Isolation of a 31 kDa form of inhibin from bovine follicular fluid

Abstract

The introduction of a pH 4.75 precipitation step to a previously described purification procedure from bovine follicular fluid (bFF) resulted in the isolation of a 31 kDa form of inhibin, in addition to 58 kDa inhibin. The procedure was monitored by an in vitro bioassay based on the suppression of the FSH cell content by pituitary cells in culture. The 31 kDa form was purified 5550-fold with approximately 5% recovery. On SDS-polyacrylamide gel electrophoresis a single band was detected with a molecular weight of 31 000 ± 1500 (mean ± SD) which upon reduction gave 2 subunits of 20 200 ± 300 and 14 800 ± 600. The biological activity expressed on mg protein basis was similar for both 31 kDa and 58 kDa inhibin although on a molar basis the 58 kDa inhibin was 2–3 times higher. A high degree of cross-reaction was observed between both forms in a radioimmunoassay of bovine inhibin using an antiserum raised against the larger form with either iodinated 31 kDa or 58 kDa inhibin as tracer. Based on the subunit composition of the 31 kDa and 58 kDa inhibin, their similar cross-reaction in a radioimmunoassay system and the apparent generation of the 31 kDa inhibin following a pH precipitation step, it is concluded that 31 kDa inhibin is a smaller form of the 58 kDa inhibin resulting from a shortening of the 43 kDa subunit to a 20 kDa subunit. The observation on a molar basis that 58 kDa inhibin has 2–3 times the biological activity of 31 kDa inhibin suggests that the extension of the 20 kDa subunit to 43 kDa does contribute to the biological activity of the molecule.