Purification and characterization of acetylcholinesterase from oriental fruit fly [Bactrocera dorsalis (Hendel)] (Diptera: Tephritidae).
An acetylcholinesterase (AChE, EC 3.1.1.7) was purified from the head of the insecticide susceptible oriental fruit fly, Bactrocera dorsalis (Hendel), by affinity chromatography of Triton X-100 extract. The degree of purification was about 8183-fold with recoveries of 52%. The molecular mass of purified AChE was 116 kDa for its native protein (nonreduced form) and 61 kDa for its subunits (reduced form) as revealed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), suggesting that the homodimer of AChE linked with disulfide bonds. Nondenaturing PAGE of the purified AChE revealed only one molecular form. The maximum velocities (V(max)) for hydrolyzing acetylthiocholine (ATC), propionylthiocholine, and S-butyrylthiocholine were 833.3, 222.2, and 57.5 micromol/min/mg, and the Michaelis constants (K(m)) were 87.9, 26.9, and 195.3 microM, respectively. More than 97% of AChE activity was inhibited by 10 microM eserine or BW284C51, but only 53% of the activity was inhibited by ethopropazine at the same concentration. On the basis of the substrate and inhibitor specificities, the purified enzyme appeared to be a true AChE. Nevertheless, the purified AChE exhibited some distinctive characteristics including (i) a lack of the substrate inhibition phenomenon when using ATC as the hydrolyzing substrate and (ii) a higher V(m) value for ATC than AChE from other insect species. These biochemical properties may show that AChE purified from the oriental fruit fly may have structural differences from those of other insect species.
Hsiao
Yi-Min
YM
Center for Research and Development, Chungtai Institute of Health Sciences and Technology, 11 Pu-tzu Lane, Pei-tun District 40605, Takun, Taichung, Taiwan, Republic of China. ymhsiao@chtai.ctc.edu.tw
Lai
Jing-Ying
JY
Liao
Hsiu-Ying
HY
Feng
Hai-Tung
HT
D000110:Acetylcholinesterase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D002846:Chromatography, Affinity; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008970:Molecular Weight; D013379:Substrate Specificity; D033621:Tephritidae / Q000201:enzymology*
D004220:Disulfides; D000110:Acetylcholinesterase