Purification and characterization of acetyl-cholinesterase from the Colorado potato beetle, Leptinotarsa decemlineata (say)

Abstract

Acetylcholinesterase (AChE) was purified from an insecticide-susceptible strain of Colorado potato beetle by affinity chromatography following extraction with Triton X-100. The purification factor and yield were 39,000-fold and 51%, respectively. AChE activity was significantly inhibited by higher concentrations of acetylthiocholine (ATC) and acetyl-β-methylthiocholine (AβMTC), and moderately inhibited by propionylthiocholine (PTC). AChE activity was not inhibited by S-butyrylthiocholine (BTC). Substrate specificity constants ( κ cat K m ) of AChE for ATC, AβMTC and PTC were 21-, 25- and 13-fold, respectively, higher than that for BTC. Turnover numbers ( κ cat) of the native AChE were 23,000 min −1 for ATC, 14,000 for AβMTC, 8000 for PTC and 700 for BTC. AChE activity was effectively inhibited by 0.1 μM concentrations of eserine and BW284C51, but not by ethopropazine. The purified AChE consisted of two different molecular forms. The major form (92%) was a hydrophilic dimer, whereas the minor form (8%) was an amphiphilic dimer. Both molecular forms had virtually identical molecular weights (130,000 for native AChE and 65,000 for subunits) and isoelectric points (pI 7.3). The amphiphilic form of the AChE was insensitive to the action of phospholipase C, suggesting that this form has a hydrophobic domain that has been structurally modified. Amino acid analysis indicated that the mole percentages of amino acids of the enzyme were highly comparable to those of the previously reported AChE from Drosophila.