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?
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