Characterization and Solubilization of Gonadotropin Receptor of Bovine Corpus Luteum

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Plasma membranes isolated from bovine corpora lutea showed specific binding with 125I-human chorionic gonadotropin and 125I-human luteinizing hormone; unlabeled hormones competitively inhibited the binding. The binding of the 125I-human chorionic gonadotropin to the receptor was a saturable phenomenon and the half-saturation was attained at a concentration of 4 x 10-10 m . Maximum hormone receptor binding was obtained within 15 min at pH 7.2 and 37°. The rate constants of association and dissociation of human chorionic gonadotropin to the receptor determined at 37° were 2.8 x 106 m -1 s -1 and 2.1 x 10-5 s -1, respectively. The dissociation constant calculated from these rates was 8.0 x 10-10 m . At 4° the rate of association was extremely slow and the hormone receptor complex was stable up to 48 hours. The dissociation constants obtained from equilibrium data were calculated to be 1.5 x 10-10 m for human chorionic gonadotropin and the number of binding sites were approximately 6.3 x 10-15 per mg of tissue. The binding of 125I-human chorionic gonadotropin to the plasma membranes was optimum at pH 7.2 and was reduced at acidic and basic pH. The binding was also inhibited at high concentrations of CaCl2, MgCl2, and NaCl, and in the presence of guanidine HCl and urea. The inhibition of binding at pH 5 and 9 and up to 1 m concentration of various ions was reversible. Treatment of plasma membrane with phospholipase C, neuraminidase, glucosidase, trypsin, and α-chymotrypsin did not decrease the binding of 125I-human chorionic gonadotropin. Pepsin and phospholipase A inhibited the binding of 125I-human chorionic gonadotropin to plasma membranes. The plasma membranes were solubilized in detergents. The proteins reprecipitated from the solution by ethanol-ammonium acetate retained the ability to bind 125I-human chorionic gonadotropin. Solubilization of the plasma membranes in 6 m guanidine HCl resulted in the separation of protein and lipid components. Fractionation of the protein component was achieved by gel filtration on a column of Sepharose 4B in 6 m guanidine HCl and ion exchange chromatography on DEAE-cellulose in 3 m urea. The receptor activity of the purified fractions could be reconstituted by emulsification with the lipid fraction, indicating the requirement of lipids to retain the native conformation of the receptor. The molecular weight of the active material estimated from Sepharose 4B column in 6 m guanidine HCl and polyacrylamide gel electrophoresis in sodium dodecyl sulfate was between 30,000 to 70,000. This partially purified protein may represent the regulatory subunit of the gonadotropin receptor.