MOLECULAR MECHANISMS OF OXIDATIVE STRESS IN AGING MUSCLE

说明(申请人摘要)：氧化损伤，尤其是对蛋白质的损伤， 被广泛认为是导致肌肉功能丧失的主要原因 衰老。研究人员观察到线粒体氧化作用的减少 肌肉中的能力和酶活性与年龄可能有直接关系 线粒体产生自由基氧化剂。几个关键的代谢 酶--磷酸果糖激酶(限速糖酵解酶)，两个关键 线粒体酶(柠檬酸合成酶和乌头酸酶)和一个电子 运输链酶(细胞色素c氧化酶)--已显示出下降 随着年龄的增长，骨骼肌和心肌也会发生变化。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，活性自由基物种的来源 对蛋白质氧化损伤的影响还知之甚少，主要是因为 用于研究蛋白质氧化的非特异性方法。因此，我们将 使用灵敏的分析方法(气相色谱和质谱仪) 探索金属催化氧化途径的各自作用， 酪氨酸基介导的途径，以及活性氮途径， 产生特定的非天然氨基酸，即o-酪氨酸，m-酪氨酸， O‘-二氨基酪氨酸和3-硝基酪氨酸。通过这种方法，调查人员将 能够确定以下内容：1)哪些自由基物种负责 对于蛋白质损伤？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)蛋白质损伤的水平 尿液中的氧化氨基酸与线粒体和/或 细胞液？5)氧化蛋白质修饰是否影响酶功能和 线粒体功能能力？为了实现这些目标，这些 调查人员将确定氧化剂的产生，总的氧化蛋白和 幼体的脂质损伤、特定的酶活性和线粒体功能， 成年的，和年老的老鼠。他们还将测量纯化酶的酶活性。 以及氧化酶的损伤量。此外，他们将确定是否 卡路里限制和终身自愿两种保护性干预措施 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 要回答以下问题：1)线粒体是不是对 蛋白质随年龄增长而氧化？2)是受氧化剂影响的关键代谢酶 随着年龄的增长，肌肉的功能会受到损害并导致功能衰退？ 酶活性反映了酶蛋白的损失或非活性物质的积累 酶的形式？4)酶功能和线粒体功能 通过限制卡路里和/或日常锻炼来恢复体能？

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?