Heterologous expression, purification, and biochemical characterization of a greenbug ( Schizaphis graminum ) acetylcholinesterase encoded by a paralogous gene ( ace‐1 )

Acetylcholinesterase is a critical enzyme in the regulation of cholinergic neurotransmission in insects. To produce Schizaphis graminum acetylcholinesterase‐1 for structure–function analysis, we constructed a recombinant baculovirus to infect Sf 9 cells, which secreted the soluble protein at a final concentration of 4.0 mg/L. The purified enzyme had an apparent M r of 70 and 130 kDa in the reducing and nonreducing SDS‐polyacrylamide gels, respectively, indicating that it formed a dimer via an intermolecular disulfide bond. The fresh enzyme had a specific activity of 245 U/mg, which stabilized at a lower level (115 U/mg) in storage. The Michaelis constant and maximum velocity were 88.3 ± 9.6 μM and 133.2 ± 1.6 U/mg for acetylthiocholine iodide, 113.9 ± 12.5 μM and 106.4 ± 3.0 U/mg for acetyl(β‐methyl)thiocholine iodide, 68.9 ± 7.8 μM and 76.7 ± 1.0 U/mg for propionylthiocholine iodide, and 201.1 ± 21.0 μM and 4.4 ± 0.1 U/mg for S ‐butyrylthiocholine iodide, respectively. The IC 50 values (5 min, room temperature) of ethopropazine, BW284C51, carbaryl, eserine, malaoxon, and paraoxon were 102, 1.66, 0.94, 0.20, 0.061, 0.016 μM, respectively. The bimolecular reaction constants ( k i ) were (6.50 ± 0.40) × 10 4 for carbaryl, (1.00 ± 0.16) × 10 5 for eserine, (4.70 ± 0.13) × 10 5 for malaoxon, and (9.06 ± 0.23) × 10 5 M −1 min −1 for paraoxon. The enzyme was also inhibited by one of its products, choline, at concentrations higher than 20 mM, suggesting that choline bound to an anionic site and regulated the enzymatic activity. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:51–59, 2010; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20311