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

T. Tobin, Amar K. Sen

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88 Scopus citations


Specific [3H]ouabain binding to guinea pig kidney (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 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 Pi 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 Ki 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.

Original languageEnglish
Pages (from-to)120-131
Number of pages12
JournalBBA - Enzymology
Issue number1
StatePublished - Jan 14 1970

Bibliographical note

Funding Information:
Tlus work was ~upported by grant MA-2485 from the Medical Research Council of Canada We are grateful to Drs J T F Wong and P Seeman fl)r helpful criticism in the preparation of this manuscript We x~isll to thank Mi~s Catharine Yates and Mr V\]jay Khanna fl~r excellent technical assistance

ASJC Scopus subject areas

  • General Medicine


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