The long-term goal of this investigation is to elucidate the mechanisms by which molecular oxidative damage causes losses in cellular functions during the aging process. In the current view, age-related attenuation in physiological functions is due to the accumulation of randomly- inflicted molecular oxidative damage. The proposed study will test an alternate hypothesis that """"""""senescence-associated losses in cellular functions are due to the accrual of oxidative damage to specific proteins."""""""" The validity of this hypothesis will be tested in different tissues of the mouse by determining whether an age-associated increase in oxidative damage and a loss in catalytic activity involve only a limited number of proteins and whether the amount of such damage and loss of protein function are related to the life expectancy of the animals. Life expectancy of the mice will be experimentally varied by caloric restriction. Protein oxidation will be detected by the presence of carbonyl groups.
Specific aims are: (1) Immunochemically detect carbonylated proteins in mitochondrial, microsomal and cytosolic fractions of various tissues in aged (24-month old) mice. (2) Quantify the age-associated increase in carbonylation of specific proteins in ad libitum-fed (AL) and calorically restricted (CR) mice. Select the proteins showing attenuation of carbonylation by CR (referred to here as 'CR-sensitive carbonylated proteins'.) (3) Purify and identify by microsequencing the 'CR-sensitive carbonylated proteins'. (4) Determine if carbonylated proteins lose catalytic activity during aging. (5) Elucidate mechanisms of carbonylation and loss of catalytic activity by determining the source of carbonylation and its effect on catalytic activity of the proteins. Results of this study should provide significant new information by indicating how oxidative molecular damage to specific proteins is mechanistically linked to senescence-associated losses in cellular functions. Results will also provide a test of the validity of oxidative stress hypothesis of aging.
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