Molecular and Cellular Endocrinology, 0 1992 Elsevier Scientific Publishers

MOLCEL

201

83 (1992) 20 l-209 Ireland, Ltd. 0303.7207/92/$05.00

02693

Osmolytes improve the reconstitution of luteinizing hormone/ chorionic gonadotropin receptors into proteoliposomes J. Kolena, Institute of Expc’rimmtal

K. MatejEikovi

Endocrinology,

(Received

Key words:

Luteinizing (Rat ovary)

hormone/human

chorionic

Slowk

and G. henkelovii of Scicv~ces. Bratisiul,a.

Acudetny

23 July 1991: accepted

gonadotropin

human

23 October

receptor;

Czechosloc~akiu

1991)

Reconstitution;

Adenylate

cyclase;

Spin label;

Lipid;

Summary Rat ovarian membrane luteinizing hormone/ human chorionic gonadotropin (LH/hCG) receptor was reconstituted into proteoliposomes. The ability of sodium cholate to extract and reconstitute hCG binding activity was dependent on the protein/detergent ratio. Trypsinization of the LH/hCG receptor containing proteoliposomes indicated that approximately 57% of hCG binding sites were oriented extravesicularly. The presence of 20% glycerol or other osmolytes during reconstitution increased the accessibility of LH/hCG receptors but not the activity of adenylate cyclase in proteoliposomes. This beneficial effect was independent of any specific detergent or its presence during detergent solubilization of proteins. Dynamic properties of membranes were monitored by electron spin resonance of 16-, 12-, and 5-doxyl stearic acid. Reconstituted proteoliposomes contain less ordered membrane lipids than do native membranes. Addition of glycerol before reconstitution increased the order of lipid bilayer and shifted it to the physical state of the native membrane. These findings are consistent with the hypothesis that a rise in membrane ordering increases the accessibility of membrane-bound LH/hCG receptors.

Introduction Biological membranes are dynamic structures containing a variety of components capable of influencing each other. It is generally believed that membrane structure and molecular order and dynamics are essential for the maintenance of membrane function. Reconstituted membrane proteins in phospholipid liposomes provide a suit-

Correspondence to: Dr. Jaroslav Kolena, Institute of Experimental Endocrinology, Slovak Academy of Sciences, Vlarska 3, 833 06 Bratislava, Czechoslovakia.

able model for study of several aspects of the structure and function of naturally occurring membranes. Recently this laboratory (Kolena et al., 1989) reported successful reconstitution of luteinizing hormone/ human chorionic gonadotropin (LH/hCG) receptors and adenylate cyclase system from sodium cholate-solubilized rat ovarian membranes. Proteoliposomes were formed after removal of the detergent by absorption onto Bio-Beads SM-2 resin. The functional interaction between receptor and effector was restored. Adenylate cyclase was stimulated by hCG. As an extension to previous reconstitution protocols we will show the effect of detergent concen-

202

tration on insertion of binding activity into proteoliposomes and the orientation of the receptor within the bilayer. Recently, Maloney and Ambudkar (1989) described protein stabilizing agents osmolytes (glycerol, sucrose, glucose, glycine, etc.) - which preserve activity during detergent solubilization and reconstitution of membrane protein. Recovery activity of proton- and calcium-motive ATPases from S. Iuctis and rat liver membrane-bound glucose-6-phosphatase in proteoliposomes was feasible only if glycerol was present during protcin solubilization. The favorable effect of glycerol on the preservation of biological activity of the LH/hCG receptor during purification and that of sucrose during homogenization and storage preparations of the adenylate cyclase system are well known (Birnbaumer et al., 1976; Metsikko et al., 1990). Data are presented here to document the beneficial effect of osmolytes on the spontaneous insertion of LH/hCG receptor into proteoliposomes with concomitant physical changes associated with the reconstitution of binding activity. Materials

and methods

Materials Purified hCG (CR 123; 12,780 U/mg> was generously supplied by NIAMDD (NIH, Bethesda, MD, USA). Na12”I was purchased from the Radiochemical Centre (Amersham, UK). Bio-Beads SM-2 were from Bio-Rad, and they were methanol-activated prior to use (Holloway, 1973). Creatine phosphate, creatine kinase, GTPr-S, trypsin (activity 11,680 BAEE units/mg protcin) and trypsin inhibitor were obtained from Boehringer-Mannheim. Pregnant mare’s serum gonadotropin (PMSG) and hCG (Praedyn) were from Spofa, Prague. Stearic acid spin probes with dimethyloxazolidinyl group (16 DSA, 12 DSA and 16 DSA), and all other chemicals were from Sigma. Methods Preparation of proteoliposomes. Crude membrane preparations were obtained from superovulated rat ovaries (Wistar strain, aged 26 days) 5 days after PMSG and hCG priming (Parlow,

1961). Homogenates of ovaries (100 mg/ml) in ice-cold buffer A (25 mM NaH,PO,, 1 mM EDTA, 40 mM NaCI, pH 7.4) containing 20% sucrose were filtered through six layers of surgical gauze, centrifuged at 1000 x g for 15 min, and the supernatant was further centrifuged at 20,000 Xg for 30 min (Kolena et al., 1986). Plasma membranes were prepared by the method of EkStrom and Hunzicker-Dunn (1989). The membrane preparations in buffer A were layered over a cushion of 45% (w/w) sucrose (9 ml membrane/16 ml cushion) and centrifuged at 60,000 X g for 60 min at 4°C. The interface of membranes on top of sucrose was sucked off, diluted 6-fold, and centrifuged at 20,000 X g for 30 min. To solubilized gonadotropin receptor approximately 10 mg membrane proteins were stirred with 0.5 ml of 20 mM sodium cholate in buffer A at 4°C for 60 min. The solution was then centrifuged at 20,000 x g for 30 min. The solubilized membrane protein was applied to a Bio-Beads SM-2 column (1 x 8 cm), previously equilibrated with buffer A. The same buffer was used for elution. After 5-fold dilution the turbid fraction containing proteoliposomes was centrifuged at 160,000 x g for 60 min (Kolena, 1989). Osmolytes were added to membranes before solubilization or before application of solubilized membranes to a resin column. hCG binding assay. Aliquots of 0.1 ml of ovarian membranes or proteoliposomes were incubated 16 h at 24°C with 0.1 ml buffer A + 1 mg ml ’ bovine serum albumin (BSA) with or without lOO-fold excess of unlabeled hCG and 0.1 ml [‘2sI]hCG (l-l.5 ng, spec. act. about 60 FCi pg.-‘). After incubation and ccntrifugation, the membrane pellets were washed twice with buffer A (Kolena, 1976). Hormone-receptor complex in proteoliposomes was precipitated twice with polyethylene glycol (Sebokova and Kolena, 1984). The results are expressed as specific binding per mg protein. Adenylate cyclase actkity. Adenylate cyclase activity was assayed at 30°C for 25 min (Hwang and Ryan, 1981; Kolena, 1989). The assay system contained 25 mM Tris-HCI (pH 7.4), 5 mM MgCl,, 1 mM EDTA, 0.3 mM 3-isobutylmethylxanthine, 1 mM ATP, 20 mM phosphocreatine, 100 U/ml creatine kinase, and 30-40 pg protein

203 of proteoliposomes in a final volume of 400 ~1. When present, GTP-y-S, NaF and hCG were at 0.1 mM, 10 mM and 0.1 pg/assay, respectively. The assay was terminated by heating to 100°C for 3 min, followed by addition of 0.1 ml ice-cold 30% trichloroacetic acid. After centrifugation at 1500 x g for 15 min. the supernatant was extracted 3 times with water-saturated diethyl ether, and lyophilized. The residue was dissolved in water and the CAMP content estimated by protein binding assay (Kolena et al., 1986). Electron spin resonance 03~) measurements. Samples for ESR measurements were prepared as follows: 10 pg of spin probe were mixed with 0.06 ml of membrane or proteoliposome preparation (3 mg protein). ESR spectra were recorded using an ESR-230 X-band spectrometer (ZWG AdW, Berlin, Germany) with a microwave power output of 4 mW, frequency 9.5 GHz, and a magnetic field 3300 GS (Kolena and OndriaS, 1984). Since the outer splitting in most of our spectra could not be measured due to a low signal to noise ratio, the value of A I was used as a parameter characterizing the spin label ordering. The order parameter decreased linearly with the and A.O.l (Gaffney, 1976). At S < 0.1 and A i > 1 .l mT the spin label motion was nearly isotropic and its rate of molecular reorientation could be described by rotational correlation time as calculated by Schreier et al. (1978). The membranes were labeled with an experimentally determined low probe/ membrane protein concentration to avoid spin-spin interaction effects on the hyperfine splittings. Other methods. Cholesterol was assayed enzymatically (Sate, 1984). Phospholipids were determined calorimetrically (as dipalmitoyl phosphatidylcholine) in a complex with ammonium ferrothiocyanate (Stewart, 1980). Protein was determined by the method of Lowry et al. (1951). Student’s t-test was used for statistical evaluation. Results Sodium cholate was used for solubilization of ovarian membranes and reconstitution of the LH/hCG receptor into proteoliposomes as this

~~~~~,

10 DETERGENT

30

20 CONCENTRATION

O

OL

Fig. 1. Solubilization

and reconstitution

activity into proteoliposomes, cholate

concentration

(left)

08

LLLx&x$

ImHi

plotted

of LH/hCG

as a function

and detergent

12 (5)

l6

binding of sodium

to protein

ratio

(right). Data are means + SE of three determinations.

detergent, having a high critical micelle concentration (14 mM), is almost completely removed from protein and lipids on a column of hydrophobic resin Bio-Beads SM-2. During this process, the LH/hCG receptor and adenylate cyclase are inserted into the newly formed bilayer (Kolena, 1989). Fig. 1 shows the recovery of LH/hCG binding activity as a function of sodium cholate concentration. The figure illustrates that recovered optimum activities are different for the two protein concentrations. At a higher protein concentration (6.9 mg ml-‘) an optimum detergent/ protein ratio is found at approx. 0.7. At a lower value of protein (4.6 mg ml- ‘) the optimum shifts towards a higher value of the detergent/protein ratio (0.9). The detergent/protein ratio 0.7 in the case of the initial protein concentration of 4.6 mg ml-’ leads to a cholate concentration which is not strongly solubilizing. On the other hand, the results suggest that the initial concentration of protein should be high (or detergent/protein ratio low) when labile protein is solubilized with maintenance of receptoric function. Since electrostatic interactions are a wellknown characteristic of intramolecular forces in membranes, a higher ionic strength of the buffer could contribute to dissociate complexes held together and increase solubilization of membranes. Rising of KC1 in the buffer from 0.1 to 0.5 M increased the binding activity in proteoliposomes; however, when related to protein, LH/hCG receptors were not changed (data not shown). LH/hCG receptor can be inserted into the bilayer or trapped inside of proteoliposomes.

204

Therefore, the orientation of the reconstituted LH/hCG receptor was tested. Fig. 2 shows that after treatment of proteoliposomes with a low sodium cholate concentration (0.005-0.025%) the amount of hCG binding is constant and reflects the binding sites oriented externally. Upon increasing the concentration of the detergent to 0.05%, hCG binding increased reflecting the exposition of binding sites oriented internally. Maximum binding of hCG was observed at 0.1% sodium cholate (P < 0.001 comparing to 0.025% and lower concentrations). At higher concentrations of the detergent hCG binding was inhibited. In further experiments LH/hCG receptors exposed to the surface were destroyed by trypsinization (Table 1). For this purpose proteoliposomes were incubated with increasing concentrations of trypsin, followed by incubation with trypsin inhibitor. The proteoliposomes were then separated by centrifugation, and hCG binding was determined in the presence or absence of 0.1% detergent. The results showed that 200 and 600 pg ml- ’ trypsin proteolyzed 22% and 57%, respectively, of the accessible LH/hCG receptors incorporated into proteoliposomes. Comparable results were obtained when trypsinization of proteoliposomes was carried out after exposure of intravesicular binding sites to 0.1% sodium

I

-

(1

I1111111

0.01 Sodium

0.1 cholate (%wlv~

10

Fig. 2. Orientation of hCG binding activity in proteoliposomes. Reconstituted vesicles were incubated with the indicated concentrations of sodium cholate and [‘2’I]hCG for lh h at 24°C. They were then assayed for specific binding activity as described under Methods. The values are means k SE of four determinations.

TABLE

1

EFFECT OF TRYPSIN LIPOSOMES

ON hCG BINDING

TO PROTEO-

Proteoliposomes were incubated with trypsin for 30 min at 25°C followed by addition of 3-fold amounts of trypsin inhibitor for 10 min. After centrifugation at lhf.I,flflflX g for 60 min, proteoliposomes were assayed for hCG binding in the presence or absence of 0.1% sodium cholate. Data are the meansf SE of four estimations (repeated twice). Treatment

Trypsin

[“‘l]hCG Trypsin Ttypsin + 0.1% sodium cholate

tpg/ml)

bound

201.6+

8.5

236.8 +

1I .4

(fmol mgg’)

156.6+4.1

164.6 f 3.6

87.0 * 2.x

101.5+3.8

cholate. However, the trypsin susceptibility of the LH/hCG receptor of native membrane preparations was considerably higher. Incubation of membranes with 200 pg of trypsin destroyed all hCG binding sites. A further series of experiments was performed to determine the stabilizing effect of osmolytes on the reconstitution of LH/hCG receptors into proteoliposomes. Addition of 20% glycerol, sucrose and glucose at the time of sodium cholate solubilization increased hCG binding to proteoliposomes by more than 60% (Fig. 3). The same beneficial effect was observed when osmolytes were added to detergent solubilized proteins before the application on the Bio-Beads SM-2 column. There were no differences whether osmolytes were added before clarifying detergent extraction (centrifugation at 20,000 X g for 30 min) or immediately before the reconstitution (data not shown). It seemed important to test whether proteoliposomes treated by osmolytes are capable of increasing ligand binding and also activating the adenylate cyclase system. Stimulation of adenylate cyclase was not changed by glycerol in any step of its addition during the process of reconstitution (Fig. 4). On the other hand, the presence of sucrose during solubilization increased the activation of adenylate cyclase by GTP-y-S, GTP-y-S + hCG or NaF (P < 0.01). However, no beneficial effect of sucrose was

205

cant Emulphogene After

solublllzotion

Fig. 3. The stimulatory action of osmolytes on the reconstitution of hCG binding activity into proteoliposomes. Ovarian membranes were solubilized in the absence (control) or presence of 20% (v/v) osmolytes in 20 mM sodium cholate or osmolytes were added into solubilized membrane protein before the application of the detergent on Bio-Beads SM-2 column (right part). Shown are mean values of four estimations. These results were confirmed in five independent exper-

+ GLY

0

Octyl-fl-gluc+ooslde GLY

Trlton

X-l

0 l

GLY

Fig. 5. The effects of various detergents on the formation of proteoliposomes. Ovarian membranes were solubilized in 1% Emulphogene BC 720, octyl-P-glucoside or Triton X-100 in the absence or presence of 20% (v/v) glycerol (GLY). The detergent was removed using Bio-Beads SM-2. hCG binding activity in proteoliposomes was assayed as described under Methods. Means f SE of four estimations are shown.

iments.

achieved when it was added to the solubilized preparation before sodium cholate removal. The stimulatory effect of osmolytes is not restricted to the cholate detergent; enhancement of

//

Control

Glycerol

Sucrose

Sucrose after solub

Fig. 3. The effect of osmolytes on adenylate cyclase activity of proteoliposomes. Adenylate cyclase was determined in the absence of stimulants (basal activity) and in the presence of GTP+ (0.1 mM), hCG (0.1 pg) and NaF (10 mM). For details see legend to Fig. 3. Each value is the mean+ SE of four estimations. The experiments were repeated 4 times with similar results.

hCG binding activity by glycerol was also found for other detergents, Emulphogene BC 720, octylP-glucoside and Triton X-100 (Fig. 5). However, sodium cholate was used as the solubilizing detergent in our further experiments, since proteoliposomes prepared by solubilization with any other detergent had not the LH/hCG receptor coupled to G, protein and adenylate cyclase activity (Kolena, 1989). To determine whether osmolytes can modify the physical state of the proteoliposomes, the order of the lipid matrix was investigated by electron spin resonance. Fig. 6 shows that the hyperfine splitting parameter A i for ovarian membranes and proteoliposomes was the lowest with 5 DSA (doxyl stearic acid), intermediate with 12 DSA, and highest with 16 DSA, reflecting the known mobility of the acyl chain in membranes (McConnel and McFarland, 1972). Parameter A I is significantly higher (P < 0.001) for proteoliposomes than for ovarian membranes. In comparing the temperature and the ordering of proteoliposomes as measured by spin probes 16 DSA, 12 DSA and 16 DSA, the formation of proteoliposomes was found to have similar effects on parameter A 1 as the membranes were warmed by about 12, 7 and 4”C, respectively. Proteoliposomes reconstituted in the presence of

glycerol have lower A I values (16 DSA and 12 DSA P < 0.001; 5 DSA P < 0.05>, but they are still different from those measured for membranes with the spin probes 16 DSA and 12 DSA (P < 0.001). However, the values of A I for 16 DSA were outside the range of the linear relationship between the order parameter and A i . We therefore evaluated the correlation times from spectra of this spin label. Proteoliposomes labeled with 16 DSA had significantly (P < 0.01) lower correlation times than did ovarian membranes (data not shown). These results indicate that the order of proteoliposomes reconstituted in the presence of glycerol is closer to the order of native membrane lipids. Determination of lipids showed higher concentrations of total and free cholesterol, as well as phospholipids in pro-

Membranes

_

Glycerol

Control

1.31

Fig. 7. Concentrations of total (open column) and free (hatched column) cholesterol and phospholipids in ovarian membranes (n = 4). proteoliposomes (control, II = 12) and proteoliposomes reconstituted in the presence of 21% (v/v) glycerol (n = 12). Meansf SE are shown. The experiment was repeated with similar results.

1.2

16 DSA

teoliposomes than in membranes (Fig. 7). Glycerol had no effect on the lipid contents in proteoliposomes. However, the molar ratio of cholesterol to phospholipids was significantly lower (P < 0.01) in proteoliposomes (0.344 + 0.008) than in ovarian membranes (0.401 k 0.020), although glycerol left this ratio unchanged (0.320 _t 0.018).

1.1

c E ,' 1.0 12 LISA

Discussion 0 91

5 OSA

0.0

I

5

I

15 25 TEMPERATURE

1

35

45

I"0

Fig. 6. Temperature dependence of the hyperfine splitting parameter A I of spin probes 5 DSA (doxyl stearic acid), 12 DSA and 16 DSA in ovarian membranes (W.. ‘. ~1, proteoliposomes (O - - - - - - 01 and proteoliposomes + glycerol (@p 0). Solubilization of membranes was done as described in the caption to Fig, 3. The experiments were repeated 2-3 times.

In this paper we have shown that the LH/hCG receptor from rat ovarian membranes can be solubilized in an active form by sodium cholate and reconstituted into proteoIiposomes after removal of the detergent. To date no general methods of solubilizing and reconstituting transmembrane proteins are available. The conditions for solubilization of a functionally active receptor system have to be found empirically. During our initial attempts to solubilize and reconstitute the LH/hCG receptor, several detergents (Triton X100, Emulgophene BC 720, Lubrol PX, sodium

207

cholate and octyl+glucoside) were used (Kolena, 1989). With regard to binding activity, the best results were obtained with Triton X-100; however, except for sodium cholate preparations, adenylate cyclase was not coupled to the receptor. The ability of sodium cholate to extract and reconstitute hCG binding activity was dependent on the protein/ detergent ratio. The optimal concentration of cholate was approx. 20 mM at the higher level of protein. An increase in cholate concentration destroyed the binding activity. At high detergent to protein or lipids ratios, lipids are separated from proteins, and pure proteindetergent micelles are formed (Pitt-Rivers and Impiombato, 1968) and the receptor may be inactivated. During biosynthesis, the receptor in the cell is oriented simultaneously in the bilayer. During reconstitution, the receptor and effector protein must leave their soluble state after detergent solubilization and incorporate into the bilayer. This process does not necessarily result in the right location of the receptor within the bilayer. In reconstituted proteoliposomes approximately 57% of the hCG binding sites were digested by trypsin and were, therefore, presumably oriented externally on the proteoliposomes. In some other studies higher proteolytic digestion of receptors incorporated into liposomes was demonstrated. Trypsinization of proteoliposomes containing insulin receptor proteolyzed approx. 75% binding sites (Sweet et al., 19851, and protease treatment of reconstituted epidermal growth factor receptor destroyed more than 95% of specific binding sites (Panayotou et al., 1985). However, a loss of receptor binding sites after digestion does not necessarily mean that the receptor and the effector are in the desired juxtaposition in the bilayer. Permeabilization of proteoliposomes with 0.1% sodium cholate exposed further intravesicular binding activity. At a higher detergent concentration, there was a marked inhibition of hCG binding. In comparison with native membranes an appreciable part of LH/hCG binding sites remained associated with proteoliposomes and was protected from proteolytic degradation. This may be due to different lipid environments in which the receptor is embedded in proteoliposomes. The change of the physical state of the mem-

brane lipids can influence not only the accessibility of the receptor for hCG (Kolena et al., 1986, 1990; present studies), but also for the molecule of trypsin. The differential trypsin sensitivity reflecting a conformational difference of the receptor in various environment has been shown for the insulin receptor (Pilch and Czech, 1980). Glycerol and other osmolytes showed a beneficial effect on reconstitution of LH/hCG receptors into proteoliposomes. Osmolytes represent a group of chemically different compounds including some polyols, sugars and amino acids, which are known to retain cell volume and turgor pressure (Yancy et al., 1982). Moreover, these substances stabilize the protein structure against thermal denaturation (Arakawa and Timasheff, 1985). However, the stimulator-y action of osmolytes on the incorporation of the LH/hCG receptor into proteoliposomes was not solely dependent on their presence during detergent solubilization of membranes; the effect was the same when the osmolytes were present merely during the formation of proteoliposomes. Earlier studies with functional reconstitution of bacterial anion exchange proteins into proteoliposomes had shown that recovery of activity was feasible only if osmolytes were used during protein solubilization (Ambudgar and Maloney, 1986). A similar stabilizing effect of sucrose on activation of ovarian adenylate cyclase was associated only with the detergent extraction procedure, but not with the incorporation into proteoliposomes. The orientation of adenylate cyclase enzyme centers toward the inner space of the proteoliposomes and a lower permeability of membranes for small molecules like ATP may be a reason for lacking of increased hormonal responsiveness of proteoliposomes in formation of CAMP in the presence of osmolytes. It has been demonstrated that addition of membrane-permeabilizing compounds like alamethicine to closed cardiac sarcolemmal vesicles resulted in the increase of adenylate cyclase activity as well as that of the degree of activation of this enzyme by a P-adrenergic agonist (Jones et al., 1980). ESR experiments indicate that the beneficial effect of glycerol on reconstitution of hCG binding activity may be related to the modified physical state of the lipids in the bilayer. Proteolipo-

somes formed from cholate-solubil~ed ovarian membranes contain less ordered membrane lipids than do native membranes. The increase in parameter A I was interpreted as a decrease in the order of lipids at the given depth within the membrane. The greatest changes in membrane ordering, expressed on a temperature scale, were in the hydrophobic membrane part at approx. the C,, carbon level. Differences in the molecular order of natural membranes are closely correlated with differences in their choIesterol/ pbospholipid molar ratio. Under physiological conditions a decrease of this ratio is associated with a disordering of membrane lipids (Shinitzky and Inbar, 1976). Therefore, the decrease. in the order of proteoliposome bilayer may be the result of an alteration in the cholesterol/phospholipid molar ratio; this is not the case with proteoliposomes prepared in the presence of glycerol where the molar ratio remained unchanged. In spite of the differences in the dynamic properties of proteoliposomes, the reconstitution was not associated with any alteration of the relative affinity of the receptor for hCG (Kolena, 1989). Addition of glycerol before the reconstitution of proteoliposomes increased the order of lipid bilayer and shifted it to the physical state of the native membrane. The ordering of the lipid environment in which the receptor is embedded can affect the accessibility of the latter. Thus, the exposure of /3-adrenergic receptors in rabbit reticulocytes (Strittmatter et al., 1979) and those for serotonin in mouse brain membranes (Heron et al., 1980) was enhanced by increased membrane ordering. We reported earlier that LH/ hCG receptors are increased after the incorporation of cholesteryl-hemisuccinate into rat testicular membranes (Kolena et al., 1986; Kolena and Kasal, 1989) and a positive correlation between the order of membrane lipids and the number of LH/hCG receptors during the formation of rat corpora lutea (Kolena et al., 1990). The increased accessibility of LH/hCG receptors in proteoliposomes reconstituted with glycerol may, therefore, be in line with the concept of vertical displacement of membrane proteins (Borochov and Shinitzky, 1976). According to this concept, the bulk of membrane proteins becomes more exposed to the aqueous medium by increasing the

membrane order. The relationship between the action of glycerol on membrane order and the subsequent changes in hormone binding to receptor remains unknown. Studies on calf brain tubulin (Timasheff et al., 1976) and egg albumin (Bull and Breese, 1978) as a model system have suggested that glycerol may act by decreasing the surface tension of aqueous solvents and causing preferential hydration of protein. Entering of glycerol into the water lattice and strengthening of the solvent structure ~~cDuffie et al., 1962) could result in an enhancement of the hydrophobicity of the receptor protein. The cell receptor for LH/hCG is thought to be an integral protein containing seven transmembrane segments (McFarland et al., 1989) associated with the membrane by strong hydrophobic bonds. These hydrophobic bonds can play an important role in the process of receptor activation at this level by ahering the lipids-receptor interaction. Interaction of glycerol with water leaves the solubilized protein in an exceedingly hydrated state that is thermodynamically disadvantageous. Therefore, this system has the tendency to reduce this state by minimizing the disadvantageous proteinsolvent contact, and to maximize the proteinprotein contact, which is induced by favoring the folded or compact state (Gekko and Timasheff, 1981). Morever, protein-protein association should be advantageous to the functioning of membrane proteins, which must retain their hydrophilic domains (Ambudkar and Maloney, 1986). Since the binding of hCG to LH/hCG receptors occurs through the hydrophilic extracelIular domains, these domains may be responsible both for the binding of the hormone and for the interaction with the transmembrane domains to mediate signal transduction. Osmolytes appear, therefore, to maintain the proper spatial configuration of solubilized LH/hCG receptor in the native state with active domains and desired orientation during reincorporation of the receptor into the new Iipid bilayer.

This investigation received financial support from the WHO Special Program of Research, Development, and Research Training in Human

209

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