Occlusion of 22Na+ and 86Rb+ in membrane-bound and soluble protomeric alpha beta-units of Na,K-ATPase.
In this work, we examined occlusion of 22Na+ and 86Rb+ in membranous and detergent-solubilized Na,K-ATPase from outer renal medulla. Optimum conditions for occlusion of 22Na+ were provided by formation of the phosphorylated complex from the beta,gamma-bidentate complex of chromium (III) with ATP (CrATP). Release of occluded cations occurred at equally slow rates in soluble and membrane-bound Na,K-ATPase. Values of 22Na+ occlusion as high as 11 nmol/mg of protein were measured, corresponding to 1.8-2.7 mol of Na+/mol of phosphorylated Na,K-ATPase as determined by 32P incorporation from [gamma-32P]CrATP. Maximum capacity for phosphorylation from [gamma-32P]CrATP was 6 nmol/mg of protein and equal to capacities for binding of [48V]vanadate and [3H]ouabain. The stoichiometry for occlusion of Rb+ was close to 2 Rb+ ions/phosphorylation site. In an analytical ultracentrifuge, the soluble Na+- or Rb+-occluded complexes showed sedimentation velocities (S20,w = 6.8-7.4) consistent with monomeric alpha beta-units. The data show that soluble monomeric alpha beta-units of Na,K-ATPase can occlude Rb+ or Na+ with the same stoichiometry as the membrane-bound enzyme. The structural basis for occlusion of cations in Na,K-ATPase is suggested to be the formation of a cavity inside a monomeric alpha beta-unit constituting the minimum protein unit required for active Na,K-transport.
D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D002414:Cations, Monovalent; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D007679:Kidney Medulla / Q000201:enzymology; D007700:Kinetics; D010042:Ouabain / Q000378:metabolism; D010766:Phosphorylation; D011868:Radioisotopes; D012413:Rubidium / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D012979:Sodium Radioisotopes; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D014461:Ultracentrifugation; D014638:Vanadates; D014639:Vanadium / Q000378:metabolism
D002414:Cations, Monovalent; D011868:Radioisotopes; D012979:Sodium Radioisotopes; D014639:Vanadium; D014638:Vanadates; D010042:Ouabain; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium