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ATP and its analogues act on the minimal functional unit of Na, K-ATPase, the alpha beta protomer, with high and low affinity effects. Fluorescein isothiocyanate (FITC) irreversibly blocks the high affinity, or catalytic, ATP site, and yet the surviving K+-phosphatase activity of soluble FITC-modified alphabeta protomers can be photoinactivated by 2'(3')-O-trinitrophenyl (TNP)-8N3-ADP (Ward, D. G., and Cavieres, J. D. (1998) J. Biol. Chem. 273, 14277-14284). We have now used TNP-8N3-[alpha-32P]ADP as a photoaffinity label for Na,K-ATPase. The native enzyme can be photolabeled at 5 microM TNP-8N3-[alpha-32P]ADP, and ATP or FITC treatment prevents labeling of the alpha chain. At 25 microM, however, TNP-8N3-[alpha-32P]ADP can be incorporated in the FITC-modified alpha chain, concurrently with the inactivation of the K+-phosphatase activity, to an extrapolated level of 0.5-1.2 mol of 32P-probe per mol of alpha chain. Photoinactivation and labeling are prevented by TNP-ADP, vanadate, or strophanthidin and are promoted by Na+ or Mg2+, but not K+. The cation effects suggest that the fluorescein-modified enzyme incorporates the TNP-8N3-[alpha-32P]ADP. Mg complex preferentially, and the free probe when in the E1 enzyme form and after occupation of a low-affinity Na+ site. Partial trypsinolysis reveals that the point of TNP-8N3-[alpha-32P]ADP attachment is on the C-terminal 58-kDa fragment of the FITC-modified alpha chain. The affinity labeling of the fluorescein enzyme by TNP-8N3-[alpha-32P]ADP endorses the view that two nucleotide sites can be occupied simultaneously in each alpha subunit of Na,K-ATPase.

1. Thallium (Tl) inhibits the ouabain‐sensitive K influx in human red cells in high‐Na medium. At 1 m Mexternal K concentration [Ko], the ouabain‐sensitive K influx decreases steadily with increasing Tl concentration, up to 0·9 m Moutside; at 0·17 m M‐Ko, however, Tl stimulates the ouabain‐sensitive K influx below 0·1 m M‐Tloand inhibits it at higher concentrations.

1. The activation by Na ions and the effect of the anti‐L antibody on the sodium pump of low‐potassium type (LK) erythrocytes, have been studied by measuring ouabain‐sensitive ATPase activity of red cell membranes of LK goats. The experimental data were first corrected for incomplete occupation of the external K sites of the pump, using a saturation function obtained from influx experiments.

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.

The Na,K-ATPase activity of the sodium pump exhibits apparent multisite kinetics toward ATP, a feature that is inherent to the minimal enzyme unit, the alpha beta protomer. We have argued that this should arise from separate catalytic and noncatalytic sites on the alpha beta protomer as fluorescein isothiocyanate (FITC) blocks a high affinity ATP site on all alpha subunits and yet the modified Na, K-ATPase retains a low affinity response to nucleotides (Ward, D. G., and Cavieres, J. D. (1996) J. Biol. Chem. 271, 12317-12321). We now find that 2'(3')-O-(2,4,6-trinitrophenyl)8-azido-adenosine 5'-diphosphate (TNP-8N3-ADP), a high affinity photoactivatable analogue of ATP, can inhibit the K+-phosphatase activity of the FITC-modified enzyme during assays in dimmed light. The inhibition occurs with a Ki of 140 microM at 20 mM K+; it requires the adenine ring as 2'(3')-O-(2,4 6-trinitrophenyl) (TNP)-UDP or TNP-uridine are less potent and 2,4,6-trinitrobenzene-sulfonate is ineffective. Under irradiation with UV light, TNP-8N3-ADP inactivates the K+-phosphatase activity of the fluorescein-enzyme and also its phosphorylation by [32P]Pi. The photoinactivation process is stimulated by Na+ or Mg2+, and is inhibited by K+ or excess TNP-ADP. In the presence of 50 mM Na+ and 1 mM Mg2+, TNP-8N3-ADP photoinactivates with a K0.5 of 15 microM. Furthermore, TNP-8N3-ADP photoinactivates the FITC-modified, solubilized alpha beta protomers, even more effectively than the membrane-bound fluorescein-enzyme. These results strongly suggest that catalytic and allosteric ATP sites coexist on the alpha beta protomer of Na,K-ATPase.

1. We have developed a procedure for preparing resealed red cell ghosts that contain ADP but very little ATP. 2. The procedure involves (i) lysis of the cells in a very large volume of lysing solution, (ii) resuspension of the ghosts in a small volume, (iii) the incorporation into the ghosts, before they are resealed, of the adenylate kinase inhibitor P1,P5‐di(adenosine‐5'‐)pentaphosphate (AP5A) and of hexokinase, and (iv) the removal of traces of ATP, formed by residual adenylate kinase activity, by the addition of glucose. 3. Measurements of sodium efflux from ghosts prepared in this way show that sodium‐sodium exchange through the sodium pump does not occur in the absence of ATP even if ADP is present. 4. The beta:gamma imido analogue of ATP (AMP.PNP), which is incapable of phosphorylating sodium, potassium‐ATPase, cannot replace ATP in supporting sodium‐sodium exchange. 5. These findings support the hypothesis that the outward movement of sodium ions through the sodium pump is associated with the transfer of a phosphoryl group from ATP to the enzyme, and that the inward movement of sodium ions through the pump is associated with the return of a phosphoryl group from the phosphoenzyme to ADP.