Co(NH₃)₄ATP has been used to probe ATP binding sites in the sodium pump. The purified enzyme (J¿rgensen, 1974) binds the analogue at two sites, with K_D = 0.1 µM and 0.4-0.6 mM (Scheiner-Bobis et al. 1987; Ward & Cavieres, 2003). At the latter, binding is followed by slow non-covalent trapping and inactivation of the K⁺-dependent reactions of the pump cycle. We have reported (Ward et al. 1997) that low concentrations of ATP and ATP analogues like Co(NH₃)₄ATP itself promote the release of the occluded [³H]- or [λ³³P]-labelled Co(NH₃)₄ATP. The time courses fit the sum of two decaying exponentials. At 20°C, the amplitude for the fast exponential increases hyperbolically with the ATP concentration, with K_0.5 = 3.4 µM, and 1/t{special} for the slow exponential increases with K_0.5 = 1.9 µM. This indicates that ATP binds to the Co(NH₃)₄ATP-occluded enzyme and accelerates a slow deocclusion reaction; the released analogue would then bind back to the pump at equilibrium, with a very high affinity, and under ATP competition.

The high-affinity rebinding was exposed by passive dilution of the suspension of Co(NH₃)₄[³H]ATP-enzyme (no nucleotide added), as this caused Co(NH₃)₄[³H]ATP release; we estimated K_D = 11 nM from the mass action. Alternatively, we released Co(NH₃)₄[³H]ATP with ATP and removed the latter on DEAE-Sephadex. When we presented the released analogue to the washed depleted enzyme (still ‘K⁺-inactive’), we estimated K_D = 26 nM. With the native Na⁺,K⁺-ATPase instead, K_D was 70 nM, not too different from the high affinity towards fresh analogue. The partially inactivated, depleted pump could also bind [λ³³P]ATP and fresh Co(NH₃)₄[³H]-ATP with K_D values of 330 and 450 nM, respectively.

These results suggest that ATP and other nucleotides bind at the extant high affinity site of the Co(NH₃)₄ATP-enzyme, promoting Co(NH₃)₄ATP deocclusion and a decrease of Co(NH₃)₄ATP rebinding affinity at the modified regulatory site. By extension, these findings imply that negative co-operativity between the two ATP sites plays an important role in the reaction of native Na⁺,K⁺-ATPase.

This work was supported by research grants from The Wellcome Trust and the Medical Research Council.