Interactions between solubilized cytochrome P-450 and hepatic microsomes.

Solubilizedcytochromes 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[ulpyrene 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 NADPHcytochrome 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.The endoplasmic reticulum of liver cells contains a monoxygenase system which catalyzes the biotransformation of steroids, fatty acids, drugs, carcinogens, insecticides, anes-* This work was supported by Grant CA-16788 from the National Cancer Institute and Grant 472 from the Nutrition Foundation.This is Paper 2 in the series of studies on "Interactions between Solubilized Cytochrome P-450 and Hepatic Microsomes."Paper 1 is Ref. 7.