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Determination of the reactive site (s) of kininogens for cysteine proteases using technique of site-directed mutagenesis

Research Project

Project/Area Number 02670114
Research Category

Grant-in-Aid for General Scientific Research (C)

Allocation TypeSingle-year Grants
Research Field General medical chemistry
Research InstitutionShiga university of Medical Science

Principal Investigator

OHKUBO Iwao  Shiga University of Medical Science, Professor, 医学部, 教授 (80152073)

Project Period (FY) 1990 – 1991
Project Status Completed (Fiscal Year 1991)
Budget Amount *help
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1991: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1990: ¥1,300,000 (Direct Cost: ¥1,300,000)
KeywordsMutagenesis / kininogens / cysteine protease inhibitor / reactive site
Research Abstract

High and low molecular weight kininogens strongly inactivate cysteine proteases such as cathepsin B, H and L, calpains I and II, papain, and ficin in noncovalent and noncompetitive manners. It is thought that both kininogens play important physiological roles in the regulation of cysteine proteases. There are two inhibitory domains on the common heavy chain of both kininogens. It is speculated that domains 2 and 3 contain two regions as potential candidates for the reactive site of cysteine proteases : Gln-Val-Val-Ala-Gly (QVVAG) located at approximately two-thirds of the distance from the N-terminus of the domains and Gly located at 44 amino acid residues upstream from the QVVAG region.
In this project, using a cDNA coding human low molecular weight kininogen, we have attemed to express the wild type protein and proteins with deletions of domains 2 and 3, and with deletions or substitutions of the QVVAG and Gly regions, in eukaryotic cells such as BHK, COS and CHO.
Although the amounts of the wild type protein and two proteins with deletions of domains 2 or 3 (<50ng/ml) expressed by the induction with dexamethasone in BHK cells were very low, we collected these mediums from the cells (totally about 5 L each), partially purified these three proteins by using Red-Sepharose and Mono Q column chromatographies, and finally obtained about 3 ug of each protein. Now, we are attempting to analyze their kinetic parameters, and determine the real reactive site (s) of kininogens for cysteine proteases.

Report

(3 results)
  • 1991 Annual Research Report   Final Research Report Summary
  • 1990 Annual Research Report
  • Research Products

    (9 results)

All Other

All Publications (9 results)

  • [Publications] M.Sasaki M.Kunimatsu I.Ohkubo: "Calpain and Kininogen mediated inflammation" Biomed.Biochem.Acta. 50. 499-508 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] S.Asakura R.W.Hurly K.Skorstengaard I.Ohkubo D.F.Mosher: "Inhibition of cell adhesion by high molecular weight kininogen" J.Cell Biol.116. 465 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] M.Sasaki,M.Kunimatsu I.Ohkubo: "Role of calpains and kininogens in inflammation" Akademiai Kiad,Butapest. (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Makato Sasaki, Mitoshi kunimatsu and Iwao Ohkubo: "Calpain and kininogen mediated inflammation" Biomed. Biochem. Acta. 50. 499-508 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Shinji Asakura, Randy W. Hurly, Karana Skorstengaard, Iwao Ohkubo and Deane F. Mosher: "Inhibition of cell adhesion by high molecular weight kininogen" J. Cell Biol.116. 465-476 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Makoto Sasaki, Mitoshi Kunimatsu and Iwao Ohkubo: "Role of calpains and kininogens in inflammation" Akademiai Kiado, Butapest. (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] M.Sasaki,M.Kunimatsu,I.Ohkubo: "Calpain and kininogen mediated inflammation" Biomed.Biochem.Acta. 50. 499-508 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] S.Asakura,R.W.Hurly,K.Skorstengaard,I.Ohkubo,D.F.Mosher: "Inhibition of cell adhesion by high molecular weight kininogen" J.Cell Biol.116. 465-476 (1992)

    • Related Report
      1991 Annual Research Report
  • [Publications] M.Sasaki,M.Kunimatsu,I.Ohkubo: "Role of calpains and kininogens in inflammation" Akademial Kiado,Butapest. (1992)

    • Related Report
      1991 Annual Research Report

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Published: 1990-04-01   Modified: 2016-04-21  

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