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.
- MeSH
- ferrikyanidy metabolismus MeSH
- glycerolfosfátdehydrogenasa chemie metabolismus MeSH
- glycerolfosfáty metabolismus MeSH
- krysa rodu rattus MeSH
- mitochondrie enzymologie metabolismus MeSH
- peroxid vodíku metabolismus MeSH
- reaktivní formy kyslíku metabolismus MeSH
- savci MeSH
- sukcinátdehydrogenasa chemie metabolismus MeSH
- transport elektronů * MeSH
- ubichinon metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
OBJECTIVES: The aim of this study was to assess the effect of various flavonoids on the NADPH:cytochrome P450 oxidoreductase (CYPOR) activity in respect of the reduction of different electron acceptors as well as to study an impact of flavonoids on monooxygenation of a model substrate of cytochrome P450 (CYP). DESIGN: The modulation of CYPOR activity was determined spectrophotometrically based on the time course of the reduction of different electron acceptors. The CYP reduction was monitored via its complex formation with CO, having pronounced the absorption maximum at 450 nm. Finally, effect of CYPOR stimulation by 7,8benzoflavone (ANF) on 7pentoxyresorufin Odepentylation was assayed in the microsomal monooxygenation system using the fluorimetric detection of formed resorufin. RESULTS: The stimulation of CYPOR activity via ANF was found to be associated with following electron acceptors: cytochrome c, potassium ferricyanide, cytochrome b5, but not with CYP. Surprisingly, 5,6benzoflavone, a position isomer of ANF, was ineffective in the CYPOR stimulation as well as the other flavonoids tested. In microsomal preparations, ANF did not markedly enhance the reaction rate of monooxygenation of CYP2B4 model substrate. CONCLUSION: Our results document that among all of the tested flavonoids only ANF is able to stimulate CYPOR activity, however, the ANF-mediated stimulation of CYPOR has no impact on the oxidative metabolism catalyzed by CYP system.
- MeSH
- antioxidancia farmakologie MeSH
- benzoflavony chemie metabolismus MeSH
- beta-naftoflavon chemie metabolismus MeSH
- časové faktory MeSH
- cytochromy b5 metabolismus MeSH
- cytochromy c metabolismus MeSH
- ferrikyanidy metabolismus MeSH
- flavonoidy chemie farmakologie MeSH
- fluorometrie MeSH
- králíci MeSH
- kyselina lipoová farmakologie MeSH
- mikrozomy účinky léků enzymologie metabolismus MeSH
- NADPH-cytochrom c-reduktasa metabolismus MeSH
- oxaziny metabolismus MeSH
- oxid uhelnatý metabolismus MeSH
- oxidace-redukce účinky léků MeSH
- spektrofotometrie MeSH
- systém (enzymů) cytochromů P-450 metabolismus MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
