title: Effect of ouabain binding on the fluorescent properties of the Na+/K+-ATPase.
abstract: The influence of occupancy by ouabain of its specific binding site on the stability and conformation of the Na+/K+-ATPase has been investigated. When native Na+/K+-ATPase is exposed to guanidinium chloride or diluted acid, tryptophanyl fluorescence falls to 50% of the initial value. If ouabain is bound, higher concentrations of GdmCl or acidity are needed to reach the same decrease in fluorescence. The rotational diffusion coefficient (relaxation time), shows higher values for the Na+/K+-ATPase (ouabain) complex compared to the enzyme alone, suggesting an increase in molecular asymmetry. This observation is confirmed by the Stern-Volmer analysis that shows an increase in the accessibility of the fluorophores in the Na+/K+-ATPase (ouabain) (KSV = 15.6 M-1) with respect to the native enzyme (KSV = 12.5 M-1). Iodine perturbation of the enzyme labelled with FITC, demonstrates a decrease in the accessibility of the fluorescein probe in the Na+/K+-ATPase(ouabain) (KSV = 4 M-1) compared to the Na+/K+-ATPase (KSV = 7 M-1) indicating that after ouabain binding this site of the enzyme is less exposed to the solvent. These data, in agreement with other reports, suggest an allosteric effect of ouabain binding on the Na+/K+-ATPase conformation.
authors.affiliation: Istituto di Medicina Sperimentale C.N.R., Roma, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Grimaldi
authors.forename: E
authors.initials: E
authors.lastname: Pascale
authors.forename: D
authors.initials: D
authors.lastname: Pozzi
authors.forename: M
authors.initials: M
authors.lastname: D'Onofrio
authors.forename: M G
authors.initials: MG
authors.lastname: Giganti
authors.forename: R
authors.initials: R
authors.lastname: Verna
mesh_terms: D020106:Acrylamide; D000178:Acrylamides; D000818:Animals; D005453:Fluorescence; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007454:Iodides; D010042:Ouabain / Q000378:metabolism*; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013552:Swine; D014364:Tryptophan
chemical_list: D000178:Acrylamides; D006146:Guanidines; D007454:Iodides; D020106:Acrylamide; D010042:Ouabain; D014364:Tryptophan; D000254:Sodium-Potassium-Exchanging ATPase; D019791:Guanidine