Role of the self-association of beta subunits in the oligomeric structure of Na+/K+-ATPase.
The two subunits of Na(+)/K(+)-ATPase that are essential for function are alpha and beta. Previous cross-linking studies on the oligomeric structure of the membrane-bound enzyme identified alpha,beta and alpha,alpha associations, but only the former and not the latter could be detected after solubilization. To study the possibility of direct beta,beta association, the purified membrane enzyme and a trypsin-digested enzyme that occludes cations and contains an essentially intact beta and fragments of alpha were subjected to oxidative cross-linking in the presence of Cu(2+)-phenanthroline. Resolution of products on polyacrylamide gels, N-terminal analysis and reactivity with anti-beta antibody showed that, in addition to previously identified products (e.g. alpha,alpha and alpha,beta dimers), a beta,beta dimer, most likely linked through intramembrane Cys(44) 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 beta,beta association was not due to random collisions. In the digested enzyme, K(+) but not Na(+) enhanced beta,beta dimer formation. The alternative cross-linking of beta-Cys(44) to a Cys residue of a transmembrane alpha-helix was antagonized specifically by K(+) or Na(+). The findings (i) indicate the role of beta,beta association in maintaining the minimum oligomeric structure of (alpha,beta)(2), (ii) provide further support for conformation-dependent flexibilities of the spatial relations of the transmembrane helices of alpha and beta and (iii) suggest the possibility of significant differences between the quaternary structures of the P-type ATPases that do and do not contain a beta subunit.
Ivanov
Alexander V
AV
Department of Pharmacology, Medical College of Ohio, 3035 Arlington Avenue, Toledo, OH 43614-5804, U.S.A. aivanov@mco.edu
Modyanov
Nikolai N
NN
Askari
Amir
A
D000818:Animals; D002384:Catalysis; D002412:Cations; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D003902:Detergents / Q000494:pharmacology; D004072:Digitonin / Q000494:pharmacology; D019281:Dimerization; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D007202:Indicators and Reagents / Q000494:pharmacology; D007477:Ions / Q000378:metabolism; D007679:Kidney Medulla / Q000201:enzymology; D008024:Ligands; D010100:Oxygen / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D013552:Swine; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology
D002412:Cations; D003432:Cross-Linking Reagents; D003902:Detergents; D007202:Indicators and Reagents; D007477:Ions; D008024:Ligands; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase; D003545:Cysteine; D004072:Digitonin; D011188:Potassium; D010100:Oxygen