Martin Sarah F, Hattersley Neil, Samuel Ifor D W, Hay Ronald T, Tatham Michael H
Biophotonics Collaboration, School of Physics and Astronomy, University of St. Andrews, North Haugh, St. Andrews KY16 9SS, Scotland, UK.
Anal Biochem. 2007 Apr 1;363(1):83-90. doi: 10.1016/j.ab.2006.12.018. Epub 2007 Jan 4.
Dynamic modification of proteins with the small ubiquitin-like modifier (SUMO) affects the stability, cellular localization, enzymatic activity, and molecular interactions of a wide spectrum of protein targets. We have developed an in vitro fluorescence-resonance-energy-transfer-based assay that uses bacterially expressed substrates for the rapid and quantitative analysis of SUMO paralog-specific C-terminal hydrolase activity. This assay has applications in SUMO protease characterization, enzyme kinetic analysis, determination of SUMO protease activity in eukaryotic cell extracts, and high-throughput inhibitor screening. In addition, while demonstrating such uses, we show that the SUMO-1 processing activity in crude HeLa cell extracts is far greater than that of SUMO-2, implying that differential maturation rates of SUMO paralogs in vivo may be functionally significant. The high degree of structural conservation across the ubiquitin-like protein superfamily suggests that the general principle of this assay should be applicable to other post-translational protein modification systems.
用小泛素样修饰物（SUMO）对蛋白质进行动态修饰，会影响多种蛋白质靶点的稳定性、细胞定位、酶活性及分子相互作用。我们开发了一种基于体外荧光共振能量转移的检测方法，该方法使用细菌表达的底物，用于快速定量分析SUMO旁系同源物特异性C末端水解酶活性。此检测方法可应用于SUMO蛋白酶特性鉴定、酶动力学分析、真核细胞提取物中SUMO蛋白酶活性的测定以及高通量抑制剂筛选。此外，在展示这些用途的同时，我们发现HeLa细胞粗提物中的SUMO-1加工活性远高于SUMO-2，这意味着SUMO旁系同源物在体内的不同成熟速率可能具有功能意义。泛素样蛋白超家族中高度的结构保守性表明，该检测方法的一般原理应适用于其他蛋白质翻译后修饰系统。
