Acetylcholinesterase of Haematobia irritans (Diptera: Muscidae): baculovirus expression, biochemical properties, and organophosphate insensitivity of the G262A mutant.
This study reports the baculovirus expression and biochemical characterization of recombinant acetylcholinesterase from Haematobia irritans (L.) (rHiAChE) and the effect of the previously described G262A mutation on enzyme activity and sensitivity to selected organophosphates. The rHiAChE was confirmed to be an insect AChE2-type enzyme with substrate preference for acetylthiocholine (Km 31.3 microM) over butyrylthiocholine (Km 63.4 microM) and inhibition at high substrate concentration. Enzyme activity was strongly inhibited by eserine (2.3 x 10(-10) M), BW284c51 (3.4 x 10(-8) M), malaoxon (3.6 x 10(-9) M), and paraoxon (1.8 x 10(-7) M), and was less sensitive to the butyrylcholinesterase inhibitors ethopropazine (1.1 x 10(-6) M) and iso-OMPA (4.1 x 10(-4) M). rHiAChE containing the G262A substitution exhibited decreased substrate affinity for both acetylthiocholine (Km 40.9 microM) and butyrylthiocholine (Km 96.3 microM), and exhibited eight-fold decreased sensitivity to paraoxon, and approximately 1.5- to 3-fold decreased sensitivity to other inhibitors. The biochemical kinetics are consistent with previously reported bioassay analysis, suggesting that the G262A mutation contributes to, but is not solely responsible for observed phenotypic resistance to diazinon or other organophosphates.
Knipling-Bushland U.S. Livestock Insects Research Laboratory, USDA-ARS, 2700 Fredericksburg Road, Kerrville, TX 78028, USA. kevin.temeyer@ars.usda.gov
Kevin B
KB
Temeyer
Danett K
DK
Brake
Kristie G
KG
Schlechte
D000110:Acetylcholinesterase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016367:Baculoviridae / Q000201:enzymology; D007305:Insecticide Resistance / Q000235:genetics*; D007306:Insecticides; D009117:Muscidae / Q000201:enzymology* / Q000235:genetics; D010755:Organophosphates; D017354:Point Mutation; D011994:Recombinant Proteins / Q000378:metabolism
D007306:Insecticides; D010755:Organophosphates; D011994:Recombinant Proteins; D000110:Acetylcholinesterase