Liu H, Kehrer J P
Division of Pharmacology and Toxicology, University of Texas at Austin, USA.
Free Radic Biol Med. 1996;20(3):433-42. doi: 10.1016/0891-5849(95)02093-4.
Factors affecting the reduction of GSSG by rat liver mitochondria after a t-butyl hydroperoxide-induced (t-BOOH) oxidative stress were studied. The amounts of ADP and mitochondrial protein were adjusted to consume less than 50% of the available oxygen during the 8-min experimental period. A 4-min treatment of mitochondria with 24 nmol t-BOOH/mg protein (60 microM) oxidized 91% of total glutathione. In the presence of glutamate/malate, succinate or ascorbate/N,N,N',N'-tetramethyl-p- phenylenediamine (TMPD) (state 4 respiration), 84, 84, and 28% of the GSSG formed during t-BOOH treatment was reduced after 4 min, respectively. A similar extent of reduction was seen during state 3 respiration (1.5 mM ADP) with glutamate/malate, but no reduction occurred during state 3 respiration with either succinate or ascorbate/TMPD. The succinate-supported reduction of GSSG was completely blocked by rotenone, antimycin A, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), or 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). In contrast, oligomycin potentiated GSSG reduction using either glutamate/malate or succinate as substrates. Rotenone partially blocked glutamate/malate-supported GSSG reduction. NADPH levels were altered in direct proportion to the effects on GSSG reduction. The current data indicate that the reduction of GSSG in oxidatively-stressed isolated rat liver mitochondria occurs most efficiently during state 4 respiration and is independent of ATP synthesis. Both transhydrogenation and the transmembrane proton gradient appear to be important in NADPH regeneration and consequent GSSG reduction.
研究了叔丁基过氧化氢（t-BOOH）诱导的氧化应激后影响大鼠肝脏线粒体还原谷胱甘肽二硫化物（GSSG）的因素。将二磷酸腺苷（ADP）和线粒体蛋白的量进行调整，以使在8分钟的实验期间消耗的氧气量低于可用氧气的50%。用24 nmol t-BOOH/mg蛋白（60 microM）处理线粒体4分钟，可氧化91%的总谷胱甘肽。在存在谷氨酸/苹果酸、琥珀酸或抗坏血酸/N，N，N'，N'-四甲基对苯二胺（TMPD）（状态4呼吸）的情况下，t-BOOH处理期间形成的GSSG在4分钟后分别有84%、84%和28%被还原。在以谷氨酸/苹果酸进行状态3呼吸（1.5 mM ADP）时，观察到类似程度的还原，但在以琥珀酸或抗坏血酸/TMPD进行状态3呼吸时未发生还原。鱼藤酮、抗霉素A、羰基氰化物对三氟甲氧基苯腙（FCCP）或1，3-双（2-氯乙基）-1-亚硝基脲（BCNU）完全阻断了琥珀酸支持的GSSG还原。相比之下，寡霉素增强了以谷氨酸/苹果酸或琥珀酸为底物时的GSSG还原。鱼藤酮部分阻断了谷氨酸/苹果酸支持的GSSG还原。烟酰胺腺嘌呤二核苷酸磷酸（NADPH）水平的变化与对GSSG还原的影响成正比。目前的数据表明，在氧化应激的离体大鼠肝脏线粒体中，GSSG的还原在状态4呼吸期间最有效，且与三磷酸腺苷（ATP）合成无关。转氢作用和跨膜质子梯度似乎在NADPH再生及随后的GSSG还原中都很重要。
