title: Interaction between ouabain and the phosphorylated intermediate of Na,K-ATPase.
abstract: After phosphorylation of electric eel Na,K-ATPase by Na+, Mg2+, and ATP was terminated by removing the unbound Mg2+, the phosphoenzyme was able to bind ouabain upon the addition of 2 mM ouabain under certain conditions. This binding was demonstrated by a 50% inhibition of ATPase after the removal of unbound ouabain by a Sephadex G-50 column (ouabain trapping method). At 4 degrees, this ouabain binding was observed on the K+- sensitive phosphoprotein (E2P) formed in the presence of 10 mM Na+ but was not observed on the ADP-sensitive phosphoprotein (E1P) formed in the presence of 1000 mM Na+. The increase in the dephosphorylation rate of E2P with various concentrations of K+ paralleled the decrease in inhibition by the addition of 2 mM ouabain after the termination of phosphorylation. In 50-200 mM Na+, the eel enzyme used here formed the E1P-rich phosphoprotein, but this phosphoprotein could bind with ouabain, even though the presence of ADP or oligomycin, which prevents the conversion of E1P to E2P, partially interfered with this ouabain binding. At 25 degrees, ouabain binding with E1P-rich phosphoprotein was observed in higher yield (up to 71%), but in each of these cases ADP or oligomycin strongly inhibited ouabain binding. Moreover, ouabain binding with E2P-rich phosphoprotein did not significantly change with temperature, but ouabain binding with E1P-rich phosphoprotein increased more than 6 times at temperatures from 4 degrees to 25 degrees. From these results, it can be concluded that E2P can bind with ouabain in the absence of free Mg2+ whereas E1P cannot, and that the interconversion between E1P and E2P can be stimulated with ouabain binding and accelerated with elevation of temperature. ADP- and K+ -insensitive phosphoprotein probably is only a minor intermediate for ouabain binding.
mesh_terms: D000818:Animals; D003080:Cold Temperature; D004557:Electric Organ / Q000201:enzymology; D004593:Electrophorus; D066298:In Vitro Techniques; D010042:Ouabain / Q000494:pharmacology*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013997:Time Factors
chemical_list: D010750:Phosphoproteins; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium