鲫鱼酸性磷酸酶酶学特性及不同效应物对酶活力的影响  

PROPERTIES OF ACID PHOSPHATASE FROM CARASSIAS AURATUS AND EFFECTS OF SEVERAL EFFECTORS ON THE ACTIVITY OF THE ENZYME

作　　者：高举[1] 赵欣平[1] 詹付凤[1] 张营[1] 余同[1] 

机构地区：[1]四川大学生命科学学院生物资源与生态环境教育部重点实验室,成都610065

出　　处：《水生生物学报》2010年第2期394-401,共8页Acta Hydrobiologica Sinica

基　　金：成都市科技局(05GGSF220)资助

摘　　要：经NaAc-HAc缓冲液(pH5.0)抽提,正丁醇处理,硫酸铵分级沉淀,DEAE-32离子交换层析,SephadexG-150凝胶过滤纯化,从鲫鱼内脏中分离纯化出电泳纯的酸性磷酸酶。该酶提纯倍数为30.82,比活力195.06U/mg。研究表明,该酶催化对硝基苯磷酸二钠水解反应,最适pH4.8,pH小于4和大于7时不稳定;最适温度45℃,温度高于50℃不稳定;米氏常数为0.23mmol/L,利用SDS-PAGE测定酶亚基分子量为33.3kD。化学修饰剂SUAN、PMSF、DTT、NBS对该酶活力影响不大,BrAc和IAc有明显抑制作用。金属离子对该酶催化活力有不同影响,Na+、K+、Ni2+、Co2+影响不显著,Mg2+、Ca2+、Ba2+、Mn2+有激活作用,Ag+、Cu2+、Pb2+、Cd2+有抑制作用,其中Mg2+、Ca2+、Pb2+、Cd2对鲫鱼酸性磷酸酶荧光光谱的影响表明金属离子对酶活力的影响与酶构象改变有关。Acid phosphatase （ACP） is one kind of enzyme, which is not absolutely specific. It can catalyze the hydroly-sis of phosphoric acid ester chemical compound in acid environment. ACP activities are involved in a variety of metabolic processes, such as molecule permeability, growth and cell differentiation. In ecotoxicology, this enzyme has been used as an indicator of intoxication because of its sensitivity to metallic salts. So the objective of this paper is to characterize the ACP from Carassias auratus, to analyze the in vitro effects of different metal ions on the enzyme activity and to estimate its potential use as a stress biomarker. This paper mainly deals with isolating, purifying ACP from viscera of Carassias auratus and studying its characters and preparing for its application. ACP was prepared and purified by means of the following techniques： n-butyl alcohol extraction, 0.3 and 0.8 amonanium sulfate precipitation, ion ex-change chromatography on DEAE-32 column and gel filtration chromatography on Sephadex G-150. The preparation was shown to be homogenous on polyacrylamide gel electrophoresis. The specific activity of the enzyme was 195.06 unit per mg protein. Its molecular weight was determined to be about 33.3kD on SDS-PAGE. The kinetic characters of the enzyme had been studied. The enzyme showed an optimum pH of 4.8 with p-nitrophenylphosphate as substrate, however, high catalytic activity of the enzyme was within the pH range of 4.0—5.5. The optimal catalytic reaction tem-perature was 45℃, and the activity of the enzyme decreased quickly when the temperature was above 55℃. The Micha-elis-Menten constant （Km） was 0.23mmol/L. Chemical modification was used to illuminate the essential amino acid in the catalytic activity of ACP from Carassias auratus. The results revealed that succinic anhydride, phenylmethylsulfony fluoride, dithiothreitol and N-bromosuccinimide had no effect on the activity of the enzyme, while bromoacetic acid and iodoacetic acid inhibited the enzyme. The inhibition of

关 键 词：酸性磷酸酶 纯化 酶学性质 化学修饰 金属离子 鲫鱼 

分 类 号：Q556[生物学—生物化学]