Partial purification of the ouabain-binding component and of Na,K-ATPase from human red cell membranes.

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.