Institute of Biochemistry, Carleton University, Ottawa, Ontario, Canada.
J Enzyme Inhib Med Chem. 2009 Dec;24(6):1319-31. doi: 10.3109/14756360902852586.
In mammals, aging is linked to a decline in the activity of citrate synthase (CS; E.C. 2.3.3.1), the first enzyme of the citric acid cycle. We used 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), a water-soluble generator of peroxyl and alkoxyl radicals, to investigate the susceptibility of CS to oxidative damage. Treatment of isolated mitochondria with AAPH for 8-24 h led to CS inactivation; however, the activity of aconitase, a mitochondrial enzyme routinely used as an oxidative stress marker, was unaffected. In addition to enzyme inactivation, AAPH treatment of purified CS resulted in dityrosine formation, increased protein surface hydrophobicity, and loss of tryptophan fluorescence. Propyl gallate, 1,8-naphthalenediol, 2,3-naphthalenediol, ascorbic acid, glutathione, and oxaloacetate protected CS from AAPH-mediated inactivation, with IC(50) values of 9, 14, 34, 37, 150, and 160 muM, respectively. Surprisingly, the antioxidant epigallocatechin gallate offered no protection against AAPH, but instead caused CS inactivation. Our results suggest that the current practice of using the enzymatic activity of CS as an index of mitochondrial abundance and the use of aconitase activity as an oxidative stress marker may be inappropriate, especially in oxidative stress-related studies, during which alkyl peroxyl and alkoxyl radicals can be generated.
在哺乳动物中，衰老与柠檬酸合酶（CS；EC 2.3.3.1）活性的下降有关，CS 是柠檬酸循环的第一个酶。我们使用 2,2'-偶氮双（2-脒基丙烷）二盐酸盐（AAPH），一种水溶性过氧自由基和烷氧自由基生成剂，来研究 CS 对氧化损伤的敏感性。用 AAPH 处理分离的线粒体 8-24 小时会导致 CS 失活；然而，线粒体酶顺乌头酸酶的活性不受影响，顺乌头酸酶通常被用作氧化应激标志物。除了酶失活外，AAPH 处理纯化的 CS 还导致二酪氨酸形成、蛋白质表面疏水性增加和色氨酸荧光丧失。没食子酸丙酯、1,8-萘二醇、2,3-萘二醇、抗坏血酸、谷胱甘肽和草酰乙酸能保护 CS 免受 AAPH 介导的失活，其 IC50 值分别为 9、14、34、37、150 和 160 μM。令人惊讶的是，抗氧化剂表没食子儿茶素没食子酸酯（epigallocatechin gallate）不能防止 AAPH 对 CS 的失活作用，反而会导致 CS 失活。我们的结果表明，目前将 CS 的酶活性用作线粒体丰度的指标，以及将顺乌头酸酶活性用作氧化应激标志物的做法可能是不恰当的，特别是在与氧化应激相关的研究中，在此期间可能会产生烷基过氧自由基和烷氧自由基。
