Inhibin/activin β-subunit monomer: Isolation and characterization

David M. Robertson, Lynda M. Foulds, Michelle Prisk, Mark P. Hedger

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Abstract

Using an activin RIA that showed limited cross-reaction with inhibin, activin immunoactivity was monitored throughout the isolation of activin from bovine follicular fluid and side-fractions during the isolation of human recombinant inhibin. Two peaks of activin immunoactivity were identified in both materials and isolated to homogeneity by dye affinity chromatography, hydrophobic interaction and gel permeation chromatography, and reverse phase HPLC. The purified proteins in all four peaks had terminal amino acid sequences identical to those of the inhibin/activin β-subunit. The molecular masses determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing (and reducing) conditions were 25 and 15 and 15 and 15 kilodaltons (kDa) for each pair of proteins from both sources. Based on these criteria, the bovine and human recombinant 25-kDa proteins correspond to the inhibin/activin βA-subunit dimer (activin-A), while the 15-kDa proteins correspond to the inhibin/activin βA-subunit monomer. The activity of the monomer was 17% of the activity of the dimer in the activin RIA. Based on this level of cross-reaction and the proportion of monomer to dimer immunoactivity found after reverse phase HPLC of bovine follicular fluid, it is estimated that the levels of monomer in bovine follicular fluid are 25-60% those of the dimer. The biological activities of the human recombinant activin monomer and dimer were investigated in two different cell culture systems. In a rat pituitary cell system the activity of the activin monomer was 19% of the activity of the dimer in stimulating FSH release, while in rat thymocyte cultures the activity of the monomer was 45% the activity of the dimer in suppressing lectin-stimulated [3H]thymidine uptake. It is concluded that the βA-subunit monomer is found in bovine follicular fluid at a level 25-60% that of the βA-subunit dimer (activin-A). The monomer displays in vitro responses similar to those of the dimer, although the monomer is less active (18-45%) than the dimer. It is unclear if dimerization of the monomer is a necessary prerequisite for biological activity.

Original languageEnglish
Pages (from-to)1680-1687
Number of pages8
JournalEndocrinology
Volume130
Issue number3
DOIs
Publication statusPublished - 1992

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