Purification of SUMO conjugating enzymes and kinetic analysis of substrate conjugation.
Purification of SUMO conjugating enzymes and kinetic analysis of substrate conjugation.
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DOI：
10.1007/978-1-59745-566-4_11
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发表时间：
2009
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影响因子：
--
通讯作者：
Lima, Christopher D
中科院分区：
文献类型：
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作者：
Yunus, Ali A;Lima, Christopher D
SUMO与蛋白质底物的缀合需要专用的E2泛素缀合酶（Ubc 9）和相关的E3连接酶的协同作用。虽然Ubc9可以直接识别和修饰SUMO修饰共有位点内的底物赖氨酸残基，但E3连接酶可以在体外和体内SUMO缀合期间重定向特异性并提高缀合速率。在本章中，我们将描述用于纯化SUMO结合酶的方法和可用于体外分析SUMO结合的模型底物。我们还将描述在E3依赖性或E3非依赖性底物缀合期间提取动力学参数的方法。
SUMO conjugation to protein substrates requires the concerted action of a dedicated E2 ubiquitin conjugation enzyme (Ubc9) and associated E3 ligases. Although Ubc9 can directly recognize and modify substrate lysine residues that occur within a consensus site for SUMO modification, E3 ligases can redirect specificity and enhance conjugation rates during SUMO conjugation in vitro and in vivo. In this chapter, we will describe methods utilized to purify SUMO conjugating enzymes and model substrates which can be used for analysis of SUMO conjugation in vitro. We will also describe methods to extract kinetic parameters during E3-dependent or E3-independent substrate conjugation.