PT  - JOURNAL ARTICLE
AU  - ATSUNOBU YODA
AU  - SHIZUKO YODA
TI  - Suppression by Sodium, Potassium or Nucleotides of Binding between Cardiac Steroid (Digoxigenin) and Sodium- and Potassium-Dependent Adenosine Triphosphatase Formed in the Presence of Magnesium and Phosphate
DP  - 1979 Jul 01
TA  - Molecular Pharmacology
PG  - 120--134
VI  - 16
IP  - 1
4099  - http://molpharm.aspetjournals.org/content/16/1/120.short
4100  - http://molpharm.aspetjournals.org/content/16/1/120.full
SO  - Mol Pharmacol1979 Jul 01; 16
AB  - The cardiotonic steroid binding to (Na+ + K+)-ATPase formed in the presence of the Mg2+ and Pi (type II binding) is suppressed by the Na+, K+, or nucleotides in different ways. In the ouabain binding at real equilibrium, such effects were difficult to detect because of the high affinity of its sugar moiety to the enzyme. The apparent number of binding sites according to Scatchard plots of digoxigenin binding and the association rate of digoxigenin were reduced by the Na+ at pH 6.5 and 8.5. This Na+-effect was delayed in the initial phase at pH 6.5, when the Na+ was added after the phosphorylation of the enzyme was accomplished by the Mg2+ and Pi. Therefore, the Na+ seems to bind with the nonphosphorylated form of the enzyme, and to reduce level of the phosphorylated enzyme, the active form for digoxigenin binding. In contrast to the Na+, the K+ increased the apparent binding constant according to Scatchard plot of digoxigenin binding at pH 6.5 and 8.5; the K+ seems to bind with the phosphorylated form of enzyme, causing the insensitive complex not to bind with digoxigenin. Unlike either the Na+ or K+, triphosphonucleotides suppressed the type II digoxigenin binding only partially. The potencies of ATP and β,γ-methylene ATP were similar; both were most potent among the triphosphonucleotides (its half-maximum concentration [unknown] 0.2 mM). GTP and ITP were less potent while AMP was almost ineffective. ADP was as effective as ATP in less than 0.8 mM, but in higher concentrations, ADP increased the inhibition of digoxigenin binding, reducing the increment of the increase, while ATP showed saturation. According to Scatchard plots, ATP decreased only the apparent number of binding sites below pH 8.0, but it increased only the apparent binding constant above pH 8.0. β,γ-Methylene ATP reduced the association rate of digoxigenin at pH 7.0 and 8.3, but did not seem to change the level of phosphorylated protein by the Mg2+ and Pi and the dissociation rate of digoxigenin-enzyme complex at both pHs. Therefore, such nucleotide-effect seems to be one of the low-affinity effects of ATP, and seems to include some conformational changes of phosphorylated active form produced by the Mg2+ and Pi, reducing its affinity to cardiotonic steroids.