This project will continue to investigate the molecular factors that determine the mechanism of the binding of steroid hormones to specific binding sites on macromolecules. New affinity labeling (site-directed irreversible inhibitor) analogs of steroid hormones will be synthesized for use as reagents in these studies. Included is a new series of estradiol derivatives with 14C-bromoacetate side chains. They will be used to affinity label the active site of 17Beta-estradiol dehydrogenase, isolated from human placenta. New progesterone and cholesterol analogs with 14C-bromoacetate side chains will be synthesized and then used to affinity label the active site of cytochrome P-450 (steroid-specific) side chain cleavage enzymes, recently isolated from porcine testis and adrenals. 20Alpha-Hydroxysteroid oxidoreductase (20Alpha-HSD) from ovine fetal erythrocytes will be isolated and characterized by methods developed in the PI's laboratory. Then the active site of 20Alpha-HSD will be affinity labeled with substrate analogs (described above). Peptides from each of the 14C-labeled active sites will be isolated following proteolytic digestion of the affinity labeled enzymes. The peptides will be subjected to amino acid sequence analysis. The amino acid configurations of the enzyme active sites will be compared for clues to the relationships among configurations, steroid binding characteristics, and enzyme catalysis. The isolated ovine fetal 20Alpha-HSD will be injected into rabbits to generate antibodies. The antibodies will be used to measure by radioimmunoassay 20Alpha-HSD in blood from lamb fetuses in utero. This will give a profile of 20Alpha-HSD production in fetal blood throughout pregnancy. The 20AlphaHSD antibody will also be used to compare the structural relationship between ovine and bovine fetal 20Alpha-HSD. Affinity labeling steroids which rapidly inhibit ovine 20Alpha-HSD in vitro will be infused through a cannula into lamb fetuses in utero in an attempt to block this enzyme activity in vivo. This may reveal the role of 20Alpha-HSD in fetal development. Continuation of the in vitro affinity labeling experiments with the enzymes (above) are expected to further our understanding of the molecular factors which promote steroid binding and catalysis at the enzyme active sites. Greater insights into these factors which control estradiol- and progesterone-specific enzyme reactions will advance molecular concepts of steroid biosynthesis. This information is expected to provide foundation for developing sex steroids that may be useful in controlling reproduction and also fetal development.
