Tyrosine nitration and altered protein pool levels of glyceraldehyde-3-phosphate dehydrogenase following ischemia/reperfusion: The influence of aging in glycolytic skeletal muscle

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Title: Tyrosine nitration and altered protein pool levels of glyceraldehyde-3-phosphate dehydrogenase following ischemia/reperfusion: The influence of aging in glycolytic skeletal muscle
Author: Christopher Eric Bailey
Abstract: Aging and skeletal muscle ischemia /reperfusion (I /R ) injury both lead to skeletal \r \nmuscle dysfunction , evidenced by decreased contractile force generation , particularly in \r \nglycolytic muscle . The deficits in I /R are more severe and persistent in aged animals . \r \nPrevious studies in our lab led us to hypothesize that the expression of the glycolytic \r \nenzyme glyceraldehyde -3 -phosphate dehydrogenase may be altered following I /R . We \r \nfurther hypothesized that aging would enhance the oxidative stress and oxidative damage \r \nexperienced by the muscle . GAPDH protein levels were measured by Western blotting . \r \nWe observed that the enzyme is significantly decreased at 3 and 5 days of reperfusion in \r \nthe young muscle , while the enzyme was significantly decreased in the aged muscle at 1 , \r \n3 , 5 , and 7 days . Using PCR , we compared GAPDH mRNA levels at 5 days reperfusion \r \nand found that the I /R tissue from both young and old have significant increases in \r \nGAPDH transcript at this time point compared to control , suggesting that the protein \r \ndeficit is not due to decreased transcription . Finally , we examined tyrosine nitration . A \r \nvii \r \nspot selected following 2D gel electrophoresis and nitrotyrosine western blotting of \r \nyoung and old muscle lysate was identified as GAPDH by mass spectrometry . We \r \ncompared tyrosine nitration over the time course of reperfusion . While total tyrosine \r \nnitration does not increase in the I /R tissue in the young , nitration of GAPDH is \r \nsignificantly increased at 1 and 3 days reperfusion . In contrast to the young , total \r \ntyrosine nitration in the aged muscle was significantly increased at 1 , 3 , and 5 days of \r \nreperfusion , with significant increases in nitration of GAPDH at the same time points . \r \nWe conclude that GAPDH protein levels are decreased following I /R , which could \r \ninterfere with metabolism and ATP generation . Further , this decrease is not likely \r \ntranscriptionally mediated . Based on the increases in tyrosine nitration , we propose that \r \noxidative modification enhances the degradation of GAPDH following I /R , and that the \r \npersistence of decreased GAPDH in the aged muscle is due to the prolonged increases in \r \noxidative modification seen in that age group . This suggests that the aged muscle \r \nexperiences greater oxidative stress , protein modification , and GAPDH degradation , \r \npossibly contributing to the decreased muscle function reported in the literature .
URI: http : / /hdl .handle .net /2152 .3 /128
Date: 2009-06-09

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Tyrosine nitration and altered protein pool levels of glyceraldehyde-3-phosphate dehydrogenase following ischemia/reperfusion: The influence of aging in glycolytic skeletal muscle. Doctoral dissertation, The University of Texas Medical Branch. Available electronically from http : / /hdl .handle .net /2152 .3 /128 .

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