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

• Published in: Archives of insect biochemistry and physiology 1998, Vol.39 (3), p.118-125
• Main Authors: Gao, J.R. (Kansas State University, Manhattan.), Rao, J.V, Wilde, G.E, Zhu, K.Y
• Format: Article
• Language: English
• Subjects: ACETILCOLINESTERASA; ACETYLCHOLINESTERASE; Acetylcholinesterase - isolation & purification; Acetylcholinesterase - metabolism; ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Animals; CARBARIL; CARBARYL; CARBOFURAN; CARBOFURANO; CHLORPYRIFOS-METHYL; CHLORPYRIFOS-METHYL OXON; Cholinesterase Inhibitors; Chrysomelidae; CLORPIRIFOS-METIL; Coleoptera - enzymology; CONTROL DE INSECTOS; DIABROTICA VIRGIFERA; enzyme inhibition; ENZYME INHIBITORS; ENZYME KINETICS; ENZYMIC ACTIVITY; INHIBICION; INHIBIDORES DE ENZIMAS; INHIBITEUR D'ENZYME; INHIBITION; INSECT CONTROL; INSECTICIDAS; INSECTICIDE; INSECTICIDES; Kinetics; LUTTE ANTIINSECTE; MALAOXON; PARAOXON; PURIFICACION; PURIFICATION; Substrate Specificity; SUBSTRATES; western corn rootworm
• ISSN: 0739-4462; 1520-6327
• DOI: 10.1002/(SICI)1520-6327(1998)39:3<118::AID-ARCH4>3.0.CO;2-6

Acetylcholinesterase (AChE, EC 3.1.1.7) was purified from western corn rootworm (WCR, Diabrotica virgifera) beetles by affinity chromatography. The purification factor reached over 20,000-fold with a specific activity of 169.5 micromole/min/mg and a yield of 23%. The Vmax values for hydrolyzing 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 micromole/min/mg, respectively, and Km values were 19.7, 18.5, 14.1, and 11.0 micromolar, respectively. The first three substrates showed significant inhibition to the AChE at higher concentrations, whereas BTC showed inhibition at the concentrations of 0.25-2 mM but activation at 4 mM. AChE activity was almost completely inhibited by 1 micromolar eserine and BW284C15, respectively, but only 12% of AChE activity were inhibited by ethopropazine at the same concentration. Then results suggested that the purified AChE from WCR was a typical inset AChE. Insecticides or their oxidative metabolites, chlorpyrifos-methyl oxon, carbofuran, carbaryl, malaoxon, and paraoxon, used in in vitro kinetic study exhibited high inhibition to AChE purified from WCR. However, chlorpyrifos-methyl oxon and carbofuran showed at least 36- and 4-fold, respectively, higher inhibitory potency than the remaining insecticides examined. Results from our in vitro inhibition of AChE agreed quite well with the previously published in vivo bioassay data