The milestone publication of Mason et al. (Nature 318: 659, 1985) dictates a redefinition of inhibin chemistry and the unsolved aspects remaining. This proposal has applied those advances in our own lab as well as the new information from the Mason report to redirect our studies to these new goals. Our studies will utilize the genomic cosmid libraries we have prepared from pig, human, horse, cow, mouse, and chicken to evaluate the distribution of the inhibin gene(s) in these species. We will also evaluate the expression of the gene in selected tissues by means of Northern blot probes of the mRNA. 1) The carbohydrate moieties of inhibin (composition and structure). 2) The disulfide placements in the inhibin molecule. For these two studies we will use selective protein degradation methods to isolate the glycopeptides and disulfide-containing peptides, respectively. Appropriate pre-treatments will be applied to simplify the approach as much as possible (e.g. reduciton and alkylation of the cystine residues, followed by subunit separation in the case of the glycopeptide study). This type of study requires the native hormone (e.g. it cannot be done with recombinant DNA methods). We will characterize the higher molecular weight forms of inhibin (greater than 32,000). These higher molecular weight forms are obtained in good yield in the course of our purification of the 32,000 molecular weight form, and have been consistently observed in at least 4 other reports from labs that provide a good accounting of inhibin activity. Availability of our highly purified inhibin preparations now makes it possible to develop newer, more sensitive (and expediant) assays. Thus we will develop (1) radioimmunoassays, (2) ELISA-type assays (a form of immunoassay that couples enzyme-antibody to bypass the need for radioactive tracers), and (3) radioligand assays. The latter type of assay has been very useful in our functional group substitution experiments to learn something of the mechanism of action of thee glycoprotein hormones.
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