Ubiquinol regeneration by plasma membrane ubiquinone reductase

Several enzyme systems have been proposed to play a role in the maintenance of ubiquinol in membranes other than the inner mitochondrial membrane. The aim of this study was to investigate the mechanisms involved in NADH-drive n regeneration of antioxidant ubiquinol at the plasma membrane. Regeneratio n was measured by quantifying the oxidized and reduced forms of ubiquinone by electrochemical detection after separation by highperformance liquid chr omatography. Plasma membrane incubation with NADH resulted in the consumpti on of endogenous ubiquinone, and a parallel increase in ubiquinol levels. T he activity showed saturation kinetics with respect to the pyridine nucleot ides and was moderately inhibited by p-hydroxymercuribenzoate. Only a sligh t inhibition was achieved with dicumarol at concentrations reported to full y inhibit DT-diaphorase. Salt-extracted membranes displayed full activity o f endogenous ubiquinol regeneration, supporting the participation of an int egral membrane protein. In liposomes-reconstituted systems, the purified cy tochrome b(5) reductase catalyzed the reduction of the natural ubiquinone h omologue coenzyme Q(10) at rates accounting for the activities observed in whole plasma membranes, and decreased the levels of lipid peroxidation. Our data demonstrate the role of the cytochrome b(5) reductase in the regenera tion of endogenous ubiquinol.