Assay of deoxyhypusine synthase activity.
Assay of deoxyhypusine synthase activity.
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DOI：
10.1007/978-1-61779-034-8_12
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Park, Myung Hee
中科院分区：
文献类型：
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作者：
Wolff, Edith C;Lee, Seung Bum;Park, Myung Hee
脱氧羟腐胺赖氨酸合酶催化一种不寻常的蛋白质修饰反应。将一部分亚精胺共价连接到一种真核蛋白eIF 5A（真核起始因子5A）的一个特定赖氨酸残基上，形成脱氧羟腐胺赖氨酸残基。该试验测量[1，8 - 3 H]亚精胺掺入eIF 5A蛋白的放射性。该酶对eIF 5A前体蛋白具有特异性，对短肽（<50个氨基酸）不起作用。本章描述了反应的最佳条件和产物含脱氧羟腐胺赖氨酸的eIF 5A的四种检测方法。第一种，也是最具体的方法是在通过离子交换色谱法分离蛋白质水解产物后，测量蛋白质水解产物中[3 H]脱氧羟腐胺赖氨酸的量。然而，这种方法需要一些专门的设备。第二种方法是彻底洗涤后计数TCA沉淀蛋白中的放射性。第三种方法涉及通过SDS-PAGE分离后测定含[3 H]脱氧羟腐胺赖氨酸的eIF 5A条带中的放射性。第四种方法是滤膜结合试验。重要的是尽量减少[3 H]亚精胺与测定混合物中蛋白质的非特异性结合，特别是对于方法2和4，如本章中的比较图所示。
Deoxyhypusine synthase catalyzes an unusual protein modification reaction. A portion of spermidine is covalently added to one specific lysine residue of one eukaryotic protein, eIF5A (eukaryotic initiation factor 5A) to form a deoxyhypusine residue. The assay measures the incorporation of radioactivity from [1,8-3H]spermidine into the eIF5A protein. The enzyme is specific for the eIF5A precursor protein and does not work on short peptides (<50 amino acids). Optimum conditions for the reaction and four detection methods for the product, deoxyhypusine-containing eIF5A, are described in this chapter. The first, and most specific, method is the measurement of the amount of [3H]deoxyhypusine in the protein hydrolysate after its separation by ion exchange chromatography. However, this method requires some specialized equipment. The second method is counting the radioactivity in TCA-precipitated protein after thorough washing. The third method involves determining the radioactivity in the band of [3H] deoxyhypusine-containing eIF5A after separation by SDS-PAGE. The fourth method is a filter-binding assay. It is important to minimize nonspecific binding of [3H]spermidine to proteins in the assay mixture, especially for methods 2 and 4, as illustrated in a comparison figure in the chapter.