Cook J C, Chock P B
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3454-7. doi: 10.1073/pnas.92.8.3454.
Ubiquitin-activating enzyme, E1, is the first enzyme in the pathway leading to formation of ubiquitin-protein conjugates. E1 exists as two isoforms in human cells which are separable by electrophoresis. These isoforms migrate with apparent molecular sizes of 110 kDa and 117 kDa in SDS/polyacrylamide gels. Immunoprecipitation of E1 from lysates of HeLa cells metabolically labeled with [32P]phosphate indicated the presence of a phosphorylated form of E1 which migrates at 117 kDa. Phospho amino acid analysis identified serine as the phosphorylated residue in E1. Phosphorylated E1 was also detected in normal and transformed cells from another human cell line. Phosphatase-catalyzed dephosphorylation of E1 in vitro did not eliminate the 117-kDa E1 isoform detected by Coomassie staining after SDS/polyacrylamide gel electrophoresis, thereby demonstrating that phosphorylation is not the sole structural feature differentiating the isoforms of E1. These observations suggest new hypotheses concerning mechanisms of metabolic regulation of the ubiquitin conjugation pathway.
泛素激活酶E1是通向泛素-蛋白质缀合物形成途径中的第一种酶。E1在人类细胞中以两种同工型存在，可通过电泳分离。这些同工型在SDS/聚丙烯酰胺凝胶中的表观分子大小分别为110 kDa和117 kDa。从用[32P]磷酸盐进行代谢标记的HeLa细胞裂解物中免疫沉淀E1，表明存在一种迁移率为117 kDa的磷酸化形式的E1。磷酸氨基酸分析确定丝氨酸是E1中的磷酸化残基。在来自另一种人类细胞系的正常细胞和转化细胞中也检测到了磷酸化的E1。体外磷酸酶催化的E1去磷酸化并没有消除SDS/聚丙烯酰胺凝胶电泳后考马斯亮蓝染色检测到的117-kDa E1同工型，从而证明磷酸化不是区分E1同工型的唯一结构特征。这些观察结果提出了关于泛素缀合途径代谢调节机制的新假设。
