The objectives of the research proposed are to define the biochemistry, function, and physiological roles of the 17beta-hydroxysteroid dehydrogenases (17beta-HSDs), a group of isozymes crucial to the biosynthesis and activation/inactivation of sex steroid hormones. Over the last four years, we isolated and characterized cDNAs and genes that encode two distinct isozymes of 17beta-HSD, designated type 2 and type 3, the biochemical properties and tissue distributions of which are different and distinct. These findings pose a number of questions relating to the physiological roles of the two isozymes, and how their different biochemical properties influence these roles. The initial goal of the studies proposed is to conduct detailed analyses of the tissue distribution, cell type-specific expression and ontogeny of the two isozymes. These analyses will be accomplished by immunoblotting, immunohistochemistry, RNA blotting, in situ mRNA hybridization, and 17beta-HSD isozyme activity assays. The second objective is to purify the type 2 isozyme to homogeneity. The kinetic, pharmacological, and structural properties of the isozyme will be determined and related to its intracellular localization and function. The third goal is to identify and characterize key mutations in the 17beta-HSD type 3 gene among male pseudohermaphrodites caused by 17beta-HSD deficiency. some of these mutations give a gent that encodes an enzyme with modified Michaelis-Menten constants for substrate or cofactor; the study of these modifications will provide important information concerning the structural domains of the protein. The identification of mutations will be accomplished using the polymerase chain reaction followed b single strand DNA conformation polymorphism and sequence analysis of amplified DNA. The final objective of this research is to expand the structure-function information derived from naturally occurring mutations by creating, and expressing a limited number of site-directed mutations in the 17beta-HSD type 3 cDNA using the baculovirus system. These studies will be focused on identifying amino acid sequences that form the cofactor and substrate binding domains of the protein. Detailed kinetic, pharmacological and structure properties of the purified mutant enzymes will be compared with those of the normal enzyme.
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