Cross-linking of the erythrocyte (Na+,K+)-ATPase. Chemical cross-linkers induce alpha-subunit-band 3 heterodimers and do not induce alpha-subunit homodimers.
Earlier studies (Periyasamy, S. M., Huang, W.-H., and Askari, A. (1983) J. Biol. Chem. 258, 9878-9885) suggested that Cu2+ and o-phenanthroline induced the formation of cross-linked homodimers between alpha-subunits of the erythrocyte (Na+,K+)-ATPase. This was interpreted as indicating that alpha-subunits existed in close proximity in native erythrocyte membranes. The alpha-subunit and band 3 monomers have similar molecular weights (M(r) approximately 100,000) and exist in the membrane in molar ratios of approximately 1:3000 alpha-subunit:band 3. We explored the possibility that alpha-subunit and band 3 could be induced to form heterodimeric structures in the presence of cross-linking reagents. Using methods similar to those employed in the above-cited reference we demonstrated that cross-linked dimers containing phosphorylated alpha-subunits had proteolytic sensitivity that was inconsistent with the formation of alpha-subunit homodimers and fully consistent with heterodimer formation between alpha-subunit and band 3. The data also indicated that alpha-subunit-band 3 heterodimer formation is dependent on the conformational state of the (Na+,K+)-ATPase. Using the appropriate reagents we obtained cross-linked products which were consistent with heterodimer formation between alpha- and beta-subunits of the (Na+,K+)-ATPase. Our data argue against a close association between pairs of (Na+,K+)-ATPase alpha-subunits in the human red cell membrane.
Martin
D W
DW
Department of Medicine, State University of New York, Stony Brook 11794-8151.
Sachs
J R
JR
D003300:Copper / Q000494:pharmacology*; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006801:Humans; D046911:Macromolecular Substances; D008970:Molecular Weight; D010449:Peptide Mapping; D010618:Phenanthrolines / Q000494:pharmacology*; D010766:Phosphorylation; D000254:Sodium-Potassium-Exchanging ATPase / Q000097:blood* / Q000302:isolation & purification; D013327:Strophanthidin / Q000494:pharmacology
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D010618:Phenanthrolines; D013327:Strophanthidin; D003300:Copper; D000254:Sodium-Potassium-Exchanging ATPase; C025205:1,10-phenanthroline