Liu M C, Yu S, Sy J, Redman C M, Lipmann F
Proc Natl Acad Sci U S A. 1985 Nov;82(21):7160-4. doi: 10.1073/pnas.82.21.7160.
[35S]Sulfate labeling of the human hepatoma cell line HepG2 showed it to contain many sulfated proteins of diverse molecular weight range. The isolation of tyrosine O-sulfate indicated the supernatant fraction to contain a 5- to 7-fold higher level than the cellular fraction at the end of a 24-hr incubation. The proteins in the supernatant fraction were immunoprecipitated and examined for sulfation. Of 15 proteins tested, 7 were found to be sulfated as indicated by [35S]sulfate incorporation into proteins separated by NaDodSO4/PAGE and detected by autoradiography. The 35S-labeled bands were excised from the dried gel and subjected to extensive Pronase hydrolysis and the hydrolysates were analyzed for tyrosine [35S]sulfate by a two-dimensional procedure combining high-voltage electrophoresis and thin-layer chromatography [Liu, M. C. & Lipmann, F. (1984) Proc. Natl. Acad. Sci. USA 81, 3695-3698]. Of the sulfated proteins, three--fibrinogen, alpha-fetoprotein, and fibronectin--were found to contain tyrosine O-sulfate. The simultaneous presence of carbohydrate-bound sulfate, however, could not be exactly determined, but the other four [35S]sulfate-containing proteins--alpha 1-antitrypsin, alpha 1-antichymotrypsin, alpha 2-macroglobulin, and transferrin--did not reveal any tyrosine O-sulfate and might be sulfated on their carbohydrate moieties.
对人肝癌细胞系HepG2进行[35S]硫酸盐标记，结果显示其含有许多分子量范围各异的硫酸化蛋白。酪氨酸O - 硫酸盐的分离表明，在24小时孵育结束时，上清液部分所含水平比细胞部分高5至7倍。对上清液部分的蛋白质进行免疫沉淀并检测其硫酸化情况。在检测的15种蛋白质中，通过[35S]硫酸盐掺入经十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（NaDodSO4/PAGE）分离并经放射自显影检测的蛋白质中，发现有7种被硫酸化。从干燥的凝胶中切下35S标记的条带，用链霉蛋白酶进行广泛水解，然后通过结合高压电泳和薄层色谱的二维方法分析水解产物中的酪氨酸[35S]硫酸盐[刘，M.C. & 利普曼，F.（1984年）美国国家科学院院刊81，3695 - 3698]。在硫酸化蛋白质中，发现纤维蛋白原、甲胎蛋白和纤连蛋白这三种含有酪氨酸O - 硫酸盐。然而，无法确切确定是否同时存在与碳水化合物结合的硫酸盐，但其他四种含[35S]硫酸盐的蛋白质——α1 - 抗胰蛋白酶、α1 - 抗糜蛋白酶、α2 - 巨球蛋白和转铁蛋白——未显示任何酪氨酸O - 硫酸盐，可能是其碳水化合物部分被硫酸化。
