Purification and primary structure of Cl^- pump

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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.