The membrane potential (DeltaPsim) dependence of the generation of reactive oxygen species (ROS) in isolated guinea-pig brain mitochondria respiring on NADH-linked substrates (glutamate plus malate) was addressed. Depolarization by FCCP was without effect on H(2)O(2) formation in the absence of bovine serum albumin (BSA). Addition of BSA (0.025%) to the assay medium hyperpolarized mitochondria by 6.1 +/- 0.9 mV (from 169 +/- 3 to 175.1 +/- 2.1 mV) and increased the rate of H(2)O(2) formation from 207 +/- 4.5 to 312 +/- 12 pmol/min/mg protein. Depolarization by FCCP (5-250 nM) in the presence of BSA decreased H(2)O(2) formation but only to the level observed in the absence of BSA. Rotenone stimulated the formation of H(2)O(2) both in the absence and presence of BSA. It is suggested that H(2)O(2) formation in mitochondria supported by NADH-linked substrates is sensitive to changes in DeltaPsim only when mitochondria are highly polarized and even then, 60% of ROS generation is independent of DeltaPsim. This is in contrast to earlier reports on the highly DeltaPsim sensitive ROS formation related to reverse electron flow observed in well-coupled succinate-supported mitochondria.

译文

解决了在NADH连接的底物 (谷氨酸加苹果酸) 上呼吸的分离的豚鼠脑线粒体中活性氧 (ROS) 产生的膜电位 (DeltaPsim) 依赖性。在不存在牛血清白蛋白 (BSA) 的情况下,FCCP的去极化对H(2)O(2) 的形成没有影响。通过6.1 +/- 0.9 mV (从169 +/- 3到175.1 +/- 2.1 mV) 将BSA (0.025%) 添加到测定培养基超极化线粒体中,并将H(2)O(2) 的形成速率从207 +/- 4.5增加到312 +/- 12 pmol/min/mg蛋白。在BSA存在下通过FCCP (5-250 nM) 去极化降低了H(2)O(2) 的形成,但仅达到在不存在BSA的情况下观察到的水平。在不存在和存在BSA的情况下,鱼藤酮均刺激H(2)O(2) 的形成。建议仅当线粒体高度极化时,由NADH连接的底物支持的线粒体中H(2)O(2) 的形成才对DeltaPsim的变化敏感,即使这样,ROS生成的60% 也独立于DeltaPsim。这与先前关于在充分耦合的琥珀酸支持的线粒体中观察到的与反向电子流有关的高度DeltaPsim敏感的ROS形成的报道相反。

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