Incorporation of cytochrome b5 into rat liver microsomal membranes : Impairment of cytochrome P-450-dependent mixed function oxidase activity

Abstract

Cytochrome b 5 was purified to electrophoretic homogeneity from the liver microsomes of untreated rats and reincorporated into liver microsomes from phenobarbital-treated rats, resulting in an approximate three-fold enrichment of the cytochrome b 5 specific content (1.5 nmol haemoprotein · mg −1 protein). Our results have shown that the N-demethylation of benzphetamine was progressively inhibited in cytochrome b 5-fortified microsomal preparations. Using stopped flow, visible difference spectrophotometry, the NADPH-driven reduction kinetics of cytochrome P-450 were examined in the modified microsomes over the first few seconds of reaction. Increasing the amount of incorporated cytochrome b 5 resulted in a progressive inhibition of the initial, fast phase reduction rate constant of microsomal cytochrome P-450, both in the absence and presence of the type I substrate benzphetamine. Although the initial rate of NADPH-driven cytochrome b 5 reduction was the same for both native and cytochrome b 5-fortified microsomes, the extent of cytochrome b 5 reduction was greater in the fortified microsomes. If cytochrome b 5 has a positive role to play in cytochrome P-450-dependent mixed function oxidase activity either as an effector or in electron transfer or both, the former haemoprotein must be already present in sufficient concentrations in the native microsomes.