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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD018210-07
Application #
3315229
Study Section
Biochemical Endocrinology Study Section (BCE)
Project Start
1984-03-01
Project End
1992-02-29
Budget Start
1990-03-01
Budget End
1992-02-29
Support Year
7
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Texas MD Anderson Cancer Center
Department
Type
Other Domestic Higher Education
DUNS #
001910777
City
Houston
State
TX
Country
United States
Zip Code
77030
Moore, K H; Dunbar, B S; Bousfield, G R et al. (1994) Initial characterization of equine inhibin. Biol Reprod 51:63-71
Moore, K H; Dunbar, B S; Bousfield, G R et al. (1990) The heterogeneity of porcine 32,000 Mr inhibin alpha-subunit: a gel electrophoresis and immunoblot study. Endocrinology 127:1477-86
Gordon, W L; Bousfield, G R; Ward, D N (1989) Comparative binding of FSH to chicken and rat testis. J Endocrinol Invest 12:383-92
Bousfield, G R; Liu, W K; Ward, D N (1989) Effects of removal of carboxy-terminal extension from equine luteinizing hormone (LH) beta-subunit on LH and follicle-stimulating hormone receptor-binding activities and LH steroidogenic activity in rat testicular Leydig cells. Endocrinology 124:379-87
Sugino, H; Takio, K; Ward, D N (1989) Reevaluation of the amino acid sequence of porcine follitropin. J Protein Chem 8:197-219
Shome, B; Parlow, A F; Liu, W K et al. (1988) A reevaluation of the amino acid sequence of human follitropin beta-subunit. J Protein Chem 7:325-39
Bousfield, G R; Ward, D N (1988) Selective proteolysis of ovine lutropin or its beta subunit by endoproteinase Arg-C. Properties of the Arg beta 43 cleaved hormone. J Biol Chem 263:12602-7
Ward, D N; Wen, T; Bousfield, G R (1987) Sulfate and phosphate analysis in glycoproteins and other biologic compounds using ion chromatography. Application to glycoprotein hormones and sugar esters. J Chromatogr 398:255-64
Bousfield, G R; Liu, W K; Sugino, H et al. (1987) Structural studies on equine glycoprotein hormones. Amino acid sequence of equine lutropin beta-subunit. J Biol Chem 262:8610-20
Gordon, W L; Liu, W K; Akiyama, K et al. (1987) Beta-microseminoprotein (beta-MSP) is not an inhibin. Biol Reprod 36:829-35

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