A Cycle for Ouabain Inhibition of Sodium- and Potassium-dependent Adenosine Triphosphatase

Abstract

Abstract Physiological ligands of (sodium-potassium)-dependent adenosine triphosphatase influenced the sensitivity of the enzyme to the inhibitor, ouabain. Phosphorylation of the native enzyme is accelerated by sodium ions, while potassium ions accelerate dephosphorylation. Exposure of the phosphoenzyme to ouabain slowed its turnover and rendered it insensitive to potassium ion. This action of ouabain did not require free magnesium ion and was accelerated by high concentrations of sodium ion. The ouabain-treated phosphoenzyme gradually yielded a dephosphoenzyme which could not be rephosphorylated from adenosine triphosphate but could incorporate inorganic phosphate. The native dephosphoenzyme on incubation with ouabain lost its ability to be phosphorylated by adenosine triphosphate. This effect was accelerated by magnesium ion or magnesium ion plus inorganic phosphate and could be prevented but not reversed by sodium ion at 0°. At 23° or above it was reversible with or without sodium ion. The inhibited dephosphoenzyme incorporated inorganic phosphate in the presence of magnesium ion. All phosphorylated forms of the enzyme yielded identical patterns on electrophoresis and radioautography of proteolytic digests. A cyclic reaction sequence for inhibition of the enzyme by ouabain is proposed and related to physiological transport of sodium and potassium ions.