草鱼肝胰脏碱性磷酸酶的分离纯化及性质的研究

Purification and Some Characterization of Phosphatase from Grass Carp(Ctenopharyngodon idellus)

为获得草鱼肝胰脏的碱性磷酸酶,并阐明其性质,利用Tris-HCl(pH 7.5)缓冲液抽提、正丁醇脱脂、硫酸铵分级沉淀分离和葡聚糖凝胶(Sephadex G-150)等步骤对草鱼肝胰脏碱性磷酸酶进行分离纯化,测定碱性磷酸酶的动力学性质,研究金属离子对碱性磷酸酶活力的影响。试验结果表明,得到提取倍数为7.3倍、比活力为949.8U/mg的碱性磷酸酶,酶液经SDS-PAGE呈一条带,其亚基的分子量约54ku。动力学测定结果显示,该酶催化对硝基磷酸苯二钠水解反应的最适pH为10.33,最适温度为38℃,米氏常数为9.03mmol/L,最大反应速度为1.5μmol/(L·min)。Li+、Na+、K+对碱性磷酸酶活力影响不大;Ba2+、Ca2+、Mg2+、Mn2+能激活碱性磷酸酶活力;EDTA、Cu2+和Zn2+抑制碱性磷酸酶活力。研究结果可为碱性磷酸酶在鱼类脂质代谢中的作用提供参考和依据。

The properties of alkaline phosphatase （AKP） were studied in hepatopancreas of grass carp Ctenopharyngodon idellus .In our study ,the isolation and purification of AKP were conducted by some processes of Tris-HCl（pH 7 .5）extraction ,n-butanol degreasing ,ammonium sulfate fractional precipitati-on and gel filtration （Sephadex G -150） ,and the kinetic properties of AKP and the effects of metal ions on the enzyme activity were studied .The molecular weight of the protein was about 54 ku by SDS-PAGE , with 7 .3 fold purification and specific activity of 949 .8U/mg .Kinetic assay showed that the optimum pH of the enzyme in catalyzed nitrobenzene disodium phosphate （PNPP） hydrolysis reaction was 10 .33 ,the optimum temperature was 38 ℃ ,the Michaelis constant （Km） value was 9 .03 mmol /L and the maximum reaction rate （Vmax） was 1 .5μmol /（L · min） .The findings showed that Li＋ ,Na＋ ,K＋ had no effects on the activity of AKP ,and Ba2＋ ,Ga2＋ ,Mg2＋and Mn2＋ led to activation of the enzyme activity ,but EDTA , Cu2＋ and Zn2＋ inhibited the enzyme activity .Together ,we successfully isolated and purified the AKP from hepatopancreas of the grass carp and stuied the properties of AKP .Our findings might provide some refer-ence for the understanding of the function of AKP in the lipid metabolism of fish .