In a biochemical sense, the mixed-function oxidase system represents the first line of defence between man and his environment. This system is responsible for the early metabolic steps in they detoxification of xenobiotics as well as the metabolic activation of certain chemical carcinogens. Hence, the response of the cell to foreign compounds and the eventual fate of the cell depends upon the activity level of these key enzymes. This proposal is an extension of our ongoing studies dealing with the structure. function, and regulation of NADPH-cytochrome P450 oxidoreductase (P450R) and the cytochrome 3A family. Four major areas will be addressed during the next granting period. The first will examine those structural features of the reductase molecule that are responsible for maintaining optimal alignment and integrity of the flavin domains. Three specific regions will be analyzed. These are the interconnecting flavin domain, the carboxyl terminal region that is important for the structural and functional character of the FAD/NADPH binding domains and is the port of entry for electrons from NADPH, and the hinge region close to the FMN domain. The second area will focus on identify the recognition site(s) on the surface of P450R responsible for the selective binding of cytochrome P450 and other protein substrates such as cytochrome b5 and heme oxygenase. Third, the regulation of the P450R and CYP3A23 genes will be investigated in order to better understand the factors controlling the cellular levels of these enzymes. Fourth, the biological role of P450R will be evaluated by studying the developmental and tissue-specific expression using in situ hybridization and immunohistochemistry along with the creation of a mouse strain with a null allele at the P450R locus.
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