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生細胞における蛋白質分解過程の可視化解析

Research Project

Project/Area Number 10878130
Research Category

Grant-in-Aid for Exploratory Research

Allocation TypeSingle-year Grants
Research Field Cell biology
Research InstitutionTokyo University of Pharmacy and Life Science

Principal Investigator

高橋 健治  東京薬科大学, 生命科学部, 教授 (70011533)

Co-Investigator(Kenkyū-buntansha) 西井 亘  東京薬科大学, 生命科学部, 助手 (30287461)
工藤 佳久  東京薬科大学, 生命科学部, 教授 (20080179)
中村 健  東京薬科大学, 生命科学部, 助手 (50227906)
Project Period (FY) 1998 – 2000
Project Status Completed (Fiscal Year 2000)
Budget Amount *help
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 2000: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1999: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1998: ¥800,000 (Direct Cost: ¥800,000)
Keywords細胞内プロテオリシス / 可視化解析 / 二重蛍光基質 / FRET / 海馬スライス培養 / カスパーゼ1 / 細胞死 / グルタミン酸刺激 / 蛍光基質 / アクリロダン標識基質 / 細胞内注入 / コラゲナーゼ / カスパーゼ / 二重蛍光標識基質 / 細胞内プロテアーゼ / FRET(蛍光共鳴エネルギー転移) / CAAXモチーフ特異的プロテアーゼ
Research Abstract

細胞内プロテアーゼの一つであるカスパーゼ1は細胞死におけるシグナル伝達で重要な役割を担うとされ、また基質特異性や活性発現の時期等が厳密に制御されている.本研究ではこのプロテアーゼをモデル酵素に送び.細胞内プロテオリシスの可現化解析を試みた.まず.可視化用蛍光基質をデザイン,調製した.すなわち、ペプチドYVADAPCのN末端アミノ基にテトラメチルローダミン,C末端システインのSH基にアクリロダンを導入した二重蛍光基質を合成し.DA間の切断により蛍光スペクトルの変化と増大が起こることを確認し.これがFRET(蛍光共鳴エネルギー移動)によるものであることを示した.次いで,本基質をグルタミン酸で刺激したラット脳・海馬スライス培養を用いたカスパーゼ1活性の可視化に有効であることを示した。すなわち、ラット海馬スライス培養に10mMグルタミン酸を1時間添加することで細胞死を誘導し,測定1時間前に本蛍光基質を加え.経時的に蛍光を観察したところ.CA1およびCA3領域において活性が経時的に上昇し、5〜7.5時間で最大になることが確認された,この結果は,海馬の細胞死においてカスパーゼ1が何らかの関与をしていることを示唆するものである.また,本方法はマイクロインジェクション等の特別な手法を用いなくても蛍光基質が試料表面に添加するだけで細胞内に導入されるものであり、細胞抽出物では測定できないレベルの微少の活性を検知できる事,また細胞組織を破壊せずに比較的インタクトな状態で細胞内プロテアーゼ活性を検出できるという点で優れており、応用範囲が広いものと考えられる.

Report

(3 results)
  • 2000 Annual Research Report
  • 1999 Annual Research Report
  • 1998 Annual Research Report
  • Research Products

    (13 results)

All Other

All Publications (13 results)

  • [Publications] Tomofumi Fujino: "Indentification of the Cleavage Sites of Oxidized Protein That Are Susceptible to Oxidized Protein Hydrolase (OPH) in the Primary and Tertiary Structures of the Pretein"Journal of Biochemistry. 127. 1087-1093 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Xiang-Ping Huang: "Identification of a Glutamic Acid and an Aspartic Acid Residue Essential for Catalytic Activity of Aspergillopepsin II,a Non-pepsin Type Acid Proteinase"Journal of Biological Chemistry. 275. 26607-26614 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Atsushi Kimura: "Identification of Porcine Follipsin as Plasma Kallikrein,and Its Possible Involvement in the Production of Bradykinin within the Follicles of Porcine Ovaries"Molecular Reproduction and Development. 57. 79-87 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Hideshi Inoue: "Effects of Cys 10 mutation to Ala in glutathione transferase from Escherichia coli"Journal of Organometallic Chemistry. 611. 593-595 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Masaki Kojima: "Structure-Function Relationship of Ribonuclease T1 Based on Molecular Structure"Bioimages. 8. 45-55 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Gwang-Ho Jeohn: "Purification and characterization of a detergent-requiring membrane-bound metalloendopeptidase from porene brain"European Journal of Biochemistry. 260. 318-324 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Masashi Matsushima: "Purification and further characterization of enteropeptidase from porcine duodenum"Journal of Biochemistry. 125. 947-951 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Satoru Watanabe: "Molecular cloning of the Lon gene from Thermus thermophilus HB8 and characterization of its gene product"European Journal of Biochemistry. 266. 811-819 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Masaki Kojima: "ph-Dependent unfolding of aspergillopepsin II studied by small-angle X-ray scattering"Biochemistry. 39. 1364-1372 (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] Xiang-Ping Huang: "Identification of a glutamicacid and an asparfic acid residue essential for catalytic activity of aspergllopepsin II ,a non-pepsin type acid proteinase"Journal of Biological Chemistry. (in press). (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] Satoshi Yonezawa: "Cathepsin E Gene in Mouse" Biomedical Research. 19. 327-334 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Gwang-Ho Jeohn: "Purification and Characterization of a Detergent-requiring Membrane-bound Metalloendo peptidase from Porcine Brain" European Journal of Biochemistry. 259. 1-8 (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] Masashi Matsushima: "Purification and Further Characterization of Enteropeptidase from Porcine Duodenum" Journal of Biochemistry. 125 (in press). (1999)

    • Related Report
      1998 Annual Research Report

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Published: 1998-04-01   Modified: 2016-04-21  

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