IDENTIFICATION OF LUTEINIZING-HORMONE RECEPTOR-BINDING INHIBITOR IN BOVINE CORPORA-LUTEA

A 7000 g supernatant, obtained during the purification of luteinizing hormone (LH) receptor from bovine corpora lutea homogenate, was concen trated by ultrafiltration. The filtrate, containing < 50 000 molecular weight material, exhibited LH receptor binding inhibitor (LH-RBI) act ivity. The filtrate was ultrafiltered sequentially through Amicon PM-1 0, PM-30 and UM-2 filters to yield a LH-RBI-containing fraction in the higher molecular weight range of 30 000-10 000 and a LH-RBI-containin g fraction in the lower molecular weight range of 10 000-1000. The hig her molecular weight LH-RBI fraction was purified on Sephadex G-25 and the lower molecular weight LH-RBI fraction was purified on Sephadex G -50. Both the high- and the low-molecular-weight LH-RBI species inhibi ted the binding of I-125-labeled human chorionic gonadotropin (hCG) to bovine corpora lutea and to rat Leydig cell membrane receptors. Simil arly, the production of testosterone by hCG-stimulated rat Leydig cell s was inhibited in a dose-response manner by both the high- and the lo w-molecular-weight LH-RBI species. The LH-RBI activity in the low-mole cular-weight species was stable at 4 degrees C for up to 6 months and at temperatures up to 90 degrees C for 15 mins, whereas the LH-RBI act ivity of the high-molecular-weight species was stable at 4 degrees C f or 15 months and unstable at 60 degrees C after 15 min. The 7000 g sup ernatant provided a much-needed source to obtain larger than previousl y reported quantities of LH-RBI for isolation as well as for structure and function studies.