Interactions between solubilized cytochrome P-450 and hepatic microsomes.
Solubilized cytochromes P-450 and P-448 have been prepared from liver microsomes of phenobarbital- and 3-methylcholanthrene-pretreated rats, respectively. These hemoproteins can bind to microsomes and increase the microsomal monoxygenase activities. The binding of cytochrome P-450 enhances the microsomal benzphetamine demethylase activity, whereas cytochrome P-448 enhances the ethoxycoumarin dealkylase and benzo[a]pyrene hydroxylase activities. The added cytochrome P-450 is believed to be incorporated into the microsomal membrane, and the enriched microsomes can be separated from the free hemoprotein by gel filtration. A correlation between the increased cytochrome P-450 content and the enhanced catalytic activity of the microsomes is shown. Several lines of evidence suggest that the exogenous cytochrome P-450 molecules become catalytically active only when they are incorporated into the membrane. By measuring the enhanced ethoxycoumarin dealkylase activity, the rate of the proposed incorporation of cytochrome P-448 into microsomes can be measured, and the temperature dependence of the rate is reported. The addition of cytochromes P-448 and P-450 causes a great increase in the monoxygenase activities of microsomes which have been treated with linoleic acid hydroperoxide. The hydroperoxide treatment denatures almost all the cytochrome P-450 molecules in the microsomes but retains most of the NADPH-cytochrome P-450 reductase activity. Experiments with such microsomes indicate that the added cytochrome P-450 molecules, after incorporation into the membrane, have a direct access to the reductase molecules and are able to receive electrons directly from the latter. The present results are consistent with a nonrigid model for the organization of cytochrome P-450 and NADPH-cytochrome P-450 reductase in the microsomal membrane.
D000818:Animals; D001665:Binding Sites; D003374:Coumarins; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005019:Ethyl Ethers; D007700:Kinetics; D008297:Male; D008748:Methylcholanthrene / Q000494:pharmacology; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*; D010634:Phenobarbital / Q000494:pharmacology; D011485:Protein Binding; D051381:Rats; D012995:Solubility
D003374:Coumarins; D005019:Ethyl Ethers; D008748:Methylcholanthrene; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D010634:Phenobarbital