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1996 Fiscal Year Final Research Report Summary

Analysis of function and strueture of receptor protein for storageprotein SPI in Bomyxmori.

Research Project

Project/Area Number 07454229
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field 動物生理・代謝
Research InstitutionTOKYO METROPOLITAN UNIVERSITY

Principal Investigator

IZUMI Susumu  TOKYO METROPOLITIAN UNIVERSTTY Graduate school of science, Associate professor, 理学研究科, 助教授 (10145659)

Co-Investigator(Kenkyū-buntansha) TOMINO Shiro  TOKYO METROPOLITTAN UNIVERSTTY Graduate School of science, Professor, 理学研究科, 教授 (30101075)
Project Period (FY) 1995 – 1996
Keywordssilk worm / fat body / plasma protein / storage protein / receptor / cDNA / cloning / imunohistochemistry
Research Abstract

1) The binding of storage protein to the plasma membrane fraction was studied using the fat body preparation from the fifth instar day-9 larvae. SP1 binding to the plasma membrane was optimal at pH 5 in the presence of divalent cations and apparent Km for SP1 was determined to be 3.5 x 10^<-8> M.The competition experiments of SP1 binding strongly suggested that SP1 binds to the plasma membrane of fat body with high specificity, and that SP1 and SP2 might be recognized by each distinct receptor. Solubilization and characterization of the SP1-binding protein were performed from the fat body plasma membrane fraction to identify and purify the SP1-binding protein. Immunoprecipitate and ligand blot assay demonstrated that a l00 kDa protein in the fat body membrane fraction is SPl binding protein.
2) SP1-binding proteins was separated by the gel filtration on an Ultrogel ACA34 column, affinity chromatography on an anti-SP1 antibody-Sepharose column in the presence of Ca^<++> and preparative g … More el electrophoresis, and an antibody was raised against the purified SP1 binding protein. Immunoblotting result show that the a protein with mol.wt.l00k revealed only in the fat body tissue from the fifth instar day-3 larvae to silk-spinning and disappears at pupal development. Electron microscopic analyzes suggested that the purified SP1-binding protein is a membrane protein locating at the plasma membrane reticular system and basal lamina.
3) A cDNA expression library was constructed from the fat body mRNA of 5-day old fifth instar female larvae. Screening of the library with the affinity-purified antibodies against the SP1-binding protein yielded seven immuno-positive clones. The cDNA library was rescreened using labeled cDNA as a probe. By screening 5 x 10^5 pfu, a 3.5 kb cDNA-clone was isolated.The 5' terminal region of SP1-binding protein mRNA was cloned by the method of rapid amplification of cDNA end. There is an open reading frame coding for 1,025 amino acid residues in the cloned region of SP1-binding protein cDNA and deduced peptide was a molecular weight of 114,000 that is a little higher than molecular weight 100,000 estimated with SDS polyacrylamide gel electrophoresis. Primary structure deduced from mRNA sequence revealed that SP1-binding protein has two putative membrane-spanning structures. Less

  • Research Products

    (12 results)

All Other

All Publications (12 results)

  • [Publications] Mine, Eriko: "The fat body cell-free system for fissue-specific franscription of plasma protein of Bombyx mori." Nucleic Acids Res. 23. 2648-2653 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Sakurai, Hiroshi: "An invitro franscription system from BmN cells of the silkworm Bombyx mori." Methods in Molecular Biol.37. 233-244 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Hayakawa, Yoichi: "Molecular cloning and characterization of cDNA for insect biogenic peptide,growth blocking peptide" FEBS Letters. 376. 185-189 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Wang, Jiming: "Purification and cDNA cloning of SPI receptor of Bombyx mori." Zoological Science. 13supplement. 48-48 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Mine, Eriko: "In vitro transcuiption system from cultured cells and fat body tissue of the silkworm,Bombyx mori" Molecular Biotechnology. 8. 79-88 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Nakato, Hiroshi: "Purification snd cDNA cloning of evolutionally couserved lavval cuticle protein of the silkworm,Bombyx mori." Insect Biochem.Molec.Biol. 27. 701-709 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Eriko Mine et al.: "The fat body cell free system for tissue-specific transcription of plasma protein of Bombyx mori." Nucleic Acid Res.23. 2648-2653 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Hiroshi Sakurai et al.: "An in vitro transcription system from BmN cells of the silkworm, Bombyx mori." Methods in Molecular Biol.37. 233-244 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Youichi Hayakawa et al.: "Molecular cloning and characterization of cDNA for insect biogenic peptide, growth blocking peptide." FEBS Letters. 376. 185-189 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Jinming Wang et al.: "Purification and cDNA cloning of SP1 receptor of Bombyx mori." Zoological Science. 13 Supplement. 48-48 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Eriko Mine et al.: "ln vitro transcription systems from cultured cells and fat body tissue of the silkworm, Bombyx mori." Molecular Biotechnology. 8. 79-88 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Hiroshi Nakato el al.: "Purification and cDNA cloning of evolutionally conserved larval cuticle protein of the silkworm Bombyx mori." Insect Biochem.Molec.Biol.27. 701-709 (1997)

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

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Published: 1999-03-16  

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