Molecular Pharmacology, Vol 10, 963-974, Copyright © 1974 by the American Society for Pharmacology and Experimental Therapeutics

Organic Solvent Extraction of Liver Microsomal Lipid

II. Effect on the Metabolism of Substrates and Binding Spectra of Cytochrome P-450

¹ Department of Biochemistry and Drug Metabolism, Hoffman-La Roche, Inc., Nutley, New Jersey 07110

Extraction of lyophilized liver microsomes from 3-methylcholanthrene- or phenobarbital-treated rats with 1-butanol and acetone removed all the neutral lipids and 80% of the phospholipids; extraction with acetone alone removed all the neutral lipds but only trace amounts of the phospholipids. Recoveries of cytochrome P-450 and NADPH-cytochrome c reductase were 80-90%. Using this extraction procedure we examined several properties of the liver microsomal hydroxylating enzyme system and report the following. (a) Extraction of microsomes with 1-butanol and acetone decreased both 3,4-benzpyrene hydroxylase and benzphetamine N-demethylase activities. Addition of synthetic phosphatidylcholine to the extracted microsomes restored enzymatic activity to control levels. (b) Extraction of microsomes from phenobarbital-treated rats with 1-butanol and acetone increased the magnitude of both the hexobarbital- and benzphetamine-induced type I binding spectra and the ethanol- and phenacetin-induced reverse type I binding spectra. (c) Extraction of microsomes from 3-methylcholanthrene-treated rats with 1-butanol and acetone eliminated the hexobarbital-induced reverse type I binding spectra. (d) Extraction of microsomes from phenobarbital-treated rats with acetone greatly decreased the initial fast phase in the reduction kinetics of NADPH-dependent cytochrome P-450 reductase. Addition of 1 mM benzphetamine to the extracted microsomes restored the initial fast phase. These results indicate that while lipid is required for enzymatic activity, it is not essential for the binding of substrates to cytochrome P-450. In addition, the phenacetin- and ethanol-induced reverse type I binding spectra are not due simply to the displacement of endogenous substrates from cytochrome P-450.

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