Rearrangements of the membrane-associated segments of Na⁺/K⁺ -ATPase upon phosphorylation, ion-occlusion and ouabain binding

Abstract

The reaction cycle of the Na⁺/K⁺-ATPase includes several intermediate stages, which are produced by the binding of different physiological ligands accompanied by conformational changes of the enzyme (2). Recently we have shown that extensive proteolytic digestion in the presence of various physiological ligands could be a useful tool in studies of rearrangements of membrane-associated segments of Na⁺/K⁺ -ATPase (10). Distinctive conformational changes were revealed by N-terminal amino acid sequence analysis of the digests following SDS PAGE (10). The changes in cleavage patterns result from alterations in domain-domain interactions of the protein. Here, we exploit this approach to shed light on events which occur following ouabain binding. We addressed this question by a comparative study of the posttryptic products obtained after extensive degradation of Na⁺/K⁺-ATPase after phosphorylation, in the presence of potassium or Mg,Pi,ouabain.