This proposal represetns a continuation of our efforts to isolate, purify, and characterize isozymes of cytochrome P-450 which are endogenous to liver microsomes of the untreated animal. Such procedures wil utilize sequential steps of hydrophobic, cationic and anionic column chromatography. Two forms of cytochrome P-450 have already been characterized and three more have reached a stage of purity whereby separation procedures will gear up for sufficient amounts for characterization. Attempts will be made to discern the role of these isozymes in maintaining the homeostasis of the animal; compounds of endogenous origin, e.g., testosterone, progesterone, estradiol, cholesterol and fatty acids will be examined as substrates. The type I binding site, the region where substrates bind, will be studied to determine its dimension. Substrates of increasing size will be used until restriction of carbon monoxide binding is observed. Pressure studies for volume change mesurements will also be performed. The amino acid composition of the type I site will be determined using amino acid reagents and measuring their affect on substrate binding and protection from reagents by substrates. The mechanism of action of the P-450 system will be studied in steps, assessing the influence of spin state of the ferric cytochrome and reductase binding on the kinetics of reduction. The oxygen sensitivity of the reaction will also be measured. These spectral studies will be backed by development of thermodynamic models and mathematical modeling. The roles of cytochrome b5 in P-450 monooxygenation will also be studied and attempts will be made to explain the mechanism of NADH synergism. In addition, differences in influence of cytochrome b5 on the NADPH-dependent metabolism of different substrates will be examined to determine whether metabolite patterns are altered. Attemps will be made to learn the reason for observed differences.
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