Purification and properties of detergent-solubilized pig kidney trehalase.
Purification and properties of detergent-solubilized pig kidney trehalase.
复制标题
洗涤剂溶解的猪肾海藻糖酶的纯化和性质。
DOI：
10.1016/0003-9861(87)90307-9
复制
发表时间：
1987
影响因子：
3.9
通讯作者：
Yoneyama,Y
中科院分区：
文献类型：
--
作者：
Yoneyama,Y
在蛋白水解酶抑制剂存在下，用Triton X-100和脱氧胆酸钠从猪肾皮质刷状缘膜上增溶海藻糖酶(α，α-trehalase，EC 3.2.1.28)。通过凝胶过滤、离子交换层析、ConA-Sepharose层析、苯基-SepharoseCL-4B疏水作用层析、Tris-Sepharose6B亲和层析和羟基磷灰石层析，将肾酶纯化3060倍。用Tris-Sepharose6B吸附污染蛋白。根据氨基端氨基酸分析，纯度估计为99%或更高。纯化后的酶比活力为278个单位/mg蛋白，银染后显示一条主带，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法测得其相对分子质量为8万。纯化的海藻糖酶是一种糖蛋白，每摩尔海藻糖酶含有2摩尔的氨基葡萄糖。Tris、HgCl2和根皮苷对肾脏海藻糖酶有抑制作用，Ki值分别为3.8mM、11μm和2.4mM。在反应混合物中加入Cl-−可保护酶不被HgCl2失活。计算出海藻糖的表观Km值为2.1 mm。肾脏海藻糖酶对海藻糖有高度专一性，最适pH为5.9。经层析聚焦测定，等电点在pH 4.7~4.4之间。
Trehalase (α,α-trehalase, EC 3.2.1.28) was solubilized from the brush border membrane of pig kidney cortex by Triton X-100 and sodium deoxycholate in the presence of inhibitors of proteolytic enzymes. The kidney enzyme was purified 3060-fold using gel filtration, ion exchange chromatography, Con A-Sepharose chromatography, phenyl-Sepharose CL-4B hydrophobic interaction chromatography, Tris-Sepharose 6B affinity chromatography, and hydroxylapatite chromatography. Tris-Sepharose 6B was utilized to absorb contaminant proteins. Purity was estimated as 99% or greater, based on amino-terminal amino acid analysis. The purified enzyme had a specific activity of 278 units/ mg protein, showed one major band after silver staining, and had an estimated molecular weight of 80,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was a glycoprotein and contained 2 mol of glucosamine per mole of trehalase. Kidney trehalase was inhibited by Tris, HgCl2, and phlorizin withKivalues of 3.8 mm, 11 μm, and 2.4 mm, respectively. Inclusion of Cl−in the reaction mixture protected the enzyme from inactivation by HgCl2. The apparentKmfor trehalose was calculated to be 2.1 mm. Kidney trehalase was highly specific for trehalose and exhibited an optimal pH of 5.9. The isoelectric point was between pH 4.7 and 4.4, as measured by chromatufocusing.