Generation of Superoxide by the Microsomal Mixed-Function Oxidase System
Part of the
Basic Life Sciences
book series (BLSC, volume 49)
The microsomal mixed-function oxidase (MFO) system catalyzes the oxidation of numerous endogenous and exogenous compounds using NADPH as its electron donor. During its oxidation of drug substrates and even in the absence of substrates, this electron transport system has been demonstrated to produce O2·̄, H2O2, and excess H2O. We have investigated the ability of several purified forms of cytochrome P450 to produce O2·̄ when incubated with purified NADPH cytochrome P450 reductase. Addition of cytochrome P450 isozymes to the purified reductase resulted in increased rates of O2·̄ formation, assessed using either EPR spin trapping techniques or acetylated cytochrome c reduction. Conditions necessary for optimal rates of O2·̄ production were similar to those previously shown to be necessary for optimal reconstitution of MFO activity (phospholipid, detergent-solubilized reductase, cytochrome P450:reductase ratios of greater than 3:1). Cytochrome P450d addition to incubations of reductase resulted in greater rates of O2·̄ production than either cytochrome P450c or P450b, especially at equimolar concentrations of cytochrome P450 isozymes and reductase. The contribution of cytochrome P450d to microsomal O2·̄ generation was addressed by investigating O2·̄ production by microsomes isolated from isosafrole-treated rats.
KeywordsLiver Microsome Superoxide Production Cytochrome P450 Reductase NADPH Oxidation NADPH Cytochrome P450 Reductase
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
A. Astrom and J.W. DePierre, Metabolism of 2-acetylaminofluorene by eight different forms of cytochrome P-450 isolated from rat liver, Carcinogenesis
6:113 (1985).PubMedCrossRefGoogle Scholar
D.J. Waxman and C. Walsh, Phenobarbital-induced rat liver cytochrome P450: Purification and characterization of two closely related isozymic forms, J. Biol. Chem.
257:10446 (1982).PubMedGoogle Scholar
Y. Yasukochi and B.S.S. Masters, Some properties of a detergent-solubilized NADPH-cytochrome C (cytochrome P450) reductase purified by biospecific affinity chromatography, J. Biol. Chem.
251:5337 (1976).PubMedGoogle Scholar
K. Wada and K. Okunuki, Studies on chemically modified cytochrome c. I. The acetylated cytochrome c, J. Biochem.
64:667 (1968).PubMedGoogle Scholar
L.A. Morehouse, C.E. Thomas, and FS.D. Aust, Superoxide generation by NADPH-cytochrome P450 reductase: The effect of iron chelators and the role of superoxide in microsomal lipid peroxidation, Arch. Biochem. Biophys.
232:366 (1984).PubMedCrossRefGoogle Scholar
P.P. Tamburini, I. Jansson, L.V. Favreau, W.L. Backes, and J.L. Schenkman, Differences in the spectral interactions between NADPH-cytochrome P450 reductase and a series of cytochrome P450 enzymes, Biochem. Biophys. Res. Comm.
137:437 (1986).PubMedCrossRefGoogle Scholar
M. Dickins, U.K. Elcombe, S.J. Moloney, K.J. Netter, and J.W. Bridges, Further studies on the dissociation of the isosafrole metabolite-cytochrome P450 complex, Biochem. Pharmacol.
28:231 (1979).PubMedCrossRefGoogle Scholar
M. Murray, K. Hetnarski, and G.F. Wilkinson, Selective inhibitory interactions of alkoxymethylene dioxybenzenes towards monooxvgenase activity in rat hepatic microsomes, Xenobiotica
15:369 (1985).PubMedCrossRefGoogle Scholar
© Plenum Press, New York 1988