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 Sf9 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 microM and 133.2 +/- 1.6 U/mg for acetylthiocholine iodide, 113.9 +/- 12.5 muM and 106.4 +/- 3.0 U/mg for acetyl(beta-methyl)thiocholine iodide, 68.9 +/- 7.8 microM and 76.7 +/- 1.0 U/mg for propionylthiocholine iodide, and 201.1 +/- 21.0 microM 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 microM, respectively. The bimolecular reaction constants (k(i)) were (6.50 +/- 0.40) x 10(4) for carbaryl, (1.00 +/- 0.16) x 10(5) for eserine, (4.70 +/- 0.13) x 10(5) for malaoxon, and (9.06 +/- 0.23) x 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.
Department of Entomology and Plant Pathology, Oklahoma State University, 127 Noble Research Center, Stillwater, OK 74078, USA.
Picheng
P
Zhao
Kun Yan
KY
Zhu
Haobo
H
Jiang
D000110:Acetylcholinesterase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000122:Acetylthiocholine / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001042:Aphids / Q000201:enzymology* / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D002794:Choline / Q000378:metabolism; D002800:Cholinesterase Inhibitors / Q000378:metabolism; D019281:Dimerization; D004795:Enzyme Stability; D017344:Genes, Insect; D006863:Hydrogen-Ion Concentration; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014162:Transfection
D002800:Cholinesterase Inhibitors; D019476:Insect Proteins; D011993:Recombinant Fusion Proteins; C543539:acetylthiocholine iodide; D000122:Acetylthiocholine; D000110:Acetylcholinesterase; D002794:Choline