The overall goal of the research project is an improved understanding of the structure-function relationships and control mechanisms for mammalian cytochrome P-450. Such information is essential for an adequate evaluation of the role of this large and versatile family of cytochromes in the metabolism of steroids and prostaglandins and in the conversion of environmental chemicals either to more toxic substances, including carcinogens, or to detoxified products.
The specific aims are as follows: (a) to isolate and characterize the remaining unidentified or inadequately identified P-450 isozymes of rabbit liver and determine their physical and chemical properties, including amino acid sequences. The cytochromes to be purified include one for which this laboratory has already obtained the predicted sequence from an isolated gene (P-450z) and other P-450's that are induced by clofibrate, flavonoids, cholestyramine, debrisoquine, or starvation, are sex-specific, or are responsible for the oxygenation of steroids and prostaglandins in various positions. Attempts to crystallize these membrane-bound enzymes for the purpose of X-ray diffraction will be continued, and additional genes, particularly of the """"""""phenobarbital family,"""""""" will be isolated and characterized. A related goal is to carry out site-specific mutagenesis of selected P-450 amino acid residues that are likely to play a role in P-450 catalysis or the binding of substrates and electron donors; (b) to determine the substrate specificity of liver microsomal P-450's under conditions more typical of the in vivo situation, with partially limiting substrate and electron carrier concentrations. The rates of competing reactions in which 0-2 is converted to H-20-2 or undergoes 4-electron reduction with the presumed formation of water will also be determined; and (c) to study factors that control the activity of the P-450 system, such as induction and repression, possible allosteric effectors, and cytochrome b-5. The effects of b-5 are striking in that it stimulates some reactions and inhibits others. The mechanism, which apparently involves electron transfer to or from b-5 by the Fe-II-0-2 complexes of the P-450 isozymes, will be studied by stopped flow spectrophotometry. Preliminary results indicate that b-5 reacts differently with different P-450's and controls the rate of formation of several other oxygen-containing species from the ferrous dioxygen complex.
Showing the most recent 10 out of 54 publications
