Quantification of NADH:ubiquinone oxidoreductase (complex I) content in biological samples.
Quantification of NADH:ubiquinone oxidoreductase (complex I) content in biological samples.
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NADH的定量：生物样品中的泛酮氧化还原酶（复杂I）含量。
DOI：
10.1016/j.jbc.2021.101204
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发表时间：
2021-10
期刊：
影响因子：
--
通讯作者：
Galkin A
中科院分区：
文献类型：
--
作者：
Ansari F;Yoval-Sánchez B;Niatsetskaya Z;Sosunov S;Stepanova A;Garcia C;Owusu-Ansah E;Ten V;Wittig I;Galkin A
线粒体能量代谢的损伤与线粒体和核DNA突变、神经退行性和心血管疾病、糖尿病和衰老相关的人类遗传疾病有关。线粒体复合物I结构和活性的改变已被证明在帕金森病和缺血/再灌注组织损伤中起关键作用，但在评估给定样品中该酶复合物的含量方面仍然存在显著困难。本文介绍了一种利用非变性聚丙烯酰胺凝胶电泳结合黄素荧光扫描测定制剂中复合物I和α-酮戊二酸脱氢酶复合物绝对含量的新方法。我们发现，在脑线粒体中复合物I的含量为19 ± 1 pmol/mg蛋白质，而在不同的小鼠组织中变化高达10倍。与测量NADH依赖性比活性一起，我们的方法还可以准确测定复合物I催化周转率，计算为小鼠脑线粒体制备物中NADH：泛醌还原酶的104 min−1。测定小鼠脑和牛心脏线粒体的α-酮戊二酸脱氢酶复合物含量分别为65 ± 5和123 ± 9 pmol/mg蛋白。我们的方法也可以扩展到培养细胞，并且我们证明单个人胚肾293细胞中存在约90 × 103个复合物I分子。测定复合物I含量的能力应提供有价值的工具，以研究突变生物体、培养物中的细胞或人活检中体内处理后样品中的酶状态。
Impairments in mitochondrial energy metabolism have been implicated in human genetic diseases associated with mitochondrial and nuclear DNA mutations, neurodegenerative and cardiovascular disorders, diabetes, and aging. Alteration in mitochondrial complex I structure and activity has been shown to play a key role in Parkinson's disease and ischemia/reperfusion tissue injury, but significant difficulty remains in assessing the content of this enzyme complex in a given sample. The present study introduces a new method utilizing native polyacrylamide gel electrophoresis in combination with flavin fluorescence scanning to measure the absolute content of complex I, as well as α-ketoglutarate dehydrogenase complex, in any preparation. We show that complex I content is 19 ± 1 pmol/mg of protein in the brain mitochondria, whereas varies up to 10-fold in different mouse tissues. Together with the measurements of NADH-dependent specific activity, our method also allows accurate determination of complex I catalytic turnover, which was calculated as 104 min−1 for NADH:ubiquinone reductase in mouse brain mitochondrial preparations. α-ketoglutarate dehydrogenase complex content was determined to be 65 ± 5 and 123 ± 9 pmol/mg protein for mouse brain and bovine heart mitochondria, respectively. Our approach can also be extended to cultured cells, and we demonstrated that about 90 × 103 complex I molecules are present in a single human embryonic kidney 293 cell. The ability to determine complex I content should provide a valuable tool to investigate the enzyme status in samples after in vivo treatment in mutant organisms, cells in culture, or human biopsies.
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影响因子：
3.5
作者：
Borna NN;Kishita Y;Sakai N;Hamada Y;Kamagata K;Kohda M;Ohtake A;Murayama K;Okazaki Y
通讯作者：
Okazaki Y
影响因子：
16.6
作者：
Bridges HR;Fedor JG;Blaza JN;Di Luca A;Jussupow A;Jarman OD;Wright JJ;Agip AA;Gamiz-Hernandez AP;Roessler MM;Kaila VRI;Hirst J
通讯作者：
Hirst J
影响因子：
4.4
作者：
Cox, Juergen;Neuhauser, Nadin;Mann, Matthias
通讯作者：
Mann, Matthias
影响因子：
8.8
作者：
Garcia CJ;Khajeh J;Coulanges E;Chen EI;Owusu-Ansah E
通讯作者：
Owusu-Ansah E
影响因子：
2.9
作者：
Blinova, Ksenia;Levine, Rodney L.;Boja, Emily S.;Griffiths, Gary L.;Shi, Zhen-Dan;Ruddy, Brian;Balaban, Robert S.
通讯作者：
Balaban, Robert S.