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Development of Synthetic Fluorogenic Peptide Substrates for Blood Clotting Proteases

Research Project

Project/Area Number 63870017
Research Category

Grant-in-Aid for Developmental Scientific Research

Allocation TypeSingle-year Grants
Research Field Pathological medical chemistry
Research InstitutionKyushu University

Principal Investigator

IWANAGA Sadaaki  Kyushu Univ., Fac. of Sci., Professor, 理学部, 教授 (90029942)

Co-Investigator(Kenkyū-buntansha) KIMURA Terutoshi  Peptide Institute, Inc., Chief, 研究部長 (30072771)
MIYATA Toshiyuki  Kyushu Univ., Fac. of Sci., Assistant, 理学部, 助手 (90183970)
榊原 俊平  ペプチド研究所, 所長 (50072765)
川畑 俊一郎  九州大学, 理学部, 助手 (90183037)
Project Period (FY) 1988 – 1989
Project Status Completed (Fiscal Year 1989)
Budget Amount *help
¥9,600,000 (Direct Cost: ¥9,600,000)
Fiscal Year 1989: ¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1988: ¥6,500,000 (Direct Cost: ¥6,500,000)
KeywordsBllod clotting protease / Factor VII / Mouse glandular kallikrein / Factor VII-monoclonal antibody / Synthetic fluorogenic substrate / Synthetic chromogenic substrate / 蛍光性ペプチド / 凝固プロテアーゼ / VII因子 / IX因子 / セリンプロテアーゼ / マウス顎下腺カリクレイン
Research Abstract

The utility of the synthetic chromogenic and fluorogenic peptide substrates for specific assay of various proteases in body fluid has been established, because of their high sensitivities. The specific substrates for alpha-thrombin, factor Xa, plasma kallikrein, protein Ca, urokinase, factor XIIa, factor XIa, plasmin and limulus clotting enzyme developed by our group are now commercially available. The purpose of this project is to kind specific and sensitive substrates for factors VIIa and IXa and glandular kallikreins and to realize these synthetic substrates for determinations of various proteases in body fluids. The results are as follows:
1. Boc-Met-Ala-Arg4-methylcoumaryl-7-amide was found to be the most sensitive substrate for mouse glandular (submaxillary) kallikrein. the substrate comprised the amino acid sequence adjacent to the NH_2- and COOH-terminal regions of the kinin moiety found in mouse low molecular weight kininogen. The mouse kallikrein cleaved this substrate (Km = 2 … More 30 muM, kcat = 1.4s^<-1>) approximately 80-fold faster than hog glandular kallikreins.
2. We newly synthesized a chromogenic substrate of Boc-Leu-Thr-arg-p-nitrobenzylester for factor VIIa. This substrate was found to be most sensitive one for factor VIIa among the previously eynthesized 74 peptide substrates. This finding made it possible to investigate the molecular interaction between factro VII and tissue factor participated in the extrinsic coagulation system.
3. A murine monoclonal antibody (designated VII-M31) directed against bovine factor VII was prepared and characterized as the first step for specific assay of factor VII in body fluid. The antibody VII-M31 possessed a strong affinity only for factor VII in the presence of Ca^<2+> without any reactivities to prothrombin, factor X, factor IX, protein C, protein S and protein Z. Denaturation of factor VII by urea and SDS all its reduction with 2-mercaptoethanol did not destroy the antigenic site, measured by immunoblotting method. Moreover, the antibody bound specifically to the Gla-containing peptide corresponding to the NH_2-terminal 23-50 residues of factor VII. These results indicated that VII-M31 is available for specific enzyme-linked immunoassay of factor VII, in combination with the synthetic peptide substrate described above. Less

Report

(3 results)
  • 1989 Annual Research Report   Final Research Report Summary
  • 1988 Annual Research Report
  • Research Products

    (37 results)

All Other

All Publications (37 results)

  • [Publications] Nishimura,H.: "Identification of a Disaccharide(xyl-Glc)and a Trisaccharide(xyl2-Glc)O-Glycosidically Linked to a Serine Residue in the First Epidermal Growth Factor-like" J.Biol.Chem.Domain of Human Factors VII and Protein Z and Bovine Protein Z.264. 20320-20325 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Miyata,T.: "Fibrinogen Nagoya:a Replacement of Glutamine-329 by Arginine in the γ-Chain That Impairs the Polymerization of Fibrin Monomer." J.Biochem. 105. 730-735 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Sakai,T.: "Blood Clotting Factor IX Kashihara:Amino Acid Substitution of Valine182 by Phenylalanine" J.Biochem.105. 756-759 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Miyata,T.: "Factor XII Washington D.C:Inactive Factor XIIa Resulting from Replacement of Cysteine-571 by Serine." Proc.Natl.Acad.Sci.,U.S.A.86. 8319-8322 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Suehiro,K.: "Blood Clotting Factor IX BM Nagoya:Substitution of Arginene-180 by Tryptophan and Its activation by Chymotrypsin" J.Biol.Chem.264. 21257-21265 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Sugimoto,M.: "Factor IX Kawachinagano:Impaired function of the Gladomain caused by attached propeptide region due to substitution of arginine by glutamine at position" Br.J.Haematol.72. 216-221 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] 徳永文稔,岩永貞昭: "抗リポ多糖因子:その構造と生物活性「代謝」26巻No.5" 中山書店(東京), 11 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] 宮田敏行,岩永貞昭: "組織因子「BIO medica」4巻,NO.12" 北隆館(東京), 6 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Miyata, T. et al.: "Factor XII Washington D.C.: Inactive Factor XIIa Resulting from Replacement of Cysteine-571 by Serine." Proc. Natl. Acad. Sci., U.S.A., 86, 8319-9322.1989.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Tokunaga, F. et al.: "The Factor V-Activating Enzyme (RVV-V) from Russell's Biper Venom: Identification of Isoproteins, RVV-Valpha, -beta and -Vgamma, and Their Complete Amino Acid Sequences." J. Biol. Chem. 263, 14741-17481.1988.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Takeya, H. et al.: "Bovine Factor VII: Its Purification and Complete Amino Acid Sequence." J. Biol. Chem. 263, 14858-14877.1988.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Enjyoji, K. et al.: "Purification and Characterization of RatT-kininogens Isolated from Adjuvant-treated Rat Plasma: Identification of Three kinds of T-kininogens." J. Biol. Chem. 263(2), 973-979.1988.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Enjyoji, K. et al.: "Purification and Characterization of Two Kinds of Low-Molecular-Weight Kininogens from Rat (Non-inflamed) Plasma: One Is Resistant and Another Is Sensitive to Rat Glandular Kallikreins." J. Biol. Chem. 263(2), 959-964.1988.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Kawano, Y. et al.: "Localization of the Pyridoxal Phosphate Binding Site at the COOH-terminal Region of Erythrocyte Band 3 Protein." J. Biol. Chem. 263(17), 8232-8238.1988.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Kawabata, S. et al.: "Highly Sensitive Peptide-4-methylcoumary-7-amide Substrates for Blood Clotting Proteases and Tyrpsin." Eur. J. Biochem. 172(2), 17-25.1988.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Miyata, T. et al.: "Antimicrobial Peptides, Isolated from the Horseshoe Crab Hemocytes Tachyplesin II, Polyphemusins I and II: Chemical Structure and Biological Activity." J. Biochem. 106, 663-668.1989.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Uragoh, K. et al.: "A Novel Immunohistochemical Method for in vivo Detection of Endotoxin Using Horseshoe Crab Factor C." J. Histochem. Cytochem. 36(10), 1275-1283.1989.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Nakamura, T. et al.: "Synthesis and Property of Tachyplesin I, a Lipopolysaccharide Binding Peptide, from Tachypleus tridentatus." FEBS Lett. 252, 121-124.1989.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Nakamura, T. et al.: "Tachyplesin, a Class of Antimicrobial Peptide from the Hemocytes of the Horseshoe Crab (Tachypleus tridentatus)." J. Biol. Chem. 263, 16709-16713.1988.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Nakamura, T. et al.: "Intracellular Serine-protease Zymogen, Factor C, from Horseshoe Crab Hemocytes: Its Activation by Synthetic Lipid A Analogues and Acidic Phospholipids." Eur. J. Biochem. 176, 89-94.1988.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Nakamura, T. et al.: "Interaction between Lipopolysaccharide and Intracellular Serine Protease Zymogen, Factor C, from Horseshoe Crab (Tachypleus tridentatus) Hemocytes." J. Biochem. 103(2), 370-374.1988.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Nishimura,H.: "Indentification of a Disaccharide(Xyl-Glc)and a Trisaccharide(Xyl_2-Glc)O-Glycosidically Linked to a Serine Residue in the First Epidermal Growth Factor-like Domain of Human Factors VII and IX and Protein Z and Boving Protein Z." J.Biol.Chem.264. 20320-20325 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Miyata,T.: "Fibrinogen Nagoya:a Replacement of Glutamine-329 by Arginine in the γ-Chain That Impairs the Polymerization of Fibrin Monoment" J.Biochem.105. 730-735 (1089)

    • Related Report
      1989 Annual Research Report
  • [Publications] Sakai,T.: "Blood Clotting Factor IX Kashihara:Amino Acid Substitution of Valine^<182> by Phenylalanine." J.Biochem.105. 756-759 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Miyata,T.: "Facter XII Washington D.C.:Inactive Factor XIIa Resulting from Replacement of Cysteine-571 by Serine." Proc.Natl.Acad.Sci.,U.S.A.86. 8319-8322 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Suehiro,K.: "Blood Clotting Factor IX B_M Nagoya:Substitution of Arginine-180 by Tryptophan and Its activation by Chymotrypsin." J.Biol.Chem.264. 21257-21265 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Sugimoto,M.: "Factor IX Kawachinagano:impaired function of the Gladomain caused by attached proprptide region due to substitution of arginine by glutamine at position" Br.J.Haematol.72. 216-221 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] 徳永文稔,岩永貞昭: "抗リポ多糖因子:その構造と生物活性「代謝」26巻No.5" 中山書店(東京), 11 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] 宮田敏行,岩永貞昭: "組織因子「BIOmedica」4巻No.12" 水隆館(東京), 6 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Kawabata,S.: Eur.J.Biochem.172. 17-25 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] Morita,T.: J.Biochem.104. 368-374 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] Takeya,H.: J.Biol.Chem.263. 14868-14877 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] Tokunaga,F.: J.Biol.Chem.263. 17471-17481 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] Hase,S.: J.Biochem.104. 867-868 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] Sugimoto,M.: J.Biochem.104. 878-880 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] 岩永貞昭 他 編集: "ビタミンK:医学・生物学領域における新展開" メディカル・ジャーナル社, 1-332 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] 西村仁 他: "実験医学(特集)" 羊土社, 1236-1243 (1988)

    • Related Report
      1988 Annual Research Report

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Published: 1988-04-01   Modified: 2016-04-21  

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