App H, Holzer H
Biochemisches Institut, Universität Freiburg, Federal Republic of Germany.
J Biol Chem. 1989 Oct 15;264(29):17583-8.
Neutral trehalase was purified from stationary yeast ABYS1 mutant cells deficient in the vacuolar proteinases A and B and the carboxypeptidases Y and S. The purified electrophoretically homogeneous preparation of phosphorylated neutral trehalase exhibited a molecular mass of 160,000 Da on nondenaturing gel electrophoresis and of 80,000 Da on sodium dodecyl sulfate-gel electrophoresis. Maximal activity (114 mumol of trehalose min-1 x mg-1 at 37 degrees C) was observed at pH 6.8-7.0. The apparent Km for trehalose was 34.5 mM. Among seven oligosaccharides studied, the enzyme formed glucose only from trehalose. Neutral trehalase is located in the cytosol. A polyclonal rabbit antiserum raised against neutral trehalase precipitates the enzyme in the presence of protein A. The antiserum does not react with acid trehalase. Dephosphorylation by alkaline phosphatase from Escherichia coli of the active phosphorylated enzyme is accompanied by greater than or equal to 90% inactivation. Rephosphorylation by incubation with the catalytic subunit of beef heart protein kinase is accompanied by reactivation and incorporation of 0.85 mol of phosphate/mol subunit (80,000 Da). The phosphorylated amino acid residue was identified as phosphoserine.
从缺乏液泡蛋白酶A和B以及羧肽酶Y和S的静止酵母ABYS1突变细胞中纯化出中性海藻糖酶。纯化后的磷酸化中性海藻糖酶在非变性凝胶电泳上显示分子量为160,000 Da，在十二烷基硫酸钠凝胶电泳上显示分子量为80,000 Da。在pH 6.8 - 7.0时观察到最大活性（37℃下为114 μmol海藻糖·min⁻¹·mg⁻¹）。海藻糖的表观Km为34.5 mM。在所研究的七种寡糖中，该酶仅从海藻糖生成葡萄糖。中性海藻糖酶位于细胞质中。用针对中性海藻糖酶产生的兔多克隆抗血清在蛋白A存在下沉淀该酶。该抗血清不与酸性海藻糖酶反应。来自大肠杆菌的碱性磷酸酶对活性磷酸化酶进行去磷酸化伴随着≥90%的失活。与牛心蛋白激酶的催化亚基一起孵育进行再磷酸化伴随着再活化以及每摩尔亚基（80,000 Da）掺入0.85摩尔磷酸盐。磷酸化的氨基酸残基被鉴定为磷酸丝氨酸。
