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 microM; Ki' = 10 microM). 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 MnCl2 of 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.