High-affinity 86Rb-binding and structural changes in the alpha-subunit of Na+,K+-ATPase as detected by tryptic digestion and fluorescence analysis.
High-affinity 86Rb-binding has been related to tryptic cleavage and fluorescence from intrinsic and extrinsic probes in order to examine the relationship of cation binding to structural transitions in the alpha-subunit of pure membrane-bound Na+,K+-ATPase from the outer renal medulla. Native Na+,K+-Atpase binds two Rb+ ions per alpha-subunit (12.3 nmol/mg protein) with high affinity (Kd = 7.5 microM) in 25 mM Tris-HCl, pH 7.5. Enzyme with one molecule of covalently attached fluorescein per alpha-subunit has the same capacity (12.8 nmol/mg protein) but a much lower affinity for Rb+ (Kd = 29.2 microM). The changes in conformational state of the protein are correlated with occupancy of the high-affinity sites for Rb+, also at concentrations of Rb+ below the Kd. Titration at varying ionic strength suggests that the E2-form is the relaxed or native conformation of the alpha-subunit. Changes in tryptic digestion pattern and in fluorescence are parallel events both in the conditions of the binding assay and at physiological ionic strength. Reversible blocking of sulfhydryl groups with Thimerosal (ethylmercurythiosalicylate) abolishes the fluorescence responses to K+ or Rb+ without affecting the capacity or the affinity for binding of 86Rb. The demonstration of high-affinity binding of Rb+ without coupling to a conformational change suggests that the E1-form of the protein exposes sites for tight binding of K+ or Rb+ at the cytoplasmic membrane surface.
D000818:Animals; D001665:Binding Sites; D066298:In Vitro Techniques; D007679:Kidney Medulla / Q000201:enzymology; D007700:Kinetics; D011487:Protein Conformation; D012413:Rubidium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013552:Swine; D013849:Thimerosal; D014357:Trypsin; D014364:Tryptophan
D013849:Thimerosal; D014364:Tryptophan; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium