title: Binding to the high-affinity substrate site of the (Na+ + K+)-dependent ATPase.
abstract: The (Na+ + K+)-dependent ATPase exhibits substrate sites with both high affinity (Km near 1 microM) and low affinity (Km near 0.1 mM) for ATP. To permit the study of nucleotide binding to the high-affinity substrate sites of a canine kidney enzyme preparation in the presence as well as absence of MgCl2, the nonhydrolyzable beta-gamma imido analog of ATP, AMP-PNP, was used in experiments performed at 0-4 degrees C by a centrifugation technique. By this method the KD for AMP-PNP was 4.2 microM in the absence of MgCl2. Adding 50 microM MgCl2, however, decreased the KD to 2.2 microM; by contrast, higher concentrations of MgCl2 increased the KD until, with 2 mM MgCl2, the KD was 6 microM. The half-maximal effect of MgCl2 on increasing the KD occurred at approximately 1 mM. This biphasic effect of MgCl2 is interpreted as Mg2+ in low concentrations favoring AMP-PNP binding through formation at the high-affinity substrate sites of a ternary enzyme-AMP-PNP-Mg complex; inhibition of nucleotide binding at higher MgCl2 concentrations would represent Mg2+ acting through the low-affinity substrate sites. NaCl in the absence of MgCl2 increased AMP-PNP binding, with a half-maximal effect near 0.3 mM; in the presence of MgCl2, however, NaCl increased the KD for AMP-PNP. KCl decreased AMP-PNP binding in the presence or absence of MgCl2, but the simultaneous presence of a molar excess of NaCl abolished (or masked) the effect of KCl. ADP and ATP acted as competitors to the binding of AMP-PNP, although a substrate for the K+-dependent phosphatase reaction also catalyzed by this enzyme, p-nitrophenyl phosphate, did not. This lack of competition is consistent with formulations in which the phosphatase reaction is catalyzed at the low-affinity substrate sites.
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D004285:Dogs; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
chemical_list: D015636:Magnesium Chloride; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium