Search

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.

The high‐affinity E1ATP site of Na+/K+‐ATPase labeled with fluorescein 5′‐isothiocyanate and its E2ATP site labeled with erythrosin 5′‐isothiocyanate (ErITC), as was shown recently [Linnertz et al. (1998) J. Biol. Chem. 273, 28813–28821], reside on separate and adjacent catalytic α subunits. This paper provides evidence that specific labeling of the E2ATP binding site with ErITC resulted in a modification of the Cys549residue in the tryptic fragment with the sequence Val545‐Leu‐Gly‐Phe‐Cys549‐His550. Hence, Cys549is part of or close to the low‐affinity E2ATP binding site of Na+/K+‐ATPase.