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 rootworm (WCR, Diabrotica virgifera virgifera) beetles by affinity chromatography. The purification factor reached over 20,000-fold with a specific activity of 169.5 mumol/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 mumol/min/mg, respectively, and K(m) values were 19.7, 18.5, 14.1, and 11.0 microM, 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 nM but activation at > 4 mM. AChE activity was almost completely inhibited by 1 microM eserine and BW284C15, respectively, but only 12% of AChE activity were inhibited by ethopropazine at the same concentration. These results suggested that the purified AChE from WCR was a typical insect 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.
Gao
J R
JR
Department of Entomology, Kansas State University, Manhattan 66506, USA.
Rao
J V
JV
Wilde
G E
GE
Zhu
K Y
KY
D000110:Acetylcholinesterase / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002800:Cholinesterase Inhibitors; D001517:Coleoptera / Q000201:enzymology*; D007306:Insecticides; D007700:Kinetics; D013379:Substrate Specificity
D002800:Cholinesterase Inhibitors; D007306:Insecticides; D000110:Acetylcholinesterase