牛小肠碱性磷酸酶部分性质研究  

Some characterization of alkaline phosphatase from bovine intestine

作　　者：余同[1] 张营[1] 赵欣平[1] 高举[1] 杨梅强[1] Xin-Ping Mei-Qiang 

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610064

出　　处：《四川大学学报（自然科学版）》2009年第6期1838-1844,共7页Journal of Sichuan University(Natural Science Edition)

基　　金：“十一五”国家科技支撑计划重点项目(BAF07B01)

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.Mg^(2+)、Mn^(2+)和Ca^(2+)对酶有不同程度的激活作用,Zn^(2+)和EDTA对酶有抑制作用.随着Mg^(2+)、Zn^(2+)和EDTA浓度增加,270nm处紫外吸收值增加.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 EDT A concentrations.

关 键 词：肠碱性磷酸酶 性质研究 ALKALINE PHOSPHATASE enzyme SDS-PAGE OPTIMUM temperature absorption spectra specific activity SEPHADEX molecular weight EDTA 最大反应速度 不稳定 The OPTIMUM 紫外吸收值 最适温度 抑制作用 小肠 水解反应 浓度增加 

分 类 号：N55[自然科学总论]