The hepatic monooxygenase system consisting of NADPH-cytochrome P-450 reductase and cytochrome P-450 is involved in the oxidative metabolism of xenobiotics including therapeutic drugs, carcinogens, and environmental pollutants such as industrial wastes and pesticides. This enzyme system is also involved in the metabolism of endogenous compounds such as steroids and fatty acids. Both enzymes are intimately associated with the microsomal membrane and this forms the basis upon which this proposal is founded. From previous studies of this system as well as studies of other membrane bound enzymes, it appears that the lipid components of membranes influence the properties of membrane bound proteins. The objective of the studies outlined in this proposal is the elucidation of lipid-protein and protein-protein interactions within the microsomal membrane which influence the substrate specificity and the rate of hydroxylation catalyzed by the cytochrome P-450 system. To achieve this objective, experiments are outlined which involve the systematic in vitro modification of the lipid composition of the microsomal membrane and subsequent investigation of alterations in substrate binding and kinetic parameters. Microsomal fractions isolated from untreated rats as well as from rats pretreated with phenobarbital, 3-methylcholanthrene, and pregnenolone-16 alpha-carbonitrile will be used. Several techniques not previously utilized for the study of the hepatic monooxygenase system will be employed including phospholipid substitution of native and organic solvent extracted microsomal membranes. Phospholipid substitution will be achieved through the use of detergent or phospholipid exchange proteins. These studies should yield results which will be applicable to other membrane bound enzymes in addition to the hepatic monooxygenase system.
NIH ExPorter
NIH ExPorter