【摘要】 用正丁醇抽提、硫酸铵分级沉淀、EAE-32柱层析和Sephadex G-150凝胶过滤,从牛小肠中分离纯化出牛小肠碱性磷酸酶（BIAP）.提纯倍数为50.69,比活力为48.87U/mg蛋白.酶液经SDS-PAGE呈现单一条带,且不含DNA核酸酶.该酶催化对硝基苯磷酸二钠（p-NPP）水解反应的最适pH值为9.7,pH小于6.5大于11.5均不稳定;最适温度为45℃,高于50℃不稳定.45℃,pH 9.7时K_m值为0.29mmol/L,最大反应速度(V_max)为4.6μmol/（L·min）.利用SDS-PAGE测定酶亚基的分子量为66 kD.Mg²⁺、Mn²⁺和Ca²⁺对酶有不同程度的激活作用,Zn²⁺和EDTA对酶有抑制作用.随着Mg²⁺、Zn²⁺和EDTA浓度增加,270nm处紫外吸收值增加.更多还原

【Abstract】 An alkaline phosphatase purified from bovine intestine by following procedures:n-butyl alcohol extraction,ammonium sulfate precipation,ion-exchange chromatography on DEAE-32 column,fellowed by gel filtration through Sephadex G-150 and ion-exchange chromatography on DEAE-32.The purification multiple was 50.69 and the specific activity of the enzyme was 48.87U/mg.The preparation was formed a single band on SDS-PAGE and not containing the DNA nuclease.The optimum pH and optimum temperature for the enzyme to catalyze the hydrolysis of phenylphosphoric acid disodium salt （p-NPP） were pH 9.7 and 45 ℃ ,The enzyme is stable in the range of pH from 6.5 to 11.5 and at the temperature below 50 ℃.Michaelis-Menten constant（K_m） is 0.29 mmol/L and the maximum velocity (V_max) is 4.6 μmol/（L·min） at pH 9.7 and 45 ℃.Its molecular weight was determined to be about 66 kD on SDS-PAGE.Mg²⁺ ,Mn²⁺ and Ca²⁺ activated the enzyme while Zn²⁺ and EDTA inhibited the enzyme. The ultraviolet absorption spectra peak at 270 nm of the enzyme increased with increasing Mg²⁺、 Zn²⁺ and EDT A concentrations.更多还原