Forshaw Thomas E, Conway Myra E
Faculty of Health and Applied Sciences, University of the West of England, Bristol, UK.
Department of Applied Sciences, University of the West of England, Bristol, UK.
Methods Mol Biol. 2019;1990:71-84. doi: 10.1007/978-1-4939-9463-2_6.
The human branched-chain aminotransferase (hBCAT) enzymes play an integral role in brain glutamate and branched-chain amino acid (BCAA) metabolism. Optimal hBCAT activity is dependent on the oxidation state of their redox reactive thiols, where post-translational modification by nitric oxide (NO) and glutathione results in reversible inhibition. Incubation of the cytosolic isoform (hBCATc) with S-nitrosating agents was found to inhibit in both a time and dose dependent manner through formation of a mixture of products including cysteine-nitric oxide (SNO) and S-glutathionylation. Mechanistic details of these redox interactions were studied using labeling with fluorescein-5-maleimide and confirmed via mass spectrometry and Western blot analysis. Though the mitochondrial isoform (hBCATm) was inhibited by nitrosating agents adduct formation could only be observed by DTNB titration as neither SNO, S-glutathionylation or disulfide bond formation could be detected. These studies revealed that the two isoforms of hBCAT, namely hBCATc and hBCATm, were differently regulated by S-nitrosation or S-glutathionylation pointing to distinct functional/mechanistic responses to GSNO modification. Detection of these adducts is essential for studies into the effect of NO on cells and the redox proteome which can offer insight into several pathological states and normal functioning of the cell.
人类支链氨基转移酶（hBCAT）在大脑谷氨酸和支链氨基酸（BCAA）代谢中发挥着不可或缺的作用。hBCAT的最佳活性取决于其氧化还原反应性硫醇的氧化状态，其中一氧化氮（NO）和谷胱甘肽的翻译后修饰会导致可逆抑制。发现用亚硝基化剂孵育胞质同工型（hBCATc）会通过形成包括半胱氨酸-一氧化氮（SNO）和S-谷胱甘肽化在内的产物混合物，以时间和剂量依赖性方式抑制。使用5-马来酰亚胺荧光素标记研究了这些氧化还原相互作用的机制细节，并通过质谱和蛋白质印迹分析得到证实。尽管线粒体同工型（hBCATm）被亚硝基化剂抑制，但只能通过DTNB滴定观察到加合物的形成，因为未检测到SNO、S-谷胱甘肽化或二硫键形成。这些研究表明，hBCAT的两种同工型，即hBCATc和hBCATm，受S-亚硝基化或S-谷胱甘肽化的调节不同，这表明对GSNO修饰有不同的功能/机制反应。检测这些加合物对于研究NO对细胞和氧化还原蛋白质组的影响至关重要，这可以深入了解细胞的几种病理状态和正常功能。
