PURIFICATION AND PARTIAL CHARACTERIZATION OF GLUTATHIONE S-TRANSFERASES FROM INSECTICIDE-RESISTANT AND LINDANE-INDUCED SUSCEPTIBLE SPODOPTERA-LITTORALIS (BOISD) LARVAE
PURIFICATION AND PARTIAL CHARACTERIZATION OF GLUTATHIONE S-TRANSFERASES FROM INSECTICIDE-RESISTANT AND LINDANE-INDUCED SUSCEPTIBLE SPODOPTERA-LITTORALIS (BOISD) LARVAE
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DOI：
10.1016/0965-1748(93)90054-v
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发表时间：
1993-06-01
影响因子：
3.8
通讯作者：
ECHAUBARD, M
中科院分区：
文献类型：
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作者：
LAGADIC, L;CUANY, A;ECHAUBARD, M
对敏感品系斜纹夜蛾的四龄幼虫单次局部应用非致死剂量的林丹后，细胞内GST活性被跟踪。1-氯-2，4-二硝基苯(CDNB)偶联GSTs处理8h达到高峰，为1.5倍，1，2-二氯-4-硝基苯(DCNB)偶联酶处理12h达到高峰(1.3倍)。一株抗性品系的CDNB结合GST活性是未诱导敏感幼虫的两倍。用谷胱甘肽-琼脂糖柱亲和层析法对两种幼虫的GST进行了纯化，纯化倍数为30倍，产率为50-70%。以CDNB为底物，对纯化的GSTs进行了表征。无论林丹诱导与否，抗性品系和敏感品系CDNB和还原型谷胱甘肽(GSH)的表观K(M)和V(Max)值均有显著差异。林丹处理只改变了敏感幼虫的CDNB和GSH的K(M)值。两株GSTs的一维SDS-PAGE均显示一条表观分子量为27 kDa的条带。
Cytosolic GST activity was followed after a single topical application of a non-lethal dose of lindane to fourth instar larvae of a susceptible strain of Spodoptera littoralis (Boisd.). The induction reached its peak 8 h after treatment for 1-chloro-2,4-dinitrobenzene (CDNB) conjugating GSTs (1.5-fold), and 12 h after the treatment for 1,2-dichloro-4-nitrobenzene (DCNB) conjugating enzymes (1.3-fold). CDNB conjugating GST activity in an insecticide-resistant strain of S. littoralis was twice that measured in the non-induced susceptible larvae. No significant difference was observed with DCNB.GSTs of both larvae were purified by affinity chromatography using a glutathione-agarose column, which resulted in a 30-fold purification and a yield of 50-70%. Purified GSTs were characterized using CDNB as the substrate. Apparent K(m) and V(max) values calculated for CDNB and reduced glutathione (GSH) were significantly different between the resistant strain and the susceptible one, whether induced by lindane or not. The lindane treatment modified only K(m) values for CDNB and GSH in susceptible larvae. Monodimensional SDS-PAGE of GSTs purified from both strains revealed a large band of 27 kDa apparent molecular weight.