High rates of substrate hydroxylation by human cytochrome p450 3A4 in reconstituted membranous vesicles: Influence of membrane charge

CYP3A4 represents the most important form of human cytochrome P450 active in drug metabolism. Reconstitution of this enzyme has in the past been a major problem, Using purified cDNA-expressed CYP3A4 incorporated into membranous vesicles made from microsomal phospholipids, rates of nifedipine and testesterone oxidation of about 60 nmol/nmol P450/min were achieved, whereas similar reconstitution into dilauroylphosphatidylcholine micelles was unsuccessful. A higher V-max for nifedipine oxidation was obtained in negatively charged vesicles as compared to neutral membranes, whereas the membrane charge did not influence the K-m. It is concluded that the native function of CYP3A4 requires a negatively charged microsomal membrane. (C) 1996 Academic Press, Inc.