Molecular Pharmacology, Vol 9, 278-281, Copyright © 1973 by the American Society for Pharmacology and Experimental Therapeutics

Ouabain Binding to Sodium- and Potassium-Dependent Adenosine Triphosphatase: Inhibition by the ,-Methylene Analogue of Adenosine Triphosphate

¹ Department of Pharmacology, Michigan State University, East Lansing, Michigan 48823

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 ,-methylene ATP (adenylylmethylenediphosphonate) to support [³H]ouabain binding. ,-Methylene ATP is an analogue of ATP in which the - and -phosphates are linked by a methylene group. It is not hydrolyzed by the (Na⁺ + K⁺)-ATPase. In the presence of Na⁺ and Mg⁺⁺, ,-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 P_i, the addition of ,-methylene ATP was inhibitory, in contrast to the stimulation produced by ATP. These results show that ,-methylene ATP binds to the (Na⁺ + K⁺)-ATPase and that this interaction does not support [³H]ouabain binding.

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ACKNOWLEDGMENT The authors would like to thank Mrs. Annie Han for excellent technical assistance.