Cys577 is a conformationally mobile residue in the ATP-binding domain of the Na,K-ATPase α-subunit

2-[4'-Maleimidylanilino]naphthalene 6-sulfonic acid (MIANS) irreversibly inactivates Na,K-ATPase in a time- and concentration-dependent manner. Inactivation is prevented by 3 mM ATP or low K+ (<1 mM); the protective effect K+ is reversed at higher concentrations, This biphasic effect was also observed with K+ congeners, In contrast, Na+ ions did not protect. MIANS inactivation disrupted high affinity ATP binding. Tryptic fragments of MIANS-labeled protein were analyzed by reversed phase high performance liquid chromatography, ATP clearly protected one major labeled peptide peak. This observation was confirmed by separation of tryptic peptides in SDS-polyacrylamide gel electrophoresis revealing a single fluorescently-labeled peptide of similar to 5 kDa, N-terminal. amino acid sequencing identified the peptide (V(545)LGFCH...), This hydrophobic peptide contains only two Cys residues in all sodium pump alpha-subunit sequences and is found in the major cytoplasmic loop between M4 and M5, a region previously associated with ATP binding. Subsequent digestion of the tryptic peptide with V8 protease and N-terminal amino acid sequencing identified the modified residue as Cys(577). The cation-dependent change in reactivity of Cys(577) implies structural alterations in the ATP-binding domain following cation binding and occlusion in the intramembrane domain of Na,K-ATPase and expands our knowledge of the extent to which cation binding and occlusion are sensed in the ATP hydrolysis domain.