High‐performance affinity chromatography method for identification of L‐arginine interacting factors using magnetic nanobeads

Abstract

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.

High‐performance affinity purification for identification of 15‐deoxy‐Δ12,14‐PGJ2 interacting factors using magnetic nanobeads

“…Affinity-purified PGJIFs were separated by SDS-PAGE and gels subjected to silver staining as previously described (Shevchenko et al, 1996;Hiramoto et al, 2010). The specific protein bands were excised, reduced with 10 mM DTT followed by alkylation with 55 mM iodoacetamide.…”

“…This enables the rapid and efficient purification of binding proteins for capsaicin and L-arginine (Kuramori et al, 2009;Hiramoto et al, 2010). In this study we apply this system for identification of PGJ2 target proteins.…”

Arginine-induced insulin secretion in endoplasmic reticulum

“…Insulin secretion was determined as previously described [2]. Briefly, after cultivation of NIT-1 cells, medium was replaced with Fe12 K medium without arginine for 10 min.…”

“…Main action of arginine for insulin secretion might not be the substrates of these enzymes. The mechanism of arginine-induced insulin secretion is not known yet [2]. The minor mechanism is most probably arginine binds to G Protein-Coupled Receptor, Class C, Group 6, Member A (GPRC6A) and activates PKA signaling to granule of b cells.…”

Prohibitins, novel vitamin K2 target factors in osteoblast

“…FG beads were prepared as previously described [13,18]. Epoxy groups on FG beads were aminolyzed by NH 4 OH and coupled to ethylene glycol diglycidyl ether (EGDE) to produce FGNEGDE beads.…”