Molecular Pharmacology, Vol 6, 213-220, Copyright © 1970 by the American Society for Pharmacology and Experimental Therapeutics

Properties of a Solubilized Form of the Cytochrome P-450-Containing Mixed-Function Oxidase of Liver Microsomes

¹ Department of Biological Chemistry, Medical School, The University of Michigan, Ann Arbor, Michigan 48104

The carbon monoxide-sensitive liver microsomal mixed-function oxiodase has recently been solubilized and resolved into three components: cytochrome P-450, TPNH-cytochrome P-450 reductase, and a heat-stable fraction. Of a variety of compounds tested as possible substrates in the reconstituted rat liver enzyme system, d-benzphetamine (N-benzyl-N,-dimethylphenylethylamine) was the most active. A series of n-alkanes (hexane, heptane, octane, nonane, decane, dodecane, tetradecane, and hexadecane) were shown to serve as substrates, as well as compounds such as cyclohexane, ethylmorphine, hexobarbital, aminopyrine, and norcodeine, which are known to undergo hydroxylation in intact microsomal suspensions. Trilaurin, diolein, and triolein appeared to be active, whereas certain other lipids, including a series of fatty acids, and aniline were not.

In the reconstituted rabbit liver enzyme system, hexane was the most active substrate, followed by cyclohexane and benzphetamine. A series of fatty acids front hexanoate to palmitoleate were found to undergo hydroxylation, in contrast to the results obtained in the rat liver enzyme system.

An absolute requirement for the heat-stable lipid fractoin was shown for the hydroxylation of the various drugs as well as of several alkanes. A study of the stoichiometry of benzphetamine demethylation resulting from hydroxylation of the methyl group and liberation of the resulting hydroxymethyl group as formaldehyde) indicated that equimolar amounts of TPNH and molecular oxygen were consumed and of formaldehyde were produced. The results corresponded to the stoichiometry expected of a mixed-function oxidase, provided that catalase was added to the enzyme system. In the absence of catalase, a significantly greater oxygen uptake was observed.

The K_m of benzphetamine was 1.8 x 10^-4 in the reconstituted enzyme system, and the K_s of benzphetamine, which was unaltered in the absence of the heat-stable lipid fraction, was 5.5 x 10^-4 M. Aniline and octante were shown to act as competitive inhibitors with respect to benzphetamine.

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ACKNOWLEDGMENT We wish to acknowledge the technical assistance of Miss Joanne Heidema.

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