IMMUNOPHARMACOLOGICAL IDENTIFICATION AND REGULATION OF CYTOCHROME P-450

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The goal of this research project is to understand the function and physiological regulation of the various forms of cytochrome P-450 using immunologically-based methodologies. The approach being undertaken is to generate anti-peptide antibodies, which permit the creation of antibodies which are specific for single P-450 enzyme and not a given P-450 family. The development of P-450 specific antibodies permits us to more precisely define the role of that P-450 enzyme in the metabolism of selected substrates. Current research is focused on the development of polyclonal and monoclonal antibodies against sequences unique to P450 IA2. This P-450 enzyme is a key enzyme in the metabolism of heterocyclic amine carcinogens. Several unique regions have been identified in P-450 IA2. Currently, there is no available antibody which specifically recognizes P-450 IA2. Another aspect to this work has been the elucidation of the extent to which P-450 IA2 is involved in the metabolism of acetanilide. Using monoclonal antibody 1-7-1, which recognizes both P-450 lAl and IA2, and cDNA-expressed mouse IAI and mouse and human IA2, we were able to demonstrate that IA2 is the major P-450 form responsible for the metabolism of acetanilide to its hydroxylated metabolites. P450s IAI, IIB1 and IIB2 did not metabolize acetanilide. MAb 1-7-1 inhibited 80% of the acetanilide hydroxylase activity of both mouse and human cDNA expressed IA2, though 24-fold more antibody was needed to achieve maximal inhibition of human IA2 as compared to that needed for mouse IA2. MAb 1-7-1 inhibited the acetanilide hydroxylase activity of 3-methylcholanthrene-induced liver microsomes by 80% also.