Oxidative and nitrative modifications of α-enolase in cardiac proteins from diabetic rats

Many studies have reported that oxidative and nitrative stress might be important in the pathogenesis of diabetes. By means of immunoprecipitation analysis, α-enolase (EC 4.2.1.11, 2-phospho-d-glycerate hydrolyase) was identified as the important target for oxidative and nitrative modifications in diabetic cardiac proteins. The levels of protein carbonyls and 3-nitrotyrosine residues in α-enolase (biomarkers of oxidative and nitrative damage, respectively) from cardiac proteins of diabetic rats were determined and compared with age-matched controls. After 6 weeks of streptozotocin administration, the cardiac proteins from diabetic rats showed: (a) the levels of α-enolase expression and nitration were clearly increased, whereas (b) the enolase activity and oxidation status were not significantly changed. By means of immunoprecipitation and liquid chromatography–tandem mass spectrometry analysis, it was found that Tyr 257 and Tyr 131 of α-enolase were the most susceptible to nitration in diabetic rat heart. Further studies in vitro revealed a significant contribution of protein tyrosine nitration to the inactivation of enolase. These results suggest that tyrosine nitration of α-enolase could contribute to an impaired glycolytic activity in diabetic cardiomyopathy. Meanwhile, the up-regulation of α-enolase expression could be a protective mechanism to neutralize oxidative and nitrative stress in diabetes.