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^-泵的纯化：使用非离子去污剂MEGA-10，我们成功地溶解了Cl^-泵（Cl^--ATPase），而其对抑制剂依他尼酸的敏感性没有任何变化。柱层析（CM-Sepharose和MonoQ-HPLC）和聚丙烯酰胺电泳的溶解的Cl^-泵产生的520 kDa和580 kDa的蛋白质与Cl^-泵的活性。2. C ^-运输重建Cl^-泵：溶解的Cl^-泵被有效地重建在卵磷脂与磷脂酰肌醇-4-磷酸。在K^+离子载体和40 mM K^+（或质子载体）存在的情况下，通过重构Cl^-泵的ATP依赖性Cl^-转运增强，表明Cl^-转运是产电的。3.Cl ^-泵的亚基：Cl ^-泵的SDS-聚丙烯酰胺电泳显示至少有4个亚基（110 kDa、97 kDa、67 kDa、47 kDa）。在家兔体内产生的抗Cl ^-泵抗体通过与60 kDa亚基结合而抑制Cl^-ATP酶活性。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

Hara,m.: "Uneven distribution of intracellular Cl^- in rat hippocampal neurons" Neuroscience Letters. 143. 135-138 (1992)

Hara,m.：“大鼠海马神经元细胞内 Cl^- 分布不均匀”《神经科学快报》。

Inoue,m.: "Ethacrynic acid-induced convulsions and brain neurotransmitters in mice" European Journal of Pharmacology. 221. 135-137 (1992)

Inoue,m.：“依他尼酸引起的小鼠惊厥和脑神经递质”欧洲药理学杂志。

S.Ohnishi et al: "Delayed shortening and shrinkage of cochlear outer hair cells." Am.J.Physiol.

S.Ohnishi 等人：“耳蜗外毛细胞延迟缩短和收缩。”

C.Inagaki et al.: "Eletrogenic Chloride Transporters in Biological Membranes." Springer-Verlag, (1992)

C.Inagaki 等人：“生物膜中的电氯化物转运蛋白”。

Ohnishi,S: "Regulation of Cl^‐ conductance in delayed shortening and shrinkage of outer hair cells." Acta otolaryngol(Stockh). Suppl500. 42-45 (1993)

Ohnishi, S：“外毛细胞延迟缩短和收缩中的 Cl^- 电导调节”（Acta otolaryngol）（Suppl500）。