ROS generation and multiple forms of mammalian mitochondrial glycerol-3-phosphate dehydrogenase |
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Authors: | Tomá &scaron Mrá ček,Eli&scaron ka Holzerová Zdeněk Drahota,Nikola Ková řová Marek Vrbacký ,Pavel Je&scaron inaJosef Hou&scaron těk |
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Affiliation: | Institute of Physiology Academy of Sciences of the Czech Republic v.v.i., Vídeňská 1083, 14220 Prague 4, Czech Republic |
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Abstract: | Overproduction of reactive oxygen species (ROS) has been implicated in a range of pathologies. Mitochondrial flavin dehydrogenases glycerol-3-phosphate dehydrogenase (mGPDH) and succinate dehydrogenase (SDH) represent important ROS source, but the mechanism of electron leak is still poorly understood. To investigate the ROS production by the isolated dehydrogenases, we used brown adipose tissue mitochondria solubilized by digitonin as a model. Enzyme activity measurements and hydrogen peroxide production studies by Amplex Red fluorescence, and luminol luminescence in combination with oxygraphy revealed flavin as the most likely source of electron leak in SDH under in vivo conditions, while we propose coenzyme Q as the site of ROS production in the case of mGPDH. Distinct mechanism of ROS production by the two dehydrogenases is also apparent from induction of ROS generation by ferricyanide which is unique for mGPDH. Furthermore, using native electrophoretic systems, we demonstrated that mGPDH associates into homooligomers as well as high molecular weight supercomplexes, which represent native forms of mGPDH in the membrane. By this approach, we also directly demonstrated that isolated mGPDH itself as well as its supramolecular assemblies are all capable of ROS production. |
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Keywords: | AA, antimycin A BAT, brown adipose tissue CoQ, coenzyme Q DCPIP, 2,6-dichlorophenolindophenol FeCN, ferricyanide, potassium hexacyanoferrate(III) GP, sn-glycerol-3-phosphate mGPDH, mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase HAR, hexaammineruthenium(III) chloride HQNO, 2-n-heptyl-4-hydroxyquinoline N-oxide HRP, horseradish peroxidase MXT, myxothiazol OXPHOS, oxidative phosphorylation ROS, reactive oxygen species SDH, succinate dehydrogenase |
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