| Project/Area Number |
07454229
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
動物生理・代謝
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| Research Institution | TOKYO METROPOLITAN UNIVERSITY |
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Principal Investigator |
IZUMI Susumu TOKYO METROPOLITIAN UNIVERSTTY Graduate school of science, Associate professor, 理学研究科, 助教授 (10145659)
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| Co-Investigator(Kenkyū-buntansha) |
TOMINO Shiro TOKYO METROPOLITTAN UNIVERSTTY Graduate School of science, Professor, 理学研究科, 教授 (30101075)
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| Project Period (FY) |
1995 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥7,700,000 (Direct Cost: ¥7,700,000)
Fiscal Year 1996: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1995: ¥5,600,000 (Direct Cost: ¥5,600,000)
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| Keywords | silk worm / fat body / plasma protein / storage protein / receptor / cDNA / cloning / imunohistochemistry / エレクトロポレーション |
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| 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
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