HIGH-RATES OF SUBSTRATE HYDROXYLATION BY HUMAN CYTOCHROME-P450 3A4 INRECONSTITUTED MEMBRANOUS VESICLES - INFLUENCE OF MEMBRANE CHARGE

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