Characterization of NADPH-dependent ubiquinone reductase activity in rat liver cytosol: effect of various factors on ubiquinone-reducing activity and discrimination from other quinone reductases.
Cytosolic NADPH-dependent ubiquinone reductase (NADPH-UQ reductase) accounted for about 68% of the total ubiquinone (UQ) reductase activity in rat liver homogenate [Takahashi, T. et al. (1995) Biochem. J. 309, 883-890]. We investigated the effects of various factors on this enzyme activity in rat liver cytosol with the aim of elucidating its physiological roles. The NADPH-UQ reductase in rat liver cytosol catalyzed the reduction of UQ to UQH2 with concomitant oxidation of equimolar NADPH. The optimal pH was around 7.4, and the optimal temperatures were about 28 degrees C for NADH and about 37 degrees C for NADPH. NADH, deamino NADH, and deamino NADPH were much less active hydrogen donors than NADPH, whereas reduced nicotinamide mononucleotide, ascorbate, erythorbate, reduced glutathione, and cysteine were inactive. As the hydrogen acceptor, UQ-9 had the highest Vmax/Km among the long-chain UQ homologues tested. FAD and FMN stimulated the activity. Anionic detergents, Mg2+ and Sr2+ also enhanced the activity. Rotenone, malonic acid, antimycin A, and KCN, which inhibit mitochondrial and microsomal electron transfer enzymes, superoxide dismutase, and acetylated cytochrome c had no effect on the NADPH-UQ reductase activity. These results indicated that the NADPH-UQ reductase in rat liver cytosol is a flavoprotein that reduces UQ-10 by a two-electron reduction mechanism and is distinguishable from known microsomal and mitochondrial enzymes, as well as DT-diaphorase [EC 1.6.99.2].
Takahashi
T
T
Faculty of Pharmaceutical Sciences, Kobe Gakuin University.
Okamoto
T
T
Kishi
T
T
D000227:Adenine Nucleotides / Q000494:pharmacology; D000818:Animals; D003600:Cytosol / Q000201:enzymology*; D003902:Detergents / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005486:Flavin Mononucleotide / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000494:pharmacology; D008099:Liver / Q000201:enzymology*; D008297:Male; D008670:Metals; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010084:Oxidation-Reduction; D011808:Quinone Reductases / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013482:Superoxide Dismutase / Q000494:pharmacology; D014451:Ubiquinone / Q000378:metabolism*
D000227:Adenine Nucleotides; D003902:Detergents; D004791:Enzyme Inhibitors; D008670:Metals; D014451:Ubiquinone; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D013482:Superoxide Dismutase; D016660:NAD(P)H Dehydrogenase (Quinone); D011808:Quinone Reductases