An acetylcholinesterase purified from the greenbug (Schizaphis graminum) with some unique enzymological and pharmacological characteristics

An acetylcholinesterase (AChE, EC 3.1.1.7) was purified from the greenbug, Schizaphis graminum (Rondani). The maximum velocities (V-max) for hydrolyzi ng acetylthiocholine (ATC), acetyl-(beta -methyl) thiocholine (A beta MTC), propionylthiocholine, and S-butyrylthiocholine were 78.0, 67.0, 37.4, and 2.3 mu mol/min/mg, and the Michaelis constants (K-m) were 57.6, 60.6, 31.3, and 33.4 muM, respectively. More than 98% of AChE activity was inhibited b y 10 muM eserine or BW284C51, but only 7% of the activity was inhibited by ethopropazine at the same concentration. Based on the substrate and inhibit or specificities, the purified enzyme appeared to be a true AChE. Nondenatu ring polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing of the purified AChE revealed three molecular forms. The isoelectric points we re 7.3 for the major form and 6.3 and 7.1 for two minor forms. The major fo rm of purified AChE showed molecular masses of 129 kDa for its native prote in and 72 kDa for its subunits on SDS-PAGE. However, the purified AChE exhi bited some distinctive characteristics including: (1) lack of affinity to t he affinity ligand 3-(carboxyphenyl) ethyldimethyl ammonium, which has been used widely in purification of AChE from various insect species; and (2) 2 0-200-fold higher substrate-inhibition thresholds for ATC and A beta MTC th an AChE from other insect species. These biochemical properties may reflect structural differences of AChE purified from the greenbug compared with th at from other insect species. (C) 2001 Elsevier Science Ltd. All rights res erved.