Shift to the Na+ from of Na+/K+‐transporting ATPase due to modification of the low‐affinity ATP‐binding site by Co(NH3)4ATP

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Abstract
1. Inactivation of purified Na+/K+-transporting ATPase by the MgATP complex analogue Co(NH3)4ATP, which binds to the low-affinity ATP-binding site, results in the concomitant inhibition of the K+-activated p-nitrophenylphosphatase, which is considered to be a partial reaction catalyzed by the enzyme in the E2 conformational state. 2. Complete inactivation of Na+/K+-transporting ATPase by Co(NH3)4ATP does not alter the ADP/ATP exhange reaction which is considered to be part of the catalytic activity in the E1 conformation. 3. The enzyme binds eosin at the high-affinity ATP-binding site as measured by the change in eosin fluorescence. Eosin binding to the Co(NH3)4ATP-inactivated enzyme is, in contrast to the untreated enzyme, not stimulated by Na+. Inactivation by Co(NH3)4ATP increased the half-maximal opposing effect of K+ on eosin binding from 1.1 mM in the control to 43.2 mM in the almost completely inactive enzyme. No wosin fluorescence changes were observed when the Co(NH3)4ATP-inactivated enzyme was treated subsequently with CrATP. This MgATP complex analogue forms a stable compled at the high-affinity ATP-binding site. CrATP thus abolishes eosin binding. It is concluded, that Co(NH3)4ATP interacts with Na+/K+ -transporting ATPase in the E2 conformation and arrests it there. This effects eosin binding to the high-affinity ATP-binding site, since the K+ sensitivity is lost. A possible interpretation of these differing effects of Co(NH3)4ATP induces the Na+ formm (E1 form) in the corresponding .alpha.,.beta. protomer, as is indicated by the unaffected ADP/ATP exchange and the response of the eosin fluorescence on Na+ and K+.