Modulatory effect of two cardioglycosides on reconstituted Na⁺/K⁺-ATPase in proteoliposomes

1. 1. Na,K-ATPase was extracted from Cavia cobaya kidneys, solubilized with nonionic detergent C₁₂E₈ (octaethyleneglycol dodecyl monoether) in mixed lipid-detergent-protein micelles. The Na,K-ATPase specific activity was 30-35 IU/mg protein. 2. 2. The enzyme was reconstituted in vesicles, made of phosphatidylethanolamine and cholesterol: an enhancement of +60% in specific activity was obtained. 3. 3. Two different vesicle-types were carried out: open liposomes (partially organized membranes) and closed liposomes. 4. 4. Proteoliposomes were employed for measuring the modulatory effect of two cardioglycosides: ouabain and digoxin. 5. 5. Inhibition of the Na,K-ATPase activity revealed apparent K_i of 1.25μM for ouabain and 0.25μ M for digoxin in open liposomes, and apparent K_i, of 0.75μ M for ouabain and of 1.75μ M for digoxin in closed liposomes. 6. 6. Maximum enhancement of enzymatic activity was found at concentrations of 5-0.5 nM for ouabain and 5-1 nM for digoxin in open liposomes, and 25-1 nM for both digoxin and ouabain in closed liposomes.