Premium
High‐performance affinity chromatography method for identification of L‐arginine interacting factors using magnetic nanobeads
Author(s) -
Hiramoto Masaki,
Maekawa Naoya,
Kuge Takeshi,
Ayabe Fumiaki,
Watanabe Atsushi,
Masaike Yuka,
Hatakeyama Mamoru,
Handa Hiroshi,
Imai Takeshi
Publication year - 2010
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.1334
Subject(s) - chemistry , chromatography , arginine , glycidyl methacrylate , hela , monomer , biochemistry , cell , amino acid , polymer , organic chemistry
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 © 2009 John Wiley & Sons, Ltd.