PURIFICATION AND CHARACTERIZATION OF HIGH-MOLECULAR-WEIGHT FORMS OF INHIBIN FROM BOVINE FOLLICULAR-FLUID

High molecular mass forms [95 kilodaltons (kDa)] of bovine inhibin-A as well as the known forms of intermediate (55 kDa) and low (32 kDa) mass were purified from bovine follicular fluid by ion exchange chromatography on DEAE-Sepharose, immunoaffinity chromatography using a monoclonal antibody directed against bovine 32-kDa inhibin-A, gel permeation HPLC on TSK-gel, and reverse phase HPLC. The 95-kDa inhibin-A had similar suppressive activity on FSH secretion from cultured rat anterior pituitary cells as the 55- and 32-kDa inhibins. There is, however, a possibility that the inhibin activity detected with larger forms may be due to that of the 32-kDa form that results from proteolytic processing during incubation with rat pituitary cells. Both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis using monoclonal antibodies specific for 32-kDa inhibin alpha- or beta(A)-subunits revealed that the 95-kDa inhibin preparation contained two forms of inhibin (105 and 95 kDa), which were composed of either a 50- or a 40-kDa alpha-subunit linked by a disulfide bond(s) to a 55-kDa beta(A)-subunit. Amino-terminal sequence analysis showed that the 50-kDa alpha-subunit and the 55-kDa beta(A)-subunit were generated by removal of a signal peptide from each corresponding primary translation product [the first NH2-terminal 17 residues of the inhibin alpha-subunit (residues 1-360) and the first 20 residues of the inhibin beta(A)-subunit (residues 1-425)] and suggested that the 40-kDa alpha-subunit. On the basis of our findings, we propose that in bovine follicular fluid, the larger 105-kDa form of inhibin is processed successively to form the lowest molecular mass form, 32 kDa inhibin, through the smaller 95- and 55-kDa forms.