A fluorometric method for the determination of functional (Na,K)-ATPase and cardiac glycoside receptors.
A method to measure high-affinity binding sites for fluorescent ligands is described. The method is applied to the determination of (Na,K)-ATPase using the fluorescent ouabain derivative anthroylouabain (P. A. G. Fortes (1977) Biochemistry 16, 531-540). The method consists of measurements of the fluorescence intensities of a saturating concentration of anthroylouabain in the presence and absence of (Na,K)-ATPase and ouabain. These data and the fluorescence enhancement factor upon anthroylouabain binding are used to calculate the concentration of binding sites. The measurements can be done in a few minutes and 10 to 100 pmoles of ouabain sites is sufficient for accurate determinations. Because phosphorylation of (Na,K)-ATPase is necessary to bind anthroylouabain, only functional enzyme is detected by this method.
D000818:Animals; D000873:Anthracenes; D004285:Dogs; D004557:Electric Organ / Q000201:enzymology; D004593:Electrophorus; D007668:Kidney / Q000201:enzymology; D008024:Ligands; D010042:Ouabain / Q000031:analogs & derivatives; D011955:Receptors, Drug / Q000032:analysis*; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D000873:Anthracenes; D008024:Ligands; D011955:Receptors, Drug; C028996:cardiac glycoside receptors; D010042:Ouabain; C013814:anthroylouabain; D000254:Sodium-Potassium-Exchanging ATPase