草鱼酸性磷酸酯酶的性质及金属离子对其活性的影响  

Characterization of acid phosphatase from Ctenopharyngodon idellus and effects of metal ions on the enzyme activity

作　　者：杨立红[1] 肖波[1] 王晓洁[1] 侯月利[1] 王佳[1] 孙静[1] 

机构地区：[1]鲁东大学生命科学学院,山东烟台264025

出　　处：《中国水产科学》2010年第5期969-976,共8页Journal of Fishery Sciences of China

基　　金：山东省烟台市科学技术发展计划项目(2007152);鲁东大学"校引进人才基金"项目(LY20083307);鲁东大学学科建设项目(2009)

摘　　要：以草鱼肝脏为材料提取一种酸性磷酸酯酶(ACPase,EC.3.1.3.2),进一步用(NH4)2SO4沉淀分离,Sephadex G-200柱纯化,经聚丙烯酰胺凝胶电泳纯度鉴定呈现单一酶蛋白带,该酶紫外吸收峰为240nm和280nm,等电点为4.8,相对分子质量为100046.1。该酶水解对硝基苯磷酸二钠(pNPP)的最适pH值为4.4,最适温度为45℃,并有热激活现象,热激活温度为60℃。耐热实验表明,该酶在最适温度下耐热时间为33min,最适温度下20min时表现出最大活性。在60℃下耐热时间为25min,15min时表现最大活性。酶促反应初速度为0.0475μmol/min,最大反应速度为0.153μmol/min。米氏常数Km值为3.56×10-3mol/L。金属离子对酶活性影响的研究结果表明,K+对酶有激活作用,Li+、Na+对酶的活性没有明显影响;碱土金属离子Mg2+、Ca2+和Ba2+对酶有激活作用,其激活程度由大到小依次为:Ba2+、Mg2+、Ca2+;Mn2+对酶有激活作用,Zn2+、Cu2+、Co2+则有抑制作用;重金属离子Hg2+、Pb2+、Ag+和Cd2+对酶有抑制作用,其中Hg2+的抑制效应最强。Mg2+和Cu2+的作用机制研究结果表明,Mg2+对酶具有混合型激活作用,Cu2+对酶具有竞争性抑制作用。An acid phosphatase（ACPase,E.C.3.1.3.2）was extracted from liver of Ctenopharyngodon idellus.Then it was precipitated by ammonium sulfate and purified on sephadex G-200,and a single band of ACPase appeared in the SDS-PAGE gel.The maximal UV absorbance of the enzyme was under 240 nm and 280 nm,and the isoelectric point was 4.8.When the acid phosphatase was used to hydrolyze pNPP,the optimal pH was 4.4,and the optimal temperature was 45℃.Furthermore,hydrolyzation of the enzyme could be activated by heating,and the temperature of heat activation was 60℃.Thermo-stability experiment showed that activity of the enzyme could last for 33min at optimal temperature,and the maximal activity was revealed at 20min.The activity of the enzyme could last for 25min at 60℃,and the maximal activity was revealed at 15min.The initial velocity of the enzymatic reaction was 0.0475μmol/min and the maximal velocity was 0.153μmol/min.The Michaelis constant（K m）was 3.56×10^-3mol/L.Effects of metal ions on the enzyme activity indicated that the enzyme could be activated by K^ ＋ obviously,but could not be affected by Li^ ＋ or by Na ^＋.The positive bivalent alkaline earth metal ions of Mg^2＋,Ca^2＋and Ba^2＋could activate the enzyme,and the activation ability was as follows：Ba^2＋〉Mg^2＋〉Ca^2＋.The transition metal ions Mn^2＋could activate the enzyme.However,Zn^2＋,Cu^2＋and Co^2＋inhibited the enzyme.The heavy metal ions Hg^2＋,Pb^2＋,Ag^＋and Cd^2＋also inhibited the enzyme,respectively,and the inhibitory effect of Hg^2＋was the strongest.The mechanism of Mg^2＋or Cu^2＋affecting the enzyme indicated that Mg^2＋could make mixed activation on enzyme,but Cu^2＋displayed competitive inhibition on the enzyme.

关 键 词：草鱼 酸性磷酸酯酶 金属离子 活性 

分 类 号：Q556[生物学—生物化学] S917[农业科学—水产科学]