The complexes of ouabain with sodium and potassium activated adenosine triphosphatase formed with various ligands: relationship to the complex formed in the beating heart

T. Akera, D. Ku, T. Tobin, T. M. Brody

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35 Scopus citations

Abstract

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 3 different forms of the ouabain enzyme complex prepared in vitro with rat brain enzyme. The ouabain enzyme complex formed with Mg2+ and Pi 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 Pi, 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 Pi 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.

Original languageEnglish
Pages (from-to)101-114
Number of pages14
JournalMolecular Pharmacology
Volume12
Issue number1
StatePublished - 1976

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

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