Acta Scientiarum Naturalium Universitatis Pekinensis

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Studies on Purification and Properties of Glutathione S-transferase from Asian corn Borer

MA Suyong, ZHOU Xianwan, ZHANG Tingfang   

  1. College of Life Sciences, Peking University, Beijing, 100871
  • Received:1998-04-21 Online:1999-07-20 Published:1999-07-20



  1. 北京大学生命科学学院,北京,100871

Abstract: Glutathione S-transferase(GST) was purified from larvae of Asian corn borer(Ostrina furnacalis) by Sephadex G-75, DEAE-Sepharose Fast Flow and reduced glutathione(GSH) affinity chromatography. The specific activity determined by 2, 4-Dinitrochlorobenzene(CDNB) and GSH as substrates is 1070 U/mg protein. The purification fold is 1138 and the total recovered activity is 22.9%. SDS-PAGE and PAGE showed one band and the molecular weight of the subunit is 25kD and is 50kD determined by gel exclusion, which indicates that the enzyme is composed of two identical subunits. The pI point of the enzyme is 8.2. The kinetics constants of the purified GST toward substrate were investigated. For CDNB, Km is 0.15mmol/L, kcat is 783s-1, and for GSH, Km is 0.35mmol/L, kcat is 783s-1. The reaction mechanism of GST for bisubstrate is sequential mechanism by kinetic research. The optimum temperature is 41oC and the optimum pH range is 6.3-6.9. The enzyme is inhibited by Cl- and 3, 5-oic Acid. The kind of inhibition by Cl- is competitive for CDNB and non-competitive for GSH, respectively. The kind of inhibition by 3, 5-Dinitrobenzoic Acid is competitive for CDNB and un-competitive for GSH, respectively.

Key words: glutathione S-transferase(GST), Asian corn borer, reduced glutathione(GSH), 4-dinitrochlorobenzene(CDNB), kinetics

摘要: 以亚洲玉米螟(Ostrinia furnacalis)为材料,经Sephadex-G75、DEAE-Sepharose Fast Flow、还原型谷胱甘肽(GSH)为配基的亲和层析,分离纯化得到玉米螟谷胱甘肽转硫酶(GST)。以2,4-二硝基氯苯(CDNB)为底物测得其比活为1070U/mg蛋白,收率为22.9%,提纯1138倍。经SDS-PAGE和PAGE鉴定,得到的GST为一条带,亚基的分子量为25kD,经凝胶过滤测得其全酶分子量为50kD,说明该酶是由两个相同亚基组成的。IEF测得主带等电点为8.2。该酶最适温度为41℃,最适pH范围为6.3~6.9。经动力学测定该酶双底物反应机制为序列机制,对CDNB的Km值为0.15mmol/L,对GSH的Km值为0.35mmol/L,两种底物的kcat均为783s-1。Cl-对底物CDNB有竞争性抑制作用,对GSH是非竞争性抑制作用;3,5-二硝基苯甲酸(DNB)对CDNB有竞争性抑制作用,对GSH是反竞争性抑制作用。

关键词: 谷胱甘肽转硫酶(GST), 玉米螟, 还原型谷胱甘肽(GSH), 4-二硝基氯苯(CDNB), 动力学

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