MOLECULAR MECHANISMS OF OXIDATIVE STRESS IN AGING MUSCLE

描述（申请人摘要）：氧化损伤，特别是对蛋白质的氧化损伤， 被广泛认为是肌肉功能丧失的主要原因， 衰老研究人员已经观察到线粒体氧化能力的降低， 能力和酶活性与年龄的肌肉，这可能直接相关 线粒体产生自由基氧化剂。几个关键代谢 酶-磷酸果糖激酶（限速糖酵解酶），两个关键 线粒体酶（柠檬酸合成酶和顺乌头酸酶）和一个电子 运输链酶（细胞色素c氧化酶）--已经显示出下降 骨骼肌和心肌的年龄变化。产生这种现象的机制 下降是未知的，可能与氧化损伤有关， 线粒体老化此外， 对蛋白质氧化损伤的贡献知之甚少，主要是因为 研究蛋白质氧化的非特异性方法。所以我们会 使用灵敏的分析方法（气相色谱和质谱）， 探索金属催化氧化途径， 酪氨酰自由基介导的途径和活性氮途径， 产生特定的非天然氨基酸，即，邻酪氨酸，间酪氨酸， o ′-二酪氨酸和3-硝基酪氨酸。通过这种方法，研究人员将 能够确定以下内容：1）哪些自由基物种负责 对蛋白质的破坏2)蛋白质损伤主要是由线粒体引起的吗？ 3)蛋白质损伤在肌肉中累积到什么程度？4)做水平的 尿液中的氧化氨基酸与线粒体中的氧化氨基酸相关和/或 胞质溶胶？和5）氧化蛋白质修饰是否影响酶功能， 线粒体功能能力？为了实现这些目标，这些 研究人员将确定氧化剂的产生，总的氧化蛋白质和 脂质损伤、特定酶活性和年轻人的线粒体功能， 成年和老年大鼠。他们还将测量纯化酶的酶活性 和氧化酶损伤的数量。此外，他们将确定，如果 两种保护性干预措施，热量限制和终身自愿 运动，可以减弱肌肉蛋白质氧化，恢复酶功能 和线粒体功能。这些实验将提供第一个证据 为了回答以下问题：1）线粒体是 蛋白质氧化与衰老2)是受氧化剂影响的关键代谢酶 随着年龄的增长，肌肉的功能会下降吗？3)并 酶活性反映酶蛋白的损失或非活性物质的积累 酶的形式？酶功能和线粒体功能 通过热量限制和/或日常锻炼恢复能力？

DESCRIPTION (Applicant's abstract): Oxidative damage, particularly to proteins, is widely believed to be a major cause of the loss of muscle function during senescence. Researchers have observed decreases in mitochondrial oxidative capacity and enzyme activity with age in muscle, which may be directly related to free radical oxidant production by mitochondria. Several key metabolic enzymes -- phosphofructokinase (rate limiting glycolytic enzyme), two key mitochondrial enzymes (citrate synthase and aconitase) and one electron transport chain enzyme (cytochrome c oxidase) -- have been shown to decline with age in skeletal muscle and heart muscle. The mechanism that produces this decline is unknown and may be related to oxidative damage that occurs with aging in mitochondria. Moreover, the sources of reactive radical species that contribute to protein oxidative damage are poorly understood, primarily because of the non-specific methods used to study protein oxidation. Therefore, we will use sensitive analytical methods (gas chromatography and mass spectrometry) to explore the respective roles of the metal-catalyzed oxidation pathway, the tyrosyl radical mediated pathway, and the reactive nitrogen pathway, which generate specific unnatural amino acids, i.e., o-tyrosine, m-tyrosine, o'-dityrosine, and 3-nitrotyrosine. With this approach the investigators will be able to determine the following: 1) which radical species are responsible for protein damage? 2) is protein damage predominantly caused by mitochondria? 3) to what extent does protein damage accumulate in muscle? 4) do the levels of oxidized amino acids in the urine correlate with those in mitochondria and/or cytosol? And 5) does oxidative protein modification affect enzyme function and mitochondrial functional capacity? To accomplish these aims, these investigators will determine oxidant production, overall oxidative protein and lipid damage, specific enzyme activity, and mitochondrial function of young, adult, and old rats. They will also measure enzyme activity of purified enzymes and the amount of oxidative enzyme damage. Furthermore, they will determine if two protective interventions, caloric restriction and life-long voluntary exercise, can attenuate muscle protein oxidation and restore enzymatic function and mitochondrial function. These experiments will provide the first evidence to answer the following questions:1) are mitochondria major contributors to protein oxidation with aging? 2) are key metabolic enzymes affected by oxidant damage and contribute to the functional decline in muscle with age? 3) does enzyme activity reflect loss of enzyme protein or the accumulation of inactive forms of enzymes? And 4) can enzyme function and mitochondrial functional capacity be restored with caloric restriction and/or daily exercise?