Reduced nicotinamide adenine dinucleotide-dependent reduction of tertiary amine N-oxide by liver microsomal cytochrome P-450

Abstract

NADH-supported reduction of tertiary amine N-oxides in rat liver microsomes was investigated with imipramine N-oxide, tiaramide N-oxide and N, N-dimethylaniline N-oxide as substrate in the presence of NADH. The reductase activity is sensitive to carbon monoxide. NADH-cytochrome b 5 reductase or cytochrome b 5, solubilized by trypsin or subtilisin, showed no N-oxide reductase activity. The NADH-dependent reduction of tertiary amine N-oxides was markedly inhibited by antibody to NADH-eytochrome b 5 reductase and antibody to cytochrome b 5. These results confirmed that NADH-dependent N-oxide reduction was catalyzed by cytochrome P-450 and the reducing equivalent for the N-oxide reduction was transferred from NADH to cytochrome P-450 mainly via NADH-cyto-chrome b 5 reductase and cytochrome b 5 in the microsomal membranes. NADH-dependent N-oxide reductase is also sensitive to oxygen and 4 μM oxygen gave 50 per cent inhibition with imipramine N-oxide. Kinetic study shows that K m values for the reduction of imipramine N-oxide, tiaramide N-oxide and N, N-dimethylaniline N-oxide were 0.05 mM, 0.14 mM and 0.16 mM, respectively. NADH-dependent N-oxides reductase activity is affected by azo and nitroso compounds and hydrazide, although the degree of inhibition was rather weak compared with those of NADPH-dependent activity. Furthermore, NADH-dependent reduction of tertiary amine N-oxide was only slightly affected by n-octylamine, 2,4-diehloro-6-phenylphenoxyethylamine and aniline. NADH-dependent N-oxide reductase activity in liver microsomes was less sensitive to phenobarbital or 3-methyleholanthrene pretreatment than NADPH-dependent activity. Some characteristics of NADH-dependent N-oxide reductase activity were discussed and compared with those of NADPH-dependent activity.