Purification, Biochemical And Toxicological Characterization Of Acetylcholinesterase In Delia Antiqua

Onion maggot (Delia antiqua) is an insect pest of the Anthomyiidae, Diptera, widely distributed in Asia, Europe and North America. It is a worldwide underground pests, and have a great impact on the yield and quality to Liliaceae vegetables. Acetylcholinesterase (AChE, EC3.1.1.7) maintains normal nerve impulse transmission by catalytic hydrolysis of the neurotransmitter acetylcholine (ACh) in the synaptic gap. And is the target of organic phosphorus and carbamate insecticides. Therefore, the research has important scientific significance on biochemical properties of AChE onion maggot and its resistance, rational design, rational use of pesticides and so on. However, there were no information about purification, the resistance and the nature of onion maggot AChE at present. In this paper, Acetylcholinesterase(AChE) of the onion maggot, separated from the crude extract of its head, was purified to electrophoretic homogeneity by procainamide-ECH-Sepharose 4B affinity chromatography. Then the properties of crude extract and purified enzyme were studied, and the various factors that affect AChE activity were discussed to provide the scientific basis for further study. The results showed that the optimal extraction buffer for acetylcholinesterase from onion maggot head was, pH7.5, 0.1 mol/L PBS, containing 1% TritonX-100, with 30% of the 1M NaCl. The AChE was purified 81.8-fold with a yield of 15.1%. The molecular weight of the purified enzyme was 58.4Kda, measured by SDS-PAGE. The optimum temperature of the purified AChE was 35℃. It exhibited optimum activity at pH7.5. The Michaelis constant for acetylthiocholine iodide was 0.175mmol/L. The optimum concentration of substrate was 1.5mM, and the enzyme could be inhibited by high level of acetylthiocholine iodide. The median inhibiting concentration (IC₅₀) of physostigmine to crude extract was IC₅₀=7.58×10^-8, and that of physostigmine to purified enzyme was IC₅₀=1.26×10^-8. Eserine concentration of the purified enzyme inhibition was significantly lower than that of crude enzyme solution, indicating that a number of other elements play a protective role. Finally, the properties and kinetic parameters of AChEs from the dichlorvos resistant groups and susceptible groups were different by in vitro enzyme kinetic experiments. The kinetic parameters for AChEs from the dichlorvos resistant groups and susceptible groups showed that the Michaelis constant (Km) and maximum reaction velocity (Vmax) ratios of the dichlorvos resistant groups to susceptible groups were 2.20 and 2.06, respecttively. The bimolecular rate constant (ki) of AChE from the dichlorvos resistant groups was much smaller than that of AChE from susceptible groups. The differences in ki may be mainly attributed to the decreased affinity of these compounds for the dichlorvos resistant groups AChE, which indicated that the dichlorvos resistant groups AChE might be qualitatively altered. The changes of the properties and kinetic parameters of AchEs may be due to changes in its gene mutations, caused by changes in the structure of the active site, which is not sensitive to these insecticides.