| Project/Area Number |
14580633
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Structural biochemistry
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| Research Institution | Tokyo Metropolitan Foundation for Research on Aging and Promotion of Human Welfare |
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Principal Investigator |
IWASHITA Yoshiko Tokyo Metropolitan Foundation for Research on Aging and Promotion of Human Welfare, Tokyo Metropolitan Institute of Gerontology, Biomembrane Research Group, Senior Research Scientist, 東京都老人総合研究所・生体膜機能研究グループ, 副参事研究員 (50111498)
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| Co-Investigator(Kenkyū-buntansha) |
INOMATA Mitsushi Tokyo Metropolitan Foundation for Research on Aging and Promotion of Human Welfare, Tokyo Metropolitan Institute of Gerontology, Biomembrane Research Group, Senior Research Scientist, 福祉振興財団・東京都老人総合研究所・生体膜機能研究グループ, 主任研究員 (30142649)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2003: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2002: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Keywords | CHOLESTEROL / MICRODOMAIN / LIPID RAFTS / PERFRINGOLYSIN O / JURKAT CELLS / SIGNALING / ラフト / 脂質マイクロドメイン / 脂質ミクロドメイン |
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| Research Abstract |
We prepared a non-cytolytic derivative of perfringolysin O (θ-toxin), a cholesterol-binding cytolysin, as a probe for cholesterol, and demonstrated that the probe (BCθ; protease-nicked and biotinylated θ-toxin) binds selectively to cholesterol in cholesterol-rich membrane domains of intact cells. By electron microscopic analysis, we showed that BCθ binds to a subpopulation of membranes among those recovered in detergent-insoluble, low-density fractions (raft fractions). The results indicate that raft fractions contain heterogeneous populations of membranes. Therefore we further isolated BCθ-bound membranes and characterized them. Raft fractions were prepared from BCθ-bound Jurkat T cells and fractionated into BCθ-bound and -unbound membranes using avidin-magnet beads. BCθ-bound membranes have higher cholesterol contents than unbound fractions. Flotillin, a typical raft marker protein, and Src-family protein kinases, such as Lck and Fyn, in raft fractions were almost exclusively recovered from BCθ-bound fractions. Among molecules that participate in T-cell signaling, LAT was recovered from BCθ-bound fractions, while CD3 and Zap70 were recovered from unbound fractions. These results indicate that raft fractions are heterogeneous in molecular composition, and reveal that BCθ is useful for the subfractionation of cholesterol-rich membranes in raft fractions and for detecting molecules localized in them.
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