INVESTIGATION OF ION-BINDING TO THE CYTOPLASMIC-BINDING SITES OF THE NA,K-PUMP

A dual-wavelength fluorimeter was constructed, which used two light em itting diodes (LEDs) to excite the fluorescence dye RH 421 alternately with two different wavelengths. The ratio of the emissions at the two excitation wavelengths provided a drift-insensitive signal, which all owed detection of very small changes of the fluorescence intensity. Th ose small changes were induced by ion binding and release in conformat ion E(1) of the Na,K-ATPase, Titration experiments were performed to d etermine equilibrium dissociation constants (+/- standard deviation) f or each step in the complete binding and release sequence: 0.12 +/- 0. 01 mM (E(2)(K-2) <-> KE(1)), 0.08 +/- 0.01 mM (KE(1) <-> E(1)), 3.0 +/ - 0.2 mM (NaE(1)<-> E(1)), 5.2 +/- 0.4 mM (Na(2)E(1) <-> NaE(1)) and 6 .5 +/- 0.4 mM (Na(3)E(1) <-> Na(2)E(1)) at pH 7.2 and T = 16 degrees C . These numbers show that the affinities of the binding sites exposed to the cytoplasm, are higher for K+ than for Naf ions, similar to what was found on the extracellular side. The physiological requirement fo r extrusion of Na+ from the cytoplasm, and for import of K+ from the e xtracellular medium seems to be facilitated not by favorable binding a ffinities in state E(1) but by the two ATP-driven reaction steps of th e cycle, E(2)(K-2) + ATP --> K(2)E(1) . ATP and Na(3)E(1) . ATP <-> (N a-3) E(1)-P, which border the ion exchange reactions at the binding si tes in conformation E(1).