罗非鱼肌肉乙酰胆碱酯酶的纯化、性质及农药敏感性研究

以罗非鱼脑组织为材料，研究了乙酰胆碱酯酶的分离纯化方法、性质及杀虫剂对酶的抑制作用。结果表明，当脑组织的TritonX-100抽提物采用(NH4)2SO4分级盐析、Sephadex G-100凝胶过滤和DEAE-纤维素DE23离子交换柱层析的分离纯化步骤后，可获得纯化的乙酰胆碱酯酶，酶的比活力达到20.628U.mg-1, 纯化倍数139，回收率22.1%。酶作用最适温度为35～40℃，最适pH为7.5～8.0，对碘化硫代乙酰胆碱的Km值为0.183mmol.L-1，有过量底物抑制现象，最佳底物浓度为6mmol.L-1。被测4种农药中，有机磷类杀虫剂对罗非鱼脑AChE的抑制能力高于氨基甲酸酯类，抑制能力分别为：敌敌畏 > 辛硫磷 > 三唑磷 > 灭多威， 4种杀虫剂对罗非鱼脑AChE的I50值均小于5μg.mL-1。

Purification and Characteristics of Muscle Acetylcholinesterase of Oreochromis aurea and Its Sensitivity to Insecticides

The decline of enzymatic activity of acetylcholinesterase(AChE) is a significant biochemical indicator to agricultural chemical pollution in aquatic environment.Muscular tissues of Oreochromis aurea were tested on to study purification methods,characteristics of acetylcholinesterase(AChE) and its sensitivity to insecticides.The result indicated AChE was purified from the Triton X-100 extract by(NH4)2SO4 fractional precipitation,Sephadex G-100 gel filtration and DEAE-cellulose through DE23 ion exchange chromatography,with the specific activity of this purified AChE was 21.34 U/mg,purification fold 118,and recovery rate 22.08%.The optimal condition of AChE activity determined by multi-factor orthogonal method:pH value of the reaction system was 7.5;temperature was 40℃;reaction time was 10min;concentration of substrate was 7 mmol/L.This purified acetylcholinesterase(AChE) hydrolyzed agent transport and communication interface(ATCI) at high rate with Michaelis constant(Km) 0.229 mmol/L,there being inhibition of excess substrate.Four insecticides(dichlorvos,phoxim,triazophos,and methomyl) exhibited strong inhibitions on this AChE with IC50 values less than 5×10-6 mol/L.Compared to carbamate insecticide,the inhibition potential of organophosphate was higher,and the inhibition potential was:dichlorvos> phoxim>triazophos>methomyl.The inhibition of four insecticides(dichlorvos,phoxim,triazophos,and methomyl) on muscle acetylcholinesterase(AChE)of O.aurea showed a good dose-effect relation,with their correlation coefficients all above 0.93.The above results suggest that it is possible to use muscle AChE in O.aurea as a biochemical marker to monitor the contamination of insecticides in aquatic environment.