The proposed research is concerned with cytochrome P-450-containing liver microsomal enzyme system which catalyzes the hydroxylation of steroids, fatty acids, and prostaglandins as well as a variety of foreign compounds, including drugs, petroleum products, anesthetics, pesticides, and carcinogens. Those isozymes of rabbit liver microsomal cytochrome P-450 (P-450LM) not previously obtained in homogeneous form will be purified and characterized by electrophoretic and immunological methods as well as by amino acid analysis, peptide mapping, kinetic constants, spectrometric techniques (visible, UV, CD, and EPR), and analysis for end-groups, carbohydrates, and lipids. The complete amino acid sequence of each P-450 will be undertaken, beginning with phenobarbital-inducible P-450LM2, 5,6-benzoflavone-inducible P-450LM4, and constitutive P-450LM3b, which are available in the largest amounts. NADPH-cytochrome P-450 reductase will be further studied to determine the individual roles of FMN and FAD. The activities of the cytochromes will be compared in the reconstituted enzyme system and in liposomes and with activities enhanced in microsomes by induction. If time permits, similar studies will be carried out with P-450 systems in other organelles or tissues, as well as with the P-450-containing monooxygenase system of Candida tropicalis and the nonheme iron-containing monooxygenase system of Pseudomonas oleovorans.
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