Isolation of the luteinizing hormone-chorionic gonadotropin receptor in high yield from bovine corpora lutea. Molecular assembly and oligomeric nature.

The luteinizing hormone-human chorionic gonadotropin (LH-hCG) receptor was isolated from 8-liter batches of a supernatant (7,000 X g) of bovine corpora lutea homogenate from 1,200 ovaries obtained biweekly from the Wampole Laboratories, Princeton, NJ. The supernatant was concentrated by ultrafiltration to remove an inhibitor of hormone-receptor binding and inert proteins of M, < 50,000. The concentrate was fractionated by ultracentrifugation in a stepwise sucrose density gradient to remove light density lipid and high density inactive protein fractions. The LH-hCG receptor fraction was solubilized in 0.5% Triton X-100 and treated with petroleum ether to remove neutral lipids and low affinity, high capacity binding sites. The solubilized LH-hCG receptor fraction was purified by gel filtration on Sepharose 6B to separate the adenylate cyclase, on Sepharose 4B to separate the 5’-nucleotidase activity, and on Ultrogel AcA 34 to remove excess Triton X-100 and to concentrate the receptor for final purification by zone electrophoresis on cellulose columns. From each batch, 14.6 & 0.0046 mg of the LHhCG receptor was obtained in a highly purified and stable state. The receptor contained a binding capacity of 2682 pmol of hCG/mg of protein and an affinity constant (Kd) of 0.76 X 10”’ liter M-’. The amino acid composition of the LH-hCG receptor revealed a predominance of aspartic and glutamic acid residues and a low content of cysteine. The receptor contained approximately 10% carbohydrates, which were composed of sialic acid, mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine. The LH-hCG receptor produced in the rabbit antibodies, the presence of which was demonstrated by 1) a quantitative precipitin test, 2) inhibition of the binding capacity of the receptor to plasma membranes from bovine corpora lutea, and 3) in vitro inhibition of the production of testosterone by hCGstimulated Leydig cells. In sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, the LH-hCG receptor migrated as a single entity with an approximate M, = 240,000. After treatment of the receptor with 2% SDS, 1% mercaptoethanol for 1% min at 100 “C, the disc gel electropho-