Purification And Characterization Of Acetylcholinesterase, A Kind Of Pesticide Target

Acetylcholinesterase in the nervous system of insects is one of the most important enzymes, in the central and peripheral nervous system and the acetylcholine (ACh) receptor involved nerve-nerve and nerve-muscle synaptic transmission between the action potential, and its main function is catalyzed hydrolysis cationic neurotransmitter acetylcholine. At present, in the natural products active material, the AChE ester moves the spot inhibitor physostigmine was studied clearly。Used as templates for the synthesis of carbamate pesticide, has become a major pesticide production of pesticides, organophosphates, carbamate pesticides on acetylcholinesterase (ACHE), a strong inhibition They mainly to the rapid and irreversible part of the activity of AChE, and the enzyme deactivation, which led to poisoning. Therefore, extraction and purification of natural products for pest and acetylcholinesterase in vitro selection and the pesticide is to develop new high-throughput screening generation of pesticides is very effective. This paper mainly for the extraction of the crude enzyme solution, Using a Sephadex G25 chromatography. DEAE-Sepharose Fast Flow ion-exchange chromatography and Sephacryl S 200 gel filtration chromatography. Acetylcholinesterase(AChE) of Bornbyx mori Linaeus, separated from the crude extract, was purified to electrophoretic homogeneity by Sephadex G-25 chromatography,DEAE-Sepharose Fast Flow ion-exchange chromatography and Sephacryl S-200 gel filtration, respectively. Silkworm is a model organism lepidopteran. Then studied the properties of crude extract and purified enzyme. The results show that the enzyme acetylcholinesterase from Silkworm head optimum conditions for the extraction buffer is , 1% Trition X-100, pH 8.0. It is found that substrate (ATChI) hydrolysis of the phenomenon at the Alkaline pH conditions, This occurs in response to the pH of greater than 8.5 .The Molecular weight of the purified enzyme was 77.8 KDa, measured by SDS-PAGE.The optimum temperature of the AChE was 37℃,above which the enzyme would be unstable. It exhibited optimum activity at pH 7.5.The Michaelis constant for acetylthiocholine iodide was 0.392 mmol/L. The optimum concentration of substrate was 1.6 mmol/L,and the enzyme could be inhibitied by high level of acetylthiocholine iodide. Eserine concentration of the purified enzyme inhibition was significantly lower than that of crude enzyme solution, it is shows a number of other elements plays on a protective role. The half inhibitory concentration of Stellera chamaejasme is 0.002 mg/m...