論文 - 詳細
| RRC ID | 6546 |
|---|---|
| 著者 | Ikeda K, Yukihiro Hiraoka B, Iwai H, Matsumoto T, Mineki R, Taka H, Takamori K, Ogawa H, Yamakura F. |
| タイトル | Detection of 6-nitrotryptophan in proteins by Western blot analysis and its application for peroxynitrite-treated PC12 cells. |
| ジャーナル | Nitric Oxide |
| Abstract |
We have previously reported on the formation of 6-nitrotryptophan by the reaction of reactive nitrogen species with a tryptophan residue in human Cu, Zn-superoxide dismutase (SOD) (F. Yamakura et al., J. Biochem. 138 (2005) 57-69). Here, we report on the preparation of anti-6-nitrotryptophan antiserum by using synthesized 6-nitrotryptophan-conjugated keyhole limpet hemocyanin as an antigen and the purification of the antibody by using a 6-nitrotryptophan-conjugated affinity column. The purified antibody was immunoreactive with 6-nitrotryptophan residue containing Cu, Zn-SOD but not immunoreactive with Cu, Zn-SOD, Mn-SOD, bovine serum albumin, and 3-nitrotyrosine residue containing Mn-SOD. Nitro group of 6-nitrotryptophan was reduced by sodium hydrosulfite to form 6-aminotryptophan as a major product. The reduced 6-nitrotryptophan residues lost its immunoreactivity with the antibody. We detected different immunoreactive bands between using antibody for 6-nitrotryptophan residues and that for 3-nitrotyrosine residues in crude extracts of neuron-like PC12 cells treated with peroxynitrite by a Western blot analysis. Western blot analysis for two-dimensional gel electrophoresis showed nine intensively stained immunoreactive spots for 6-nitrotryptophan residues in the peroxynitrite-treated PC12 cells, which were subjected to trypsin digestion and LC-ESI-MS/MS analysis. We identified M2 pyruvate kinase, elongation factor 2, mitochondrial aconitase, pyruvate carboxylase, and heat shock protein HSP90alpha as candidates for 6-nitrotryptophan residues containing proteins, with peptide coverage over 10%, in crude extracts of peroxynitrite-treated PC12 cells. |
| 巻・号 | 16(1) |
| ページ | 18-28 |
| 公開日 | 2007-2-1 |
| DOI | 10.1016/j.niox.2006.04.263 |
| PII | S1089-8603(06)00059-0 |
| PMID | 16765071 |
| MeSH | Animals Antibodies / analysis Antibody Formation Antibody Specificity Blotting, Western Chromatography, Liquid Electrophoresis, Polyacrylamide Gel Mass Spectrometry PC12 Cells Peroxynitrous Acid / pharmacology* Proteins / chemistry Proteins / metabolism* Rats Tryptophan / analogs & derivatives* Tryptophan / analysis Tryptophan / immunology Tryptophan / metabolism |
| IF | 3.311 |
| 引用数 | 31 |
| WOS 分野 | BIOCHEMISTRY & MOLECULAR BIOLOGY CELL BIOLOGY |
| オルトメトリクス指標 |
オルトメトリクス指標項目
|
| 最多言及媒体 | Patent(IFI CLAIMS) |
| 各媒体での言及数の合計 | 1 |
| 過去6か月間でのオルトメトリクス指標の変動値 | 0.0 |
| リソース情報 | |
| ヒト・動物細胞 | |