Pyridine nucleotide cofactor requirements of indicine N-oxide reduction by hepatic microsomal cytochrome P-450

Abstract

Indicine N-oxide is reduced to indicine under anaerobic conditions by rat hepatic microsomal fraction in the presence of either NADH or NADPH. CO completely inhibits reduction, indicating the involvement of cytochrome P-450. In contrast to cytochrome P-450-catalyzed oxidations, for which NADH is about 15 per cent as effective as NADPH, NADH is 80 per cent as effective as NADPH in supporting indicine N-oxide reduction. In the presence of 3 mM NADH, the K m for indicine N-oxide is 0.37 mM, and the V max is 2.65 nmoles indicine formed/min/mg; with 3 mM NADPH the K m is 0.51 mM, and the V max is 3.00 nmoles/min/mg. NADH- and NADPH-dependent indicine N-oxide reduction appear to involve different pathways. NADH-supported reduction is inhibited 48 per cent by 0.5 mM KCN and 45 per cent by 0.8 M acetone, while NADPH-supported reduction is inhibited 3 per cent by 0.5 mM KCN and stimulated 28 per cent by 0.8 M acetone. The ability of NADH and NADPH at saturating concentrations to support indicine N-oxide reduction is additive, although this effect is not seen with phenobarbital- or 3-methylcholanthrene-pretreated animals. Phenobarbital pretreatment produces a selective increase in the V max for NADPH-dependent reduction, to 5.75 nmoles/min/mg, but has no significant effect upon K tm with NADPH or upon either the K m for the f max for NADH-supported indicine N-oxide reduction. Pretreatment with 3-methylcholanthrene has no significant effect upon the K m or V max for NADPH- or NADH-supported reduction. A possible explanation for the observations is a form of cytochrome P-450 which can accept electrons from NADH and catalyze indicine N-oxide reduction but which does not contribute to oxidative microsomal drug metabolism