Molecular Pharmacology, Vol 10, 626-633, Copyright © 1974 by the American Society for Pharmacology and Experimental Therapeutics

Cardiac Glycoside Interaction with Solubilized Myocardial Sodium- and Potassium-Dependent Adenosine Triphosphatase

¹ Cardiac Unit and Laboratory of Physical Biochemistry, Massachusetts General Hospital, and Departments of Medicine and Biochemistry, Harvard Medical School, Boston, Massachusetts 02114

Canine myocardial microsomal membranes were exposed under optimum binding conditions to 8 nM[³H]ouabain, a concentration producing only partial inhibition of the (Na⁺ + K⁺)-ATPase activity in this preparation. Microsomal membrane components were then solubilized with the nonionic detergent Lubrol-WX. After centrifugation at 100,000 x g for 1 h and gel permeation chromatographiy on Sepharose 6B, [³H]ouabain was found exclusively in fractions containing (Na⁺ + K⁺)-ATPase activity, and closely paralleled the enzyme activity profile. In Lubrol-solubilized preparations, bound [³H]ouabain penetrated the gel with a component of apparent molecular weight 600,000. Sucrose density gradient centrifugation and liquid isoelectric focusing of Lubrol-solubilized preparations also resulted in close correspondence between the presence of [³H]ouabain and (Na⁺ + K⁺)-ATPase activity. Lubrol-solubilized (Na⁺ + K⁺)-ATPase interacted with ouabain in a manner similar to the membrane-bound enzyme, as judged by identical half-maximal inhibitory concentrations of 60 nM. Thus solubilization of myocardial microsomal membrane components resulted in preservation of ouabain binding and did not disclose any highaffinity receptor separable from (Na⁺ + K⁺)-ATPase by these techniques.

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ACKNOWLEDGMENTS The authors are grateful to Dr. A. Donny Strosberg for assistance in isoelectric focusing experiments, and to Miss Patricia Lando for skilled technical assistance.