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Recombinant expression and biochemical characterization of the catalytic domain of acetylcholinesterase-1 from the African malaria mosquito, Anopheles gambiae
- Jiang, Haobo, Liu, Siwei, Zhao, Picheng, Pope, Carey
- Insect biochemistry and molecular biology 2009 v.39 no.9 pp. 646-653
- Anopheles gambiae, insect pests, hematophagous insects, gene expression, protein synthesis, recombinant proteins, acetylcholinesterase, enzyme activity, protein subunits, insecticide resistance, mechanism of action, defense mechanisms, phenotype, Baculoviridae, inhibitory concentration 50, dosage, reaction kinetics, enzyme inhibition, enzyme inhibitors, protein binding
- Acetylcholinesterases (AChEs) and their genes from susceptible and resistant insects have been extensively studied to understand the molecular basis of target site insensitivity. Due to the existence of other resistance mechanisms, however, it can be problematic to correlate directly a mutation with the resistant phenotype. An alternative approach involves recombinant expression and characterization of highly purified wild-type and mutant AChEs, which serves as a reliable platform for studying structure-function relationships. We expressed the catalytic domain of Anopheles gambiae AChE1 (r-AgAChE1) using the baculovirus system and purified it 2,500-fold from the conditioned medium to near homogeneity. While K(M)'s of r-AgAChE1 were comparable for ATC, AβMTC, PTC, and BTC, V(max)'s were substantially different. The IC₅₀'s for eserine, carbaryl, paraoxon, BW284C51, malaoxon, and ethopropazine were 8.3, 72.5, 83.6, 199, 328, and 6.59 x 10⁴ nM, respectively. We determined kinetic constants for inhibition of r-AgAChE1 by four of these compounds. The enzyme bound eserine or paraoxon stronger than carbaryl or malaoxon. Because the covalent modification of r-AgAChE1 by eserine occurred faster than that by the other compounds, eserine is more potent than paraoxon, carbaryl, and malaoxon. Furthermore, we found that choline inhibited r-AgAChE1, a phenomenon related to the enzyme activity decrease at high concentrations of acetylcholine.