Tekman Bilge, Ozdemir Hasan, Senturk Murat, Ciftci Mehmet
Atatürk University, Arts and Science Faculty Department of Chemistry, TR-25240, Erzurum-Turkey.
Comp Biochem Physiol C Toxicol Pharmacol. 2008 Aug;148(2):117-21. doi: 10.1016/j.cbpc.2008.04.005. Epub 2008 Apr 22.
Glutathione reductase (E C: 1.8.1.7; GR) was purified from rainbow trout (Oncorhynchus mykiss) liver, and some characteristics of the enzyme were investigated. The purification procedure consisted of four steps: preparation of homogenate, ammonium sulfate fractionation, affinity chromatography on 2',5'-ADP Sepharose-4B and gel filtration chromatography on Sephadex G-200. The enzyme, with a specific activity of 27.45 U/mg protein, was purified 1,654-fold with a yield of 41%. Optimal pH, stable pH, optimal temperature, optimum ionic strength, molecular mass, KM and Vmax values for GSSG and NADPH were also determined for the enzyme. In addition, Ki values and inhibition types were determined for GSH and NADP+. Additionally, inhibitory effects of metal ions (Cd+2, Cu+2, Pb+2, Hg+2, Fe+3 and Al+3) on glutathione reductase were investigated. Ki constants and IC50 values for metal ions were determined by Lineweaver-Burk graphs and plotting activity % vs. [I], respectively. IC50 values of Cd+2,Cu+2, Pb+2, Hg+2, Fe+3 and Al+3 were 0.0655, 0.082, 0.122, 0.509, 0.797 and 0.804 mM, and the Ki constants for Cd+2 and Cu+2 were 0.104+/-0.001, 0.117+/-0.001, respectively.
谷胱甘肽还原酶（EC：1.8.1.7；GR）从虹鳟（Oncorhynchus mykiss）肝脏中纯化得到，并对该酶的一些特性进行了研究。纯化过程包括四个步骤：匀浆制备、硫酸铵分级分离、2',5'-ADP琼脂糖-4B亲和层析以及葡聚糖凝胶G-200凝胶过滤层析。该酶的比活性为27.45 U/mg蛋白质，纯化了1654倍，产率为41%。还测定了该酶的最适pH、稳定pH、最适温度、最佳离子强度、分子量、GSSG和NADPH的KM和Vmax值。此外，还测定了GSH和NADP+的Ki值和抑制类型。另外，研究了金属离子（Cd+2、Cu+2、Pb+2、Hg+2、Fe+3和Al+3）对谷胱甘肽还原酶的抑制作用。通过Lineweaver-Burk图和绘制活性%与[I]的关系曲线分别测定了金属离子的Ki常数和IC50值。Cd+2、Cu+2、Pb+2、Hg+2、Fe+3和Al+3的IC50值分别为0.0655、0.082、0.122、0.509、0.797和0.804 mM，Cd+2和Cu+2的Ki常数分别为0.104±0.001、0.117±0.001。
