Department of Biochemistry, Obafemi Awolowo University, Ile-Ife, Nigeria.
Fish Physiol Biochem. 2013 Dec;39(6):1657-63. doi: 10.1007/s10695-013-9817-3. Epub 2013 Jun 9.
Purified juvenile catfish (Clarias gariepinus) glutathione transferase (cgGST) was denatured in vitro and renatured in the absence and presence of different concentrations of endogenous or xenobiotic model substrates. Protein transitions during unfolding and refolding were monitored by activity measurement as well as changes in protein conformation using UV difference spectra at 230 nm. Gdn-HCl at 0.22 M caused 50 % inactivation of the enzyme and at 1.1 M, the enzyme was completely unfolded. Refolding of cgGST main isozyme was not completely reversible at higher concentrations of Gdn-HCl and is dependent on protein concentration. An enzyme concentration of 30 μg/ml yielded 40 % percentage residual activity in the presence of glutathione (GSH), regardless of the concentration that was present as opposed to 30 % obtained in its absence. The xenobiotic model substrate, lindane, appears to have no effect on the refolding of the enzyme. In summary, our results show that GSH assists in the refolding of cgGST in a concentration-independent manner and may be involved in the same function in vivo whereas the xenobiotic model substrate does not.
纯化的幼年鲶鱼（Clarias gariepinus）谷胱甘肽转移酶（cgGST）在体外变性，并在有无不同浓度的内源性或外源模型底物的情况下复性。通过活性测量以及在 230nm 处使用紫外差光谱监测蛋白质构象变化来监测展开和重折叠过程中的蛋白质转变。Gdn-HCl 浓度为 0.22M 时，酶的失活率为 50%，而 1.1M 时，酶完全展开。在较高浓度的 Gdn-HCl 下，cgGST 主要同工酶的重折叠不是完全可逆的，并且依赖于蛋白质浓度。在存在谷胱甘肽（GSH）的情况下，酶浓度为 30μg/ml 时，酶的残留活性百分比为 40%，而在不存在 GSH 的情况下，酶的残留活性百分比为 30%。外源模型底物林丹似乎对酶的重折叠没有影响。总之，我们的结果表明，GSH 以浓度独立的方式辅助 cgGST 的重折叠，并且可能在体内参与相同的功能，而外源模型底物则没有。
