金属离子对牛小肠碱性磷酸酶的影响  

Effects of Metal Ions on Alkaline Phosphatase of Calf Intestine

作　　者：张营[1] 余同[1] 赵欣平[1] 高举[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：《四川动物》2009年第6期835-838,842,共5页Sichuan Journal of Zoology

基　　金："十一五"国家科技支撑计划重点项目;项目编号:2006BAF07B01

摘　　要：经Tris-HCl缓冲液抽提,硫酸铵分级分离沉淀,2次DEAE-32柱层析和SephadexG-150凝胶过滤,从牛小肠中得到碱性磷酸酶(ALP),提纯倍数为50.69倍,比活为48.87U/mg,酶液经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测呈现单一条带。该酶催化底物对硝基苯磷酸二钠(pNPP)水解反应的最适pH值为9.7,最适温度为45℃。研究了多种金属离子对牛小肠碱性磷酸酶活性的影响,结果表明:一价金属离子Na+、K+对酶活力没有影响;不同的二价金属离子对酶的影响不同,过渡态金属离子中,Ni2+、Co2+对酶的活力影响不大,Mg2+、Ca2+、Mn2+对酶有不同程度的激活作用,Zn2+、Cu2+对酶有抑制作用;重金属离子Pb2+、Cd2+对酶的活力起抑制作用。Alkaline phosphatase （ALP, EC 3 · 1 · 3 · 1 ） is a non-specific phosphomononoesterase that functions through the phosphoseryl intermediate to produce a free group to other alcohols. ALPs have been extensively investigated and widely used as biochemical and diagnostic reagents, among which ALP from calf intestine is an ideal one for its extensive uses in genetic engineering. We choose calf intestine as a resource of ALP for the purpose of applying ALP to biochemical and clinical reagents. Calf intestine ALP was separated by extraction of n-butyl alcohol, amonanium sulfate precipitation, ion ex- change through DEAE-32 chromatography column, gel filtration chromatography through Sephadex G-150 column and DE- AE-32 chromatography column. ALP was detected as one band through SDS-Polyacrylamide gel electrophoresis, indicating its homogeneity in components. The ultimate specific activity of purified enzyme was measured as 48.87 U/mg. Study of various metal ions demonstrates that the positive monovalent cations Na^＋ , K^＋ had no effect on the enzymes; Ni^2＋ , Co^2＋ had no obvious effect on the enzyme, Mg^2＋ , Mn^2＋ , Ca^2＋ activated the enzyme while Zn^2＋ , Cu^2＋ , Pb^2＋ , Cd^2＋ inhibited the enzyme.

关 键 词：牛小肠 碱性磷酸酶 金属离子 

分 类 号：Q55[生物学—生物化学]