| Project/Area Number |
12470483
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Chemical pharmacy
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| Research Institution | Kumamoto University |
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Principal Investigator |
NAKAYAMA Hitoshi Kumamoto University, Graduate School of Medical and Pharmaceutical Sciences, Professor, 大学院・医学薬学研究部, 教授 (70088863)
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| Co-Investigator(Kenkyū-buntansha) |
KAWAI Nobufumi Jichi Medical College, Dept. of Physiology, Professor Emertis, 名誉教授 (00073065)
KUNIYASU Akihiko Kumamoto University, Graduate School of Medical and Pharmaceutical Sciences, Associate Professor, 大学院・医学薬学研究部, 助教授 (90241348)
MASUDA Katsuyoshi Suntory Co., Research Institute for Bioorganic Chemistry, Research Fellow, 研究員 (30190359)
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥13,500,000 (Direct Cost: ¥13,500,000)
Fiscal Year 2002: ¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2001: ¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2000: ¥6,300,000 (Direct Cost: ¥6,300,000)
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| Keywords | photoaffinity labeling / anti-peptide antibodies / TOF-MS / ESI-MS / MS / photolabeled peptide fragments / identifying binding sites / TDF-MS / ラベル化ペプチド解析 / 神経特異的ハチ青 |
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| Research Abstract |
In this project we aim to develop a method for rapid identification of the target molecules and ligand binding sites with high resolution in the recourse of immunoaffinity purification by anti-ligand antibodies, followed by moderen mass spectrometry. We planed to develop the method for identifying the binding site(s) of glutathione conjugate in cMOAT, an ATP-driven drug trasporter. During the passed 3 years, we obtained the results as follows. (1) We raised a antibody against hapten having analogous structure to photolabeling reagent of TOY-SG, a glutathione derivative. The antibody has a high titer to able to recognize hapten at sub-picomole level. (2) cMOAT protein that was photolabeled by TOY-SG was prepared and purified. We established the conditions of Lys-C digestion, which gave the photolabeled fragments of 3-4 kDa, proper size for MS nalysis. The photolabeled fragments were subjected to immunoaffinify purification using the anti-hapten antibody, but the yield was as low as 〜5%, which was not applicable for further MS analysis. (3) We fractionated the Lys-C fragments by HPLC as an alternative method and the fractions containing photolabeled fragments was analyzed by MALDI-TOF MS. We revealed two new photolabeled sites that located near nucleotide binding sites. The sites had not identified by any other methods such as site-directed mutagenesis. (4) Efforts to raise anti-hapten antibodies with much higher titer are still required.
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