Purification and studies on characteristics of cholinesterases from Daphnia magna  

作　　者：Yan-xia YANG Li-zhi NIU Shao-nan LI 

机构地区：[1]Institute of Pesticide and Environmental Toxicology,College of Agriculture and Biotechnology,Zhejiang University

出　　处：《Journal of Zhejiang University-Science B(Biomedicine & Biotechnology)》2013年第4期325-335,共11页浙江大学学报（英文版）B辑（生物医学与生物技术）

基　　金：Project supported by the Zhejiang Provincial Natural Science Foundation of China(No.LY12B07008);the Department of Education of Zhejiang Province,China(No.20070138)

摘　　要：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 V max /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 pseudocholinesterase,and it also suggested that Daphnia magna contained multiple types of ChE in their bodies.Due to their significant value in both economy and ecology, Daphnia had long been employed to inves- tigate 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 pro- cedure, i.e., Triton X-100 extraction, ammonium sulfate precipitation, and diethylaminoethyl （DEAE）-SepharoseTM- 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 （Vrnax）/Michaelis constant （Km）=8.428 L/（min-mg protein）] to acetylthiocholine iodide （ATCh） [with VrnaxlKm=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-allyldimethytammoniumphenyl） pentan-3-one dibromide （BW284C51）. Result of the study suggested that the purified ChE was more like a type of pseudocholinesterase, and it also suggested that Daphnia magna contained multiple types of ChE in their bodies.

关 键 词：CRUSTACEA PSEUDOCHOLINESTERASE CHOLINESTERASE Substrate preference Selective inhibitors 

分 类 号：Q55[生物学—生物化学]