Rb+ occlusion in renal (Na+ + K+)-ATPase characterized with a simple manual assay.
This paper describes properties of a simple manual assay for Rb+ occlusion on renal (Na+ + K+)-ATPase. Rb+ occlusion is measured by applying the enzyme plus Rb+ (86Rb) mixture to a Dowex-50 cation exchange column at 0 degree C, and eluting the enzyme with occluded Rb+ using an ice-cold sucrose solution. The enzyme-Rb+ complex is quite stable at 0 degree C. This method is useful for measuring Rb+ occlusion under equilibrium binding conditions and slow rates of dissociation of the enzyme-Rb+ complex. The stoichiometry of Rb+ occluded per phosphorylation site is 2. Rb+ saturation curves are strictly hyperbolic, suggesting that the two Rb+ sites have very different affinities, one in the micromolar range and one in the tens of millimolar range. ATP shifts the Rb+ saturation curves to the right (control K0.5 100-200 microM; plus ATP, K0.5 0.8-1.4 mM, in a 100 mM Tris-HCl medium, pH 7.0) and reduces the maximal level occluded (control approx. 4 nmol/mg; plus ATP approx. 3 nmol/mg protein). Thus, as expected, ATP shifts the E(1)2Rb+-E2(2Rb+)occ equilibrium towards E1. Sodium ions at concentrations of up to 30 mM compete with the rubidium ions, KNa = 1.86 mM in the Tris-HCl medium. Na+ at higher concentrations (30-100 mM) has an added non-competitive antagonistic effect. At room temperature, Rb+ dissociates slowly from the enzyme, kobs = 0.08 s-1, in the presence of either Rb+ (20 mM) or Na, (100 mM). As expected, dissociation is greatly accelerated by ATP, the rate being to fast to be measured by this technique. (Na+ + K+)-ATPase proteolyzed selectively by chymotrypsin in a Na+ medium, occludes Rb+. For control and proteolyzed (Na+ + K+)-ATPase the Rb+ saturation curves are similar and the rates of dissociation of the enzyme-Rb+ complex are identical. The chymotryptic split appears to disrupt antagonistic interactions between cation and ATP binding domains, while the E1-E2 conformational transition of the unphosphorylated protein probably remains.
Shani
M
M
Biochemistry Department, Weizmann Institute of Science, Rehovot, Israel.
Goldschleger
R
R
Karlish
S J
SJ
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D002414:Cations, Monovalent; D002918:Chymotrypsin / Q000378:metabolism; D003080:Cold Temperature; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D012413:Rubidium / Q000378:metabolism*; D012414:Rubidium Radioisotopes; D012964:Sodium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013552:Swine
D002414:Cations, Monovalent; D010710:Phosphates; D012414:Rubidium Radioisotopes; D000255:Adenosine Triphosphate; D012964:Sodium; D002918:Chymotrypsin; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium