Purification and properties of human chorionic gonadotropin/lutropin receptor from plasma-membrane and soluble fractions of bovine corpora lutea.
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 properties, salts and gangliosides. The receptor preparations obtained from the plasma-membrane 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 hu man chorionic gonadotropin. The purified receptor from the plasma-membrane and the soluble fractions contained binding capacities of 901000 and 87000 fmol of human chorionic gonadotropin/mg of protein. Yields of 0.02 and 0.22mg of protein were obtained from 250 g of bovine corpora lutea, which represents a 10000- and 1000-fold increase respectively in the specific binding with 125I-labelled human chorionic gonadotropin. Immunization of rabbits with a partially purified receptor fraction generated antibodies that specifically inhibited the binding of the 125I-labelled human chorionic gonadotropin to the receptor.
D000818:Animals; D002417:Cattle; D002458:Cell Fractionation; D002462:Cell Membrane / Q000032:analysis; D006063:Chorionic Gonadotropin / Q000302:isolation & purification / Q000378:metabolism; D002850:Chromatography, Gel; D003338:Corpus Luteum / Q000032:analysis*; D004798:Enzymes / Q000494:pharmacology; D005260:Female; D005732:Gangliosides / Q000494:pharmacology; D006801:Humans; D007986:Luteinizing Hormone / Q000302:isolation & purification / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D011974:Receptors, LH; D012492:Salts / Q000494:pharmacology
D006063:Chorionic Gonadotropin; D004798:Enzymes; D005732:Gangliosides; D011956:Receptors, Cell Surface; D011974:Receptors, LH; D012492:Salts; D007986:Luteinizing Hormone