CAJ | 학술논문

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48.87 U/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4.6 μmol/(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,270 nm处紫外吸收值增加.
용정정순추제、류산안분급침정、EAE-32주층석화Sephadex G-150응효과려, 종우소장중분리순화출우소장감성린산매(BIAP).제순배수위50.69,비활력위48.87 U/mg단백.매액경SDS-PAGE정현단일조대,차불함DNA핵산매.해매최화대초기분린산이납(p-NPP)수해반응적최괄pH치위9.7,pH소우6.5대우11.5균불은정;최괄온도위45 ℃,고우50 ℃불은정. 45 ℃,pH 9.7시K_m치위0.29mmol/L,최대반응속도(V_(max))위4.6 μmol/(L·min).이용SDS-PAGE측정매아기적분자량위66 kD. Mg~(2+)、Mn~(2+)화Ca~(2+)대매유불동정도적격활작용,Zn~(2+)화EDTA대매유억제작용.수착Mg~(2+)、Zn~(2+)화EDTA농도증가,270 nm처자외흡수치증가.
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.