中国仓鼠肝脏中半乳糖激酶的分离、纯化及部分特性鉴定

Isolation, purification and partial characterization of galactokinase from Chinese hamster liver.

Talbot B, Thirion J P

Int J Biochem. 1982;14(8):719-26. doi: 10.1016/0020-711x(82)90008-8.

Abstract

Galactokinase was purified from Chinese hamster liver. The purification process consisted of an initial biphasic partition separation followed by column chromatography on DEAE-cellulose. DEAE-Sephadex, Sephacryl S-200 and hydroxyapatite. 2. The enzyme was stabilized during the purification procedure by the inclusion of 10% glycerol, 1 mM phosphate and 20 mM beta-mercaptoethanol in all the buffer solutions. 3. Chromatography on hydroxyapatite separated two forms of galactokinase, one of which was purified to homogeneity using gel electrophoresis. 4. The purified galactokinase has a mol. wt of approximately 60,000 as determined by SDS gel electrophoresis and molecular sieving column chromatography. 5. The pH optimum is 7.4 and the Km for galactose is 1.16 x 10(-4) M.

摘要

半乳糖激酶是从中国仓鼠肝脏中纯化得到的。纯化过程包括最初的双相分配分离，随后依次在DEAE - 纤维素、DEAE - 葡聚糖凝胶、Sephacryl S - 200和羟基磷灰石上进行柱层析。2. 在整个纯化过程中，通过在所有缓冲溶液中加入10%甘油、1 mM磷酸盐和20 mMβ - 巯基乙醇来稳定该酶。3. 在羟基磷灰石上进行层析分离出两种形式的半乳糖激酶，其中一种通过凝胶电泳纯化至同质。4. 通过SDS凝胶电泳和分子筛柱层析测定，纯化的半乳糖激酶分子量约为60,000。5. 最适pH为7.4，半乳糖的Km值为1.16×10⁻⁴ M。