STUDIES ON A LOW-MOLECULAR WEIGHT FOLLICLE-STIMULATING-HORMONE BINDING INHIBITOR FROM HUMAN-SERUM

Low molecular weight components of human serum, prepared by dialysis through Spectrapor no. 1 membrane (mol wt retention, 8000), interfered with the interaction of human [125I]FSH ([125]hFSH) and hormone-specific receptors in testes of mature rats in a dose-related manner. A partial purification of the FSH binding inhibitor (FSH-BI) was obtained by sequential molecular sieving of serum dialysates through columns of Sephadex G-25, G-50, and G-10. The most active fraction, G-10-1, emerged slightly retarded by the Sephadex G-10-column, indicating a molecular weight of about 700. FSH-BI activity passed through an Amicon UM-2 membrane on dialysisultrafiltration (mol wt retention, 1000) but not through an Amicon UM-05 membrane (mol wt retention, 500), in agreement with the size estimate obtained from the gel filtration experiments. Preincubation of rat testis membrane receptors with G-10-1 resulted in a 50% diminution in binding of subsequently added [125]hFSH compared to preincubation with buffer alone. The inhibitor fraction also facilitated dissociation of preformed hormone-membrane receptor complex, although to a lesser degree. Fraction G-10-1 strongly inhibited the binding of [125I]-hFSH to membrane-bound receptors in calf testes or bovine granulosa cells. It also inhibited [125I]hFSH binding to detergentsolubilized, hormone-and tissue-specific calf testis receptors, whereas bovine LH and bovine serum albumin at high concentrations did not. Incubation of [125I]hFSH with G-10-1, followed by dialysis, did not affect the binding of radioligand to testis receptors. No evidence for a dissociating or fragmenting effect of G-10-1 on [I25I]hFSH could be discerned by gel filtration of [125I]hFSH G-10-1 incubates through Sephadex G-100. Lineweaver-Burke analysis of [125I]hFSH binding to particulate rat testis receptors in the presence or absence of varying concentrations of G-10-1 indicated a mixed competitive-noncompetitive type of binding inhibition. These results suggest that G-10-1 may interfere with [125I]hFSH binding through an interaction with the FSH receptor, although a possible interaction with the hormone as well cannot be ruled out at present. FSH-BI activity of fraction G-10-1 was stable after incubation at 56 C for 1 h but decreased significantly (75%) after incubation at 105 C for 24 h. FSH-BI activity was neither adsorbed by charcoal nor soluble in diethyl ether. It was, however, markedly diminished (72%) after treatment with N-bromosuccinimide. FSH-BI activity was also lost after incubation of fraction G-10-1 with immobilized carboxypeptidase Y (by 70%) or with immobilized aminopeptidase M (by 60%), suggesting that the inhibitor may be at least partially peptide in composition. © 1979 by The Endocrine Society.