Purification and Characterization of Glutathione S-transferase from Diamondback Moth, Plutella xylostella L.

Abstract

The glutathione-S-transferase (GSTase) had been suggested as a possible metabolic enzyme related to insecticide resistance in the diamondback moth (DBM ), Plutella xylostella L.and other insects. Although the GSTase was studied in many insects, it had not been thoroughly investigated in the DBM. In this study, GSTase was purified either by the traditional protein purification method or the affinity chromatographic method. The affinity chromatographic method was proven to offer a much simpler purification procedure and resulted in higher yield and purer grade of GSTase. The purification factors of GSTase by the affinity chromatography are 32.1 and 24.9 for resistant and susceptible DBM strains. The total activity of GSTase is higher in the resistant DBM, which was agreed with the previous observation that the organophosphate-resistant DBM possessed 2-4 fold higher GSTase activity than the susceptible DBM. After affinity chromatogram-phic purification, only a single protein band was obtained in the polyacrylamide gel electrophoresis study, and the GSTase activity staining indicated the active zone is close but not totally matched the location of protein band. The explanation is unknown and needs further investigation. The molecular weight of DBM larval GSTase was measured at 45,000-46,500 daltons compared to other standard calibration proteins in the gel filtration columns, and is similar to the molecular weight of GSTases in housefly. Glutathione-S-轉移酶在小菜蛾以及其他昆蟲體內，被認為可能與抗藥性有關，尤其是有機磷殺蟲劑中之甲基或乙基的解裂，而使殺蟲劑在到達毒理作用點以前因分解而失效。小菜蛾之抗藥性極困擾蔬菜之蟲害防治，而有關有機磷抗性之機制，迄今仍不完全瞭解，故就此蟲之Glutathione-S-轉移酶進行純化與定性。本研究以兩種方式進行該酶之純化，一為傳統之「鹽析」及「分子篩分離」以及「離子交換」方法，一為利用新發展之「親和性分析柱」方式，結果發現後者之純化步驟簡單，回收率及酶之純度均較傳統之純化方法為佳，純化倍數為25 －32倍之間，同時測得小菜蛾之Glutathione-S-轉移酶分子量介於45,000～46,500 daltons之間，而且，此酶之活性在抗性品系小菜蛾高過感性品系2倍以上。