Characterization of soluble LH/hCG receptor from bovine corpora lutea

Gonadotropin receptor protein was isolated from bovine corpora lutea after sonication (20 kHz, 6 X 15 s at 4 degrees C) of plasma membranes. The yield of binding proteins after repeating the sonication three times was about 26%. After a single cooling to -20 degrees C of sonicated preparation the specific binding of hCG decreased by 20% and no further changes were observed up to 10 days. The same decrease of specific binding by 20% was found after keeping the preparation at 4 degrees C up to 3 days. Maximum inhibition of [125I]hCG binding to a soluble gonadotropin receptor was found at a concentration of 3 X 10(-9) mol 1(-1). Scatchard analysis showed one type of high affinity binding sites characterized by an association constant Ka = 14.6 +/- 3.7 1 nmol-1 which did not differ from that for a particulate receptor (Ka = 15.6 +/- 1.51 nmol-1). However, the binding capacity of soluble LH/hCG receptor was less (Bmax = 11.3 +/- 2.1 fmol mg-1) than that of membrane bound receptor (Bmax = 31.7 +/- 1.8 fmol mg-1). With the aid of affinity chromatography on hCG-CNBr-Sepharose-4-B a homogeneous fraction was isolated which showed specific binding of [125I]hCG of 510 fmol mg-1 and molecular weight of about 60 000 as estimated with SDS polyacrylamide gel electrophoresis in 12-20% gradient gel.