Stability and ligand sensitivity of [³H]ouabain binding to (Na⁺ + K⁺)-ATPase

Specific [³H]ouabain binding to guinea pig kidney (Na⁺ + K⁺)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) at 37° was determined. At less than 1·10^-6 M ouabain specific binding to the native enzyme was negligible. Specific binding occurred in two ways. Binding was stimulated by Mg²⁺ or Mg²⁺ and P_i which greatly increased the affinity of the enzyme for ouabain. This binding was saturated at 10 μM ouabain and reduced by Na⁺ or EDTA. The Na⁺ inhibition showed cooperative effects, apparent K_i values between 2.5 and 70 mM and appeared to be indirect. K⁺ at low concentrations stimulated, then at higher concentrations inhibited this binding. The inhibition appeared to be competitive. Binding in the presence of 200 mM Na⁺ required ATP and Mg²⁺. In the presence of ATP and Mg²⁺, binding was stimulated by 0-16 mM Na⁺ and further increased by 200 mM Na⁺. SrCl₂ or ADP did not support binding in the presence of Na⁺. The half-life of the enzyme-ouabain complex was 9 h at 0°. On warming, the rate of dissociation increased exponentially with temperature to t _{1 2} = 3 min at 37°. The experiments suggest that there is one type of specific binding site and that the binding pathways are reciprocally related with respect to Na⁺. They suggest different binding sites and conformations of maximum affinity for Na⁺ and ouabain. The Na⁺-stimulated pathway requires phosphorylation. Phosphorylation and/or ouabain binding appear to alter the affinity of the Na⁺ sites. Any single non-covalent bond type appears insufficient to explain the enzyme-ouabain interaction.