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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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Recommended Citation Recommended Citation Olson Joan Carlyn Repression of β-galactosidase synthesis in Escherichia coli by salicylates (1972) Dissertations and Theses Paper 1125 httpsdoiorg1015760etd1125

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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

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

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

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

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

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

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

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

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

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

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)

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

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

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

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

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

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

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

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

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

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

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