摘要
二氢硫辛酰胺脱氢酶(dihydrolipoamide dehydrogenase,DLDH)是线粒体3个α-酮酸脱氢酶复合物(丙酮酸脱氢酶复合物、α-酮戊二酸脱氢酶复合物、支链氨基酸脱氢酶复合物)的关键成分,属于吡啶依赖性二硫化物氧化还原酶类,对活性氮自由基(reactive nitrogen species,RNS)和活性氧自由基(reactive oxygenspecies,ROS)造成的氧化修饰非常敏感。本研究探索由Angeli盐所产生的RNS对DLDH的修饰作用及机制。将大鼠脑线粒体分离,与不同浓度的Angeli盐作用,应用分光光度计、蓝色胶、基于二维电泳的蛋白质组学等手段,测定DLDH酶活性。结果显示,Angeli盐呈浓度依赖性方式灭活DLDH,过氧亚硝酸盐在同样条件下对DLDH酶活性无抑制作用,说明Angeli盐对DLDH的作用可能是非随机的。由于Angeli盐在生理pH条件下可分解为硝基阴离子(nitroxyl anion,HNO)和一氧化氮(nitric oxide,NO),故进一步分析了Angeli盐对DLDH的灭活作用是否由HNO引起,结果证实确实如此。最后,二维电泳Western blot结果显示,Angeli盐对DLDH的灭活伴随着DLDH蛋白质的S-亚硝基硫醇形成,提示S-亚硝基硫醇化可能是导致DLDH酶失活的原因。综上,本研究为研究Angeli盐灭活DLDH的机制提供了新证据。
Dihydrolipoamide dehydrogenase （DLDH） is a key component of 3 mitochondrial α-keto acid dehydrogenase complexes including pyruvate dehydrogenase complex, α-ketoglutarate dehydrogenase complex, and branched chain amino acid dehydrogenase complex. It is a pyridine-dependent disulfide oxidoreductase that is very sensitive to oxidative modifications by reactive nitrogen species （RNS） and reactive oxygen species （ROS）. The objective of this study was to investi- gate the mechanisms of DLDH modification by RNS derived from Angeli＇s salt. Studies were conducted using isolated rat brain mitochondria that were incubated with varying concentrations of Angeli＇s salt followed by spectrophotometric enzyme assays, blue native gel analysis, and 2-dimensional gel-based proteomic approaches. Results show that DLDH could be inactivated by Angeli＇s salt in a concentration dependent manner and the inactivation was a targeting rather than a random process as peroxynitrite did not show any detectable inhibitory effect on the enzyme＇s activity under the same exper- imental conditions. Since Angeli＇s salt can readily decompose at physiological pH to yield nitroxyl anion （HNO） and nitric oxide, further studies were conducted to determine the actual RNS that was responsible for DLDH inactivation. Results indicate that it was HNO that exerted the effect of Angeli＇s salt on DLDH. Finally, two-dimensional Western blot analysis indicates that DLDH inactivation by Angeli＇s salt was accompanied by formation of protein S-nitrosothiols, Suggesting that S-nitrosylation is likely the cause of loss in enzyme＇s activity. Taken together, the present study provides insights into mechanisms of DLDH inactivation induced by HNO derived from Angeli＇s salt.
出处
《生物物理学报》
CAS
CSCD
北大核心
2012年第4期341-350,共10页
Acta Biophysica Sinica
基金
supported by a grant from NIH(AG022550)~~
