COMPETITIVE INTERACTIONS BETWEEN CYTOCHROMES P450 2A6 AND 2E1 FOR NADPH-CYTOCHROME P450 OXIDOREDUCTASE IN THE MICROSOMAL-MEMBRANES PRODUCEDBY A BACULOVIRUS EXPRESSION SYSTEM

The present study investigated the interactions between cytochrome P45 0 (P450) enzymes and the NADPH: cytochrome oxidoreductase (OR) in the microsomal membrane. Microsomes containing human cytochrome P450 2A6 ( h2A6) coexpressed with human OR (hOR) via a baculovirus expression sys tem displayed coumarin hydroxylase activity with apparent K-m and V-ma x values of 0.41 mu M and 405 nmol/min/nmol P450, respectively. Incorp oration of purified rat liver cytochrome bs (b(5)) into the microsomes increased the V-max 2.5-fold, but did not affect the K-m. The N-nitro sodimethylamine (NDMA) demethylase activity of human cytochrome P450 2 E1 (h2E1) coexpressed similarly was characterized previously. Coumarin was shown not to be a substrate nor an inhibitor of h2E1, and NDMA wa s not a substrate nor an inhibitor of h2A6. In microsomes containing h 2A6, h2E1, and hOR (M-h2A6-h2E1-hOR) obtained from a triple expression system, the two P450 enzymes were shown to compete with each other fo r interaction with hOR. In incubations with M-h2A6-h2E1-hOR, the prese nee of a h2A6 substrate (coumarin) decreased NDMA demethylase activity by a maximum of 47%, and the presence of a h2E1 substrate (NDMA) decr eased coumarin hydroxylase activity by a maximum of 19%. This substrat e-induced competition between h2A6 and h2E1 was decreased by the addit ion of purified b(5). In the absence of a substrate, the NADPH-depende nt H2O2 formation was high in both M-h2A6-h2E1-hOR and M-h2E1-hOR, but low in M-h2AG-hOR. The addition of NDMA had little effect on the H2O2 formation in M-h2A6-h2E1-hOR and M-h2E1-hOR. The addition of coumarin , however, slightly decreased H2O2 formation in M-h2A6-h2E1-hOR, but d rastically increased H2O2 formation in M-h2A6-hOR. These results sugge st that the presence of a h2A6 substrate decreased the electron flow t o h2E1 in M-h2A6-h2E1-hOR. The activities of coumarin hydroxylase and NDMA demethylase of M-h2A6-h2E1-hOR were decreased and increased, resp ectively by an increase in ionic strength. The ionic. strength, howeve r, did not drastically change the substrate-induced competition betwee n h2A6 and h2E1 for hOR. The results demonstrate the usefulness of the coexpression system for mechanistic studies and illustrate that the i nteraction of monooxygenase enzymes in the microsomal membrane is regu lated by the presence. of substrates and b(5). (C) 1997 Academic Press .