Histochemical staining and quantification of dihydrolipoamide dehydrogenase diaphorase activity using blue native PAGE
Histochemical staining and quantification of dihydrolipoamide dehydrogenase diaphorase activity using blue native PAGE
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DOI：
10.1002/elps.200600574
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发表时间：
2007-04-01
期刊：
影响因子：
2.9
通讯作者：
Forster, Michael J.
中科院分区：
文献类型：
--
作者：
Yan, Liang-Jun;Yang, Shao-Hua;Forster, Michael J.
哺乳动物线粒体二氢脂酰胺脱氢酶（DLDH, EC 1.8.1.4）在体内催化NAD（+）依赖的二氢脂酰胺氧化，也可以在体外作为二磷酸酶催化烟酰胺腺嘌呤二核苷酸（还原形式）（NADH）依赖的电子接受分子如泛醌和硝基蓝四唑（NBT）的还原。在本文中，我们报告了一种基于凝胶的方法，使用蓝色天然PAGE （BN-PAGE）进行组织化学染色和定量DLDH diaphorase活性。大鼠脑线粒体提取物作为DLDH的来源，用非梯度BN-PAGE（9%）分离，然后以NADH为电子供体，NBT为电子受体进行脱氢酶活性染色。结果表明，DLDH脱氢酶活性染色与蛋白量和时间有关。此外，这种凝胶内活性染色方法被证明与传统的分光光度法（以二氢脂酰胺为底物）测量DLDH脱氢酶活性的方法是一致的。该方法被应用于测定除脑外的几种大鼠组织中的DLDH脱氢酶活性水平，结果表明所有检查组织的DLDH脱氢酶活性水平相似。最后，我们评估了n -乙基马来酰亚胺（NEM）和一氧化氮等硫醇反应试剂对DLDH脱氢酶活性的影响，结果表明，使用这种方法，可以在不去除可能干扰分光光度法测量DLDH脱氢酶活性的硫醇反应试剂的情况下测定DLDH脱氢酶活性。凝胶法也可以作为分离线粒体DLDH的一种手段，在研究DLDH翻译后修饰时，可以通过质谱技术对线粒体DLDH进行分析。
Mammalian mitochondrial dihydrolipoamide dehydrogenase (DLDH, EC 1.8.1.4) catalyzes NAD(+)-dependent oxidation of dihydrolipoamide in vivo and can also act as a diaphorase catalyzing in vitro nicotinamide adenine dinucleotide (reduced form) (NADH)-dependent reduction of electron-accepting molecules such as ubiquinone and nitroblue tetrazolium (NBT). In this paper, we report a gel-based method for histochemical staining and quantification of DLDH diaphorase activity using blue native PAGE (BN-PAGE). Rat brain mitochondrial extracts, used as the source of DLDH, were resolved by nongradient BN-PAGE (9%), which was followed by diaphorase activity staining using NADH as the electron donor and NBT as the electron acceptor. It was shown that activity staining of DLDH diaphorase was both protein amount- and time-dependent. Moreover, this in-gel activity-staining method was demonstrated to be in good agreement with the conventional spectrophotometric method that measures DLDH dehydrogenase activity using dihydrolipoamide as the substrate. The method was applied to determine levels of DLDH diaphorase activity in several rat tissues other than the brain, and the results indicated a similar level of DLDH diaphorase activity for all the tissues examined. Finally, the effects of thiol-reactive reagents such as N-ethylmaleimide (NEM) and nitric oxide donors on DLDH diaphorase activity were evaluated, demonstrating that, with this method, DLDH diaphorase activity can be determined without having to remove these thiol-reactive reagents that may otherwise interfere with spectrophotometric measurement of DLDH dehydrogenase activity. The gel-based method can also be used as a means to isolate mitochondrial DLDH that is to be analyzed by mass spectral techniques in studying DLDH post-translational modifications.