Role of the self-association of β subunits in the oligomeric structure of Na+/K+-ATPase

Abstract

The two subunits of Na+/K+-ATPase that are essential for function are α and β. Previous cross-linking studies on the oligomeric structure of the membrane-bound enzyme identified α,β and α,α associations, but only the former and not the latter could be detected after solubilization. To study the possibility of direct β,β association, the purified membrane enzyme and a trypsin-digested enzyme that occludes cations and contains an essentially intact β and fragments of α were subjected to oxidative cross-linking in the presence of Cu2+-phenanthroline. Resolution of products on polyacrylamide gels, N-terminal analysis and reactivity with anti-β antibody showed that, in addition to previously identified products (e.g. α,α and α,β dimers), a β,β dimer, most likely linked through intramembrane Cys44 residues of two chains, is also formed. This dimer was also noted when digitonin-solubilized intact enzyme, and the trypsin-digested enzyme solubilized with digitonin or polyoxyethylene 10-laurylether were subjected to cross-linking, indicating that the detected β,β association was not due to random collisions. In the digested enzyme, K+ but not Na+ enhanced β,β dimer formation. The alternative cross-linking of β-Cys44 to a Cys residue of a transmembrane α-helix was antagonized specifically by K+ or Na+. The findings (i) indicate the role of β,β association in maintaining the minimum oligomeric structure of (α,β)2, (ii) provide further support for conformation-dependent flexibilities of the spatial relations of the transmembrane helices of α and β and (iii) suggest the possibility of significant differences between the quaternary structures of the P-type ATPases that do and do not contain a β subunit.