【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。更多还原

【Abstract】 Alkaline phosphatase(ALP,EC 3·1·3·1) is a non-specific phosphomononoesterase that functions through the phosphosery1 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 exchange through DEAE-32 chromatography column,gel filtration chromatography through Sephadex G-150 column and DEAE-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;Ni2+,Co2+ had no obvious effect on the enzyme,Mg2+,Mn2+,Ca2+ activated the enzyme while Zn2+,Cu2+,Pb2+,Cd2+ inhibited the enzyme.更多还原