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Functional Analysis of Tyrosinekinase in B Cells with Fluorescent Molecular Rotars

Research Project

Project/Area Number 08672483
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Physical pharmacy
Research InstitutionSuzuka University of Medical Science and Technology (1997)
Nagoya City University (1996)

Principal Investigator

NOJI Masahide  Suzuka University of Medical Science and Technology, Faculty of Medical Nutriology, Professor, 医療栄養学科, 教授 (70080190)

Co-Investigator(Kenkyū-buntansha) FURUNO Tadahide  Nagoya City University, Faculty of Pharmaceutical Sciences, Lecturer, 薬学部, 講師 (80254308)
Project Period (FY) 1996 – 1997
Project Status Completed (Fiscal Year 1997)
Budget Amount *help
¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1997: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1996: ¥700,000 (Direct Cost: ¥700,000)
Keywordstyrosinekinase / phosphorylated peptides / monoclonal antibody / ITAM / Image analysis / リン酸化チロシン / IgEレセプター / チロシンキナーゼ / 可視化解析 / RBL-2H3
Research Abstract

The IgE receptors (Fc epsilon RI) of rat basophilic leukemia cells (RBL-2H3) contain a consensus sequence termed immunoreceptor tyrosine-based activation motif (ITAM) in beta and gamma subunits. In ITAM there exist two phosphotyrosines, which play an important role in signal transduction cascade.
In this study, we have synthesized tyrosine phosphorylated ITAM peptides for beta and gamma subunits in rat IgE receptors, that is, (DRLYEELNHVYSPIYSELC) and (DAVYTGLNTRNQETYETLC) for beta and gamma subunits, respectively, where Y represents phosphorylated tyrosine. These phosphorylated peptides were synthesized by solid-phase Fmoc chemistry by using Fmoc-Tyr-(PO_3Me_2).
[Image Analysis of ITAM in RBL-2H3 Cells]
Among the obtained monoclonal antibodies against the peptides containing phosphorylated tyrosines, J-59 and J-69 monoclonal antibodies recognized proteins existing near cell membranes and cytoplasm after the antigen stimulation of the cells, especially the later one binds more strongly to proteins existing near cell membranes than the former. Encouraged by this result, time course of J-69 recognition for the membrane proteins after the stimulation was pursued by analyzing images obtained by a confocal fluorescence microscopy. In 5 and 15minutes after the stimulation J-69 recognized more preferentially ITAM near cell membranes. However, the fluorescence intensity decreased remarkably in 1 hr and 2 hr later, indicating the removal of antibodies from the membrane protein. This phenomenon can be explained by taking account of dephosphorylation of the gamma subunits by phosphatase with the resultant J-69 removal.

Report

(3 results)
  • 1997 Annual Research Report   Final Research Report Summary
  • 1996 Annual Research Report

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Published: 1996-04-01   Modified: 2016-04-21  

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