PURIFICATION AND BIOCHEMICAL CHARACTERIZATION OF GLUTATHIONE S-TRANSFERASES IN BACTROCERA MINAX (DIPTERA: TEPHRITIDAE)
PURIFICATION AND BIOCHEMICAL CHARACTERIZATION OF GLUTATHIONE S-TRANSFERASES IN BACTROCERA MINAX (DIPTERA: TEPHRITIDAE)
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DOI：
10.1653/024.095.0309
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发表时间：
2012-09-01
影响因子：
1.4
通讯作者：
Wang, Jin-Jun
中科院分区：
文献类型：
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作者：
Chen, Er-Hu;Dou, Wei;Wang, Jin-Jun
采用谷胱甘肽-琼脂糖亲和层析法从柑橘实蝇（Bactrocera minax）的3个发育阶段分离纯化了谷胱甘肽S-转移酶（GST），并分别以1-氯-2，4-二硝基苯（CDNB）和还原型谷胱甘肽（GSH）为底物，采用米氏动力学方法对其进行了表征。与三龄幼虫和成虫相比，蛹中GST对CDNB的比活性最高。不同发育阶段酶的比活力不同，但纯化得率相近。SDS-PAGE显示所有3个阶段在23 kDa处只有一条带。成虫GST对CDNB的Km值最高，蛹GSH对CDNB的Km值最高。3个阶段的CDNB结合的最适温度和pH分别为37 ℃和7.5。抑制动力学研究表明，依他尼酸、溴磺灵、马来酸二乙酯、二硫化四乙基秋兰姆和姜黄素对B中纯化的GST具有较好的抑制作用。minax。此外，蛹表现出最高的催化能力的基础上的V-max。CDNB和GSH的值，这可能表明在蛹阶段比其他2个发育阶段具有更高的GST解毒能力。
Glutathione S-transferases (GSTs) were purified from 3 developmental stages of Bactrocera minax through glutathione-agarose affinity chromatography, and characterized subsequently using the Michaelis-Menten kinetics toward the artificial substrates 1-chloro-2, 4-dinitrobenzene (CDNB) and reduced glutathione (GSH), respectively. Compared to the counterparts of third instar larva and adult, the highest specific activity of the purified GSTs towards CDNB was observed in the pupae. Although the specific activities of purified enzymes varied among 3 developmental stages, the purification yields were similar. SDS-PAGE revealed only one band at 23 kDa for all 3 stages. GSTs of the adults exhibited the highest K-m value towards CDNB, while for GSH the pupae possessed the highest K-m. The optimum temperature and pH for CDNB conjugation of the 3 stages were 37 degrees C and 7.5, respectively. Inhibition kinetics showed that ethacrynic acid, bromosulfalein, diethyl maleate, tetraethylthiuram disulfide and curcumin possessed excellent inhibitory effects on purified GSTs in B. minax. Moreover, the pupa showed the highest catalytic capability based on V-max. values for both the CDNB and GSH, which may suggest a potentially higher GSTs detoxification ability in the pupal stage than the other 2 developmental stages.