Occlusion of the K+-Congener Tl+ through the Phosphointermediates of Na,K-ATPase

ROSSI, R. C.; NØRBY, J. G.

The Sodium Pump: Recent Developments

The Rockefeller University Press

Año: 1991; p. 419 - 422

The current model for the reaction mechanism of Na,K-ATPase proposes a cycle with Na+- and Mg2+-dependent phosphorylation by ATP and a K+-dependent dephosphorylation, accompanied by conformational transitions. Evidence has been found that K+ and its congeners, after promoting dephosphorylation, could remain tightly bound to the pump. This phenomenon, called occlusion, was interpreted as the expression of a transport intermediate in which the cations are "travelling" across the membrane and therefore are temporarily inaccessible to the aqueous solutions. The present work aimed at characterizing quantitatively the K+-transport steps at 20 °C using 204Tl+ as a probe for K+. With this goal we measured, in parallel experiments, the time courses of both phosphoenzyme formation and breakdown, and Tl+-occlusion via the dephosphorylation pathway. To measure occlusion, we have developed a quenching-and-washing chamber that allows the rapid isolation on a filter of the cation tightly bound to the enzyme from the free cation in solution. Results show that the time course of dephosphorylation is mirrored by that of Tl+ occlusion, with a ratio of two Tl+ per unit of enzyme that undergoes dephosphorylation. This would indicate that: (a) in the presence of Na+ and ATP, the Na,K/Pump becomes reactive to Tl+ only after the appearance of a phosphorylated intermediate; (b) the Tl+-dependent dephosphorylation requires the binding of two Tl+ per phosphoenzyme unit; (c) if any intermediate exists between the reactive phosphoenzyme with two Tl+ bound and the Tl+-occluded dephosphoform, its concentration is so small that it cannot be detected.