Characteristics of a Soluble Gonadotropin Receptor from the Rat Testis

Abstract The gonadal receptor for luteinizing hormone LH and chorionic gonadotropin was extracted in soluble form from a particulate binding fraction of the interstitial cells of the rat testis by treatment with the nonionic detergent Triton X-100. During binding studies with the soluble receptor and 125I-labeled human chorionic gonadotropin (hCG), receptor-bound and free forms of the hormone were separated by a double precipitation procedure with polyethylene glycol. The soluble gonadotropin receptors retained hormonal specificity and high affinity for LH and hCG, and showed rapid and reversible gonadotropin binding during incubation with 10-11 m 125I-hCG. The initial rate of binding of hCG by soluble receptors was higher at 34° than at 24° or 4°, but degradation of receptors occurred more rapidly at the higher temperature with corresponding loss of binding activity. The equilibrium association constant of the soluble hormone-receptor complex at 24° (0.5 to 1 x 1010 m-1) was detectably lower than that of the particulate receptors for hCG (2.4 x 1010 m-1). The optimum pH for gonadotropin binding was 7.4, and no effects of buffer composition, ionic strength, or calcium concentration upon binding were demonstrable. Exposure of particulate and soluble receptors to trypsin caused loss of gonadotropin binding activity, indicating the protein nature of an essential component of the receptor site. In addition, a significant role of phospholipid in the structural and functional properties of the receptor was suggested by the reduced binding activity observed after treatment of particulate and soluble receptors with phospholipase A, and by the aggregation which occurred after exposure of the soluble receptors to phospholipase C. Gel filtration and density gradient centrifugation of the free receptors, and the receptor-hormone complex formed by equilibration of the soluble receptors with 125I-hCG, were performed in solutions containing 0.1% Triton. The soluble receptor and receptor-hormone complex showed adsorption to Sepharose 6B during gel filtration, and were quantitatively bound by blue dextran. For these reasons, blue dextran could not be used as a front marker during gel filtration studies, and 0.01% bovine serum albumin was included in buffers employed for chromatography on Sepharose 6B. The distribution coefficient (Kav) of the receptorhormone complex on Sephadex G-200 was 0.09, and that of free hCG was 0.33. On columns of Sepharose 6B, the Kav of the free receptors and the receptor-hormone complex was 0.32, and that of free hCG was 0.56. By reference to the behavior of standard proteins during filtration on Sepharose 6B, the hydrodynamic radius of the receptor was calculated to be 64 A. Sucrose density gradient centrifugation showed that the sedimentation constant of the free receptor was 6.5 S, and that of the hormone-receptor complex was 7.5 S. Dialysis of the complex to remove Triton X-100 caused conversion to an 8.8 S form, but no aggregation occurred. The density of the 7.5 S hormone-receptor complex in cesium chloride gradients was 1.289. From these values, the molecular weights of the 6.5 S (free) and 7.5 S (combined) forms of the receptor were calculated to be 194,000 and 224,000, respectively, and the axial ratios (prolate) of the two forms were 12 and 10.2, respectively. The properties of the gonadotropin receptor extracted by Triton X-100 were consistent with those of a highly asymmetric molecule, predominantly of protein nature, with a minor but functionally important phospholipid component. The retention of high specificity and affinity by the solubilized gonadotropin receptors indicates that the receptor macromolecules possess relatively high conformational stability, and provides an approach to the structural analysis of the hormone binding site.

K,, of the free receptors and the receptor-hormone complex was 0.32, and that of free hCG was 0.56.
By reference to the behavior of standard proteins during filtration on Sepharose 6B, the hydrodynamic radius of the receptor was calculated to be 64 A. Sucrose density gradient centrifugation showed that the sedimentation constant of the free receptor was 6.5 S, and that of the hormone-receptor complex was 7.5 S. Dialysis of the complex to remove Triton X-100 caused conversion to an 8.8 S form, but no aggregation occurred.
The density of the 7.5 S hormone-receptor complex in cesium chloride gradients was 1.289. From these values, the molecular weights of the 6.5 S (free) and '7.5 S (combined) forms of the receptor were calculated to be 194,000 and 224,000, respectively, and the axial ratios (prolate) of the two forms were 12 and 10.2, respectively. This procedure has been previously shown to release interstitial cell fragments ITit high binding affinity for LH and hCG (1, 2). Further release of interstitial cell particles could be effected by mixing the mass of dispersed tubules for 5 to 10 min with a magnetic stirrer.
After filtration through cotton wool, the particulate suspension was centrifuged at 120 x g for 20 min to remove intact cells and tissue fragments. The supernatant solution n-as then centrifuged at 27,000 X g for 30 min to sediment particles with gonadotropin binding activity, and the Fellets were resuspended in 1% Triton X-100 at 4" for 30 min.
After dilution to 0.1 c/0 Triton with phosphate-buffered saline, the solution WE centrifuged at 27,000 x g for 20 min to remove undissolved particles. Treatment of the particulate binding fraction from 10 testes with 0.5 ml of 1 o0 Triton X-100 estractcd the majority of the gonadotropin binding sites, forming a solution with total protein content of 4 mg/lO testes. The solubiliecd binding sites were not sedimented by further centrifugntion at 360,000 x g for 3 hours. In some experiments, t,esticular binding particles T%-cre preincubated \vith '*"I-hCG to label the receptor sites prior to extraction n ith detergent. For this purpose, interstitial cell particles (40 mg) n-ere incubated at 4" for 16 hours with 600,000 cpm (20 ne;) of 1251-labcl~d hCG, then extensively washed with phosphate:buEercd saline to remove free hCG and recovered by centrifugation at 20,000 x g. Under these conditions, 40 to 60% of the labeled hCG was taken up by the particulate fraction and remained bound during storngc of the particles at 4" for several days.
Such labeled particles were of particular value for determination of the rate and efficacy of procedures for receptor solubilization. Also, the reccptor~hormonc complex 2 Triton X-100 is a. polyoxSethylene alkylphenol derivative containing 9 to 10 moles of ethylene oxide per mo!e of p-tertoctylphenol.
The average molecular XT-eight of the condensate is about 620; the molecular n-eight of the micelles formed by the detergent molecules in dilute solution is close to 90,000 (18). extracted from such particles proved to be much more stable than the soluble receptor extracted from testis particles not previously labeled with hCG.
After gentle shaking for 20 s, the iodination mixture lx-as transferred to a column of cellulose or ic'cl)harose-coilcaiiavaliil A for purification of the labeled hormone. I%oth methods provide tracer of suitable properties for receptor binding studies, but the more lengthy affinity chromatography procedure employing Sepharose-concannvalin A gives tracer of slightly but significantly higher binding, and is the preferred method.
Purijcation by Cellulose Chromatograplay-A small column of cellulose Tvas prepared by lightly packing Whatman CF60 powdered cellulose to occupy 2 ml in a disposable 6-ml syringe barrel, after first using the plunger to punch out and deposit a circle of glass filter paper in the end of the empty barrel.
After transfer of the iodination mixture to the dry cellulose, the column IT--as washed four times with 4-ml aliquots of cold phosphatebuffered saline, pH 7.4, then the labeled hormone eluted with 4-ml aliquots of 2% bovine y-globulin or bovine serum albumin in phosphate-buffered saline.
The method is rapid and simple, and most of the labeled hormone was eluted in the first tIT-o fractions.
The tracer JT-as then diluted in phosphate-buffered saline and stored frozen in l-ml aliquots.
PuriJcafion by Seplzarose-concan.avalin A Chromatography-The affinity of the carbohydrate moieties of glycoprotein hormones for concanavalin A has been applied to cstraction of gonadotropins from plasma and urine and to the purification of the iodinated hormones after labeling with '221 (8). In this method, the labeled hormone is extracted from the iodination mixture during passage through a small column of Sepharose-concanavalin A and then eluted with solutions containing 0.2 M mcthylglucopyranoside or mcthylmannopyranoside. The selectivity of this procedure for the carbohydrate portion of the labeled molecule cont,rasts with that of the cellulose absorption method for the protein component of the gonadotropin molecules. Sepharose-concanavalin A M as prepared as previously described by coupling 250 mg of concanavalin A to 25 g of cyanogen bromide-activated Sepharose 6B (8). For purification of 1251labeled hCG, the iodination mixture was transferred to a column (5 X 140 mm) of Sepharose-concanavalin A, and free iodide and damaged hormone were rluted with 12 ml of phosphate-buffered saline containing 1 mg per ml of bovine y-globulin. The labeled hormone Teas then eluted with the same solution containing 0.2 BI methylglucopyranoside.
The tracer TTith highest binding activity was more highly retarded, appearing in Fractions 25 to 33 of the eluant.
Tracer prepared by a combination of the tn-o procedures gives the highest specific uptake by testicular receptors, and the lowest nonspecific blank value.
By gel filtration, polyacrylamide gel electrophorcsis, and elcctrofocusing, the physical properties of '*51-labeled hCG have been shown to bc identical with those of the unlabeled hormone. Complete retention of the full biological nctiTity of the original hormone has also been confirmed after labeling, by in vivo biological assays such as the ventral prostate weight and ovarian ascorbic acid depletion assays (9), and by a more sensitive in vitro assay based on the production of testosterone by the isolated by guest on March 24, 2020 http://www.jbc.org/ Downloaded from rat testis (10). The specific activity of each preparation of labeled hCG was determined by solid phase radioimmunoassay (II), by self-displacement, and comparison with standards of the unlabeled hormone.
The usual specific activity of 1251-hCG employed for binding studies was 60 to 100,000 dpm per ng, or approximately 50 $Zi per pg.

Assay of Receptor Binding
The relatively large size of the labeled ligand (mol u-t 37,000) restricted the range of separation procedures for isolation of receptor-bound tracer hormone after binding studies with lzjIlabeled hCG.
Adsorbent procedures for removal of the free tracer were not applicable, and precipitation methods to isolate the bound complex gave relatively high nonspecific values caused by partial coprccipitation of free hCG. The hormone-receptor complex could be adsorbed by cellulose membranes, but again the nonspecific binding of free hCG was moderately high. A satisfactory method for isolation of the bound complex was established with polyethylene glgcol, employed at a final concentration of 10 7 '% w/v. Separation of antibody-and receptorbound ligands of smaller molecular weight has been previously performed by a single precipitation with polyethylene glycol (5, 12). For the hCG-receptor comples, precipitation with polyethylene glycol followed by immediate redissolving in 0.1 y0 Triton X-100 and a second precipitation with polyethylene glycol was necessary to achieve a satisfactory blank value. In detail, 0.5.ml aliquots of receptor solution were mixed with 0.1 ml of phosphatc-buffered saline containing no hCG, or known quantities of unlabeled hCG, followed by 0.1 ml of phosphatebuffered saline containing 50,000 cpm of 1251-labcled hCG.
?;Sonspecific binding was determined from tubes in which the labeled hormone was incubated with receptor in the presence of 20 pg of unlabeled hCG, and also in the absence of soluble receptor. After the assay tubes (containing 0.7 ml) were kept at 4" for 16 hours, 1 mg of bovine y-globulin (0.2 ml of 5 mg per ml of solution) was added as carrier, followed by 0.5 ml of 30% polyethylene glycol (w/v) in phosphate-buffered saline. The tubes were centrifuged at 1500 x g for 10 min at 4", and the supernatants v,crc aspirated.
The precipitates were redissolved in 0.9 ml of O.lc;/; Triton X-100 in phosphate-buffered saline, and after standing for 10 mill at 4" were reprccipitated \I-ith 0.5 ml of 30 7; polyethylene glycol. After further centrifugation and aspiration of the supernatants, the bound hormone present in the precipitates was determined by counting the radioactivity remaining in the assay tubes in an automatic y-spectrometer with efficiency of 50% for Y.
By this procedure, the nonspecific binding present in tubes containing excess mass, or no rcccptor, was usually belolv 1 c/;, of the added radioactivity, and the specific binding ranged from 20 to 40%, depending on the concentration of receptor present.

Gel Filtration
Columns of Sephades G-200 (2.5 x 100 cm) and Sepharose 6B (0.9 x 100 cm) Tvere equilibrated nit11 50 m&r Tris-HCl buffer, pII 7.4, containing 0.1 a/c Triton X-100, and all separations were performed at 4'. To minimize adsorption of the soluble receptors to agarose, the buffer employed for gel filtration studies with Sepharose 61s also contained 0.01% bovine serum albumin.
The columns were calibrated with blue dextran and 3Hz0 to define Vt, and markers of the following standard proteins were employed, either unlabeled or labeled with 14C or lZjI : thyroglobulin, apoferritin, human y-globulin, bovine serum albumin, human chorionic gonadot,ropin, and myoglobin.
In each experi-ment 3Hz0 and two or three reference proteins were added to the aliquot of soluble receptor applied to the column, and the values for K av of the receptor-hormone complex, the free hormone, and in some experiments the free receptors, were determined.
Blue dextran could not be used to determine V0 during gel filtration of the soluble receptor, as it exhibited high affinity for the receptors and caused virtually all of t'he bound radioactivity to appear in the void volume.
However, a small peak of radioactivity with detectable absorbance at 280 nm was consistently present in the void volume of both columns during gel filtration of soluble receptor preparations which had been equilibrated with lZ51-hCG.
This minor peak of aggregated material was shown to coincide with the position of the blue dextran peak, and was used to indicate the void volume of the columns during gel filtration studies of the soluble receptors.
In addition to the relatively weak adsorption of the solubilized receptors to Sepharose 6B during gel filtration on long columns of agarose, the free receptors were also found to be strongly adsorbed by were prepared m-ith an LKB gradient former model 11300 employing a linear gradient profile and reservoirs containing 5% and 20% sucrose in 50 mM Tris-HCl buffer, pH 7.4. After passage through a small mixing chamber the stream was split by a three-channel Buchler polystaltic pump and directed into cellulose nitrate centrifuge tubes (yia inch diameter x 334 inches).
Sucrose gradients were stored at 4" for 3 to 4 hours before use, but could be used immediately if necessary since the procedure gave extremely linear and reproducible gradients. Sample solutions of up to 0.5 ml were applied after addition of protein markers, usually bovine serum albumin and 7 S human y-globulin.
Other marker proteins included apoferritin and thyroglobulin, and free '2jI-hCG was usually present in the sample.
After centrifugation at 38,000 rpm for 18 hours at 4", fractions were collected for timed intervals by aspiration through a glass capillary connected to the Buchlcr polystaltic pump. A total of 43 fractions, each of 0.35 ml, was usually collected from each tube.
The position of protein peaks was determined by measurement of optical density at 280 nm, and labeled peaks were located by counting the radioactivit'y present in each fraction. To determine the density of the soluble hormone receptor, the hormone-receptor complex, and free hCG, centrifugation was performed in isopycnic density gradients of cesium chloride in the presence of 0.1% Trit'on.
Samples of 0.4 ml were layered on 4.5 ml of 50% (w/v) ccsium chloride solution in 50 m&r Tris-IICl buffer, pI1 7.4, containing 0.1% Triton X-100, and centrifuged for 40 hours at 50,000 rpm in an SW 65 rotor at 4". Thirt,y-six fractions wcrc collected from each tube and densities were determined by measurement of refractive index at 25".

Solubilixafion oj Tesfis Recepfors with Triton X-100
After extraction of particulate binding fractions with 1% Triton X-100, negligible binding activity remained in the undissolved material recoverctl by ccntrifugation at 27,000 x g for 30 min.
The small pellet resulting from further centrifugation of the solution for 60 min at 360,000 X g was also devoid of by guest on March 24, 2020 http://www.jbc.org/ Downloaded from significant binding activity. Determination of the recovery of prelabeled binding sites, from testis particles incubated with 1251-hCG before detergent extraction, confirmed that more than 90 70 of the labeled sites were extracted with 1 70 Triton X-100 and that 80 70 of the sites were extracted by 0.1 7. Triton.
By these criteria, Triton X-100 appears to be a relatively effective agent for extraction of the testicular receptors. However, the recovery of unlabeled receptor sites during Triton extraction, as measured by quantitative binding studies of the particu1at.e and soluble binding sites with i2jI-hCG, revealed significant loss of binding activity after detergent extraction.

Stability of Soluble Receptors
Apart from the difference in recovery of binding sites according to the free or charged nature of the gonadotropin receptors, the receptors solubilized by Triton X-100 were significantly less stable than the particulate binding fractions during storage or exposure to elevated temperature.
The binding activity of the detegent-extracted receptors was reduced by 50 7* during storage for 24 hours at O-4", and incubation of the preparation for 20 min at 34" caused a similar loss of binding capacity.
Retention of binding activity during incubation at 34" was not altered by the presence of t.rypsin inhibitor (2 mg per ml) and was slightly increased (+ 12 70) in the presence of Trasylol (Table I).
Whether the loss of binding capacity of the soluble receptor during incubation at 34" in O.lG//o Triton results from a direct effect of the detergent, or from cnzymic degradation, has not yet been determined.
However, the relative lability of the uncharged soluble receptor is in marked contrast with the much higher stability of the aolnbilized hormone-receptor complex and of the original particulate rcccptors.

Binding Studies
The specific uptake of 1251-labeled hCG by soluble gonadotropin receptors increased serially with rising concentration of the solubilizcd testis preparation (Fig. 1). Conversely, saturation of receptor sites by increasing quantities of lab&d or unlabeled hCG was readily demonstrable, with an approximate binding capacity of 10P2 moles per mg of soluble protein.
The equilibrium binding of '251-1zCG by solubilized testis receptors showed a relatively sharp pH optimum at pH 7.4 (Fig. 2). The rate and extent of hormone binding were markedly influenced by tempcratnre, but were not affected by variations in calcium concentrnt,ion.
Uiucling n-as progressively inhibited by in- Of the added lz51-hCG, 31ci, x-as bound after addition of 500 ~1 (400 pg of protein) of the solubilized gonadotropin binding fraction, and deviation from linearit,y is observed above 250 ~1 (200 pg of protein). 1% Triton X-100 were added to 0.4 ml of buffer solutions to give a series of pH values between 5.6 and 9.4 (pH 5.6 to 6.6, 0.1 RI acetate buffer; pH 6.9 to 7.4, 0.1 JI phosphate buffer; pH 7.6 to 9.4, 0.1 M Tris-HCl buffer).
Maximum binding was attained at pH 7.4 upon incubation with 'ZjI-hCG for 16 hours at 4". 24" and 4". Due to the more rapid degradation of receptors during incubation at 34", greater binding occurred at the lower temperatures.
At 24", uptake of l251-hCG continued to rise until a maximum was reached at 6 hours; at 4", comparable levels of binding were attained after 24 hours (Fig. 3). The second order association rate constant calculated from the binding velocity and the estimated binding capacity of the soluble testis receptor was 6.1 X lo5 M-' mine1 at 4'.
Dissociation of 1251-labeled hCG from soluble receptor-hormone complexes, formed during equilibration with 1251-hCG at 4" for 24 hours, was determined at various intervals after the addition of 20 pg of unlabeled hCG and further incubation at 4". The results indicated that dissociation occurred extremely slowly, with a first order dissociation rate constant of 1.2 x 10d4 min+. The equilibrium constant determined from the association and dissociation rate constants at 4" was 0.5 X lOlo M-'. Equilibrium Binding-The rapid degradation of soluble receptors at 34" precluded the determination of valid equilibrium constants at higher temperatures.
At 4" and 24", studies on the binding of lz51-hCG at equilibrium were performed during incubation with increasing concentrations of unlabeled hCG, and association constants were determined from Scatchard plots of the binding-inhibition data (Fig. 4). The values determined in this way for the association constant (K,) of the soluble receptor and Y-labeled hCG were 0.5-l x lOlo M-'.

Gel Filtration and Density Gradient Centrijugation
The elution profile obtained by gel filtration of the 1251-hCGreceptor complex on Sephadex G-ZOO in 0.1% Triton X-100 showed a prominent peak of radioactivity, which was coincident with the void volume indicated by blue dextran, and a retarded peak of free hCG with K,, = 0.33 (Fig. 5). The elution pattern obtained by gel filtration on Sepharose 6B also exhibited a major radioactive component which was coincident with the front peak of blue dextran, and a peak of free hCG with K,, = 0.56.
These results suggested that the receptor-hormone complex was absorbed to the front marker of blue dextran during gel filtration; this was confirmed by gel filtration performed in the absence of blue dextran (Figs. 6 and 7).
On Sephadex G-200, a more retarded peak of radioactivity on Sepharose 6B, in the absence of blue dextran. A small peak of radioactivity was present at the void volume, coincident with a minor and constant peak of aggregated protein.
The hCG-receptor complex was eluted as a shoulder preceding the major peak of free hCG. Bottom, fractions corresponding to the hCG-receptor peak (top) were pooled and concentrated B-fold with dry Sephadex G-50. An aliquot containing 6000 cpm was then subjected to further gel filtration on Sepharose 6B. The elution profile shows two clearly separated radioactive peaks corresponding to free hCG and the hormone-receptor complex.
emerged immediately following the void volume, and could be completely abolished by preceding incubation with excess unlabeled hCG (Fig. 6). When hormone-receptor complex containing 3.6 mg of protein and 81,400 cpm of bound hCG (specific activity 22.2 cpm per fig) was fractionated on Sephadex G-200, the bound radioactivity was eluted close to the void volume (Fig. 6) ; the specific activity of the 10 fractions across the peak was relatively constant at 192 f 18 (S.D.) cpm per pg, indicating a purification of 8.5.fold during gel filtration, and the over-all recovery was 93 %. On Sepharose 6B, gel filtration in the absence of blue dextran showed a small front peak of aggregated material, followed by two closely adjacent peaks of receptor-bound and free hCG (Fig. 7). The receptor-hormone complex appears as a shoulder on the larger peak of free hCG in the upper part of Fig. 7 and is more clearly separated after concentration and refiltration of this portion of the peak as shown in the lower part of the figure, with K ay = 0.32 in contrast to the K,, = 0.56 of free hCG. By  7, top). Two discrete peaks of radioactivity were resolved, corresponding to free hCG and the hormone-receptor complex, which was 98% precipitable by polyethylene glycol. those of reference proteins by the method of Laurent and Killander (13) the hydrodynamic radius of the complex was estimated to be 64 A. A further aliquot of the pooled receptor-hormone peak was subjected to density gradient centrifugation in 5 to 20% sucrose for 16 hours at 190,000 x g, giving the sedimentation pattern shown in Fig. 8. The mean sedimentation coefficient of the soluble receptor-hormone complex calculated by comparison with reference proteins (14) was determined to be 7.5 f 0.35 (S.D.) in 10 separate experiments.
The free gonadotropin receptor was also subjected to gel filtration on Sepharose 6B, in 50 mM Tris-HCl buffer, pH 7.4, containing 0.1 y. Triton X-100 and 0.01 To bovine serum albumin. After elution, 0.5-ml aliquots of each fraction were incubated with lz51-hCG tracer, in the presence and absence of excess unlabeled hCG to determine specific binding. After 16 hours at 4", separation of receptor-bound and free tracer hCG was performed by precipitation with polyethylene glycol, giving a sharp peak of binding activity wit,h K,, = 0.31 as shown in Fig. 9.
Sucrose density gradient centrifugation of the free gonadotropin receptor gave the sedimentation pattern shown in Fig. 10, revealing a single peak of binding activity with sedimentation coefficient of 6.5 S.
Calculation of Physical Parameters of Gonadotropin Receptor-The values for the Stokes radius (a) and sedimentation coefficients (s) of the receptor and hormone-receptor complex were employed to citlculate the molecular weight (M) and frictional ratio (j/so) of the components from the equations (15)(16)(17): where 17 is the viscosity of the solvent, N is Avogadros number, fi is the partial specific volume, p is the solvent density (0.9876 g per cm), and 6 is the solvation factor. For the gonadotropin receptor-hormone complex, the partial specific volume derived comparison of the K,, of the receptor-hormone complex with from the density of the molecule in cesium chloride gradients was calculated to be 0.776 cm per g. The relative viscosity (VT) of the solvent containing 0.1% Triton X-100 was determined in an Oswald viscometer to be 0.950, and the viscosity coefficient (7) calculated from the formula 7 = r]r.qo was 0.0094 poise. This value, which does not take into account the possible effect of slippage, is almost identical with that determined for dilute Triton X-100 solutions by Kushner and Hubbard (18).
The molecular weight of the free receptors calculated from Equation 1 was 194,000, and that of the receptor-hormone complex was 224,200. The difference between these values (30,000) is in reasonable agreement with the molecular weight of 37,000 for hCG determined by structural analysis (21), and with the molecular weight of 38,000 calculated from Equation 1 and the values determined for sedimentation coefficient (2.9 S) and Stokes radius (34 9) of the labeled hCG molecule.
The frictional ratios of the receptor and the receptor-hormone complex calculat.ed from Equation 2 (neglecting the solvation fact,or) were 1.64 and 1.56, corresponding to axial ratios (prolate) After fractionation, loo-p1 aliquots were incubated ?I-ith 5000 cpm of lz51-hCG in the presence or absence of 10-T M hCG for 16 hours at 4", and the bound radioactivity was determined by precipitation with polyethylene glycol. 6979 of 12.0 and 10.2, respectively. The results of these calculations are summarized in Table II. Effects oj Enzyme Treatment and Reducing Agents The binding properties of interstitial cell particles and Tritonsolubilized gonadotropin receptors were examined following exposure to trypsin, neuraminidase, phospholipase A and phospholipase C, and to various reducing agents. The uptake of lz51-labeled hCG by the particulate binding fraction was slightly enhanced by pretreatment with neuraminidase, and significantly reduced by exposure to trypsin and phospholipase A. Testis particles treated with phospholipase C showed unaltered or sometimes slightly reduced uptake of 1251-hCG (Table III).
Treatment of the solubilized receptors with trypsin also caused substantial loss of binding activity, and reduced binding was again apparent after exposure to Phospholipase A. By contrast, neuraminidase caused marked increase in gonadotropin binding by soluble receptors, and phospholipase C had a similar effect as determined by polyethylene glycol precipitation.
The patterns  (19,20). From these values, maximal hydrated specific volumes 0.768 and 0.776 were derived for free hCG and the hormonereceptor complex, respectively; the latter estimate was employed also for calculation of the molecular weight of the free receptor. c Axial ratios were derived by interpolation of f/f0 values into the series for prolate ellipsoids described by Schachman (17).  Fig. 11. The presence of aggregated material after treatment with neuraminidase and phospholipase C is apparent in the lower (Zest) part of the gradient (Fig. 12), and the magnitude of the 7.5 S receptor peak is slightly increased by both enzymes in comparison to the control. The enhanced binding after neuraminidase treatment of the soluble receptor is attributable to desialylation of the labeled gonadotropin, since asialo-hCG has been shown to possess higher affinity for gonadotropin receptors than the natire hormone (9). The marked reduction of the gonadotropin-receptor peak after exposure to phospholipase A is also apparent in this figure, and the degradation caused by incubating the untreated soluble receptor preparation at 34" is clearly obvious in contrast to the binding activity of the same solution kept at 4". Addition of 1.0 mM dithiothreitol during preincubation of the soluble receptor at 34" for 20 min significantly impaired the subsequent binding of W-hCG, to less than 50% of the control value.
A similar but less marked effect on binding (-20%) was also observed when dithiothreitol Ivas added to cont,rol tubes incubated at 4" for 16 hours.
Similar effects were demonstrable during incubation of the soluble receptor with 7 mar mercaptoethanol, suggesting that the integrity of disulfide bonds is an important factor in hormone-receptor interaction (Table III). UISCUSSION The testicular gonadotropin receptors solubilized by detergent extraction with Triton X-100 retain hormonal specificity for LH and hCG and possess high affinity for these hormones.
Khile these properties are retained to a remarkable degree in the soluble receptors, the susceptibility of such receptors to degradation ITas markedly apparent during these studies and probably accounts for the detectable loss of receptor affinity as well as for the substantial fall in binding capacity during incubation of the soluble receptor preparation.
Degradation of receptors was more rapid at 34" than at lower temperatures, and was more apparent in free receptors than in those previously combined with the trophic hormone.
Such receptor degradation may be a consequence of intrinsic instability of the solubilized binding sites, or may be due to nonspecific enzymatic degradation of the receptors. The effect of trypsin and other enzymes upon the binding properties of the Triton-solubilized receptor has clearly indicated that a protein component is essential for binding activity, and degradation by proteases present in the solubilizecl testis particles may contribute to the observed loss of binding activity at 34". If this is so, the protective effect of combination with hCG may reflect the reduced susceptibility of the more ordered structure to proteolytic attack. The increased binding caused by neuraminidase treatment of the soluble receptor preparation is probably due to desialylation of the gonadotropin during subsequent incubation, since asialo-hCG is known to show higher affinity for gonadal binding sites t.han the natire molecule (9). Similar esperimcnts performed with particulate binding fractions and neuraminidase showed only slight enhancement of h<'G uptake, possibly caused by nonspecific modification of the charge of the receptors following dcsialylation.
The actions of phospholipases A and C upon receptor binding arc of interest in comparison to the effects observed on binding of glucagon and insulin by liver and fat. cell membranes.
Glucagon binding to liver and fat cell membranes is reduced or abolished by digestion with phospholipase C (22, 23), whereas insulin binding by liver and fat cell membranes is substantially increased by treatment with phosyholipase C and phospholipase A (23). This enhancement of insulin binding has been attributed to unmasking of insulin reccptors by removal of phospholipids from the cell membrane, with increased accessibility of the receptors to insulin as n-e11 as to protcolytic enzymes such as trypsin.
Howel-er, no effect of phospholipase digestion upon the binding activity or physical properties of solubilized insulin receptors was observed, consistent wit.11 the probable absence of membrane phospholipids from the soluble insulin receptor (24). The effects of phospholipase treatment of the particulate gonadotropin receptors arc unlike by guest on March 24, 2020 http://www.jbc.org/ Downloaded from either of those noted nith glucagon and insulin, but are inter-