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.
Institut für Biochemie und Endokrinologie, Fachbereich Veterinärmedizin, Justus-Liebig-Universität Giessen, Federal Republic of Germany.
E
E
Buxbaum
W
W
Schoner
D009597:4-Nitrophenylphosphatase / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001693:Biological Transport, Active; D003035:Cobalt / Q000494:pharmacology*; D004801:Eosine Yellowish-(YS) / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000187:drug effects; D006801:Humans; D007668:Kidney / Q000201:enzymology; D008274:Magnesium / Q000494:pharmacology*; D017616:Magnesium Compounds; D010042:Ouabain / Q000494:pharmacology; D010710:Phosphates / Q000494:pharmacology*; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology*; D011983:Receptors, Purinergic / Q000187:drug effects; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism; D013552:Swine
D017616:Magnesium Compounds; D010710:Phosphates; D000644:Quaternary Ammonium Compounds; D011983:Receptors, Purinergic; C066354:cobalt tetraammine phosphate; D003035:Cobalt; C030781:magnesium phosphate; D010042:Ouabain; D000255:Adenosine Triphosphate; D012964:Sodium; D009597:4-Nitrophenylphosphatase; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D004801:Eosine Yellowish-(YS)