ANALYSIS OF CHANGES IN MITOCHONDRIAL PROTEINS WITH AGE IN WILDTYPE AND MNSOD KNOCKOUT MICE
H. Van Remmen, J. Chang, T. Shibatani, W. Ward, J. E. Cornell and A. Richardson
University of Texas Health Science Center San Antonio,
Department of Physiology,
7703 Floyd Curl Dr.,
San Antonio, Texas 78229-3900
The accumulation of oxidative damage with age has been suggested as a primary factor in the age-related decline of physiological function and in the pathogenesis of age-related diseases. Mitochondria are a primary source of free radical production and therefore are also particularly susceptible to oxidative damage. We are interested in analyzing alterations in mitochondrial proteins with age in wildtype (WT) mice and mice with reduced activity of the mitochondrial antioxidant enzyme, manganese superoxide dismutase (MnSOD). Using blue native gels we have analyzed changes in the level and activity of specific mitochondrial electron transport complexes in heart mitochondria isolated from young (6 month) and old (29-30 month) WT and MnSOD knockout (KO) mice. The activity of Complex V decreases 30% between 6 and 29 months in WT mice. Complex V activity is also 30% lower in heart mitochondria from young MnSOD KO mice compared to young WT mice. The activity decreases an additional 20% in the old KO mice. The level of Complex V protein is approximately 20% lower in the young KO and both the old WT and old KO mice. The activities of Complexes I and IV were not altered with age or in the KO mice. To analyze global changes in mitochondrial proteins, we have developed a 2-DE system for analyzing the profile of skeletal muscle mitochondrial proteins as a function of age in WT and MnSOD KO mice. We have optimized the separation of muscle mitochondrial proteins and achieved high gel to gel reproducibilty. Using protein concentrations ranging from 10 micrograms to 200 micrograms, we are able to visualize 300 to 500 proteins and at least 80% of the protein spots show a linear increase in fluorescence with increasing protein concentration, R2 of >0.8. The coefficient of variance (CV) was less than 50%. Thus, we have established a system in which we can reproducibly separate and quantify protein expression in mouse hindlimb skeletal muscle mitochondria. These two techniques are powerful tools to analyze the effect of age and oxidative stress on alterations in the level and activity of mitochondrial proteins.
Key words:
mitochondria, proteins, oxidative stress, MnSOD
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