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Study on structure and function of myofibril-bound serine proteinase (MBP) using molecular cloning

Research Project

Project/Area Number 13660203
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Fisheries chemistry
Research InstitutionNagasaki University

Principal Investigator

HARA Kenji  Nagasaki University, Fac. of Fish., Professor, 水産学部, 教授 (10039737)

Co-Investigator(Kenkyū-buntansha) OSATOMI Kiyoshi  Nagasaki University, Fac. of Fish., Professor, 水産学部, 助教授 (40253702)
TACHIBANA Katsuyasu  Nagasaki University, Fac. of Fish., Professor, 水産学部, 教授 (20171712)
ISHIHARA Tadashi  Nagasaki University, Fac. of Fish., Professor, 水産学部, 教授 (40039722)
Project Period (FY) 2001 – 2003
Project Status Completed (Fiscal Year 2003)
Budget Amount *help
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2003: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2002: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2001: ¥1,500,000 (Direct Cost: ¥1,500,000)
KeywordsMyofibril-bound serine protease / Molecular cloning / Amino acid sequence / Carp / Lizard fish / Trypsin type protease / Modori / Serine proteinase inhibitor / mRNA発現 / 筋原繊維結合型セリンプロテアーゼ / N末端アミノ酸配列 / 火戻り / 精製 / プローブ
Research Abstract

Myofibril-bound serine proteinases (MBP) from carp and lizard fish were purified. Proteolysis of the two MBPs on myofibril proteins and their gel formation ability were investigated. MBPs readily decomposed myosin heavy chain as indicated by SDS-PAGE. In the preparation of kamaboko, the gel formation ability was diminished by addition of MBPs. The degradation effects of MBPs on actin, α-actinin and tropomyosin were studied by the imrnunoblotting method. Because of its myofibril-bound and myofibril protein degradation characteristics, MBP was regarded as the proteinase most probably involved in the modori effect.
A full-length cDNA clone encoding was amplified according to reverse transcription-polymerase chain reaction of RNA from ordinary muscle of carp. The sequence consisted of a 33 by 5'-non-coding region and a 726 by open reading frame which was followed by a 250 by 3'-non-coding region : The predicted protein consisted of 242 amino acids which was possibly processed to an active e … More nzyme of 222 amino acids that showed some similarity to other member of serine protease family. MBP contained the catalytic triad (His-60, Asp-106, and Ser-196) of trypsin-like proteases that has been characterized as the active site. Cys 167:181 (Met loop), and cys 192:216 (Ser loop), Compared to the amino acid compositions reported for trypsin-type serine proteases from all other sources, MBP molecule contained significant amounts of Lys (27 amino acid residues) per mole enzyme.
A novel myofibril-bound serine proteinase inhibitor (MBSPI) was purified to homogeneity from the skeletal muscle of lizard fish. MBSPI was a monomeric protein with the molecular mass of 50 kDa as estimated by SDS-PAGE and gel filtration. MBSPI reveals high inhibition specificity toward a myofibril-bound serine proteinase (MBSP) purified from lizard fish muscle. No inhibition is detected toward bovine trypsin, bovine chymotrypsin, two trypsins from carp hepatopancreas and a serine proteinase isolated from the sarcoplasmic fraction of white croaker muscle. It does not exert any inhibitory activity toward a myofibril-bound serine proteinase from carp muscle. Less

Report

(4 results)
  • 2003 Annual Research Report   Final Research Report Summary
  • 2002 Annual Research Report
  • 2001 Annual Research Report
  • Research Products

    (6 results)

All Other

All Publications (6 results)

  • [Publications] K.osatomi 他5名: "Molecular cloning of novel myofibriL-bound serin proteinase (MBSP)from ordinary muscle of carp Cyprinus cergio"Fisheries Science. 68. 1615-1616 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] M.Ohkubo 他5名: "Purification and characterization of myofibriL-bound serine protease from lizard fish (saurida undosquamis)muscle."Comp.Biochem.PhysioL.Part B. 137. 139-150 (2004)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] 原 研治(分担): "かまぼこ その科学と技術"山澤正勝・關伸男・福田裕 編集. 377 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] K.Osatomii, M-J.Cao, K.Tsunemoto, M.Ohkubo, K.Hara, T.Ishihara: "Molecular cloning of novel myofibril-bound serine prpteinase(MBP) from ordinary muscle of carp Cyprinus carpio"Fisheries Science. 68. 1615-1616 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] M.Ohkubo, K.Miyagawa, K.Osatomi, K.Hara, Y.Nozaki, T.Ishihara: "Purification and characterization of myofibril-bound serine protease from lizard fish (Saurida undosquarnis) muscle"ComBiochem.Physiol. Part B. 137. 139-150 (2004)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Kenji Hara: "Modori caused by proteases Kamaboko, Its chemical and technique(Editing by M.Yamasawa, N.Seki, H.Fukuda)"Kouseisha Kouseikaku. 77-87 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary

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Published: 2001-04-01   Modified: 2016-04-21  

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