The existence of a lysosomal redox chain and the role of ubiquinone

Several studies concerning the distribution of ubiquinone (UQ) in the cell report a preferential accumulation of this biogenic quinone in mitochondria , plasma membranes, Golgi vesicles, and lysosomes, Except for mitochondria, no recent comprehensive experimental evidence exists on the particular fun ction of UQ in these subcellular organelles, The aim of a recent study was to elucidate whether UQ is an active part of an electron-transfer system in lysosomes, In the present work, a lysosomal fraction was prepared from a l ight mitochondrial fraction of rat liver by isopycnic centrifugation, The p urity of our preparation was verified by estimation of the respective marke r enzymes. Analysis of lysosomes for putative redox carriers and redox proc esses in lysosomes was carried out by optical spectroscopy, HPLC, oxymetry, and ESR techniques. UQ was detected in an amount of 2.2 nmol/mg of protein in lysosomes, Furthermore, a b-type cytochrome and a flavin-adenine dinucl eotide (FAD) were identified as other potential electron carriers, Since NA DH was reported to serve as a substrate of UQ redox chains in plasma membra nes, we also tested this reductant in lysosomes, Our experiments demonstrat e a NADH-dependent reduction of UQ by two subsequent one-electron-transfer steps giving rise to the presence of ubisemiquinone and an increase of the ubiquinol pool in lysosomes, Lysosomal NADH oxidation was accompanied by an approximately equimolar oxygen consumption, suggesting that O-2 acts as a terminal acceptor of this redox chain. DMPO/(OH)-O-. spin adducts were dete cted by ESR in NADH-supplemented lysosomes, suggesting a univalent reductio n of oxygen. The kinetic analysis of redox changes in lysosomes revealed th at electron carriers operate in the sequence NADH > FAD > cytochrome b > ub iquinone > oxygen. By using the basic spin label TEMPAMINE, we showed that the NADH-related redox chain in lysosomes supports proton accumulation in l ysosomes, In contrast to the hypothesis that UQ in lysosomes is simply a wa ste product of autophagy in the cell, we demonstrated that this lipophilic electron carrier is a native constituent of a lysosomal electron transport chain, which promotes proton translocation across the :Lysosomal membrane, (C) 2000 Academic Press.