Full Text:   <2189>

CLC number: Q89

On-line Access: 2013-04-03

Received: 2012-04-08

Revision Accepted: 2012-10-15

Crosschecked: 2013-03-04

Cited: 5

Clicked: 3480

Citations:  Bibtex RefMan EndNote GB/T7714

-   Go to

Article info.
1. Reference List
Open peer comments

Journal of Zhejiang University SCIENCE B 2013 Vol.14 No.4 P.325-335


Purification and studies on characteristics of cholinesterases from Daphnia magna

Author(s):  Yan-xia Yang, Li-zhi Niu, Shao-nan Li

Affiliation(s):  Institute of Pesticide and Environmental Toxicology, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029, China

Corresponding email(s):   snli@zju.edu.cn

Key Words:  Crustacea, Pseudocholinesterase, Cholinesterase, Substrate preference, Selective inhibitors

Yan-xia Yang, Li-zhi Niu, Shao-nan Li. Purification and studies on characteristics of cholinesterases from Daphnia magna[J]. Journal of Zhejiang University Science B, 2013, 14(4): 325-335.

@article{title="Purification and studies on characteristics of cholinesterases from Daphnia magna",
author="Yan-xia Yang, Li-zhi Niu, Shao-nan Li",
journal="Journal of Zhejiang University Science B",
publisher="Zhejiang University Press & Springer",

%0 Journal Article
%T Purification and studies on characteristics of cholinesterases from Daphnia magna
%A Yan-xia Yang
%A Li-zhi Niu
%A Shao-nan Li
%J Journal of Zhejiang University SCIENCE B
%V 14
%N 4
%P 325-335
%@ 1673-1581
%D 2013
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B1200113

T1 - Purification and studies on characteristics of cholinesterases from Daphnia magna
A1 - Yan-xia Yang
A1 - Li-zhi Niu
A1 - Shao-nan Li
J0 - Journal of Zhejiang University Science B
VL - 14
IS - 4
SP - 325
EP - 335
%@ 1673-1581
Y1 - 2013
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B1200113

Due to their significant value in both economy and ecology, Daphnia had long been employed to investigate in vivo response of cholinesterase (ChE) in anticholinesterase exposures, whereas the type constitution and property of the enzyme remained unclear. A type of ChE was purified from Daphnia magna using a three-step procedure, i.e., Triton X-100 extraction, ammonium sulfate precipitation, and diethylaminoethyl (DEAE)-Sepharose™-Fast-Flow chromatography. According to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), molecular mass of the purified ChE was estimated to be 84 kDa. Based on substrate studies, the purified enzyme preferred butyrylthiocholine iodide (BTCh) [with maximum velocity (Vmax)/Michaelis constant (Km)=8.428 L/(min·mg protein)] to acetylthiocholine iodide (ATCh) [with Vmax/Km=5.346 L/(min·mg protein)] as its substrate. Activity of the purified enzyme was suppressed by high concentrations of either ATCh or BTCh. Inhibitor studies showed that the purified enzyme was more sensitive towards inhibition by tetraisopropylpyrophosphoramide (iso-OMPA) than by 1,5-bis(4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284C51). Result of the study suggested that the purified ChE was more like a type of cholinesterase%29&ck%5B%5D=abstract&ck%5B%5D=keyword'>pseudocholinesterase, and it also suggested that Daphnia magna contained multiple types of ChE in their bodies.

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article


[1]Acey, R.A., Bailey, S., Healy, P., Chang, J., Unger, T.F., Hudson, R.A., 2002. A butyrylcholinesterase in the early development of the brine shrimp (Artemia salina) larvae: a target for phthalate ester embryotoxicity? Biochem. Biophys. Res. Commun., 299(4):659-662.

[2]Barata, C., Baird, D.J., Soares, A.M.V.M., Guilhermino, L., 2001. Biochemical factors contributing to response variation among resistant and sensitive clones of Daphnia magna Straus exposed to ethyl parathion. Ecotoxic. Environ. Safety, 49(2):155-163.

[3]Belzunces, L.P., Colin, M.E., 1991. Differential response of Apis mellifera acetylcholinesterase towards pirimicarb. Neuroreport, 2(5):265-268.

[4]Bocquené, G., Roig, A., Fournier, D., 1997. Cholinesterases from the common oyster (Crassostrea gigas): evidence for the presence of a soluble acetylcholinesterase insensitive to organophosphate and carbamate inhibitors. FEBS Lett., 407(3):261-266.

[5]Bourguet, D., Raymond, M., Fournier, D., Malcolm, C.A., Toutant, J.P., Arpagaus, M., 1996. The existence of two acetylcholinesterases in the mosquito Culex pipiens (Diptera: Culicidae). J. Neurochem., 67(5):2115-2123.

[6]Bradford, M.M., 1976. Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein dye binding. Anal. Biochem., 72(1-2):248-254.

[7]Brestkin, A.P., Maizel, E.B., Moralev, S.N., Novozhilov, K.V., Sazonova, I.N., 1985. Cholinesterases of aphids—1: Isolation, partial purification and some properties of cholinesterases from spring grain aphid Schizaphis gramina (Rond.). Insect Biochem., 15(2):309-314.

[8]Carvalho, F.D., Machado, I., Martinez, M.S., Soares, A., Guilhermino, L., 2003. Use of atropine-treated Daphnia magna survival for detection of environmental contamination by acetylcholinesterase inhibitors. Ecotoxicol. Environ. Safety, 54(1):43-46.

[9]Chuiko, G.M., 2000. Comparative study of acetylcholinesterase and butyrylcholinesterase in brain and serum of several freshwater fish: specific activities and in vitro inhibition by DDVP, an organophosphorus pesticide. Comp. Biochem. Physiol. Part C: Pharmacol. Toxicol. Endocrinol., 127(3):233-242.

[10]Coelho, S., Oliveira, R., Pereira, S., Musso, C., Domingues, I., Bhujel, R.C., Soares, A.M.V.M., Nogueira, A.J.A., 2011. Assessing lethal and sub-lethal effects of trichlorfon on different trophic levels. Aquat. Toxicol., 103(3-4):191-198.

[11]Dámasio, J., Guilhermino, L., Soares, A.M.V.M., Riva, M.C., Barata, C., 2007. Biochemical mechanisms of resistance in Daphnia magna exposed to the insecticide fenitrothion. Chemosphere, 70(1):74-82.

[12]den Besten, P.J., Valk, S., van Weerlee, E., Nolting, R.F., Postma, J.F., Everaarts, J.M., 2001. Bioaccumulation and biomarkers in the sea star Asterias rubens (Echinodermata: Asteroidea): a North Sea field study. Mar. Environ. Res., 51(4):365-387.

[13]Diamantino, T.C., Almeida, E., Soares, A.M.V.M., Guilhermino, L., 2007. Characterization of cholinesterases from Daphnia magna Straus and their inhibition by zinc. Bull. Environ. Contam. Toxicol., 71(2):219-225.

[14]Doctor, B.P., Blick, D.W., Caranto, G., Castro, C.A., Gentry, M.K., Larrison, R., Maxwell, D.M., Murphy, M.R., Schutz, M., Waibel, K., et al., 1993. Cholinesterases as scavengers for organophosphorus compounds: protection of primate performance against soman toxicity. Chem. Biol. Int., 87(1-3):285-293.

[15]Duquesne, S., 2006. Effects of an organophosphate on Daphnia magna, at suborganismal and organismal levels: implications for population dynamics. Ecotoxicol. Environ. Safety, 65(2):145-150.

[16]Duquesne, S., Küster, E., 2010. Biochemical, metabolic, and behavioural responses and recovery of Daphnia magna after exposure to an organophosphate. Ecotoxicol. Environ. Safety, 73(3):353-359.

[17]Elhalwagy, M.E.A., Farid, H.E.A., Gh, F.A.A., Ammar, A.E., Kotb, G.A.M., 2010. Risk assessment induced by knapsack or conventional motor sprayer on pesticides applicators and farm workers in cotton season. Environ. Toxicol. Pharm., 30(2):110-115.

[18]Ellman, G.L., Courtney, K.D., Andres, V., 1961. Featherstone RM, a new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharm., 7(2):88-95.

[19]Forget, J., Beliaeff, B., Bocquene, G., 2003. Acetylcholinesterase activity in copepods (Tigriopus brevicornis) from the Vilaine River estuary, France, as a biomarker of neurotoxic contaminants. Aquat. Toxicol., 62(3):195-204.

[20]Gälli, R., Rich, H.W., Scholtz, R., 1994. Toxicity of organophosphate insecticides and their metabolites to the water flea Daphnia magna, the microtox test and an acetylcholinesterase inhibition test. Aquat. Toxicol., 30(3):259-269.

[21]Guilhermino, L., Lopes, M.C., Carvalho, A.P., Soares, A.M.V.M., 1996. Inhibition of acetylcholinesterase activity as effect criterion in acute tests with juvenile Daphnia magna. Chemosphere, 32(4):727-738.

[22]Guo, S., Cao, S., Cheng, Y., 1993. Studies on the purification and properties of duck serum choline esterase. Prog. Biochem. Biophys., 20(2):119-124 (in Chinese).

[23]Ioannidis, P.M., Grafius, E.J., Wierenga, J.M., Whalon, M.E., Hollingworth, R.M., 1992. Selection, inheritance and characterization of carbofuran resistance in the Colorado potato beetle (Coleoptera: Chrysomelidae). Pest. Sci., 35(3):215-222.

[24]Jemec, A., Drobne. D., Tisler, T., Trebse, P., Ros, M., Sepcic, K., 2007. The applicability of acetylcholinesterase and glutathione S-transferase in Daphnia magna toxicity test. Comp. Biochem. Physiol. Part C: Pharmacol. Toxicol. Endocrinol., 144(4):303-309.

[25]Jung, J.H., Kim, S.J., Lee, T.K., Shim, W.J., Woo, S., Kim, D.J., Han, C.H., 2008. Biomarker responses in caged rockfish (Sebastes schlegeli) from Masan Bay and Haegeumgang. South Korea Mar. Poll. Bull., 57(6-12):599-606.

[26]Kato, Y., Tanaka, T., Miyata, T., 2004. Comparison of kinetic properties of a hydrophilic form of acetylcholinesterase purified from strains susceptible and resistant to carbamate and organophosphorus insecticides of green rice leafhopper (Nephotettix cincticeps Uhler). Pest. Biochem. Physiol., 79(2):64-73.

[27]Kaufer, D., Friedman, A., Seidman, S., Soreq, H., 1999. Anticholinesterase induce multigenic transcriptional feedback response suppressing cholinergic neurotransmission. Chem. Biol. Int., 119-120:349-360.

[28]Khattab, A.D., Ali, L.S., 2007. Immunoassays for avian butyrylcholinesterase: implications for ecotoxicological testing and clinical biomonitoring. Environ. Toxicol. Pharm., 24(3):275-285.

[29]Khattab, A.D., Walker, C.H., Mackness, M.I., Saphier, P.W., 1993. Purification and immunological characterization of pigeon serum butyrylcholinesterase: implications on environmental monitoring and toxicological testing of birds. Biochem. Pharm., 45(5):991-998.

[30]Kirby, M.F., Morris, S., Hurst, M., Kirby, S.J., Neall, P., Tylor, T., Fagg, A., 2000. The use of cholinesterase activity in flounder (Platichthys flesus) muscle tissue as a biomarker of neurotoxic contamination in UK estuaries. Mar. Poll. Bull., 40(9):780-791.

[31]Kousba, A.A., Poet, T.S., Timchalk, C., 2003. Characterization of the in vitro kinetic interaction of chlorpyrifos-oxon with rat salivary cholinesterase: a potential biomonitoring matrix. Toxicology, 188(2-3):219-232.

[32]Laemmli, U.K., 1970. Cleavage of structural proteins during assembly of head of bacteriophage-T4. Nature, 227:680-685.

[33]Lenz, D.E., Maxwell, D.M., Koplovitz, I., Clark, C.R., Capacio, B.R., Cerasoli, D.M., Federko, J.M., Luo, C.Y., Saxena, A., Doctor, B.P., et al., 2005. Protection against soman or VX poisoning by human butyrylcholinesterase in guinea pigs and cynomolgus monkeys. Chem. Biol. Int., 157:205-210.

[34]Li, S., Ji, S., Wang, L., 2006. Study on the isolation, purification and the properties of cholinesterase from horse serum. Sci. Technol. Food Ind., 27(9):91-93 (in Chinese).

[35]Li, S.N., Tan, Y.J., 2011. Hormetic response of cholinesterase from Daphnia magna in chronic exposure to triazophos and chlorpyrifos. J. Environ. Sci., 23(5):852-859.

[36]Maxwell, D.M., Saxena, A., Gordon, R.K., Doctor, B.P., 1999. Improvements in scavenger protection against organophosphorus agents by modification of cholinesterases. Chem. Biol. Int., 120:419-428.

[37]Mehrani, H., 2004. Simplified procedures for purification and stabilization of human plasma butyrylcholinesterase. Proc. Biochem., 39(7):877-882.

[38]Nolan, J., Schnitzerling, H.J., Schuntner, C.A., 1972. Multiple forms of acetylcholinesterase from resistantand susceptible strains of the cattle tick Boophilus microplus (Can). Pest. Biochem. Physiol., 2(1):85-94.

[39]Peng, X., Tao, K., Teng, Y., Hang, X., Hou, T., 2008. Purification and characterization of acetylcholinesterase, a kind of pesticide target. J. Sichuan Univ. (Nat. Sci. Ed.), 45(1):189-193 (in Chinese).

[40]Principato, G.B., Talesa, V., Giovannini, E., Pascolini, R., Gabriella, R., 1988. Characterization of the soluble cholinesterase from Squilla mantis. Comp. Biochem. Physiol. Part C: Pharmacol. Toxicol. Endocrinol., 90:413-416.

[41]Printes, L.B., Fellowes, M.D.E., Callaghan, A., 2008. Clonal variation in acetylcholinesterase biomarkers and life history traits following OP exposure in Daphnia magna. Ecotoxicol. Environ. Safety, 71(2):519-526.

[42]Printes, L.B., Fernandes, M.N., Espíndola, E.L.G., 2011. Laboratory measurements of biomarkers and individual performances in Chironomus xanthus to evaluate pesticide contamination of sediments in a river of southeastern Brazil. Ecotoxicol. Environ. Safety, 74(3):424-430.

[43]Quintaneiro, C., Monteiro, M., Pastorinho, R., Soares, A.M.V.M., Nogueira, A.J.A., Morgado, F., Guilhermino, L., 2006. Environmental pollution and natural populations: a biomarkers case study from the Iberian Atlantic coast. Mar. Poll. Bull., 52(11):1406-1413.

[44]Raveh, L., Grunwald, J., Marcus, D., Papier, Y., Cohen, E., Ashani, Y., 1993. Human butyrylcholinesterase as a general prophylactic antidote for nerve agent toxicity: in vitro and in vivo quantitative characterization. Biochem. Pharm., 45(12):2465-2474.

[45]Sáenz, L.A., Seibert, E.L., Zanette, J., Fiedler, H.D., Curtius, A.J., Ferreira, J.F., de Almeida, E.A., Marques, M.R.F., Bainy, A.C.D., 2010. Biochemical biomarkers and metals in Perna perna mussels from mariculture zones of Santa Catarina, Brazil. Ecotoxicol. Environ. Safety, 73(5):796-804.

[46]Sanchez-Hernandez, J.C., Fossi, M.C., Leonzio, C., Focardi, S., Barra, R., Gavilan, J.F., Parra, O., 1998. Use of biochemical biomarkers as a screening tool to focus the chemical monitoring of organic pollutants in the Biobio river basin (Chile). Chemosphere, 37(4):699-710.

[47]Stien, X., Percic, P., Gnassia-Barelli, M., Romeo, M., Lafaurie, M., 1998. Evaluation of biomarkers in caged fishes and mussels to assess the quality of waters in a bay of the NW Mediterranean Sea. Environ. Poll., 99(3):339-345.

[48]Sturm, A., Hansen, P.D., 1999. Altered cholinesterase and monooxygenase levels in Daphnia magna and Chironomus riparius exposed to environmental pollutants. Ecotoxicol. Environm. Safety, 42(1):9-15.

[49]Talesa, V., Contenti, S., Principato, G.B., Pascolini, R., Giovannini, E., Rosi, G., 1992. Cholinesterases from Maia verrucosa and Palinurus vulgaris: a comparative study. Comp. Biochem. Physiol. Part C: Pharmacol. Toxicol. Endocrinol., 101(3):499-503.

[50]Tripathi, R.K., O′Brien, R.D., 1972. Effect of organophosphates in vivo upon acetylcholinesterase isozyme from housefly head and thorax. Pest. Biochem. Physiol., 2(4):418-424.

[51]van Oosterom, J., King, S.C., Negri, A., Humphrey, C., Mondon, J., 2010. Investigation of the mud crab (Scylla serrata) as a potential bio-monitoring species for tropical coastal marine environments of Australia. Mar. Poll. Bull., 60(2):283-290.

[52]Vesela, S., Kuca, K., Jun, D., 2006. Toxicity of the nerve agent tabun to Daphnia magna, a new experimental species in military toxicology. Chem. Ecol., 22(2):175-180.

[53]Vesela, S., Kuca, K., Jun, D., 2008. Daphnia intoxicated by nerve agent tabun can be treated using human antidotes. Environm. Toxicol. Pharm., 25:329-333.

[54]Villatte, F., Bachmann, T.T., 2002. How many genes encode cholinesterase in arthropods? Pest. Biochem. Physiol., 73(2):122-129.

[55]Wang, Y.X., Boeck, A.T., Duysen, E.G., van Keuren, M., Saunders, T.L., Lockridge, O., 2004. Resistance to organophosphorus agent toxicity in transgenic mice expressing the G117H mutant of human butyrylcholinesterase. Toxicol. Appl. Pharmcol., 196(3):356-366.

[56]Wierenga, J.M., Hollingworth, R., 1993. Inhibition of altered actylcholinesterases from insecticide-resistant Colorado potato beetle (Coleoptera: Chrysomelidae). J. Econ. Entomol., 86:673-679.

[57]Zhang, X., Shi, Y., Zhang, H., 1999. A preliminary study of the purification acetylcholinesterase from human cerehellum by immuno-affinity chromatography. J. First Mil. Med. Univ., 19(2):165-167 (in Chinese).

[58]Zhang, Y., Yang, B., 2006. Separation and purification of Recombinant Nippostrongylus brasiliensis acetylcholinesterase from culture medium of genetic engineering Pichia pastoris. Chin. J. Chrom., 24(1):39-41 (in Chinese).

[59]Zhou, M., Zhang, C., Richard, P., Haugland, R.P., 2000. Choline oxidase: a useful tool for high-throughput assays of acetylcholinesterase, phospholipase D, phosphayidylcholine-specific phospholipase C, and sphingomyelinase. Proc. SPIE, 3926:166-171.

[60]Zhu, K.Y., Brindley, W.A., 1992. Molecular properties of acetylcholinesterase purified from Lygus hesperus Knight (Hemiptera: Miridae). Insect Biochem. Mol. Biol., 22(3):253-260.

[61]Zhu, K.Y., Clark, M., 1994. Purification and characterization of acetyl-cholinesterase from the Colorado potato beetle, Leptinotarsa decemlineata (Say). Insect Biochem. Mol. Biol., 24(5):453-461.

[62]Zhu, X.S., Meng, F.P., He, D.H., 2006. Purification and partial biochemical characterization of acetylcholinesterase from Scomberomorus Niphonius (Cuvier). J. Qingdao Univ. (E&T), 21(2):35-40 (in Chinese).

Open peer comments: Debate/Discuss/Question/Opinion


Please provide your name, email address and a comment

Journal of Zhejiang University-SCIENCE, 38 Zheda Road, Hangzhou 310027, China
Tel: +86-571-87952783; E-mail: cjzhang@zju.edu.cn
Copyright © 2000 - Journal of Zhejiang University-SCIENCE