Identification of high affinity binding sites for inhibin on ovine pituitary cells in culture

The aim of this study was to identify and characterize binding sites for in hibin in primary cultures of ovine anterior pituitary cells. Recombinant hu man 31-kDa inhibin A was iodinated by an optimized lactoperoxidase procedur e. Fractionation of the labeled protein by gel filtration chromatography on Sephadex G-100 in 0.1 M HCl yielded two immunoactive peak regions, the sec ond of which was bioactive as assessed by in vitro bioassay, with a ratio o f bioactivity/immunoactivity of 0.62-0.77 and an iodine incorporation ratio of 1.7-2.0 mol I-125/mol inhibin. The specific binding of purified [I-125] inhibin to cultured ovine pituitary cells varied with time, temperature, an d cell number. Displacement of the tracer by unlabeled inhibin, as assessed by Scatchard analysis, revealed two binding sites with average K-d values of 0.28 and 3.9 nM and with approximately 250 and 3100 binding sites/anteri or pituitary cell, respectively. There was little cross-reaction between in hibin and activin A (<2%), transforming growth factor-beta (<0.2%), or foll istatin (much less than 0.1%), Examination of cell lines that were not expe cted to have inhibin receptors showed that there was no specific binding of inhibin to human leukemia (Jurkat) cells, whereas the binding to human emb ryonic kidney (293) cells was displaced by both inhibin and activin with a similar degree of crossreaction, which suggests binding to an activin recep tor. It is concluded that inhibin-binding sites with high affinity and spec ificity have been identified on ovine pituitary cells, consistent with both inhibin action on the pituitary and the presence of the putative inhibin r eceptor.