Mechanism of 2-naphthylamine oxidation catalysed by pig liver microsomes.

1. In pig liver microsomes 2-naphthylamine-dependent NADPH oxidation, oxygen reduction, and hydroxylamine formation are linear with time for several minutes. A sharp increase in NADPH oxidation and oxygen uptake then coincides with an abrupt loss of hydroxylamine from the medium. 2. The initial rate of 2-naphthylamine N-oxidation correlates with the microsomal concentration of mixed-function amine oxidase and the extent of linear accumulation of hydroxylamine is dependent on microsomal NADPH-cytochrome c reductase activity and concentration of lipid (microsomes). 3. Antisera to NADPH-cytochrome c reductase markedly decreased hydroxylamine accumulation during incubation but had no effect on the rate of 2-naphthylamine N-oxidation. 4. A system duplicating all of the kinetic properties of the microsomal 2-naphthylamine oxidase was constructed with two purified flavoproteins, (mixed-function amine oxidase and NADPH-cytochrome c reductase) and a lipid phase (erythrocyte ghosts or synthetic lecithin liposomes). 5. By independently varying the concentrations of each component in the reconstituted system, the contribution of each to the observed kinetics was defined. 6. In addition to the initial N-oxidation of 2-naphthylamine, at least six other reactions contribute to the kinetic patterns of 2-naphthylamine oxidation catalysed by the reconstituted system.