人精浆酸性磷酸酶的纯化与特性分析

Purification and characterization of human seminal plasma acid phosphatase.

作者信息

Nag Das S K, Bhattacharyya A, Bhattacharyya A K

出版信息

Ital J Biochem. 1986 Mar-Apr;35(2):71-85.

Abstract

A 81-fold purification of human seminal plasma acid phosphatase was obtained by a three-step procedure, involving ammonium sulfate precipitation, DEAE-cellulose chromatography and Sephadex G-200 gel filtration. Homogeneity of the preparation during purification steps was tested by polyacrylamide gel electrophoresis and only one major band was obtained after the final step. The pH optimum for the activity of the purified enzyme was 5.6 and thermal stability was obtained even up to 40 degrees C. PNPP was the most specific synthetic substrate. The Km of purified seminal acid phosphatase towards PNPP was 1.5 X 10(-3) M. Among the metal ions tested, Hg+2 showed an I50 value of 4.2 X 10(-7) M. Studies with PCMB, PMSF and EDTA did not show any inhibition, whereas NaF and L(+)tartrate, at 1 mM concentration, inhibited the enzyme by 95% and 85%, respectively.

摘要

通过三步程序实现了人精浆酸性磷酸酶81倍的纯化，该程序包括硫酸铵沉淀、DEAE - 纤维素色谱和Sephadex G - 200凝胶过滤。在纯化步骤中，通过聚丙烯酰胺凝胶电泳测试制剂的纯度，最后一步后仅获得一条主要条带。纯化酶活性的最适pH为5.6，甚至在高达40℃时仍具有热稳定性。对硝基苯磷酸二钠（PNPP）是最特异的合成底物。纯化的精浆酸性磷酸酶对PNPP的米氏常数（Km）为1.5×10（-3）M。在所测试的金属离子中，Hg +2的半数抑制浓度（I50）值为4.2×10（-7）M。对氯汞苯甲酸（PCMB）、苯甲基磺酰氟（PMSF）和乙二胺四乙酸（EDTA）的研究未显示出任何抑制作用，而1 mM浓度的氟化钠（NaF）和L（+）酒石酸盐分别使该酶的活性抑制95%和85%。