Abstract
The liver microsomal enzyme system which hydroxylates fatty acids, hydrocarbons, and a variety of drugs and other foreign compounds was previously solubilized and resolved into three components: cytochrome P-450, NADPH-cytochrome P-450 reductase, and phospholipid. The two enzymes were partially purified from pyrophosphate-treated, cholate-solubilized liver microsomes of phenobarbital-treated rabbits by fractionation with polyethylene glycol 6000 and DEAE-cellulose column chromatography in the presence of Renex-690, a nonionic detergent. These steps were followed by treatment with Amberlite XAD-2 and calcium phosphate gel. Cytochrome P-450 preparations purified to a content as high as 15 nmoles per mg of protein were free of cytochrome b5 and contained no significant amount of NADPH-cytochrome c reductase, NADH-cytochrome c reductase, nonheme iron, other metals as measured by neutron activation analysis, or labile sulfide. The molecular weight was judged to be about 280,000 by gel exclusion chromatography and sucrose density gradient centrifugation. Polyacrylamide gel electrophoresis of the partially purified cytochrome P-450 preparation treated with sodium dodecyl sulfate and mercaptoethanol showed the presence of two major polypeptide bands, one with a molecular weight of 47,000 to 49,000 and the other 52,000 to 53,000. The component of lower molecular weight corresponded to the major polypeptide induced by phenobarbital treatment, as shown by electrophoresis of the proteins in normal and induced microsomes. The partially purified preparation accepted 2 electrons from dithionite per molecule of cytochrome P-450 under anaerobic conditions, indicating the presence of another electron acceptor in addition to the iron atom of the hemeprotein. The partially purified NADPH-cytochrome P-450 reductase preparations, which catalyzed the reduction of 8.6 to 10.0 µmoles of cytochrome c per min per mg of protein at 30°, were free of cytochrome P-450 and contained only a trace of NADH-cytochrome c reductase. FMN and FAD were found to be present in equimolar amounts, and labile sulfide was absent. The reconstituted enzyme system was active in the hydroxylation of fatty acids (laurate), hydrocarbons (hexane, cyclohexane, and octane), drugs (benzphetamine, hexobarbital, and ethylmorphine), and aniline. The presence of both phosphatidylcholine and deoxycholate was necessary for maximal activity.
Highlights
During gel chromatography a small amount of the cytochrome p-450 was converted to cytochrome p-420, whereas no such denaturation was observed during the centrifugation experiment
In Experiment 2, a comparison was made of liver microsomes from normal and induced animals, and it is apparent that treatment with phenobarbital greatly increased the concentration of the polypeptide band indicated by the arrow
Cytochrome P-450 purified to a concentration of 13.2 nmoles per mg of protein by the procedures described in the present paper was titrated with dithionite under anaerobic conditions with CO present, and the formation of the reduced CO complex was followed by the appearance of the characteristic peak at 450 nm
Summary
The liver microsomal enzyme system which hydroxylates fatty acids, hydrocarbons, and a variety of drugs and other foreign compounds was previously solubilized and resolved into three components: cytochrome P-450, NADPH-cytochrome P-450 reductase, and phospholipid. The two enzymes were partially purified from pyrophosphate-treated, cholate-solubilized liver microsomes of phenobarbital-treated rabbits by fractionation with polyethylene glycol 6000 and DEAE-cellulose column chromatography in the presence of. These steps were followed by treatment with Amberlite XAD-2 and calcium phosphate gel. Cytochrome P-450 preparations purified to a content as high as 15 nmoles per mg of protein were free of cytochrome bS and contained no significant amount of NADPH-cytochrome c reductase, NADH-cytochrome c reductase, nonheme iron, other metals as measured by neutron activation analysis, or labile sulfide. The molecular weight was judged to be about 280,000 by gel exclusion chroma.tography and sucrose density gradient centrifugation
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