Localization of Cytochrome P450 in Membranes: Reconstituted Systems

Abstract

Since the successful isolation of the components of the cytochrome P450 dependent monooxygenase system by Lu and Coon (1968), their reconstitution to an enzymatically active system was established by the use of phosphatidylcholines by Strobel et al. (1970). Among various acyl derivatives of glycerol-3-phosphorylcholine the dioleoyl-derivative was most effective. The most commonly used and convenient system for reconstitution in numerous studies over the years and until now for routine work consists of the appropriate purified cytochrome P450 (0.3–1µM), a 1.5 molar excess of purified NADPH-cytochrome P450 reductase (reductase) and 30 µM l-α-dilauroylglyceryl-3-phosphatidylcholine (DLPC). This system was described by Guengerich et al. (1982) for eight different rat liver cytochrome P450 forms for use with numerous substrates. Generally the enzymatic reaction is started by the addition of NADPH or an NADPH-regenerating system. This simple reconstitution system has been found to be very reproducible, so that data from different laboratories can often be easily compared if the same incubation conditions have been used.