水生生物学报
水生生物學報
수생생물학보
ACTA HYDROBIOLOGICA SINICA
2010年
2期
394-401
,共8页
高举%赵欣平%詹付凤%张营%余同
高舉%趙訢平%詹付鳳%張營%餘同
고거%조흔평%첨부봉%장영%여동
酸性磷酸酶%纯化%酶学性质%化学修饰%金属离子%鲫鱼
痠性燐痠酶%純化%酶學性質%化學脩飾%金屬離子%鯽魚
산성린산매%순화%매학성질%화학수식%금속리자%즉어
Acid phosphatase%Purification%Properties%Chemical modification%Metal ions%Carassias auratus
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pH 4.8,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0.23 mmol/L,利用SDS-PAGE测定酶亚基分子量为33.3 kD.化学修饰剂SUAN、PMSF、DTT、NBS对该酶活力影响不大,BrAc和IAe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~+、Ni~(2+)、Co~(2+)影响不显著,Mg~(2+)、Ca~(2+)、Ba~(2+)、Mn~(2+)有激活作用,Ag~+、Cu~(2+)、pb~(2+)、Cd~(2+)有抑制作用,其中Mg~(2+)、Ca~(2+)、pb~(2+)、Cd~2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.
經NaAc-HAc緩遲液(pH5.0)抽提,正丁醇處理,硫痠銨分級沉澱,DEAE-32離子交換層析,SephadexG-150凝膠過濾純化,從鯽魚內髒中分離純化齣電泳純的痠性燐痠酶.該酶提純倍數為30.82,比活力195.06U/mg.研究錶明,該酶催化對硝基苯燐痠二鈉水解反應,最適pH 4.8,pH小于4和大于7時不穩定;最適溫度45℃,溫度高于50℃不穩定;米氏常數為0.23 mmol/L,利用SDS-PAGE測定酶亞基分子量為33.3 kD.化學脩飾劑SUAN、PMSF、DTT、NBS對該酶活力影響不大,BrAc和IAe有明顯抑製作用.金屬離子對該酶催化活力有不同影響,Na~+、K~+、Ni~(2+)、Co~(2+)影響不顯著,Mg~(2+)、Ca~(2+)、Ba~(2+)、Mn~(2+)有激活作用,Ag~+、Cu~(2+)、pb~(2+)、Cd~(2+)有抑製作用,其中Mg~(2+)、Ca~(2+)、pb~(2+)、Cd~2對鯽魚痠性燐痠酶熒光光譜的影響錶明金屬離子對酶活力的影響與酶構象改變有關.
경NaAc-HAc완충액(pH5.0)추제,정정순처리,류산안분급침정,DEAE-32리자교환층석,SephadexG-150응효과려순화,종즉어내장중분리순화출전영순적산성린산매.해매제순배수위30.82,비활력195.06U/mg.연구표명,해매최화대초기분린산이납수해반응,최괄pH 4.8,pH소우4화대우7시불은정;최괄온도45℃,온도고우50℃불은정;미씨상수위0.23 mmol/L,이용SDS-PAGE측정매아기분자량위33.3 kD.화학수식제SUAN、PMSF、DTT、NBS대해매활력영향불대,BrAc화IAe유명현억제작용.금속리자대해매최화활력유불동영향,Na~+、K~+、Ni~(2+)、Co~(2+)영향불현저,Mg~(2+)、Ca~(2+)、Ba~(2+)、Mn~(2+)유격활작용,Ag~+、Cu~(2+)、pb~(2+)、Cd~(2+)유억제작용,기중Mg~(2+)、Ca~(2+)、pb~(2+)、Cd~2대즉어산성린산매형광광보적영향표명금속리자대매활력적영향여매구상개변유관.
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 amonauium 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 (Kin) 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 metal ions 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 Ni~(2+) had no effect on the enzyme; Mg~(2+), Ca~(2+), Ba~(2+) and Mn~(2+) activated the enzyme while Cu~(2+), Pb~(2+) and Cd~(2+) inhibited the enzyme. The aviation of Mg~(2+) and Ca~(2+) and the inhibition of Pb~(2+) and Cd~(2+) were depended on the concentration of metal ions. Fluorescence spectra of ACP which was incubated with Pb~(2+), Cd~(2+), Mg~(2+) and Ca~(2+) 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.
