Conformational changes of renal sodium plus potassium ion-transport adenosine triphosphatase labeled with fluorescein.
We studied conformational changes of purified renal sodium plus potassium ion-transport adenosine triphosphatase (ATP phosphohydrolase, EC 3.6.1.3) labeled with fluorescein isothiocyanate. Fluorescein covalently binds to the alpha-subunit of the enzyme and inhibits the ATPase but not the p-nitrophenylphosphatase activity. Four unphosphorylated and three phosphorylated conformations were distinguished by the level of fluorescence and by the rate of its change (relative fluorescence is shown in percentages). Fluorescence of the ligand-free form (E1, 100%) was increased by Na+ (E1.Na form, 103%) and quenched by K+ (E2.K, 78%) at a site of high affinity (K0.5 for K+ = 0.07 mM). Mg2+ did not alter fluorescence of E1 or E1.Na but raised that of E2.K (E2.K.Mg form, 85-90%). Addition of excess Na+ to the E2.K.Mg form restored high fluorescence but the rate of transition from E2.K.Mg to E1.Na became progressively slower with increasing Mg2+ concentration. Two phosphorylated conformations, (E2-P).Mg (82%) and (E2-P).Mg.K (82%) were differentiated by a faster turnover of the latter form. A third conformation, (E2-P).Mg.ouabain, had the lowest fluorescence (56%) and its formation allowed the binding of ouabain to the phosphoenzyme. Reversible blocking of sulfhydryl groups with thimerosal inhibited the formation of E2.K and (E2-P).Mg.ouabain but not that of the other conformations of the fluorescein-enzyme. The thimerosal-treated fluorescein-enzyme retained K+-p-nitrophenylphosphatase activity, inhibition of this activity by ouabain and ouabain binding. The unphosphorylated enzyme had low (K0.5 = 1.2 mM) and the phosphoenzyme had high affinity (K0.5 = 0.03 - 0.09 mM) for Mg2+ in the absence of nucleotides. Since low and high affinity for Mg2+ alternates as the enzyme turns over, Mg2+ may be bound and released sequentially during the catalytic cycle.
D000818:Animals; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007679:Kidney Medulla / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D010042:Ouabain / Q000494:pharmacology; D010766:Phosphorylation; D011189:Potassium Chloride / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D012965:Sodium Chloride / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase; D013050:Spectrometry, Fluorescence; D013552:Swine; D013849:Thimerosal / Q000494:pharmacology; D013861:Thiocyanates
D005452:Fluoresceins; D005456:Fluorescent Dyes; D013861:Thiocyanates; D015636:Magnesium Chloride; D013849:Thimerosal; D012965:Sodium Chloride; D010042:Ouabain; D011189:Potassium Chloride; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate; D008274:Magnesium