Purification, Biochemical And Toxicological Characterization Of Acetylcholinesterase From Liposcelis Entomophila And L. Bostrychophila (Psocoptera: Liposcelididae)

This paper was supported by the National Natural Science Foundation (30871631) and the Specialized Research Fund for the Doctoral Program of Higher Education (200806350009). On the basis of AChE purification of different field populations of Liposcelis entomophila Enderlein and L. bostrychophila Badonnel through the affinity chromatography method, the comparative biochemical and toxicological characterizations were systematically analyzed, respectively. The main results are as follows:1. Purification of AChE from different field populations of L. entomophila and L. bostrychophilaAcetylcholinesterase (AChE) from the different field populations of L entomophila and L. bostrychophila were purified by affinity chromatography (procainamide affinity column). The overall purification factors and yields were 587-fold and 61.4% for Beibei (BB) population, respectively. The AChE of Guanghan (GH) population was purified 786-fold with a yield of 41.3%, and Wuhan (WH) population was purified 700-fold with a yield of 29.8%. For L. bostrychophila, the BB population was purified 497-fold with a yield of 61.2%, and Mianyang (MY) population was purified 616-fold with a yield of 48%.The purity of elution product from different field populations was confirmed by SDS-PAGE via the silver staining method, and the affinity purified enzyme of L. entomophila and L. bostrychophila appeared as one AChE band. The molecular mass of the purified AChE of L. entomophila is estimated at about 55 kDa, while the purified AChE of L. bostrychophila is estimated at about 56 kDa.2. Biochemical and toxicological characterization of AChE from different field populations of L. entomophila and L. bostrychophila2.1 Biochemical and toxicological characterization of AChE from three field populations of L. entomophilaAChE from three populations of L. entomophila reached its activity peak at pH 7.0. Based on parameter estimations, the optimal pH for the purified AChE activity is 7.36,7.21, and 7.22 for BB, GH and WH populations, respectively. AChE from three populations of L. entomophila reached its activity peak at 37.0℃. Based on parameter estimations, the optimal temperature for the purified AChE activity is 37.3℃,38.0℃, and 39.2℃for BB, GH and WH populations, respectively.The susceptibility of inhibitions against three field populations of L. entomophila was assayed by using the pesticide-membrane method. The result showed that the potential resistance threat of L. entomophila in Sichuan Province is greater than in Hubei Province and Chongqing Municipality. Compared to the other two populations, WH population possessed the highest specific activity of purified AChE. Kinetic analyses showed that the purified AChE from GH population expressed a significant lower affinity to the substrate, and higher catalytic activity toward ATChI (i.e. higher Km and V_max values) than BB and WH populations. In vitro inhibition studies of AChE suggested that the five inhibitors (aldicarb, eserine, BW284C51, omethoate, and propoxur) all possessed strong inhibitory effects and eserine appeared to be the best inhibitor against purified AChE. According to the bimolecular rate constants (ki), the purified AChE from GH population was least sensitive to all inhibitors except for omethoate than the other two populations.2.2 Biochemical and toxicological characterization of AChE from three field population of L. bostrychophilaAChE from BB and MY populations of L. bostrychophila reached its activity peak at pH 7.0. Based on parameter estimations, the optimal pH for the purified AChE is 7.33,7.27 for BB and MY population, respectively.AChE from two populations of L. bostrychophila reached its activity peak at 37.0℃. Based on parameter estimations, the optimal temperature for the purified AChE is both 36.8℃for BB and MY populations.In comparison of the biochemical and toxicological characterizations of AChE from BB and MY populations, the results showed that there was a signigicant difference Between BB and MY populations to the kinetic parameters. The purified AChE had an apparent K_m value of 0.37 mM and a V_max of 673.93 nmol/min/mg protein in MY population, respectively. The appatent K_m and the V_max were 1.32- and 1.14-fold higher, respectively, in BB population. Kinetic analyses showed that the purified AChE from MY population expressed a significant lower affinity to the substrate, and higher catalytic activity toward ATChI (i.e. higher K_m and V_max values) than BB populations. In vitro inhibition studies of AChE showed that the sensibility of AChE to six inhibiotors from the most to the least potent inhibitors were eserine> paraoxon-ethyl> propoxur> alsicarb> BW284C51> omethoate, as determined by comparison of their bimolecular rate constants (k_i), and that the k_i values in MY population were lower than those in BB population.The differences in AChE among different populations may be caused by insensitive AChE. While the insensitive AChE may be attributed partially to the differences in control practices for psocids among three locations.