牛小肠碱性磷酸酶部分性质研究

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.Km值为0.29mmol/L.45℃，pH9.7时最大反应速度（Vmax）为4.6μmol/(L•min).利用SDS-PAGE测定酶亚基的分子量为66KD. 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.

Some characterization of alkaline phosphatase from bovine intestine

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. Mg~(2+)、Mn~(2+) and Ca~(2+) activated the enzyme while Zn~(2+) and EDTA inhibited the enzyme. The ultraviolet absorption spectra peak at 270 nm of the enzyme increased with increasing Mg~(2+)、Zn~(2+) and EDTA concentrations.