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.
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.
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