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
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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。Abstract: Acid phosphatase (ACP) is one kind of enzyme, which is not absolutely specific. It can catalyze the hydrolysis 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 exchange 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 temperature was 45℃, and the activity of the enzyme decreased quickly when the temperature was above 55℃. The Michaelis-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 bromoacetic acid and iodoacetic acid on the activity of ACP from Carassias auratus indicated that there were histidine residues at the active site of the enzyme. Different metal ions had different effects on ACP from Carassias auratus. The positive monovalent metalions K+ and Na+ had no effect on the enzyme, while Ag+ inhibited the enzyme; The positive bivalent cations had different effects on the enzyme: Co2+ and Ni2+ had no effect on the enzyme; Mg2+, Ca2+, Ba2+ and Mn2+ activated the enzyme while Cu2+, Pb2+ and Cd2+ inhibited the enzyme. The aviation of Mg2+ and Ca2+ and the inhibition of Pb2+ and Cd2+ were depended on the concentration of metal ions. Fluorescence spectra of ACP which was incubated with Pb2+, Cd2+, Mg2+ and Ca2+ showed that effects of metal ions on the enzyme might be related to a conformational change on the enzyme. Thus these characters of ACP suggest that the enzyme is suitable to be used as a stress biomarker, but more knowledge of the physicochemical and kinetic characteristics of the enzyme activity of ACP from Carassias auratus is necessary before it can be employed as biochemical indicator of stress due to heavy metals.
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Keywords:
- Acid phosphatase /
- Purification /
- Properties /
- Chemical modification /
- Metal ions /
- Carassias auratus
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