Molecular Pharmacology, Vol 9, 336-349, Copyright © 1973 by the American Society for Pharmacology and Experimental Therapeutics

Calcium Ion and Sodium- and Potassium-Dependent Adenosine Triphosphatase: Its Mechanism of Inhibition and Identification of the E₁—P Intermediate

¹ Department of Pharmacology, Michigan State University, East Lansing, Michigan 48823

Calcium ion inhibits (Na⁺ + K⁺)-ATPase (EC 3.6.1.3) primarily by reducing the rate of the phosphorylation step. Inhibition of this step is competitive with respect to Na⁺ and noncompetitive with respect to Mg⁺⁺. Although Ca⁺⁺ acts to reduce the rate of the phosphorylation step, high concentrations are required to reduce the steady-state levels of phospho-enzyme, and Ca⁺⁺ alone stimulates a slow phosphorylation of the enzyme. In the presence of Ca⁺⁺ and Mg⁺⁺ the phospho-enzyme formed is sensitive to K⁺ and ouabain, and its steady-state levels are low when K⁺ is present. If the concentration of Mg⁺⁺ is low, of the phosphoenzyme formed in the presence of Ca⁺⁺ is insensitive to K⁺ and ouabain, and reacts readily with ADP. The reactivity of the Ca⁺⁺-dependent phospho-enzyme with ADP is reduced by the addition of Mg⁺⁺; conversely, the reactivity of the Mg⁺⁺-dependent phospho-enzyme with ADP is increased by high concentrations of Na⁺. Ca⁺⁺ also inhibits the Mg⁺⁺- and P_[unknown]-dependent pathway of [³H]ouabain binding. The identification of an ADP-sensitive intermediate in the reaction cycle of this enzyme is reported. Its transformation to a K⁺-sensitive intermediate is influenced by Na⁺, Mg⁺⁺, and Ca⁺⁺ in a manner consistent with proposed reaction mechanisms for this enzyme.

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ACKNOWLEDGMENTS The authors would like to thank Mrs. Annie Han for excellent technical assistance, and Mr. Ralph Evans, who assisted as a summer student.