Purification and kinetic analysis of acetylcholinesterase from western corn rootworm, Diabrotica virgifera virgifera (Coleoptera : Chrysomelidae)

Acetylcholinesterase (AChE, EC 3.1.1.7) was purified from western corn root worm (WCR, Diabrotica virgifera virgifera) beetles by affinity chromatograp hy. The purification factor reached over 20,000-fold with a specific activi ty of 169.5 mu mol/min/mg and a yield of 23%. The V-max values for hydrolyz ing acetylthiocholine (ATC), acetyl-(beta-methyl)thiocholine (A beta MTC), propionylthiocholine (PTC), and S-butyrylthiocholine (BTC) were 184.8, 140. 5, 150.2, and 18.8 mu mol/min/mg, respectively, and K-m values were 19.7, 1 8.5, 14.1, and 11.0 mu M, respectively. The first three substrates showed s ignificant inhibition to the AChE at higher concentrations, whereas ETC sho wed inhibition at the concentrations of 0.25-2 mM but activation at >4 mM. AChE activity was almost completely inhibited by 1 mu M eserine and BW284C1 5, respectively, but only 12% of AChE activity were inhibited by ethopropaz ine at the same concentration. These results suggested that the purified AC hE, from WCR was a typical insect AChE. Insecticides or their oxidative met abolites, chlorpyrifos-methyl oxon, carbofuran, carbaryl, malaoxon, and par aoxon, used in in vitro kinetic study exhibited high inhibition to AChE pur ified from WCR. However, chlorpyrifos-methyl oxon and carbofuran showed at least 36- and 4-fold, respectively, higher inhibitory potency than the rema ining insecticides examined. Results from our in vitro inhibition of AChE a greed quite well with the previously published in vivo bioassay data. (C) 1 998 Wiley-Liss, Inc.