来自丝状线虫鹿丝状线虫胞质谷胱甘肽-S-转移酶的纯化及生化特性分析
Purification and biochemical characterization of cytosolic glutathione-S-transferase from filarial worms Setaria cervi.
作者信息
Ahmad Rumana, Srivastava Arvind K, Walter Rolf D
机构信息
Division of Biochemistry, P.O. Box No. 173, Central Drug Research Institute, Chattar Manzil Palace, Lucknow-226001, India.
出版信息
Comp Biochem Physiol B Biochem Mol Biol. 2008 Nov;151(3):237-45. doi: 10.1016/j.cbpb.2008.03.019. Epub 2008 Apr 8.
The present study reports the purification and characterization of GST from cytosolic fraction of Setaria cervi. GST activity was determined in various subcellular fractions of bovine filarial worms S. cervi (Bubalus bubalis Linn.) and was found to be localized mainly in the cytosolic and microsomal fractions. The soluble enzyme from S. cervi was purified to homogeneity using a combination of salt precipitation, centrifugation, cation exchange and GSH-Sepharose affinity chromatography followed by ultrafiltration. SDS-PAGE analysis revealed a single band and activity staining was also detected on PAGE gels. Gel filtration and MALDI-TOF studies revealed that the native enzyme is a homodimer with a subunit molecular mass of 24.6 kDa. Comparison of kinetic properties of the parasitic and mammalian enzymes revealed significant differences between them. The substrate specificity and inhibitor profile of cytosolic GST from S. cervi appeared to be different from GST from mammalian sources.
本研究报告了从牛丝状线虫胞质部分纯化和鉴定谷胱甘肽S-转移酶（GST）的过程。测定了水牛丝状线虫（Bubalus bubalis Linn.）各亚细胞部分的GST活性，发现其主要定位于胞质和微粒体部分。采用盐析、离心、阳离子交换和谷胱甘肽-琼脂糖亲和层析结合超滤的方法，将来自牛丝状线虫的可溶性酶纯化至同质。SDS-PAGE分析显示有一条单一的条带，并且在PAGE凝胶上也检测到活性染色。凝胶过滤和基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）研究表明，天然酶是一种同二聚体，亚基分子量为24.6 kDa。寄生酶和哺乳动物酶动力学性质的比较显示它们之间存在显著差异。牛丝状线虫胞质GST的底物特异性和抑制剂谱似乎与哺乳动物来源的GST不同。
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