Two proteins with structural characteristics similar to peptide sequences identified in the inhibin alpha-subunit precursor sequence have been isolated from bovine follicular fluid. A side-fraction from the purification of bovine follicular fluid inhibin with high levels of inhibin immunoactivity relative to its inhibin bioactivity was fractionated through a sequence of procedures which included triazine dye affinity and phenyl-Sepharose chromatography, gel permeation chromatography on Sephadex G-100, reverse phase HPLC, and preparative polyacrylamide gel electrophoresis. The first of the two proteins identified had a molecular mass of 25-26K under reducing and nonreducing conditions and a NH2-terminal sequence identical to that of 43K inhibin alpha-subunit and showed minimal activity (less than 2% activity) compared with bovine 31K inhibin in either the inhibin in vitro bioassay or the RIA. These data suggest that this protein is the alpha 1-166 sequence of the bovine inhibin alpha-subunit (designated alpha N-subunit), most likely released after processing of either the inhibin alpha-subunit precursor or the 43K alpha-subunit involved in the conversion of 58K to 31K inhibin. The other protein identified (designated pro-alpha C-subunit) has a molecular mass of 27K under nonreducing conditions and 20K and 6K under reducing conditions. It is inactive in the in vitro bioassay, although highly reactive in the inhibin RIA, and has NH2-termini identical to the pro sequence of the inhibin alpha-subunit precursor and the 20K alpha-subunit sequence. These results suggest that pro-alpha C is a disulfide-linked structure and may represent an intermediate in the dimerisation of alpha- and beta-subunits to form inhibin while the alpha N-subunit is probably a proteolytic product of either the alpha-subunit precursor or 58K inhibin.
Anterior pituitary extracts from intact and 4 week postcastration male and female rats were electrofocused in sucrose density gradients within the pH range 3.5-10. Column fractions were combined to cover this pH range in 0.5 pH units and assayed for LH by in vitro bioassay and RIA and for FSH by radioreceptor assay and RIA. The pH distribution of bioactive LH was altered after castration in both sexes, with the proportion of recovered activity in the alkaline pH range increasing (P less than 0.01) from 52-57% in the intact animal to 71-73% after castration. In addition, significantly more bioactivity was recovered in the pH range 7-9.5 with the male (37%) than with either of the female (diestrous, 30% or proestrous, 28%) groups (P less than 0.05). FSH receptor binding activity was located in the pH region 3.5-6.5. Significantly less receptor binding activity was recovered in the pH range 3.5-4.5 with the female groups (39% and 37% diestrous and proestrous, respectively) than the male group (61%; P less than 0.05, P less than 0.01). The distribution of immunoreactive LH and FSH was similar to that observed with the LH in vitro bioassay and FSH radioreceptor assay. It is concluded that the charge distribution of pituitary gonadotropins is altered according to the sex of the animal and after castration. These findings provide further evidence that the type of gonadotropin produced by the pituitary is under endocrine control.
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