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-driven regeneration of antioxidant ubiquinol at the plasma membrane. Regeneration was measured by quantifying the oxidized and reduced forms of ubiquinone by electrochemical detection after separation by highperformance liquid chromatography. Plasma membrane incubation with NADH resulted in the consumption of endogenous ubiquinone, and a parallel increase in ubiquinol levels. The activity showed saturation kinetics with respect to the pyridine nucleotides and was moderately inhibited by p-hydroxymercuribenzoate. Only a slight inhibition was achieved with dicumarol at concentrations reported to fully inhibit DT-diaphorase. Salt-extracted membranes displayed full activity of endogenous ubiquinol regeneration, supporting the participation of an integral membrane protein. In liposomes-reconstituted systems, the purified cytochrome b(5) reductase catalyzed the reduction of the natural ubiquinone homologue 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 regeneration of endogenous ubiquinol.