Detection of a ouabain-induced structural change in the sodium, potassium-adenosine triphosphatase.
The structural consequences of ouabain interaction with a highly purified Na+K+-ATPase preparation, isolated from the outer medulla of porcine kidneys, were examined. The apparent heat capacity vs. temperature profile of the enzyme was obtained with a newly designed differential scanning calorimeter. The profile was characterized by a major endothermic transition at 55.3 degrees C. This transition appeared to correspond to irreversible protein denaturation since it was associated with loss of enzyme activity and the transition was not present in claorimetric profiles obtained after the initial scan of a sample. Interaction of ouabain with its receptor surface on the Na+, K+-ATPase shifted the endothermic transition from 55.3 to 59.5 degrees C and decreased the width of the transition. This indicated that the ouabain-Na+, K+-ATPase complex was more stable with respect to temperature and that the apparent cooperative nature of the transition was greater for the complex than for the untreated enzyme. The effects of the ouabain-enzyme interaction were examined with the fluorescence probe, 8-anilino-1-naphthalenesulfonic acid. The fluorescence of this dye in the presence of the enzyme was monitored as a function of temperature. These measurements also suggested that ouabain induces the formation of a more stable enzyme conformation. Incubation of the enzyme for 10 min at 53 degrees C with and without ouabain and measurement of remaining enzyme activity after the dissociation of bound ouabain confirmed the conclusions from the fluorescence and scanning calorimeter experiments.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000817:Anilino Naphthalenesulfonates; D000818:Animals; D002151:Calorimetry; D004789:Enzyme Activation / Q000187:drug effects; D007668:Kidney / Q000201:enzymology*; D007679:Kidney Medulla / Q000201:enzymology*; D010042:Ouabain / Q000494:pharmacology*; D011188:Potassium / Q000494:pharmacology; D011487:Protein Conformation; D012964:Sodium / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013696:Temperature
D000817:Anilino Naphthalenesulfonates; D010042:Ouabain; D012964:Sodium; D000251:Adenosine Triphosphatases; D011188:Potassium