Purification and characterization of acetylcholinesterase from cotton aphid (Aphis gossypii Glover).
A simple and effective method was set up to purify acetylcholinesterase (AChE, EC3.1.1.7) from the cotton aphid, Aphis gossypii Glover. The procedure involved filtration on a sephadex G-25 column, separation with sephadex G-200 and procainamide affinity column. AChE from both susceptible and resistant strains were purified to a single band as resolved on denaturing polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity increased by 35,100- and 33,680-fold with a yield of 30.3 and 29.8%, respectively. The molecular mass of the purified AChE was about 63,500 Dalton as determined by SDS-PAGE. However, three bands resolved on PAGE gel electrophoresis, leading to the inference that native AChE exists in three forms. The optimum conditions for measuring the activity of purified AChE with kinetic method were 0.02M phosphate buffer, pH7.2, 0.02 mM 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and 25 degrees C. Investigation also revealed that crude extract and purified AChE had different kinetic characteristics and inhibitory properties. They responded differently to varied DTNB, ATChI, and phosphate buffer ion concentrations, as well as pH, temperature, and inhibitors. The purified AChE was more sensitive to eserine, methamidophos, and pirimicarb. Especially for resistant aphids, the sensitivity of purified AChE to methamidophos and pirimicarb was enhanced 6.43 and 11.73 times, respectively. We infer that one or more factors in the crude extract from the resistance strain have more influence on AChE sensitivity. Further study is needed to investigate the basis of these observations.
Li
Fei
F
Key Laboratory of Monitoring and Management of Plant Diseases and Pests, Ministry of Agriculture, Nanjing Agricultural University, Nanjing, China.
Han
Zhaojun
Z
D000110:Acetylcholinesterase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001042:Aphids / Q000201:enzymology* / Q000378:metabolism; D002800:Cholinesterase Inhibitors / Q000494:pharmacology; D002846:Chromatography, Affinity; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D011342:Procainamide / Q000737:chemistry; D013696:Temperature
D002800:Cholinesterase Inhibitors; D004228:Dithionitrobenzoic Acid; D000110:Acetylcholinesterase; D011342:Procainamide