Liu M C, Suiko M
Arch Biochem Biophys. 1987 May 15;255(1):162-7. doi: 10.1016/0003-9861(87)90306-7.
Tryptic fragments of [35S]sulfate-labeled 3Y1 secreted fibronectin were fractionated by hydroxylapatite column chromatography and examined using sodium dodecyl sulfate gel electrophoresis, followed by autoradiography. Radioactive bands containing tyrosine-O-[35S]sulfate were detected at 17- and 40-kDa positions under reducing conditions. Under nonreducing conditions, the 17-kDa band was no longer present and new bands at 57- and 80-kDa positions appeared, indicating a disulfide linkage between the two smaller fragments in the native state. These fragments exhibited binding affinity toward fibrin and could be immunoprecipitated by the monoclonal antifibronectin Fib-2 domain antibody. These results suggested that the tyrosine sulfation site in 3Y1 secreted fibronectin is located in the C-terminal fibrin-binding (Fib-2) domain, being within 17 kDa of the C-terminus.
对经[35S]硫酸盐标记的3Y1分泌型纤连蛋白的胰蛋白酶片段进行羟基磷灰石柱层析分离，然后用十二烷基硫酸钠凝胶电泳检测，接着进行放射自显影。在还原条件下，在17 kDa和40 kDa位置检测到含有酪氨酸-O-[35S]硫酸盐的放射性条带。在非还原条件下，17 kDa条带不再出现，在57 kDa和80 kDa位置出现新条带，表明在天然状态下两个较小片段之间存在二硫键连接。这些片段对纤维蛋白表现出结合亲和力，并且可以被抗纤连蛋白Fib-2结构域单克隆抗体免疫沉淀。这些结果表明，3Y1分泌型纤连蛋白中的酪氨酸硫酸化位点位于C末端纤维蛋白结合（Fib-2）结构域，在C末端的17 kDa范围内。
