Molecular Pharmacology, Vol 12, 1007-1018, Copyright © 1976 by the American Society for Pharmacology and Experimental Therapeutics

Sodium- and Potassium-Dependent Adenosine Triphosphatase of Electric Organ: Interaction with Ouabain in Situ, in a Membrane Fraction, and in the Solubilized Form

¹ Déepartment de Pharmacologie, Ecole de Médecine, 1211 Genève 4, Switzerland

(Na⁺ + K⁺)-ATPase (EC 3.6.1.3) of the electric organ of Torpedo marmorata has been studied in situ, in an enriched membrane-bound fraction, and after solubilization. In situ, ouabain did not modify cholinergic transmission of a single impulse but impaired the ability of the tissue to maintain repetitive activity. Pieces of electric organ were incubated in the presence of [³H]ouabain. As much as 0.7-0.9 nmole was bound per gram of tissue, even after prolonged washing. In the membrane fraction, ouabain binding was measured in the presence of ATP and Na⁺. There was a good correlation between the amount of bound glycoside and the degree of enzymatic inhibition. Binding in the presence of Mg²⁺ and P_i was more stable and corresponded to 3.13 nmoles of sites per gram of starting tissue. Torpedo (Na⁺ + K⁺)-ATPase could be solubilized using the nonionic detergent Lubrol W, and was subjected to gel filtration and sucrose gradient centrifugation. The solubilized enzyme behaved as a homogeneous molecular species having a Stokes radius of 10.7 nm and a sedimentation coefficient of 10.5 S, indicating an apparent molecular weight of 475,000. The activity of the solubilized (Na⁺ + K⁺)-ATPase was the same as that for the enzyme inserted normally in the membrane: about 30 molecules of ATP hydrolyzed per site per second at 37°.

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ACKNOWLEDGMENTS We are grateful to L. Hirt for technical assistance, to G. Jones and R. Zubler for helpful discussions and criticism, to Mrs. M. Chmouliovsky and N. Collet for help in this work, and to the staff of the Marine Station of Arcachon for providing Torpedo.