Partial Purification of the Ouabain-Binding Component and of Na,K-ATPase from Human Red Cell Membranes

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Abstract
Ghosts of human red cells were incubated with tritiated ouabain in the presence of ATP, Mg, and Na, conditions under which ouabain binds with high specificity to Na:K transport sites. The labeled membranes were solubilized with sodium dodecyl sulfate and dialyzed. Sodium dodecyl sulfate solubilizes most of the membrane protein and leaves most of the tritiated-ouabain bound to a solubilized component. Solubilized Na,K-ATPase could also be obtained after dialysis. The solubilized membranes were centrifuged in a sucrose density gradient. The ouabain-membrane complex and the Na,K-ATPase sedimented faster than the bulk of the protein. The ouabain-membrane complex and the Na,K-ATPase appeared to be identical and were purified about eightfold relative to the starting material. These results represent a step toward the isolation and characterization of the cation transport mechanism in red cell membranes.

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