Hydroxylation of benzphetamine and other drugs by a solubilized form of cytochrome P-450 from liver microsomes: lipid requirement for drug demethylation.

A solubilized hepatic microsomal enzyme system previously shown to catalyze the w-hydroxylation of fatty acids also catalyzes the hydroxylation of drugs. Benzphetamine, aminopyrine, ethylmorphine, hexobarbital, norcodeine, and p-nitroanisole undergo aerobic demethylation in the presence of NADPH and the resolved enzyme system. The required submicrosomal components for benzphetamine demethylation, as determined either by formaldehyde liberation or by NADPH oxidation, are cytochrome P-450, NADPH-cytochrome P-450 reductase, and a heat-stable lipid fraction. Similar requirements were shown for the oxidation of aminopyrine, ethylmorphine, and hexobarbital. Laurate and benzphetamine were found to be mutually inhibitory, as would be expected if a common "methyl hydroxylase" were involved. The solubilized cytochrome P-450 preparation exhibits a difference spectrum in the presence of benzphetamine with a peak at 392 ml and a trough at 427 rnp and difference spectra with aniline and hexobarbital typical of those obtained with the microsomal bound .form of this hemoprotein. Cytochrome P-450, the carbon monoxide-binding pigment of microsomes (l), serves as a mixed function oxidase effecting the hydroxylation of steroids and the oxidative demethylation and hydroxylation of drugs in animal tissues (2-4). Attempts to solubilize this pigment by a variety of techniques resulted in the extensive formation of P-420 (l), an altered form possessing no catalytic activity. Recent studies in our laboratory on the w-hydroxylation of fatty acids in liver microsomes led to "solubilization" of the enzyme system by the criterion that enzymatic activity remained in