Solubilization and Some Characteristics of the Follitropin Receptor from Calf Testis

Immature calf testes were found to be an unusually rich source of follitropin (FSH) receptors. Particulate fractions derived from such testes bound 32% of added biologically active radiolabeled human FSH (12”I-labeled hFSH) and had a binding capacity of 52 x lo-l4 mol/mg of protein. These values are considerably higher than those previously reported for FSH receptors in other types of beef or mature and immature rat testis. Solubilization of the receptor was achieved by extraction with Triton X-100. Its presence in detergent extracts of immature calf testes was demonstrated by gel filtration and sucrose density gradient centrifugation experiments wherein the position of 12”I-labeled hFSH.soluble receptor complex was determined after incubation of radioligand with soluble receptor in the absence and presence of lOOO-fold excess unlabeled hormone. Separation of free ‘““I-labeled hFSH from that bound to soluble receptor was done by double precipitation with polyethylene glycol, with inclusion of appropriate controls to correct for co-precipitation of free 12”I-labeled hFSH with the hor-mone.receptor complex. Binding of the lZZI-labeled hFSH to soluble receptor was pH- and temperature-dependent, being maximal at pH 7.5

Immature calf testes were found to be an unusually rich source of follitropin (FSH) receptors. Particulate fractions derived from such testes bound 32% of added biologically active radiolabeled human FSH (12"I-labeled hFSH) and had a binding capacity of 52 x lo-l4 mol/mg of protein. These values are considerably higher than those previously reported for FSH receptors in other types of beef or mature and immature rat testis. Solubilization of the receptor was achieved by extraction with Triton X-100. Its presence in detergent extracts of immature calf testes was demonstrated by gel filtration and sucrose density gradient centrifugation experiments wherein the position of 12"I-labeled hFSH.soluble receptor complex was determined after incubation of radioligand with soluble receptor in the absence and presence of lOOO-fold excess unlabeled hormone. Separation of free '""I-labeled hFSH from that bound to soluble receptor was done by double precipitation with polyethylene glycol, with inclusion of appropriate controls to correct for co-precipitation of free 12"I-labeled hFSH with the hormone.receptor complex. Binding of the lZZI-labeled hFSH to soluble receptor was pH-and temperature-dependent, being maximal at pH 7.5 and 24" after 3 h of incubation, and could be completely inhibited by excess unlabeled hormone. Large amounts (5000 ng) of other pituitary hormones did not inhibit binding of 12"I-labeled hFSH to soluble receptor beyond that attributable to trace contamination with native hFSH. The characteristics of the '2"I-labeled hFSH binding inhibition curve obtained with graded doses of unlabeled hormone were similar whether solubilized or particulate receptors were utilized.
Triton X-100 extracts of particulate fractions from such nongonadal tissue as liver, kidney, and spleen did not result in solubilization of factors capable of binding W-labeled hFSH, demonstrating the tissue specificity of the Triton X-100~solubilized testicular receptor. The binding capacity of solubilized receptor derived from immature calf testes, 19 X lo-l4 mollmg of protein, was significantly (64%) less than that seen with particulate receptor prior to detergent extraction, but at least as high as that seen for FSH particulate receptors in larger beef testis or testis from mature or immature rats. Various trinucleotides (1 mM) inhibited the binding of 1Z51-labeled hFSH and also promoted dissociation of specifically bound i2"I-labeled hFSH from preformed hormone. soluble receptor complex, * Recipient of United States Public Health Service Grant HD-08228.
in a manner analogous to that previously reported for membrane-bound receptors of rat testis. Scatchard analysis of binding data indicated one class of high affinity binding sites with an affinity constant (K,) of 2.1 x IO3 M-'. This is 2fold greater than the K, seen for interaction of radioligand with particulate receptors. Based on gel filtration experiments, the Stokes radius of the solubilized receptor was estimated to be 47 A and that of the hormone receptor complex, 50 A. The sedimentation coefficient of the free solubilized receptor was estimated to be 6.3, whereas that of the hormone.receptor complex was estimated at 7.4. These values allowed calculation of molecular masses of 146,000 daltons for the free receptor and 183,000 for the hormone.receptor complex. The difference between these estimates, 37,000, is consistent with current molecular weight estimates of hFSH and suggests a binding of 1 molecule of hFSH per molecule of solubilized receptor. The diffusion coefficient of the free receptor was estimated at 4.35 x 1OW cm* set' and that of the hormone.receptor complex, 4.06 x lo-' cm2 see-'. The frictional ratio of the free receptor was estimated at 1.30 and that of the hormone receptor complex, 1.34.
These studies represent initial reports on successful solubilization of hormone and tissue-specific FSH receptors from testis and form the basis for further studies on purification and chemical characterization of the solubilized receptor.
In an earlier report in this journal, we characterized the interaction of biologically active radiolabeled human follitropin with hormone-specific receptors present in crude homogenates of testes from mature rats (1) and, recently, with receptors in highly purified tubule membranes derived from the same source (2). In general, however, fundamental questions regarding receptor chemistry can best be answered through studies with solubilized receptors. Although many studies have appeared on solubilization and purification of lutropin receptors from testes and ovary (3-61, little information has heretofore been available on solubilization of FSH' receptors from gonadal tissue. The latter problem is complicated by the primary need for a reliable method to separate receptor-bound FSH from free hormone, thereby allowing accurate and spe-  (11). The specific activity and recovery of labeled hormone are calculated as described by Greenwood et al. (12). The mass of 12SI-labeled hFSH in these studies was determined by radioimmunoassay with '"'I-labeled hFSH as described elsewhere (2). After iodination, the labeled hormone is diluted with 0.05 M Trisl HCl buffer, pH 7.5, containing 5 rnivr MgCl, and 0.1% egg albumin (twice crystallized), to a final concentration of 1.5 x 1Om'5 mol (5 ng) in 50 ~1 and stored frozen until used. The specific activity of the lZhIlabeled hFSH ranged from 10 to 15 pCi/pg of radioligand, with 1 ng representing about 10,000 cpm.

Solubilization of FSH Receptor from Calf Testes
Six to eight frozen calf testes (each weighing from 3.5 to 6.5 g prior to removal of the tunica) were defrosted, the tunica was removed, and the remaining tissue (about 1.5 g) was minced and then homogenized in 0.05 M Tris/HCl buffer, pH 7.5, containing 0.25 M sucrose (5 ml of buffer/g of tissue), with a polytron homogenizer (Brinkmann type PT-101, at maximal speed for 30 s. The homogenate was filtered through two layers of cheesecloth, and the filtrate was centrifuged at 120 x g for 10 min to remove nuclei and tissue fragments. The supernatant solution was then centrifuged at 32,000 x g for 30 min. These testicular pellets were used either for solubilization or as the receptor source for binding assays. When used for the latter purpose, they will hereafter be referred to as the "particulate fraction." For the binding assay utilizing the testicular particulate fraction, various quantities of the latter were incubated with 10 ng of '2"I-labeled hFSH for 16 h at 24" in the presence and absence of a 1000-fold excess of unlabeled hormone, followed by the usual sequence of centrifuging, washing, and counting of the pellet. For solubilization, the pellets were suspended in 1.0 ml of 1% Triton X-100 in 0.05 M Tris/HCl buffer, pH 7.5, at 4" and then incubated with gentle agitation for 90 min at the same temperature. The suspension was then diluted 1:lO with Tris/HCl buffer and centrifuged at 300,000 x g for 90 min. When a more concentrated solution of the solubilized receptor was needed, detergent was re-moved from the extract by agitation with Bio-Beads SM-2 for 2 h at 4" as described by Holloway (13)  at 300,000 x g for 90 min, and the amount of radioactivity in the supernatant was counted on an Auto-Gamma counter as an index of receptor complex solubilization.
As is discussed below, gel filtration, sucrose density gradient centrifugation, and polyethylene glycol precipitation experiments were performed to verify the relationship between counts solubilized by detergent and the presence of the 12"I-labeled hFSH receptor complex.

Detection of Receptor Activity
Gel Filtration -Aliquots of detergent-solubilized, buffer-diluted receptor derived from the previously described testicular particulate fraction, containing from 200 to 400 pg of protein, were incubated for 6 h or overnight at 24" with 10 ng of Y-labeled hFSH in a total volume of 0.7 ml. The buffer used for incubation was 0.025 M Tris/ HCl, pH 7.5, containing 0.1% (w/v) egg albumin and 5 rnM MgCl,. At the end of the incubation period, 0.5 ml of cold buffer containing 0.1% Triton X-100 was added, and the mixture was applied on a column (1.5 x 60 cm) of Sephadex G-200 previously equilibrated and developed with the solvent buffer containing 0.1% Triton X-100. The radioactivity of the eluted fractions was counted in an Auto-Gamma counter.
The peak of radioactivity emerging near the column void volume, as determined by calibration with blue dextran, was considered to represent the 'Y-labeled hFSH receptor complex. A peak of radioactivity emerging with aK,, of 0.32 was considered to represent the free or unbound hormone on the basis of the elution position of 'Y-labeled hFSH, run in separate experiments on the same column.

Precipitation with Polyethylene
Glycol -Aliquots (0.4 ml) of the soluble receptor preparation were incubated for 6 h or sometimes overnight at 24" with 10 ng of 'Y-labeled hFSH in 25 mM Tris/HCl buffer, pH 7.5, made 0.1% with egg albumin and 5 rnM MgCl, in a final volume of 0.8 ml. Nonspecific binding was determined in the presence of 1 x 10m7 M (1000-fold excess) unlabeled hFSH. After incubation, 0.2 ml of 5.mg/ml bovine y-globulin was added, followed by 1.0 ml of 25% polyethylene glycol (w/v) in 0.02 M phosphate, 0.15 M NaCl buffer, pH 7.5. The tubes were left in ice for 20 min and then centrifuged at 10,000 x g for 10 min at 4". The resulting precipitates were redissolved in 1.0 ml of 0.1% Triton X-100 in phosphate-buffered saline as above and, after standing at 4" for 10 min, were reprecipitated by the addition of 1.0 ml of polyethylene glycol and a final incubation of 20 min in an ice bath. The tubes were then centrifuged and decanted, and the pellets were counted. Some co-precipitation of free Y-labeled hFSH with the presumed FSH receptor complex usually occurs and results in relatively high apparent nonspecific binding values in the assay. In order to correct for this and allow more accurate assessment of nonspecific binding, we introduced '29-labeled hFSH precipitation control tubes, which were routinely included in each assay. These control tubes were carried under identical conditions as the assay tubes and contained the same amount of buffer, receptor, and cold hormone, except that the 12ZI-labeled hFSH was added at the end of the incubation period and was immediately followed by precipitation with polyethylene glycol.
In equation 1, n is the system viscosity in ergs/cm3, N is Avagodro's number, 6 is the partial specific volume assumed to be 0.72, and p is the density of the medium in g/cm3. The diffusion coefficient (D) was also calculated from knowledge of the Stokes radius and diffusion coefficient by the relationship:  Decapsulated immature calf testes were utilized as the source of the particulate fraction, prepared as described in the text. The 32,000 x g receptor pellet was extracted with 0.05 M Tris/HCl buffer, pH 7.5, made 1% with Triton X-100 for 90 min at 4" and then removed by centrifugation at 300,000 x g for 2 h. The supernatant was diluted to 0.1% Triton X-100, and aliquots were incubated at 24" with 10 ng of 'Y-labeled hFSH for 6 h and cooled in ice. Then approximately 1 ml was applied to a column of Sephadex G-200 (1.5 x 60 cm) equilibrated and developed at 4" in 0.05 M TrislHCl buffer, pH 7.5, containing 0.1% Triton X-100 and 0.1% egg albumin. Fractions of 1 ml were collected, and radioactivity was counted. The 12"1labeled hFSH receptor complex (Curue A) was eluted near the void volume (Fraction 30 determined with dextran blue), followed by another peak (B) of radioactivity, presumably free Y-labeled hFSH. Curve C, a separate experiment in which the soluble receptor was incubated with lOOO-fold molar excess of unlabeled hormone prior to addition of '2"I-labeled hFSH. The position of radioactivity in this experiment (K,, = 0.32) corresponds to that obtained after filtration of Y-labeled hFSH also run in separate experiment.
tion seen in numerous experiments ( Table  I). The amount of hormone binding to immature calf testis was significantly greater than to testis from immature or mature rats ( Table I). Analysis of binding data shown in Fig. 1  As can be seen in Fig. 1 (Fig. 7). There was a marked reduction in binding capacity but concomitant increased binding affinity of the receptor after solubilization with detergent (Table IV). The binding capacity decreased from 52 x lo-'* mol/mg of protein in the particulate fraction to 19 x lo-l4 mol/mg of protein for the soluble receptor. The association constant of the solubilized receptor was greater than that of the particulate receptor fraction (Table IV). An increase in affinity of receptor for hormone after solubilization has been seen in other systems, such as with rabbit mammary gland receptors for prolactin (25).

Some
Physical Properties of Solubilized Receptors -The sedimentation coefficient of the solubilized receptor, either free or associated with Y-labeled hFSH, was estimated by ultracentrifugation through sucrose density gradients as recommended by Martin and Ames (16). The results are summarized in Fig. 8 tor was estimated to be 6.3 x 10-l" s, and that of the lZ51labeled hFSH . receptor complex, 7.4 x 10-l" s. Based on the relationship between experimental determination of the K,, and Stokes radius of reference proteins (171, the Stokes radius (a) of the free receptor was estimated to be 47& whereas that of the Y-labeled hFSH . solubilized receptor complex was estimated at 50 A (Fig. 9). By use of Equation 1, these values allowed calculations of a molecular mass (M) of 146,000 daltons for the free receptor and 183,000 daltons for the hormone.receptor complex. The difference between these values, 37,000 daltons, is consistent with current estimates of the molecular mass of human FSH (26), 38,000 daltons, and implies a binding of 1 molecule of hormone per molecule of solubilized receptor. The diffusion coefficient and frictional ratios of the free and hormone-bound solubilized receptor were calculated through use of Equations 2 and 3 and are given in Table IV, along with a summary of the various other physical properties described above. The sedimentation and gel filtration patterns of the marker proteins utilized in these studies were apparently not affected by the presence of 0.1% detergent, since their .s~,~,~ and K,, values were not significantly different when determined in the presence and absence of 0.1% Triton X-100. However, in the case of the free receptor and the hormone.receptor complex, aggregation in the absence of detergent did seem to occur, and their solutions tended to become slightly turbid on standing. Therefore, it was ultimately necessary to carry out the experiments in the presence of detergents. DISCUSSION Our studies indicate that immature calf testes are an especially rich source of FSH receptors for solubilization and purification studies. Binding of lZSI-labeled hFSH to crude calf membrane receptors averaged about 32% of added radioligand (Table Il. This is an unusually high figure and may be contrasted with the 12 to 15% binding previously reported for crude membrane receptor preparations from rat (11) and mature beef (21) testes. The binding capacity of the 32,000 x g particulate fraction derived from immature calf testes is significantly greater than that seen with similar particulate fractions from larger beef and mature or immature rat testis (Table I). Solubilization was achieved by treating calf testicular particulate fractions with 1% Triton X-100, a nonionic detergent widely utilized for such purposes. The soluble nature of the binding component present in Triton X-100 extracts of calf testis was evident from its failure to sediment at 300,000 x g over a period of 90 min.
The polyethylene glycol double precipitation method utilized by Dufau et al. (3) for separation of receptor-bound from free radiolabeled hCG proved effective in separating free Ylabeled hFSH from receptor-bound hormone. One modification in our use of the polyethylene glycol assay was the addition of a control tube to account for co-precipitation of free Y-labeled hFSH by the polyethylene glycol. This was found to be important in allowing correct assessment of nonspecific binding that, if uncorrected for this variable, becomes spuriously high (Fig. 2).
The binding characteristics of the soluble receptor appear very similar to those previously reported for FSH receptors in highly purified membranes derived from mature rat testes (2). The soluble receptor, however, appears less stable than the membrane-bound receptor and more susceptible to loss of binding activity on standing, especially at 37". This could be due to intrinsic instability of the receptor after detachment from the membrane or loss of an essential stabilizing component such as a phospholipid (2) or a ganglioside (28). Nonspecific enzymatic degradation at the higher temperature is also a possibility.
Hormone specificity of the FSH receptor was retained after solubilization. Only unlabeled FSH inhibited "Y-labeled hFSH binding in a dose-related manner. Very high concentrations of a variety of protein and peptide hormones, such as hCG, prolactin, thyrotrophin, adrenocorticotropic hormone, and hLH did not inhibit receptor binding of Y-labeled hFSH. The inhibition seen at high concentrations (5000 ng) of hLH is attributed to contamination of such fractions with small amounts of intact FSH. The tissue specificity of the FSH receptor solubilized from calf testes by Triton X-100 was demonstrated by the fact that similarly prepared detergent extracts of such bovine nongonadal tissue as liver, kidney, and spleen did not bind lz51-labeled hFSH to any significant degree (Table II). Bhalla et al. (23) have recently studied the interaction of