Ouabain binding to sodium and potassium dependent adenosine triphosphatase: inhibition by the β,γ methylene analogue of adenosine triphosphate

To determine the mechanism of nucleotide dependent, Na⁺ stimulated binding of [³H]ouabain to (Na⁺ + K⁺)-ATPase (EC 3.6.1.3.), we tested the ability of β, γ methylene ATP (adenylylmethylenediphosphonate) to support [³H]ouabain binding. β, γ Methylene ATP is an analogue of AN ANALOGUE OF ATP in which the β and γ phosphates are linked by a methylene group. It is not hydrolyzed by the (Na⁺ + K⁺)-ATPase. In the presence of Na⁺ and Mg⁺⁺, β, γ methylene ATP did not support [³H]ouabain binding to rat brain (Na⁺ + K⁺)-ATPase and it inhibited ATP dependent binding. When [³H]ouabain binding to guinea pig kidney (Na⁺ + K⁺)-ATPase was determined in the presence of Na⁺, Mg⁺⁺, and Pi, the addition of β, γ methylene ATP was inhibitory, in contrast to the stimulation produced by ATP. The results show that β, γ methylene ATP binds to the (Na⁺ + K⁺)-ATPase and that this interaction does not support [³H]ouabain binding.