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 phoximtriazophosmethomyl.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.