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1998 Fiscal Year Final Research Report Summary

Expression of mutant smooth muscle myosin and sliding mechanism

Research Project

Project/Area Number 08458215
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Biophysics
Research InstitutionNational Cardiovascular Center Research Institute

Principal Investigator

ONISHI Hirofumi  National Cardiovascular Center Research Institute, Department of Structural Analysis, Section Chief, 循環器形態部, 室長 (80092542)

Project Period (FY) 1996 – 1998
KeywordsSmooth muscle myosin / Protein expression / Site-directed mutagenesis / Baculovirus / ATP hydrolysis
Research Abstract

Earlier kinetic studies have revealed that the ATP hydrolysis of myosin is a multi-transition process with many intermediates. From crystallographic comparisons of myosin heads with different nucleotide analogues, Fisher et al. have proposed that in one of these transitions, M・ATP to M・ADP・P,a rotation around Ile466-Ala467-Gly468 (of chicken smooth muscle myosin) occurs and that the rotation allows formation of a salt-bridge between Glu470 and Arg247 in the narrow 50 kDa cleft [Biochemistry 34,8960-8972(1995)]. Here, we made mutants of smooth muscle heavy meromyosin (HMM ; N-terminal truncated myosin fragment) to test whether both rotational movement and salt-bridge formation really occur in the normal ATP hydrolysis or not Smooth muscle 11MM mutants were expressed by using a baculovirus-cultured insect cell system. All mutant HMMs expressed here decorated actinfilaments, forming the characteristic arrowheads and were dissociated from actin by the addition of ATP.Therefore, their mutat … More ional effects on functions are neither global nor gross. A mutant G468A exhibited no ATPase activity, no initial phosphate burst, and no Trp fluorescence enhancement upon adding ATP.A mutant E470A also exhibited no ATPase activity and no phosphate burst, but it showed the Trp fluorescence enhancement corresponding to MATP^*. To test whether this failure of hydrolysis is due to the loss of a proton acceptor or to the loss of an essential salt-bridge suggested by Fisher et al., we made a double-mutant HMM (E470R/R247E) and the constituent single-mutant HMMs (E470R and R247E). The double-mutant HMM, like wild-type HMM, had ATPase activity, a phosphate burst, and a high-level Trp fluorescence enhancement corresponding toM・ADP P^<**>, whereas the two single-mutant HMMs had no ATPase activity, no phosphate burst, and no fluorescence enhancement This result suggests that whether in the normal or in the inverted direction, an intact salt-bridge is necessary for ATP hydrolysis. Thus, we propose that both the rotation at Gly468 and the salt-bridge formation really occur in the ATP hydrolytic process. Less

  • Research Products

    (10 results)

All Other

All Publications (10 results)

  • [Publications] Onishi, H: "Functional transitions in myosin : Role of highly conserved Gly and Glu residues in the active site." Biochemistry. 36(13). 3767-3772 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Onishi, H.: "Functional transitions in myosin : Formation of a critical salt-bridge and transmission of effect to the sensitive tryptophan." Proc.Natl.Acad.Sci.USA. 95(12). 6653-6658 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Katoh,K.: "Isolation and contraction of the stress fiber." Mol.Biol.Cell. 9(7). 1919-1938 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Onishi, H.: "Smooth muscle myosin : Amino acid residues responsible for the hydrolysis of ATP. In“Mechanism of work production and work absorption in muscle"" Plenum Publishing Corp. New York and London, 99-104 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Onishi, H.: "Structure and function of smooth muscle myosin as revealed by baculovirus-Sf9 cell expression system. In“Molecular mechanism and the disorder of smooth muscle contraction"" Randes Bioscience, Georgetown, 1-13 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Onishi, H.: "Functional transitions in myosin : Role of highly conserved Gly and Glu residues in the active site." Biochemistry. 36(13). 3767-3772 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Onishi, H.: "Functional transitions in myosin : Formation of a critical salt-bridg and transmission of effect to the sensitive tryptophan." Proc.Natl.Acad.Sci.USA. 95(12). 6653-6658 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Katho, K.: "Isolation and contraction of the stress fiber." Mol.Biol.Cell. 9(7). 1919-1938 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Onishi, H.: Smooth muscle myosin : Amino acid residues responsible for the hydrolysis of ATP.In"Mechanism of work production and work absorption in muscle (eds.Sugi, H.and Pollack, G.)". Plenum Publishing Corp.New York and London, 99-104 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Onishi, H.: Structure and function of smooth muscle myosin as revealed by baculovirus-Sf9 cell expression system. In"Molecular mechanism and the disorder of smooth muscle contraction(eds.Kohama, K.and Sasaki, Y.)". Randes Bioscience Georgetown, 1-13 (1999)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1999-12-08  

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