REGULATION AND FUNCTION OF P450III GENE FAMILY ISOZYMES

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The overall aim of this research is to clarify the functions and regulation of the proteins coded by the steroid-inducible cytochrome P450 family (cytochrome P450III gene) which is found in humans and other species. Although the contributions of individual members of this family toward the metabolism of substrates are unclear, previous studies have indicated that this family of cytochrome P450 plays a pivotal role in the metabolism of certain endobiotics (steroids), clinically important drugs (cyclosporin A and macrolide antibiotics), and widely occurring environmental contaminants (aflatoxin B1). Despite the high amino acid sequence homology among members of this gene family (e.g., 89% amino acid homology for two isozymes, PCNa and PCNb) we propose that it is possible to produce monoclonal antibodies specific for individual members of this family. It is important to distinguish between these isozymes as we hypothesize that there are sufficient structural differences between them to markedly influence the substrate specificity and regioselectivity of metabolism by these isozymes. These differences between isozymes in the types of metabolites formed in combination with differences in the extrahepatic distribution may influence the target organ specificity of carcinogens and toxicants. The specific aims of this project are: 1) to produce and purify a battery of specific-, epitope distinct- and inhibitory-monoclonal antibodies against rat PCNa, PCNb and PCNc some of which are positive on Western blots, 2) to use these antibodies to evaluate whether or not PCNc is a third member of the steroid-inducible family of P450, 3) to use differential immunoaffinity chromatography to isolate new members of this gene family for production of isozyme specific monoclonal antibodies, 4) to clone new members of this family for sequencing information and construction of specific probes, 5) to use these antibodies to quantitate the levels of individual isozymes in microsomes from the liver and extrahepatic tissues and thus provide an understanding of the regulation of these proteins, 6) to use these antibodies to assess the contributions of individual isozymes toward the microsomal metabolism of selected substrates, 7) to determine which of the human proteins (HLp, HLp2 and HLp3) of the cytochrome P450III gene family are structural and/or functional homologs of the rat, 8) to examine selected monkey tissue samples for expression of P450III family homologs. Factors which influence the activation of foreign compounds, including the induction of these enzymes which metabolize many compounds, directly influence human health and the risk from xenobiotic exposure.