1992 Fiscal Year Final Research Report Summary
Studies on the roles of kinesin in the function of cells by monoclonal antibodies.
| Project/Area Number |
02670158
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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 |
Experimental pathology
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| Research Institution | SAPPORO MEDICAL UNIVERSITY |
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Principal Investigator |
HATTORI Atsuo Sapporo Medical University, Dept. Pathol., Instructor, 医学部・病理学, 助手 (90208538)
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| Co-Investigator(Kenkyū-buntansha) |
SAWADA Norimasa Sapporo Medical University, Dept. Pathol., Assistant Professor, 医学部・病理学, 講師 (30154149)
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| Project Period (FY) |
1990 – 1992
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| Keywords | Kinesin / Microtubule motor protein / Microtubule / Tubulin |
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| Research Abstract |
Tubulin was purified from hog brain homogenates by polymerization and depolymerization followed by DEAE column chromatography. Kinesin was copurified from hog brain with Taxol and DEAE-purified tubulin by the method of Wagner with slight modification (M.C.Wagner et al. Cell Motil. Cytoskel. 12, 195-215 1989). Twenty mug kinesin was obtained from 100g of hog brain. Vmax of ATPase activity of purified kinesin was 0.152mumol/min/mg in the presence of microtubles. Immunoblot analysis showed that the protein was 124kD on SDS-PAGE corresponding to the reported MW of the heavy chain of kinesin. Using this protein, mouse monoclonal antibodies were established. Clones producing antibodies against protein were screened by Western blot method. The immunoglobulin subclasses of the antibodies were determined to be Ig M, kappa. By immunocytochemistry, it was shown that kinesin is localized diffusely throughout the cytoplasm of neurons. On the other hand, in cultured fibroblasts, neuroblasts-blasts a
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nd hepatocytes, kinesin was localized along the microtubules as small granules. In the brain, kinesin was found more abundantly in the neural cells than the Glial cells. Kinesin was localized as small granules like Nissle's body in neural cells and also in the dendric processes. In the liver, kinesin was found mostly in the cytoplasm of bile duct epithelia, but found only weakly in the cytoplasm of hepatocytes. Immunoelectron microscopic examination of cultured neuroblastoma cells revealed that kinesin is mostly localized to the membranes of cytoplasmic organelles such as lysosomes and mitochondria. Madin-Darby Canine Kidney (MDCK) cells were kept at 4ﾟC for 30 min followed by a 30 min incubation at 37ﾟC in the presence of 33muM nocodazole to depolymerize microtubules. Localization of kinesin in the MDCK cells was not significantly affected by this treatment. Also, treating MDCK cells with 20kappaM Taxol polymerize tubulin did not affect the localization of kinesin in MDCK cells. Treatment of MDCK cells with Brefeldin A or Vanadate did not affect the localization of kinesin. Less
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Research Products
(8 results)
