Resolution of the Cytochrome P-450-containing ω-Hydroxylation System of Liver Microsomes into Three Components

Abstract

Abstract The enzyme system in liver microsomes which catalyzes the ω-hydroxylation of fatty acids in the presence of molecular oxygen and a reduced pyridine nucleotide has been solubilized and resolved into fractions containing cytochrome P-450, a cytochrome P-450 reductase, and a heat-stable factor. The apparent Km values of TPNH and DPNH are 2.0 x 10-5 m and 6.3 x 10-4 m, respectively. As a further indication of the role of cytochrome P-450, ω-hydroxylation is enhanced in microsomes prepared from animals previously treated with phenobarbital and is inhibited in the presence of carbon monoxide, with partial reversal of the inhibition by exposure to light. The soluble preparation of cytochrome P-450 exhibits a carbon monoxide difference spectrum, an electron paramagnetic resonance spectrum, and substrate difference spectra similar to those previously attributed to the microsome-bound form. The difference spectrum obtained in the presence of laurate, with a peak at about 388 mµ and a trough at 419 mµ, is of the type given by hexobarbital rather than aniline. The Ks of laurate, determined from difference spectra, is 6.3 x 10-4 m, and the Km, determined in the complete hydroxylation system, is 4.4 x 10-5 m. The partially purified reductase was shown to catalyze electron transfer from TPNH to cytochrome P-450 under anaerobic conditions. The TPNH-cytochrome c reductase activity of the enzyme preparations was found to be unrelated to their ability to reduce cytochrome P-450, and therefore to function in the hydroxylation system. The activity of the heat-stable fraction is attributed to a lipid component which enhances the rate of the hydroxylation reaction.