Gen. Pharmac. Vol. 12. pp. 439 to 444. 1981 0306-3623/81/060439-06102.00/0 Printed in Great Britain. All rights reserved Copyright © 1981 Pergamon Press Ltd

ALIPHATIC ALCOHOLS AND THE INHIBITION BY THIOL CHELATING AGENTS OF THE BINDING OF

I-3H]DIHYDROALPRENOLOL TO MYOCARDIAL fl-ADRENOCEPTORS

ESAM MOUSTAFA and MERVYN WONG Department of Biochemistry, University of Auckland, New Zealand

(Received 26 March 1981)

Abstract-- l . Binding of [3H]dihydroalprenolol to ovine or rat myocardial fl-adrenoceptors was inhibited by parahydroxymercuribenzoate and mersalyl to a greater degree than by N-ethylmaleimide.

2. The inhibition by the thiol chelating agents was enhanced by low concentrations of aliphatic alcohols in the order: n-butanol > n-propanol > ethanol > methanol. Tert-butanol caused 39To of the effect given by n-butanol at equimolar concentration.

3. The increased inhibition caused by n-butanol was concentration dependent with a maximum at about 0.2 M.

4. Membranes which have been preincubated with n-butanol followed by washing required further addition of butanol in order to observe the enhanced inhibition of the binding by thiol chelating agents.

5. The data are discussed in relation to the hydrophobic environment in the membrane protein in which thiol groups required for the binding of the antogonist to its receptors may be situated.

I N T R O D U C T I O N

Binding of fl-adrenergic agents to fl-receptors in various tissues is inhibited by thiol chelating agents, (Guellaen & Hanoune, 1979; Stadel & Lefkowitz, 1979; Vauquelin & Maguire, 1980). Thiol groups in some soluble proteins such as human haemoglobin (Cecil & Thomas, 1965) and fumarase (Robinson et al., 1967) are situated in a hydrophobic region of the molecule. This has been demonstrated by the differ- ence in reactivity with thiol chelating agents of vary- ing chemical nature and by the enhancement of the reactivity of the thiol groups by low concentrations of aliphatic alcohols known to weaken the hydrophobic interactions in the protein molecule (Schrier & Scher- aga, 1962; von Hippel & Wong, 1965). No infor- mation is available concerning the degree of reactivity of different thiol chelating agents with the thiol groups required for the fl-adrenoceptor binding in relation to the environment within the membrane protein in which the sulphydryl groups are situated.

The work presented here shows that thiol groups required for the binding of I-3H]dihydroalprenolol to myocardial fl-adrenoceptors are more reactive with parahydroxymercuribenzoate (PHMB) and o-[(3-hy- droxymercuri-2-methoxypropyl)-carbamoyl] phenoxy- acetate (mersalyl) than with N-ethylmaleimide (NEM). The present work also examines the effect of aliphatic alcohols on the inhibition by thiol chelating agents of the binding of [3H]dihydroalprenolol to myocardial fl-adrenoceptors.

MATERIALS AND METHODS

Chemicals

[3H]Dihydroalprenolol (46Ci/mmol) was supplied by New England Nuclear Co. All other chemicals were of analytical grade and were purchased from Sigma Co.,

439

except Tris which was obtained from British Drug Houses. Tris buffers were adjusted to the appropriate pH by addition of HCI at either 2-4 or 37 °.

Preparation of membranes from ovine and rat heart

Immediately after slaughter, hearts from sheep (about 1 yr old) were collected in ice cold Tris-sucrose, MgCI 2 buffer (50mM Tris HCI, pH7.5; 0.25 M sucrose; 1 mM MgCI2). The procedure used to prepare ventricular mem- branes was modified slightly from that described by Ciaraldi & Marinetti (1978). The entire operation was carried out at 2-4~C using ice-cold reagents. Ventricular tissue (6 gram aliquots) was minced and homogenised in Tris-sucrose buffer using a polytron homogeniser (3 x 3 sec bursts at setting 6). The homogenate was centrifuged at 700 9 for 12 min and the supernatant was centrifuged at 8000g for 12 rain. The supernatant was then centrifuged at 30,000@ for 15rain and the pellet was washed once in 75mM Tris-HCl buffer containing 25 mM MgCI 2, pH 7.5 and resuspended in the same buffer (8-12 mg protein/ml). The membrane preparation was snap frozen in liquid nitrogen and stored under the same conditions. No significant loss of specific binding of [3HI dihydroalprenolol to fl-adreno- ceptors was observed for periods up to 3 months.

The same method was used to prepare membranes from ventricles excised from hearts removed from rats after mild ether anaesthesia.

Determination of [3H]dihydroalprenolol binding to heart membranes

The reaction mixture (total volume 150/A) contained Tris-HC1 buffer (ll.25/tmoles, pH7.5), MgCI 2 (3.75 /~motes) and membrane preparation (50/.d, 13 l-187 pg pro- tein). After preincubation for 0.5 min at 37 °, [aH]dihydro- alprenolol (l.97-2.15pmoles) alone or with 1.5nmoles (-)propranoiol was added and the mixture incubated for 4 min at 37 °. The reaction was stopped by the addition of 5 ml of ice-cold Tris-HCl buffer (75 raM, pH 7.5) contain- ing 25 mM MgCl2 (referred to hereafter as Tris-Mg 2+ buffer) and the solution was immediately filtered under reduced pressure through a 24 mm glass microfibre filter (Whatman, GF/C). The filter was rapidly washed twice

440 ESAM MOUSTAFA and MERVYN WONG

with 5 ml aliquots of ice-cold Tris-Mg 2 ÷ buffer. Filtration and washing were done within 15 sec. The filter was trans- ferred to a scintillation vial, dried at 37C and counted at an efficiency of 30% in 6 ml of scintillation fluid (0.5,°/, PPO and 0.01",, POPOP in a mixture of triton X 100 and toluoL 1:2 by vol.). Nonspecific binding was defined as the amount of [3H]dihydroalprenolol bound to the mem- branes when the incubation was done in the presence of propranolol. The level of nonspecific binding was not sig- nificantly affected by (-)propranolol at concentrations between 5 and 10/~M. Maximum specific binding was achieved within 4 min.

Determination of paratlydroxymercuribenzoate in .stock sol- utions

Concentrations of PHMB in stock solutions were deter- mined according to the method described by Suzuki et al. ( 1 9 7 9 ) .

Protein determination Proteins were measured by the method described by

Markwell et al. (1978).

RESULTS

Binding of [3 H]dihydroalprenolol to oeine myocardial fl-adrenoceptors

The amount of [3H]dihydroalprenolol bound specifically to fl-adrenoceptors was 78-82% of the total binding. The specific binding fulfilled the major criteria established for fl-adrenoceptors (Williams & Lefkowitz, 1978) i.e., it was saturable, association and dissociation were rapid, the rate of displacement of dihydroalprenolol by L- was larger than by D-isomers of fl-adrenergic agents, and the displacement by several fl-agonists followed the pattern characteristic to fl-adrenoceptors (results are not shown).

Inhibition by thiol chelating agents of the binding of [3 H]dihydroalprenolol to myocardial fl-adrenoceptors

PHMB, N E M and mersalyl when present during the 4 minute incubation of the membranes with [3H]dihydroalprenolol, inhibited the specific binding of the fl-antagonist to its receptors (Fig. l) but had no significant effect on nonspecific binding (results not shown). It will be seen that 1 mM P H M B caused almost complete inhibition whereas 1 mM mersalyl caused about 59% inhibition and N E M at the same concentration caused only about 28%. Any increase of N E M concentration above 1 mM resulted in no further increase in the inhibition of the binding of [3H]dihydroalprenolol to the fl-adrenoceptors when NEM was incubated with the membranes for 4 min (Fig. l). The effect of N E M was time dependent as shown by the results of experiments in which mem- branes were preincubated at 18 ° in Tris-Mg 2+ buffer, pH 7.5 without (control) or with N E M (20mM) for periods up to 6 hr. After the appropriate preincuba- tion time, [3H]dihydroalprenolol was added and the binding was determined. The results (Fig. 2) show that the inhibition by N E M of the binding, after an initial very rapid inhibition (about 26%), was a slow time- dependent process with 62% inhibition achieved 6 hr after addition of NEM to the reaction mixture.

Effect of aliphatic alcohols on inhibition by thiol chelat- ing1 agents of the binding of [3H]dihydroalprenolol to myocardial fl-adrenoceptors

Ovine myocardial membranes were preincubated for 60min at 18 ° in Tris-Mg 2+ buffer, pH 7.5 with or without N E M (20 mM) in the presence or absence of 0.2 M methanol, ethanol, n-propanol, n-butanol and tert-butanol. After preincubation, [3H]dihydroalpre- nolol was added and the binding of the fl-antagonist

300

el

E ;ZOO - - o - o

'°°1\ " • I 2

o

Fig. 1. Effect of PHMB, mersalyl and NEM on the binding of [3H]dihydroalprenolol to ovine myocar- dial fl-adrenoceptors. Reaction mixture (total volume 150/~1) contained membranes (157~g protein), Tris-HCl (11.25/~mole, pH 7.5), MgC12 (3,75/~mole), [3H]dihydroalprenolol (2.07 pmole) and varying concentrations of each of the thiol chelating agents. After incubation at 37C for 4 rain binding of the labelled antogonist to fl-adrenoceptors was determined as described under methods, SE bars are not

shown when they occur within the experimental points. • PHMB, A Mersalyl. O NEM.

Concentration (raM)

Aliphatic alcohols and the inhibition by thiol chelating agents 441

c

E 200

I O 0

I I I I I I I

I 2 3 4 5 6

Time ( H r )

Fig. 2. Time course of the inhibition of NEM of the bind- ing of [3H]dihydroalprenolol to ovine myocardial fl-adre- noceptors. For experimental conditions, see the text. SE bars are not shown when they occur within the experimen-

tal points. O Control, • +20 mM NEM.

was measured. The results presented in Table 1 show that aliphatic alcohols increased the degree of inhi- bition by N E M in the order n-butanol > n-propranol > ethanol > methanol. Tert-butanol had much less effect on the degree of inhibition than n-butanol (Table 1).

n-Butanol also increased the degree of inhibition by P H M B and mersalyl of the binding of the labelled antagonist to ovine or rat myocardial fl-adrenocep- tors. For this to be demonstrated lower concen- trations of the two thiol chelating agents had to be used (0.1 mM and 0.25mM respectively). It will be seen (Table 2) that the increase in the degree of inhi- bition by thiol reagents was observed whether ovine or rat heart membranes were used.

Table 1. Effect of aliphatic alcohols on the inhibition by NEM of the binding of [3H]dihydroalprenolol to ovine

myocardial fl-adrenoceptors

Treatment ~ Inhibition*

NEM (20 mM) 20 mM NEM + methanol (0.2 M) 20 mM NEM + ethanol (0.2 M) 20 mM NEM + n-propanol (0.2 M) 20 mM NEM + n-butanol (0.2 M) 20 mM NEM + tert-butanol (0.2 M)

11.8 ___ 1.7 15.7 + 1.9 18.3 +_ 2.0 21.5 -4- 2.1 71.0 + 4.6 28.0 _+ 2.9

* Corrected for the effect of alcohol alone. Results of two experiments, each of which was run in triplicate.

Membranes (180 #g protein) were preincubated for 60 min at 18 ~ in Tris-Mg ÷ buffer, pH 7.5 with no additions (con- trol), with NEM (20mM), with the appropriate alcohol (0.2 M) or with NEM (20 raM) plus the appropriate alcohol (0.2 M). After preincubation [3H]dihydroalprenolol was added (final concentration 11.6 nM). The reaction mixture was further incubated at 37 ~ for 4 rain and the binding of the labelled antagonist was measured as described under methods.

Table 2. Effect of n-butanol on the inhibition by three thiol chelating agents of the binding of [3H]dihydroalprenolol

to rat and ovine myocardial fl-adrenoceptors

Treatment 0% Inhibition*

Ovine heart membranes n-Butanol (0.17 M1 8.1 + 0.9 NEM (20mM) 25.2 _+ 2.1 NEM (20 mM) + n-butanol (0.17 M) .63.5 + 6.8 PHMB (0.I mMl 27.1 + 2.3 PHMB (0.1 raM) + n-butanol (0.17 M) 60.7 + 5.1 Mersalyl (0.25 mM) 65.2 + 6.1 Mersalyl (0.25 mMl + n-butanol (0.17 M) 85.9 ___ 8.6

Rat heart membranes n-Butanol (0.17 M) 10.0 _+ l.l NEM (20 mMl 2.2 + 0.3 NEM (20 mM) + n-butanol (0.17 Mt 30.8 + 2.4 PHMB (0.1 mM) 12.1 _+ 1.1 PHMB (0.1 mM) + n-butanol (0.17 M) 33.8 _+ 2.5 Mersalyl (0.25 mM) 40.2 + 4.3 Mersalyl (0.25 mM) + n-butanol (0.17 M) 70.7 _+ 8.1

* Results of two experiments each of which was run in triplicates.

Rat or ovine myocardial membranes (202-220~ug pro- tein) in a total volume of 150gl were preincubated for 60 min at 18 ° in Tris-Mg 2+ buffer, pH 7.5 containing:

(a) n-butanol alone (0.17 MI; (b) the appropriate thiol chelating agent alone; (c) n-butanol (0.17 M) + the appropriate thiol chelating

agent. After preincubation [~H]dihydroalprenolol was added and the reaction mixture was further incu- bated for 4 min at 37 and the binding of the labelled antagonist to fl-adrenoceptors was determined as de- scribed under methods.

Effect of n-butanol concentration

The increase in the degree of inhibition by N E M of the binding of [3H]dihydroalprenolol to myocardial fl-adrenoceptors is dependent on the concentration of butanol (Fig. 3). The inhibition of the binding by butanol alone is relatively low at concentrations lower than 0.19 M above which a sharp increase in the inhibition occurred.

Effect of n-butanol on the time course of inhibition by NEM of the binding of [3H]dihydroalprenolol to fl-adr enoc e p tor s

Membranes were preincubated at 18 ~ for various periods of time in the presence of (1) buffer alone, (2) 0.16M butanol in buffer, (3) 2 0 m M NEM in buffer, and (4) n-butanol 10.16 M) plus N E M (20 mM). After the appropriate preincubation period, [~H]dihydro- alprenolol was added and the binding of the labelled antagonist to myocardial fl-adrenoceptors was measured. The results in Fig. 4 indicate that n-butanol increased the rate of inhibition by N E M of the bind- ing of [3H]dihydroalprenolol to fl-adrenoceptors. After 60 min preincubation with N E M plus n-butanol, binding of the labelled antagonist was 57°/, of the control corrected for the inhibition by N E M and n-butanol individually.

Reversibility of the action of n-butanol

Membranes (4.1 mg protein/ml) in a total volume of 2ml were preincubated for 60rain at 18 ° in the

442 ESAM MOUSTAFA and MERVYN WONG

50O

q

I 0 0

I ! oi, 02 0'.'3

E N- Butanol ] ( M )

Fig. 3. Effect of n-butanol concentration on the inhibition by NEM of the binding of [3H]dihydroalprenoiol to ovine fl-adrenoceptors. Experimental conditions are described in the text. SE bars are not shown when they occur within the experimental points. O n-butanol, • n-butanol + NEM.

absence (control) and presence of 0.16 M n-butanol. The preincubation was terminated by the addition of 5 ml of ice-cold Tris-Mg 2+ buffer, pH 7.5. The mix- ture was immediately centrifuged at 30,000g for 15 min and the sedimented membranes were washed twice by resuspension in 5 ml of ice-cold Tris-Mg 2+ buffer. The washed membranes were resuspended in ice-cold Tris-Mg 2+ buffer to the original protein con- centration. The butanol-pretreated membranes, in 150td reaction mixtures were again preincubated for

60 min at 18 ° with or without 20 mM NEM. [aH]Di- hydroalprenolol was then added and the binding of the antagonist was determined. The results in Fig. 5 show that the binding of the antagonist to/~-adreno- ceptors was not significantly affected by the preincu- bation of membranes with n-butanol followed by washing. NEM caused about the same degree of inhi- bition with either control membranes or membranes which have been preincubated with n-butanol fol- lowed by washing. It will be also seen that further addition of n-butanol was required to membranes which had been pretreated with butanol followed by washing, in order to demonstrate the enhancement of inhibition by thiol chelating agents in the presence of the alcohol.

DISCUSSION

The reactivity of thiol groups in proteins with che- lating agents varies according to the environment in the protein molecule in which the thiol groups are situated, and the chemical nature of the thiol chelat- ing agents (Liu, 1977). The higher degree of reactivity of thiol groups of fumarase with mercurials than with NEM was interpreted by Robinson et al. (1967) as due to the hydrophobic nature of mercury which makes it more capable of interfering with the inter- actions in the hydrophobic regions which hinder the reaction of thiol groups with the chelating agents. The results reported here show that the two mercury com- pounds PHMB and mersalyl caused higher rate of inhibition of the binding of myocardial fl-adrenocep- tars to I-3H]dihydroalprenolol than that given by NEM which binds thiol groups across a double bond. If the information relating to thiol groups in soluble proteins such as fumarase can be extrapolated to membrane sulphydryl groups, the data presented here suggest that the thiol groups necessary for the binding

300

-/

20G

I 0 0

I I I 0 I I I I I H 2 0 30 6 0 9 0

T l m t ( rain )

Fig. 4. Effect of n-butanol on the time course of inhibition by NEM of the binding of [3H]dihydro- alprenolol to ovine myocardial fl-adrenoceptors. Experimental conditions are described in the text. SE bars are now shown when they occur within the experimental points. ~ +n-butanol (0.17 M), U3 [] + NEM (20 mM), A - A + n-butanol (0.17 M) + NEM (20 mM), O O Summation of indi-

vidual effect of 0.17 M n-butanol and 20 mM NEM.

Aliphatic alcohols and the inhibition by thiol chelating agents 443

._=

OD E

o E

v

"r r~

2OO

I00

Control

\ ~ J

\ \ J

"% "N !

\ N J

N - Butanol Pmtreated

I

O IM o '" o 2 + " ' z z o ~÷,., z z g ~ . a ~ z ®

Fig. 5. Effect of pretreatment of ovine myocardial membrane with n-butanol followed by washing on the inhibition of NEM of the binding of [aH]dihydroalprenolol to ovine myocardial fl-adrenocoptors.

Experimental conditions are described in the text.

of the labelled antagonist to fl-adrenoceptors are close to the hydrophobic region of the membrane protein.

The results in the present work show that the degree of inhibition by NEM is increased by aliphatic alcohols in the order n-butanol > n-propranol > eth- anol > methanol. The longer the carbon chain the more hydrophobic the alcohol becomes. The increase in hydrophobicity exerts a greater effect on the inter- actions which hinder the reaction between thiol groups and thiol chelating agents (Schrier & Scher- aga, 1962). This may be further evidence that thiol groups necessary for the binding of myocardial fl-adrenoceptors to the antagonist are in a hydro- phobic region of the membrane protein. It is interest- ing to note that tert-butanol which has the same mol- ecular weight as n-butanol had much less effect on the degree of inhibition by NEM of the binding of [3H]dihydroalprenolol to myocardial fl-adrenocep- tars. This may be explained by the fact that n-butanol is sparingly water soluble (2.ng/100ml at 25 :) whereas tert-butanol is completely miscible with water, making it less effective in disrupting hydro- phobic interactions in the membrane protein. Simi- larly, the presence of thioi groups required for the action of yeast alcohol dehydrogenase (Heitz et al. 1968) and D-amino acid oxidase (Fonda & Anderson, 1969) was suggested because the inactivation of the two enzymes by N-alkylmaleimide increased as the chainlength of the N-alkylmaleimide increased.

The effect of n-butanol on the inhibition was seen to be reversible. This indicates that the alcohol does not cause permanent change in the structure of the membrane protein.

Using the sensitivity towards NEM as a structural probe, Bottari et al. (1979), Vauquelin & Maguire (1980) have recently demonstated that fl-adrenergic agonists cause a rapid and reversible change of the fl-adrenoceptors in membranes of turkey erythrocytes and of S 49 lymphoma cells which increased the sensi-

tivity of the receptors to inactivation by NEM. The results presented here show an increased sensitivity of myocardial fl-adrenoceptors to NEM caused by ali- phatic alcohol similar to that in other systems induced by fl-agonists.

Acknowledgement--This work was supported by a grant from the Auckland Medical Research Foundation. We wish to thank Dr R. Hughes for reading the manuscript.

REVERENCES

BOTTARI S. G. et al. (1979) The fl-adrenergic receptor of turkey erythrocyte membrane: Conformational modifi- cation by fl-adrenergic agonists. Biochem. Biophys. Res. Commun. 86, 1331-1338.

CEOL R. & THOMAS M. A. W. (1965) Nature of the unreac- tive sulphydryl groups in human haemoglobin. Nature 206, 1317-1320.

OARALDI T. P. & MARINETTI G. V. (19781 Hormone action at the membrane level. VII1 Adrenergic receptors in rat heart and adipocytes and the modulation by thyroxine. Biochim. biophys. Acta. 541, 334--346.

FONDA M. L. & ANDERSON B. M. (1969) D-Amino acid oxidase. IV Inactivation by maleimides. J. Biol. Chem. 244, 666--674.

GUELLAEN G. & HANOUNE J. (1979) Thiol reactivity and molecular individuality of :t and fl-adrenoreceptors in rat liver plasma membranes. Biochim. biophy,s. Acta. 587, 618-627.

HEITZ J. R., ANDERSON C. D. & ANDERSON B. M. (1968} Inactivation of yeast alcohol dehydrogenase by N-alkyl- maleimides. Archs Biochem. Biophy.s. 127, 627-636.

LIU T-Y. (1977) The role of sulphur in proteins. In The Proteins (Edited by NEURATH H. & HILL R. L.) 3rd edn, Vol. 3, pp. 362-365. Academic Press, New York.

MARKWELL M. A. K. et al. (1978) A modification of the Lowry Procedure to simplify protein determination in membrane and lipoprotein samples. Analyt Bioehem. 87, 206-210.

ROnXNSON G. W. et al. (1967) The thiol groups of fumarase. J. biol. Chem. 242, 2709-2718.

444 ESAM MOUSTAFA and MERVYN WONG

SCHRIER E. E. & SCHERAGA H. A. (1962) The effect of aqueous alcohol solutions on the thermal transition of ribonuclease. Biochim. biophys. Acta. 64, 406-408.

STADEL J. M. & LEFKOWITZ R. J. (1979) Multiple reactive sulphydryl groups modulate the function of adenylate cyclase coupled beta-adrenergic receptors. Molec. Phar- mac. 16, 709-718.

SuzuKI M., COOMBS T. L. & VALLEE B. L. (1969) Determi- nation of protein thiol groups by atomic absorption spectrometry. Anal. Biochem. 32, 106-117.

VAUQUELIN G. & MAGUIRE M. E. (1980) Inactivation of

fl-adrenergic receptors by N-ethylmaleimide in S 49 lym- phoma ceils. Agonist induction of functional receptor heterogeneity. Molec. Pharmacol. 18, 362-369.

VON HIPPEL P. H. & WONG K-Y. (1965) On the conforma- tional stability of globular proteins. The effects of various electrolytes and nonelectrolytes on the thermal ribonuclease transition. J. biol. Chem. 240, 3909- 3923.

WILLIAMS L. T. & LEFKOWITZ R. J. (1978) Receptor binding studies in adrenergic pharmacology, p. 43, Raven Press, New York.