Chimeric Ca2+‐ATPase/Na+,K+‐ATPase molecules

FEBS Letters

Andersen, Jens Peter¹  Nørregaard, Anders¹  Vilsen, Bente¹ 

[1]Danish Biomembrane Research Centre, Institute of Physiology, University of Aarhus, Ole Worms Allé 160, DK-8000 Aarhus C, Denmark

关键词: Ca2+ occlusion;    Ca2+-pump;    Chimera;    Na+;    K+-pump;    Thapsigargin;    Ca2+-ATPase;    Ca2+-activated adenosine triphosphatase;    C12E8;    octaethyleneglycol monododecyi ether;    E1P;    ADP-sensitive phosphoenzyme intermediate;    E2P;    ADP-insensitive phosphoenzyme intermediate;    EGTA;    [ethylenebis(oxyethylene-nitrilo)] tetraacetic add;    M1-M10;    putative transmembrane segments numbered from the NH2-tenninal end of the peptide;    MES;    2-(N-morpholino) ethanesulionic acid;    MOPS;    3-(N-morpholino)propane- sulfonic acid;    Na+K+-ATPase;    Na+- and K+-activated adenosine triphosphatase;    TES;    N-tris[hydroxymethyymethyl-2-aminoethanesulfonic acid;    SDS;    sodium dodecyl sulfate;    SR;    sarcoplasmic reticulum;   

DOI  :  10.1016/0014-5793(93)80813-A

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Chimeric molecules consisting of parts from the sarcoplasmic reticulum Ca²⁺-ATPase and the Na⁺,K⁺-ATPase were expressed in COS-1 cells and analysed functionally. One chimera, in which most of the central cytoplasmic loop was derived from the Na⁺,K⁺-ATPase, while the transmembrane segments and the minor cytoplasmic loop came from the Ca²⁺-ATPase, was able to occlude Ca²⁺ and to be phosphorylated from ATP with normal apparent affinity for Ca²⁺ and ATP. This chimera also displayed normal sensitivity to thapsigargin, but was unable to undergo the transition from ADP-sensitive to ADP-insensitive phosphoenzyme and to transport Ca²⁺. The other chimera, which consisted of the NH₂-terminal two-thirds of Na⁺,K⁺-ATPase and the COOH-terminal one-third of Ca²⁺-ATPase, was unable to phosphorylate from ATP, but phosphorylated from inorganic phosphate in a Ca²⁺-inhibitable and thapsigargin-insensitive reaction. These results can be explained in terms of a structural model in which the non-conserved residues in the central cytoplasmic domain of the Ca²⁺-ATPase are without major importance for the binding and occlusion of Ca²⁺, but are involved in the E1P→E2P conformational changes of the phosphoenzyme, whereas residues in transmembrane segments on both sides of the central cytoplasmic domain are involved in formation of the Ca²⁺-binding sites. The data moreover show that thapsigargin sensitivity is dependent on residues in the NH₂-terminal one-third of the Ca²⁺-ATPase molecule.

【 授权许可】

Unknown