High-performance affinity chromatography method for identification of L-arginine interacting factors using magnetic nanobeads
High-performance affinity chromatography method for identification of L-arginine interacting factors using magnetic nanobeads
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DOI：
10.1002/bmc.1334
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发表时间：
2010-06-01
影响因子：
1.8
通讯作者：
Imai, Takeshi
中科院分区：
文献类型：
--
作者：
Hiramoto, Masaki;Maekawa, Naoya;Imai, Takeshi
L-精氨酸通过一组复杂且高度调控的途径表现出广泛的生物活性，这些途径在全身和细胞水平上仍不完全清楚。本研究的目的是开发和验证 L-精氨酸相互作用因子 (AIF) 的有效纯化系统。我们最近开发了由磁铁矿颗粒/甲基丙烯酸缩水甘油酯（GMA）-苯乙烯共聚物/覆盖的GMA组成的新型磁性纳米珠（FG珠）。与市售磁珠相比，这些纳米珠在纯化效率方面表现出更高的性能。在这项研究中，我们通过将 L-AME 缀合到这些纳米珠的表面，新开发了 L-精氨酸甲酯 (L-AME) 固定珠。首先，我们展示了诱导型一氧化氮合酶，它结合并使用 L-精氨酸作为底物，特异性结合 L-AME 固定珠。其次，我们使用该亲和色谱系统从 HeLa 细胞粗提物中新鉴定出磷酸果糖激酶、RuvB-like 1 和 RuvB-like 2 作为 AIF。这里提供的数据表明，L-AME 固定珠是直接从粗细胞提取物中纯化 AIF 的有效工具。我们期望本方法可用于从各种类型的细胞中纯化 AIF。版权所有 (C) 2009 John Wiley & Sons, Ltd.
L-Arginine exhibits a wide range of biological activities through a complex and highly regulated set of pathways that remain incompletely understood at both the whole-body and the cellular levels. The aim of this study is to develop and validate effective purification system for L-arginine interacting factors (AIFs). We have recently developed novel magnetic nanobeads (FG beads) composed of magnetite particles/glycidyl methacrylate (GMA)-styrene copolymer/covered GMA. These nanobeads have shown higher performance compared with commercially available magnetic beads in terms of purification efficiency. In this study, we have newly developed L-arginine methyl ester (L-AME)-immobilized beads by conjugating L-AME to the surface of these nanobeads. Firstly, we showed that inducible nitric oxide synthase, which binds and uses L-arginine as a substrate, specifically bound to L-AME-immobilized beads. Secondly, we newly identified phosphofructokinase, RuvB-like 1 and RuvB-like 2 as AIFs from crude extracts of HeLa cells using this affinity chromatographic system. The data presented here demonstrate that L-AME-immobilized beads are effective tool for purification of AIFs directly from crude cell extracts. We expect that the present method can be used to purify AIFs from various types of cells. Copyright (C) 2009 John Wiley & Sons, Ltd.