Purification and characterization of a safener-induced glutathione S-transferase from wheat (Triticum aestivum)
Purification and characterization of a safener-induced glutathione S-transferase from wheat (Triticum aestivum)
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DOI：
10.1034/j.1399-3054.1999.106103.x
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发表时间：
1999-05-01
影响因子：
6.4
通讯作者：
Scalla, R
中科院分区：
文献类型：
--
作者：
Pascal, S;Scalla, R
栽培小麦的基因组是六倍体，因此预期在该生物体中存在大量谷胱甘肽S-转移酶（GST，EC www.example.com）同工酶。首次采用反相高效液相色谱法（RP-HPLC）对小麦CST亚基进行了分析。在根和地上部组织中，亚基4、8和9组成型表达，而亚基2、3和5可被除草剂安全剂萘二甲酸酐（NA）诱导。然而，这六个主要亚基在根和地上部的分布之间存在显著差异。一个主要的GST同工酶纯化从植物的芽处理NA。硫酸铵沉淀，疏水相互作用色谱（HIC）和亲和色谱的组合导致纯化的表观产率为4.6%，对1-氯-2，4-二硝基苯（CDNB）的比活性增加了48倍。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析显示在24.5kDa处的单一条带。通过非变性PAGE估计分子量为49.5 kDa。这些结果表明该酶以二聚体形式存在。通过天然等电聚焦（IEF）测定pI为5.2。通过2-D电泳分析显示单一斑点，具有5.8 - 5.9的pi。然而，通过RP-HPLC的进一步分析表明，这两个亚基是不同的。它们通过电喷雾电离质谱（ESI-MS）表征和鉴定为亚基2和3，分子量分别为24924 +/-3和24958 +/-5 Da。因此，GST（2 - 3）显然是由亚基2和3组成的异二聚体。CDNB的表观K-M值为424 μ M，谷胱甘肽（GSH）的表观K-M值为228 μ M。GST（2 - 3）代谢除草剂氟草醚，确定除草剂的K-M为22 μ M。
The genome of cultivated wheat is hexaploid, and in consequence a large number of glutathione S-transferase (GSTs, EC 2.5.1.18) isozymes is expected in that organism. Wheat CST subunits were first analyzed by reverse-phase high performance liquid chromatography (RP-HPLC). In root and shoot tissues, subunits 4, 8, and 9 were constitutively expressed whereas subunits 2, 3, and 5 were inducible by the herbicide safener naphthalic anhydride (NA). Significant differences were observed, however, between the distributions of these six major subunits in roots and shoots. A major GST isozyme was purified from the shoots of plants treated by NA. A combination of ammonium sulphate precipitation, hydrophobic interaction chromatography (HIC) and affinity chromatography resulted in purification with an apparent yield of 4.6% and a 48-fold increase in specific activity toward 1-chloro-2,4-dinitrobenzene (CDNB). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a single band at 24.5 kDa. Molecular mass estimated by nondenaturing PAGE was 49.5 kDa. These results suggest that the enzyme exists as a dimer. A pI of 5.2 was determined by native isoelectric focusing (IEF). Analysis by 2-D electrophoresis showed a single spot, with a pi of 5.8-5.9. However, further analysis by RP-HPLC revealed that the two subunits were different. They were characterized and identified by electrospray ionization mass spectrometry (ESI-MS) as subunits 2 and 3, molecular masses 24924 +/- 3 and 24958 +/- 5 Da, respectively. Therefore, GST(2-3) is apparently a heterodimer consisting of subunits 2 and 3. Apparent K-M values were 424 mu M for CDNB and 228 mu M for glutathione (GSH). GST(2-3) metabolized the herbicide fluorodifen, and a K-M of 22 mu M was determined for the herbicide.