title: Stabilisation of Na,K-ATPase structure by the cardiotonic steroid ouabain.
abstract: Cardiotonic steroids such as ouabain bind with high affinity to the membrane-bound cation-transporting P-type Na,K-ATPase, leading to complete inhibition of the enzyme. Using synchrotron radiation circular dichroism spectroscopy we show that the enzyme-ouabain complex is less susceptible to thermal denaturation (unfolding) than the ouabain-free enzyme, and this protection is observed with Na,K-ATPase purified from pig kidney as well as from shark rectal glands. It is also shown that detergent-solubilised preparations of Na,K-ATPase are stabilised by ouabain, which could account for the successful crystallisation of Na,K-ATPase in the ouabain-bound form. The secondary structure is not significantly affected by the binding of ouabain. Ouabain appears however, to induce a reorganization of the tertiary structure towards a more compact protein structure which is less prone to unfolding; recent crystal structures of the two enzymes are consistent with this interpretation. These circular dichroism spectroscopic studies in solution therefore provide complementary information to that provided by crystallography.
authors.affiliation: Institute of Structural and Molecular Biology, Birkbeck College, University of London, London, UK.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Miles
authors.forename: Natalya U
authors.initials: NU
authors.lastname: Fedosova
authors.forename: Søren V
authors.initials: SV
authors.lastname: Hoffmann
authors.forename: B A
authors.initials: BA
authors.lastname: Wallace
authors.forename: Mikael
authors.initials: M
authors.lastname: Esmann
mesh_terms: D002316:Cardiotonic Agents; D002462:Cell Membrane / Q000737:chemistry*; D004789:Enzyme Activation; D004795:Enzyme Stability; D010042:Ouabain / Q000737:chemistry*; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000648:ultrastructure*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D002316:Cardiotonic Agents; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase