REGULATION AND FUCTION OF P450 3A GENE FAMILY ISOZYMES

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DESCRIPTION: This is the first revision of a competitive continuation. The work is divided into four specific aims: SA1. To produce and purify a battery of Mab reagents directed against rat P450 3A1, 3A2 and 3Ax, and the related human P450s 3A3, 3A4, 3A5 and 3A7. It is proposed that members of the desired product set will be competent in ELISA assays, western blotting, inhibitory towards P450 activity in microsomal extracts, and that several antibodies binding to distinct epitopes will be found for each P450, to facilitate interspecies comparison of P450 homologues, for membrane topology analysis and immunocytochemical analysis. SA2. To study the age and sex specific regulation of 3A1 and 3A2 in the rat at the mRNA and protein level. SA3. To clone and characterize the cDNA for a novel 3A isotope (3Ax), recently identified by the principal investigator, and to study its expression as a function of age, sex and tissue type, as described in SA2. SA4. To use the inhibitory Mab s obtained in SA1, to selectively inhibit P450 expression in rat and human microsomal extracts, so as to establish the function of the various isotopes in the metabolism of selected substrates (CsA, progesterone, and others).