Cross-linking of the erythrocyte (Na+,K+)-ATPase. Chemical cross-linkers induce alpha-subunit-band 3 heterodimers and do not induce alpha-subunit homodimers

• Published in: The Journal of biological chemistry Vol. 267; no. 33; pp. 23922 - 23929
• Main Authors: MARTIN, D. W, SACHS, J. R
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 25-11-1992
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Copper - pharmacology; Cross-Linking Reagents - pharmacology; Enzymes and enzyme inhibitors; Erythrocyte Membrane - enzymology; Fundamental and applied biological sciences. Psychology; Humans; Hydrolases; Macromolecular Substances; Molecular Weight; Peptide Mapping; Phenanthrolines - pharmacology; Phosphorylation; Sodium-Potassium-Exchanging ATPase - blood; Sodium-Potassium-Exchanging ATPase - isolation & purification; Strophanthidin - pharmacology; Human; Band 3 protein; Radiolabelling; Enzyme; Na,K-ATPase; Red blood cell; Crosslinking; Subunit; Dimerization
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)35925-8

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.