Immunochemical Study on the Route of Electron Transfer from NADH and NADPH to Cytochrome P-450 of Liver Microsomes

Publisher Website
Google Scholar
Abstract
Rabbit antibodies were prepared against NADPH-cytochrome P-450 reductase (fpT), NADH cytochrome b5 reductase (fpD), cytochrome P and cytochrome b of rat liver microsomes. The four antibodies were purified by affinity chromatography with antigen-conjugated Sepharose. Monovalent fragments of the antibodies against fpT and cytochrome b5 were also prepared. These antibody preparations were used as specific inhibitors in elucidating the electron pathway from NADPH or NADH to cytochrome P450 for drug oxidation reactions catalyzed by liver microsomes from normal, pheriobarbital (PB)-treated, and 3-methylcholanthrene (MC)-treated mice. Drug oxidation activities of the liver microsomes from PB-treated rats were also examined and compared with those of mouse liver microsomes. The drugs used were 7-ethoxycoumarin, benzo(a)pyrene, benzphetamine, and aniline. The antibody against fpT (anti-fpT IG) strongly inhibited both NADPH-supported and NADH-supported oxidation reactions of all substrates. On the other hand, the antibody against fpD (anti-fpD IG) did not inhibit NADPH-supported reactions but significantly in hibited the NADH-supported oxidation of drugs, except for aniline. The NADH-supported oxidation of 7-ethoxycoumarin, in which the concentration of NADH was lower, was less sensitive to anti-fpT 10 and highly sensitive to anti-fpD IG. The dependence of the inhibitory effects of these two antibodies on the concentration of NADH was confirmed; the results suggested that the relative contributions of fpT and fpD to the electron flow from NADH to cytochrome P-450 vary with the concentration of NADH. Judging from the inhibitory effects of antibody, cytochrome b5 participates in both NADH-supported and NADPH supported drug oxidation reactions, but the extent of its involvement in the reactions varies significantly according to the type of drug and the induced states of the animals. Inhibition studies using fpT-depleted microsomes suggested that the fpD-cytochrome b5 couple can support NADH-dependent drug oxidation at a slow but detectable rate. Since purified fpr and cytochrome P-450 could reconstitute not only NADPH-supported but also NADH supported oxidation of benzphetamine, the fpD-cytochrome b5 couple and fpT can separately support NADH-dependent drug oxidation reactions, although these two electron pathways seem to cooperate in intact microsomes.