Assay of deoxyhypusine hydroxylase activity.
Assay of deoxyhypusine hydroxylase activity.
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DOI：
10.1007/978-1-61779-034-8_13
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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：
Park, Jong Hwan;Wolff, Edith C;Park, Myung Hee
真核翻译起始因子5A （eIF5A）是唯一含有一种特殊氨基酸的细胞蛋白，即hypusine [Nε-（4-氨基-2-羟基丁基）赖氨酸]。eIF5A及其hypusine/脱氧hypusine修饰对真核细胞增殖至关重要。Hypusine是在翻译后由脱氧Hypusine合酶和脱氧Hypusine羟化酶催化的两个酶促步骤形成的。脱氧hypusine羟化酶催化eIF5A中间蛋白eIF5A（Dhp）中脱氧hypusine残基的立体特异性羟化。该酶对该蛋白完全特异，不作用于短肽（<50个氨基酸）。该分析测量了放射性标记的脱氧碱基到eIF5A中碱基的转化。本章描述了反应的最佳条件和两种检测方法，该产品含有hypusine eIF5A。第一种也是最可靠的方法是用离子交换色谱法测定从[3H]脱氧hypusine中分离出来的蛋白水解液中[3H]hypusine的量。这种方法需要专门的设备。第二种方法是基于计算反应混合物经高碘酸钠氧化后的总TCA可溶性放射性，因为高碘酸氧化后，放射性标记的4-氨基-2-羟基丁基部分被切割并从蛋白质中释放为放射性标记的β-丙醛和甲醛。
The eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains an unusual amino acid, hypusine [Nε-(4-amino-2-hydroxybutyl)-lysine]. eIF5A and its hypusine/deoxyhypusine modification are vital for eukaryotic cell proliferation. Hypusine is formed posttranslationally by two enzymatic steps catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase. Deoxyhypusine hydroxylase catalyzes a stereo-specific hydroxylation of the deoxyhypusine residue in the eIF5A intermediate protein, eIF5A(Dhp). The enzyme is totally specific for this protein and does not act on short peptides (<50 amino acids). The assay measures the conversion of the radiolabeled deoxyhypusine residue to a hypusine residue in eIF5A. Optimum conditions for the reaction and two detection methods for the product, hypusine-containing eIF5A, are described in this chapter. The first, and most reliable, method is the measurement of the amount of [3H]hypusine in the protein hydrolysate after its separation from [3H]deoxyhypusine, by ion exchange chromatography. This method does require specialized equipment. The second method is based on counting the total TCA soluble radioactivity after sodium periodate oxidation of the reaction mixture, since the radiolabeled 4-amino-2-hydroxy butyl moiety of the hypusine residue is cleaved and is released from protein as radiolabeled β-propionaldehyde and formaldehyde by periodate oxidation.