Parnaik V K, Luzio G A, Grahame D A, Ditson S L, Mayer R M
Carbohydr Res. 1983 Sep 16;121:257-68. doi: 10.1016/0008-6215(83)84022-1.
Dextransucrase was treated with [14C]sucrose, and the product applied to gel-permeation columns. In the absence of the detergents SDS and Triton X-100, poor recovery of enzyme was observed; however, that enzyme which was recovered was labeled. In the presence of detergents, recovery was increased, but the material appeared to be a large aggregate (mol. wt. greater than 5 X 10(6) ). In addition, the ratio of D-glucose to enzyme suggested that a polymer had been formed. Disc-gel electrophoresis in the presence of a mixture of SDS and Triton X-100 showed similar results, and indicated that the aggregate was disrupted upon treatment with dextranase. Native enzyme that had been immobilized on hydroxylapatite could also be labeled with [14C]sucrose, and the labeling followed saturation kinetics. The labeled protein could be released from the gel with 8M urea, but was aggregated. Radioactive sugars, free from protein, could be released by heating the labeled enzyme. The sugars released consisted of a mixture of D-glucose with oligosaccharides having an average chain-length of 17 D-glucosyl residues. The significance of these observations is discussed.
用[¹⁴C]蔗糖处理葡聚糖蔗糖酶，并将产物应用于凝胶渗透柱。在没有去污剂十二烷基硫酸钠（SDS）和聚山梨醇酯80（Triton X - 100）的情况下，观察到酶的回收率很低；然而，回收的酶被标记了。在有去污剂的情况下，回收率提高了，但该物质似乎是一个大聚集体（分子量大于5×10⁶）。此外，D - 葡萄糖与酶的比例表明形成了一种聚合物。在SDS和Triton X - 100混合物存在下的圆盘凝胶电泳显示了类似的结果，并表明用葡聚糖酶处理后聚集体被破坏。固定在羟基磷灰石上的天然酶也可以用[¹⁴C]蔗糖标记，并且标记遵循饱和动力学。标记的蛋白质可以用8M尿素从凝胶中释放出来，但会聚集。不含蛋白质的放射性糖可以通过加热标记的酶释放出来。释放的糖由D - 葡萄糖与平均链长为17个D - 葡糖基残基的寡糖的混合物组成。讨论了这些观察结果的意义。
