Purification and properties of human chorionic gonadotropin/lutropin receptor from plasma-membrane and soluble fractions of bovine corpora lutea

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Abstract
Plasma-membrane and soluble fractions containing human chorionic gonadotropin/lutropin receptor were prepared from bovine corpora lutea by ultracentrifugation. The plasma-membrane and soluble fractions were studied for physicochemical poperties, namely pH, protein concentration, temperature, time and effects of various enzymes, salts and gangliosides. The receptor preparations obtained from the plasma-membrane and soluble fractions appeared to be identical in their properties. Each fraction was purified individually by sucrose-density-gradient centrifugation, which resulted in a partial dissociation of the hormone-binding subunit from the intact functional receptor unit, which consists of both hormone-binding (regulatory) and adenylate cyclase-associated (catalytic) subunits. The fractions containing the functional receptor unit were further purified by gel filtration on Sepharose-6B and chromatography on concanavalin A-Sepharose. The receptor was finally purified by affinity chromatography on a column of controlled-pore glass covalently coupled to human chorionic gonadotropin. The purified receptor from the plasma-membrane and the soluble fractions contained binding capacities of 901,000 and 87,000 femtomol of human chorionic gonadotropin/mg protein. Yields of 0.02 and 0.22mg protein were obtained from 250 g bovine corpora lutea, which represents a 10,000- and 1000-fold increase respectively in the specific binding with 125I-labeled human chorionic gonadotropin. Immunization of rabbits with a partially purified receptor fraction generated antibodies that specifically inhibited the binding of the 125I-labeled human chorionic gonadotropin to the receptor.