The membrane-bound fraction of purified Na,K-ATPase was characterized following extensive proteolytic digestion in the presence of various physiological ligands which stabilize different conformational states of the sodium pump. There are distinctive conformational changes of the protein which are revealed by amino-terminal amino acid sequence analysis of the digests following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The changes in cleavage patterns result from alterations in domain-domain interactions of the protein. We provide evidence in the α-subunit for (i) tight interaction between part of the cytoplasmic ATP binding domain and the membrane-bound portion of the protein; (ii) involvement of the cytoplasmic loop between M2 and M3 in structural rearrangements upon phosphorylation or ion binding; (iii) generation of the same digested products when either ouabain or potassium (rubidium) is present. Similarly, evidence is provided for conformational sensitivity of the extracellular domain of the β-subunit. The position of the tryptic cleavage point in the β-subunit is altered depending on whether the α-subunit is phosphorylated or whether rubidium ions are occluded. Based upon the conformationally dependent patterns of exposure and protection of different tryptic cleavage sites in the α- and β-subunits we propose a model for intraprotein interactions within the α- subunit and between α- and β-subunits following the binding of physiological ligands to the Na,K-ATPase.
|Original language||English (US)|
|Number of pages||10|
|Journal||Journal of Biological Chemistry|
|State||Published - Feb 11 1994|
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