RECONSTITUTION PREMIXES FOR ASSAYS USING PURIFIED RECOMBINANT HUMAN CYTOCHROME-P450, NADPH-CYTOCHROME P450 REDUCTASE, AND CYTOCHROME B(5)

The development of enzyme and buffer premixes for in vitro biotransfor mation assays is described. The protein premixes contain a mixture of three recombinant human proteins, cytochrome P450 (P450) 3A4, NADPH-P4 50 reductase, cytochrome bet and liposomes. The buffer premix contains reagents which, when diluted, provide for optimal metabolic activity with selected P450 3A4 substrates. P450 3A4 premixes were competent in the oxidation of known substrates including testosterone, midazolam, nifedipine, erythromycin, benzphetamine, and amitriptyline. Premixes s tored at -80 degrees C for 2 months and those that underwent an additi onal five freeze/thaw cycles were able to hydroxylate testosterone at turnover rates similar to freshly prepared reconstitution mixes. In ad dition, premixes stored unfrozen at 4 degrees C for 2 weeks showed no significant loss in the rate of testosterone 6 beta-hydroxylation by P 450 3A4. Premixes prepared with and without reduced glutathione, a com ponent which had previously been found to be important for P450 3A4 re actions, were equally efficient at carrying out testosterone hydroxyla tion under these conditions. Kinetic parameters determined for the met abolism of testosterone, amitriptyline, nifedipine, and benzphetamine using P450 3A4 premixes were compared with human pooled microsomes and insect microsomes prepared from cells infected with a baculovirus con taining two cDNA inserts coding for P450 3A4 and NADPH-P450 reductase. Each format gave different V-max and K-m values indicating different catalytic efficiencies. Analysis of P450 1A2 premixes which contained different lipid concentrations indicated that V-max and K-m could be a ltered. The availability of human P450 recombinant enzymes and the dev elopment of the P450 premixes that remain active after being stored fr ozen should allow for rapid identification of novel P450 substrates an d inhibitors and the development of large-scale screening assays. (C) 1997 Academic Press.