| Project/Area Number |
12680644
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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 |
Functional biochemistry
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| Research Institution | Tokyo Metropolitan Institute of Gerontology |
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Principal Investigator |
IWASHITA YOSHIKO Tokyo Metropolitan Institute of Gerontology, Department of Protein Biochemistry, Research Associate, 蛋白質生化学部門, 室長 (50111498)
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| Co-Investigator(Kenkyū-buntansha) |
NAKAMURA MEGUMI Tokyo Metropolitan Institute of Gerontology, Department of Protein Biochemistry, Research Associate, 蛋白質生化学部門, 研究助手 (10159072)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2001: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2000: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Keywords | CHOLESTEROL / LIPID DOMAIN / RAFTS / PERFRINGOLYSINO / PLATELET / PLASMA MEMBRANE / コレステロール結合毒素 / β-シクロデキストリン |
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| Research Abstract |
Recent studies indicate that sphingolipid- and cholesterol-rich microdomains (rafts) exist in the plasma membrane, where specific proteins assemble and play a role in signal transduction and many other cellular events. Cholesterol depletion causes disassembly of the raft-associated proteins, suggesting an essential role of cholesterol in the structural maintenance and function of rafts. However, no tool has been available for the detection and monitoring of raft cholesterol in living cells. Here we show that a protease-nicked and biotinylated derivative (BCθ) of perfringolysin O (θ-toxin) binds selectively to cholesterol-rich microdomains of intact cells, the domains that fulfill the criteria of rafts. We fractionated the homogenates of non-treated and Triton X-100-treated platelets after incubation with BCθ on a sucrose gradient. BCθ was predominantly localized in the floating low-density fractions (FLDF) where cholesterol, sphingomyelin and Src family kinases are enriched. Immunoelectron microscopy demonstrated that BCθ binds to a sub-population of vesicles in FLDF. Depletion of 35% cholesterol from platelets with cyclodextrin, which accompanied 76% reduction in cholesterol from FLDF, almost completely abolished BCθ binding to FLDF. The staining patterns of BCθ and filipin in human epidermoid carcinoma A431 cells with and without cholesterol depletion suggest that BCθ binds to specific membrane domains on the cell surface, whereas filipin binding is indiscriminate to cell cholesterol. Further, BCθ binding does not cause any damage to cell membranes, indicating that BCθ is a useful probe for the detection of membrane rafts in living cells.
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