Irzyk G P, Fuerst E P
Department of Crop and Soil Sciences, Washington State University, Pullman 99164-6420.
Plant Physiol. 1993 Jul;102(3):803-10. doi: 10.1104/pp.102.3.803.
A glutathione S-transferase (GST) isozyme from maize (Zea mays Pioneer hybrid 3906) treated with the dichloroacetamide herbicide safener benoxacor (CGA-154281) was purified to homogeneity and partially characterized. The enzyme, assayed with metolachlor as a substrate, was purified approximately 200-fold by ammonium sulfate precipitation, anion-exchange chromatography on Mono Q resins, and affinity chromatography on S-hexylglutathione agarose from total GST activity present in etiolated shoots. The purified protein migrated during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) as a single band with a molecular mass of 27 kD. Using nondenaturing PAGE, we determined that the native protein has a molecular mass of about 57 kD and that the protein exists as a dimer. Two-dimensional electrophoresis revealed only a single protein with an isoelectric point of 5.75 and molecular mass of 27 kD. These results further suggest that the protein exists as a homodimer of two identical 27-kD subunits. The enzyme was most active with substrates possessing a chloroacetamide structure. trans-Cinnamic acid and 1-chloro-2,4-dinitrobenzene were not effective substrates. Apparent Km values for the enzyme were 10.8 microM for the chloroacetamide metolachlor and 292 microM for glutathione. The enzyme was active from pH 6 to 9, with a pH optimum between 7.5 and 8. An apparently blocked amino terminus of the intact protein prevented direct amino acid sequencing. The enzyme was digested with trypsin, and the amino acid sequences of several peptide fragments were obtained. The sequence information for the isolated GST we have designated "GST IV" indicates that the enzyme is a unique maize GST but shares some homology with maize GSTs I and III.
用二氯乙酰胺类除草剂安全剂苯氧喹啉（CGA - 154281）处理玉米（先锋杂交种3906）后，分离纯化得到一种谷胱甘肽S - 转移酶（GST）同工酶，并对其进行了部分特性分析。以异丙甲草胺为底物测定该酶活性，通过硫酸铵沉淀、Mono Q树脂阴离子交换色谱和S - 己基谷胱甘肽琼脂糖亲和色谱，从黄化苗中存在的总GST活性中纯化该酶约200倍。纯化后的蛋白质在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（PAGE）中迁移为一条分子量为27 kD 的单带。采用非变性PAGE，我们确定该天然蛋白质的分子量约为57 kD，且该蛋白质以二聚体形式存在。二维电泳仅显示一种等电点为5.75、分子量为27 kD的单一蛋白质。这些结果进一步表明该蛋白质以两个相同的27 - kD亚基的同型二聚体形式存在。该酶对具有氯乙酰胺结构的底物活性最高。反式肉桂酸和1 - 氯 - 2,4 - 二硝基苯不是有效的底物。该酶对氯乙酰胺类异丙甲草胺的表观Km值为10.8 μM，对谷胱甘肽的表观Km值为292 μM。该酶在pH 6至9具有活性，最适pH在7.5至8之间。完整蛋白质的氨基末端明显被封闭，无法直接进行氨基酸测序。用胰蛋白酶消化该酶，获得了几个肽段的氨基酸序列。我们将分离得到的GST命名为“GST IV”，序列信息表明该酶是一种独特的玉米GST，但与玉米GST I和III有一些同源性。
