The role of simultaneously existing ATP‐binding sites in the catalytic process of Na+/K+‐ATPase is unclear. In order to learn whether blocking the E1 ATP site affects the properties of the E2 ATP site, the E1 ATP site was inactivated by either fluorescein 5′‐isothiocyanate, the non‐phosphorylating Cr(H2O)4‐AdoPP [CH2]P or the phosphorylating Cr(H2O)4ATP. The properties of the remaining E2ATP site were studied by measuring ‘backdoor phosphorylation’ in the presence of ouabain, or K+‐activated hydrolysis of p‐nitrophenyl phosphate. The involvement of the E2ATP site was further tested by the effects of Co(NH3)4ATP, a specific inactivator of this site. When the E1ATP site was inactivated by fluorescein 5′‐isothiocyanate or the non‐phosphorylating Cr(H2O)4AdoPP [CH2]P, backdoor phosphorylation and the activity of K+‐activated p‐nitrophenylphosphatase remained unchanged. Both processes were lost, however, when the E2ATP site was additionally inactivated by Co(NH3)4ATP. Inactivation of the E1ATP site by fluorescein 5′‐isothiocyanate or Cr(H2O)4 AdoPP [CH2]P decreased the affinity of the P‐nitrophenyl‐phosphatase activity of the E2ATP site for the substrate p‐nitrophenyl phosphate by four times. This is consistent with a former report showing that dephosphorylation in a fluorescein 5′‐isothiocyanate‐inactivated Na+/K+‐ATPase has a lowered sensitivity for ATP [Scheiner‐Bobis, G., Antonipillai, J. & Farley, R. A. (1993) Biochemistry 32, 9592–9599]. Inactivation of the E1ATP site by the phosphorylating Cr(H2O)4ATP, however, led to a loss of the property of the E2ATP site to hydrolyse K+‐dependent p‐nitrophenyl phosphate and to achieve backdoor phosphorylation. Evidently, ATP sites coexist in Na+/K+‐ATPase, and binding of ATP to one site affects the property of the other site [Scheiner‐Bobis, G., Esmann, M. & Schoner, W. (1989) Eur. J. Biochem. 183, 173–178]. Although the enzyme can be phosphorylated from both ATP sites, phosphorylation of the E1ATP site excludes the phosphorylation of the E2ATP site.

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The role of simultaneously existing ATP-binding sites in the catalytic process of Na+/K(+)-ATPase is unclear. In order to learn whether blocking the E1ATP site affects the properties of the E2ATP site, the E1ATP site was inactivated by either fluorescein 5'-isothiocyanate, the non-phosphorylating Cr(H2O)4AdoPP[CH2]P or the phosphorylating Cr(H2O)4ATP. The properties of the remaining E2ATP site were studied by measuring 'backdoor phosphorylation' in the presence of ouabain, or K(+)-activated hydrolysis of p-nitrophenyl phosphate. The involvement of the E2ATP site was further tested by the effects of Co(NH3)4ATP, a specific inactivator of this site. When the E1ATP site was inactivated by fluorescein 5'-isothiocyanate or the non-phosphorylating Cr(H2O)4AdoPP[CH2]P, backdoor phosphorylation and the activity of K(+)-activated p-nitrophenylphosphatase remained unchanged. Both processes were lost, however, when the E2ATP site was additionally inactivated by Co(NH3)4ATP. Inactivation of the E1ATP site by fluorescein 5'-isothiocyanate or Cr(H2O)4AdoPP[CH2]P decreased the affinity of the p-nitrophenylphosphatase activity of the E2ATP site for the substrate p-nitrophenyl phosphate by four times. This is consistent with a former report showing that dephosphorylation in a fluorescein 5'-isothiocyanate-inactivated Na+/K(+)-ATPase has a lowered sensitivity for ATP [Scheiner-Bobis, G., Antonipillai, J. & Farley, R. A. (1993) Biochemistry 32, 9592-9599]. Inactivation of the E1ATP site by the phosphorylating Cr(H2O)4ATP, however, led to a loss of the property of the E2ATP site to hydrolyse K(+)-dependent p-nitrophenyl phosphate and to achieve backdoor phosphorylation. Evidently, ATP sites coexist in Na+/K(+)-ATPase, and binding of ATP to one site affects the property of the other site [Scheiner-Bobis, G., Esmann, M. & Schoner, W. (1989) Eur. J. Biochem. 183, 173-178]. Although the enzyme can be phosphorylated from both ATP sites, phosphorylation of the E1ATP site excludes the phosphorylation of the E2ATP site.

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