Purification and primary structure of Cl^- pump

1.氯离子泵的纯化：用非离子洗涤剂MEGA-10成功地溶解了氯离子泵(Cl^-ATPase)，且不改变其对抑制剂乙丙烯酸的敏感性。柱层析(CM-Sepharose和MonoQ-Hplc)和聚丙烯酰胺凝胶电泳法分别得到520 kDa和580 kDa具有氯泵活性的蛋白质。2.重组氯泵的C~+转运：溶解的氯泵在卵磷脂中与磷脂酰肌醇-4-磷酸有效地重组。在K~(++)离子载体和40 mM K~(++)或原载体的存在下，重组的氯离子泵对ATP依赖的氯离子的转运被增强，表明氯离子的转运是电生的。重组的520 kDa蛋白具有比质膜囊泡高20倍的依赖于ATP的氯离子转运活性。3.氯泵的亚基：氯泵的SDS-PAGE显示至少4个亚基(110 kDa、97 kDa、67 kDa、47 kDa)。兔抗氯泵抗体通过与60 kDa亚基结合，抑制氯离子-ATPase活性。蛋白质测序仪分析表明，SPGGGSISIPSGITH的N-端氨基酸序列是正确的。

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.