Biochemical studies on cytochrome P-450 solubilized from liver microsomes: partial purification and mechanism of catalysis.

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The cytochrome P-450 of the hepatic endoplasmic reticulum is of particular interest to biochemists and pharmacologists, because of its inducibility by many agents and its remarkably broad substrate specificity. Indeed, it is probably no exaggeration to state that it is the most versatile biological catalyst known. Not only physiological substrates, such as steroids and fatty acids, but also many foreign compounds, including drugs, anesthetics, petroleum products, insecticides, and so on, are attacked by molecular oxygen in the presence of NADPH as an electron donor and cytochrome P-450 as the catalyst. The hydroxylation of foreign substances is generally viewed as an important initial step in their detoxication, but in some instances the compounds so modified may become more toxic or even carcinogenic. Although much has been learned about the inducibility, specificity, and other properties of this enzyme system through work with liver microsomal suspensions, it seems likely that some of the more puzzling questions will not be answered without purification and characterization of the individual components. Two of the more difficult questions are: whether one or more forms of cytochrome P-450 account for the broad specificity, and whether oxygen undergoes activation in the presence of cytochrome P-450 to produce some species more reactive toward these substrates, most of which are lipophilic in nature and are often attacked at positions that do not appear to be highly chemically reactive. Hopefully, the conclusions reached will be applicable to the membranous system of microsomes and will prove useful to our understanding of the disposition of drugs and other foreign substances in the intact organism. Several years ago, this laboratory reported the resolution of the liver microsoma1 enzyme system by treatment with deoxycholate in the presence of stabilizing agents and ion-exchange chromatography of the resulting extract.'.* The three components that were found to be necessary for the hydroxylation of a variety of substrates are cytochrome P-450, NADPH-cytochrome P-450 reductase, and a heat-stable, chloroform-soluble fraction which contains phosphatidylcholine as the active component.'*' The reductase, which has NADPH-cytochrome c reducing activity, has been partially purified, and the lipid factor may be replaced by synthetically prepared phosphatidylcholines. This paper is concerned with the properties of the cytochrome P-450 fraction, which has recently been partially purified but is not yet homogeneous, and with the possible role of superoxide in substrate hydroxylation in this reconstituted enzyme system. As has been described elsewhere, the cytochrome P-450 is SO~U* This research was supported by Grant GB-30419X from the National Science Foundation and Grant AM-10339 from the United States Public Health Service.