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 E 2ATP site, i.e. the K +-activated para-nitrophenylphosphatase activity, in an enzyme whose high-affinity E 1ATP site had been blocked by fluorescein 5′isothiocyanate (FITC). It also reduced the activity of the E 1ATP site, i.e. the Na +-activated protein phosphorylation, in an enzyme whose low-affinity E 2ATP site had been blocked by Co(NH 3) 4PO 4. Fluorescence quenching experiments with KI, CsCl and MnCl 2of the NBD-Cl-labeled Na +/K +-ATPase revealed two differently accessible types of fluorophores depending on the ATP site: The E 2ATP site apparently differs from the E 1ATP site in that it is more open because the fluorophore labeling in the E 2ATP site was sterically better accessible for quenchers.