Mráček Tomáš, Holzerová Eliška, Drahota Zdeněk, Kovářová Nikola, Vrbacký Marek, Ješina Pavel, Houštěk Josef
Institute of Physiology Academy of Sciences of the Czech Republic v.v.i., Vídeňská 1083, 14220 Prague 4, Czech Republic.
Biochim Biophys Acta. 2014 Jan;1837(1):98-111. doi: 10.1016/j.bbabio.2013.08.007. Epub 2013 Aug 30.
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.
活性氧（ROS）的过度产生与一系列病理状况有关。线粒体黄素脱氢酶甘油-3-磷酸脱氢酶（mGPDH）和琥珀酸脱氢酶（SDH）是重要的ROS来源，但电子泄漏的机制仍知之甚少。为了研究分离出的脱氢酶产生ROS的情况，我们以洋地黄皂苷增溶的棕色脂肪组织线粒体为模型。通过Amplex Red荧光法、鲁米诺发光法结合氧电极法进行的酶活性测量和过氧化氢生成研究表明，在体内条件下，黄素是SDH中最可能的电子泄漏源，而我们认为辅酶Q是mGPDH产生ROS的位点。铁氰化物对mGPDH产生ROS具有独特的诱导作用，这也表明两种脱氢酶产生ROS的机制不同。此外，使用天然电泳系统，我们证明mGPDH可形成同型寡聚体以及高分子量超复合物，这些是mGPDH在膜中的天然形式。通过这种方法，我们还直接证明了分离出的mGPDH本身及其超分子组装体都能够产生ROS。
