No abstract

Regular physical exercise retards a number of age-associated disorders, in spite of the paradox that free radical generation is significantly enhanced with exercise. Eight weeks of treadmill running resulted in nearly a 40% increase in maximal oxygen uptake in both middle-aged (20-month-old) and aged (30-month-old) rats. The age-associated increase in 8-hydroxy-2'-deoxyguanosine (8-OHdG) content was significantly attenuated in gastrocnemius muscle by exercise. The 8-OHdG repair, as measured by the excision of 32P-labeled damaged oligonucleotide, increased in muscle of exercising animals. The reactive carbonyl derivatives (RCD) of proteins did not increase with aging. However, when the muscle homogenate was exposed to a mixture of 1 mM iron sulfate and 50 mM ascorbic acid, the muscle of old control animals accumulated more RCD than that of the trained or adult groups. The chymotrypsin-like activity of proteasome complex increased in muscle of old trained rats. We suggest that regular exercise-induced adaptation attenuates the age-associated increase in 8-OHdG levels, and increases the activity of DNA repair and resistance against oxidative stress in proteins.

No abstract

In accordance with their manifold tasks, various dysfunctions of mitochondria are critically involved in a large number of diseases and the aging process. This has inspired considerable efforts to identify all the mitochondrial proteins by denaturing approaches, notably, the standard gel-based method employing isoelectric focusing. Because a significant part of the mitochondrial proteome is membrane-associated and/or functions as homo- or heterooligomeric protein complexes, there is an urgent need to detect and identify mitochondrial proteins, both membranous and soluble ones, under conditions preserving protein-protein interactions. Here, we investigated mitochondria of five different rat organs (kidney, liver, heart, skeletal muscle, and brain) solubilized with digitonin, enabling the quantitative extraction of the five oxidative phosphorylation (OXPHOS) complexes. The analysis by blue-native (BN)-PAGE recovered the OXPHOS complexes to a large extent as supercomplexes and separated many other protein complexes and individual proteins which were resolved by subsequent 2D SDS-PAGE revealing the tissue-diverse mitochondrial proteomes. Using MS peptide mass fingerprinting, we identified in all five organs 92 nonredundant soluble and membrane-embedded non-OXPHOS proteins, among them, many as constituents of known mitochondrial protein complexes as well as novel ones such as the putative "stomatin-like protein 2 complex" with an apparent mass of ca. 1800 kDa. Interestingly, the identification list included 36 proteins known or presumed to be localized to nonmitochondrial compartments, for example, glycolytic enzymes, clathrin heavy chain, valosin-containing protein/p97, VoV1-ATPase, and Na,K-ATPase. We expect that more than 200 distinct non-OXPHOS proteins of digitonin-solubilized rat mitochondria separated by 2D BN/SDS-PAGE, representing a partial "protein interactome" map, can be identified.