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Expression, characterization, and localization of acetylcholinesterase-1 from the African malaria mosquito, Anopheles gambiae

Liu, Siwei
Abstract
Findings and Conclusions:
Acetylcholinesterases (AChEs) play an essential role in neurotransmission at cholinergic synapses in vertebrates and invertebrates. Anopheles gambiae has two AChE genes, ace1 and ace2. The properties of AgAChEs remain unknown, and the complex resistance mechanisms can cause the problem to directly correlate a mutation with the resistance phenotype. The purposes of this study are to express and purify AChE1, characterize it in vitro, and localize its expression in vivo. In this study, a cDNA fragment of AChE1 from an A. gambiae EST was subcloned and expressed. The optimized three-step purification scheme took approximately eight hours and yielded 51% of the protein with a specific activity of 523U/mg. A pH of 7.0-8.0 is the best range for AgAChE1 reaction with acetylcholine. The enzyme size is 65 kDa and 130 kDa on SDS-polyacrylamide gels under reducing and nonreducing conditions, respectively. AgAChE1 hydrolyzes ATC 14-fold faster than BTC. The IC50, ki, and Kd demonstrated that AgAChE1 is highly sensitive to inhibition by BW284C51 instead of ethopropazine, and the affinity of BW284C51 is greater than that of ethopropazine. These findings indicate that Ag AChE1 is a true AChE, which exerts the physiological function of ACh hydrolysis at cholinergic synapses. In situ hybridization and immunohistochemistry showed that ace 1 is expressed mainly in the central nervous system. The procedures of Ag AChE1 purification and asymmetric PCR for making ISH probes could be used for similar studies in other insect species. The data are useful for understanding Ag AChE1 and for developing selective insecticides to control the African malaria mosquito.
Date
2008-12