Immunochemical Study on the Electron Pathway from NADH to Cytochrome P-450 of Liver Microsomes

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Abstract
Rabbit antibodies were prepared against NADPH-cytochrome c reductase, NADH-cytochrome b5 reductase, cytochrome b5 and cytochrome P-450 of rat liver microsomes. The antibodies were purified by adsorption on antigen-conjugated Sepharose and used in elucidating the pathway of electron transfer from NADH to cytochrome P-450 in the oxidation of drugs catalyzed by mouse liver microsomes. These redox enzymes of rat liver microsomes were immunochemically identical with the corresponding enzymes of mouse liver microsomes as determined by Ouchterlony double diffusion tests and inhibition studies on microsomal reductase activities. The antibody against NADPH-cytochrome c reductase strongly inhibited all NADH-supported drug oxidation reactions examined; these were 7-ethoxycoumarin O-deethylation, benzo(a)pyrene hydroxylation, benzphetamine N-demethylation and aniline hydroxylation. The antibody against cytochrome b5 inhibited NADH-supported 7-ethoxycoumarin O-deethylase, benzo(a)pyrene hydroxylase and benzphetamine N-demethylase, but did not inhibit aniline hydroxylase. The antibody against NADH-cytochrome b5 reductase inhibited NADH-supported 7-ethoxycoumarin O-deethylase and benzo(a)pyrene hydroxylase, but did not inhibit benzphetamine N-demethylase or aniline hydroxylase. NADPH- and NADH-supported aniline hydroxylase was not inhibited by the antibody against cytochrome P-450 which was prepared against phenobarbital-induced cytochrome P-450. The other 3 reactions were inhibited by this antibody. The participation of NADPH-cytochrome c reductase in NADH-supported drug oxidation reactions was further confirmed by the selective removal of this reductase from microsomes, which was accomplished by mild trypsin digestion. NADPH- and NADH-supported oxidations of aniline by trypsin-treated microsomes decreased in parallel with the loss of NADPH-cytochrome c reductase from microsomes. The decrease of 7-ethoxycoumarin O-deethylase activity also almost paralleled that of the reductase activity. The high Km value for NADH and the competitive inhibition by NADP+ of NADH-supported drug oxidation reactions suggested that NADPH-cytochrome c reductase accepts electrons directly from NADH and transfers them to cytochrome P-450. These inhibition profiles and kinetic evidence confirmed that NADPH-cytochrome c reductase is an essential component in the electron flow from NADH to cytochrome P-450 for drug oxidations; NADH-cytochrome b5 reductase and cytochrome b5 participate in NADH-supported oxidation of some, but not all, drugs.