| Project/Area Number |
06670367
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Immunology
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| Research Institution | Tokyo Metropolitan Institute for Neuroscience |
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Principal Investigator |
OGIMOTO Mami Tokyo Metropolitan Institute for Neuroscience, Department of Microbiology and lmmunology, Staff Scientist, 微生物学・免疫学研究部門, 主事 (80158609)
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| Co-Investigator(Kenkyū-buntansha) |
KATAGIRI Tatsuo Tokyo Metropolitan Institute for Neuroscience, Department of Microbiology and lm, 微生物学・免疫学研究部門, 主事 (00233742)
YAKURA Hidetaka Tokyo Metropolitan Institute for Neuroscience, Department of Microbiology and lm, 微生物学・免疫学研究部門, 副参事 (60166486)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1995: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1994: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Keywords | CD45 / Receptor-type tyrosine phosphatase / CD45 ligand / CD45-binding molecule / レセプター型チロシンフォスファターゼ / リガンド / バキュロウィルスベクター |
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| Research Abstract |
Lymphocyte activation, proliferation, and cell death by apoptosis are regulated by protein tyrosine phosphorylation.One of the key playrs in these processes is a receptor-type protein tyrosine phosphatase (PTP), CD 45. To understand the function of CD 45 in terms of the regulation of cell-cell interaction and of the PTP activity identification of the physiological ligand (s) for CD45is crucial.In this proiect we tried to identify CD45-binding proteins as a first step toward this goal.
1) Purification of native CD45 from the WEHI-231 B cell line
Native CD45 was purified from the WEHI-231 B cell line, with molecular-sieve, wheat germ agglutinin and spectrin affinity chromatographies.The gold-colloid staining of the purified CD45 separated by SDS-PAGE revealed that only one band with Mr of >200 kDa is visualized, suggesting that CD45 has been purified to homogeneity.
2) ldentification of CD45-binding proteins in spleen lysates
Mouse spleen was homogenized and separated into soluble and membrane-bound fractions.Each fraction was applied to a CD45-bound column, and then eluted from the bound fractions, respectively, contain 70kDa protein and three proteins of 54 kDa, 60 kdA and 66 kDa.
3) Effect of CD45-binding proteins on CD45 PTP activity
Next, how the interaction of DC45 and CD45-binding protein affects enzymatic activity of CD45 was analyzed.Purified CD45 was incubated 1 hr on ice with CD45-binding proteins from each fraction and the CD45 PTP activity was measured sing a phosphotyrosine-containing oligopeptide as a substrate.
4) Cells that can bind to CD45
Finally, it was determined which spleen cell population is bound to CD45.FACS analysis with FITC-CD45 and PE-alpha-Thy-1 demonstrated that CD45 can bind a part of both T and B cells.
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