Quenching of 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole‐modified Na+/K+‐ATPase reveals a higher accessibility of the low‐affinity ATP‐binding site

7‐Chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD‐Cl) labeled Na+/K+‐ATPase covalently with two different inactivation constants (Ki=2.5 μM; Ki′=10 μM). It apparently modified the two different ATP‐binding sites of the enzyme since it decreased the activity of the E2ATP site, i.e. the K+‐activated para‐nitrophenylphosphatase activity, in an enzyme whose high‐affinity E1ATP site had been blocked by fluorescein 5′isothiocyanate (FITC). It also reduced the activity of the E1ATP site, i.e. the Na+‐activated protein phosphorylation, in an enzyme whose low‐affinity E2ATP site had been blocked by Co(NH3)4PO4. Fluorescence quenching experiments with KI, CsCl and MnCl2of the NBD‐Cl‐labeled Na+/K+‐ATPase revealed two differently accessible types of fluorophores depending on the ATP site: The E2ATP site apparently differs from the E1ATP site in that it is more open because the fluorophore labeling in the E2ATP site was sterically better accessible for quenchers.