1988 Fiscal Year Final Research Report Summary
Studies on the molecular mechanism of endocytic process of biological cells using very fine ferrite particles and high-gradient magnetic separation technique.
| Project/Area Number |
62440091
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| Research Category |
Grant-in-Aid for General Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Research Field |
生物物性学
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| Research Institution | Kyoto University |
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Principal Investigator |
OHNISHI Shun-ichi Faculty of Science, Kyoto University, 理学部, 教授 (00025272)
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| Co-Investigator(Kenkyū-buntansha) |
SATO Satoshi Faculty of Science, Kyoto University, 理学部, 助手 (30183049)
ITO Tadanao Faculty of Science, Kyoto University, 理学部, 助教授 (90093187)
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| Project Period (FY) |
1987 – 1988
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| Keywords | Endocytosis / Endosomes / Ferrite / Magnetic separation / membrane process / 膜融合 |
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| Research Abstract |
To elucidate the molecular mechanism of dynamic endocytic membrane process, we have developed a new method for isolation of ligand-specific endosomes at different stages of intracellular transport. Very fine ferrite particles with the edge length of 5nm were dispersed by coating with oleic acid and further coated with phosphatidylcholine and asialoganglioside. Some acylated marker enzymes such as horse-radish preoxidase (HRP) could also be conjugated. They were used as ligands of receptor-mediated endocytosis of rat hepatocyte. When these ligands were introduced to liver by perfusion, most of them were taken by the parenchymal cells. The ligand-containing endosomes were separated by high-gradient magnetic separation (HGMS) techique. The liver homogenate was applied to the HGMS column which contained mesh-works of ferritic stainless wires. When these wires are placed in a magnetic field, very steep magnetic gradient is generated around the wires and this attracts the ferrite-containing endosomes. After washing extensively with buffer, the endosomes were easily recovered by simply turning off the magnet. When the endosomes were isolated after 5 min-perfusion, they peaked at density of 1.05 and contained both ligands and receptors. By 15 min-perfusion, another population of endosome with the density of 1.08 lacking receptors but containing the ligands was isolated. By longer perfusion, ligand-containing lysosomes with the density higher than 1.12 were isolated. We detected several specific proteins in these different population of endosomes. These proteins would play important roles in critical steps such as directed transport of vesicles, fusion, fission and sorting of ligands and receptors. Further characterization of these proteins are under progress. Several monoclonal antibodies specific to endosome protein components were prepared and they were used for elucidation of in vivo function of endosome components.
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