The measurement of ouabain binding and some related properties of digitonin-treated (Na +, K +)-ATPase

Abstract

1. 1. Digitonin-treated membrane preparations purified from dog kidney lose their (Na +, K +)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity, but the K +-phosphatase and Na +-dependent ADP-ATP exchange activities survive and remain ouabain-sensitive. Because the enzyme preparations consist largely of pure (Na +, K +)-ATPase, these effects of digitonin must be intrinsic to the Na + pump. 2. 2. Concomitant with these enzymatic changes, digitonin treatment alters the sensitivity of the phosphatase and exchange activities to ouabain. 3. 3. Attempts to measure ouabain binding by the usual centrifugation or filtration methods proved unsuccessful. A filtration method involving a double 0.01 μm filter and omitting water washes is necessary to demonstrate ouabain binding. Under these conditions, ouabain binding capacity appears to be unchanged in the presence of digitonin, but the apparent dissociation constant is doubled. 4. 4. Ouabain binding is rendered more reversible by digitonin treatment, since washing filters with water removes a large fraction of bound ouabain without affecting the retention of exchange activity. 5. 5. The double filter method traps essentially all of the ADP-ATP exchange activity on the filter. However, a large and somewhat variable proportion of the K +-phosphatase activity passes through the filter. Sodium dodecyl sulfate polyacrylamide gel analysis of the filtrate shows that a small amount of filtrable protein catalyzes this phosphatase activity at greatly increased turnover rates. Both subunits of the (Na +, K +)-ATPase are present in this latter protein fraction.