Isolation of bovine follicular fluid inhibin of about 32 kDa

Abstract

Inhibin of about 32 kDa from bovine follicular fluid (bFF) was purified by using chromatographies operated under protein-dissociating conditions, which we have established for our previous purification of porcine follicular fluid (pFF) inhibin. On a gel filtration, bFF inhibin activity was eluted at 3 distinct regions corresponding to apparent molecular weight of 96, 55 and 32 kDa, representing 17%, 27% and 24% of the total inhibin activity in the follicular fluid, respectively. The smallest inhibin, named 32 kDa bFF inhibin, that evidently suppressed the basal secretion of FSH from the pituitary cells, was purified to homogeneity with a 5330-fold purification factor in a recovery yield of ca. 11%. 32 kDa bFF inhibin thus purified consists of 2 polypeptide chains (A-chain: 20 kDa and B-chain: 13 kDa), linked by disulfide bridges. N-terminal sequences were Ser-Thr-Pro-Pro- for the A-chain and Gly-Leu-Glu-Cys- for the B-chain. The identical N-terminal sequences were also found in 32 kDa pFF inhibin, except that Pro-3 of the bFF A-chain is substituted by Ala in the 20 kDa chain of pFF inhibin.