Joint Study on Post-Translational Protein Tyrosine Sulfation

蛋白质的硫酸化是通过硫酸盐的共价附着进行的翻译后修饰。在已经提出的许多可能的功能作用中，酪氨酸硫酸化参与细胞内蛋白质的分选和运输得到了我们最近在牛肝脏中发现的高尔基定位175 k Da的膜结合酪氨酸- o -硫酸结合蛋白的有力支持。本文描述了酪氨酸- o -硫酸盐（TyrS）受体的结合特性。采用TyrS- affi -gel亲和层析、羟基磷灰石层析和电洗脱相结合的方法从牛肝脏中高度纯化了TyrS受体。在还原条件下，经SDS-PAGE测定，纯化后的受体表观分子量为175k Da。我们以P3-X63-Ag8-Ul细胞作为融合伙伴细胞，建立了对纯化受体反应的小鼠单克隆抗体。获得了两个分泌抗TyrS受体单克隆抗体的稳定杂交瘤克隆（r1 -1, R2-4）。我们分析了含TyrS受体的膜结合蛋白在溶解后与TyrS的结合。在悬浮液中加入胰蛋白酶，并对混合物进行消化。为了检测配体的结合特异性，将反应混合物与TyrS-Affi-Gel 10孵育。然后进行SDS-PAGE分析。很明显，65k、63k和59k的三个片段具有与TyrS结合的能力。在SDS-PAGE上，TyrS受体以双蛋白带迁移，表观分子量约为175K。为了获得有关这些蛋白和三个片段的结构形式的信息，我们研究了V8肽图。这些TyrS受体的部分V8蛋白酶定位相似，显示同源性。TyrS是一种具有n型碳水化合物链的糖蛋白。因此，TyrS受体通过糖苷酶对其碳水化合物链进行了不同程度的修饰，表明其反应性发生了变化或失去了与TyrS结合的能力。结果表明，TyrS受体是一种分子量为175k的糖蛋白。该蛋白的N连接碳水化合物残基在识别tyr中非常重要，并且在受体活性中具有功能贡献。少

The sulfation of protein is a post-translational modification by covalent attachment of sulfate. Among a number of possible funcitonal roles that have been proposed, the involvement of tyrosine sulfation in intracellular protein sorting and transport received strong support from our recent finding of Golgi located 175 k Da membrane-bound tyrosine-O-sulfate binding protein from bovine liver.Here we described the bindingproperties fo the tyrosine-O-sulfate (TyrS) receptor.TyrS receptor was highly purified from bovine liver using a combination of TyrS-Affi-gel 10 affinity chromatography, hydroxylapatite chromatography and electroelution. The purified receptor exhibited an apparent molecular weight of 175k Da as determined by SDS-PAGE under reducing conditions. We established mouse monoclonal antibodies reactive to the purified receptor using P3-X63-Ag8-Ul cells as fusion partner cells. Two stable hybridoma clone secreting anti TyrS receptor monoclonal antibodies were obtained (R3-1, R2-4) … More .We analyzed for the binding against TyrS,after solubilization the membrane-bound protein containing TyrS receptor. Trypsin was added to the suspension and the mixture was digested. To examine the ligand binding specificity, the reaction mixtures were incubated with TyrS-Affi-Gel 10.Then we analyzed by SDS-PAGE.It was apparent that three fragments of 65k, 63k and 59k Da have the ability to bind against TyrS.TyrS receptor migrated as double protein bands with apparent molecular weights of ca. 175K upon SDS-PAGE.In order to obtain information concerning the sturctural form of these proteins and three fragments we investigated V8 peptide maps. Partial V8 protease mapping of these TyrS receptor were similar showing the homology. TyrS is a glycoprotein which has N-liked carbohydrate chain. So, TyrS receptor variously trimmed their carbohydrate chains by glycosidase showing the change of reactivity or loss of the ability to bind against TyrS.It is concluded that TyrS receptor is a glycoprotein which is having a molecular weight of 175 K.The N linked carbohydrate residue of this protein is very important in recognition of the TyrS and has a functional contribution in receptor activity. Less