1993 Fiscal Year Final Research Report Summary
Purification and primary structure of Cl^- pump
| Project/Area Number |
03454148
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
General pharmacology
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| Research Institution | Kansai Medial University |
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Principal Investigator |
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| Co-Investigator(Kenkyū-buntansha) |
URIU Toshiko 関西医科大学, 医学部, 助手 (40203608)
HARA Mitsuyoshi 関西医科大学, 医学部, 助手 (50192282)
OTANI Hitomi 関西医科大学, 医学部, 助手 (40140272)
INOUE Masafumi 関西医科大学, 医学部, 講師 (90203257)
OMORI Kyoko Kansai Medical University, Medicine, professor., 医学部, 講師 (90152256)
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| Project Period (FY) |
1991 – 1993
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| Keywords | Cl^- pump / Brain / primary structure / neuron |
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| Research Abstract |
1.Purification of Cl^- pump : Using a non-ionic detergent, MEGA-10, we successfully solubilized Cl^- pump (Cl^--ATPase) without any changes in its sensitivity to an inhibitor, ethacrynic acid. Column chromatography (CM-Sepharose and MonoQ-HPLC) and polyacrylamide electrophoresis of solubilized Cl^- pump yielded 520kDa and 580kDa protein with Cl^- pump activity.
2.C^- transport by reconstituted Cl^- pump : Solubilized Cl^- pump was effectively reconstituted in lecithin with phosphatidylinositol-4-phosphate. ATP-dependent Cl^- transport by reconstituted Cl^- pump was enhanced in the presence of K^+ ionophore and 40mM K^+, or a protonophore, suggesting that the Cl^- transport is electrogenic. Reconstituted 520kDa protein showed 20 times as high ATP-dependent Cl^- transport activity as that in plasma membrane vesicles.
3.Subunits of Cl^- pump : SDS-polyacrylamide electrophoresis of Cl^- pump showed at least 4 subunits (110kDa, 97kDa, 67kDa, 47kDa). Anti-Cl^- pump antibody raised in the rabbit inhibited Cl^--ATPase activity by binding to 60kDa subunit. Analysis by a protein sequencer showed the N-terminal amino acid sequence of SPGGGSISIPSGITH.
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[Publications] Inoue, M., Hara, M., Zeng, X.-T., Hirose, T., Ohnishi, S., Yasukura, T., Uriu, T., Minato, A.and Inagaki, C.: "An ATP-driven Cl^- pump regulates Cl^- concentrations in rat hippocampal neurons." Neurosci.Lett.134. 75-78 (1991)
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[Publications] Hara, M., Inoue, M., Yasukura, T., Ohnishi, S., Mikami, Y.and Inagaki, C.: "Uneven distribution of intracellular C^- in rat hippocampal neurons." Neurosci.Lett.143. 135-138 (1992)
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[Publications] Ohnishi, S., Hara, M., Inoue, M., Yamashita, T., Kumazawa, T., Minato, A.and Inagaki, C.: "Delayd shortening and shrinkage of cochlear outer hair cells." Am.J.Physiol.263. C1088-C1095 (1992)
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