Trépanier J M, Seargeant L E, Stinson R A
Biochem J. 1976 Jun 1;155(3):653-60. doi: 10.1042/bj1550653.
Alkaline phosphatase from human liver was purified to homogeneity. The purification procedure included solubilization with butanol, fractionation with acetone, and chromatography on concanavalin A-Sepharose, DEAE-cellulose, Sephadex G-200 and DEAE-Sephadex. Purity was established by standard and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The isoelectric point of the protein was determined to be 4.0. Sephadex-gel filtration gave a mol.wt. of 146000, although a higher value was obtained in the presence of 100mM-NaC1. The subunit mol.wt. 76700, was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Neuraminidase treatment resulted in two enzyme-activity bands on isoelectric-focused gels with isoelectric points of 6.6 and 6.8. The desialylated enzyme gave only one protein band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with a subunit molecular weight indistinguishable from that of the non-neuraminidase-treated protein. The desialylated enzyme was more readily denatured by sodium dodecyl sulphate in the presence of mercaptoethanol than was the native enzyme.
人肝脏碱性磷酸酶被纯化至同质。纯化过程包括用丁醇溶解、用丙酮分级分离，以及在伴刀豆球蛋白A - 琼脂糖、二乙氨基乙基纤维素、葡聚糖凝胶G - 200和二乙氨基乙基葡聚糖上进行层析。通过标准和十二烷基硫酸钠/聚丙烯酰胺凝胶电泳确定纯度。该蛋白质的等电点测定为4.0。葡聚糖凝胶过滤得到的分子量为146000，尽管在100mM氯化钠存在下获得了更高的值。亚基分子量76700通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳确定。神经氨酸酶处理在等电聚焦凝胶上产生两条酶活性带，等电点分别为6.6和6.8。去唾液酸化酶在十二烷基硫酸钠/聚丙烯酰胺凝胶电泳上仅产生一条蛋白带，其亚基分子量与未用神经氨酸酶处理的蛋白无法区分。在巯基乙醇存在下，去唾液酸化酶比天然酶更容易被十二烷基硫酸钠变性。
