Complex formation between deoxyhypusine synthase and its protein substrate, the eukaryotic translation initiation factor 5A (eIF5A) precursor.
Complex formation between deoxyhypusine synthase and its protein substrate, the eukaryotic translation initiation factor 5A (eIF5A) precursor.
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DOI：
10.1042/bj3400273
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发表时间：
1999-05
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影响因子：
--
通讯作者：
Y. B. Lee;Y. Joe;E. Wolff;E. Dimitriadis;M. Park
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文献类型：
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作者：
Y. B. Lee;Y. Joe;E. Wolff;E. Dimitriadis;M. Park
脱氧马尿苷合成酶催化单个细胞蛋白（真核起始因子 5A (eIF5A) 的前体）中马尿苷 [Nepsilon-(4-氨基-2-羟丁基) 赖氨酸] 翻译后合成的第一步。脱氧马尿苷合酶以四聚体形式存在，具有四个潜在的活性位点。通过使用多组氨酸标记 (His.Tag) ec-eIF5A 的亲和层析、凝胶迁移率变换方法和分析超速离心，检查人脱氧马尿苷合成酶与其蛋白底物、人重组 eIF5A 前体 (ec-eIF5A) 之间稳定复合物的形成。脱氧马尿苷合酶被固定在树脂上的 His.Tag-ec-eIF5A 选择性保留。在非变性条件下通过电泳将脱氧马尿苷合酶和ec-eIF5A的复合物与游离酶和蛋白质底物分离。通过复合物的 N 端氨基酸测序，复合物中两种组分的化学计量估计为 1 个脱氧马尿苷合酶四聚体与 1 个 ec-eIF5A 单体。平衡超速离心数据进一步支持了这一 1:1 比例，并表明该酶与 ec-eIF5A (Kd</=0.5 nM) 存在非常强的相互作用。复合物的形成不依赖于 NAD+ 或亚精胺，并且发生在 pH7.0-9.2 范围内。在 pH7.0-9.2 且含有 1 mM 亚精胺的完全反应混合物中，还检测到了酶-产物复合物以及含脱氧马尿苷的产物（修饰的 ec-eIF5A）。
Deoxyhypusine synthase catalyses the first step in the post-translational synthesis of hypusine [Nepsilon-(4-amino-2-hydroxybutyl) lysine] in a single cellular protein, the precursor of eukaryotic initiation factor 5A (eIF5A). Deoxyhypusine synthase exists as a tetramer with four potential active sites. The formation of a stable complex between human deoxyhypusine synthase and its protein substrate, human recombinant eIF5A precursor (ec-eIF5A), was examined by affinity chromatography using polyhistidine-tagged (His.Tag) ec-eIF5A, by a gel mobility-shift method, and by analytical ultracentrifugation. Deoxyhypusine synthase was selectively retained by His.Tag-ec-eIF5A immobilized on a resin. The complex of deoxyhypusine synthase and ec-eIF5A was separated from the free enzyme and protein substrate by electrophoresis under non-denaturing conditions. The stoichiometry of the two components in the complex was estimated to be 1 deoxyhypusine synthase tetramer to 1 ec-eIF5A monomer by N-terminal amino acid sequencing of the complex. Equilibrium ultracentrifugation data further supported this 1:1 ratio and indicated a very strong interaction of the enzyme with ec-eIF5A (Kd</=0.5 nM). Formation of the complex was not dependent on NAD+ or spermidine and occurred at pH7.0-9.2. An enzyme-product complex, as well as the deoxyhypusine-containing product (modified ec-eIF5A), was also detected at pH7.0-9.2 in a complete reaction mixture containing 1 mM spermidine.