Identification of luteinizing hormone receptor binding inhibitor in bovine corpora lutea

Abstract

Rathnam P, Lin S, Saxena BB. Identification of luteinizing hormone receptor binding inhibitor in bovine corpora lutea. Eur J Endocrinol 1995;132:213–17. ISSN 0804–4643 A 7000 g supernatant, obtained during the purification of luteinizing hormone (LH) receptor from bovine corpora lutea homogenate, was concentrated by ultrafiltration. The filtrate, containing < 50 000 molecular weight material, exhibited LH receptor binding inhibitor (LH-RBI) activity. The filtrate was ultrafiltered sequentially through Amicon PM-10, 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-containing fraction in the lower molecular weight range of 10 000–1000. The higher 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 inhibited the binding of 125I-labeled human chorionic gonadotropin (hCG) to bovine corpora lutea and to rat Leydig cell membrane receptors. Similarly, the production of testosterone by hCG-stimulated rat Leydig cells was inhibited in a dose–response manner by both the high- and the low-molecular-weight LH-RBI species. The LH-RBI activity in the low-molecular-weight species was stable at 4°C for up to 6 months and at temperatures up to 90°C for 15 mins, whereas the LH-RBI activity of the high-molecular-weight species was stable at 4°C for 15 months and unstable at 60°C after 15 min. The 7000 g supernatant provided a much-needed source to obtain larger than previously reported quantities of LH-RBI for isolation as well as for structure and function studies. Brij B Saxena, Cornell Univ. Medical College, Rm A-267, 1300 York Ave, New York, NY 10021, USA