BLOCKING OF NA+/K+ TRANSPORT BY THE MGPO4 COMPLEX ANALOG CO(NH3)4PO4 LEAVES THE NA+/NA+-EXCHANGE REACTION OF THE SODIUM-PUMP UNALTERED AND SHIFTS ITS HIGH-AFFINITY ATP-BINDING SITE TO A NA+-LIKE FORM

Inactivation of Na+/K+‐ATPase activity by the MgPO4 complex analogue Co(NH3)4PO4 leads, in everted red blood cell vesicles, to the parallel inactivation of 22Na+/K+ flux and 86Rb+/Rb+ exchange, but leaves the 22Na+/Na+‐exchange activity and the uncoupled ATP‐supported 22Na+ transport unaffected. Furthermore, inactivation of purified Na+/K+‐ATPase by Co(NH3)4PO4 leads to a parallel decrease of the capacity of the [3H]ouabain receptor site, when binding was studied by the Mg2+/Pi‐supported pathway (ouabain‐enzyme complex II) but the capacity of the ouabain receptor site was unaltered, when the Na+/Mg2+/ATP‐supported pathway (ouabain‐enzyme complex I) was used. No change in the dissociation constants of either ouabain receptor complex was observed following inactivation of Na+/K+‐ATPase. When eosin was used as a marker for the high‐affinity ATP‐binding site of the E1 conformation, formation of stable E′2· Co(NH3)4PO4 complex led to a shift in the high‐affinity ATP‐binding site towards the sodium form. This led to an increase in the dissociation constant of the enzyme complex with K+, from 1.4 mM with the unmodified enzyme to 280 mM with the Co(NH3)4PO4‐inactivated enzyme. It was concluded, that the effects of Co(NH3)4PO4 on the partial activities of the sodium pump are difficult to reconcile with an α,β‐protomeric enzyme working according the Albers‐Post scheme. The data are consistent with an α2,β2 diprotomeric enzyme of interacting catalytic subunits working with a modified version of the Albers‐Post model. Copyright © 1990, Wiley Blackwell. All rights reserved