The overall goal of the reseach project is to determine the role of P-450 oxygenases in alcohol metabolism and toxicity. Our laboratory has recently isolated a new form of P-450 (isozyme 3a) from liver microsomes of rabbits chronically treated with ethanol and shown that this cytochrome has relatively high activity in ethanol oxidation in the reconstituted enzyme system.
The specific aims are: (a) to characterize ethanol-inducible P-450 from various sources and determine its quantitative role in ethanol oxidation; (b) to determine whether alcohol-inducible P-450 catalyzes the oxidation of other hydroxy compounds of biological or toxicological importance; (c) to examine other aspects of the P-450 oxygenase system such as alterations in biosynthesis or interaction of the components as an explanation of the metabolic effects of ethanol; and (d) to elucidate the mechanism of ethanol oxidation by P-450 isozyme 3a in comparison to that of the other less active isozymes and of the reductase alone. The extent to which ethanol-inducible P-450 participates in ethanol oxidation will be determined by the inhibitory effect of isozyme 3a antibodies added to microsomal suspensions. An attempt will be made to find a specific inhibitor of ethanol oxidation by P-450-LM3a for studies in tissue slices and intact animals, and blood ethanol clearance in vivo in normal and alcohol-induced rabbits will be compared with the in vitro data on P-450-mediated alcohol oxidation. Although catalysis by P-450 is probably not a major pathway, this cytochrome may play a significant role in ethanol oxidation in individual organelles, tissues, or species, or in animals or man chronically exposed to ethanol. Studies to determine the mechanism of electron transfer and oxygen activation by the various P-450 isozymes will include attempts to account for the O2 and NADPH consumed in excess of known products formed, stopped flow spectral identification of active oxygen species derived from the O2-ferrous complex, examination of the role of peroxy compounds substituted for NADPH and O2, determination of the rate-limiting step(s), and site-directed mutagenesis to determine the function of specific amino acid residues in the enzyme.
Showing the most recent 10 out of 44 publications
