Purification and primary structure of Cl^- pump

Cl^-泵的净化及主要结构

• 批准号: 03454148
• 负责人: INAGAKI Chiyoko
• 金额: $ 1.28万
• 依托单位: Kansai Medial University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-03454148/
• 关键词: Cl^- pump; Brain; primary structure; neuron; Cl^--ATPase; エタクリン酸

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.

1. cl^ - 泵的实用：使用非离子洗涤剂MEGA-10，我们成功溶解了Cl^ - 泵（Cl^ - ATPase），而没有其对抑制剂乙酸乙酸的敏感性的任何变化。圆柱色谱（CM-塞帕罗斯和MonoQ-HPLC）和聚丙烯酰胺的溶解Cl^ - 泵的聚丙烯酰胺电泳产生了520kDa和580KDA蛋白，具有Cl^ - 泵活性。2.c^ - 通过重新构成的Cl^ - 泵^ - 泵^-Cl^ - 泵py：溶解的cl^ - 在lecite-lecity-liceStiTed-lecite-liceStiTy-lecity-phospityyy中有效地磷酸化磷酸化。在K^+离子载体和40mm K^+的存在下，通过重构Cl^ - 泵的ATP依赖性Cl^ - 或质子团增强了泵，这表明Cl^ - 转运是电的。重建的520KDA蛋白显示出高度依赖ATP的Cl^ - 转运活性的20倍。3。cl^ - 泵的subunits Cl^ - cl^ - 泵的SDS-聚丙烯酰胺电泳至少显示4个亚基（110KDA，97KDA，97KDA，67KDA，67KDA，47KDA，47KDA）。抗Cl^ - 在兔中升高的泵抗体通过与60kDa亚基结合，抑制了ATPase活性。通过蛋白质测序仪分析显示SPGGGSISIPSGITH的N末端氨基酸序列。

项目成果

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