Gonadotropin Binding Factor(s)

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 251, No. 16, Issue of August 25, pp , 1976 Printed in U.S.A. Gonadotropin Binding Factor(s) EXTRACTION OF HIGH AFFINITY GONADOTROPIN BINDING SITES FROM RAT TESTIS AND PARTIAL CHARACTERIZATION OF THEIR INTERACTION WITH HUMAN FOLLITROPIN, LUTROPIN, AND CHORIOGONADOTROPIN* (Received for publication, March 3, 1975, and in revised form, October 24, 1975) VINOD K. BHALLA,$ JOYCE HASKELL, HARRY GRIER, AND VIRENDRA B. MAHESH From the Department of Endocrinology, Medical College of Georgia, Augusta, Georgia Factor(s) that bind gonadotropins have been extracted from rat testis by 30% ethanol (v/v) in water and their interaction with human lutropin (hlh) and human follitropin (hfsh) have been investigated by a new assay using dextran-coated charcoal. These studies reveal that: 1. Maximal binding of gonadotropin with soluble factors was observed over a broad range of ph from 6.0 to 8.0 with a relative decline in binding at extremes of ph. The binding was independent of the ionic strength of the buffer and reached equilibrium within 5 min at 4, 27, and The soluble factors have marked thermostability, a point of distinction from detergent-solubilized receptors. 3. The equilibrium dissociation constant (K,) of %hfsh binding to the soluble factor was 6.0 * 0.58 x lo-l0 M, consistent with the values obtained from the membrane binding studies. Similarly, the Kd value for *4-hLH to the soluble factor(s) was 3.33 l 0.3 x 10m9 M, comparable to the values obtained from the membrane binding studies. Hill plots demonstrated a lack of a cooperative relationship with an apparent Hill coefficient of for hlh and for hfsh. Furthermore, two classes of binding sites for *51-human choriogonadotropin (hcg) were clearly discernible by both Lineweaver-Burk and Hill plots with an equilibrium dissociation constant of x lo- M and 1.35 * 1.2 x lo- M. The apparent Hill coefficient of interaction of Y-hCG with the soluble factors was found to be for high affinity and 1.09 for low affinity binding sites. 4. The binding of %hlh and ** I-hFSH with respect to concentrations of soluble factor(s) was found to be a saturable process, yielding an expected 4.4-fold higher K, for hlh (294 * 13.8 pg/ml) compared to hfsh ( rg/ml). These findings are comparable with the equilibrium dissociation constants, thus confirming a 5-fold higher affinity of hfsh as compared to hlh for the soluble factors, i.e. the ratio of 3.0 x lo- M to 6.0 x lo-l0 M versus the ratio of 294 pg/ml to 66.6 pg/ml. 5. The hormone specificity of the interaction has been studied by using radiolabeled hfsh, hlh, hcg, prolactin, growth hormone, and bovine serum albumin. The binding of FSH at low factor concentrations was found to be 5. to lo-fold greater than prolactin, growth hormone, and albumin. 6. The soluble factors are found in higher concentration in testis compared to liver, kidney, and blood. 7. The effect of ethanol upon solubilization of the factor(s) has been investigated. The factor(s) can be extracted with buffer or water alone. However, 10 to 25%. of ethanol (v/v) facilitates the process of solubilization. The treatment with 70% ethanol (v/v) or more did not extract any factor activity from testes. The factor(s) were insoluble in petroleum ether, chloroform, absolute ethanol, methanol, or lipid solvent. 8. Finally the effect of soluble factors on classical membrane binding was investigated. Increasing concentrations of the soluble factors resulted in graded inhibition of specific binding of 2JI-hLH and I-hFSH to fresh testicular homogenates. These studies implicate a possible regulatory role of soluble factors in hlh and hfsh activation of testis, more particularly as inhibitors of gonadotropin binding to testicular receptors. *This work was supported by General Research Support Grant with gonadotropin binding with factor(s) from testis. The first paper is 5SOl RR from National Institutes of Health and Training Grant Ref. 10. A preliminary presentation of a part of these studies was made HD from the National Institute of Child Health and Human at the Annual Meeting of the Society for the Study of Reproduction Development, National Institutes of Health, United States Public (see Ref. 1). Health Service, Bethesda, Maryland. This is Paper 2 of a series dealing $ To whom all correspondence should be addressed. 4947

2 4948 Binding of Gonadotropin to Testicuh- Soluble Factor(s) Recent evidence suggests that human lutropin and human follitropin interact with membrane receptors to activate ade- nylate cgclase (2-6). The specific binding of hlh and hfsh to membrane receptors of rat testes has been documented by us and other investigators (2-4, 8, 9). During the course of our studies to measure the binding of hlh and hfsh to particulate testicular receptors, we reported the extraction of specific factors from the targe tissue that interfere with gonadotropin binding to particulate receptors (10). These factor(s) may be involved in modulating the target tissue response to gonadotropins by acting as inhibitors of gonadotropin binding to particulate receptors. The characteristics of hlh and hfsh binding to soluble factors, however, could not be studied in detail earlier due to poor sensitivity of the previous methods adapted to detect such binding (10). The present investigation was. therefore, undertaken to develop a more sensitive method for the measurement of hormone-factor interactions with hlh and hfsh. Such a method could also be of considerable use in monitoring large scale factor purification from testis. A dextran-coated charcoal method was utilized, and optimum conditions were established for the separation of factor-bound hlh and hfsh from free hormones. This technique was used to examine the hormone and tissue specificity of soluble factor(s) that bind gonadotropins with high affinity. The role of ethanol and buffer in solubilization of these factors from rat testicular tissue was investigated. Furthermore, the inhibitory effect of soluble factors in binding of hlh and hfsh to membrane-bound receptors was investigated, and existence of binding sites other than those present in particulate fractions was established. From these studies, we concluded that the soluble factors are constantly released from the tissue during in vitro incubations and that they bind gonadotropins with high affinity. The release of these high affinity binding sites (factors) from the tissue have not been taken into account in classical binding studies using particulate receptors previously reported. Therefore, these observations might explain the low total binding of the added tracer at saturating concentrations of the particulate receptors (2-4, 8, 9) and other discrepancies that have been observed in binding studies by other investigators (11, 12). MATERIALS AND METHODS Mature rats (200 to 250 g) of the Sprague-Dawley strain were utilized unless otherwise noted. Chloramine-T was purchased from Eastman Kodak, magnesium chloride and sodium metabisulfite from Mallinckrodt, sucrose from Fisher Scientific Co., egg albumin (twice crystallized) from Schwarz/Mann, carrier free NaIlSI from Amersham Searle Corporation, neutral charcoal (Norit) from Amend Drug & Chemical Co., N. J., and dextran (60,060 to 90,000) from Nutritional Biochemical Corp., Ohio. Use of Hormones-Highly purified hfsh (1575-C) and hlh (LER 966) were supplied by Dr. Leo E. Reichert, Emory University, Atlanta, Ga. Both these hormones were prepared and bioassayed according to the methods already reported in Ref. 8. The hfsh preparation had an FSH activity of 3608 IU/mg and an LH activity of 100 IU/mg. The hlh utilized in this studv has an LH activitv of 4680 IU/me and FSH activity of 1.9 IU/mgY Radioiodination of hfsh and hlh-iodination of hfsh and hlh were carried out by the chloramine-t method (13), modified to allow the retention of biologic activity of LH and FSH as reported previously (8, 10). After iodination, the quality of freshly radioiodinated hlh and hfsh was tested in radioligand receptor assay as described (8, 10). Preparation of 1500 x g Pellet of Rat Testes and Binding Stud- The abbreviations used are: LH, lutropin; FSH, follitropin; CG, choriogonadotropin; h, human. These abbreviations are in compliance with the recent recommendations of IUPAC-IUB Commission on Biochemical Nomenclature (7). ies-rats were killed by CO, asphyxiation immediately prior to use. The testes were removed, decapsulated, and suspended in 0.01 M phosphate buffer, ph 7.5, containing 5 mm MgC12, 0.1 M sucrose, and 0.1% egg albumin in a ratio of 2 ml of buffer/g of starting tissue. Subsequent homogenization was carried out in a Teflon pestle tissue grinder (Arthur L. Thomas, Philadelphia, Pa.) and the 1500 x g pellet was prepared as described earlier (10). Binding with 1500 x g Pellet-All binding studies were carried out in a total volume of 1 ml which consisted of 50 ~1 of laheled hfsh or hlh (5 ng), 450 ~1 of 1506 x g pellet suspension (45 mg, wet tissue weight), and 500 ~1 of 0.01 M phosphate buffer, ph 7.5 (8, 10). A 606fold excess of respective cold hormone was used for the estimation of nonspecific binding (8, IO). Specific binding was calculated from the relationship (B, - B,)/B, x 100 where B, is the total counts hound (expressed as counts per min) to 1500 x g pellet (45 mg of tissue, wet weight) and B, (nonspecific binding) is the total counts bound in tubes containing excess cold hormone. Calculations of specific moles of hormone bound to the receptor were based upon molecular weight of 33,000 for LH and FSH. All determinations were carried out in duplicate and the variation of repiicate determination did not exceed 5%. These procedures are described in detail in our earlier publications (8, lo). Extraction of Soluble Factor(s) from Rat Testes-Unless otherwise stated, the soluble factor(s) from rat testes were extracted according to the following procedure. Rats were killed by CO, asphyxiation. Denuded testes (50 g) were homogenized and resuspended in 200 ml of 30% ethanol (v/v) in H,O. The extract was then incubated at 37 for 1 hour after which the whole solution was kept at 4 for 48 hours prior to centrifugation. The sedimentable material was removed by centrifugation at 12,000 x g for 30 min, followed by a second centrifugation at 48,000 x g for 30 min. Further centrifugation at 300,000 x g for 1 hour did not yield any additional sedimentable material. Our preliminary studies clearly revealed that heating this extract at 100 under the conditions described in Table I did not result in any detectable loss of activity. Consequently, heated extract was used for subsequent work. The heated extract was colorless and free of Soret bands near 400 nm, indicating the absence of contaminating heme proteins compared to the crude unheated extract. Unless otherwise stated, subsequent studies were performed utilizing a single soluble factor preparation. Twenty-five microliters of this preparation had a dry weight of rg and contained 52 pg of protein as measured by the method of Lowry et al. (14) using bovine serum albumin as the standard. Preparation of Deztran-coated Charcoal-Norit, a neutral charcoal (6.25 g), and dextran (0.625 g) were suspended in 1 liter of the respective buffers for hlh and hfsh (see below) and allowed to stay overnight at 4. The supernatant was then decanted to remove the fine particles. The remaining charcoal suspension was resuspended in the respective buffers to make up the original volume. The weight of the fine particles was considered negligible compared to the total amount of charcoal. Binding of hlh or hfsh with Soluble Factor(s)-A new method has been developed to assess the interaction of hlh and hfsh with soluble factor(s) using dextran-coated charcoal. All the reactions were carried TABLE I Comparison of equilibrium dissociation constants of unheated and heated soluble factor(s) The unheated preparation was extracted from rat testis as described under Materials and Methods. The heated preparation was prepared by heating the extract on boiling water until reflux. The water condenser was used to keep ethanol from evaporating. The heated extract was centrifuged at 20,000 x g for 10 min. The supernatant was then used for subsequent work. The assay conditions are described under Fig. 7. The assay was run over a wide range of unheated and heated factor concentration with constant amount of Y-hLH or Y-hFSH (2.5 ng). a S.E. Factor Unheated Heated 2 I-hLH Kd I?-hFSH &nl, dry weight 333 zt * * * 4

3 Binding of Gonadotropin to Testicular Soluble Factor(s) out under equilibrium conditions at 4. The binding studies were carried out in glass test tubes (12 x 75 mm). To each tube was added?-hlh or *%hfsh, the soluble factor, and 0.01 M phosphate buffer, ph 7.5, in a total volume of 1 ml. Appropriate concentrations of the hormones and soluble factors in various experiments have been described in the legends of figures and tables. All reactions were carried out in duplicate with soluble factor(s) and without M soluble factor(s). The reaction was allowed to proceed for 1 hour at 4, after which charcoal/dextran mixture (10/l, w/w) was added (1.274 mg in 200 pl of buffer). The optimum ionic strength of the buffer for suspension of the charcoal was different for the two hormones (0.5 phosphate buffer, ph 7.5, for hlh and 0.01 phosphate buffer, ph 7.5, for hfsh1. The studies to arrive at these conditions have been discussed (see below). After addition of dextramcharcoal the tubes were kept at 4 for 30 min; followed by centrifugation at 1500 x g for 15 min. After centrifugation the supernatant was discarded and the pellet counted. In the absence of the soluble factor, 85 to 90% of the added tracer was absorbed by the charcoal and constituted free labeled hormone. However, in the presence of the factor, the counts per min bound to the charcoal were reduced depending upon the concentration of the factor. Therefore, the binding is defined as B, B, where B, is the total counts per min absorbed to charcoal in the absence of factor(s) and B, is the total counts per min bound in the presence of the soluble factor. Variation of replicate determinations did not exceed 5&. Analysis and Determination of Equilibrium Dissociation Constants- The method of unweighed least squares has been used in estimating parameters of various lines of regression reported in this paper. The relative quality of fit to the data was assessed by applying the t test for goodness of fit and by considering the significance of parameter values derived from each regression. The standard errors (S.E.) were also calculated and the confidence limits of the intercepts were derived. RESULTS Properties of I-hLH and Z I-hFSH Binding to Soluble Factor(~) Extracted from Rat Testes The binding of * I-hLH and iz51-hfsh was maximal within 10 min at O, 27, and 37. Extended incubation for 1 hour at 4 did not result in a detectable increase in binding (data not shown). Consequently, a l-hour incubation period was utilized for subsequent work to ensure equilibrium. The optimum ph was around 6.5 to 8.0 with a relative decline in binding at extremes of ph. The preparation of dextran-coated charcoal solution has been described under Materials and Methods. Increasing the concentration of charcoal above 200 ~1 (1.274 mg) resulted in a decrease of * I-hLH binding to the factor and, therefore, 200 ~1 of charcoal suspension were used for precipitating lz51-lh as well as lz51-fsh (data not shown). Although the buffer concentration requirements for in uitro incubations of I-hLH and I-FSH binding to the factor(s) were less stringent, ionic strength of the buffer in which the charcoal was suspended had an appreciable effect in the assessment of bound and free hormone concentrations (data not shown). For example, addition of charcoal in 0.5 M phosphate buffer resulted in 3.2-fold higher binding of 1-hLH to the factor as compared to addition of charcoal in 0.1 M phosphate buffer, ph 7.5 (data not shown). The binding of * I-hFSH to the factor was unaltered under these conditions. This difference of * I-hLH binding to the factor is not so much due to the total adsorption of the labeled hormone as to the nature of hlh-factor complex, which tends to adsorb in the charcoal at low ionic strength of the buffer (data not shown). Measurement of Affinity2 of Interaction OF Effect of Increasing Concentrations of lz51-hfsh I-hLH upon Binding-The binding of hfsh to soluble factor(s) was The terms affinity and avidity are used synonymously and relate to the energy of reaction. Numerically the affinity (K.) of the interaction FIG. 1. Double reciprocal plots of bound ZSI-hFSH concentrations uwsus free lz51-hfsh concentrations at two fixed levels of soluble factor(s). A, 50 pl of soluble factor(s), and B, of soluble factor(s). The experimental data at two fixed levels of soluble factor is shown in the inset. Increasing concentration of izsi-hfsh was incubated with and without soluble factor(s) at each hormone concentration in a total volume of 1 ml containing 0.01 M phosphate buffer, ph 7.5. The incubations were carried out for 1 hour at 27 and the binding was determined as described under Materials and Methods. z I-hFSH (2.5 ng) contained 9183 cpm. The regression of lines A and B which are the reciprocal of bound 12 I-FSH on the reciprocal of free $1-FSH arey = 0.317~ and y = 0.193x , respectively. The correlation coefficient of both A and B is The confidence of the intercept and the slope of the lines is at the p <O.OOl level giving K, values of 5.97 x lo- M and 5.23 x 10-l M. Based on four different observations, the mean K, value is 6.0 x 10. M with a S.E. of * 0.58 x 10. O M (99% confidence limits 4.0 x lo- M to 7.6 x lo- M). hyperbolic with respect to Labeled hormone concentrations (Fig. 1). A typical double reciprocal plot (16) with Y-hFSH as a variable at two different fixed levels of soluble factor(s) (Fig. 1) revealed intersecting lines at the ordinate axis with a single crossover point on the horizontal line yielding an apparent equilibrium dissociation constant (K,,) of x lo-l0 M. Heterogeneity of the factor(s) population is very apparent from a Scatchard plot (17); two classes of binding sites are discernible with K, values of x 10-l M and 6.0 * 0.6 x 10mLO M (Fig. 2). The Kd value for the low affinity and high capacity binding sites is in good agreement with the observed value from the double reciprocal plot (Fig. 1). The significance of high affinity sites is hard to ascertain due to the low binding at low I-hFSH concentrations. In an attempt to determine the cooperativity of such an interaction, the same experimental data has been used to construct a Hill plot (18). The apparent Hill coefficient was indicating that hfsh interacts with soluble factor in 1:l ratio (Fig. 3). The apparent Kd value calculated from the Hill plot was found to be 7 x 10- M, which is in agreement with the reported K,, values from double reciprocal and Scatchard plots (see Figs. 1 and 2). Similarly, the Kd value for hlh to soluble factor has been found to be 3.33 * 0.3 x 10e9 M by the double reciprocal plot of the antigen or the hormone for antibody in classical radioimmunoassay must equal avidity (K.) of the interaction of the antibody for the antigen (15). However, expression of such a relationship in molarity (M) presumes pure hormone and pure antibody and the knowledge of their respective molecular weights. In the present studies, the soluble factor preparation is in crude form and the two terms have been defined separately to avoid confusion. The affinity of the hormone for the factor (K,) is obtained from hormone saturation curves and the avidity of the factor for the hormone (K,) is obtained from the factor saturation curves. All the data are expressed in terms of equilibrium dissociation constants (I(,).

4 4950 Binding of Gonadotropin to Testicular Soluble Factor(s) (Fig. 4) and the Hill plot (data not shown) with an apparent Hill coefficient of 1.071, thus indicating that hlh also interacts with soluble factor in 1:l ratio. At extremely low concentrations of Y-hLH, another class of binding sites was demonstrable by the double reciprocal and Scatchard plots with a K, value similar to that obtained for YhFSH (data not shown). The extent of the concentration of the high affinity binding sites was approximately 1 to 2% of the total number of binding sites, Therefore, this class of binding sites was assigned to FSH contamination in LH preparation. Effect of Increasing Concentration of hcg upon Binding- The binding of Y-hCG to the soluble factor(s) was measured as a function of increasing concentrations of Y-hCG (I.5 x 10-l M to 2.7 x 10m9 M). Two classes of binding sites for hcg with a Hill coefficient close to unity were apparent in the Hill plot (Fig. 5) and the double reciprocal plot (Fig. 6, A and B). The B,,, values for the Hill plot were taken from the double reciprocal plot (Fig. 6, A and Bl. The high affinity binding class for Y-hCG constituted approximately 5% of the total number of binding sites with an apparent Kd value of 2.4 j; 0.5 x 10-l M. At higher concentrations of the hormone, however, a deviation from linearity was observed for a short range which was once again restored to linearity over a wide range of high Y-hCG concentrations (Fig. 6, A and B), thus indicating a second class of binding sites with a Kd value of 1.35 * I.2 x 10e9 M. As will be discussed subsequently, we feel this aspect should be rigorously pursued. The high affinity class of binding sites may correspond to hfsh-like activity in hcg preparations. Measurement of Avidity of Interaction Effect of Increasing Concentration of Soluble Factor(s) upon Binding-The effect of soluble factor(s) concentration upon I I IO I-hFSti BOUND, IO- M FIG. 2. Scatchard plot (17) of the binding of I-hFSH to soluble factor(s). The data is obtained from the experiment described in Fig. 1 at a fixed concentration of the soluble factor(s) (50 ~1). The relationship used for Scatchard analysis is [boundl/[free] = l/k, ([binding sites] - [bound]). The high and low affinity equilibrium dissociation constants are x 10-l M and 6.0 * 0.6 x 10m O~, respectively. The correlation coefficient of the line for high affinity binding sites is 0.88 (p <O.OOl) and of low affinity sites is 0.95 (p <O.OOl). FIG. 4. Double reciprocal plots of bound [ I-hLH concentrations uersus free * I-hLH concentrations at two fixed levels of ethanol soluble factor(s). A, 25 ~1 of ethanol soluble factor(s); B, 50 ~1 of ethanol soluble factor(s). The inset shows the experimental data. Soluble factor (A = 25 ~1 and B = 50 ~1) was incubated with increasing concentrations of Y-hLH in a total volume of 1 ml containing 0.01 M phosphate buffer, ph 7.5. The incubations were carried out with and without ethanol soluble factor at each concentration of Y-hLH. The reaction tubes were then incubated at 27 for 1 hour and the binding was determined as described under Material and Methods. %hlh (2.5 ng) contained 17,800 cpm. The regression of reciprocal of bound 9-LH on free I-LH in A and B is y = 2.063~ and y = 1.170~ with a correlation coefficient of and 0.994, respectively. The x intercepts yield KG value of 3.49 x 1O-9 M and 3.77 x 10e9 M. The extimated Kd value of five different experiments is 3.33 * 0.3 x 10. M (99% confidence limits 2.7 x 10-O M to 3.96 x 10m9 M). FIG. 3. Hill plot (18) of * I-hFSH binding. The data are taken from the experiment described in Fig. 1. The correlation coefficient is (~~0.001). The estimated x intercept from regression of log[b/(b,.. - B)] on log of free B I-FSH (M) yields a value of FIG. 5. Hill plot of the binding of I-hCG to soluble factor(s). I-hCG (CR 117) was iodinated under the conditions described (8, 10). Increasing concentrations of I-hCG were incubated with and without 50 ~1 of soluble factor in a total volume of 1 ml in 0.01 M phosphate buffer and the reaction was carried out as described under Materials and Methods. The B,., values were obtained from double reciprocal plots (Fig. 6, A and B). The regression of log(bl(b,., - B)] on log of free * I-hCG in nanograms/ml for high and low affinity sites yields n intercept value of and 1.654, respectively, with a correlation coefficient of 0.99 in both cases. The estimated K, value for high affinity binding sites is 2.4 * 0.5 x 10-l M and low affinity binding sites is 1.35 * 1.2 x lo-* M.

5 Binding of Gonadotropin to Testicular Soluble Factor(s) 4951 FIG. 6. The double reciprocal plot of I-hCG over a wide range of hcg concentration. A, represents linearity over low concentration range of hcg with a deviation from linearity at high 2 I-hCG concentrations. The solid line in A has a correlation coefficient of 0.99 (p < 0.001). A part of the dotted line in A is amplified in B to indicate its linear nature. The dotted part in B is linear with a correlation coefficient of 0.99 (p < 0.001). Thus, A represents two classes of I FIG. 7. Effect of the increasing concentrations of soluble factor(s) upon I-hFSH binding (left) and Y-hLH binding (right). * I-hFSH or I-hLH was incubated with increasing concentration of soluble factor(s) in a total volume of 1 ml containing 0.01 M phosphate buffer, ph 7.5. The incubations were carried out for 1 hour at 27 after which dextran-coated charcoal was added (see Materials and Methods ). The assay was run in duplicate with and without soluble factor(s). Control tubes contained increasing concentrations of ethanol corresponding to the test samples. Such results clearly indicated that the presence of ethanol up to 30% (v/v) had no detectable effect upon association of * I-hFSH or 2sI-hLH with soluble factor(s) or upon the total free hormone adsorption by dextran-coated charcoal. These the binding of hfsh and hlh was found to be a saturable process, indicating that all the labeled hormones added have been taken up by the soluble factor(s) (Fig. 7). The double reciprocal plot of the binding as a function of soluble factor concentration yielded the apparent K, value of 66.6 * 4 &ml for hfsh and 294 f 13.8 pg/ml for hlh. It is evident from these saturation curves that the Kd value for hfsh is 4.4.fold lower than hlh, thus consistent with the lower K, of hfsh (6.0 k 0.58 x 10-l M, Fig. 1) than that of hlh (3.3 l 0.3 x 10m9 M, Fig. 4). A Hill plot (Fig. 8) exhibits a straight line with an apparent Hill coefficient of for hfsh and for hlh and a K, value of 63 rg/ml for hfsh and 350 &ml for hlh. The Hill slope, however, does flatten out at higher log [s] value (above + 0.6) which is unreliable due to the small differences between counts per min bound and counts per min left to bind. Similarly, the displacement curve of F/T x 100 uersus log of [s] where F is the counts per min absorbed to char- I free I-hCG t nqlml) binding sites, the low affinity binding site being amplified in B. The calculations of equilibrium dissociation constants of Z51-hCG for the soluble factor is based upon a molecular weight of 33,000 for hcg. The concentrations of high and low affinity binding sites of hcg are highly dependent upon the ionic strength of the buffer in which charcoal is suspended. I studies, as well as others, also showed that a liberal excess of dextran-coated charcoal was present. The regression of reciprocal of bound labeled hormone on soluble factor in case of * I-hFSH (left) and z I-hLH (right) are y = 2.71 x IO-% x 10e5 and y = 0.021x x lo-, respectively, with correlation coefficient >0.99 in both cases. Based on five different observations, the average Kd value is rg/ml for FSH (left) and Kg/ml for LH (right). lzsi- FSH or 1-LH concentrations were 2.5 rig/ml. Note that the range of soluble factor concentrations to obtain a linear dose response relationship of hormone binding to soluble factors are different for FSH (left) and LH (Right). coal in the presence of the soluble factor and Tis the counts per min absorbed in the absence of the soluble factor, yielded a sigmoid curve (Fig. 9). An inhibition of 50% corresponds to a direct Kd value of 322 rg for hlh and 80 pg/ml for hfsh, thus substantiating the values of double reciprocal and Hill plots and providing yet another method to assess the K,. The bimolecular nature of the reaction seems apparent from Fig. 10 where increasing the concentration of lz51-hfsh or * I-hLH from 2.5 ng to 20 ng at fixed soluble factor concentrations resulted in proportional increase of B,,, with a Kd of 66.6 * 3.98 pg/ml for hfsh and pg/ml for hlh which are in accordance with the results obtained earlier (Fig. 7). Hormone and Tissue Specificity of Interaction Hormone specificity of interaction was shown by labeling hfsh, hcg, hlh, prolactin, growth hormone, and bovine serum albumin under the conditions outlined in Table II. The I

6 4952 Binding of Gonadotropin to Testicular Soluble Factor(s) labeled proteins were then used as radioligands to ascertain their avidity of interaction with the soluble factor (Fig. 11). At low substrate concentrations, the formation of Y-FSH-factor complex was 5- to IO-fold higher than prolactin, growth hormone, and bovine serum albumin. Estradiol, testosterone, or aldosterone did not show any detectable binding to the soluble factor(s) preparation (data not shown). Tissue specificity of the interaction was demonstrated by extracting the soluble factor under identical conditions from variety of rat tissues, such as testis, kidney, liver, and blood. The soluble factor preparations from such tissues were then used in the experiment described (Fig. 12). We have preferred multiple point determinations for ascertaining the amount of soluble factor protein required for half-maximal binding of Y-hFSH or Y-hLH. It should be pointed out, however, that the chemical nature of these soluble factors are not known at the present time. Therefore, basing the results on protein concentrations might not be the most appropriate way to express the data. Also, in view of our recent findings with respect to the heterogeneity of the soluble factor population, this aspect needs further investigation (see below). Based upon these experiments, it is reasonable to assume that the relative FIG. 8. Hill plot of the data shown in Fig. 7. The abscissa represents total factor concentration in micrograms/ml. The correlation coefficient is >0.99 in both cases (p < 0.001). FIG. 9. Displacement curve of the data shown in Fig. 7 for lz51- hfsh and I-hLH. The ordinate axis represents F/T x 109 for either * I-hLH or Y-hFSH over a wide range of the factor concentration. F represents counts adsorbed to charcoal in the presence of the soluble factor(s) and T represents counts adsorbed to charcoal in the absence of the soluble factor(s) at any given factor concentration. Fifty per cent inhibition corresponds to the Kd value for hfsh or hlh in micrograms/ml. concentration of these soluble factor(s) is greater in testis compared to liver, kidney, or blood, reflected by lower K, value for testicular extract which must be considered as an index of purity under these circumstances. Role of These Factors in Inhibition of Specific Binding of ZSZ-hFSH or 25Z-hLH to x g Pellet of Rat Testis In an attempt to elucidate the role of soluble factor(s) in the classical membrane binding assay, an experiment was performed where 1-hLH or Y-hFSH and fresh testicular homogenate were incubated with increasing amounts of soluble factor (Fig. 13). The factor preparation was lyophilized to remove ethanol. As can be seen in Fig. 13, the increase in the concentration of the soluble factor(s) resulted in graded inhibition of specific binding of hlh or hfsh to fresh testicular membranes. This may indicate an inhibitory role of soluble factors at the level of hormone binding to its specific particular receptors. The nonspecific binding was corrected by using an excess of appropriate cold hormones. In such experiments, soluble factors seem to act as noncompetitive inhibitor of gonadotropin binding. It should be pointed out, however, that a true soluble receptor is also expected to be a noncompetitive inhibitor. Therefore, the net effect of soluble factors like true soluble receptors is to make it seem as if less hormone is present to bind to particular receptors. Hence, the relationship of the soluble factors versus soluble receptors is unclear at the present time. Role of Percentage of Ethanol (v/v) in Solubilization of Factors We now have evidence to support the idea that the factor(s) leaches out of intact testes or testicular membranes in aqueous media or buffer during in vitro incubations The ethanol (30%, v/v, in water or buffer) only seems to facilitate the process of solubilization (Fig. 14) as reflected by a 2-fold lower K,, value in the ethanol extract as compared to buffer or water extracts of rat testes. Similar results were obtained for hlh (data not shown). Solubility Properties of Factors in Organic Solvents The factor is insoluble in petroleum ether (40 to 60), chloroform, ethanol, methanol, or lipid solvent (chloroform/ methanol, 3/l). It can be lyophilized and redissolved in buffer or water without detectable loss of activity (data not shown). DISCUSSION The present report demonstrates the extraction of high affinity binding sites for hlh, hfsh, and hcg from target tissues. The apparent dissociation constants for hlh, hfsh, and HCG are remarkably similar to the values reported from studies using membrane receptors of testicular or ovarian origin (Table III). The equilibrium dissociation constant of hfsh for testicular receptors (8) is in excellent agreement with the calculated Kd of hfsh with soluble factor(s) (Table III). Also, the Kd of hlh-factor interaction ( x 10m9 M) is the same as that calculated by Gospodarowicz using LH and bovine corpus luteum membranes (21) and Cole et al. using LH and corpus luteum (23, 24). Discrepancies between the relative affinities of LH for testicular or ovarian receptors obtained by different laboratories, in some instances (Table III), are hard to explain. It should, however, be kept in mind that the variations within the laboratories might be due to the hormone preparations used, the method of labeling the hormone, or the method determining the binding of the hormone to the receptors. In our

7 Binding of Gonadotropin to Testicular Soluble Factor(s) Frc. 10. Avidity of the factor for Y-hFSH (left) and 2 I-hLH (right). Soluble factor (10 pl, 50 ~1, 100 ~1, and 400 pl for FSH and 50 ~1, 200 ~1, 300 ~1, and 406 ~1 for hlh1 was incubated with indicated concentrations of Y-hFSH or Y-hLH ranging from 2.5 to 20 ng in a total volume of 1 ml. The experiment was then carried out under the conditions described under Materials and Methods. At each concentration of I-hFSH or lasi-hlh appropriate controls contained equivalent amount of ethanol with soluble factor(s). Y-hFSH (2.5 ng) contained 10,986 cpm and I-hLH (2.5 ng) contained 21,183 cpm. The correlation coefficient in all cases is >0.99 (p < 0.001). TABLE II Conditions of iodination of proteins The proteins were iodinated according to the method of Greenwood et al. (13) with the modifications described below. The free iodine was then separated from labeled protein through Sephadex G-100 and the specific activity and recovery calculated as described (8, IO). After iodination, labeled proteins were diluted with buffer (0.01 M phosphate buffer, ph 7.5, containing 5 mm MgCl,, 0.1 M sucrose, and 0.05% egg albumin) to a final concentration of 10 ng in 100 al and stored frozen in separate vials until thawed for use. P&T rg P&Y s PWM 1. hlh hcg Prolactin Growth hor mane 5. Bovine serum albumin n Conditions for iodination of hfsh were same as described (8, 10). Immunochemical grade human prolactin, hcg, and human growth hormone were provided by the National Institute of Arthritis, Metabolism, and Digestive Diseases Hormone Distribution Program. Bovine serum albumin was purchased from Sigma Chemical Co. b The reaction temperature was 4. experience, the concentration of the receptor ~ersu.s the amount of the labeled hormone used is critical for analysis of the data. Human choriogonadotropin (hcg1 is a placental hormone, secreted during pregnancy, and is structurally different from hlh (30-35). Although it has been found to resemble hlh in biological activity (33). it has long been suggested (34, 35) that hcg might contain both FSH and LH activity in the same molecule. We have recently reported that purified hcg (CR 115) shows a I-hFSH uptake inhibitory activity of 0.9% of that of pure hfsh in radioligand receptor assay of FSH (36). This was later confirmed by other investigators using four different hcg preparations (37). In most of these studies, hcg is frequently used as a tracer for investigating the properties of hlh and testicular or ovarian receptors (3, 11, 25-29). The FIG. 11. Hormone specificity of interaction. hfsh, hcg, hlh, bovine serum albumin (LISA), prolactin (fill, and growth hormone (GH) were iodinated under the conditions described in Table II. Under the conditions described, hfsh, hlh. and hcg retain their bioactivity. Although the biological activity of 1251-prolactin and I-growth hormone have not been determined, the adapted conditions for radioidination are extremely mild. A constant amount of 151-hormone (2.5 ngl was separately incubated with and without factor over a wide range of factor concentrations in a total volume of 1 ml. The experiment was then carried out under the conditions described (see Materials and Methods ). In the absence of the factor, the total adsorption of the I-hormone to the charcoal was as follows: hfsh 75% hlh 72.4%, hcg 73.6%, bovine serum albumin 23.6%, prolactin 60% and growth hormone 83%. relative receptor affinity for hcg is always higher (4 to 5 times) than observed for hlh (25-27). Rao has shown that bovine LH does not compete as well as hcg for bovine plasma membrane receptors using l*y-hcg as tracer (25), a finding which was later confirmed by Papainannou and Gospodarowicz using luteal plasma membrane (27). This observation that hcg has higher affinity for membrane LH receptors is once again clearly visible in our present studies. Two classes of binding sites for hcg are clearly discernible from the data shown in Figs. 5 and 6 with relative Kd value of 2.4 it 0.5 x 10-l M and x loa M. The high affinity binding class is approximately 4% of the total binding sites calculated on the basis of B,,, in double reciprocal plots (Figs. 5 and 6). We feel, on the basis of these studies, that binding to high affinity binding sites for hcg is due to hfsh-like activity in the hcg molecule. Based upon the measurement of the equilibrium dissociation

8 Binding of Gonadotropin to Testicular Soluble Factor(s) FIG. 12. Tissue specificity of interaction. Equal amounts of freshly obtained testes, liver, and kidney were suspended in 0.01 M phosphate buffer containing 30% ethanol (v/v) and homogenized. The resulting homogenate was incubated at 37 for I hour, followed by centrifugation at 20,ooO x g for 10 min. The supernatants from each tissue were used to assess the interaction of z51-hfsh over a wide range of factor concentration under the conditions described in Fig. 7. The results are based upon protein concentration. The K, values are 15 rg/ml in testes, in kidney, 222 pglml in liver, and 1000 wg/ml in blood for Z I-hFSH (left) and the Kd values are 46.5 Fg/ml in testes, 303 &ml in kidney, 666 pg/ml in liver, and 5000 fig/ml in blood for I-hLH (right). The regression coefficient is >0.99 (p < 0.001). 10, Factor (pg potemlml) FIG. 13. Inhibition of * I-hLH or 1-hFSH binding to testicular receptors in the presence of soluble factor(s). Testicular homogenate (1500 x g pellet) was prepared as described under Materials and Methods. Testes homogenate (45 mg, wet tissue, weight) was incubated with 5 ng of Y-hLH or Y-hFSH at indicated concentration of soluble factor. At each concentration of the factor, the triplicate tubes were run with and without the excess cold hormone for determination of nonspecific binding. The incubation period was 3 hours at 37 after which these tubes are centrifuged and the supernatants discarded. The 1500 x g pellet was washed and recentrifuged as described earlier (8). The specific binding was calculated as described under Materials and Methods. The correlation coefficient for Y-LH is 0.91 and Y-FSH is t $ 20 & constants from hormone saturation curves, hfsh had 5-fold greater affinity for the soluble factor than hlh which has been confirmed by subsequent measurement of the avidity of interaction of factors for hfsh and hlh. The binding of FSH and LH as a function of soluble factor(s) yielded the apparent K, value of pg/ml for hfsh compared to 294 f 13.8 fig/ml for hlh. In our present studies, we have also shown an apparent lack of cooperativity in the Hill plots over a wide range of either hormone (Fig. 3) or factor concentrations (Fig. 8). However, an allosteric nature of the interaction cannot be completely ruled out until further studies are performed on isolation of hlh or hfsh factor(s). The present work, however, provides the preliminary investigations with regard to our understanding of this phenomenon. 1 -I FIG. 14. Effect of ethanol upon solubilization of factors. Rat testes were homogenized to prepare 1,500 x g pellet as described under Materials and Methods. The homogenate (45 mg) was treated with 1 ml of buffer (0.01 M phosphate buffer) containing 5 rnm sucrose in case of control (none) and 30% ethanol (v/v) in above mentioned phosphate buffer. The incubations were then carried out at 4O for 1 hour. After centrifugation, the 1,500 x g pellet was used for classical membrane binding studies as described under Materials and Methods. The specific binding of Y-LH or I-FSH to the 1,500 x g pellet treated with buffer alone was 85% and SO%, respectively. The specific binding of Y-LH and Y-FSH to ethanol-treated pellet decreased below 10% of untreated controls (data not shown; see ref. 10). This decrease in gonadotropin binding to ethanol-treated 1,500 x g pellet versus untreated controls correlated in part with the appearance of higher concentration of soluble factor(s) in ethanol-treated supernatants (see below). The supernatants of both sets of experiments were recentrifuged at 20,000 x g and the experiment was then performed with the factors (in supernatant) over a wide range of factor concentrations as described under Materials and Methods. *%hfsh or lasi-hlh (2.5 ng each) was used as a ligand. The correlation coefficient in both cases is < 0.001). The ph or ionic strength requirements are less stringent in the present studies compared with studies with particulate receptors (8, 9), a finding in common with other investigators (29, 38) who have solubilized hcg and TSH receptors with nonionic detergents. Although the hormone specificity of interaction has been shown at critical substrate concentrations (Fig. ll), we have been unable to show greater than 20% of the inhibition of Y-hormone binding to the soluble factors by

9 Binding of Gonadotropin to Testicular Soluble Factor(s) 4955 TABLE III Affinity constants for hlh, hfsh, hcg receptor interactions H0rmlXle TiSSW Investigators Reference Equilibrium dissociation constants (IQ FSH LH hcg Testes Testes Testes Corpus Ovary luteum Luteoma Corpus luteum Ovary Corpus luteum Testes Corpus luteum Testes Bhalla & Reichert Means & Vaitukaitis Moudgal et al. Gospodarowicz Lee & Ryan Lee & Ryan Cole et al. Cole et al. Lee & Ryan I%30 Catt et al. Papainannou & Gospcdarowicz Leidenberger & Reichert x lo- M 6.0 x IO- 7.0 x 10-e 1.5 x 10-n 1.13 x 10-Q; 3.0 x 10-Q 3.6 x 1Om8 4.9 x lo-* 4.1 x 10-Q 9.8 x x 10-l 1.2 x lo x lo- 5 x lo- 0 hfsh Soluble receptors No report hcg Soluble receptors Dufau et al x (from testes) FSH Soluble factors Bhalla et al. This report 5.6 x lo- I ; 6.0 x lo- LH Soluble factors Bhalla et al. This report 6.0 x lo-lob; 3.3 x 10m9 hcg Soluble factors Bhalla et al. This report 2.4 x 10-lLb; 1.35 x lo-* y In the case of the interaction of FSH with the soluble factors, a third class of high affinity binding site has been observed at very high concentrations of * I-FSH with a Kd value of 3 x 10m9 M. This class of binding site has been tentatively assigned to LH contamination in FSH preparations. D For interpretations of these classes of binding sites-see text. co-addition of the respective unlabeled hormone.31t is not clear whether this is due to different binding behavior of the iodinated and noniodinated hormones or due to their properties toward charcoal adsorption method. If the former is the case, then the binding sites being measured in the present studies are not truly for the unlabeled hormone but rather for the Y-hormone; however, the significance of the lz51-hormone binding should not be underestimated since the labeled hormones used in our studies do retain their biologic activity and have been demonstrated to interact specifically with physiologic particulate receptors of either testicular or ovarian origin with high affinities (Table III). If the latter is true, the preparation of unlabeled iodohormones and their subsequent use in competitive displacement studies using dextran-coated charcoal method might circumvent the problem in methodology provided we assume that the relative affinities of the labeled and unlabeled hormones for the soluble factors are identical and that the incorporation of iodine atom within the hormone is a prerequisite for their absorption to the charcoal. Also, additional possibilities must be considered where the interference due to heterogeneous populations of soluble factor can cause erroneous results. In this context, it is interesting to note that we have been able to resolve the activity into dialyzable and nondialyzable fractions. The dialyzable frac- 3 Unpublished observation. Efforts to study the mechanism of reaction of 12sI-hormone binding to the soluble factor are in progress. The possibility that the initial interaction of lz51-hormone with the soluble factor is rapidly reversible with the ultimate formation of some irreversible complex cannot be excluded. In view of heterogeneous population of the soluble factor in crude extracts of rat testis, it is felt that dissociation experiments should only be planned after achieving a reasonable purification of soluble factor. tion has been purified 66-fold with complete retention of hfsh binding activity. It has a molecular weight of about 5000 and might turn out to be similar to the inhibitors of gonadotropins.6 Another fraction has been obtained during purification which binds to LH with much greater specificity than crude soluble factor preparation. The binding of LH as a function of soluble purified factor yields lower apparent Kd for LH than FSH, thus once indicating that two separate soluble factors exist in crude soluble extract of rat testis. Further work to explore the hormone specificity of these fractions and their respective affinities to gonadotropin is in progress. It may or may not be the true receptor but the present investigations clearly demonstrate its essential role in gonadotropin binding to the target receptors, exemplified by the fact that addition of this factor(s) preparation to the incubation medium containing I-hLH or I-hFSH and freshly prepared particulate receptors results in inhibition of specific binding of the labeled tracer to the membrane receptors. This finding can, in part, be explained by the fact that the factor binds to the hormone with high affinity and thus makes it seem as if less tracer is present for binding to the particulate receptors. In such experiments, since these factors inhibit the binding of hlh or hfsh to fresh testicular membrane, they may be called inhibitors of gonadotropin binding. However, we feel the term inhibitors should be restrictively used until the physiologic role of the soluble factor(s) as gonadotropin inhibitors is conclusively established. In particular, evidences are V. K. Bhalla, unpublished observations. The K, value for the purified dialyzable fraction in case of FSH is 444 ng of protein/ml; dialyzable fraction 20 fig of protein/ml and original extract prior to dialysis 29.3 Kg of protein/ml. BDarrel Ward, personal communication and Yang et al. (39).

10 4956 Binding of Gonadotropin to Testicular Soluble Factor(s) required to ascertain whether or not soluble factors are different from soluble receptors. The concentration of the soluble factors is greater in testes, compared to other tissues such as liver, kidney, or seminal vesicles and blood. However, this lack of absolute tissue specificity is also reflected in in uioo studies where administration of gonadotropins results in incorporation of the hormone in nongonadal tissues, such as liver and kidney (4, 40-42). Furthermore, Lee and Ryan have recently reported the solubilization of LH receptors from nongonadal tissues, such as liver, spleen, and kidney (43). Therefore, the relationship of those receptors with soluble factor(s) is unknown at the present time. The effect of ethanol upon solubilization was reinvestigated by using dextran-coated charcoal method which is more sensitive and reliable than those reported previously (10). These studies clearly indicate that soluble factors leach out from the membrane in aqueous media or buffer under the in oitro incubation conditions of membrane binding assays (Fig. 14). Ethanol (30%, v/v, either in water or in 0.01 M phosphate buffer, ph 7.5) facilitates the process of solubilization of these factors. The repeated washing of the membranes with appropriate buffers reduces the concentration of the soluble factors but does not result in their complete extraction without reducing the specific binding of the gonadotropin to the membranes. Therefore, the observation of constant release of these factors during the in vitro incubations of homogenate or membranes in classical membrane binding studies might explain, in part, the low total binding of the added tracer at saturating membrane concentrations (2-4, 8, 9). In such studies, slow release of the soluble factors from the membranes might, in turn, interact with the added tracer in the incubation medium, thus making less tracer available to bind to the membrane receptors. These studies, therefore, provide a possible explanation for some discrepancies that are observed by other investigators in classical membrane binding studies (11, 12). The physiologic significance of these soluble factors is not known at this time. Their unique characteristics of thermostability, high affinity for gonadotropins, partial tissue specificity, hormone specificity, and their constant release from homogenates during in vitro incubation medium in the absence of added hormone might suggest their possible regulatory function. It will be of interest to compare the properties of these factors with recently reported inhibitors of gonadotropins (44-47) and known heat-stable inhibitors of adenylate cyclase (48, 49), and protein kinase (50-52). Further studies, however, are needed to explain this phenomenon. Acknowledgments-We wish to thank Dr. Leo E. Reichert, Emory University, Atlanta, Georgia, and the National Institute of Arthritis, Metabolism, and Digestive Diseases Hormone Distribution Program for a generous gift of purified hlh (LER 960) and purified hfsh (1575-C). We also wish to thank Dr. E. D. Bransome, Chief, Section of Endocrine and Metabolic Diseases of the Department of Medicine, and Dr. T. G. Muldoon, Department of Endocrinology, Medical College of Georgia, for helpful discussions, criticism, and suggestions. We appreciate the help of Dr. T. M. Mills, Department of Endocrinology, Medical College of Georgia in statistical analysis. V. K. Bhalla, unpublished observations REFERENCES Bhalla, V. K. (1975) Meeting of the Society for the Study of Reproduction Abstract 3, 14 Means, A. R. (1973) Ado. Erp. Med. Biol. 36, Catt, K. J., and Dufau, M. L. (1973) Adu. Erp. Med. Biol. 36, Rao, C. V., and Saxena, B. B. 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