Vanadate binding to the (Na + K)-ATPase.
A particulate (Na + K)-ATPase preparation from dog kidney bound [48V]-ortho-vanadate rapidly at 37 degrees C through a divalent cation-dependent process. In the presence of 3 mM MgCl2 the Kd was 96 nM; substituting MnCl2 decreased the Kd to 12 nM but the maximal binding remained the same, 2.8 nmol per mg protein, consistent with 1 mol vanadate per functional enzyme complex. Adding KCl in the presence of MgCl2 increased binding, with a K0.5 for KCl near 0.5 mM; the increased binding was associated with a drop in Kd for vanadate to 11 nM but with no change in maximal binding. Adding NaCl in the presence of MgCl2 decreased binding markedly, with an I50 for NaCl of 7 mM. However, in the presence of MnCl2 neither KCl nor NaCl affected vanadate binding appreciably. Both the nonhydrolyzable, beta, gamma-imido analog of ATP and nitrophenyl phosphate, a substrate for the K-phosphatase reaction that this enzyme also catalyzes, decreased vanadate binding at concentrations consistent with their acting at the low-affinity substrate site of the enzyme, the presence of KCl increased the concentration of each required to decrease vanadate binding. Oligomycin decreased vanadate binding in the presence of MgCl2, whereas dimethyl sulfoxide and ouabain increased it. With inside-out membrane vesicles from red blood cells vanadate inhibited both the K-phosphatase and (Na + K)-ATPase reactions; however, with the K-phosphatase reaction extravesicular K+ (corresponding to intracellular K+) both stimulated catalysis and augmented vanadate inhibition, whereas with the (Na + K)-ATPase reaction intravesicular K+ (corresponding to extracellular K+) both stimulated catalysis and augmented vanadate binding.
D000818:Animals; D004285:Dogs; D004910:Erythrocyte Membrane / Q000201:enzymology*; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007679:Kidney Medulla / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D011485:Protein Binding; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D014638:Vanadates; D014639:Vanadium / Q000378:metabolism*
D014639:Vanadium; D015636:Magnesium Chloride; D014638:Vanadates; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium