淡色库蚊抗吡丙醚种群与敏感种群谷胱甘肽硫转移酶的生物化学性质

Biochemical characteristics of glutathione s-transferases from the pyriproxyfen-resistant and susceptible populations of Culex pipiens pallens

目的比较淡色库蚊抗吡丙醚种群和敏感种群谷胱甘肽硫转移酶(GSTs)的生物化学性质,初步探讨其生化抗性机制。方法以室内连续筛选的淡色库蚊抗吡丙醚种群及淡色库蚊敏感种群为研究对象,参照Habig等(1976)的方法测定GSTs活性,采用t检验进行样本均数差别的统计学分析。结果淡色库蚊GSTs的最适底物为1-氯-2,4-二硝基苯(CDNB)。CDNB为底物时,抗吡丙醚种群和敏感种群GSTs的酶活性分别为3.626×10-4和2.737×10-4nmol/mg protein·min。淡色库蚊GSTs水解活性随底物浓度〔CDNB和DCNB(1,2-二氯-4-硝基苯)〕的增加而升高;在一定浓度范围内,抗性种群GSTs对这2种底物的水解活性略高于敏感种群。CDNB为底物时,抗性种群GSTs的米氏常数(Km)与最大反应速度(Vmax)分别为8.01 mmol/L和4.87×102μmol/min·mg,敏感种群的Km与Vmax分别为1.11 mmol/L和3.87×102μmol/min·mg,抗性种群的Km、Vmax与敏感种群差异有统计学意义(ta=11.415,tb=6.411,均P<0.05)。DCNB为底物时,抗性种群的Km与Vmax与敏感品系差异无统计学意义(tc=0.134,td=1.280,均P>0.05)。结论 GSTs在淡色库蚊对吡丙醚抗性形成过程中可能起一定解毒代谢作用。

Objective To preliminarily explore the resistance mechanism of pyriproxyfen-resistant Culex pipiens pallens by comparing the biochemical characteristics of glutathione s-transferases(GSTs)between resistant and susceptible populations of Cx.pipiens pallens.Methods The pyriproxyfen-resistant and susceptible populations of Cx.pipiens pallens were chosen through indoor successive selection as the research objects.And the activities of GSTs were determined according to the method reported by Habig et al.(1976).Statistical analysis was thus performed using the t-test for sample mean comparison.Results The optimal substrate of GSTs from Cx.pipiens pallens was 1-chloro-2,4-dinitrobenzene(CDNB).When CDNB was used as the substrate,the activities of GSTs in the resistant and susceptible populations were 3.626×10-4 and 2.737×10-4 nmol/mg protein·min,respectively.The hydrolytic activity of Cx.pipiens pallens GSTs increased with the elevation of substrate concentrations〔i.e.,CDNB and DCNB(1,2-dichloro-4-nitrobenzene)〕.In a certain concentration range,the hydrolytic activity of GSTs in the resistant population was slightly higher than that in the susceptible population towards both substrates.When CDNB was used as the substrate,the Michaelis constant(Km)and maximum reaction rate(Vmax)of GSTs in the resistant population were 8.01 mmol/L and 4.87×102μmol/min·mg,respectively,while those in the susceptible population were 1.11 mmol/L and 3.87×102μmol/min·mg,respectively.There were significant differences between these two populations regarding Km and Vmax(ta=11.415,tb=6.411,all P<0.05).When DCNB was used as the substrate,there was no significant difference between the two populations regarding Km and Vmax of GSTs(tc=0.134,td=1.280,all P>0.05).Conclusion GSTs may play an antidotal and metabolic role in the formation of pyriproxyfen resistance in Cx.pipiens pallens.