 pH 70 The units of
enzyme in the sample can be calculated from the 1act 1 17fL mole per ml
of o-nitrophenol has a Klett reading of 045 under experirlental condishy
tions used here
To verify the assay procedure With E coJ strain 3000 enzyme
activity was confirmed to be linear in relation to time and concentrashy
tion
l-Iaterials
All chevicals were commercially obtained Cyclic adenosine
3 5-monophosphatie was obtained from Sigma and its purity 1-1as
confirmed chromatographically and spectrophotometrlcally Based on
readings vy a Cry Spectrophotometer the cyclic ffJ proved to be
99 pure b-leight Cyclic AHPs biolobical activity was confiIr4ed by
its ability to reverse glucose repression
RESULTS
Tho Effects of Salicylates on 13-GaJactosidase Synthesis
Various concentrations of aspirin were added to E coli strain
3000 cultures grmrlng on glycerol medium At a 5 x 10-3f concentration
ofacetylsa1i~lic aci~the ~fferential rate of j3-galactosidase synshy
thesis decreased (Table I) This 1-1aS accompanied by a slight decrease
in bacterial growth rate At 1 x 10-~ and 25 X 10-3M aspirin had
no significant effect on j3-galactos1dase synthesis
Sinceaspirin1s pharmacological action depends on its salicylic
acid content (1) the effect of salicylic acid on j3-galactosidase synshy
thesis ~1as detemined At all concentrations salicylic acid produced
a greater effect on the differential rate of enzyme synthesis than
aspirin (Table II) Salicylic acid also inhibited grorrth to a considershy
able extent but in all cases the effect on enzyme synthesis was
greater
Control experiments showed both aspirin and salicylic acid had
no effect on the enz~e ass~ system
Since the utjlization of glycerol requires the inducible enzoJlne
glycerol kinase kriown to be sensitive to catabolite repression (~)
it Has postulated the effect of salicylic ~cid on grouth rate resulted
from an inhibition of glYcerol kinase synthesis Such an inhibition
would have the effect of masking decreases in the differential rate
of ~-galactosidcse synthesis For this reason it was decided to use
a carbon source not subject to catabolite repreSSion and presumably
-7
TABLE I
EFFECTS OF ASPIRIN on
DrFFEIl3NTIAL RATE OF SYNTHESIS OF P-GALACTOSIDASE
Elcp 170
Aspirin (N)
Diff Rate En~ Synth (EUrilLKR)
at Control Rate
Grol-rth Rate (1gen time)
of Control Rate
1 1 -( cO3)1x10
25x10-3 5xl0-3
21 -( con3~)5x10shy
3 I -(con3- ) 5xl0shy
361 100 361 100 361 100 254 70
313 100 210 67
33bull3 100 259 78
72hr
63hr bull67hr 60br
100 87 93 825
65hr
54hr 100
83
67hr
58hr 100 87
E coli strain 3000 1-las grOl-m in glycerol raedium until log phase was obtained At -rms time the culture was divided and transferred to fiasks containing 5 x 10- H IPTa ard the indicated amount of dissolved aspirin The control flask contained water in place of the dissolved aspirin solution To insure dilutions would not 1irrdt the media flasks were also initi~ supplemented vnth 4x y~~ Broth Davis
At varioUs cell densities 09 ml samples were taken and assayed for j3-Galactosidase activit7
8
Exp No
1
2bull
3
TABLE II
EFFECT OF SALICYLIC ACID ON
DIFFER11ITIAL It4TE OF SYNTHESIS OF J3-GALACTOSID~sE
Salicylic Acid (N)
-(contro1) x 10shy
-(contro~23 387 x 10
-(contrpound)1 x 10
DtiL Rate ~~th (EUII1lJb Iffi) of
control rate
Growth Rate (ligen time) of
Control
370 62
100 n
60hr
33hr 100
283 65
100 23
71hr
44Ihr 100 62
315 206
bull100 6
60hr
2hr 100 87
Cells l-ere gr01ffl and sampled as in Table I ITith the exception that disshysolved salicylic acid was added to ce~s in log phase in place of aspirin
9
not subject to salicylic aci~ inhibition In all subsequent eXperiments
glucose lias used as carbon source
Upon the addition of 5 x lO-~ salicylic acid to cells grovID in
glucose media there gtmiddotas an ilnmecliate cessation of a-galactosidase synshy
thesis lasting over a generation of grotrth This l-laS followed by a
recovery of srnthesis but at a decreascdrate relative to the control
(fig 1) tt 1 x 10-~i salicylic acid deceased the differential rate
of enzY1e synthesis but tlere 1-TaS a)parently no lag in synthesis after
its addition
From this data it apears salicjlic acid more effectively represses
j3-galactosidase synthesis in glucose grovID cells than in cells grovm
in glycerol redia It should be noted hOHever the inhibitory effect
of salicylic acid on grot-lth rate is the srune vJhether the carbon source
is glucose or glycerol The reason for this inhibition ra1lains obscure
The strect or CYclic AlP on Heprcssion by Snlicylic Acid
If salicylic acid acts by decreasing cyclic PlW levels then cpJmiddot~
should revese the efects of se1icylates Figure 2 shms the resiuts
of rul experinent lihere 5 x lO-3M cyclic AHP and 25 x 10-31-1 salicylic
acid were added silulta1eously to glucose grotm cells It can be seen
that cPJP revJrses the repression caused by salicylic acid Hm1ever
the reversal is only partial since the dif~erential ra-ce is not restored
to the level of the control plus c~tmiddotp cells
Salicylic acid in these experi~ents also caused a decrease in
gromh rate and it lla3 further decreased when cAt-1P las added to the
cells Because of this the data from Figure 2 was replotted with time
units on the abscis3a in place of cell density (Fig 3)~ This allows
10
FIGURE 1
EFFECT OF SLICYLIC ACID ON THE DIFF3P3NrIAL RJTE OF [3-GALACTOSIDA3E SYNTHESIS
strain 3000 Was grovffi in glucose modiUI1 until log phase wst reached
Approrinate1y 1 hour follOldn~ induction rlith IPTG the culture was
separated and trs1sierred to flasks containing the indicted additions
Samples uere talten and assayed for j3-galactosidase as in Table I
Since diaBo1ved salicylic acid lias added a dilution in cell densitr
occurred The first point on the graph represents samples taken L~Jnedi-
ately c)fter the culture Was separatedand diluted
Grorth Rate Differential Enz Rate Flask A Control 75hr 308 EUllilAKR
B Salicylic Acid 68hr 180 EUlllJfoKR
(1 x 10-31)
o Sa1icylic Acid 35hr 95 EUralAlffi (5 x 10-31-1)
12
FIGlllE 2
EFF3Cr OF CYCLIC AmiddotlP 1jJ 3lLICYLIC ACID ON DIFF~JTIAL f3-01AClOSImE SYUTH33IS
IFTG was added to a log phase culture of strain 3000 grown in glucose
medium Approximately 45 minutes after induction the culture was divided
equally into flasks containing the indicated additions 32J1lplcs were
ta~en and ~-Galactosidase was assayed as previously indica~ed The
arroH at 30 Klett unts represents the time the culture ylaS divided
and trcmsferred
DifLerential Rate Growth tate Enzyme Synthesis
Flask A Control bull67hr 23 lJrrJ KIt
B Cyclic mp (5xlO-311) 57hr 315EUrr~6KR
c SalicyliQ acid 4lhr 135ZUmlKR(25xlO-)N)
D Salicy~c acid bull 35hr 25 ~1JrrJLKR 25xlO ~ 11 + CJllmiddotfP (5xlO-)~1)
+
0
t
o
oft
$+
~
Ft flJ rsi
m
m
mlt
l ~ ~
sect4( ~
m~ ~ e1
54 PI rd ISJ
aI ttl
14
FIGURE 3
EFF~T OF CYCLIC AHP UID SJLICYLIC ACID ON j)-GLACTO3IDASE SYNTIIESIS
This represents the smne experireental data as in Figure 1 In the
graph enz~e units ~re plotted in rel~tion to tL~e rather th~n in
relation to cell denitr rIle arrml indicates the time the culture
lras divided and all additions l-Tere i1ade
bull
bullbull Q
-Ld
bull
16 the effect of 321icylic acid ~o be noted indeendently of itsmiddot effect
on generlttion time Even on this graph cAli is shown to partially
relieve repression caused by salicyla~es
Previously Pastaa ~md PerL1an (21)(22) emphasized cAHP had no
effect on growth rate of E coli strain 300 rlhich 112S in contrast to
these results Their dltta homver referto using 1 x lO-3z cANP
Since 5 x 10- 3r crolic lp is believed to overcome both tranient and
catabolite reression caused by r~ucose in strain 3000 (23) the effect
of this concentration on salicylic acids repression was observed
Sirlilar results Nere obtcgtined in a second eXgerimcnt using
S x 10-31-1 salicylic acid and 5 x lO-3H Cl]~ (Fig 4) rhe degree of
reversal ias again only partial Because grorlth inhibition at this conshy
centration of salicylic acid tdth cAHP ias so great the curve is not
extended ver ffX
From these data it appears there is an interaction between the
effects of sali~Jlic acid and those of cMmiddotP Since repression proshy
duced by the higher concentration of salictlic acid seems to be less
effectively reversed by 5 x 10-3N cMP there i~ a possibility the
degree of interaction between the tlO may be concentration dependent
The 3ffect of Sdic-lates on Consti tutive Enzyme Synthesis
To eliminate the possibility salicylic acid may repress j3-galactoshy
sidase synthesis by preventing inducer upt~~e or by competing with
inducer for repressor an experiYlent vTaS carried out using strain 3300
constitutive for fo-galactosidase synthemiddotsis From Figure 5 it is evident
that 25 x 10-311 Jalicylic acid had the same effect on strain 3300 as it
had previously on strain 3000 Therefore salicylates act independently
17
B
uJ ~ l ~ l cJ J L o o- Ul t z 3 w r N Ia Z 312L fa m sa 1m m Em2 ru KLETI UNITS
EFFECT OF CYCLIC Almiddotp lm SLICYLIC ACID OU DIFFrRENfIAL j2gt-GALACTOSIDA3E SYNlHESIS
Figure 4 Strain 3000 culture las grOiID sampled and assayed for fl-g3~actosidase as in Figure 2 uitIl the exception a hig1er concentrC)tion of salic7l1ic acid i-as added after separshyation Since sliicylic acid 35 in dissolved form a dilution occurred upon its addition This uas compensated for in
J
-fA
other fiCsls by an equal dilution
flask Pt Control
Bbull Control + cAHP (5xlO-3H)
c Salicl~ic acid (5xlO H)
D salicl~c acid
Grmmiddotrth Rc1te 71hr
52hr
22hr
15hr (5xlO 3 I) + cMi (5xlO- 1middot1)
Diff Enz Rate 22 EUmlAKR
31 t1Jm1~KR
12 1UmlAIffi
16 EUmlAKR
18
FIGUR35
EFECr 01 SALICYLIC ACID OH DIIT2HE~rrAL fgt-GAL~C0SIDASE SYIJIl133I3 nr COnSrITlJlIV3TAIH 3300
strain 3300 iolaS gron in glucose medium Jllen the cells reached log
phase25 x lO-~1 snlicylic acid ~as added Cells were sa1pled ltmd
4asJared 80S in earlier experiments IPTG (5 x 10- 1-1) liaS added to
control flasks to note the degree of const~tutiveness of tile strain
The first points ngain represent sa~les trucen in~ediately poundter the
culture was divided and additions ware made
Growth Rate Diff Enz Synth Flask A Control bull89hr 65 EUrnlAKR
B Contro~ + IPTG bull89hr 65 EUmJ6KR (5xlO-~middotI)
o SalicYli~3acid bull54hr 46 ~iJ~KR (25x 10 H)
D Sa1icrli~ acid 5hhr 30 EUml~KR (25xlR--i-r) + IPTG (5xlO- M)
lSi JT1
bull 0
Fa-
I
a +
tsJ
yen
+
~
v
+
tsi IS1 m
m
tn
til sectll
m ~ ~ 2
bull
ef ~
~ ~ tsJ
uf
shy
20
of inducer uptake and of inducer-s derepression of lac mrrNA synthesis
The zrecl of 24-Di1ikophcnol ont30alactosidase Synthesis
Since salicylates are kno-Vffi to be uncouplers of oxidative phosshy
phorylation (5) it has been sJgccsted sOrle of their phaITlClcological
effects result from their uncoupling action Tis i-lould irply that
other uncouplers should preferentially inhibit ~-gilactosidase syntheshy
sis For this reason the effect of 2 4-dini trophenol (Dr~) on J-galacshy
tosidase synthesis Has observed Because DrIP is more effective in inhibishy
ting oxidative phosphorylation than salicylic acid and is consequently
more toxic a concentration of the drug i-laS found vhic uould inhibit
grOl-rth to ap~roximately the sa11e degree as 25 x 10-31gt1 salicylic acid
This was found to be 25 x 10-411 Figure 6 illustrates DNP does not
repress differential synthesis of j3-g1actosidase This implies that
althoueh DNP 1m-lers ATP levels there is no apparent effect of the drug
on lsvels of cAl-IP Also it appears salic-rlic acid is not exerting its
repression of enzyrre sJ~~hesis by inhibiting oxidative phosphorylation
21
FIGUllS 6
EFFECrl OF DJP ON DIFFif2rIlL ~GLCiO3D~S STImiddotrYliESIS
Jhen strain 3000 cells reacled log ph~se they tmiddotrere indlccd 1ith IPTG
AproxL~ately one hour later the culture was divide1 a~d tr~ferred
to flasks containing the indicated adlitions The cellsas in preVious
experirrents Tere sarlpled at various tLTIes and assayed for B-galactoshy
sidase activity Since 2h-dinitrophenol t-rag in solution its addition
resuted in dilution of cultures This dilution las equally conpensated
for in control flasks nle first points on the graph represent samples
taken innediately after sepration of the cuJture
GroYTth Rate Difierential inz Rate Flask A Control 75hr 22 L1JmlAKR
B Contro + cAll 64hr 50 EUmlAm (5y10- H) 4z
C DNP (25xlO- -I) 42hr 22 EOIraAKR
D DNP (25xl2~4zr) + 29hr 42 UmlAKR cAJP (5x10 -0
N
N
v
o
-shy-shy+
-shy
+
bull-
DISCU3SION
Salicylic acid ruld to a lesser extent aspirin cause repression
of f3-gclactosidase srnthesis in scherichia coli This effect is not
due to an inhibition of inducer uptake nor is it due ~o interference
l-rith inducers inactivation of lac repressor since strnins constitutive
for 3-gJactosidase 3J1lthesis are also repressed b~r sclicyli~ acid
In addition because dinitrophenol is liithout effect on differential
fl-galactosidase synthesis it appecrs salicrlates do not repress enzyme
s~mthesis by inhibiting oxidative phospllorylation
At these concentr2tions however salicylic acid does decrease
growth rate and direct or indirect effects of this on the observed
repression of enzyme synt~esis still ra~ain Since the differential
rate of enzyme synthesis i~ decreased and also since 25 x 10-411 DN
decreases grotrth rate aproximately the same degree middotuitl1cut having I
effects on differential synthesis salicylates inhibition of growth
is not the sole factor responsible for its effects on enZJcre synthesis
-11en 5 x 10-31 cyclic JUfP 1-ms added to cells treated Kith 25 x 10-3
and 5 x lO-3H srJicylic acid repression of differential enzye s~1lhesis
1ES pextially relieved This sugests salicllic acid represses J3-gJ~ac-
tosidase srnthesis br directly 1011erinti intIacellular levels of cyclic AMP
or by indirectly acting on asita which in turn releases cANP from the
cell Since cMP only partially relieves repression caused by wlicylic
acid and because there appears to be an inverse relationship between
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
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2
Aspirin has many other diverse actions within the body Included
among these is its abllity to prevent collagen and epinepnrine induced
aggregation of platelets Platelet ageregation occurs in two phasesa
The first is characterized by a rapid change in the shape of platelets
the second or actual agflegation of cells follol1s the release of ADP
fram platelets (10) Aspirin blocks the second phase of agcregation
(7)(24) This again relates the actions of aspirin to prostaglandins
for POE and PG~ are also known to have effects on platelet aggregation1
Other actions of aspirin are its ability to lower blood sugar in
certain diabetic ~atients (1) to alter ion transport and membrane potenshy
tial in v8ried cell types (1)(16) to reduce lipogenosis and depress
free fatty acid release in muscle tissue (1) and tomiddot uncouple oxidative
phosphorylation (5)
lnother compound cyclic adenosine )5-mollophosphate (cUP) has
also been shmmiddotrn to have a broad spectrum of effects at sites similar to
those acted upon by salicylates These include 1) the mediation by
cM~ of prostaglandins i~1ibition of platelet agsregation (7)(10)
2) the mediatio~ by Ci~1P of insulins induction of glucose upt~~e in
hep~tic cells (11) ) its co~plex effects on ion transport and ma~brnne
potentials in various cell types (18)(25)(17) and 4) its stimulation of
lipolysis in ht~ and rat fat cells (27)
Because cyclic ffi in1 tictes the (lction alld release of many hormones
it has become knowIl as a ubiquitous second messengertl bull Adenyl cyclase
is activated when an external stimulus such as a hormone comes in con-
tact 111th the cell surface The inCleae in intracellular levels of
cyclic lIP activate other specific enzymes ACTIJI s stiI1ulation of
3
adenyl cyclase in the adrenal cortex follolled by the activation of enzymes
synthesizinEcortiaol exenpliiies this (12) Also thyroid hormone secretion
is induced middothen TSH activates thyroid adenyl cyclase (13)
lhere is some evidence cM may in a sirilar manner mediate
the synthesis ~f prostaglandins (10) EPinephrine and nore~inephrine both
known to stimulcte ade~yl cyclase in certai~ cells stimulate release of
prostaglandins from the dog sp~een (14) In addition it has been shown
prostaglandins stL~u1ate adenyl cyclase in human platelets (7)
The question arose -lhether soe of the effects of aspirin might
be mediated by its effects on the ~~1P systems of various target cells
In order to determine whether a relationship exists between the action
of aspirin ald cyclic ANP the effect of salicylates on j3-galactosidase
synthesis in the bacterial ce11 was obs~rved Synthesis of (3 -galactoshy
sidase in Jscherichia coli is known to require cyclic AImiddotrp (15) In
E COli c~middot~ acts at the transcriptional level of enz~ne synthesis by
promoting initiation of ~-galactosidase nu~fA synthesis The nucleotide
is believed to activate a protein called cMP receptor protein which
in turn allows the protein to bind to the promoter reGion of the lac
operon Its presence enhances the binding oflli1A pol~erase to the
proper initiation site of lac DNA (6) COllsequentlyincreased syntheshy
sis of B-galactosi(ase occurs with optimum concenlirations of cAllP and
repreSSion occurs ~hen the ct1middotp concentration is louered
This paplusmner reorts the effects salicylates have on j3-galactosishy
dase SJIlthesis in E coli and the relationship between cAllPs and salicylic
acid t s mechanism of ac tion within the bacterial cell
YJATERIALS AND NlTHODS
Bacterial strains
For the initial experiments Escherichia coli strain 3000 (lac i +)
was used To determine salicylates effect on inducer uptake a conshy
stitutive strain 3300 (lac i -) was used Both strains llere provided by
Dr John 11 Hyers
Grmmiddotrth Conditions
Bacteria liera grown in double strength Hinimal Broth Davis
medium (Bacto) supplelllented k1th 0002 vitamin B and either 02
glucose or 05 glycerol Double strength media lIas used since this
has essentially the same constituents as the minilal medium used in
earlier experiments observing effects of cyclic AmiddotU on E coli (7)
-Before each experiment fres11 stationary cultures were prepared
from bacteria1 slants StatioIlaty cultures were inoculatr-d into
Klett Flasks to obtain an initia1 ceU density of 10-8ml ald gr01ID on
a shaker at 370 C until log phase was reached To induce fl-galactoshy
sidase -syntheSis isopropyl j3-D-thiogalactopyranoside (rrrG) was added
to the cells in log phase producing a final concentration of 5xl0-4 11
FUrther a1ditions depending on the experiment liere made to log phase
cultures
All cell denSity measurements were taken on a K1ett-~~erson
Photoelectric Colorimeter (Model 80003) using a 660 mp filter
5 ~Galactosida3e Ass~
j3-galactosidase was assayed according to the procedure of Pardee
Jacob and Honod (6) with slight Iilodification At specific timeslt
09 ml of sample lUlS ldthdrakll fro-n the cultures and added ~o tubes
containing 2 drops of 025 sodiurn dodecyl sulfate (SOO) end 2 drops
chloroform (9) Both toluene treatment and SDS - chloroform treatshy
ment were pre~narily tested to deterwine the best method of releasshy
ing enzyme from the middotcell The latter did not alter enzyme stabilitY
and proved to increase the sensitivity of the assq
The amount of enzyme present was measured spectrophotomatr1cally
using a KJett-5UlllIllerson Photoelectric Colorimeter (lmiddotiodel 600-3) witil a
420 111)I1ilter One unit of enzyme is defined as the amount producing
1 II1j1 mole of o-nitrophenol per minute at 280 0 pH 70 The units of
enzyme in the sample can be calculated from the 1act 1 17fL mole per ml
of o-nitrophenol has a Klett reading of 045 under experirlental condishy
tions used here
To verify the assay procedure With E coJ strain 3000 enzyme
activity was confirmed to be linear in relation to time and concentrashy
tion
l-Iaterials
All chevicals were commercially obtained Cyclic adenosine
3 5-monophosphatie was obtained from Sigma and its purity 1-1as
confirmed chromatographically and spectrophotometrlcally Based on
readings vy a Cry Spectrophotometer the cyclic ffJ proved to be
99 pure b-leight Cyclic AHPs biolobical activity was confiIr4ed by
its ability to reverse glucose repression
RESULTS
Tho Effects of Salicylates on 13-GaJactosidase Synthesis
Various concentrations of aspirin were added to E coli strain
3000 cultures grmrlng on glycerol medium At a 5 x 10-3f concentration
ofacetylsa1i~lic aci~the ~fferential rate of j3-galactosidase synshy
thesis decreased (Table I) This 1-1aS accompanied by a slight decrease
in bacterial growth rate At 1 x 10-~ and 25 X 10-3M aspirin had
no significant effect on j3-galactos1dase synthesis
Sinceaspirin1s pharmacological action depends on its salicylic
acid content (1) the effect of salicylic acid on j3-galactosidase synshy
thesis ~1as detemined At all concentrations salicylic acid produced
a greater effect on the differential rate of enzyme synthesis than
aspirin (Table II) Salicylic acid also inhibited grorrth to a considershy
able extent but in all cases the effect on enzyme synthesis was
greater
Control experiments showed both aspirin and salicylic acid had
no effect on the enz~e ass~ system
Since the utjlization of glycerol requires the inducible enzoJlne
glycerol kinase kriown to be sensitive to catabolite repression (~)
it Has postulated the effect of salicylic ~cid on grouth rate resulted
from an inhibition of glYcerol kinase synthesis Such an inhibition
would have the effect of masking decreases in the differential rate
of ~-galactosidcse synthesis For this reason it was decided to use
a carbon source not subject to catabolite repreSSion and presumably
-7
TABLE I
EFFECTS OF ASPIRIN on
DrFFEIl3NTIAL RATE OF SYNTHESIS OF P-GALACTOSIDASE
Elcp 170
Aspirin (N)
Diff Rate En~ Synth (EUrilLKR)
at Control Rate
Grol-rth Rate (1gen time)
of Control Rate
1 1 -( cO3)1x10
25x10-3 5xl0-3
21 -( con3~)5x10shy
3 I -(con3- ) 5xl0shy
361 100 361 100 361 100 254 70
313 100 210 67
33bull3 100 259 78
72hr
63hr bull67hr 60br
100 87 93 825
65hr
54hr 100
83
67hr
58hr 100 87
E coli strain 3000 1-las grOl-m in glycerol raedium until log phase was obtained At -rms time the culture was divided and transferred to fiasks containing 5 x 10- H IPTa ard the indicated amount of dissolved aspirin The control flask contained water in place of the dissolved aspirin solution To insure dilutions would not 1irrdt the media flasks were also initi~ supplemented vnth 4x y~~ Broth Davis
At varioUs cell densities 09 ml samples were taken and assayed for j3-Galactosidase activit7
8
Exp No
1
2bull
3
TABLE II
EFFECT OF SALICYLIC ACID ON
DIFFER11ITIAL It4TE OF SYNTHESIS OF J3-GALACTOSID~sE
Salicylic Acid (N)
-(contro1) x 10shy
-(contro~23 387 x 10
-(contrpound)1 x 10
DtiL Rate ~~th (EUII1lJb Iffi) of
control rate
Growth Rate (ligen time) of
Control
370 62
100 n
60hr
33hr 100
283 65
100 23
71hr
44Ihr 100 62
315 206
bull100 6
60hr
2hr 100 87
Cells l-ere gr01ffl and sampled as in Table I ITith the exception that disshysolved salicylic acid was added to ce~s in log phase in place of aspirin
9
not subject to salicylic aci~ inhibition In all subsequent eXperiments
glucose lias used as carbon source
Upon the addition of 5 x lO-~ salicylic acid to cells grovID in
glucose media there gtmiddotas an ilnmecliate cessation of a-galactosidase synshy
thesis lasting over a generation of grotrth This l-laS followed by a
recovery of srnthesis but at a decreascdrate relative to the control
(fig 1) tt 1 x 10-~i salicylic acid deceased the differential rate
of enzY1e synthesis but tlere 1-TaS a)parently no lag in synthesis after
its addition
From this data it apears salicjlic acid more effectively represses
j3-galactosidase synthesis in glucose grovID cells than in cells grovm
in glycerol redia It should be noted hOHever the inhibitory effect
of salicylic acid on grot-lth rate is the srune vJhether the carbon source
is glucose or glycerol The reason for this inhibition ra1lains obscure
The strect or CYclic AlP on Heprcssion by Snlicylic Acid
If salicylic acid acts by decreasing cyclic PlW levels then cpJmiddot~
should revese the efects of se1icylates Figure 2 shms the resiuts
of rul experinent lihere 5 x lO-3M cyclic AHP and 25 x 10-31-1 salicylic
acid were added silulta1eously to glucose grotm cells It can be seen
that cPJP revJrses the repression caused by salicylic acid Hm1ever
the reversal is only partial since the dif~erential ra-ce is not restored
to the level of the control plus c~tmiddotp cells
Salicylic acid in these experi~ents also caused a decrease in
gromh rate and it lla3 further decreased when cAt-1P las added to the
cells Because of this the data from Figure 2 was replotted with time
units on the abscis3a in place of cell density (Fig 3)~ This allows
10
FIGURE 1
EFFECT OF SLICYLIC ACID ON THE DIFF3P3NrIAL RJTE OF [3-GALACTOSIDA3E SYNTHESIS
strain 3000 Was grovffi in glucose modiUI1 until log phase wst reached
Approrinate1y 1 hour follOldn~ induction rlith IPTG the culture was
separated and trs1sierred to flasks containing the indicted additions
Samples uere talten and assayed for j3-galactosidase as in Table I
Since diaBo1ved salicylic acid lias added a dilution in cell densitr
occurred The first point on the graph represents samples taken L~Jnedi-
ately c)fter the culture Was separatedand diluted
Grorth Rate Differential Enz Rate Flask A Control 75hr 308 EUllilAKR
B Salicylic Acid 68hr 180 EUlllJfoKR
(1 x 10-31)
o Sa1icylic Acid 35hr 95 EUralAlffi (5 x 10-31-1)
12
FIGlllE 2
EFF3Cr OF CYCLIC AmiddotlP 1jJ 3lLICYLIC ACID ON DIFF~JTIAL f3-01AClOSImE SYUTH33IS
IFTG was added to a log phase culture of strain 3000 grown in glucose
medium Approximately 45 minutes after induction the culture was divided
equally into flasks containing the indicated additions 32J1lplcs were
ta~en and ~-Galactosidase was assayed as previously indica~ed The
arroH at 30 Klett unts represents the time the culture ylaS divided
and trcmsferred
DifLerential Rate Growth tate Enzyme Synthesis
Flask A Control bull67hr 23 lJrrJ KIt
B Cyclic mp (5xlO-311) 57hr 315EUrr~6KR
c SalicyliQ acid 4lhr 135ZUmlKR(25xlO-)N)
D Salicy~c acid bull 35hr 25 ~1JrrJLKR 25xlO ~ 11 + CJllmiddotfP (5xlO-)~1)
+
0
t
o
oft
$+
~
Ft flJ rsi
m
m
mlt
l ~ ~
sect4( ~
m~ ~ e1
54 PI rd ISJ
aI ttl
14
FIGURE 3
EFF~T OF CYCLIC AHP UID SJLICYLIC ACID ON j)-GLACTO3IDASE SYNTIIESIS
This represents the smne experireental data as in Figure 1 In the
graph enz~e units ~re plotted in rel~tion to tL~e rather th~n in
relation to cell denitr rIle arrml indicates the time the culture
lras divided and all additions l-Tere i1ade
bull
bullbull Q
-Ld
bull
16 the effect of 321icylic acid ~o be noted indeendently of itsmiddot effect
on generlttion time Even on this graph cAli is shown to partially
relieve repression caused by salicyla~es
Previously Pastaa ~md PerL1an (21)(22) emphasized cAHP had no
effect on growth rate of E coli strain 300 rlhich 112S in contrast to
these results Their dltta homver referto using 1 x lO-3z cANP
Since 5 x 10- 3r crolic lp is believed to overcome both tranient and
catabolite reression caused by r~ucose in strain 3000 (23) the effect
of this concentration on salicylic acids repression was observed
Sirlilar results Nere obtcgtined in a second eXgerimcnt using
S x 10-31-1 salicylic acid and 5 x lO-3H Cl]~ (Fig 4) rhe degree of
reversal ias again only partial Because grorlth inhibition at this conshy
centration of salicylic acid tdth cAHP ias so great the curve is not
extended ver ffX
From these data it appears there is an interaction between the
effects of sali~Jlic acid and those of cMmiddotP Since repression proshy
duced by the higher concentration of salictlic acid seems to be less
effectively reversed by 5 x 10-3N cMP there i~ a possibility the
degree of interaction between the tlO may be concentration dependent
The 3ffect of Sdic-lates on Consti tutive Enzyme Synthesis
To eliminate the possibility salicylic acid may repress j3-galactoshy
sidase synthesis by preventing inducer upt~~e or by competing with
inducer for repressor an experiYlent vTaS carried out using strain 3300
constitutive for fo-galactosidase synthemiddotsis From Figure 5 it is evident
that 25 x 10-311 Jalicylic acid had the same effect on strain 3300 as it
had previously on strain 3000 Therefore salicylates act independently
17
B
uJ ~ l ~ l cJ J L o o- Ul t z 3 w r N Ia Z 312L fa m sa 1m m Em2 ru KLETI UNITS
EFFECT OF CYCLIC Almiddotp lm SLICYLIC ACID OU DIFFrRENfIAL j2gt-GALACTOSIDA3E SYNlHESIS
Figure 4 Strain 3000 culture las grOiID sampled and assayed for fl-g3~actosidase as in Figure 2 uitIl the exception a hig1er concentrC)tion of salic7l1ic acid i-as added after separshyation Since sliicylic acid 35 in dissolved form a dilution occurred upon its addition This uas compensated for in
J
-fA
other fiCsls by an equal dilution
flask Pt Control
Bbull Control + cAHP (5xlO-3H)
c Salicl~ic acid (5xlO H)
D salicl~c acid
Grmmiddotrth Rc1te 71hr
52hr
22hr
15hr (5xlO 3 I) + cMi (5xlO- 1middot1)
Diff Enz Rate 22 EUmlAKR
31 t1Jm1~KR
12 1UmlAIffi
16 EUmlAKR
18
FIGUR35
EFECr 01 SALICYLIC ACID OH DIIT2HE~rrAL fgt-GAL~C0SIDASE SYIJIl133I3 nr COnSrITlJlIV3TAIH 3300
strain 3300 iolaS gron in glucose medium Jllen the cells reached log
phase25 x lO-~1 snlicylic acid ~as added Cells were sa1pled ltmd
4asJared 80S in earlier experiments IPTG (5 x 10- 1-1) liaS added to
control flasks to note the degree of const~tutiveness of tile strain
The first points ngain represent sa~les trucen in~ediately poundter the
culture was divided and additions ware made
Growth Rate Diff Enz Synth Flask A Control bull89hr 65 EUrnlAKR
B Contro~ + IPTG bull89hr 65 EUmJ6KR (5xlO-~middotI)
o SalicYli~3acid bull54hr 46 ~iJ~KR (25x 10 H)
D Sa1icrli~ acid 5hhr 30 EUml~KR (25xlR--i-r) + IPTG (5xlO- M)
lSi JT1
bull 0
Fa-
I
a +
tsJ
yen
+
~
v
+
tsi IS1 m
m
tn
til sectll
m ~ ~ 2
bull
ef ~
~ ~ tsJ
uf
shy
20
of inducer uptake and of inducer-s derepression of lac mrrNA synthesis
The zrecl of 24-Di1ikophcnol ont30alactosidase Synthesis
Since salicylates are kno-Vffi to be uncouplers of oxidative phosshy
phorylation (5) it has been sJgccsted sOrle of their phaITlClcological
effects result from their uncoupling action Tis i-lould irply that
other uncouplers should preferentially inhibit ~-gilactosidase syntheshy
sis For this reason the effect of 2 4-dini trophenol (Dr~) on J-galacshy
tosidase synthesis Has observed Because DrIP is more effective in inhibishy
ting oxidative phosphorylation than salicylic acid and is consequently
more toxic a concentration of the drug i-laS found vhic uould inhibit
grOl-rth to ap~roximately the sa11e degree as 25 x 10-31gt1 salicylic acid
This was found to be 25 x 10-411 Figure 6 illustrates DNP does not
repress differential synthesis of j3-g1actosidase This implies that
althoueh DNP 1m-lers ATP levels there is no apparent effect of the drug
on lsvels of cAl-IP Also it appears salic-rlic acid is not exerting its
repression of enzyrre sJ~~hesis by inhibiting oxidative phosphorylation
21
FIGUllS 6
EFFECrl OF DJP ON DIFFif2rIlL ~GLCiO3D~S STImiddotrYliESIS
Jhen strain 3000 cells reacled log ph~se they tmiddotrere indlccd 1ith IPTG
AproxL~ately one hour later the culture was divide1 a~d tr~ferred
to flasks containing the indicated adlitions The cellsas in preVious
experirrents Tere sarlpled at various tLTIes and assayed for B-galactoshy
sidase activity Since 2h-dinitrophenol t-rag in solution its addition
resuted in dilution of cultures This dilution las equally conpensated
for in control flasks nle first points on the graph represent samples
taken innediately after sepration of the cuJture
GroYTth Rate Difierential inz Rate Flask A Control 75hr 22 L1JmlAKR
B Contro + cAll 64hr 50 EUmlAm (5y10- H) 4z
C DNP (25xlO- -I) 42hr 22 EOIraAKR
D DNP (25xl2~4zr) + 29hr 42 UmlAKR cAJP (5x10 -0
N
N
v
o
-shy-shy+
-shy
+
bull-
DISCU3SION
Salicylic acid ruld to a lesser extent aspirin cause repression
of f3-gclactosidase srnthesis in scherichia coli This effect is not
due to an inhibition of inducer uptake nor is it due ~o interference
l-rith inducers inactivation of lac repressor since strnins constitutive
for 3-gJactosidase 3J1lthesis are also repressed b~r sclicyli~ acid
In addition because dinitrophenol is liithout effect on differential
fl-galactosidase synthesis it appecrs salicrlates do not repress enzyme
s~mthesis by inhibiting oxidative phospllorylation
At these concentr2tions however salicylic acid does decrease
growth rate and direct or indirect effects of this on the observed
repression of enzyme synt~esis still ra~ain Since the differential
rate of enzyme synthesis i~ decreased and also since 25 x 10-411 DN
decreases grotrth rate aproximately the same degree middotuitl1cut having I
effects on differential synthesis salicylates inhibition of growth
is not the sole factor responsible for its effects on enZJcre synthesis
-11en 5 x 10-31 cyclic JUfP 1-ms added to cells treated Kith 25 x 10-3
and 5 x lO-3H srJicylic acid repression of differential enzye s~1lhesis
1ES pextially relieved This sugests salicllic acid represses J3-gJ~ac-
tosidase srnthesis br directly 1011erinti intIacellular levels of cyclic AMP
or by indirectly acting on asita which in turn releases cANP from the
cell Since cMP only partially relieves repression caused by wlicylic
acid and because there appears to be an inverse relationship between
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
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3
adenyl cyclase in the adrenal cortex follolled by the activation of enzymes
synthesizinEcortiaol exenpliiies this (12) Also thyroid hormone secretion
is induced middothen TSH activates thyroid adenyl cyclase (13)
lhere is some evidence cM may in a sirilar manner mediate
the synthesis ~f prostaglandins (10) EPinephrine and nore~inephrine both
known to stimulcte ade~yl cyclase in certai~ cells stimulate release of
prostaglandins from the dog sp~een (14) In addition it has been shown
prostaglandins stL~u1ate adenyl cyclase in human platelets (7)
The question arose -lhether soe of the effects of aspirin might
be mediated by its effects on the ~~1P systems of various target cells
In order to determine whether a relationship exists between the action
of aspirin ald cyclic ANP the effect of salicylates on j3-galactosidase
synthesis in the bacterial ce11 was obs~rved Synthesis of (3 -galactoshy
sidase in Jscherichia coli is known to require cyclic AImiddotrp (15) In
E COli c~middot~ acts at the transcriptional level of enz~ne synthesis by
promoting initiation of ~-galactosidase nu~fA synthesis The nucleotide
is believed to activate a protein called cMP receptor protein which
in turn allows the protein to bind to the promoter reGion of the lac
operon Its presence enhances the binding oflli1A pol~erase to the
proper initiation site of lac DNA (6) COllsequentlyincreased syntheshy
sis of B-galactosi(ase occurs with optimum concenlirations of cAllP and
repreSSion occurs ~hen the ct1middotp concentration is louered
This paplusmner reorts the effects salicylates have on j3-galactosishy
dase SJIlthesis in E coli and the relationship between cAllPs and salicylic
acid t s mechanism of ac tion within the bacterial cell
YJATERIALS AND NlTHODS
Bacterial strains
For the initial experiments Escherichia coli strain 3000 (lac i +)
was used To determine salicylates effect on inducer uptake a conshy
stitutive strain 3300 (lac i -) was used Both strains llere provided by
Dr John 11 Hyers
Grmmiddotrth Conditions
Bacteria liera grown in double strength Hinimal Broth Davis
medium (Bacto) supplelllented k1th 0002 vitamin B and either 02
glucose or 05 glycerol Double strength media lIas used since this
has essentially the same constituents as the minilal medium used in
earlier experiments observing effects of cyclic AmiddotU on E coli (7)
-Before each experiment fres11 stationary cultures were prepared
from bacteria1 slants StatioIlaty cultures were inoculatr-d into
Klett Flasks to obtain an initia1 ceU density of 10-8ml ald gr01ID on
a shaker at 370 C until log phase was reached To induce fl-galactoshy
sidase -syntheSis isopropyl j3-D-thiogalactopyranoside (rrrG) was added
to the cells in log phase producing a final concentration of 5xl0-4 11
FUrther a1ditions depending on the experiment liere made to log phase
cultures
All cell denSity measurements were taken on a K1ett-~~erson
Photoelectric Colorimeter (Model 80003) using a 660 mp filter
5 ~Galactosida3e Ass~
j3-galactosidase was assayed according to the procedure of Pardee
Jacob and Honod (6) with slight Iilodification At specific timeslt
09 ml of sample lUlS ldthdrakll fro-n the cultures and added ~o tubes
containing 2 drops of 025 sodiurn dodecyl sulfate (SOO) end 2 drops
chloroform (9) Both toluene treatment and SDS - chloroform treatshy
ment were pre~narily tested to deterwine the best method of releasshy
ing enzyme from the middotcell The latter did not alter enzyme stabilitY
and proved to increase the sensitivity of the assq
The amount of enzyme present was measured spectrophotomatr1cally
using a KJett-5UlllIllerson Photoelectric Colorimeter (lmiddotiodel 600-3) witil a
420 111)I1ilter One unit of enzyme is defined as the amount producing
1 II1j1 mole of o-nitrophenol per minute at 280 0 pH 70 The units of
enzyme in the sample can be calculated from the 1act 1 17fL mole per ml
of o-nitrophenol has a Klett reading of 045 under experirlental condishy
tions used here
To verify the assay procedure With E coJ strain 3000 enzyme
activity was confirmed to be linear in relation to time and concentrashy
tion
l-Iaterials
All chevicals were commercially obtained Cyclic adenosine
3 5-monophosphatie was obtained from Sigma and its purity 1-1as
confirmed chromatographically and spectrophotometrlcally Based on
readings vy a Cry Spectrophotometer the cyclic ffJ proved to be
99 pure b-leight Cyclic AHPs biolobical activity was confiIr4ed by
its ability to reverse glucose repression
RESULTS
Tho Effects of Salicylates on 13-GaJactosidase Synthesis
Various concentrations of aspirin were added to E coli strain
3000 cultures grmrlng on glycerol medium At a 5 x 10-3f concentration
ofacetylsa1i~lic aci~the ~fferential rate of j3-galactosidase synshy
thesis decreased (Table I) This 1-1aS accompanied by a slight decrease
in bacterial growth rate At 1 x 10-~ and 25 X 10-3M aspirin had
no significant effect on j3-galactos1dase synthesis
Sinceaspirin1s pharmacological action depends on its salicylic
acid content (1) the effect of salicylic acid on j3-galactosidase synshy
thesis ~1as detemined At all concentrations salicylic acid produced
a greater effect on the differential rate of enzyme synthesis than
aspirin (Table II) Salicylic acid also inhibited grorrth to a considershy
able extent but in all cases the effect on enzyme synthesis was
greater
Control experiments showed both aspirin and salicylic acid had
no effect on the enz~e ass~ system
Since the utjlization of glycerol requires the inducible enzoJlne
glycerol kinase kriown to be sensitive to catabolite repression (~)
it Has postulated the effect of salicylic ~cid on grouth rate resulted
from an inhibition of glYcerol kinase synthesis Such an inhibition
would have the effect of masking decreases in the differential rate
of ~-galactosidcse synthesis For this reason it was decided to use
a carbon source not subject to catabolite repreSSion and presumably
-7
TABLE I
EFFECTS OF ASPIRIN on
DrFFEIl3NTIAL RATE OF SYNTHESIS OF P-GALACTOSIDASE
Elcp 170
Aspirin (N)
Diff Rate En~ Synth (EUrilLKR)
at Control Rate
Grol-rth Rate (1gen time)
of Control Rate
1 1 -( cO3)1x10
25x10-3 5xl0-3
21 -( con3~)5x10shy
3 I -(con3- ) 5xl0shy
361 100 361 100 361 100 254 70
313 100 210 67
33bull3 100 259 78
72hr
63hr bull67hr 60br
100 87 93 825
65hr
54hr 100
83
67hr
58hr 100 87
E coli strain 3000 1-las grOl-m in glycerol raedium until log phase was obtained At -rms time the culture was divided and transferred to fiasks containing 5 x 10- H IPTa ard the indicated amount of dissolved aspirin The control flask contained water in place of the dissolved aspirin solution To insure dilutions would not 1irrdt the media flasks were also initi~ supplemented vnth 4x y~~ Broth Davis
At varioUs cell densities 09 ml samples were taken and assayed for j3-Galactosidase activit7
8
Exp No
1
2bull
3
TABLE II
EFFECT OF SALICYLIC ACID ON
DIFFER11ITIAL It4TE OF SYNTHESIS OF J3-GALACTOSID~sE
Salicylic Acid (N)
-(contro1) x 10shy
-(contro~23 387 x 10
-(contrpound)1 x 10
DtiL Rate ~~th (EUII1lJb Iffi) of
control rate
Growth Rate (ligen time) of
Control
370 62
100 n
60hr
33hr 100
283 65
100 23
71hr
44Ihr 100 62
315 206
bull100 6
60hr
2hr 100 87
Cells l-ere gr01ffl and sampled as in Table I ITith the exception that disshysolved salicylic acid was added to ce~s in log phase in place of aspirin
9
not subject to salicylic aci~ inhibition In all subsequent eXperiments
glucose lias used as carbon source
Upon the addition of 5 x lO-~ salicylic acid to cells grovID in
glucose media there gtmiddotas an ilnmecliate cessation of a-galactosidase synshy
thesis lasting over a generation of grotrth This l-laS followed by a
recovery of srnthesis but at a decreascdrate relative to the control
(fig 1) tt 1 x 10-~i salicylic acid deceased the differential rate
of enzY1e synthesis but tlere 1-TaS a)parently no lag in synthesis after
its addition
From this data it apears salicjlic acid more effectively represses
j3-galactosidase synthesis in glucose grovID cells than in cells grovm
in glycerol redia It should be noted hOHever the inhibitory effect
of salicylic acid on grot-lth rate is the srune vJhether the carbon source
is glucose or glycerol The reason for this inhibition ra1lains obscure
The strect or CYclic AlP on Heprcssion by Snlicylic Acid
If salicylic acid acts by decreasing cyclic PlW levels then cpJmiddot~
should revese the efects of se1icylates Figure 2 shms the resiuts
of rul experinent lihere 5 x lO-3M cyclic AHP and 25 x 10-31-1 salicylic
acid were added silulta1eously to glucose grotm cells It can be seen
that cPJP revJrses the repression caused by salicylic acid Hm1ever
the reversal is only partial since the dif~erential ra-ce is not restored
to the level of the control plus c~tmiddotp cells
Salicylic acid in these experi~ents also caused a decrease in
gromh rate and it lla3 further decreased when cAt-1P las added to the
cells Because of this the data from Figure 2 was replotted with time
units on the abscis3a in place of cell density (Fig 3)~ This allows
10
FIGURE 1
EFFECT OF SLICYLIC ACID ON THE DIFF3P3NrIAL RJTE OF [3-GALACTOSIDA3E SYNTHESIS
strain 3000 Was grovffi in glucose modiUI1 until log phase wst reached
Approrinate1y 1 hour follOldn~ induction rlith IPTG the culture was
separated and trs1sierred to flasks containing the indicted additions
Samples uere talten and assayed for j3-galactosidase as in Table I
Since diaBo1ved salicylic acid lias added a dilution in cell densitr
occurred The first point on the graph represents samples taken L~Jnedi-
ately c)fter the culture Was separatedand diluted
Grorth Rate Differential Enz Rate Flask A Control 75hr 308 EUllilAKR
B Salicylic Acid 68hr 180 EUlllJfoKR
(1 x 10-31)
o Sa1icylic Acid 35hr 95 EUralAlffi (5 x 10-31-1)
12
FIGlllE 2
EFF3Cr OF CYCLIC AmiddotlP 1jJ 3lLICYLIC ACID ON DIFF~JTIAL f3-01AClOSImE SYUTH33IS
IFTG was added to a log phase culture of strain 3000 grown in glucose
medium Approximately 45 minutes after induction the culture was divided
equally into flasks containing the indicated additions 32J1lplcs were
ta~en and ~-Galactosidase was assayed as previously indica~ed The
arroH at 30 Klett unts represents the time the culture ylaS divided
and trcmsferred
DifLerential Rate Growth tate Enzyme Synthesis
Flask A Control bull67hr 23 lJrrJ KIt
B Cyclic mp (5xlO-311) 57hr 315EUrr~6KR
c SalicyliQ acid 4lhr 135ZUmlKR(25xlO-)N)
D Salicy~c acid bull 35hr 25 ~1JrrJLKR 25xlO ~ 11 + CJllmiddotfP (5xlO-)~1)
+
0
t
o
oft
$+
~
Ft flJ rsi
m
m
mlt
l ~ ~
sect4( ~
m~ ~ e1
54 PI rd ISJ
aI ttl
14
FIGURE 3
EFF~T OF CYCLIC AHP UID SJLICYLIC ACID ON j)-GLACTO3IDASE SYNTIIESIS
This represents the smne experireental data as in Figure 1 In the
graph enz~e units ~re plotted in rel~tion to tL~e rather th~n in
relation to cell denitr rIle arrml indicates the time the culture
lras divided and all additions l-Tere i1ade
bull
bullbull Q
-Ld
bull
16 the effect of 321icylic acid ~o be noted indeendently of itsmiddot effect
on generlttion time Even on this graph cAli is shown to partially
relieve repression caused by salicyla~es
Previously Pastaa ~md PerL1an (21)(22) emphasized cAHP had no
effect on growth rate of E coli strain 300 rlhich 112S in contrast to
these results Their dltta homver referto using 1 x lO-3z cANP
Since 5 x 10- 3r crolic lp is believed to overcome both tranient and
catabolite reression caused by r~ucose in strain 3000 (23) the effect
of this concentration on salicylic acids repression was observed
Sirlilar results Nere obtcgtined in a second eXgerimcnt using
S x 10-31-1 salicylic acid and 5 x lO-3H Cl]~ (Fig 4) rhe degree of
reversal ias again only partial Because grorlth inhibition at this conshy
centration of salicylic acid tdth cAHP ias so great the curve is not
extended ver ffX
From these data it appears there is an interaction between the
effects of sali~Jlic acid and those of cMmiddotP Since repression proshy
duced by the higher concentration of salictlic acid seems to be less
effectively reversed by 5 x 10-3N cMP there i~ a possibility the
degree of interaction between the tlO may be concentration dependent
The 3ffect of Sdic-lates on Consti tutive Enzyme Synthesis
To eliminate the possibility salicylic acid may repress j3-galactoshy
sidase synthesis by preventing inducer upt~~e or by competing with
inducer for repressor an experiYlent vTaS carried out using strain 3300
constitutive for fo-galactosidase synthemiddotsis From Figure 5 it is evident
that 25 x 10-311 Jalicylic acid had the same effect on strain 3300 as it
had previously on strain 3000 Therefore salicylates act independently
17
B
uJ ~ l ~ l cJ J L o o- Ul t z 3 w r N Ia Z 312L fa m sa 1m m Em2 ru KLETI UNITS
EFFECT OF CYCLIC Almiddotp lm SLICYLIC ACID OU DIFFrRENfIAL j2gt-GALACTOSIDA3E SYNlHESIS
Figure 4 Strain 3000 culture las grOiID sampled and assayed for fl-g3~actosidase as in Figure 2 uitIl the exception a hig1er concentrC)tion of salic7l1ic acid i-as added after separshyation Since sliicylic acid 35 in dissolved form a dilution occurred upon its addition This uas compensated for in
J
-fA
other fiCsls by an equal dilution
flask Pt Control
Bbull Control + cAHP (5xlO-3H)
c Salicl~ic acid (5xlO H)
D salicl~c acid
Grmmiddotrth Rc1te 71hr
52hr
22hr
15hr (5xlO 3 I) + cMi (5xlO- 1middot1)
Diff Enz Rate 22 EUmlAKR
31 t1Jm1~KR
12 1UmlAIffi
16 EUmlAKR
18
FIGUR35
EFECr 01 SALICYLIC ACID OH DIIT2HE~rrAL fgt-GAL~C0SIDASE SYIJIl133I3 nr COnSrITlJlIV3TAIH 3300
strain 3300 iolaS gron in glucose medium Jllen the cells reached log
phase25 x lO-~1 snlicylic acid ~as added Cells were sa1pled ltmd
4asJared 80S in earlier experiments IPTG (5 x 10- 1-1) liaS added to
control flasks to note the degree of const~tutiveness of tile strain
The first points ngain represent sa~les trucen in~ediately poundter the
culture was divided and additions ware made
Growth Rate Diff Enz Synth Flask A Control bull89hr 65 EUrnlAKR
B Contro~ + IPTG bull89hr 65 EUmJ6KR (5xlO-~middotI)
o SalicYli~3acid bull54hr 46 ~iJ~KR (25x 10 H)
D Sa1icrli~ acid 5hhr 30 EUml~KR (25xlR--i-r) + IPTG (5xlO- M)
lSi JT1
bull 0
Fa-
I
a +
tsJ
yen
+
~
v
+
tsi IS1 m
m
tn
til sectll
m ~ ~ 2
bull
ef ~
~ ~ tsJ
uf
shy
20
of inducer uptake and of inducer-s derepression of lac mrrNA synthesis
The zrecl of 24-Di1ikophcnol ont30alactosidase Synthesis
Since salicylates are kno-Vffi to be uncouplers of oxidative phosshy
phorylation (5) it has been sJgccsted sOrle of their phaITlClcological
effects result from their uncoupling action Tis i-lould irply that
other uncouplers should preferentially inhibit ~-gilactosidase syntheshy
sis For this reason the effect of 2 4-dini trophenol (Dr~) on J-galacshy
tosidase synthesis Has observed Because DrIP is more effective in inhibishy
ting oxidative phosphorylation than salicylic acid and is consequently
more toxic a concentration of the drug i-laS found vhic uould inhibit
grOl-rth to ap~roximately the sa11e degree as 25 x 10-31gt1 salicylic acid
This was found to be 25 x 10-411 Figure 6 illustrates DNP does not
repress differential synthesis of j3-g1actosidase This implies that
althoueh DNP 1m-lers ATP levels there is no apparent effect of the drug
on lsvels of cAl-IP Also it appears salic-rlic acid is not exerting its
repression of enzyrre sJ~~hesis by inhibiting oxidative phosphorylation
21
FIGUllS 6
EFFECrl OF DJP ON DIFFif2rIlL ~GLCiO3D~S STImiddotrYliESIS
Jhen strain 3000 cells reacled log ph~se they tmiddotrere indlccd 1ith IPTG
AproxL~ately one hour later the culture was divide1 a~d tr~ferred
to flasks containing the indicated adlitions The cellsas in preVious
experirrents Tere sarlpled at various tLTIes and assayed for B-galactoshy
sidase activity Since 2h-dinitrophenol t-rag in solution its addition
resuted in dilution of cultures This dilution las equally conpensated
for in control flasks nle first points on the graph represent samples
taken innediately after sepration of the cuJture
GroYTth Rate Difierential inz Rate Flask A Control 75hr 22 L1JmlAKR
B Contro + cAll 64hr 50 EUmlAm (5y10- H) 4z
C DNP (25xlO- -I) 42hr 22 EOIraAKR
D DNP (25xl2~4zr) + 29hr 42 UmlAKR cAJP (5x10 -0
N
N
v
o
-shy-shy+
-shy
+
bull-
DISCU3SION
Salicylic acid ruld to a lesser extent aspirin cause repression
of f3-gclactosidase srnthesis in scherichia coli This effect is not
due to an inhibition of inducer uptake nor is it due ~o interference
l-rith inducers inactivation of lac repressor since strnins constitutive
for 3-gJactosidase 3J1lthesis are also repressed b~r sclicyli~ acid
In addition because dinitrophenol is liithout effect on differential
fl-galactosidase synthesis it appecrs salicrlates do not repress enzyme
s~mthesis by inhibiting oxidative phospllorylation
At these concentr2tions however salicylic acid does decrease
growth rate and direct or indirect effects of this on the observed
repression of enzyme synt~esis still ra~ain Since the differential
rate of enzyme synthesis i~ decreased and also since 25 x 10-411 DN
decreases grotrth rate aproximately the same degree middotuitl1cut having I
effects on differential synthesis salicylates inhibition of growth
is not the sole factor responsible for its effects on enZJcre synthesis
-11en 5 x 10-31 cyclic JUfP 1-ms added to cells treated Kith 25 x 10-3
and 5 x lO-3H srJicylic acid repression of differential enzye s~1lhesis
1ES pextially relieved This sugests salicllic acid represses J3-gJ~ac-
tosidase srnthesis br directly 1011erinti intIacellular levels of cyclic AMP
or by indirectly acting on asita which in turn releases cANP from the
cell Since cMP only partially relieves repression caused by wlicylic
acid and because there appears to be an inverse relationship between
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
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YJATERIALS AND NlTHODS
Bacterial strains
For the initial experiments Escherichia coli strain 3000 (lac i +)
was used To determine salicylates effect on inducer uptake a conshy
stitutive strain 3300 (lac i -) was used Both strains llere provided by
Dr John 11 Hyers
Grmmiddotrth Conditions
Bacteria liera grown in double strength Hinimal Broth Davis
medium (Bacto) supplelllented k1th 0002 vitamin B and either 02
glucose or 05 glycerol Double strength media lIas used since this
has essentially the same constituents as the minilal medium used in
earlier experiments observing effects of cyclic AmiddotU on E coli (7)
-Before each experiment fres11 stationary cultures were prepared
from bacteria1 slants StatioIlaty cultures were inoculatr-d into
Klett Flasks to obtain an initia1 ceU density of 10-8ml ald gr01ID on
a shaker at 370 C until log phase was reached To induce fl-galactoshy
sidase -syntheSis isopropyl j3-D-thiogalactopyranoside (rrrG) was added
to the cells in log phase producing a final concentration of 5xl0-4 11
FUrther a1ditions depending on the experiment liere made to log phase
cultures
All cell denSity measurements were taken on a K1ett-~~erson
Photoelectric Colorimeter (Model 80003) using a 660 mp filter
5 ~Galactosida3e Ass~
j3-galactosidase was assayed according to the procedure of Pardee
Jacob and Honod (6) with slight Iilodification At specific timeslt
09 ml of sample lUlS ldthdrakll fro-n the cultures and added ~o tubes
containing 2 drops of 025 sodiurn dodecyl sulfate (SOO) end 2 drops
chloroform (9) Both toluene treatment and SDS - chloroform treatshy
ment were pre~narily tested to deterwine the best method of releasshy
ing enzyme from the middotcell The latter did not alter enzyme stabilitY
and proved to increase the sensitivity of the assq
The amount of enzyme present was measured spectrophotomatr1cally
using a KJett-5UlllIllerson Photoelectric Colorimeter (lmiddotiodel 600-3) witil a
420 111)I1ilter One unit of enzyme is defined as the amount producing
1 II1j1 mole of o-nitrophenol per minute at 280 0 pH 70 The units of
enzyme in the sample can be calculated from the 1act 1 17fL mole per ml
of o-nitrophenol has a Klett reading of 045 under experirlental condishy
tions used here
To verify the assay procedure With E coJ strain 3000 enzyme
activity was confirmed to be linear in relation to time and concentrashy
tion
l-Iaterials
All chevicals were commercially obtained Cyclic adenosine
3 5-monophosphatie was obtained from Sigma and its purity 1-1as
confirmed chromatographically and spectrophotometrlcally Based on
readings vy a Cry Spectrophotometer the cyclic ffJ proved to be
99 pure b-leight Cyclic AHPs biolobical activity was confiIr4ed by
its ability to reverse glucose repression
RESULTS
Tho Effects of Salicylates on 13-GaJactosidase Synthesis
Various concentrations of aspirin were added to E coli strain
3000 cultures grmrlng on glycerol medium At a 5 x 10-3f concentration
ofacetylsa1i~lic aci~the ~fferential rate of j3-galactosidase synshy
thesis decreased (Table I) This 1-1aS accompanied by a slight decrease
in bacterial growth rate At 1 x 10-~ and 25 X 10-3M aspirin had
no significant effect on j3-galactos1dase synthesis
Sinceaspirin1s pharmacological action depends on its salicylic
acid content (1) the effect of salicylic acid on j3-galactosidase synshy
thesis ~1as detemined At all concentrations salicylic acid produced
a greater effect on the differential rate of enzyme synthesis than
aspirin (Table II) Salicylic acid also inhibited grorrth to a considershy
able extent but in all cases the effect on enzyme synthesis was
greater
Control experiments showed both aspirin and salicylic acid had
no effect on the enz~e ass~ system
Since the utjlization of glycerol requires the inducible enzoJlne
glycerol kinase kriown to be sensitive to catabolite repression (~)
it Has postulated the effect of salicylic ~cid on grouth rate resulted
from an inhibition of glYcerol kinase synthesis Such an inhibition
would have the effect of masking decreases in the differential rate
of ~-galactosidcse synthesis For this reason it was decided to use
a carbon source not subject to catabolite repreSSion and presumably
-7
TABLE I
EFFECTS OF ASPIRIN on
DrFFEIl3NTIAL RATE OF SYNTHESIS OF P-GALACTOSIDASE
Elcp 170
Aspirin (N)
Diff Rate En~ Synth (EUrilLKR)
at Control Rate
Grol-rth Rate (1gen time)
of Control Rate
1 1 -( cO3)1x10
25x10-3 5xl0-3
21 -( con3~)5x10shy
3 I -(con3- ) 5xl0shy
361 100 361 100 361 100 254 70
313 100 210 67
33bull3 100 259 78
72hr
63hr bull67hr 60br
100 87 93 825
65hr
54hr 100
83
67hr
58hr 100 87
E coli strain 3000 1-las grOl-m in glycerol raedium until log phase was obtained At -rms time the culture was divided and transferred to fiasks containing 5 x 10- H IPTa ard the indicated amount of dissolved aspirin The control flask contained water in place of the dissolved aspirin solution To insure dilutions would not 1irrdt the media flasks were also initi~ supplemented vnth 4x y~~ Broth Davis
At varioUs cell densities 09 ml samples were taken and assayed for j3-Galactosidase activit7
8
Exp No
1
2bull
3
TABLE II
EFFECT OF SALICYLIC ACID ON
DIFFER11ITIAL It4TE OF SYNTHESIS OF J3-GALACTOSID~sE
Salicylic Acid (N)
-(contro1) x 10shy
-(contro~23 387 x 10
-(contrpound)1 x 10
DtiL Rate ~~th (EUII1lJb Iffi) of
control rate
Growth Rate (ligen time) of
Control
370 62
100 n
60hr
33hr 100
283 65
100 23
71hr
44Ihr 100 62
315 206
bull100 6
60hr
2hr 100 87
Cells l-ere gr01ffl and sampled as in Table I ITith the exception that disshysolved salicylic acid was added to ce~s in log phase in place of aspirin
9
not subject to salicylic aci~ inhibition In all subsequent eXperiments
glucose lias used as carbon source
Upon the addition of 5 x lO-~ salicylic acid to cells grovID in
glucose media there gtmiddotas an ilnmecliate cessation of a-galactosidase synshy
thesis lasting over a generation of grotrth This l-laS followed by a
recovery of srnthesis but at a decreascdrate relative to the control
(fig 1) tt 1 x 10-~i salicylic acid deceased the differential rate
of enzY1e synthesis but tlere 1-TaS a)parently no lag in synthesis after
its addition
From this data it apears salicjlic acid more effectively represses
j3-galactosidase synthesis in glucose grovID cells than in cells grovm
in glycerol redia It should be noted hOHever the inhibitory effect
of salicylic acid on grot-lth rate is the srune vJhether the carbon source
is glucose or glycerol The reason for this inhibition ra1lains obscure
The strect or CYclic AlP on Heprcssion by Snlicylic Acid
If salicylic acid acts by decreasing cyclic PlW levels then cpJmiddot~
should revese the efects of se1icylates Figure 2 shms the resiuts
of rul experinent lihere 5 x lO-3M cyclic AHP and 25 x 10-31-1 salicylic
acid were added silulta1eously to glucose grotm cells It can be seen
that cPJP revJrses the repression caused by salicylic acid Hm1ever
the reversal is only partial since the dif~erential ra-ce is not restored
to the level of the control plus c~tmiddotp cells
Salicylic acid in these experi~ents also caused a decrease in
gromh rate and it lla3 further decreased when cAt-1P las added to the
cells Because of this the data from Figure 2 was replotted with time
units on the abscis3a in place of cell density (Fig 3)~ This allows
10
FIGURE 1
EFFECT OF SLICYLIC ACID ON THE DIFF3P3NrIAL RJTE OF [3-GALACTOSIDA3E SYNTHESIS
strain 3000 Was grovffi in glucose modiUI1 until log phase wst reached
Approrinate1y 1 hour follOldn~ induction rlith IPTG the culture was
separated and trs1sierred to flasks containing the indicted additions
Samples uere talten and assayed for j3-galactosidase as in Table I
Since diaBo1ved salicylic acid lias added a dilution in cell densitr
occurred The first point on the graph represents samples taken L~Jnedi-
ately c)fter the culture Was separatedand diluted
Grorth Rate Differential Enz Rate Flask A Control 75hr 308 EUllilAKR
B Salicylic Acid 68hr 180 EUlllJfoKR
(1 x 10-31)
o Sa1icylic Acid 35hr 95 EUralAlffi (5 x 10-31-1)
12
FIGlllE 2
EFF3Cr OF CYCLIC AmiddotlP 1jJ 3lLICYLIC ACID ON DIFF~JTIAL f3-01AClOSImE SYUTH33IS
IFTG was added to a log phase culture of strain 3000 grown in glucose
medium Approximately 45 minutes after induction the culture was divided
equally into flasks containing the indicated additions 32J1lplcs were
ta~en and ~-Galactosidase was assayed as previously indica~ed The
arroH at 30 Klett unts represents the time the culture ylaS divided
and trcmsferred
DifLerential Rate Growth tate Enzyme Synthesis
Flask A Control bull67hr 23 lJrrJ KIt
B Cyclic mp (5xlO-311) 57hr 315EUrr~6KR
c SalicyliQ acid 4lhr 135ZUmlKR(25xlO-)N)
D Salicy~c acid bull 35hr 25 ~1JrrJLKR 25xlO ~ 11 + CJllmiddotfP (5xlO-)~1)
+
0
t
o
oft
$+
~
Ft flJ rsi
m
m
mlt
l ~ ~
sect4( ~
m~ ~ e1
54 PI rd ISJ
aI ttl
14
FIGURE 3
EFF~T OF CYCLIC AHP UID SJLICYLIC ACID ON j)-GLACTO3IDASE SYNTIIESIS
This represents the smne experireental data as in Figure 1 In the
graph enz~e units ~re plotted in rel~tion to tL~e rather th~n in
relation to cell denitr rIle arrml indicates the time the culture
lras divided and all additions l-Tere i1ade
bull
bullbull Q
-Ld
bull
16 the effect of 321icylic acid ~o be noted indeendently of itsmiddot effect
on generlttion time Even on this graph cAli is shown to partially
relieve repression caused by salicyla~es
Previously Pastaa ~md PerL1an (21)(22) emphasized cAHP had no
effect on growth rate of E coli strain 300 rlhich 112S in contrast to
these results Their dltta homver referto using 1 x lO-3z cANP
Since 5 x 10- 3r crolic lp is believed to overcome both tranient and
catabolite reression caused by r~ucose in strain 3000 (23) the effect
of this concentration on salicylic acids repression was observed
Sirlilar results Nere obtcgtined in a second eXgerimcnt using
S x 10-31-1 salicylic acid and 5 x lO-3H Cl]~ (Fig 4) rhe degree of
reversal ias again only partial Because grorlth inhibition at this conshy
centration of salicylic acid tdth cAHP ias so great the curve is not
extended ver ffX
From these data it appears there is an interaction between the
effects of sali~Jlic acid and those of cMmiddotP Since repression proshy
duced by the higher concentration of salictlic acid seems to be less
effectively reversed by 5 x 10-3N cMP there i~ a possibility the
degree of interaction between the tlO may be concentration dependent
The 3ffect of Sdic-lates on Consti tutive Enzyme Synthesis
To eliminate the possibility salicylic acid may repress j3-galactoshy
sidase synthesis by preventing inducer upt~~e or by competing with
inducer for repressor an experiYlent vTaS carried out using strain 3300
constitutive for fo-galactosidase synthemiddotsis From Figure 5 it is evident
that 25 x 10-311 Jalicylic acid had the same effect on strain 3300 as it
had previously on strain 3000 Therefore salicylates act independently
17
B
uJ ~ l ~ l cJ J L o o- Ul t z 3 w r N Ia Z 312L fa m sa 1m m Em2 ru KLETI UNITS
EFFECT OF CYCLIC Almiddotp lm SLICYLIC ACID OU DIFFrRENfIAL j2gt-GALACTOSIDA3E SYNlHESIS
Figure 4 Strain 3000 culture las grOiID sampled and assayed for fl-g3~actosidase as in Figure 2 uitIl the exception a hig1er concentrC)tion of salic7l1ic acid i-as added after separshyation Since sliicylic acid 35 in dissolved form a dilution occurred upon its addition This uas compensated for in
J
-fA
other fiCsls by an equal dilution
flask Pt Control
Bbull Control + cAHP (5xlO-3H)
c Salicl~ic acid (5xlO H)
D salicl~c acid
Grmmiddotrth Rc1te 71hr
52hr
22hr
15hr (5xlO 3 I) + cMi (5xlO- 1middot1)
Diff Enz Rate 22 EUmlAKR
31 t1Jm1~KR
12 1UmlAIffi
16 EUmlAKR
18
FIGUR35
EFECr 01 SALICYLIC ACID OH DIIT2HE~rrAL fgt-GAL~C0SIDASE SYIJIl133I3 nr COnSrITlJlIV3TAIH 3300
strain 3300 iolaS gron in glucose medium Jllen the cells reached log
phase25 x lO-~1 snlicylic acid ~as added Cells were sa1pled ltmd
4asJared 80S in earlier experiments IPTG (5 x 10- 1-1) liaS added to
control flasks to note the degree of const~tutiveness of tile strain
The first points ngain represent sa~les trucen in~ediately poundter the
culture was divided and additions ware made
Growth Rate Diff Enz Synth Flask A Control bull89hr 65 EUrnlAKR
B Contro~ + IPTG bull89hr 65 EUmJ6KR (5xlO-~middotI)
o SalicYli~3acid bull54hr 46 ~iJ~KR (25x 10 H)
D Sa1icrli~ acid 5hhr 30 EUml~KR (25xlR--i-r) + IPTG (5xlO- M)
lSi JT1
bull 0
Fa-
I
a +
tsJ
yen
+
~
v
+
tsi IS1 m
m
tn
til sectll
m ~ ~ 2
bull
ef ~
~ ~ tsJ
uf
shy
20
of inducer uptake and of inducer-s derepression of lac mrrNA synthesis
The zrecl of 24-Di1ikophcnol ont30alactosidase Synthesis
Since salicylates are kno-Vffi to be uncouplers of oxidative phosshy
phorylation (5) it has been sJgccsted sOrle of their phaITlClcological
effects result from their uncoupling action Tis i-lould irply that
other uncouplers should preferentially inhibit ~-gilactosidase syntheshy
sis For this reason the effect of 2 4-dini trophenol (Dr~) on J-galacshy
tosidase synthesis Has observed Because DrIP is more effective in inhibishy
ting oxidative phosphorylation than salicylic acid and is consequently
more toxic a concentration of the drug i-laS found vhic uould inhibit
grOl-rth to ap~roximately the sa11e degree as 25 x 10-31gt1 salicylic acid
This was found to be 25 x 10-411 Figure 6 illustrates DNP does not
repress differential synthesis of j3-g1actosidase This implies that
althoueh DNP 1m-lers ATP levels there is no apparent effect of the drug
on lsvels of cAl-IP Also it appears salic-rlic acid is not exerting its
repression of enzyrre sJ~~hesis by inhibiting oxidative phosphorylation
21
FIGUllS 6
EFFECrl OF DJP ON DIFFif2rIlL ~GLCiO3D~S STImiddotrYliESIS
Jhen strain 3000 cells reacled log ph~se they tmiddotrere indlccd 1ith IPTG
AproxL~ately one hour later the culture was divide1 a~d tr~ferred
to flasks containing the indicated adlitions The cellsas in preVious
experirrents Tere sarlpled at various tLTIes and assayed for B-galactoshy
sidase activity Since 2h-dinitrophenol t-rag in solution its addition
resuted in dilution of cultures This dilution las equally conpensated
for in control flasks nle first points on the graph represent samples
taken innediately after sepration of the cuJture
GroYTth Rate Difierential inz Rate Flask A Control 75hr 22 L1JmlAKR
B Contro + cAll 64hr 50 EUmlAm (5y10- H) 4z
C DNP (25xlO- -I) 42hr 22 EOIraAKR
D DNP (25xl2~4zr) + 29hr 42 UmlAKR cAJP (5x10 -0
N
N
v
o
-shy-shy+
-shy
+
bull-
DISCU3SION
Salicylic acid ruld to a lesser extent aspirin cause repression
of f3-gclactosidase srnthesis in scherichia coli This effect is not
due to an inhibition of inducer uptake nor is it due ~o interference
l-rith inducers inactivation of lac repressor since strnins constitutive
for 3-gJactosidase 3J1lthesis are also repressed b~r sclicyli~ acid
In addition because dinitrophenol is liithout effect on differential
fl-galactosidase synthesis it appecrs salicrlates do not repress enzyme
s~mthesis by inhibiting oxidative phospllorylation
At these concentr2tions however salicylic acid does decrease
growth rate and direct or indirect effects of this on the observed
repression of enzyme synt~esis still ra~ain Since the differential
rate of enzyme synthesis i~ decreased and also since 25 x 10-411 DN
decreases grotrth rate aproximately the same degree middotuitl1cut having I
effects on differential synthesis salicylates inhibition of growth
is not the sole factor responsible for its effects on enZJcre synthesis
-11en 5 x 10-31 cyclic JUfP 1-ms added to cells treated Kith 25 x 10-3
and 5 x lO-3H srJicylic acid repression of differential enzye s~1lhesis
1ES pextially relieved This sugests salicllic acid represses J3-gJ~ac-
tosidase srnthesis br directly 1011erinti intIacellular levels of cyclic AMP
or by indirectly acting on asita which in turn releases cANP from the
cell Since cMP only partially relieves repression caused by wlicylic
acid and because there appears to be an inverse relationship between
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
-
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-
5 ~Galactosida3e Ass~
j3-galactosidase was assayed according to the procedure of Pardee
Jacob and Honod (6) with slight Iilodification At specific timeslt
09 ml of sample lUlS ldthdrakll fro-n the cultures and added ~o tubes
containing 2 drops of 025 sodiurn dodecyl sulfate (SOO) end 2 drops
chloroform (9) Both toluene treatment and SDS - chloroform treatshy
ment were pre~narily tested to deterwine the best method of releasshy
ing enzyme from the middotcell The latter did not alter enzyme stabilitY
and proved to increase the sensitivity of the assq
The amount of enzyme present was measured spectrophotomatr1cally
using a KJett-5UlllIllerson Photoelectric Colorimeter (lmiddotiodel 600-3) witil a
420 111)I1ilter One unit of enzyme is defined as the amount producing
1 II1j1 mole of o-nitrophenol per minute at 280 0 pH 70 The units of
enzyme in the sample can be calculated from the 1act 1 17fL mole per ml
of o-nitrophenol has a Klett reading of 045 under experirlental condishy
tions used here
To verify the assay procedure With E coJ strain 3000 enzyme
activity was confirmed to be linear in relation to time and concentrashy
tion
l-Iaterials
All chevicals were commercially obtained Cyclic adenosine
3 5-monophosphatie was obtained from Sigma and its purity 1-1as
confirmed chromatographically and spectrophotometrlcally Based on
readings vy a Cry Spectrophotometer the cyclic ffJ proved to be
99 pure b-leight Cyclic AHPs biolobical activity was confiIr4ed by
its ability to reverse glucose repression
RESULTS
Tho Effects of Salicylates on 13-GaJactosidase Synthesis
Various concentrations of aspirin were added to E coli strain
3000 cultures grmrlng on glycerol medium At a 5 x 10-3f concentration
ofacetylsa1i~lic aci~the ~fferential rate of j3-galactosidase synshy
thesis decreased (Table I) This 1-1aS accompanied by a slight decrease
in bacterial growth rate At 1 x 10-~ and 25 X 10-3M aspirin had
no significant effect on j3-galactos1dase synthesis
Sinceaspirin1s pharmacological action depends on its salicylic
acid content (1) the effect of salicylic acid on j3-galactosidase synshy
thesis ~1as detemined At all concentrations salicylic acid produced
a greater effect on the differential rate of enzyme synthesis than
aspirin (Table II) Salicylic acid also inhibited grorrth to a considershy
able extent but in all cases the effect on enzyme synthesis was
greater
Control experiments showed both aspirin and salicylic acid had
no effect on the enz~e ass~ system
Since the utjlization of glycerol requires the inducible enzoJlne
glycerol kinase kriown to be sensitive to catabolite repression (~)
it Has postulated the effect of salicylic ~cid on grouth rate resulted
from an inhibition of glYcerol kinase synthesis Such an inhibition
would have the effect of masking decreases in the differential rate
of ~-galactosidcse synthesis For this reason it was decided to use
a carbon source not subject to catabolite repreSSion and presumably
-7
TABLE I
EFFECTS OF ASPIRIN on
DrFFEIl3NTIAL RATE OF SYNTHESIS OF P-GALACTOSIDASE
Elcp 170
Aspirin (N)
Diff Rate En~ Synth (EUrilLKR)
at Control Rate
Grol-rth Rate (1gen time)
of Control Rate
1 1 -( cO3)1x10
25x10-3 5xl0-3
21 -( con3~)5x10shy
3 I -(con3- ) 5xl0shy
361 100 361 100 361 100 254 70
313 100 210 67
33bull3 100 259 78
72hr
63hr bull67hr 60br
100 87 93 825
65hr
54hr 100
83
67hr
58hr 100 87
E coli strain 3000 1-las grOl-m in glycerol raedium until log phase was obtained At -rms time the culture was divided and transferred to fiasks containing 5 x 10- H IPTa ard the indicated amount of dissolved aspirin The control flask contained water in place of the dissolved aspirin solution To insure dilutions would not 1irrdt the media flasks were also initi~ supplemented vnth 4x y~~ Broth Davis
At varioUs cell densities 09 ml samples were taken and assayed for j3-Galactosidase activit7
8
Exp No
1
2bull
3
TABLE II
EFFECT OF SALICYLIC ACID ON
DIFFER11ITIAL It4TE OF SYNTHESIS OF J3-GALACTOSID~sE
Salicylic Acid (N)
-(contro1) x 10shy
-(contro~23 387 x 10
-(contrpound)1 x 10
DtiL Rate ~~th (EUII1lJb Iffi) of
control rate
Growth Rate (ligen time) of
Control
370 62
100 n
60hr
33hr 100
283 65
100 23
71hr
44Ihr 100 62
315 206
bull100 6
60hr
2hr 100 87
Cells l-ere gr01ffl and sampled as in Table I ITith the exception that disshysolved salicylic acid was added to ce~s in log phase in place of aspirin
9
not subject to salicylic aci~ inhibition In all subsequent eXperiments
glucose lias used as carbon source
Upon the addition of 5 x lO-~ salicylic acid to cells grovID in
glucose media there gtmiddotas an ilnmecliate cessation of a-galactosidase synshy
thesis lasting over a generation of grotrth This l-laS followed by a
recovery of srnthesis but at a decreascdrate relative to the control
(fig 1) tt 1 x 10-~i salicylic acid deceased the differential rate
of enzY1e synthesis but tlere 1-TaS a)parently no lag in synthesis after
its addition
From this data it apears salicjlic acid more effectively represses
j3-galactosidase synthesis in glucose grovID cells than in cells grovm
in glycerol redia It should be noted hOHever the inhibitory effect
of salicylic acid on grot-lth rate is the srune vJhether the carbon source
is glucose or glycerol The reason for this inhibition ra1lains obscure
The strect or CYclic AlP on Heprcssion by Snlicylic Acid
If salicylic acid acts by decreasing cyclic PlW levels then cpJmiddot~
should revese the efects of se1icylates Figure 2 shms the resiuts
of rul experinent lihere 5 x lO-3M cyclic AHP and 25 x 10-31-1 salicylic
acid were added silulta1eously to glucose grotm cells It can be seen
that cPJP revJrses the repression caused by salicylic acid Hm1ever
the reversal is only partial since the dif~erential ra-ce is not restored
to the level of the control plus c~tmiddotp cells
Salicylic acid in these experi~ents also caused a decrease in
gromh rate and it lla3 further decreased when cAt-1P las added to the
cells Because of this the data from Figure 2 was replotted with time
units on the abscis3a in place of cell density (Fig 3)~ This allows
10
FIGURE 1
EFFECT OF SLICYLIC ACID ON THE DIFF3P3NrIAL RJTE OF [3-GALACTOSIDA3E SYNTHESIS
strain 3000 Was grovffi in glucose modiUI1 until log phase wst reached
Approrinate1y 1 hour follOldn~ induction rlith IPTG the culture was
separated and trs1sierred to flasks containing the indicted additions
Samples uere talten and assayed for j3-galactosidase as in Table I
Since diaBo1ved salicylic acid lias added a dilution in cell densitr
occurred The first point on the graph represents samples taken L~Jnedi-
ately c)fter the culture Was separatedand diluted
Grorth Rate Differential Enz Rate Flask A Control 75hr 308 EUllilAKR
B Salicylic Acid 68hr 180 EUlllJfoKR
(1 x 10-31)
o Sa1icylic Acid 35hr 95 EUralAlffi (5 x 10-31-1)
12
FIGlllE 2
EFF3Cr OF CYCLIC AmiddotlP 1jJ 3lLICYLIC ACID ON DIFF~JTIAL f3-01AClOSImE SYUTH33IS
IFTG was added to a log phase culture of strain 3000 grown in glucose
medium Approximately 45 minutes after induction the culture was divided
equally into flasks containing the indicated additions 32J1lplcs were
ta~en and ~-Galactosidase was assayed as previously indica~ed The
arroH at 30 Klett unts represents the time the culture ylaS divided
and trcmsferred
DifLerential Rate Growth tate Enzyme Synthesis
Flask A Control bull67hr 23 lJrrJ KIt
B Cyclic mp (5xlO-311) 57hr 315EUrr~6KR
c SalicyliQ acid 4lhr 135ZUmlKR(25xlO-)N)
D Salicy~c acid bull 35hr 25 ~1JrrJLKR 25xlO ~ 11 + CJllmiddotfP (5xlO-)~1)
+
0
t
o
oft
$+
~
Ft flJ rsi
m
m
mlt
l ~ ~
sect4( ~
m~ ~ e1
54 PI rd ISJ
aI ttl
14
FIGURE 3
EFF~T OF CYCLIC AHP UID SJLICYLIC ACID ON j)-GLACTO3IDASE SYNTIIESIS
This represents the smne experireental data as in Figure 1 In the
graph enz~e units ~re plotted in rel~tion to tL~e rather th~n in
relation to cell denitr rIle arrml indicates the time the culture
lras divided and all additions l-Tere i1ade
bull
bullbull Q
-Ld
bull
16 the effect of 321icylic acid ~o be noted indeendently of itsmiddot effect
on generlttion time Even on this graph cAli is shown to partially
relieve repression caused by salicyla~es
Previously Pastaa ~md PerL1an (21)(22) emphasized cAHP had no
effect on growth rate of E coli strain 300 rlhich 112S in contrast to
these results Their dltta homver referto using 1 x lO-3z cANP
Since 5 x 10- 3r crolic lp is believed to overcome both tranient and
catabolite reression caused by r~ucose in strain 3000 (23) the effect
of this concentration on salicylic acids repression was observed
Sirlilar results Nere obtcgtined in a second eXgerimcnt using
S x 10-31-1 salicylic acid and 5 x lO-3H Cl]~ (Fig 4) rhe degree of
reversal ias again only partial Because grorlth inhibition at this conshy
centration of salicylic acid tdth cAHP ias so great the curve is not
extended ver ffX
From these data it appears there is an interaction between the
effects of sali~Jlic acid and those of cMmiddotP Since repression proshy
duced by the higher concentration of salictlic acid seems to be less
effectively reversed by 5 x 10-3N cMP there i~ a possibility the
degree of interaction between the tlO may be concentration dependent
The 3ffect of Sdic-lates on Consti tutive Enzyme Synthesis
To eliminate the possibility salicylic acid may repress j3-galactoshy
sidase synthesis by preventing inducer upt~~e or by competing with
inducer for repressor an experiYlent vTaS carried out using strain 3300
constitutive for fo-galactosidase synthemiddotsis From Figure 5 it is evident
that 25 x 10-311 Jalicylic acid had the same effect on strain 3300 as it
had previously on strain 3000 Therefore salicylates act independently
17
B
uJ ~ l ~ l cJ J L o o- Ul t z 3 w r N Ia Z 312L fa m sa 1m m Em2 ru KLETI UNITS
EFFECT OF CYCLIC Almiddotp lm SLICYLIC ACID OU DIFFrRENfIAL j2gt-GALACTOSIDA3E SYNlHESIS
Figure 4 Strain 3000 culture las grOiID sampled and assayed for fl-g3~actosidase as in Figure 2 uitIl the exception a hig1er concentrC)tion of salic7l1ic acid i-as added after separshyation Since sliicylic acid 35 in dissolved form a dilution occurred upon its addition This uas compensated for in
J
-fA
other fiCsls by an equal dilution
flask Pt Control
Bbull Control + cAHP (5xlO-3H)
c Salicl~ic acid (5xlO H)
D salicl~c acid
Grmmiddotrth Rc1te 71hr
52hr
22hr
15hr (5xlO 3 I) + cMi (5xlO- 1middot1)
Diff Enz Rate 22 EUmlAKR
31 t1Jm1~KR
12 1UmlAIffi
16 EUmlAKR
18
FIGUR35
EFECr 01 SALICYLIC ACID OH DIIT2HE~rrAL fgt-GAL~C0SIDASE SYIJIl133I3 nr COnSrITlJlIV3TAIH 3300
strain 3300 iolaS gron in glucose medium Jllen the cells reached log
phase25 x lO-~1 snlicylic acid ~as added Cells were sa1pled ltmd
4asJared 80S in earlier experiments IPTG (5 x 10- 1-1) liaS added to
control flasks to note the degree of const~tutiveness of tile strain
The first points ngain represent sa~les trucen in~ediately poundter the
culture was divided and additions ware made
Growth Rate Diff Enz Synth Flask A Control bull89hr 65 EUrnlAKR
B Contro~ + IPTG bull89hr 65 EUmJ6KR (5xlO-~middotI)
o SalicYli~3acid bull54hr 46 ~iJ~KR (25x 10 H)
D Sa1icrli~ acid 5hhr 30 EUml~KR (25xlR--i-r) + IPTG (5xlO- M)
lSi JT1
bull 0
Fa-
I
a +
tsJ
yen
+
~
v
+
tsi IS1 m
m
tn
til sectll
m ~ ~ 2
bull
ef ~
~ ~ tsJ
uf
shy
20
of inducer uptake and of inducer-s derepression of lac mrrNA synthesis
The zrecl of 24-Di1ikophcnol ont30alactosidase Synthesis
Since salicylates are kno-Vffi to be uncouplers of oxidative phosshy
phorylation (5) it has been sJgccsted sOrle of their phaITlClcological
effects result from their uncoupling action Tis i-lould irply that
other uncouplers should preferentially inhibit ~-gilactosidase syntheshy
sis For this reason the effect of 2 4-dini trophenol (Dr~) on J-galacshy
tosidase synthesis Has observed Because DrIP is more effective in inhibishy
ting oxidative phosphorylation than salicylic acid and is consequently
more toxic a concentration of the drug i-laS found vhic uould inhibit
grOl-rth to ap~roximately the sa11e degree as 25 x 10-31gt1 salicylic acid
This was found to be 25 x 10-411 Figure 6 illustrates DNP does not
repress differential synthesis of j3-g1actosidase This implies that
althoueh DNP 1m-lers ATP levels there is no apparent effect of the drug
on lsvels of cAl-IP Also it appears salic-rlic acid is not exerting its
repression of enzyrre sJ~~hesis by inhibiting oxidative phosphorylation
21
FIGUllS 6
EFFECrl OF DJP ON DIFFif2rIlL ~GLCiO3D~S STImiddotrYliESIS
Jhen strain 3000 cells reacled log ph~se they tmiddotrere indlccd 1ith IPTG
AproxL~ately one hour later the culture was divide1 a~d tr~ferred
to flasks containing the indicated adlitions The cellsas in preVious
experirrents Tere sarlpled at various tLTIes and assayed for B-galactoshy
sidase activity Since 2h-dinitrophenol t-rag in solution its addition
resuted in dilution of cultures This dilution las equally conpensated
for in control flasks nle first points on the graph represent samples
taken innediately after sepration of the cuJture
GroYTth Rate Difierential inz Rate Flask A Control 75hr 22 L1JmlAKR
B Contro + cAll 64hr 50 EUmlAm (5y10- H) 4z
C DNP (25xlO- -I) 42hr 22 EOIraAKR
D DNP (25xl2~4zr) + 29hr 42 UmlAKR cAJP (5x10 -0
N
N
v
o
-shy-shy+
-shy
+
bull-
DISCU3SION
Salicylic acid ruld to a lesser extent aspirin cause repression
of f3-gclactosidase srnthesis in scherichia coli This effect is not
due to an inhibition of inducer uptake nor is it due ~o interference
l-rith inducers inactivation of lac repressor since strnins constitutive
for 3-gJactosidase 3J1lthesis are also repressed b~r sclicyli~ acid
In addition because dinitrophenol is liithout effect on differential
fl-galactosidase synthesis it appecrs salicrlates do not repress enzyme
s~mthesis by inhibiting oxidative phospllorylation
At these concentr2tions however salicylic acid does decrease
growth rate and direct or indirect effects of this on the observed
repression of enzyme synt~esis still ra~ain Since the differential
rate of enzyme synthesis i~ decreased and also since 25 x 10-411 DN
decreases grotrth rate aproximately the same degree middotuitl1cut having I
effects on differential synthesis salicylates inhibition of growth
is not the sole factor responsible for its effects on enZJcre synthesis
-11en 5 x 10-31 cyclic JUfP 1-ms added to cells treated Kith 25 x 10-3
and 5 x lO-3H srJicylic acid repression of differential enzye s~1lhesis
1ES pextially relieved This sugests salicllic acid represses J3-gJ~ac-
tosidase srnthesis br directly 1011erinti intIacellular levels of cyclic AMP
or by indirectly acting on asita which in turn releases cANP from the
cell Since cMP only partially relieves repression caused by wlicylic
acid and because there appears to be an inverse relationship between
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
-
- Let us know how access to this document benefits you
- Recommended Citation
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-
RESULTS
Tho Effects of Salicylates on 13-GaJactosidase Synthesis
Various concentrations of aspirin were added to E coli strain
3000 cultures grmrlng on glycerol medium At a 5 x 10-3f concentration
ofacetylsa1i~lic aci~the ~fferential rate of j3-galactosidase synshy
thesis decreased (Table I) This 1-1aS accompanied by a slight decrease
in bacterial growth rate At 1 x 10-~ and 25 X 10-3M aspirin had
no significant effect on j3-galactos1dase synthesis
Sinceaspirin1s pharmacological action depends on its salicylic
acid content (1) the effect of salicylic acid on j3-galactosidase synshy
thesis ~1as detemined At all concentrations salicylic acid produced
a greater effect on the differential rate of enzyme synthesis than
aspirin (Table II) Salicylic acid also inhibited grorrth to a considershy
able extent but in all cases the effect on enzyme synthesis was
greater
Control experiments showed both aspirin and salicylic acid had
no effect on the enz~e ass~ system
Since the utjlization of glycerol requires the inducible enzoJlne
glycerol kinase kriown to be sensitive to catabolite repression (~)
it Has postulated the effect of salicylic ~cid on grouth rate resulted
from an inhibition of glYcerol kinase synthesis Such an inhibition
would have the effect of masking decreases in the differential rate
of ~-galactosidcse synthesis For this reason it was decided to use
a carbon source not subject to catabolite repreSSion and presumably
-7
TABLE I
EFFECTS OF ASPIRIN on
DrFFEIl3NTIAL RATE OF SYNTHESIS OF P-GALACTOSIDASE
Elcp 170
Aspirin (N)
Diff Rate En~ Synth (EUrilLKR)
at Control Rate
Grol-rth Rate (1gen time)
of Control Rate
1 1 -( cO3)1x10
25x10-3 5xl0-3
21 -( con3~)5x10shy
3 I -(con3- ) 5xl0shy
361 100 361 100 361 100 254 70
313 100 210 67
33bull3 100 259 78
72hr
63hr bull67hr 60br
100 87 93 825
65hr
54hr 100
83
67hr
58hr 100 87
E coli strain 3000 1-las grOl-m in glycerol raedium until log phase was obtained At -rms time the culture was divided and transferred to fiasks containing 5 x 10- H IPTa ard the indicated amount of dissolved aspirin The control flask contained water in place of the dissolved aspirin solution To insure dilutions would not 1irrdt the media flasks were also initi~ supplemented vnth 4x y~~ Broth Davis
At varioUs cell densities 09 ml samples were taken and assayed for j3-Galactosidase activit7
8
Exp No
1
2bull
3
TABLE II
EFFECT OF SALICYLIC ACID ON
DIFFER11ITIAL It4TE OF SYNTHESIS OF J3-GALACTOSID~sE
Salicylic Acid (N)
-(contro1) x 10shy
-(contro~23 387 x 10
-(contrpound)1 x 10
DtiL Rate ~~th (EUII1lJb Iffi) of
control rate
Growth Rate (ligen time) of
Control
370 62
100 n
60hr
33hr 100
283 65
100 23
71hr
44Ihr 100 62
315 206
bull100 6
60hr
2hr 100 87
Cells l-ere gr01ffl and sampled as in Table I ITith the exception that disshysolved salicylic acid was added to ce~s in log phase in place of aspirin
9
not subject to salicylic aci~ inhibition In all subsequent eXperiments
glucose lias used as carbon source
Upon the addition of 5 x lO-~ salicylic acid to cells grovID in
glucose media there gtmiddotas an ilnmecliate cessation of a-galactosidase synshy
thesis lasting over a generation of grotrth This l-laS followed by a
recovery of srnthesis but at a decreascdrate relative to the control
(fig 1) tt 1 x 10-~i salicylic acid deceased the differential rate
of enzY1e synthesis but tlere 1-TaS a)parently no lag in synthesis after
its addition
From this data it apears salicjlic acid more effectively represses
j3-galactosidase synthesis in glucose grovID cells than in cells grovm
in glycerol redia It should be noted hOHever the inhibitory effect
of salicylic acid on grot-lth rate is the srune vJhether the carbon source
is glucose or glycerol The reason for this inhibition ra1lains obscure
The strect or CYclic AlP on Heprcssion by Snlicylic Acid
If salicylic acid acts by decreasing cyclic PlW levels then cpJmiddot~
should revese the efects of se1icylates Figure 2 shms the resiuts
of rul experinent lihere 5 x lO-3M cyclic AHP and 25 x 10-31-1 salicylic
acid were added silulta1eously to glucose grotm cells It can be seen
that cPJP revJrses the repression caused by salicylic acid Hm1ever
the reversal is only partial since the dif~erential ra-ce is not restored
to the level of the control plus c~tmiddotp cells
Salicylic acid in these experi~ents also caused a decrease in
gromh rate and it lla3 further decreased when cAt-1P las added to the
cells Because of this the data from Figure 2 was replotted with time
units on the abscis3a in place of cell density (Fig 3)~ This allows
10
FIGURE 1
EFFECT OF SLICYLIC ACID ON THE DIFF3P3NrIAL RJTE OF [3-GALACTOSIDA3E SYNTHESIS
strain 3000 Was grovffi in glucose modiUI1 until log phase wst reached
Approrinate1y 1 hour follOldn~ induction rlith IPTG the culture was
separated and trs1sierred to flasks containing the indicted additions
Samples uere talten and assayed for j3-galactosidase as in Table I
Since diaBo1ved salicylic acid lias added a dilution in cell densitr
occurred The first point on the graph represents samples taken L~Jnedi-
ately c)fter the culture Was separatedand diluted
Grorth Rate Differential Enz Rate Flask A Control 75hr 308 EUllilAKR
B Salicylic Acid 68hr 180 EUlllJfoKR
(1 x 10-31)
o Sa1icylic Acid 35hr 95 EUralAlffi (5 x 10-31-1)
12
FIGlllE 2
EFF3Cr OF CYCLIC AmiddotlP 1jJ 3lLICYLIC ACID ON DIFF~JTIAL f3-01AClOSImE SYUTH33IS
IFTG was added to a log phase culture of strain 3000 grown in glucose
medium Approximately 45 minutes after induction the culture was divided
equally into flasks containing the indicated additions 32J1lplcs were
ta~en and ~-Galactosidase was assayed as previously indica~ed The
arroH at 30 Klett unts represents the time the culture ylaS divided
and trcmsferred
DifLerential Rate Growth tate Enzyme Synthesis
Flask A Control bull67hr 23 lJrrJ KIt
B Cyclic mp (5xlO-311) 57hr 315EUrr~6KR
c SalicyliQ acid 4lhr 135ZUmlKR(25xlO-)N)
D Salicy~c acid bull 35hr 25 ~1JrrJLKR 25xlO ~ 11 + CJllmiddotfP (5xlO-)~1)
+
0
t
o
oft
$+
~
Ft flJ rsi
m
m
mlt
l ~ ~
sect4( ~
m~ ~ e1
54 PI rd ISJ
aI ttl
14
FIGURE 3
EFF~T OF CYCLIC AHP UID SJLICYLIC ACID ON j)-GLACTO3IDASE SYNTIIESIS
This represents the smne experireental data as in Figure 1 In the
graph enz~e units ~re plotted in rel~tion to tL~e rather th~n in
relation to cell denitr rIle arrml indicates the time the culture
lras divided and all additions l-Tere i1ade
bull
bullbull Q
-Ld
bull
16 the effect of 321icylic acid ~o be noted indeendently of itsmiddot effect
on generlttion time Even on this graph cAli is shown to partially
relieve repression caused by salicyla~es
Previously Pastaa ~md PerL1an (21)(22) emphasized cAHP had no
effect on growth rate of E coli strain 300 rlhich 112S in contrast to
these results Their dltta homver referto using 1 x lO-3z cANP
Since 5 x 10- 3r crolic lp is believed to overcome both tranient and
catabolite reression caused by r~ucose in strain 3000 (23) the effect
of this concentration on salicylic acids repression was observed
Sirlilar results Nere obtcgtined in a second eXgerimcnt using
S x 10-31-1 salicylic acid and 5 x lO-3H Cl]~ (Fig 4) rhe degree of
reversal ias again only partial Because grorlth inhibition at this conshy
centration of salicylic acid tdth cAHP ias so great the curve is not
extended ver ffX
From these data it appears there is an interaction between the
effects of sali~Jlic acid and those of cMmiddotP Since repression proshy
duced by the higher concentration of salictlic acid seems to be less
effectively reversed by 5 x 10-3N cMP there i~ a possibility the
degree of interaction between the tlO may be concentration dependent
The 3ffect of Sdic-lates on Consti tutive Enzyme Synthesis
To eliminate the possibility salicylic acid may repress j3-galactoshy
sidase synthesis by preventing inducer upt~~e or by competing with
inducer for repressor an experiYlent vTaS carried out using strain 3300
constitutive for fo-galactosidase synthemiddotsis From Figure 5 it is evident
that 25 x 10-311 Jalicylic acid had the same effect on strain 3300 as it
had previously on strain 3000 Therefore salicylates act independently
17
B
uJ ~ l ~ l cJ J L o o- Ul t z 3 w r N Ia Z 312L fa m sa 1m m Em2 ru KLETI UNITS
EFFECT OF CYCLIC Almiddotp lm SLICYLIC ACID OU DIFFrRENfIAL j2gt-GALACTOSIDA3E SYNlHESIS
Figure 4 Strain 3000 culture las grOiID sampled and assayed for fl-g3~actosidase as in Figure 2 uitIl the exception a hig1er concentrC)tion of salic7l1ic acid i-as added after separshyation Since sliicylic acid 35 in dissolved form a dilution occurred upon its addition This uas compensated for in
J
-fA
other fiCsls by an equal dilution
flask Pt Control
Bbull Control + cAHP (5xlO-3H)
c Salicl~ic acid (5xlO H)
D salicl~c acid
Grmmiddotrth Rc1te 71hr
52hr
22hr
15hr (5xlO 3 I) + cMi (5xlO- 1middot1)
Diff Enz Rate 22 EUmlAKR
31 t1Jm1~KR
12 1UmlAIffi
16 EUmlAKR
18
FIGUR35
EFECr 01 SALICYLIC ACID OH DIIT2HE~rrAL fgt-GAL~C0SIDASE SYIJIl133I3 nr COnSrITlJlIV3TAIH 3300
strain 3300 iolaS gron in glucose medium Jllen the cells reached log
phase25 x lO-~1 snlicylic acid ~as added Cells were sa1pled ltmd
4asJared 80S in earlier experiments IPTG (5 x 10- 1-1) liaS added to
control flasks to note the degree of const~tutiveness of tile strain
The first points ngain represent sa~les trucen in~ediately poundter the
culture was divided and additions ware made
Growth Rate Diff Enz Synth Flask A Control bull89hr 65 EUrnlAKR
B Contro~ + IPTG bull89hr 65 EUmJ6KR (5xlO-~middotI)
o SalicYli~3acid bull54hr 46 ~iJ~KR (25x 10 H)
D Sa1icrli~ acid 5hhr 30 EUml~KR (25xlR--i-r) + IPTG (5xlO- M)
lSi JT1
bull 0
Fa-
I
a +
tsJ
yen
+
~
v
+
tsi IS1 m
m
tn
til sectll
m ~ ~ 2
bull
ef ~
~ ~ tsJ
uf
shy
20
of inducer uptake and of inducer-s derepression of lac mrrNA synthesis
The zrecl of 24-Di1ikophcnol ont30alactosidase Synthesis
Since salicylates are kno-Vffi to be uncouplers of oxidative phosshy
phorylation (5) it has been sJgccsted sOrle of their phaITlClcological
effects result from their uncoupling action Tis i-lould irply that
other uncouplers should preferentially inhibit ~-gilactosidase syntheshy
sis For this reason the effect of 2 4-dini trophenol (Dr~) on J-galacshy
tosidase synthesis Has observed Because DrIP is more effective in inhibishy
ting oxidative phosphorylation than salicylic acid and is consequently
more toxic a concentration of the drug i-laS found vhic uould inhibit
grOl-rth to ap~roximately the sa11e degree as 25 x 10-31gt1 salicylic acid
This was found to be 25 x 10-411 Figure 6 illustrates DNP does not
repress differential synthesis of j3-g1actosidase This implies that
althoueh DNP 1m-lers ATP levels there is no apparent effect of the drug
on lsvels of cAl-IP Also it appears salic-rlic acid is not exerting its
repression of enzyrre sJ~~hesis by inhibiting oxidative phosphorylation
21
FIGUllS 6
EFFECrl OF DJP ON DIFFif2rIlL ~GLCiO3D~S STImiddotrYliESIS
Jhen strain 3000 cells reacled log ph~se they tmiddotrere indlccd 1ith IPTG
AproxL~ately one hour later the culture was divide1 a~d tr~ferred
to flasks containing the indicated adlitions The cellsas in preVious
experirrents Tere sarlpled at various tLTIes and assayed for B-galactoshy
sidase activity Since 2h-dinitrophenol t-rag in solution its addition
resuted in dilution of cultures This dilution las equally conpensated
for in control flasks nle first points on the graph represent samples
taken innediately after sepration of the cuJture
GroYTth Rate Difierential inz Rate Flask A Control 75hr 22 L1JmlAKR
B Contro + cAll 64hr 50 EUmlAm (5y10- H) 4z
C DNP (25xlO- -I) 42hr 22 EOIraAKR
D DNP (25xl2~4zr) + 29hr 42 UmlAKR cAJP (5x10 -0
N
N
v
o
-shy-shy+
-shy
+
bull-
DISCU3SION
Salicylic acid ruld to a lesser extent aspirin cause repression
of f3-gclactosidase srnthesis in scherichia coli This effect is not
due to an inhibition of inducer uptake nor is it due ~o interference
l-rith inducers inactivation of lac repressor since strnins constitutive
for 3-gJactosidase 3J1lthesis are also repressed b~r sclicyli~ acid
In addition because dinitrophenol is liithout effect on differential
fl-galactosidase synthesis it appecrs salicrlates do not repress enzyme
s~mthesis by inhibiting oxidative phospllorylation
At these concentr2tions however salicylic acid does decrease
growth rate and direct or indirect effects of this on the observed
repression of enzyme synt~esis still ra~ain Since the differential
rate of enzyme synthesis i~ decreased and also since 25 x 10-411 DN
decreases grotrth rate aproximately the same degree middotuitl1cut having I
effects on differential synthesis salicylates inhibition of growth
is not the sole factor responsible for its effects on enZJcre synthesis
-11en 5 x 10-31 cyclic JUfP 1-ms added to cells treated Kith 25 x 10-3
and 5 x lO-3H srJicylic acid repression of differential enzye s~1lhesis
1ES pextially relieved This sugests salicllic acid represses J3-gJ~ac-
tosidase srnthesis br directly 1011erinti intIacellular levels of cyclic AMP
or by indirectly acting on asita which in turn releases cANP from the
cell Since cMP only partially relieves repression caused by wlicylic
acid and because there appears to be an inverse relationship between
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
-
- Let us know how access to this document benefits you
- Recommended Citation
-
- tmp1379115374pdfCOlFc
-
-7
TABLE I
EFFECTS OF ASPIRIN on
DrFFEIl3NTIAL RATE OF SYNTHESIS OF P-GALACTOSIDASE
Elcp 170
Aspirin (N)
Diff Rate En~ Synth (EUrilLKR)
at Control Rate
Grol-rth Rate (1gen time)
of Control Rate
1 1 -( cO3)1x10
25x10-3 5xl0-3
21 -( con3~)5x10shy
3 I -(con3- ) 5xl0shy
361 100 361 100 361 100 254 70
313 100 210 67
33bull3 100 259 78
72hr
63hr bull67hr 60br
100 87 93 825
65hr
54hr 100
83
67hr
58hr 100 87
E coli strain 3000 1-las grOl-m in glycerol raedium until log phase was obtained At -rms time the culture was divided and transferred to fiasks containing 5 x 10- H IPTa ard the indicated amount of dissolved aspirin The control flask contained water in place of the dissolved aspirin solution To insure dilutions would not 1irrdt the media flasks were also initi~ supplemented vnth 4x y~~ Broth Davis
At varioUs cell densities 09 ml samples were taken and assayed for j3-Galactosidase activit7
8
Exp No
1
2bull
3
TABLE II
EFFECT OF SALICYLIC ACID ON
DIFFER11ITIAL It4TE OF SYNTHESIS OF J3-GALACTOSID~sE
Salicylic Acid (N)
-(contro1) x 10shy
-(contro~23 387 x 10
-(contrpound)1 x 10
DtiL Rate ~~th (EUII1lJb Iffi) of
control rate
Growth Rate (ligen time) of
Control
370 62
100 n
60hr
33hr 100
283 65
100 23
71hr
44Ihr 100 62
315 206
bull100 6
60hr
2hr 100 87
Cells l-ere gr01ffl and sampled as in Table I ITith the exception that disshysolved salicylic acid was added to ce~s in log phase in place of aspirin
9
not subject to salicylic aci~ inhibition In all subsequent eXperiments
glucose lias used as carbon source
Upon the addition of 5 x lO-~ salicylic acid to cells grovID in
glucose media there gtmiddotas an ilnmecliate cessation of a-galactosidase synshy
thesis lasting over a generation of grotrth This l-laS followed by a
recovery of srnthesis but at a decreascdrate relative to the control
(fig 1) tt 1 x 10-~i salicylic acid deceased the differential rate
of enzY1e synthesis but tlere 1-TaS a)parently no lag in synthesis after
its addition
From this data it apears salicjlic acid more effectively represses
j3-galactosidase synthesis in glucose grovID cells than in cells grovm
in glycerol redia It should be noted hOHever the inhibitory effect
of salicylic acid on grot-lth rate is the srune vJhether the carbon source
is glucose or glycerol The reason for this inhibition ra1lains obscure
The strect or CYclic AlP on Heprcssion by Snlicylic Acid
If salicylic acid acts by decreasing cyclic PlW levels then cpJmiddot~
should revese the efects of se1icylates Figure 2 shms the resiuts
of rul experinent lihere 5 x lO-3M cyclic AHP and 25 x 10-31-1 salicylic
acid were added silulta1eously to glucose grotm cells It can be seen
that cPJP revJrses the repression caused by salicylic acid Hm1ever
the reversal is only partial since the dif~erential ra-ce is not restored
to the level of the control plus c~tmiddotp cells
Salicylic acid in these experi~ents also caused a decrease in
gromh rate and it lla3 further decreased when cAt-1P las added to the
cells Because of this the data from Figure 2 was replotted with time
units on the abscis3a in place of cell density (Fig 3)~ This allows
10
FIGURE 1
EFFECT OF SLICYLIC ACID ON THE DIFF3P3NrIAL RJTE OF [3-GALACTOSIDA3E SYNTHESIS
strain 3000 Was grovffi in glucose modiUI1 until log phase wst reached
Approrinate1y 1 hour follOldn~ induction rlith IPTG the culture was
separated and trs1sierred to flasks containing the indicted additions
Samples uere talten and assayed for j3-galactosidase as in Table I
Since diaBo1ved salicylic acid lias added a dilution in cell densitr
occurred The first point on the graph represents samples taken L~Jnedi-
ately c)fter the culture Was separatedand diluted
Grorth Rate Differential Enz Rate Flask A Control 75hr 308 EUllilAKR
B Salicylic Acid 68hr 180 EUlllJfoKR
(1 x 10-31)
o Sa1icylic Acid 35hr 95 EUralAlffi (5 x 10-31-1)
12
FIGlllE 2
EFF3Cr OF CYCLIC AmiddotlP 1jJ 3lLICYLIC ACID ON DIFF~JTIAL f3-01AClOSImE SYUTH33IS
IFTG was added to a log phase culture of strain 3000 grown in glucose
medium Approximately 45 minutes after induction the culture was divided
equally into flasks containing the indicated additions 32J1lplcs were
ta~en and ~-Galactosidase was assayed as previously indica~ed The
arroH at 30 Klett unts represents the time the culture ylaS divided
and trcmsferred
DifLerential Rate Growth tate Enzyme Synthesis
Flask A Control bull67hr 23 lJrrJ KIt
B Cyclic mp (5xlO-311) 57hr 315EUrr~6KR
c SalicyliQ acid 4lhr 135ZUmlKR(25xlO-)N)
D Salicy~c acid bull 35hr 25 ~1JrrJLKR 25xlO ~ 11 + CJllmiddotfP (5xlO-)~1)
+
0
t
o
oft
$+
~
Ft flJ rsi
m
m
mlt
l ~ ~
sect4( ~
m~ ~ e1
54 PI rd ISJ
aI ttl
14
FIGURE 3
EFF~T OF CYCLIC AHP UID SJLICYLIC ACID ON j)-GLACTO3IDASE SYNTIIESIS
This represents the smne experireental data as in Figure 1 In the
graph enz~e units ~re plotted in rel~tion to tL~e rather th~n in
relation to cell denitr rIle arrml indicates the time the culture
lras divided and all additions l-Tere i1ade
bull
bullbull Q
-Ld
bull
16 the effect of 321icylic acid ~o be noted indeendently of itsmiddot effect
on generlttion time Even on this graph cAli is shown to partially
relieve repression caused by salicyla~es
Previously Pastaa ~md PerL1an (21)(22) emphasized cAHP had no
effect on growth rate of E coli strain 300 rlhich 112S in contrast to
these results Their dltta homver referto using 1 x lO-3z cANP
Since 5 x 10- 3r crolic lp is believed to overcome both tranient and
catabolite reression caused by r~ucose in strain 3000 (23) the effect
of this concentration on salicylic acids repression was observed
Sirlilar results Nere obtcgtined in a second eXgerimcnt using
S x 10-31-1 salicylic acid and 5 x lO-3H Cl]~ (Fig 4) rhe degree of
reversal ias again only partial Because grorlth inhibition at this conshy
centration of salicylic acid tdth cAHP ias so great the curve is not
extended ver ffX
From these data it appears there is an interaction between the
effects of sali~Jlic acid and those of cMmiddotP Since repression proshy
duced by the higher concentration of salictlic acid seems to be less
effectively reversed by 5 x 10-3N cMP there i~ a possibility the
degree of interaction between the tlO may be concentration dependent
The 3ffect of Sdic-lates on Consti tutive Enzyme Synthesis
To eliminate the possibility salicylic acid may repress j3-galactoshy
sidase synthesis by preventing inducer upt~~e or by competing with
inducer for repressor an experiYlent vTaS carried out using strain 3300
constitutive for fo-galactosidase synthemiddotsis From Figure 5 it is evident
that 25 x 10-311 Jalicylic acid had the same effect on strain 3300 as it
had previously on strain 3000 Therefore salicylates act independently
17
B
uJ ~ l ~ l cJ J L o o- Ul t z 3 w r N Ia Z 312L fa m sa 1m m Em2 ru KLETI UNITS
EFFECT OF CYCLIC Almiddotp lm SLICYLIC ACID OU DIFFrRENfIAL j2gt-GALACTOSIDA3E SYNlHESIS
Figure 4 Strain 3000 culture las grOiID sampled and assayed for fl-g3~actosidase as in Figure 2 uitIl the exception a hig1er concentrC)tion of salic7l1ic acid i-as added after separshyation Since sliicylic acid 35 in dissolved form a dilution occurred upon its addition This uas compensated for in
J
-fA
other fiCsls by an equal dilution
flask Pt Control
Bbull Control + cAHP (5xlO-3H)
c Salicl~ic acid (5xlO H)
D salicl~c acid
Grmmiddotrth Rc1te 71hr
52hr
22hr
15hr (5xlO 3 I) + cMi (5xlO- 1middot1)
Diff Enz Rate 22 EUmlAKR
31 t1Jm1~KR
12 1UmlAIffi
16 EUmlAKR
18
FIGUR35
EFECr 01 SALICYLIC ACID OH DIIT2HE~rrAL fgt-GAL~C0SIDASE SYIJIl133I3 nr COnSrITlJlIV3TAIH 3300
strain 3300 iolaS gron in glucose medium Jllen the cells reached log
phase25 x lO-~1 snlicylic acid ~as added Cells were sa1pled ltmd
4asJared 80S in earlier experiments IPTG (5 x 10- 1-1) liaS added to
control flasks to note the degree of const~tutiveness of tile strain
The first points ngain represent sa~les trucen in~ediately poundter the
culture was divided and additions ware made
Growth Rate Diff Enz Synth Flask A Control bull89hr 65 EUrnlAKR
B Contro~ + IPTG bull89hr 65 EUmJ6KR (5xlO-~middotI)
o SalicYli~3acid bull54hr 46 ~iJ~KR (25x 10 H)
D Sa1icrli~ acid 5hhr 30 EUml~KR (25xlR--i-r) + IPTG (5xlO- M)
lSi JT1
bull 0
Fa-
I
a +
tsJ
yen
+
~
v
+
tsi IS1 m
m
tn
til sectll
m ~ ~ 2
bull
ef ~
~ ~ tsJ
uf
shy
20
of inducer uptake and of inducer-s derepression of lac mrrNA synthesis
The zrecl of 24-Di1ikophcnol ont30alactosidase Synthesis
Since salicylates are kno-Vffi to be uncouplers of oxidative phosshy
phorylation (5) it has been sJgccsted sOrle of their phaITlClcological
effects result from their uncoupling action Tis i-lould irply that
other uncouplers should preferentially inhibit ~-gilactosidase syntheshy
sis For this reason the effect of 2 4-dini trophenol (Dr~) on J-galacshy
tosidase synthesis Has observed Because DrIP is more effective in inhibishy
ting oxidative phosphorylation than salicylic acid and is consequently
more toxic a concentration of the drug i-laS found vhic uould inhibit
grOl-rth to ap~roximately the sa11e degree as 25 x 10-31gt1 salicylic acid
This was found to be 25 x 10-411 Figure 6 illustrates DNP does not
repress differential synthesis of j3-g1actosidase This implies that
althoueh DNP 1m-lers ATP levels there is no apparent effect of the drug
on lsvels of cAl-IP Also it appears salic-rlic acid is not exerting its
repression of enzyrre sJ~~hesis by inhibiting oxidative phosphorylation
21
FIGUllS 6
EFFECrl OF DJP ON DIFFif2rIlL ~GLCiO3D~S STImiddotrYliESIS
Jhen strain 3000 cells reacled log ph~se they tmiddotrere indlccd 1ith IPTG
AproxL~ately one hour later the culture was divide1 a~d tr~ferred
to flasks containing the indicated adlitions The cellsas in preVious
experirrents Tere sarlpled at various tLTIes and assayed for B-galactoshy
sidase activity Since 2h-dinitrophenol t-rag in solution its addition
resuted in dilution of cultures This dilution las equally conpensated
for in control flasks nle first points on the graph represent samples
taken innediately after sepration of the cuJture
GroYTth Rate Difierential inz Rate Flask A Control 75hr 22 L1JmlAKR
B Contro + cAll 64hr 50 EUmlAm (5y10- H) 4z
C DNP (25xlO- -I) 42hr 22 EOIraAKR
D DNP (25xl2~4zr) + 29hr 42 UmlAKR cAJP (5x10 -0
N
N
v
o
-shy-shy+
-shy
+
bull-
DISCU3SION
Salicylic acid ruld to a lesser extent aspirin cause repression
of f3-gclactosidase srnthesis in scherichia coli This effect is not
due to an inhibition of inducer uptake nor is it due ~o interference
l-rith inducers inactivation of lac repressor since strnins constitutive
for 3-gJactosidase 3J1lthesis are also repressed b~r sclicyli~ acid
In addition because dinitrophenol is liithout effect on differential
fl-galactosidase synthesis it appecrs salicrlates do not repress enzyme
s~mthesis by inhibiting oxidative phospllorylation
At these concentr2tions however salicylic acid does decrease
growth rate and direct or indirect effects of this on the observed
repression of enzyme synt~esis still ra~ain Since the differential
rate of enzyme synthesis i~ decreased and also since 25 x 10-411 DN
decreases grotrth rate aproximately the same degree middotuitl1cut having I
effects on differential synthesis salicylates inhibition of growth
is not the sole factor responsible for its effects on enZJcre synthesis
-11en 5 x 10-31 cyclic JUfP 1-ms added to cells treated Kith 25 x 10-3
and 5 x lO-3H srJicylic acid repression of differential enzye s~1lhesis
1ES pextially relieved This sugests salicllic acid represses J3-gJ~ac-
tosidase srnthesis br directly 1011erinti intIacellular levels of cyclic AMP
or by indirectly acting on asita which in turn releases cANP from the
cell Since cMP only partially relieves repression caused by wlicylic
acid and because there appears to be an inverse relationship between
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
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8
Exp No
1
2bull
3
TABLE II
EFFECT OF SALICYLIC ACID ON
DIFFER11ITIAL It4TE OF SYNTHESIS OF J3-GALACTOSID~sE
Salicylic Acid (N)
-(contro1) x 10shy
-(contro~23 387 x 10
-(contrpound)1 x 10
DtiL Rate ~~th (EUII1lJb Iffi) of
control rate
Growth Rate (ligen time) of
Control
370 62
100 n
60hr
33hr 100
283 65
100 23
71hr
44Ihr 100 62
315 206
bull100 6
60hr
2hr 100 87
Cells l-ere gr01ffl and sampled as in Table I ITith the exception that disshysolved salicylic acid was added to ce~s in log phase in place of aspirin
9
not subject to salicylic aci~ inhibition In all subsequent eXperiments
glucose lias used as carbon source
Upon the addition of 5 x lO-~ salicylic acid to cells grovID in
glucose media there gtmiddotas an ilnmecliate cessation of a-galactosidase synshy
thesis lasting over a generation of grotrth This l-laS followed by a
recovery of srnthesis but at a decreascdrate relative to the control
(fig 1) tt 1 x 10-~i salicylic acid deceased the differential rate
of enzY1e synthesis but tlere 1-TaS a)parently no lag in synthesis after
its addition
From this data it apears salicjlic acid more effectively represses
j3-galactosidase synthesis in glucose grovID cells than in cells grovm
in glycerol redia It should be noted hOHever the inhibitory effect
of salicylic acid on grot-lth rate is the srune vJhether the carbon source
is glucose or glycerol The reason for this inhibition ra1lains obscure
The strect or CYclic AlP on Heprcssion by Snlicylic Acid
If salicylic acid acts by decreasing cyclic PlW levels then cpJmiddot~
should revese the efects of se1icylates Figure 2 shms the resiuts
of rul experinent lihere 5 x lO-3M cyclic AHP and 25 x 10-31-1 salicylic
acid were added silulta1eously to glucose grotm cells It can be seen
that cPJP revJrses the repression caused by salicylic acid Hm1ever
the reversal is only partial since the dif~erential ra-ce is not restored
to the level of the control plus c~tmiddotp cells
Salicylic acid in these experi~ents also caused a decrease in
gromh rate and it lla3 further decreased when cAt-1P las added to the
cells Because of this the data from Figure 2 was replotted with time
units on the abscis3a in place of cell density (Fig 3)~ This allows
10
FIGURE 1
EFFECT OF SLICYLIC ACID ON THE DIFF3P3NrIAL RJTE OF [3-GALACTOSIDA3E SYNTHESIS
strain 3000 Was grovffi in glucose modiUI1 until log phase wst reached
Approrinate1y 1 hour follOldn~ induction rlith IPTG the culture was
separated and trs1sierred to flasks containing the indicted additions
Samples uere talten and assayed for j3-galactosidase as in Table I
Since diaBo1ved salicylic acid lias added a dilution in cell densitr
occurred The first point on the graph represents samples taken L~Jnedi-
ately c)fter the culture Was separatedand diluted
Grorth Rate Differential Enz Rate Flask A Control 75hr 308 EUllilAKR
B Salicylic Acid 68hr 180 EUlllJfoKR
(1 x 10-31)
o Sa1icylic Acid 35hr 95 EUralAlffi (5 x 10-31-1)
12
FIGlllE 2
EFF3Cr OF CYCLIC AmiddotlP 1jJ 3lLICYLIC ACID ON DIFF~JTIAL f3-01AClOSImE SYUTH33IS
IFTG was added to a log phase culture of strain 3000 grown in glucose
medium Approximately 45 minutes after induction the culture was divided
equally into flasks containing the indicated additions 32J1lplcs were
ta~en and ~-Galactosidase was assayed as previously indica~ed The
arroH at 30 Klett unts represents the time the culture ylaS divided
and trcmsferred
DifLerential Rate Growth tate Enzyme Synthesis
Flask A Control bull67hr 23 lJrrJ KIt
B Cyclic mp (5xlO-311) 57hr 315EUrr~6KR
c SalicyliQ acid 4lhr 135ZUmlKR(25xlO-)N)
D Salicy~c acid bull 35hr 25 ~1JrrJLKR 25xlO ~ 11 + CJllmiddotfP (5xlO-)~1)
+
0
t
o
oft
$+
~
Ft flJ rsi
m
m
mlt
l ~ ~
sect4( ~
m~ ~ e1
54 PI rd ISJ
aI ttl
14
FIGURE 3
EFF~T OF CYCLIC AHP UID SJLICYLIC ACID ON j)-GLACTO3IDASE SYNTIIESIS
This represents the smne experireental data as in Figure 1 In the
graph enz~e units ~re plotted in rel~tion to tL~e rather th~n in
relation to cell denitr rIle arrml indicates the time the culture
lras divided and all additions l-Tere i1ade
bull
bullbull Q
-Ld
bull
16 the effect of 321icylic acid ~o be noted indeendently of itsmiddot effect
on generlttion time Even on this graph cAli is shown to partially
relieve repression caused by salicyla~es
Previously Pastaa ~md PerL1an (21)(22) emphasized cAHP had no
effect on growth rate of E coli strain 300 rlhich 112S in contrast to
these results Their dltta homver referto using 1 x lO-3z cANP
Since 5 x 10- 3r crolic lp is believed to overcome both tranient and
catabolite reression caused by r~ucose in strain 3000 (23) the effect
of this concentration on salicylic acids repression was observed
Sirlilar results Nere obtcgtined in a second eXgerimcnt using
S x 10-31-1 salicylic acid and 5 x lO-3H Cl]~ (Fig 4) rhe degree of
reversal ias again only partial Because grorlth inhibition at this conshy
centration of salicylic acid tdth cAHP ias so great the curve is not
extended ver ffX
From these data it appears there is an interaction between the
effects of sali~Jlic acid and those of cMmiddotP Since repression proshy
duced by the higher concentration of salictlic acid seems to be less
effectively reversed by 5 x 10-3N cMP there i~ a possibility the
degree of interaction between the tlO may be concentration dependent
The 3ffect of Sdic-lates on Consti tutive Enzyme Synthesis
To eliminate the possibility salicylic acid may repress j3-galactoshy
sidase synthesis by preventing inducer upt~~e or by competing with
inducer for repressor an experiYlent vTaS carried out using strain 3300
constitutive for fo-galactosidase synthemiddotsis From Figure 5 it is evident
that 25 x 10-311 Jalicylic acid had the same effect on strain 3300 as it
had previously on strain 3000 Therefore salicylates act independently
17
B
uJ ~ l ~ l cJ J L o o- Ul t z 3 w r N Ia Z 312L fa m sa 1m m Em2 ru KLETI UNITS
EFFECT OF CYCLIC Almiddotp lm SLICYLIC ACID OU DIFFrRENfIAL j2gt-GALACTOSIDA3E SYNlHESIS
Figure 4 Strain 3000 culture las grOiID sampled and assayed for fl-g3~actosidase as in Figure 2 uitIl the exception a hig1er concentrC)tion of salic7l1ic acid i-as added after separshyation Since sliicylic acid 35 in dissolved form a dilution occurred upon its addition This uas compensated for in
J
-fA
other fiCsls by an equal dilution
flask Pt Control
Bbull Control + cAHP (5xlO-3H)
c Salicl~ic acid (5xlO H)
D salicl~c acid
Grmmiddotrth Rc1te 71hr
52hr
22hr
15hr (5xlO 3 I) + cMi (5xlO- 1middot1)
Diff Enz Rate 22 EUmlAKR
31 t1Jm1~KR
12 1UmlAIffi
16 EUmlAKR
18
FIGUR35
EFECr 01 SALICYLIC ACID OH DIIT2HE~rrAL fgt-GAL~C0SIDASE SYIJIl133I3 nr COnSrITlJlIV3TAIH 3300
strain 3300 iolaS gron in glucose medium Jllen the cells reached log
phase25 x lO-~1 snlicylic acid ~as added Cells were sa1pled ltmd
4asJared 80S in earlier experiments IPTG (5 x 10- 1-1) liaS added to
control flasks to note the degree of const~tutiveness of tile strain
The first points ngain represent sa~les trucen in~ediately poundter the
culture was divided and additions ware made
Growth Rate Diff Enz Synth Flask A Control bull89hr 65 EUrnlAKR
B Contro~ + IPTG bull89hr 65 EUmJ6KR (5xlO-~middotI)
o SalicYli~3acid bull54hr 46 ~iJ~KR (25x 10 H)
D Sa1icrli~ acid 5hhr 30 EUml~KR (25xlR--i-r) + IPTG (5xlO- M)
lSi JT1
bull 0
Fa-
I
a +
tsJ
yen
+
~
v
+
tsi IS1 m
m
tn
til sectll
m ~ ~ 2
bull
ef ~
~ ~ tsJ
uf
shy
20
of inducer uptake and of inducer-s derepression of lac mrrNA synthesis
The zrecl of 24-Di1ikophcnol ont30alactosidase Synthesis
Since salicylates are kno-Vffi to be uncouplers of oxidative phosshy
phorylation (5) it has been sJgccsted sOrle of their phaITlClcological
effects result from their uncoupling action Tis i-lould irply that
other uncouplers should preferentially inhibit ~-gilactosidase syntheshy
sis For this reason the effect of 2 4-dini trophenol (Dr~) on J-galacshy
tosidase synthesis Has observed Because DrIP is more effective in inhibishy
ting oxidative phosphorylation than salicylic acid and is consequently
more toxic a concentration of the drug i-laS found vhic uould inhibit
grOl-rth to ap~roximately the sa11e degree as 25 x 10-31gt1 salicylic acid
This was found to be 25 x 10-411 Figure 6 illustrates DNP does not
repress differential synthesis of j3-g1actosidase This implies that
althoueh DNP 1m-lers ATP levels there is no apparent effect of the drug
on lsvels of cAl-IP Also it appears salic-rlic acid is not exerting its
repression of enzyrre sJ~~hesis by inhibiting oxidative phosphorylation
21
FIGUllS 6
EFFECrl OF DJP ON DIFFif2rIlL ~GLCiO3D~S STImiddotrYliESIS
Jhen strain 3000 cells reacled log ph~se they tmiddotrere indlccd 1ith IPTG
AproxL~ately one hour later the culture was divide1 a~d tr~ferred
to flasks containing the indicated adlitions The cellsas in preVious
experirrents Tere sarlpled at various tLTIes and assayed for B-galactoshy
sidase activity Since 2h-dinitrophenol t-rag in solution its addition
resuted in dilution of cultures This dilution las equally conpensated
for in control flasks nle first points on the graph represent samples
taken innediately after sepration of the cuJture
GroYTth Rate Difierential inz Rate Flask A Control 75hr 22 L1JmlAKR
B Contro + cAll 64hr 50 EUmlAm (5y10- H) 4z
C DNP (25xlO- -I) 42hr 22 EOIraAKR
D DNP (25xl2~4zr) + 29hr 42 UmlAKR cAJP (5x10 -0
N
N
v
o
-shy-shy+
-shy
+
bull-
DISCU3SION
Salicylic acid ruld to a lesser extent aspirin cause repression
of f3-gclactosidase srnthesis in scherichia coli This effect is not
due to an inhibition of inducer uptake nor is it due ~o interference
l-rith inducers inactivation of lac repressor since strnins constitutive
for 3-gJactosidase 3J1lthesis are also repressed b~r sclicyli~ acid
In addition because dinitrophenol is liithout effect on differential
fl-galactosidase synthesis it appecrs salicrlates do not repress enzyme
s~mthesis by inhibiting oxidative phospllorylation
At these concentr2tions however salicylic acid does decrease
growth rate and direct or indirect effects of this on the observed
repression of enzyme synt~esis still ra~ain Since the differential
rate of enzyme synthesis i~ decreased and also since 25 x 10-411 DN
decreases grotrth rate aproximately the same degree middotuitl1cut having I
effects on differential synthesis salicylates inhibition of growth
is not the sole factor responsible for its effects on enZJcre synthesis
-11en 5 x 10-31 cyclic JUfP 1-ms added to cells treated Kith 25 x 10-3
and 5 x lO-3H srJicylic acid repression of differential enzye s~1lhesis
1ES pextially relieved This sugests salicllic acid represses J3-gJ~ac-
tosidase srnthesis br directly 1011erinti intIacellular levels of cyclic AMP
or by indirectly acting on asita which in turn releases cANP from the
cell Since cMP only partially relieves repression caused by wlicylic
acid and because there appears to be an inverse relationship between
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
-
- Let us know how access to this document benefits you
- Recommended Citation
-
- tmp1379115374pdfCOlFc
-
9
not subject to salicylic aci~ inhibition In all subsequent eXperiments
glucose lias used as carbon source
Upon the addition of 5 x lO-~ salicylic acid to cells grovID in
glucose media there gtmiddotas an ilnmecliate cessation of a-galactosidase synshy
thesis lasting over a generation of grotrth This l-laS followed by a
recovery of srnthesis but at a decreascdrate relative to the control
(fig 1) tt 1 x 10-~i salicylic acid deceased the differential rate
of enzY1e synthesis but tlere 1-TaS a)parently no lag in synthesis after
its addition
From this data it apears salicjlic acid more effectively represses
j3-galactosidase synthesis in glucose grovID cells than in cells grovm
in glycerol redia It should be noted hOHever the inhibitory effect
of salicylic acid on grot-lth rate is the srune vJhether the carbon source
is glucose or glycerol The reason for this inhibition ra1lains obscure
The strect or CYclic AlP on Heprcssion by Snlicylic Acid
If salicylic acid acts by decreasing cyclic PlW levels then cpJmiddot~
should revese the efects of se1icylates Figure 2 shms the resiuts
of rul experinent lihere 5 x lO-3M cyclic AHP and 25 x 10-31-1 salicylic
acid were added silulta1eously to glucose grotm cells It can be seen
that cPJP revJrses the repression caused by salicylic acid Hm1ever
the reversal is only partial since the dif~erential ra-ce is not restored
to the level of the control plus c~tmiddotp cells
Salicylic acid in these experi~ents also caused a decrease in
gromh rate and it lla3 further decreased when cAt-1P las added to the
cells Because of this the data from Figure 2 was replotted with time
units on the abscis3a in place of cell density (Fig 3)~ This allows
10
FIGURE 1
EFFECT OF SLICYLIC ACID ON THE DIFF3P3NrIAL RJTE OF [3-GALACTOSIDA3E SYNTHESIS
strain 3000 Was grovffi in glucose modiUI1 until log phase wst reached
Approrinate1y 1 hour follOldn~ induction rlith IPTG the culture was
separated and trs1sierred to flasks containing the indicted additions
Samples uere talten and assayed for j3-galactosidase as in Table I
Since diaBo1ved salicylic acid lias added a dilution in cell densitr
occurred The first point on the graph represents samples taken L~Jnedi-
ately c)fter the culture Was separatedand diluted
Grorth Rate Differential Enz Rate Flask A Control 75hr 308 EUllilAKR
B Salicylic Acid 68hr 180 EUlllJfoKR
(1 x 10-31)
o Sa1icylic Acid 35hr 95 EUralAlffi (5 x 10-31-1)
12
FIGlllE 2
EFF3Cr OF CYCLIC AmiddotlP 1jJ 3lLICYLIC ACID ON DIFF~JTIAL f3-01AClOSImE SYUTH33IS
IFTG was added to a log phase culture of strain 3000 grown in glucose
medium Approximately 45 minutes after induction the culture was divided
equally into flasks containing the indicated additions 32J1lplcs were
ta~en and ~-Galactosidase was assayed as previously indica~ed The
arroH at 30 Klett unts represents the time the culture ylaS divided
and trcmsferred
DifLerential Rate Growth tate Enzyme Synthesis
Flask A Control bull67hr 23 lJrrJ KIt
B Cyclic mp (5xlO-311) 57hr 315EUrr~6KR
c SalicyliQ acid 4lhr 135ZUmlKR(25xlO-)N)
D Salicy~c acid bull 35hr 25 ~1JrrJLKR 25xlO ~ 11 + CJllmiddotfP (5xlO-)~1)
+
0
t
o
oft
$+
~
Ft flJ rsi
m
m
mlt
l ~ ~
sect4( ~
m~ ~ e1
54 PI rd ISJ
aI ttl
14
FIGURE 3
EFF~T OF CYCLIC AHP UID SJLICYLIC ACID ON j)-GLACTO3IDASE SYNTIIESIS
This represents the smne experireental data as in Figure 1 In the
graph enz~e units ~re plotted in rel~tion to tL~e rather th~n in
relation to cell denitr rIle arrml indicates the time the culture
lras divided and all additions l-Tere i1ade
bull
bullbull Q
-Ld
bull
16 the effect of 321icylic acid ~o be noted indeendently of itsmiddot effect
on generlttion time Even on this graph cAli is shown to partially
relieve repression caused by salicyla~es
Previously Pastaa ~md PerL1an (21)(22) emphasized cAHP had no
effect on growth rate of E coli strain 300 rlhich 112S in contrast to
these results Their dltta homver referto using 1 x lO-3z cANP
Since 5 x 10- 3r crolic lp is believed to overcome both tranient and
catabolite reression caused by r~ucose in strain 3000 (23) the effect
of this concentration on salicylic acids repression was observed
Sirlilar results Nere obtcgtined in a second eXgerimcnt using
S x 10-31-1 salicylic acid and 5 x lO-3H Cl]~ (Fig 4) rhe degree of
reversal ias again only partial Because grorlth inhibition at this conshy
centration of salicylic acid tdth cAHP ias so great the curve is not
extended ver ffX
From these data it appears there is an interaction between the
effects of sali~Jlic acid and those of cMmiddotP Since repression proshy
duced by the higher concentration of salictlic acid seems to be less
effectively reversed by 5 x 10-3N cMP there i~ a possibility the
degree of interaction between the tlO may be concentration dependent
The 3ffect of Sdic-lates on Consti tutive Enzyme Synthesis
To eliminate the possibility salicylic acid may repress j3-galactoshy
sidase synthesis by preventing inducer upt~~e or by competing with
inducer for repressor an experiYlent vTaS carried out using strain 3300
constitutive for fo-galactosidase synthemiddotsis From Figure 5 it is evident
that 25 x 10-311 Jalicylic acid had the same effect on strain 3300 as it
had previously on strain 3000 Therefore salicylates act independently
17
B
uJ ~ l ~ l cJ J L o o- Ul t z 3 w r N Ia Z 312L fa m sa 1m m Em2 ru KLETI UNITS
EFFECT OF CYCLIC Almiddotp lm SLICYLIC ACID OU DIFFrRENfIAL j2gt-GALACTOSIDA3E SYNlHESIS
Figure 4 Strain 3000 culture las grOiID sampled and assayed for fl-g3~actosidase as in Figure 2 uitIl the exception a hig1er concentrC)tion of salic7l1ic acid i-as added after separshyation Since sliicylic acid 35 in dissolved form a dilution occurred upon its addition This uas compensated for in
J
-fA
other fiCsls by an equal dilution
flask Pt Control
Bbull Control + cAHP (5xlO-3H)
c Salicl~ic acid (5xlO H)
D salicl~c acid
Grmmiddotrth Rc1te 71hr
52hr
22hr
15hr (5xlO 3 I) + cMi (5xlO- 1middot1)
Diff Enz Rate 22 EUmlAKR
31 t1Jm1~KR
12 1UmlAIffi
16 EUmlAKR
18
FIGUR35
EFECr 01 SALICYLIC ACID OH DIIT2HE~rrAL fgt-GAL~C0SIDASE SYIJIl133I3 nr COnSrITlJlIV3TAIH 3300
strain 3300 iolaS gron in glucose medium Jllen the cells reached log
phase25 x lO-~1 snlicylic acid ~as added Cells were sa1pled ltmd
4asJared 80S in earlier experiments IPTG (5 x 10- 1-1) liaS added to
control flasks to note the degree of const~tutiveness of tile strain
The first points ngain represent sa~les trucen in~ediately poundter the
culture was divided and additions ware made
Growth Rate Diff Enz Synth Flask A Control bull89hr 65 EUrnlAKR
B Contro~ + IPTG bull89hr 65 EUmJ6KR (5xlO-~middotI)
o SalicYli~3acid bull54hr 46 ~iJ~KR (25x 10 H)
D Sa1icrli~ acid 5hhr 30 EUml~KR (25xlR--i-r) + IPTG (5xlO- M)
lSi JT1
bull 0
Fa-
I
a +
tsJ
yen
+
~
v
+
tsi IS1 m
m
tn
til sectll
m ~ ~ 2
bull
ef ~
~ ~ tsJ
uf
shy
20
of inducer uptake and of inducer-s derepression of lac mrrNA synthesis
The zrecl of 24-Di1ikophcnol ont30alactosidase Synthesis
Since salicylates are kno-Vffi to be uncouplers of oxidative phosshy
phorylation (5) it has been sJgccsted sOrle of their phaITlClcological
effects result from their uncoupling action Tis i-lould irply that
other uncouplers should preferentially inhibit ~-gilactosidase syntheshy
sis For this reason the effect of 2 4-dini trophenol (Dr~) on J-galacshy
tosidase synthesis Has observed Because DrIP is more effective in inhibishy
ting oxidative phosphorylation than salicylic acid and is consequently
more toxic a concentration of the drug i-laS found vhic uould inhibit
grOl-rth to ap~roximately the sa11e degree as 25 x 10-31gt1 salicylic acid
This was found to be 25 x 10-411 Figure 6 illustrates DNP does not
repress differential synthesis of j3-g1actosidase This implies that
althoueh DNP 1m-lers ATP levels there is no apparent effect of the drug
on lsvels of cAl-IP Also it appears salic-rlic acid is not exerting its
repression of enzyrre sJ~~hesis by inhibiting oxidative phosphorylation
21
FIGUllS 6
EFFECrl OF DJP ON DIFFif2rIlL ~GLCiO3D~S STImiddotrYliESIS
Jhen strain 3000 cells reacled log ph~se they tmiddotrere indlccd 1ith IPTG
AproxL~ately one hour later the culture was divide1 a~d tr~ferred
to flasks containing the indicated adlitions The cellsas in preVious
experirrents Tere sarlpled at various tLTIes and assayed for B-galactoshy
sidase activity Since 2h-dinitrophenol t-rag in solution its addition
resuted in dilution of cultures This dilution las equally conpensated
for in control flasks nle first points on the graph represent samples
taken innediately after sepration of the cuJture
GroYTth Rate Difierential inz Rate Flask A Control 75hr 22 L1JmlAKR
B Contro + cAll 64hr 50 EUmlAm (5y10- H) 4z
C DNP (25xlO- -I) 42hr 22 EOIraAKR
D DNP (25xl2~4zr) + 29hr 42 UmlAKR cAJP (5x10 -0
N
N
v
o
-shy-shy+
-shy
+
bull-
DISCU3SION
Salicylic acid ruld to a lesser extent aspirin cause repression
of f3-gclactosidase srnthesis in scherichia coli This effect is not
due to an inhibition of inducer uptake nor is it due ~o interference
l-rith inducers inactivation of lac repressor since strnins constitutive
for 3-gJactosidase 3J1lthesis are also repressed b~r sclicyli~ acid
In addition because dinitrophenol is liithout effect on differential
fl-galactosidase synthesis it appecrs salicrlates do not repress enzyme
s~mthesis by inhibiting oxidative phospllorylation
At these concentr2tions however salicylic acid does decrease
growth rate and direct or indirect effects of this on the observed
repression of enzyme synt~esis still ra~ain Since the differential
rate of enzyme synthesis i~ decreased and also since 25 x 10-411 DN
decreases grotrth rate aproximately the same degree middotuitl1cut having I
effects on differential synthesis salicylates inhibition of growth
is not the sole factor responsible for its effects on enZJcre synthesis
-11en 5 x 10-31 cyclic JUfP 1-ms added to cells treated Kith 25 x 10-3
and 5 x lO-3H srJicylic acid repression of differential enzye s~1lhesis
1ES pextially relieved This sugests salicllic acid represses J3-gJ~ac-
tosidase srnthesis br directly 1011erinti intIacellular levels of cyclic AMP
or by indirectly acting on asita which in turn releases cANP from the
cell Since cMP only partially relieves repression caused by wlicylic
acid and because there appears to be an inverse relationship between
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
-
- Let us know how access to this document benefits you
- Recommended Citation
-
- tmp1379115374pdfCOlFc
-
10
FIGURE 1
EFFECT OF SLICYLIC ACID ON THE DIFF3P3NrIAL RJTE OF [3-GALACTOSIDA3E SYNTHESIS
strain 3000 Was grovffi in glucose modiUI1 until log phase wst reached
Approrinate1y 1 hour follOldn~ induction rlith IPTG the culture was
separated and trs1sierred to flasks containing the indicted additions
Samples uere talten and assayed for j3-galactosidase as in Table I
Since diaBo1ved salicylic acid lias added a dilution in cell densitr
occurred The first point on the graph represents samples taken L~Jnedi-
ately c)fter the culture Was separatedand diluted
Grorth Rate Differential Enz Rate Flask A Control 75hr 308 EUllilAKR
B Salicylic Acid 68hr 180 EUlllJfoKR
(1 x 10-31)
o Sa1icylic Acid 35hr 95 EUralAlffi (5 x 10-31-1)
12
FIGlllE 2
EFF3Cr OF CYCLIC AmiddotlP 1jJ 3lLICYLIC ACID ON DIFF~JTIAL f3-01AClOSImE SYUTH33IS
IFTG was added to a log phase culture of strain 3000 grown in glucose
medium Approximately 45 minutes after induction the culture was divided
equally into flasks containing the indicated additions 32J1lplcs were
ta~en and ~-Galactosidase was assayed as previously indica~ed The
arroH at 30 Klett unts represents the time the culture ylaS divided
and trcmsferred
DifLerential Rate Growth tate Enzyme Synthesis
Flask A Control bull67hr 23 lJrrJ KIt
B Cyclic mp (5xlO-311) 57hr 315EUrr~6KR
c SalicyliQ acid 4lhr 135ZUmlKR(25xlO-)N)
D Salicy~c acid bull 35hr 25 ~1JrrJLKR 25xlO ~ 11 + CJllmiddotfP (5xlO-)~1)
+
0
t
o
oft
$+
~
Ft flJ rsi
m
m
mlt
l ~ ~
sect4( ~
m~ ~ e1
54 PI rd ISJ
aI ttl
14
FIGURE 3
EFF~T OF CYCLIC AHP UID SJLICYLIC ACID ON j)-GLACTO3IDASE SYNTIIESIS
This represents the smne experireental data as in Figure 1 In the
graph enz~e units ~re plotted in rel~tion to tL~e rather th~n in
relation to cell denitr rIle arrml indicates the time the culture
lras divided and all additions l-Tere i1ade
bull
bullbull Q
-Ld
bull
16 the effect of 321icylic acid ~o be noted indeendently of itsmiddot effect
on generlttion time Even on this graph cAli is shown to partially
relieve repression caused by salicyla~es
Previously Pastaa ~md PerL1an (21)(22) emphasized cAHP had no
effect on growth rate of E coli strain 300 rlhich 112S in contrast to
these results Their dltta homver referto using 1 x lO-3z cANP
Since 5 x 10- 3r crolic lp is believed to overcome both tranient and
catabolite reression caused by r~ucose in strain 3000 (23) the effect
of this concentration on salicylic acids repression was observed
Sirlilar results Nere obtcgtined in a second eXgerimcnt using
S x 10-31-1 salicylic acid and 5 x lO-3H Cl]~ (Fig 4) rhe degree of
reversal ias again only partial Because grorlth inhibition at this conshy
centration of salicylic acid tdth cAHP ias so great the curve is not
extended ver ffX
From these data it appears there is an interaction between the
effects of sali~Jlic acid and those of cMmiddotP Since repression proshy
duced by the higher concentration of salictlic acid seems to be less
effectively reversed by 5 x 10-3N cMP there i~ a possibility the
degree of interaction between the tlO may be concentration dependent
The 3ffect of Sdic-lates on Consti tutive Enzyme Synthesis
To eliminate the possibility salicylic acid may repress j3-galactoshy
sidase synthesis by preventing inducer upt~~e or by competing with
inducer for repressor an experiYlent vTaS carried out using strain 3300
constitutive for fo-galactosidase synthemiddotsis From Figure 5 it is evident
that 25 x 10-311 Jalicylic acid had the same effect on strain 3300 as it
had previously on strain 3000 Therefore salicylates act independently
17
B
uJ ~ l ~ l cJ J L o o- Ul t z 3 w r N Ia Z 312L fa m sa 1m m Em2 ru KLETI UNITS
EFFECT OF CYCLIC Almiddotp lm SLICYLIC ACID OU DIFFrRENfIAL j2gt-GALACTOSIDA3E SYNlHESIS
Figure 4 Strain 3000 culture las grOiID sampled and assayed for fl-g3~actosidase as in Figure 2 uitIl the exception a hig1er concentrC)tion of salic7l1ic acid i-as added after separshyation Since sliicylic acid 35 in dissolved form a dilution occurred upon its addition This uas compensated for in
J
-fA
other fiCsls by an equal dilution
flask Pt Control
Bbull Control + cAHP (5xlO-3H)
c Salicl~ic acid (5xlO H)
D salicl~c acid
Grmmiddotrth Rc1te 71hr
52hr
22hr
15hr (5xlO 3 I) + cMi (5xlO- 1middot1)
Diff Enz Rate 22 EUmlAKR
31 t1Jm1~KR
12 1UmlAIffi
16 EUmlAKR
18
FIGUR35
EFECr 01 SALICYLIC ACID OH DIIT2HE~rrAL fgt-GAL~C0SIDASE SYIJIl133I3 nr COnSrITlJlIV3TAIH 3300
strain 3300 iolaS gron in glucose medium Jllen the cells reached log
phase25 x lO-~1 snlicylic acid ~as added Cells were sa1pled ltmd
4asJared 80S in earlier experiments IPTG (5 x 10- 1-1) liaS added to
control flasks to note the degree of const~tutiveness of tile strain
The first points ngain represent sa~les trucen in~ediately poundter the
culture was divided and additions ware made
Growth Rate Diff Enz Synth Flask A Control bull89hr 65 EUrnlAKR
B Contro~ + IPTG bull89hr 65 EUmJ6KR (5xlO-~middotI)
o SalicYli~3acid bull54hr 46 ~iJ~KR (25x 10 H)
D Sa1icrli~ acid 5hhr 30 EUml~KR (25xlR--i-r) + IPTG (5xlO- M)
lSi JT1
bull 0
Fa-
I
a +
tsJ
yen
+
~
v
+
tsi IS1 m
m
tn
til sectll
m ~ ~ 2
bull
ef ~
~ ~ tsJ
uf
shy
20
of inducer uptake and of inducer-s derepression of lac mrrNA synthesis
The zrecl of 24-Di1ikophcnol ont30alactosidase Synthesis
Since salicylates are kno-Vffi to be uncouplers of oxidative phosshy
phorylation (5) it has been sJgccsted sOrle of their phaITlClcological
effects result from their uncoupling action Tis i-lould irply that
other uncouplers should preferentially inhibit ~-gilactosidase syntheshy
sis For this reason the effect of 2 4-dini trophenol (Dr~) on J-galacshy
tosidase synthesis Has observed Because DrIP is more effective in inhibishy
ting oxidative phosphorylation than salicylic acid and is consequently
more toxic a concentration of the drug i-laS found vhic uould inhibit
grOl-rth to ap~roximately the sa11e degree as 25 x 10-31gt1 salicylic acid
This was found to be 25 x 10-411 Figure 6 illustrates DNP does not
repress differential synthesis of j3-g1actosidase This implies that
althoueh DNP 1m-lers ATP levels there is no apparent effect of the drug
on lsvels of cAl-IP Also it appears salic-rlic acid is not exerting its
repression of enzyrre sJ~~hesis by inhibiting oxidative phosphorylation
21
FIGUllS 6
EFFECrl OF DJP ON DIFFif2rIlL ~GLCiO3D~S STImiddotrYliESIS
Jhen strain 3000 cells reacled log ph~se they tmiddotrere indlccd 1ith IPTG
AproxL~ately one hour later the culture was divide1 a~d tr~ferred
to flasks containing the indicated adlitions The cellsas in preVious
experirrents Tere sarlpled at various tLTIes and assayed for B-galactoshy
sidase activity Since 2h-dinitrophenol t-rag in solution its addition
resuted in dilution of cultures This dilution las equally conpensated
for in control flasks nle first points on the graph represent samples
taken innediately after sepration of the cuJture
GroYTth Rate Difierential inz Rate Flask A Control 75hr 22 L1JmlAKR
B Contro + cAll 64hr 50 EUmlAm (5y10- H) 4z
C DNP (25xlO- -I) 42hr 22 EOIraAKR
D DNP (25xl2~4zr) + 29hr 42 UmlAKR cAJP (5x10 -0
N
N
v
o
-shy-shy+
-shy
+
bull-
DISCU3SION
Salicylic acid ruld to a lesser extent aspirin cause repression
of f3-gclactosidase srnthesis in scherichia coli This effect is not
due to an inhibition of inducer uptake nor is it due ~o interference
l-rith inducers inactivation of lac repressor since strnins constitutive
for 3-gJactosidase 3J1lthesis are also repressed b~r sclicyli~ acid
In addition because dinitrophenol is liithout effect on differential
fl-galactosidase synthesis it appecrs salicrlates do not repress enzyme
s~mthesis by inhibiting oxidative phospllorylation
At these concentr2tions however salicylic acid does decrease
growth rate and direct or indirect effects of this on the observed
repression of enzyme synt~esis still ra~ain Since the differential
rate of enzyme synthesis i~ decreased and also since 25 x 10-411 DN
decreases grotrth rate aproximately the same degree middotuitl1cut having I
effects on differential synthesis salicylates inhibition of growth
is not the sole factor responsible for its effects on enZJcre synthesis
-11en 5 x 10-31 cyclic JUfP 1-ms added to cells treated Kith 25 x 10-3
and 5 x lO-3H srJicylic acid repression of differential enzye s~1lhesis
1ES pextially relieved This sugests salicllic acid represses J3-gJ~ac-
tosidase srnthesis br directly 1011erinti intIacellular levels of cyclic AMP
or by indirectly acting on asita which in turn releases cANP from the
cell Since cMP only partially relieves repression caused by wlicylic
acid and because there appears to be an inverse relationship between
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
-
- Let us know how access to this document benefits you
- Recommended Citation
-
- tmp1379115374pdfCOlFc
-
12
FIGlllE 2
EFF3Cr OF CYCLIC AmiddotlP 1jJ 3lLICYLIC ACID ON DIFF~JTIAL f3-01AClOSImE SYUTH33IS
IFTG was added to a log phase culture of strain 3000 grown in glucose
medium Approximately 45 minutes after induction the culture was divided
equally into flasks containing the indicated additions 32J1lplcs were
ta~en and ~-Galactosidase was assayed as previously indica~ed The
arroH at 30 Klett unts represents the time the culture ylaS divided
and trcmsferred
DifLerential Rate Growth tate Enzyme Synthesis
Flask A Control bull67hr 23 lJrrJ KIt
B Cyclic mp (5xlO-311) 57hr 315EUrr~6KR
c SalicyliQ acid 4lhr 135ZUmlKR(25xlO-)N)
D Salicy~c acid bull 35hr 25 ~1JrrJLKR 25xlO ~ 11 + CJllmiddotfP (5xlO-)~1)
+
0
t
o
oft
$+
~
Ft flJ rsi
m
m
mlt
l ~ ~
sect4( ~
m~ ~ e1
54 PI rd ISJ
aI ttl
14
FIGURE 3
EFF~T OF CYCLIC AHP UID SJLICYLIC ACID ON j)-GLACTO3IDASE SYNTIIESIS
This represents the smne experireental data as in Figure 1 In the
graph enz~e units ~re plotted in rel~tion to tL~e rather th~n in
relation to cell denitr rIle arrml indicates the time the culture
lras divided and all additions l-Tere i1ade
bull
bullbull Q
-Ld
bull
16 the effect of 321icylic acid ~o be noted indeendently of itsmiddot effect
on generlttion time Even on this graph cAli is shown to partially
relieve repression caused by salicyla~es
Previously Pastaa ~md PerL1an (21)(22) emphasized cAHP had no
effect on growth rate of E coli strain 300 rlhich 112S in contrast to
these results Their dltta homver referto using 1 x lO-3z cANP
Since 5 x 10- 3r crolic lp is believed to overcome both tranient and
catabolite reression caused by r~ucose in strain 3000 (23) the effect
of this concentration on salicylic acids repression was observed
Sirlilar results Nere obtcgtined in a second eXgerimcnt using
S x 10-31-1 salicylic acid and 5 x lO-3H Cl]~ (Fig 4) rhe degree of
reversal ias again only partial Because grorlth inhibition at this conshy
centration of salicylic acid tdth cAHP ias so great the curve is not
extended ver ffX
From these data it appears there is an interaction between the
effects of sali~Jlic acid and those of cMmiddotP Since repression proshy
duced by the higher concentration of salictlic acid seems to be less
effectively reversed by 5 x 10-3N cMP there i~ a possibility the
degree of interaction between the tlO may be concentration dependent
The 3ffect of Sdic-lates on Consti tutive Enzyme Synthesis
To eliminate the possibility salicylic acid may repress j3-galactoshy
sidase synthesis by preventing inducer upt~~e or by competing with
inducer for repressor an experiYlent vTaS carried out using strain 3300
constitutive for fo-galactosidase synthemiddotsis From Figure 5 it is evident
that 25 x 10-311 Jalicylic acid had the same effect on strain 3300 as it
had previously on strain 3000 Therefore salicylates act independently
17
B
uJ ~ l ~ l cJ J L o o- Ul t z 3 w r N Ia Z 312L fa m sa 1m m Em2 ru KLETI UNITS
EFFECT OF CYCLIC Almiddotp lm SLICYLIC ACID OU DIFFrRENfIAL j2gt-GALACTOSIDA3E SYNlHESIS
Figure 4 Strain 3000 culture las grOiID sampled and assayed for fl-g3~actosidase as in Figure 2 uitIl the exception a hig1er concentrC)tion of salic7l1ic acid i-as added after separshyation Since sliicylic acid 35 in dissolved form a dilution occurred upon its addition This uas compensated for in
J
-fA
other fiCsls by an equal dilution
flask Pt Control
Bbull Control + cAHP (5xlO-3H)
c Salicl~ic acid (5xlO H)
D salicl~c acid
Grmmiddotrth Rc1te 71hr
52hr
22hr
15hr (5xlO 3 I) + cMi (5xlO- 1middot1)
Diff Enz Rate 22 EUmlAKR
31 t1Jm1~KR
12 1UmlAIffi
16 EUmlAKR
18
FIGUR35
EFECr 01 SALICYLIC ACID OH DIIT2HE~rrAL fgt-GAL~C0SIDASE SYIJIl133I3 nr COnSrITlJlIV3TAIH 3300
strain 3300 iolaS gron in glucose medium Jllen the cells reached log
phase25 x lO-~1 snlicylic acid ~as added Cells were sa1pled ltmd
4asJared 80S in earlier experiments IPTG (5 x 10- 1-1) liaS added to
control flasks to note the degree of const~tutiveness of tile strain
The first points ngain represent sa~les trucen in~ediately poundter the
culture was divided and additions ware made
Growth Rate Diff Enz Synth Flask A Control bull89hr 65 EUrnlAKR
B Contro~ + IPTG bull89hr 65 EUmJ6KR (5xlO-~middotI)
o SalicYli~3acid bull54hr 46 ~iJ~KR (25x 10 H)
D Sa1icrli~ acid 5hhr 30 EUml~KR (25xlR--i-r) + IPTG (5xlO- M)
lSi JT1
bull 0
Fa-
I
a +
tsJ
yen
+
~
v
+
tsi IS1 m
m
tn
til sectll
m ~ ~ 2
bull
ef ~
~ ~ tsJ
uf
shy
20
of inducer uptake and of inducer-s derepression of lac mrrNA synthesis
The zrecl of 24-Di1ikophcnol ont30alactosidase Synthesis
Since salicylates are kno-Vffi to be uncouplers of oxidative phosshy
phorylation (5) it has been sJgccsted sOrle of their phaITlClcological
effects result from their uncoupling action Tis i-lould irply that
other uncouplers should preferentially inhibit ~-gilactosidase syntheshy
sis For this reason the effect of 2 4-dini trophenol (Dr~) on J-galacshy
tosidase synthesis Has observed Because DrIP is more effective in inhibishy
ting oxidative phosphorylation than salicylic acid and is consequently
more toxic a concentration of the drug i-laS found vhic uould inhibit
grOl-rth to ap~roximately the sa11e degree as 25 x 10-31gt1 salicylic acid
This was found to be 25 x 10-411 Figure 6 illustrates DNP does not
repress differential synthesis of j3-g1actosidase This implies that
althoueh DNP 1m-lers ATP levels there is no apparent effect of the drug
on lsvels of cAl-IP Also it appears salic-rlic acid is not exerting its
repression of enzyrre sJ~~hesis by inhibiting oxidative phosphorylation
21
FIGUllS 6
EFFECrl OF DJP ON DIFFif2rIlL ~GLCiO3D~S STImiddotrYliESIS
Jhen strain 3000 cells reacled log ph~se they tmiddotrere indlccd 1ith IPTG
AproxL~ately one hour later the culture was divide1 a~d tr~ferred
to flasks containing the indicated adlitions The cellsas in preVious
experirrents Tere sarlpled at various tLTIes and assayed for B-galactoshy
sidase activity Since 2h-dinitrophenol t-rag in solution its addition
resuted in dilution of cultures This dilution las equally conpensated
for in control flasks nle first points on the graph represent samples
taken innediately after sepration of the cuJture
GroYTth Rate Difierential inz Rate Flask A Control 75hr 22 L1JmlAKR
B Contro + cAll 64hr 50 EUmlAm (5y10- H) 4z
C DNP (25xlO- -I) 42hr 22 EOIraAKR
D DNP (25xl2~4zr) + 29hr 42 UmlAKR cAJP (5x10 -0
N
N
v
o
-shy-shy+
-shy
+
bull-
DISCU3SION
Salicylic acid ruld to a lesser extent aspirin cause repression
of f3-gclactosidase srnthesis in scherichia coli This effect is not
due to an inhibition of inducer uptake nor is it due ~o interference
l-rith inducers inactivation of lac repressor since strnins constitutive
for 3-gJactosidase 3J1lthesis are also repressed b~r sclicyli~ acid
In addition because dinitrophenol is liithout effect on differential
fl-galactosidase synthesis it appecrs salicrlates do not repress enzyme
s~mthesis by inhibiting oxidative phospllorylation
At these concentr2tions however salicylic acid does decrease
growth rate and direct or indirect effects of this on the observed
repression of enzyme synt~esis still ra~ain Since the differential
rate of enzyme synthesis i~ decreased and also since 25 x 10-411 DN
decreases grotrth rate aproximately the same degree middotuitl1cut having I
effects on differential synthesis salicylates inhibition of growth
is not the sole factor responsible for its effects on enZJcre synthesis
-11en 5 x 10-31 cyclic JUfP 1-ms added to cells treated Kith 25 x 10-3
and 5 x lO-3H srJicylic acid repression of differential enzye s~1lhesis
1ES pextially relieved This sugests salicllic acid represses J3-gJ~ac-
tosidase srnthesis br directly 1011erinti intIacellular levels of cyclic AMP
or by indirectly acting on asita which in turn releases cANP from the
cell Since cMP only partially relieves repression caused by wlicylic
acid and because there appears to be an inverse relationship between
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
-
- Let us know how access to this document benefits you
- Recommended Citation
-
- tmp1379115374pdfCOlFc
-
+
0
t
o
oft
$+
~
Ft flJ rsi
m
m
mlt
l ~ ~
sect4( ~
m~ ~ e1
54 PI rd ISJ
aI ttl
14
FIGURE 3
EFF~T OF CYCLIC AHP UID SJLICYLIC ACID ON j)-GLACTO3IDASE SYNTIIESIS
This represents the smne experireental data as in Figure 1 In the
graph enz~e units ~re plotted in rel~tion to tL~e rather th~n in
relation to cell denitr rIle arrml indicates the time the culture
lras divided and all additions l-Tere i1ade
bull
bullbull Q
-Ld
bull
16 the effect of 321icylic acid ~o be noted indeendently of itsmiddot effect
on generlttion time Even on this graph cAli is shown to partially
relieve repression caused by salicyla~es
Previously Pastaa ~md PerL1an (21)(22) emphasized cAHP had no
effect on growth rate of E coli strain 300 rlhich 112S in contrast to
these results Their dltta homver referto using 1 x lO-3z cANP
Since 5 x 10- 3r crolic lp is believed to overcome both tranient and
catabolite reression caused by r~ucose in strain 3000 (23) the effect
of this concentration on salicylic acids repression was observed
Sirlilar results Nere obtcgtined in a second eXgerimcnt using
S x 10-31-1 salicylic acid and 5 x lO-3H Cl]~ (Fig 4) rhe degree of
reversal ias again only partial Because grorlth inhibition at this conshy
centration of salicylic acid tdth cAHP ias so great the curve is not
extended ver ffX
From these data it appears there is an interaction between the
effects of sali~Jlic acid and those of cMmiddotP Since repression proshy
duced by the higher concentration of salictlic acid seems to be less
effectively reversed by 5 x 10-3N cMP there i~ a possibility the
degree of interaction between the tlO may be concentration dependent
The 3ffect of Sdic-lates on Consti tutive Enzyme Synthesis
To eliminate the possibility salicylic acid may repress j3-galactoshy
sidase synthesis by preventing inducer upt~~e or by competing with
inducer for repressor an experiYlent vTaS carried out using strain 3300
constitutive for fo-galactosidase synthemiddotsis From Figure 5 it is evident
that 25 x 10-311 Jalicylic acid had the same effect on strain 3300 as it
had previously on strain 3000 Therefore salicylates act independently
17
B
uJ ~ l ~ l cJ J L o o- Ul t z 3 w r N Ia Z 312L fa m sa 1m m Em2 ru KLETI UNITS
EFFECT OF CYCLIC Almiddotp lm SLICYLIC ACID OU DIFFrRENfIAL j2gt-GALACTOSIDA3E SYNlHESIS
Figure 4 Strain 3000 culture las grOiID sampled and assayed for fl-g3~actosidase as in Figure 2 uitIl the exception a hig1er concentrC)tion of salic7l1ic acid i-as added after separshyation Since sliicylic acid 35 in dissolved form a dilution occurred upon its addition This uas compensated for in
J
-fA
other fiCsls by an equal dilution
flask Pt Control
Bbull Control + cAHP (5xlO-3H)
c Salicl~ic acid (5xlO H)
D salicl~c acid
Grmmiddotrth Rc1te 71hr
52hr
22hr
15hr (5xlO 3 I) + cMi (5xlO- 1middot1)
Diff Enz Rate 22 EUmlAKR
31 t1Jm1~KR
12 1UmlAIffi
16 EUmlAKR
18
FIGUR35
EFECr 01 SALICYLIC ACID OH DIIT2HE~rrAL fgt-GAL~C0SIDASE SYIJIl133I3 nr COnSrITlJlIV3TAIH 3300
strain 3300 iolaS gron in glucose medium Jllen the cells reached log
phase25 x lO-~1 snlicylic acid ~as added Cells were sa1pled ltmd
4asJared 80S in earlier experiments IPTG (5 x 10- 1-1) liaS added to
control flasks to note the degree of const~tutiveness of tile strain
The first points ngain represent sa~les trucen in~ediately poundter the
culture was divided and additions ware made
Growth Rate Diff Enz Synth Flask A Control bull89hr 65 EUrnlAKR
B Contro~ + IPTG bull89hr 65 EUmJ6KR (5xlO-~middotI)
o SalicYli~3acid bull54hr 46 ~iJ~KR (25x 10 H)
D Sa1icrli~ acid 5hhr 30 EUml~KR (25xlR--i-r) + IPTG (5xlO- M)
lSi JT1
bull 0
Fa-
I
a +
tsJ
yen
+
~
v
+
tsi IS1 m
m
tn
til sectll
m ~ ~ 2
bull
ef ~
~ ~ tsJ
uf
shy
20
of inducer uptake and of inducer-s derepression of lac mrrNA synthesis
The zrecl of 24-Di1ikophcnol ont30alactosidase Synthesis
Since salicylates are kno-Vffi to be uncouplers of oxidative phosshy
phorylation (5) it has been sJgccsted sOrle of their phaITlClcological
effects result from their uncoupling action Tis i-lould irply that
other uncouplers should preferentially inhibit ~-gilactosidase syntheshy
sis For this reason the effect of 2 4-dini trophenol (Dr~) on J-galacshy
tosidase synthesis Has observed Because DrIP is more effective in inhibishy
ting oxidative phosphorylation than salicylic acid and is consequently
more toxic a concentration of the drug i-laS found vhic uould inhibit
grOl-rth to ap~roximately the sa11e degree as 25 x 10-31gt1 salicylic acid
This was found to be 25 x 10-411 Figure 6 illustrates DNP does not
repress differential synthesis of j3-g1actosidase This implies that
althoueh DNP 1m-lers ATP levels there is no apparent effect of the drug
on lsvels of cAl-IP Also it appears salic-rlic acid is not exerting its
repression of enzyrre sJ~~hesis by inhibiting oxidative phosphorylation
21
FIGUllS 6
EFFECrl OF DJP ON DIFFif2rIlL ~GLCiO3D~S STImiddotrYliESIS
Jhen strain 3000 cells reacled log ph~se they tmiddotrere indlccd 1ith IPTG
AproxL~ately one hour later the culture was divide1 a~d tr~ferred
to flasks containing the indicated adlitions The cellsas in preVious
experirrents Tere sarlpled at various tLTIes and assayed for B-galactoshy
sidase activity Since 2h-dinitrophenol t-rag in solution its addition
resuted in dilution of cultures This dilution las equally conpensated
for in control flasks nle first points on the graph represent samples
taken innediately after sepration of the cuJture
GroYTth Rate Difierential inz Rate Flask A Control 75hr 22 L1JmlAKR
B Contro + cAll 64hr 50 EUmlAm (5y10- H) 4z
C DNP (25xlO- -I) 42hr 22 EOIraAKR
D DNP (25xl2~4zr) + 29hr 42 UmlAKR cAJP (5x10 -0
N
N
v
o
-shy-shy+
-shy
+
bull-
DISCU3SION
Salicylic acid ruld to a lesser extent aspirin cause repression
of f3-gclactosidase srnthesis in scherichia coli This effect is not
due to an inhibition of inducer uptake nor is it due ~o interference
l-rith inducers inactivation of lac repressor since strnins constitutive
for 3-gJactosidase 3J1lthesis are also repressed b~r sclicyli~ acid
In addition because dinitrophenol is liithout effect on differential
fl-galactosidase synthesis it appecrs salicrlates do not repress enzyme
s~mthesis by inhibiting oxidative phospllorylation
At these concentr2tions however salicylic acid does decrease
growth rate and direct or indirect effects of this on the observed
repression of enzyme synt~esis still ra~ain Since the differential
rate of enzyme synthesis i~ decreased and also since 25 x 10-411 DN
decreases grotrth rate aproximately the same degree middotuitl1cut having I
effects on differential synthesis salicylates inhibition of growth
is not the sole factor responsible for its effects on enZJcre synthesis
-11en 5 x 10-31 cyclic JUfP 1-ms added to cells treated Kith 25 x 10-3
and 5 x lO-3H srJicylic acid repression of differential enzye s~1lhesis
1ES pextially relieved This sugests salicllic acid represses J3-gJ~ac-
tosidase srnthesis br directly 1011erinti intIacellular levels of cyclic AMP
or by indirectly acting on asita which in turn releases cANP from the
cell Since cMP only partially relieves repression caused by wlicylic
acid and because there appears to be an inverse relationship between
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
-
- Let us know how access to this document benefits you
- Recommended Citation
-
- tmp1379115374pdfCOlFc
-
14
FIGURE 3
EFF~T OF CYCLIC AHP UID SJLICYLIC ACID ON j)-GLACTO3IDASE SYNTIIESIS
This represents the smne experireental data as in Figure 1 In the
graph enz~e units ~re plotted in rel~tion to tL~e rather th~n in
relation to cell denitr rIle arrml indicates the time the culture
lras divided and all additions l-Tere i1ade
bull
bullbull Q
-Ld
bull
16 the effect of 321icylic acid ~o be noted indeendently of itsmiddot effect
on generlttion time Even on this graph cAli is shown to partially
relieve repression caused by salicyla~es
Previously Pastaa ~md PerL1an (21)(22) emphasized cAHP had no
effect on growth rate of E coli strain 300 rlhich 112S in contrast to
these results Their dltta homver referto using 1 x lO-3z cANP
Since 5 x 10- 3r crolic lp is believed to overcome both tranient and
catabolite reression caused by r~ucose in strain 3000 (23) the effect
of this concentration on salicylic acids repression was observed
Sirlilar results Nere obtcgtined in a second eXgerimcnt using
S x 10-31-1 salicylic acid and 5 x lO-3H Cl]~ (Fig 4) rhe degree of
reversal ias again only partial Because grorlth inhibition at this conshy
centration of salicylic acid tdth cAHP ias so great the curve is not
extended ver ffX
From these data it appears there is an interaction between the
effects of sali~Jlic acid and those of cMmiddotP Since repression proshy
duced by the higher concentration of salictlic acid seems to be less
effectively reversed by 5 x 10-3N cMP there i~ a possibility the
degree of interaction between the tlO may be concentration dependent
The 3ffect of Sdic-lates on Consti tutive Enzyme Synthesis
To eliminate the possibility salicylic acid may repress j3-galactoshy
sidase synthesis by preventing inducer upt~~e or by competing with
inducer for repressor an experiYlent vTaS carried out using strain 3300
constitutive for fo-galactosidase synthemiddotsis From Figure 5 it is evident
that 25 x 10-311 Jalicylic acid had the same effect on strain 3300 as it
had previously on strain 3000 Therefore salicylates act independently
17
B
uJ ~ l ~ l cJ J L o o- Ul t z 3 w r N Ia Z 312L fa m sa 1m m Em2 ru KLETI UNITS
EFFECT OF CYCLIC Almiddotp lm SLICYLIC ACID OU DIFFrRENfIAL j2gt-GALACTOSIDA3E SYNlHESIS
Figure 4 Strain 3000 culture las grOiID sampled and assayed for fl-g3~actosidase as in Figure 2 uitIl the exception a hig1er concentrC)tion of salic7l1ic acid i-as added after separshyation Since sliicylic acid 35 in dissolved form a dilution occurred upon its addition This uas compensated for in
J
-fA
other fiCsls by an equal dilution
flask Pt Control
Bbull Control + cAHP (5xlO-3H)
c Salicl~ic acid (5xlO H)
D salicl~c acid
Grmmiddotrth Rc1te 71hr
52hr
22hr
15hr (5xlO 3 I) + cMi (5xlO- 1middot1)
Diff Enz Rate 22 EUmlAKR
31 t1Jm1~KR
12 1UmlAIffi
16 EUmlAKR
18
FIGUR35
EFECr 01 SALICYLIC ACID OH DIIT2HE~rrAL fgt-GAL~C0SIDASE SYIJIl133I3 nr COnSrITlJlIV3TAIH 3300
strain 3300 iolaS gron in glucose medium Jllen the cells reached log
phase25 x lO-~1 snlicylic acid ~as added Cells were sa1pled ltmd
4asJared 80S in earlier experiments IPTG (5 x 10- 1-1) liaS added to
control flasks to note the degree of const~tutiveness of tile strain
The first points ngain represent sa~les trucen in~ediately poundter the
culture was divided and additions ware made
Growth Rate Diff Enz Synth Flask A Control bull89hr 65 EUrnlAKR
B Contro~ + IPTG bull89hr 65 EUmJ6KR (5xlO-~middotI)
o SalicYli~3acid bull54hr 46 ~iJ~KR (25x 10 H)
D Sa1icrli~ acid 5hhr 30 EUml~KR (25xlR--i-r) + IPTG (5xlO- M)
lSi JT1
bull 0
Fa-
I
a +
tsJ
yen
+
~
v
+
tsi IS1 m
m
tn
til sectll
m ~ ~ 2
bull
ef ~
~ ~ tsJ
uf
shy
20
of inducer uptake and of inducer-s derepression of lac mrrNA synthesis
The zrecl of 24-Di1ikophcnol ont30alactosidase Synthesis
Since salicylates are kno-Vffi to be uncouplers of oxidative phosshy
phorylation (5) it has been sJgccsted sOrle of their phaITlClcological
effects result from their uncoupling action Tis i-lould irply that
other uncouplers should preferentially inhibit ~-gilactosidase syntheshy
sis For this reason the effect of 2 4-dini trophenol (Dr~) on J-galacshy
tosidase synthesis Has observed Because DrIP is more effective in inhibishy
ting oxidative phosphorylation than salicylic acid and is consequently
more toxic a concentration of the drug i-laS found vhic uould inhibit
grOl-rth to ap~roximately the sa11e degree as 25 x 10-31gt1 salicylic acid
This was found to be 25 x 10-411 Figure 6 illustrates DNP does not
repress differential synthesis of j3-g1actosidase This implies that
althoueh DNP 1m-lers ATP levels there is no apparent effect of the drug
on lsvels of cAl-IP Also it appears salic-rlic acid is not exerting its
repression of enzyrre sJ~~hesis by inhibiting oxidative phosphorylation
21
FIGUllS 6
EFFECrl OF DJP ON DIFFif2rIlL ~GLCiO3D~S STImiddotrYliESIS
Jhen strain 3000 cells reacled log ph~se they tmiddotrere indlccd 1ith IPTG
AproxL~ately one hour later the culture was divide1 a~d tr~ferred
to flasks containing the indicated adlitions The cellsas in preVious
experirrents Tere sarlpled at various tLTIes and assayed for B-galactoshy
sidase activity Since 2h-dinitrophenol t-rag in solution its addition
resuted in dilution of cultures This dilution las equally conpensated
for in control flasks nle first points on the graph represent samples
taken innediately after sepration of the cuJture
GroYTth Rate Difierential inz Rate Flask A Control 75hr 22 L1JmlAKR
B Contro + cAll 64hr 50 EUmlAm (5y10- H) 4z
C DNP (25xlO- -I) 42hr 22 EOIraAKR
D DNP (25xl2~4zr) + 29hr 42 UmlAKR cAJP (5x10 -0
N
N
v
o
-shy-shy+
-shy
+
bull-
DISCU3SION
Salicylic acid ruld to a lesser extent aspirin cause repression
of f3-gclactosidase srnthesis in scherichia coli This effect is not
due to an inhibition of inducer uptake nor is it due ~o interference
l-rith inducers inactivation of lac repressor since strnins constitutive
for 3-gJactosidase 3J1lthesis are also repressed b~r sclicyli~ acid
In addition because dinitrophenol is liithout effect on differential
fl-galactosidase synthesis it appecrs salicrlates do not repress enzyme
s~mthesis by inhibiting oxidative phospllorylation
At these concentr2tions however salicylic acid does decrease
growth rate and direct or indirect effects of this on the observed
repression of enzyme synt~esis still ra~ain Since the differential
rate of enzyme synthesis i~ decreased and also since 25 x 10-411 DN
decreases grotrth rate aproximately the same degree middotuitl1cut having I
effects on differential synthesis salicylates inhibition of growth
is not the sole factor responsible for its effects on enZJcre synthesis
-11en 5 x 10-31 cyclic JUfP 1-ms added to cells treated Kith 25 x 10-3
and 5 x lO-3H srJicylic acid repression of differential enzye s~1lhesis
1ES pextially relieved This sugests salicllic acid represses J3-gJ~ac-
tosidase srnthesis br directly 1011erinti intIacellular levels of cyclic AMP
or by indirectly acting on asita which in turn releases cANP from the
cell Since cMP only partially relieves repression caused by wlicylic
acid and because there appears to be an inverse relationship between
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
-
- Let us know how access to this document benefits you
- Recommended Citation
-
- tmp1379115374pdfCOlFc
-
bullbull Q
-Ld
bull
16 the effect of 321icylic acid ~o be noted indeendently of itsmiddot effect
on generlttion time Even on this graph cAli is shown to partially
relieve repression caused by salicyla~es
Previously Pastaa ~md PerL1an (21)(22) emphasized cAHP had no
effect on growth rate of E coli strain 300 rlhich 112S in contrast to
these results Their dltta homver referto using 1 x lO-3z cANP
Since 5 x 10- 3r crolic lp is believed to overcome both tranient and
catabolite reression caused by r~ucose in strain 3000 (23) the effect
of this concentration on salicylic acids repression was observed
Sirlilar results Nere obtcgtined in a second eXgerimcnt using
S x 10-31-1 salicylic acid and 5 x lO-3H Cl]~ (Fig 4) rhe degree of
reversal ias again only partial Because grorlth inhibition at this conshy
centration of salicylic acid tdth cAHP ias so great the curve is not
extended ver ffX
From these data it appears there is an interaction between the
effects of sali~Jlic acid and those of cMmiddotP Since repression proshy
duced by the higher concentration of salictlic acid seems to be less
effectively reversed by 5 x 10-3N cMP there i~ a possibility the
degree of interaction between the tlO may be concentration dependent
The 3ffect of Sdic-lates on Consti tutive Enzyme Synthesis
To eliminate the possibility salicylic acid may repress j3-galactoshy
sidase synthesis by preventing inducer upt~~e or by competing with
inducer for repressor an experiYlent vTaS carried out using strain 3300
constitutive for fo-galactosidase synthemiddotsis From Figure 5 it is evident
that 25 x 10-311 Jalicylic acid had the same effect on strain 3300 as it
had previously on strain 3000 Therefore salicylates act independently
17
B
uJ ~ l ~ l cJ J L o o- Ul t z 3 w r N Ia Z 312L fa m sa 1m m Em2 ru KLETI UNITS
EFFECT OF CYCLIC Almiddotp lm SLICYLIC ACID OU DIFFrRENfIAL j2gt-GALACTOSIDA3E SYNlHESIS
Figure 4 Strain 3000 culture las grOiID sampled and assayed for fl-g3~actosidase as in Figure 2 uitIl the exception a hig1er concentrC)tion of salic7l1ic acid i-as added after separshyation Since sliicylic acid 35 in dissolved form a dilution occurred upon its addition This uas compensated for in
J
-fA
other fiCsls by an equal dilution
flask Pt Control
Bbull Control + cAHP (5xlO-3H)
c Salicl~ic acid (5xlO H)
D salicl~c acid
Grmmiddotrth Rc1te 71hr
52hr
22hr
15hr (5xlO 3 I) + cMi (5xlO- 1middot1)
Diff Enz Rate 22 EUmlAKR
31 t1Jm1~KR
12 1UmlAIffi
16 EUmlAKR
18
FIGUR35
EFECr 01 SALICYLIC ACID OH DIIT2HE~rrAL fgt-GAL~C0SIDASE SYIJIl133I3 nr COnSrITlJlIV3TAIH 3300
strain 3300 iolaS gron in glucose medium Jllen the cells reached log
phase25 x lO-~1 snlicylic acid ~as added Cells were sa1pled ltmd
4asJared 80S in earlier experiments IPTG (5 x 10- 1-1) liaS added to
control flasks to note the degree of const~tutiveness of tile strain
The first points ngain represent sa~les trucen in~ediately poundter the
culture was divided and additions ware made
Growth Rate Diff Enz Synth Flask A Control bull89hr 65 EUrnlAKR
B Contro~ + IPTG bull89hr 65 EUmJ6KR (5xlO-~middotI)
o SalicYli~3acid bull54hr 46 ~iJ~KR (25x 10 H)
D Sa1icrli~ acid 5hhr 30 EUml~KR (25xlR--i-r) + IPTG (5xlO- M)
lSi JT1
bull 0
Fa-
I
a +
tsJ
yen
+
~
v
+
tsi IS1 m
m
tn
til sectll
m ~ ~ 2
bull
ef ~
~ ~ tsJ
uf
shy
20
of inducer uptake and of inducer-s derepression of lac mrrNA synthesis
The zrecl of 24-Di1ikophcnol ont30alactosidase Synthesis
Since salicylates are kno-Vffi to be uncouplers of oxidative phosshy
phorylation (5) it has been sJgccsted sOrle of their phaITlClcological
effects result from their uncoupling action Tis i-lould irply that
other uncouplers should preferentially inhibit ~-gilactosidase syntheshy
sis For this reason the effect of 2 4-dini trophenol (Dr~) on J-galacshy
tosidase synthesis Has observed Because DrIP is more effective in inhibishy
ting oxidative phosphorylation than salicylic acid and is consequently
more toxic a concentration of the drug i-laS found vhic uould inhibit
grOl-rth to ap~roximately the sa11e degree as 25 x 10-31gt1 salicylic acid
This was found to be 25 x 10-411 Figure 6 illustrates DNP does not
repress differential synthesis of j3-g1actosidase This implies that
althoueh DNP 1m-lers ATP levels there is no apparent effect of the drug
on lsvels of cAl-IP Also it appears salic-rlic acid is not exerting its
repression of enzyrre sJ~~hesis by inhibiting oxidative phosphorylation
21
FIGUllS 6
EFFECrl OF DJP ON DIFFif2rIlL ~GLCiO3D~S STImiddotrYliESIS
Jhen strain 3000 cells reacled log ph~se they tmiddotrere indlccd 1ith IPTG
AproxL~ately one hour later the culture was divide1 a~d tr~ferred
to flasks containing the indicated adlitions The cellsas in preVious
experirrents Tere sarlpled at various tLTIes and assayed for B-galactoshy
sidase activity Since 2h-dinitrophenol t-rag in solution its addition
resuted in dilution of cultures This dilution las equally conpensated
for in control flasks nle first points on the graph represent samples
taken innediately after sepration of the cuJture
GroYTth Rate Difierential inz Rate Flask A Control 75hr 22 L1JmlAKR
B Contro + cAll 64hr 50 EUmlAm (5y10- H) 4z
C DNP (25xlO- -I) 42hr 22 EOIraAKR
D DNP (25xl2~4zr) + 29hr 42 UmlAKR cAJP (5x10 -0
N
N
v
o
-shy-shy+
-shy
+
bull-
DISCU3SION
Salicylic acid ruld to a lesser extent aspirin cause repression
of f3-gclactosidase srnthesis in scherichia coli This effect is not
due to an inhibition of inducer uptake nor is it due ~o interference
l-rith inducers inactivation of lac repressor since strnins constitutive
for 3-gJactosidase 3J1lthesis are also repressed b~r sclicyli~ acid
In addition because dinitrophenol is liithout effect on differential
fl-galactosidase synthesis it appecrs salicrlates do not repress enzyme
s~mthesis by inhibiting oxidative phospllorylation
At these concentr2tions however salicylic acid does decrease
growth rate and direct or indirect effects of this on the observed
repression of enzyme synt~esis still ra~ain Since the differential
rate of enzyme synthesis i~ decreased and also since 25 x 10-411 DN
decreases grotrth rate aproximately the same degree middotuitl1cut having I
effects on differential synthesis salicylates inhibition of growth
is not the sole factor responsible for its effects on enZJcre synthesis
-11en 5 x 10-31 cyclic JUfP 1-ms added to cells treated Kith 25 x 10-3
and 5 x lO-3H srJicylic acid repression of differential enzye s~1lhesis
1ES pextially relieved This sugests salicllic acid represses J3-gJ~ac-
tosidase srnthesis br directly 1011erinti intIacellular levels of cyclic AMP
or by indirectly acting on asita which in turn releases cANP from the
cell Since cMP only partially relieves repression caused by wlicylic
acid and because there appears to be an inverse relationship between
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
-
- Let us know how access to this document benefits you
- Recommended Citation
-
- tmp1379115374pdfCOlFc
-
16 the effect of 321icylic acid ~o be noted indeendently of itsmiddot effect
on generlttion time Even on this graph cAli is shown to partially
relieve repression caused by salicyla~es
Previously Pastaa ~md PerL1an (21)(22) emphasized cAHP had no
effect on growth rate of E coli strain 300 rlhich 112S in contrast to
these results Their dltta homver referto using 1 x lO-3z cANP
Since 5 x 10- 3r crolic lp is believed to overcome both tranient and
catabolite reression caused by r~ucose in strain 3000 (23) the effect
of this concentration on salicylic acids repression was observed
Sirlilar results Nere obtcgtined in a second eXgerimcnt using
S x 10-31-1 salicylic acid and 5 x lO-3H Cl]~ (Fig 4) rhe degree of
reversal ias again only partial Because grorlth inhibition at this conshy
centration of salicylic acid tdth cAHP ias so great the curve is not
extended ver ffX
From these data it appears there is an interaction between the
effects of sali~Jlic acid and those of cMmiddotP Since repression proshy
duced by the higher concentration of salictlic acid seems to be less
effectively reversed by 5 x 10-3N cMP there i~ a possibility the
degree of interaction between the tlO may be concentration dependent
The 3ffect of Sdic-lates on Consti tutive Enzyme Synthesis
To eliminate the possibility salicylic acid may repress j3-galactoshy
sidase synthesis by preventing inducer upt~~e or by competing with
inducer for repressor an experiYlent vTaS carried out using strain 3300
constitutive for fo-galactosidase synthemiddotsis From Figure 5 it is evident
that 25 x 10-311 Jalicylic acid had the same effect on strain 3300 as it
had previously on strain 3000 Therefore salicylates act independently
17
B
uJ ~ l ~ l cJ J L o o- Ul t z 3 w r N Ia Z 312L fa m sa 1m m Em2 ru KLETI UNITS
EFFECT OF CYCLIC Almiddotp lm SLICYLIC ACID OU DIFFrRENfIAL j2gt-GALACTOSIDA3E SYNlHESIS
Figure 4 Strain 3000 culture las grOiID sampled and assayed for fl-g3~actosidase as in Figure 2 uitIl the exception a hig1er concentrC)tion of salic7l1ic acid i-as added after separshyation Since sliicylic acid 35 in dissolved form a dilution occurred upon its addition This uas compensated for in
J
-fA
other fiCsls by an equal dilution
flask Pt Control
Bbull Control + cAHP (5xlO-3H)
c Salicl~ic acid (5xlO H)
D salicl~c acid
Grmmiddotrth Rc1te 71hr
52hr
22hr
15hr (5xlO 3 I) + cMi (5xlO- 1middot1)
Diff Enz Rate 22 EUmlAKR
31 t1Jm1~KR
12 1UmlAIffi
16 EUmlAKR
18
FIGUR35
EFECr 01 SALICYLIC ACID OH DIIT2HE~rrAL fgt-GAL~C0SIDASE SYIJIl133I3 nr COnSrITlJlIV3TAIH 3300
strain 3300 iolaS gron in glucose medium Jllen the cells reached log
phase25 x lO-~1 snlicylic acid ~as added Cells were sa1pled ltmd
4asJared 80S in earlier experiments IPTG (5 x 10- 1-1) liaS added to
control flasks to note the degree of const~tutiveness of tile strain
The first points ngain represent sa~les trucen in~ediately poundter the
culture was divided and additions ware made
Growth Rate Diff Enz Synth Flask A Control bull89hr 65 EUrnlAKR
B Contro~ + IPTG bull89hr 65 EUmJ6KR (5xlO-~middotI)
o SalicYli~3acid bull54hr 46 ~iJ~KR (25x 10 H)
D Sa1icrli~ acid 5hhr 30 EUml~KR (25xlR--i-r) + IPTG (5xlO- M)
lSi JT1
bull 0
Fa-
I
a +
tsJ
yen
+
~
v
+
tsi IS1 m
m
tn
til sectll
m ~ ~ 2
bull
ef ~
~ ~ tsJ
uf
shy
20
of inducer uptake and of inducer-s derepression of lac mrrNA synthesis
The zrecl of 24-Di1ikophcnol ont30alactosidase Synthesis
Since salicylates are kno-Vffi to be uncouplers of oxidative phosshy
phorylation (5) it has been sJgccsted sOrle of their phaITlClcological
effects result from their uncoupling action Tis i-lould irply that
other uncouplers should preferentially inhibit ~-gilactosidase syntheshy
sis For this reason the effect of 2 4-dini trophenol (Dr~) on J-galacshy
tosidase synthesis Has observed Because DrIP is more effective in inhibishy
ting oxidative phosphorylation than salicylic acid and is consequently
more toxic a concentration of the drug i-laS found vhic uould inhibit
grOl-rth to ap~roximately the sa11e degree as 25 x 10-31gt1 salicylic acid
This was found to be 25 x 10-411 Figure 6 illustrates DNP does not
repress differential synthesis of j3-g1actosidase This implies that
althoueh DNP 1m-lers ATP levels there is no apparent effect of the drug
on lsvels of cAl-IP Also it appears salic-rlic acid is not exerting its
repression of enzyrre sJ~~hesis by inhibiting oxidative phosphorylation
21
FIGUllS 6
EFFECrl OF DJP ON DIFFif2rIlL ~GLCiO3D~S STImiddotrYliESIS
Jhen strain 3000 cells reacled log ph~se they tmiddotrere indlccd 1ith IPTG
AproxL~ately one hour later the culture was divide1 a~d tr~ferred
to flasks containing the indicated adlitions The cellsas in preVious
experirrents Tere sarlpled at various tLTIes and assayed for B-galactoshy
sidase activity Since 2h-dinitrophenol t-rag in solution its addition
resuted in dilution of cultures This dilution las equally conpensated
for in control flasks nle first points on the graph represent samples
taken innediately after sepration of the cuJture
GroYTth Rate Difierential inz Rate Flask A Control 75hr 22 L1JmlAKR
B Contro + cAll 64hr 50 EUmlAm (5y10- H) 4z
C DNP (25xlO- -I) 42hr 22 EOIraAKR
D DNP (25xl2~4zr) + 29hr 42 UmlAKR cAJP (5x10 -0
N
N
v
o
-shy-shy+
-shy
+
bull-
DISCU3SION
Salicylic acid ruld to a lesser extent aspirin cause repression
of f3-gclactosidase srnthesis in scherichia coli This effect is not
due to an inhibition of inducer uptake nor is it due ~o interference
l-rith inducers inactivation of lac repressor since strnins constitutive
for 3-gJactosidase 3J1lthesis are also repressed b~r sclicyli~ acid
In addition because dinitrophenol is liithout effect on differential
fl-galactosidase synthesis it appecrs salicrlates do not repress enzyme
s~mthesis by inhibiting oxidative phospllorylation
At these concentr2tions however salicylic acid does decrease
growth rate and direct or indirect effects of this on the observed
repression of enzyme synt~esis still ra~ain Since the differential
rate of enzyme synthesis i~ decreased and also since 25 x 10-411 DN
decreases grotrth rate aproximately the same degree middotuitl1cut having I
effects on differential synthesis salicylates inhibition of growth
is not the sole factor responsible for its effects on enZJcre synthesis
-11en 5 x 10-31 cyclic JUfP 1-ms added to cells treated Kith 25 x 10-3
and 5 x lO-3H srJicylic acid repression of differential enzye s~1lhesis
1ES pextially relieved This sugests salicllic acid represses J3-gJ~ac-
tosidase srnthesis br directly 1011erinti intIacellular levels of cyclic AMP
or by indirectly acting on asita which in turn releases cANP from the
cell Since cMP only partially relieves repression caused by wlicylic
acid and because there appears to be an inverse relationship between
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
-
- Let us know how access to this document benefits you
- Recommended Citation
-
- tmp1379115374pdfCOlFc
-
17
B
uJ ~ l ~ l cJ J L o o- Ul t z 3 w r N Ia Z 312L fa m sa 1m m Em2 ru KLETI UNITS
EFFECT OF CYCLIC Almiddotp lm SLICYLIC ACID OU DIFFrRENfIAL j2gt-GALACTOSIDA3E SYNlHESIS
Figure 4 Strain 3000 culture las grOiID sampled and assayed for fl-g3~actosidase as in Figure 2 uitIl the exception a hig1er concentrC)tion of salic7l1ic acid i-as added after separshyation Since sliicylic acid 35 in dissolved form a dilution occurred upon its addition This uas compensated for in
J
-fA
other fiCsls by an equal dilution
flask Pt Control
Bbull Control + cAHP (5xlO-3H)
c Salicl~ic acid (5xlO H)
D salicl~c acid
Grmmiddotrth Rc1te 71hr
52hr
22hr
15hr (5xlO 3 I) + cMi (5xlO- 1middot1)
Diff Enz Rate 22 EUmlAKR
31 t1Jm1~KR
12 1UmlAIffi
16 EUmlAKR
18
FIGUR35
EFECr 01 SALICYLIC ACID OH DIIT2HE~rrAL fgt-GAL~C0SIDASE SYIJIl133I3 nr COnSrITlJlIV3TAIH 3300
strain 3300 iolaS gron in glucose medium Jllen the cells reached log
phase25 x lO-~1 snlicylic acid ~as added Cells were sa1pled ltmd
4asJared 80S in earlier experiments IPTG (5 x 10- 1-1) liaS added to
control flasks to note the degree of const~tutiveness of tile strain
The first points ngain represent sa~les trucen in~ediately poundter the
culture was divided and additions ware made
Growth Rate Diff Enz Synth Flask A Control bull89hr 65 EUrnlAKR
B Contro~ + IPTG bull89hr 65 EUmJ6KR (5xlO-~middotI)
o SalicYli~3acid bull54hr 46 ~iJ~KR (25x 10 H)
D Sa1icrli~ acid 5hhr 30 EUml~KR (25xlR--i-r) + IPTG (5xlO- M)
lSi JT1
bull 0
Fa-
I
a +
tsJ
yen
+
~
v
+
tsi IS1 m
m
tn
til sectll
m ~ ~ 2
bull
ef ~
~ ~ tsJ
uf
shy
20
of inducer uptake and of inducer-s derepression of lac mrrNA synthesis
The zrecl of 24-Di1ikophcnol ont30alactosidase Synthesis
Since salicylates are kno-Vffi to be uncouplers of oxidative phosshy
phorylation (5) it has been sJgccsted sOrle of their phaITlClcological
effects result from their uncoupling action Tis i-lould irply that
other uncouplers should preferentially inhibit ~-gilactosidase syntheshy
sis For this reason the effect of 2 4-dini trophenol (Dr~) on J-galacshy
tosidase synthesis Has observed Because DrIP is more effective in inhibishy
ting oxidative phosphorylation than salicylic acid and is consequently
more toxic a concentration of the drug i-laS found vhic uould inhibit
grOl-rth to ap~roximately the sa11e degree as 25 x 10-31gt1 salicylic acid
This was found to be 25 x 10-411 Figure 6 illustrates DNP does not
repress differential synthesis of j3-g1actosidase This implies that
althoueh DNP 1m-lers ATP levels there is no apparent effect of the drug
on lsvels of cAl-IP Also it appears salic-rlic acid is not exerting its
repression of enzyrre sJ~~hesis by inhibiting oxidative phosphorylation
21
FIGUllS 6
EFFECrl OF DJP ON DIFFif2rIlL ~GLCiO3D~S STImiddotrYliESIS
Jhen strain 3000 cells reacled log ph~se they tmiddotrere indlccd 1ith IPTG
AproxL~ately one hour later the culture was divide1 a~d tr~ferred
to flasks containing the indicated adlitions The cellsas in preVious
experirrents Tere sarlpled at various tLTIes and assayed for B-galactoshy
sidase activity Since 2h-dinitrophenol t-rag in solution its addition
resuted in dilution of cultures This dilution las equally conpensated
for in control flasks nle first points on the graph represent samples
taken innediately after sepration of the cuJture
GroYTth Rate Difierential inz Rate Flask A Control 75hr 22 L1JmlAKR
B Contro + cAll 64hr 50 EUmlAm (5y10- H) 4z
C DNP (25xlO- -I) 42hr 22 EOIraAKR
D DNP (25xl2~4zr) + 29hr 42 UmlAKR cAJP (5x10 -0
N
N
v
o
-shy-shy+
-shy
+
bull-
DISCU3SION
Salicylic acid ruld to a lesser extent aspirin cause repression
of f3-gclactosidase srnthesis in scherichia coli This effect is not
due to an inhibition of inducer uptake nor is it due ~o interference
l-rith inducers inactivation of lac repressor since strnins constitutive
for 3-gJactosidase 3J1lthesis are also repressed b~r sclicyli~ acid
In addition because dinitrophenol is liithout effect on differential
fl-galactosidase synthesis it appecrs salicrlates do not repress enzyme
s~mthesis by inhibiting oxidative phospllorylation
At these concentr2tions however salicylic acid does decrease
growth rate and direct or indirect effects of this on the observed
repression of enzyme synt~esis still ra~ain Since the differential
rate of enzyme synthesis i~ decreased and also since 25 x 10-411 DN
decreases grotrth rate aproximately the same degree middotuitl1cut having I
effects on differential synthesis salicylates inhibition of growth
is not the sole factor responsible for its effects on enZJcre synthesis
-11en 5 x 10-31 cyclic JUfP 1-ms added to cells treated Kith 25 x 10-3
and 5 x lO-3H srJicylic acid repression of differential enzye s~1lhesis
1ES pextially relieved This sugests salicllic acid represses J3-gJ~ac-
tosidase srnthesis br directly 1011erinti intIacellular levels of cyclic AMP
or by indirectly acting on asita which in turn releases cANP from the
cell Since cMP only partially relieves repression caused by wlicylic
acid and because there appears to be an inverse relationship between
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
-
- Let us know how access to this document benefits you
- Recommended Citation
-
- tmp1379115374pdfCOlFc
-
18
FIGUR35
EFECr 01 SALICYLIC ACID OH DIIT2HE~rrAL fgt-GAL~C0SIDASE SYIJIl133I3 nr COnSrITlJlIV3TAIH 3300
strain 3300 iolaS gron in glucose medium Jllen the cells reached log
phase25 x lO-~1 snlicylic acid ~as added Cells were sa1pled ltmd
4asJared 80S in earlier experiments IPTG (5 x 10- 1-1) liaS added to
control flasks to note the degree of const~tutiveness of tile strain
The first points ngain represent sa~les trucen in~ediately poundter the
culture was divided and additions ware made
Growth Rate Diff Enz Synth Flask A Control bull89hr 65 EUrnlAKR
B Contro~ + IPTG bull89hr 65 EUmJ6KR (5xlO-~middotI)
o SalicYli~3acid bull54hr 46 ~iJ~KR (25x 10 H)
D Sa1icrli~ acid 5hhr 30 EUml~KR (25xlR--i-r) + IPTG (5xlO- M)
lSi JT1
bull 0
Fa-
I
a +
tsJ
yen
+
~
v
+
tsi IS1 m
m
tn
til sectll
m ~ ~ 2
bull
ef ~
~ ~ tsJ
uf
shy
20
of inducer uptake and of inducer-s derepression of lac mrrNA synthesis
The zrecl of 24-Di1ikophcnol ont30alactosidase Synthesis
Since salicylates are kno-Vffi to be uncouplers of oxidative phosshy
phorylation (5) it has been sJgccsted sOrle of their phaITlClcological
effects result from their uncoupling action Tis i-lould irply that
other uncouplers should preferentially inhibit ~-gilactosidase syntheshy
sis For this reason the effect of 2 4-dini trophenol (Dr~) on J-galacshy
tosidase synthesis Has observed Because DrIP is more effective in inhibishy
ting oxidative phosphorylation than salicylic acid and is consequently
more toxic a concentration of the drug i-laS found vhic uould inhibit
grOl-rth to ap~roximately the sa11e degree as 25 x 10-31gt1 salicylic acid
This was found to be 25 x 10-411 Figure 6 illustrates DNP does not
repress differential synthesis of j3-g1actosidase This implies that
althoueh DNP 1m-lers ATP levels there is no apparent effect of the drug
on lsvels of cAl-IP Also it appears salic-rlic acid is not exerting its
repression of enzyrre sJ~~hesis by inhibiting oxidative phosphorylation
21
FIGUllS 6
EFFECrl OF DJP ON DIFFif2rIlL ~GLCiO3D~S STImiddotrYliESIS
Jhen strain 3000 cells reacled log ph~se they tmiddotrere indlccd 1ith IPTG
AproxL~ately one hour later the culture was divide1 a~d tr~ferred
to flasks containing the indicated adlitions The cellsas in preVious
experirrents Tere sarlpled at various tLTIes and assayed for B-galactoshy
sidase activity Since 2h-dinitrophenol t-rag in solution its addition
resuted in dilution of cultures This dilution las equally conpensated
for in control flasks nle first points on the graph represent samples
taken innediately after sepration of the cuJture
GroYTth Rate Difierential inz Rate Flask A Control 75hr 22 L1JmlAKR
B Contro + cAll 64hr 50 EUmlAm (5y10- H) 4z
C DNP (25xlO- -I) 42hr 22 EOIraAKR
D DNP (25xl2~4zr) + 29hr 42 UmlAKR cAJP (5x10 -0
N
N
v
o
-shy-shy+
-shy
+
bull-
DISCU3SION
Salicylic acid ruld to a lesser extent aspirin cause repression
of f3-gclactosidase srnthesis in scherichia coli This effect is not
due to an inhibition of inducer uptake nor is it due ~o interference
l-rith inducers inactivation of lac repressor since strnins constitutive
for 3-gJactosidase 3J1lthesis are also repressed b~r sclicyli~ acid
In addition because dinitrophenol is liithout effect on differential
fl-galactosidase synthesis it appecrs salicrlates do not repress enzyme
s~mthesis by inhibiting oxidative phospllorylation
At these concentr2tions however salicylic acid does decrease
growth rate and direct or indirect effects of this on the observed
repression of enzyme synt~esis still ra~ain Since the differential
rate of enzyme synthesis i~ decreased and also since 25 x 10-411 DN
decreases grotrth rate aproximately the same degree middotuitl1cut having I
effects on differential synthesis salicylates inhibition of growth
is not the sole factor responsible for its effects on enZJcre synthesis
-11en 5 x 10-31 cyclic JUfP 1-ms added to cells treated Kith 25 x 10-3
and 5 x lO-3H srJicylic acid repression of differential enzye s~1lhesis
1ES pextially relieved This sugests salicllic acid represses J3-gJ~ac-
tosidase srnthesis br directly 1011erinti intIacellular levels of cyclic AMP
or by indirectly acting on asita which in turn releases cANP from the
cell Since cMP only partially relieves repression caused by wlicylic
acid and because there appears to be an inverse relationship between
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
-
- Let us know how access to this document benefits you
- Recommended Citation
-
- tmp1379115374pdfCOlFc
-
lSi JT1
bull 0
Fa-
I
a +
tsJ
yen
+
~
v
+
tsi IS1 m
m
tn
til sectll
m ~ ~ 2
bull
ef ~
~ ~ tsJ
uf
shy
20
of inducer uptake and of inducer-s derepression of lac mrrNA synthesis
The zrecl of 24-Di1ikophcnol ont30alactosidase Synthesis
Since salicylates are kno-Vffi to be uncouplers of oxidative phosshy
phorylation (5) it has been sJgccsted sOrle of their phaITlClcological
effects result from their uncoupling action Tis i-lould irply that
other uncouplers should preferentially inhibit ~-gilactosidase syntheshy
sis For this reason the effect of 2 4-dini trophenol (Dr~) on J-galacshy
tosidase synthesis Has observed Because DrIP is more effective in inhibishy
ting oxidative phosphorylation than salicylic acid and is consequently
more toxic a concentration of the drug i-laS found vhic uould inhibit
grOl-rth to ap~roximately the sa11e degree as 25 x 10-31gt1 salicylic acid
This was found to be 25 x 10-411 Figure 6 illustrates DNP does not
repress differential synthesis of j3-g1actosidase This implies that
althoueh DNP 1m-lers ATP levels there is no apparent effect of the drug
on lsvels of cAl-IP Also it appears salic-rlic acid is not exerting its
repression of enzyrre sJ~~hesis by inhibiting oxidative phosphorylation
21
FIGUllS 6
EFFECrl OF DJP ON DIFFif2rIlL ~GLCiO3D~S STImiddotrYliESIS
Jhen strain 3000 cells reacled log ph~se they tmiddotrere indlccd 1ith IPTG
AproxL~ately one hour later the culture was divide1 a~d tr~ferred
to flasks containing the indicated adlitions The cellsas in preVious
experirrents Tere sarlpled at various tLTIes and assayed for B-galactoshy
sidase activity Since 2h-dinitrophenol t-rag in solution its addition
resuted in dilution of cultures This dilution las equally conpensated
for in control flasks nle first points on the graph represent samples
taken innediately after sepration of the cuJture
GroYTth Rate Difierential inz Rate Flask A Control 75hr 22 L1JmlAKR
B Contro + cAll 64hr 50 EUmlAm (5y10- H) 4z
C DNP (25xlO- -I) 42hr 22 EOIraAKR
D DNP (25xl2~4zr) + 29hr 42 UmlAKR cAJP (5x10 -0
N
N
v
o
-shy-shy+
-shy
+
bull-
DISCU3SION
Salicylic acid ruld to a lesser extent aspirin cause repression
of f3-gclactosidase srnthesis in scherichia coli This effect is not
due to an inhibition of inducer uptake nor is it due ~o interference
l-rith inducers inactivation of lac repressor since strnins constitutive
for 3-gJactosidase 3J1lthesis are also repressed b~r sclicyli~ acid
In addition because dinitrophenol is liithout effect on differential
fl-galactosidase synthesis it appecrs salicrlates do not repress enzyme
s~mthesis by inhibiting oxidative phospllorylation
At these concentr2tions however salicylic acid does decrease
growth rate and direct or indirect effects of this on the observed
repression of enzyme synt~esis still ra~ain Since the differential
rate of enzyme synthesis i~ decreased and also since 25 x 10-411 DN
decreases grotrth rate aproximately the same degree middotuitl1cut having I
effects on differential synthesis salicylates inhibition of growth
is not the sole factor responsible for its effects on enZJcre synthesis
-11en 5 x 10-31 cyclic JUfP 1-ms added to cells treated Kith 25 x 10-3
and 5 x lO-3H srJicylic acid repression of differential enzye s~1lhesis
1ES pextially relieved This sugests salicllic acid represses J3-gJ~ac-
tosidase srnthesis br directly 1011erinti intIacellular levels of cyclic AMP
or by indirectly acting on asita which in turn releases cANP from the
cell Since cMP only partially relieves repression caused by wlicylic
acid and because there appears to be an inverse relationship between
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
-
- Let us know how access to this document benefits you
- Recommended Citation
-
- tmp1379115374pdfCOlFc
-
20
of inducer uptake and of inducer-s derepression of lac mrrNA synthesis
The zrecl of 24-Di1ikophcnol ont30alactosidase Synthesis
Since salicylates are kno-Vffi to be uncouplers of oxidative phosshy
phorylation (5) it has been sJgccsted sOrle of their phaITlClcological
effects result from their uncoupling action Tis i-lould irply that
other uncouplers should preferentially inhibit ~-gilactosidase syntheshy
sis For this reason the effect of 2 4-dini trophenol (Dr~) on J-galacshy
tosidase synthesis Has observed Because DrIP is more effective in inhibishy
ting oxidative phosphorylation than salicylic acid and is consequently
more toxic a concentration of the drug i-laS found vhic uould inhibit
grOl-rth to ap~roximately the sa11e degree as 25 x 10-31gt1 salicylic acid
This was found to be 25 x 10-411 Figure 6 illustrates DNP does not
repress differential synthesis of j3-g1actosidase This implies that
althoueh DNP 1m-lers ATP levels there is no apparent effect of the drug
on lsvels of cAl-IP Also it appears salic-rlic acid is not exerting its
repression of enzyrre sJ~~hesis by inhibiting oxidative phosphorylation
21
FIGUllS 6
EFFECrl OF DJP ON DIFFif2rIlL ~GLCiO3D~S STImiddotrYliESIS
Jhen strain 3000 cells reacled log ph~se they tmiddotrere indlccd 1ith IPTG
AproxL~ately one hour later the culture was divide1 a~d tr~ferred
to flasks containing the indicated adlitions The cellsas in preVious
experirrents Tere sarlpled at various tLTIes and assayed for B-galactoshy
sidase activity Since 2h-dinitrophenol t-rag in solution its addition
resuted in dilution of cultures This dilution las equally conpensated
for in control flasks nle first points on the graph represent samples
taken innediately after sepration of the cuJture
GroYTth Rate Difierential inz Rate Flask A Control 75hr 22 L1JmlAKR
B Contro + cAll 64hr 50 EUmlAm (5y10- H) 4z
C DNP (25xlO- -I) 42hr 22 EOIraAKR
D DNP (25xl2~4zr) + 29hr 42 UmlAKR cAJP (5x10 -0
N
N
v
o
-shy-shy+
-shy
+
bull-
DISCU3SION
Salicylic acid ruld to a lesser extent aspirin cause repression
of f3-gclactosidase srnthesis in scherichia coli This effect is not
due to an inhibition of inducer uptake nor is it due ~o interference
l-rith inducers inactivation of lac repressor since strnins constitutive
for 3-gJactosidase 3J1lthesis are also repressed b~r sclicyli~ acid
In addition because dinitrophenol is liithout effect on differential
fl-galactosidase synthesis it appecrs salicrlates do not repress enzyme
s~mthesis by inhibiting oxidative phospllorylation
At these concentr2tions however salicylic acid does decrease
growth rate and direct or indirect effects of this on the observed
repression of enzyme synt~esis still ra~ain Since the differential
rate of enzyme synthesis i~ decreased and also since 25 x 10-411 DN
decreases grotrth rate aproximately the same degree middotuitl1cut having I
effects on differential synthesis salicylates inhibition of growth
is not the sole factor responsible for its effects on enZJcre synthesis
-11en 5 x 10-31 cyclic JUfP 1-ms added to cells treated Kith 25 x 10-3
and 5 x lO-3H srJicylic acid repression of differential enzye s~1lhesis
1ES pextially relieved This sugests salicllic acid represses J3-gJ~ac-
tosidase srnthesis br directly 1011erinti intIacellular levels of cyclic AMP
or by indirectly acting on asita which in turn releases cANP from the
cell Since cMP only partially relieves repression caused by wlicylic
acid and because there appears to be an inverse relationship between
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
-
- Let us know how access to this document benefits you
- Recommended Citation
-
- tmp1379115374pdfCOlFc
-
21
FIGUllS 6
EFFECrl OF DJP ON DIFFif2rIlL ~GLCiO3D~S STImiddotrYliESIS
Jhen strain 3000 cells reacled log ph~se they tmiddotrere indlccd 1ith IPTG
AproxL~ately one hour later the culture was divide1 a~d tr~ferred
to flasks containing the indicated adlitions The cellsas in preVious
experirrents Tere sarlpled at various tLTIes and assayed for B-galactoshy
sidase activity Since 2h-dinitrophenol t-rag in solution its addition
resuted in dilution of cultures This dilution las equally conpensated
for in control flasks nle first points on the graph represent samples
taken innediately after sepration of the cuJture
GroYTth Rate Difierential inz Rate Flask A Control 75hr 22 L1JmlAKR
B Contro + cAll 64hr 50 EUmlAm (5y10- H) 4z
C DNP (25xlO- -I) 42hr 22 EOIraAKR
D DNP (25xl2~4zr) + 29hr 42 UmlAKR cAJP (5x10 -0
N
N
v
o
-shy-shy+
-shy
+
bull-
DISCU3SION
Salicylic acid ruld to a lesser extent aspirin cause repression
of f3-gclactosidase srnthesis in scherichia coli This effect is not
due to an inhibition of inducer uptake nor is it due ~o interference
l-rith inducers inactivation of lac repressor since strnins constitutive
for 3-gJactosidase 3J1lthesis are also repressed b~r sclicyli~ acid
In addition because dinitrophenol is liithout effect on differential
fl-galactosidase synthesis it appecrs salicrlates do not repress enzyme
s~mthesis by inhibiting oxidative phospllorylation
At these concentr2tions however salicylic acid does decrease
growth rate and direct or indirect effects of this on the observed
repression of enzyme synt~esis still ra~ain Since the differential
rate of enzyme synthesis i~ decreased and also since 25 x 10-411 DN
decreases grotrth rate aproximately the same degree middotuitl1cut having I
effects on differential synthesis salicylates inhibition of growth
is not the sole factor responsible for its effects on enZJcre synthesis
-11en 5 x 10-31 cyclic JUfP 1-ms added to cells treated Kith 25 x 10-3
and 5 x lO-3H srJicylic acid repression of differential enzye s~1lhesis
1ES pextially relieved This sugests salicllic acid represses J3-gJ~ac-
tosidase srnthesis br directly 1011erinti intIacellular levels of cyclic AMP
or by indirectly acting on asita which in turn releases cANP from the
cell Since cMP only partially relieves repression caused by wlicylic
acid and because there appears to be an inverse relationship between
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
-
- Let us know how access to this document benefits you
- Recommended Citation
-
- tmp1379115374pdfCOlFc
-
N
N
v
o
-shy-shy+
-shy
+
bull-
DISCU3SION
Salicylic acid ruld to a lesser extent aspirin cause repression
of f3-gclactosidase srnthesis in scherichia coli This effect is not
due to an inhibition of inducer uptake nor is it due ~o interference
l-rith inducers inactivation of lac repressor since strnins constitutive
for 3-gJactosidase 3J1lthesis are also repressed b~r sclicyli~ acid
In addition because dinitrophenol is liithout effect on differential
fl-galactosidase synthesis it appecrs salicrlates do not repress enzyme
s~mthesis by inhibiting oxidative phospllorylation
At these concentr2tions however salicylic acid does decrease
growth rate and direct or indirect effects of this on the observed
repression of enzyme synt~esis still ra~ain Since the differential
rate of enzyme synthesis i~ decreased and also since 25 x 10-411 DN
decreases grotrth rate aproximately the same degree middotuitl1cut having I
effects on differential synthesis salicylates inhibition of growth
is not the sole factor responsible for its effects on enZJcre synthesis
-11en 5 x 10-31 cyclic JUfP 1-ms added to cells treated Kith 25 x 10-3
and 5 x lO-3H srJicylic acid repression of differential enzye s~1lhesis
1ES pextially relieved This sugests salicllic acid represses J3-gJ~ac-
tosidase srnthesis br directly 1011erinti intIacellular levels of cyclic AMP
or by indirectly acting on asita which in turn releases cANP from the
cell Since cMP only partially relieves repression caused by wlicylic
acid and because there appears to be an inverse relationship between
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
-
- Let us know how access to this document benefits you
- Recommended Citation
-
- tmp1379115374pdfCOlFc
-
DISCU3SION
Salicylic acid ruld to a lesser extent aspirin cause repression
of f3-gclactosidase srnthesis in scherichia coli This effect is not
due to an inhibition of inducer uptake nor is it due ~o interference
l-rith inducers inactivation of lac repressor since strnins constitutive
for 3-gJactosidase 3J1lthesis are also repressed b~r sclicyli~ acid
In addition because dinitrophenol is liithout effect on differential
fl-galactosidase synthesis it appecrs salicrlates do not repress enzyme
s~mthesis by inhibiting oxidative phospllorylation
At these concentr2tions however salicylic acid does decrease
growth rate and direct or indirect effects of this on the observed
repression of enzyme synt~esis still ra~ain Since the differential
rate of enzyme synthesis i~ decreased and also since 25 x 10-411 DN
decreases grotrth rate aproximately the same degree middotuitl1cut having I
effects on differential synthesis salicylates inhibition of growth
is not the sole factor responsible for its effects on enZJcre synthesis
-11en 5 x 10-31 cyclic JUfP 1-ms added to cells treated Kith 25 x 10-3
and 5 x lO-3H srJicylic acid repression of differential enzye s~1lhesis
1ES pextially relieved This sugests salicllic acid represses J3-gJ~ac-
tosidase srnthesis br directly 1011erinti intIacellular levels of cyclic AMP
or by indirectly acting on asita which in turn releases cANP from the
cell Since cMP only partially relieves repression caused by wlicylic
acid and because there appears to be an inverse relationship between
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
-
- Let us know how access to this document benefits you
- Recommended Citation
-
- tmp1379115374pdfCOlFc
-
24
the concentration of salicylic acid and the effectiveness in l~ch
cAlfi) relieves its repression it is possible cHP and salicylic acid
may indirectly compete for their actions on l3-galactosidase synthesis
Thele are several possible 5ites where salicylic acid could intershy
fere with cAPs mechaniSI of action One nould be at the level of
cyclic M1P receptor protein Since interference here u~ually implies
a type at lt~losteric interaction in this case betHeen s~licrlic acid
verses cABP for the protein it is not likely to occur Another pesshy
sible site of actien weuld include salicylic acids effect at the me~brane
level Since beth compounds are knewn to influence ion new in varshy
ious cells there is a chance this m~ in turn cause a number of
effects However because ion fiOrl systems in the bacterial cell have
not been clarified it is difficult to predict direct or indirect intershy
actions batlreen salicylic acid and cAllP at this level In addition
because cAm) is lOlonll to activate many enzymes catalyzing de nevo
s)l1thesis (12)(13)(26) salicylic acid may exert its efpoundects by intershybull
fering rdth this step
These rcresent sites salicylates may act upon to e~ther release
cAHP from the cell inhibit adenyl cyclase activity stimulate phosphoshy
diesterase activity or interfere with activation of CRP Ul would
interfere lrith cAlIF fS action 11thin the cell end rlOuld consequently
serve to e~~lain salicylic acidfs repression of fl-gilactesidase synshy
thesis
Ineoncl1lsion the repression of ~galsctosidase synthesis by
salicylic acid in E coli appears to be par~ially relieved by cM1P
This raises the possibility salicylatea ray act by decreasing intrashy
cellular cAHP activities This tmiddotrould occur if the tilO competed
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
-
- Let us know how access to this document benefits you
- Recommended Citation
-
- tmp1379115374pdfCOlFc
-
25
for a con~on ion or reaction site or if salicylic acid stL~ulated the
release of cJmiddotrP fror the cell
Thene studies imply saliclyates rray a~3o have a direct effect on
lOl-rering intracellula levels of clHP in eukaryotic cells This
lould help to explain spirins diverse and cOlplex effects liithin the
body
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
-
- Let us know how access to this document benefits you
- Recommended Citation
-
- tmp1379115374pdfCOlFc
-
BIBLIOGfAPHY
middot1 L Good~an A Gilman 1970 The Pharmaco1oFical Basis of ThershyBeutics 4th Edition Hacmi11m Co 314-329
2 J Vane 1971 NltUle ne~middot~ Bio~ ~ 232
3 A Halnl~ond 1971 Science 174 48
4 J S~th A Hillis 1971 Nature NeH BioI 231 235
5 J lJiyahara R i(ar1er 1965Biochem J 97 197
6 I Pastan R Perlllan 1970 Science 169 339
1 H Shio P Ramt1e11 1972 llature lIe-1 BioI -2sect45
8 A Pardee F Jacob J Monod 1959 J Ho1 BioI 1 16$
9 M Abesnik C Levinthal 1970 Cold Spring Harbor Symp Quant BioI 35 456
10 H Shio J Shaw P Rrurrwell 1971 Ann NY Acad Sci 185 327
11 J 3xton S Lens R Ho G Robinson C Park ~ 85
12 L Garren G Gill G Walton ~ 210
13 J Durlont C tV111ems J Van Sande P Heve ~ 291
14 S Ferreira S Honcada J Vane 1971 1ature ~eT1 Biol 231 237
15 I Pastan R PerJ-nan 1968 J 3iol Chern 2h3 5420
16 H Levitan J Bcrl=0r 1972 Science 176 1423
17 R LeYin~ D iIilson 1971 Ann NY Acad Sci 185 363
18 F Hare S Jarltl ibid 351
19 C ~alson o Gigtvo1d R Doerge 1966 Textbook of Orcanic I-Iedishycinal and Plarnaceutical Chernistr-y 5~h ~i tion Lippincott Co 073-584
20 R Lcfkmdtz J Roth I Pastan 1971 AnnNY Acad Sci 185 195
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
-
- Let us know how access to this document benefits you
- Recommended Citation
-
- tmp1379115374pdfCOlFc
-
27
21 R Perlman I Pastan 1968 Biochem B1ophy Res Comr 30 656
22 R Perlman I Pastan 1968 Froc nat _cad Sci 61 1336
23 I Pa3tan R PrlzlIall 1969 N3tu-e ~ 811
24 H Sahud P Aggier 1969 NeH -ng J Eed 280 453
25 D Schnfer 1 V lst J Pols0n J Hedtke 3 3irca_ A Thakur H Goldberg 1971 Al1Il NY ~cad 3ci 85= 376
26 C Vil1ar-P~asi L Shen LG Shen ~ 74
~T ) 0 Co de d J I~ p tl land E He-r1Tlatt ibid 129
27 bull J bull Beavo h Llogers bull r JLor bull o~r bull r ltulan ~ 0U lershy
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
-
- Let us know how access to this document benefits you
- Recommended Citation
-
- tmp1379115374pdfCOlFc
-
---
APPENDIX
AB32hlVIArrONS
PG1 Prostaglamiin 1 C20H3hOSshy
PG~ Prostaglcndin pound2 C20H3205shy
PGF2 Prostaglandin F2~ PGS lI1th a reduced keto flOUP iOrring an2
~hydroxy
cAMP Cyclic adenosine 35 r -lilonophosphate
ATP Adenos~ne Si-t~phosphate
ADP Adenosine 5-diphosphate
Lcm Adrenocorticotropic hormone
TSH Tt~oid stimulating hormone
IPrG Isvpropyl ~-D-thiogalactopyranoside
Dlul Nessenzer riboneucl()ic acid
D~~ 24-Dinitrophenol
C~ ~Jclic 1~~ receptor protein
--_--- ---- -~~~---
- Repression of β-galactosidase synthesis in Escherichia coli by salicylates
-
- Let us know how access to this document benefits you
- Recommended Citation
-
- tmp1379115374pdfCOlFc
-