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 was formed by proteolytic processing of the 50-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.
Sugino
K
K
Frontier Research Program, Institute of Physical and Chemical Research (RIKEN), Saitama, Japan.
Nakamura
T
T
Takio
K
K
Miyamoto
K
K
Hasegawa
Y
Y
Igarashi
M
M
Titani
K
K
Sugino
H
H
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002417:Cattle; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005640:Follicle Stimulating Hormone / Q000378:metabolism*; D006801:Humans; D007265:Inhibins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D006080:Ovarian Follicle / Q000737:chemistry*; D010903:Pituitary Gland, Anterior / Q000187:drug effects / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D014462:Ultrafiltration / Q000379:methods
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D007265:Inhibins; D005640:Follicle Stimulating Hormone