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

Abstract

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.

Molecular Distance Measurements Reveal an (αβ)2Dimeric Structure of Na+/K+-ATPase

“…that at least 2 mol of phosphate can be incorporated into the catalytic ␣-subunit per mol of ouabain binding sites (14), is consistent with the coexistence of phosphorylated intermediates (E 1 P, E*P, and E 2 P (15)) at different places in an oligomeric enzyme. Also, the observations of phosphorylation from P i during Na ϩ -ATPase activity (16) and in an FITC-treated enzyme (10) are consistent with the possibility that Na ϩ /K ϩ -ATPase is phosphorylated from both ATP sites (12,17).…”

“…Analysis (11,12), and Na ϩ -dependent phosphorylation (frontdoor phosphorylation) detects an activity of the E 1 ATP site (1,4,21). Interference by ErITC of the E 1 ATP site can be detected from the change of the Na ϩ -dependent phosphorylation of Na ϩ /K ϩ -ATPase by the chosen isothiocyanate in an enzyme whose E 2 ATP site had been blocked with Co(NH 3 ) 4 PO 4 .…”

Phosphate binding and ATP‐binding sites coexist in Na+/K+‐transporting ATPase, as demonstrated by the inactivating MgPO4 complex analogue Co(NH3)4PO4

“…lo), ADP/ATP exchange (Fig. 12), unchanged 22Na+/Naf exchange, and uncoupled "Na+ transport in C~(NH~)~PO,-treated everted red blood cells (which lack "Na+/K+ transport and "Rb+/ Rb+ exchange) [52], and the demonstration of two ATP-binding sites (Figs 13 -15). It is clear that the (a,P)z-diprotomeric structure has also to be proven more rigorously.…”

“…Similar data have been reported for modification of the low-affinity ATP-binding site by Co(NH3),ATP [23 -251. Additionally, we recently showed that Co(NH3),P04 inactivates "Na+/K+ transport and "Rb+/Rb+ exchange but not "Na+/K+ exchange nor uncoupled 22Na+ transport in everted erythrocytes [52]. Since Co(NH3),P04 and Co(NH3),ATP, but not CrATP, differentiate between the partial activities in a similar way, it is likely that the chromium and Co(NH,), complexes per se differentiate between El and Ez, and not the nature of the ligand (ATP or phosphate).…”

Quenching of 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole‐modified Na+/K+‐ATPase reveals a higher accessibility of the low‐affinity ATP‐binding site

“…Na + -dependent phosphorylation at the EiATP site [11] and K+-activated phosphatase activity at the E 2 ATP site [12,13]. Therefore, we performed several experiments to study the effect of NBD-C1 on the enzyme with specifically blocked EiATP-(FITC-pretreated) or E 2 ATP-binding site (Co(NH 3 ) 4 P0 4 -pretreated).…”