Blocking of Na+/K+ transport by the MgPO4 complex analogue 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 alpha, beta-protomeric enzyme working according the Albers-Post scheme. The data are consistent with an alpha 2, beta 2 diprotomeric enzyme of interacting catalytic subunits working with a modified version of the Albers-Post model.