Purification and Characterisation of Rat Kidney Glutathione Reductase
Purification and Characterisation of Rat Kidney Glutathione Reductase
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DOI：
10.1007/s10930-010-9246-4
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发表时间：
2010-05-01
期刊：
影响因子：
3
通讯作者：
Ozer, Nazmi
中科院分区：
文献类型：
--
作者：
Can, Betul;Kulaksiz Erkmen, Gulnihal;Ozer, Nazmi
谷胱甘肽还原酶[GR，E.C.1.8.1.7]催化NADPH依赖的谷胱甘肽二硫化物(GSSG)还原为还原型谷胱甘肽(GSH)。因此，它是维持细胞内高[GSH]/[GSSG]比值和生理氧化还原状态的关键酶。肾脏和肝脏组织被认为是GR的丰富来源。本研究对大鼠肾糖皮质激素受体进行了纯化，并对其部分性质进行了研究。经热变性、Sephadex G25凝胶过滤、2‘，5’-ADP琼脂糖4B、PBE94柱层析，纯化2,356倍，得率16%。该酶的比活力为250U/mg蛋白，A(273)/A(463)、A(280)/A(460)、A(365)/A(460)、A(379)/A(463)的吸收比分别为7.1、6.8、1.2和1.0。每一摩尔GR亚基结合0.97摩尔FAD。大鼠肾脏GR使用NADH作为辅酶，但效率(32.7%)低于NADPH。亚基相对分子质量为53 kDa。大鼠肾脏GR的最适pH为6.5，最适温度为65℃。其活化能(Ea)和温度系数(Q(10))分别为7.02千卡/摩尔和1.42。在10~200亩M-NADPH的浓度范围内，Km((NADPH))和kcat/Km((NADPH))分别为15.3+/-A 1.4亩M和1.68×10~(-7)M~(-1)S(-1)；当GSSG为可变底物时，20~1,200亩M的Km((GSSG))和kcat/Km((GSSG)值分别为53.1+/-A，3.4亩M和4.85×10~(-1)M~(-1)S(-1)。
Glutathione reductase [GR, E.C.1.8.1.7] catalyses NADPH dependent reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH). Thus, it is the crucial enzyme to maintain high [GSH]/[GSSG] ratio and physiological redox status in cells. Kidney and liver tissues were considered as a rich source of GR. In this study, rat kidney GR was purified and some of its properties were investigated. The enzyme was purified 2,356 fold with a yield of 16% by using heat-denaturation and Sephadex G25 gel filtration, 2',5'-ADP Agarose 4B, PBE94 column chromatographies. The purified enzyme had a specific activity (Vm) of 250 U/mg protein and the ratio of absorbances at wavelengths of A (273)/A (463,) A (280)/A (460), A (365)/A (460), and A (379)/A (463), were 7.1, 6.8, 1.2 and 1.0, respectively. Each mol of GR subunit bound 0.97 mol of FAD. NADH was used as a coenzyme by rat kidney GR but with a lower efficiency (32.7%) than NADPH. Its subunit molecular weight was estimated as 53 kDa. An optimum pH of 6.5 and optimum temperature of 65 A degrees C were found for rat kidney GR. Its activation energy (Ea) and temperature coefficient (Q(10)) were calculated as 7.02 kcal/mol and 1.42, respectively. The Km((NADPH)) and kcat/Km ((NADPH)) values were found to be 15.3 +/- A 1.4 mu M and 1.68 x 10(7) M-1 s(-1) for the concentration range of 10-200 mu M NADPH and when GSSG is the variable substrate, the Km((GSSG)) and the kcat/Km((GSSG)) values of 53.1 +/- A 3.4 mu M and 4.85 x 10(6) M-1 s(-1) were calculated for the concentration range of 20-1,200 mu M GSSG.