Molecular Pharmacology, Vol 12, 101-114, Copyright © 1976 by the American Society for Pharmacology and Experimental Therapeutics

The Complexes of Ouabain with Sodium- and Potassium-Activated Adenosine Triphosphatase Formed with Various Ligands: Relationship to the Complex Formed in the Beating Heart

¹ Department of Pharmacology, Michigan State University, East Lansing, Michigan 48824

The characteristics of complexes of [³H]ouabain with (Na⁺ + K⁺)-ATPase [Mg²⁺-dependent, (Na⁺, K⁺)-activated ATP phosphohydrolase, EC 3.6.1.3.] formed in vitro in the presence of various ligands were compared using partially purified rat brain and dog heart enzyme preparations and dog heart homogenates. The differences in characteristics of the ouabain-enzyme complexes were detected by monitoring dissociation rates in media of low ionic strength. It appeared that there are at least three different forms of the ouabain-enzyme complex prepared in vitro with rat brain enzyme. The ouabain-enzyme complex formed with Mg²⁺ and P_i was stable and unaffected by K⁺ added to the dissociation mixture. The ouabain-enzyme complex formed in the presence of Mg²⁺ and ATP had an intermediary dissociation rate in the absence of K⁺ and was not stabilized by K⁺. The addition of Na⁺ to the binding mixture containing Mg²⁺ and ATP increased the ouabain binding and resulted in the formation of an unstable complex. The addition of K⁺ to the mixture after the termination of the binding reaction stabilized the complex. In contrast, the presence of K⁺ in the binding mixture with Na⁺, Mg²⁺, and ATP resulted in a K⁺-insensitive, intermediary stable complex which was similar to that formed in the presence of Mg²⁺ and ATP. With dog heart enzyme, the ouabain-enzyme complex formed in the presence of Na⁺, K⁺, Mg²⁺, and ATP had characteristics similar to that formed in the presence of Na⁺, Mg²⁺, and ATP. Both complexes were stabilized by K⁺ added to the dissociation mixture. The [³H]ouabain bound to ventricular tissue during the Langendorff perfusion of isolated puppy hearts and dissociated in vitro was stabilized by the addition of K⁺, like the complex formed in vitro in the presence of either Na⁺, Mg²⁺, and ATP or Na⁺, K⁺, Mg²⁺, and ATP, but unlike the complex formed in the presence of Mg²⁺ and P_i, which was not stabilized by K⁺. It was concluded that ouabain binds to different forms of the phosphoenzyme under various ligand conditions, resulting in different forms of the ouabain-enzyme complex, and that the ouabain-enzyme complex formed in the presence of Mg²⁺ and P_i is not a suitable model for the drug-enzyme interaction occurring in vivo. Additionally, the dissociation characteristics of the ouabain-enzyme complex formed in vitro were not influenced by the concentration of ouabain or by the duration of the drug-enzyme interaction.

Note:
ACKNOWLEDGMENTS The authors thank Mr. Roxy H.-M. So and Mrs. V.-M. K. Cheng for expert technical assistance.

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