Binding of 2'(3')-O-(2,4-6-trinitrophenyl) ADP to soluble alpha beta protomers of Na, K-ATPase modified with fluorescein isothiocyanate. Evidence for two distinct nucleotide sites.

The overall reaction of well-defined solubilized protomers of Na,K-ATPase (one alpha plus one beta subunit) retains the dual ATP dependence observed with the membrane-bound enzyme, with distinctive ATP effects in the submicromolar and submillimolar ranges (Ward, D. G., and Cavieres, J. D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5332-5336). We have now found that the K+/-phosphatase activity of the alpha beta protomers is still inhibited by 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP). What is most significant is that the TNP-ADP effect can be observed clearly with protomeric enzyme whose high affinity ATP site has been blocked covalently with fluorescein isothiocyanate. We conclude that nucleotides can bind at two discrete sites in each protomeric unit of Na,K-ATPase.