The rate of binding of [3H]ouabain to untreated membrane preparations of [Na+ +K+]-ATPase is a timperature--dependent process displaying a thermal transition close to 25degreesC. The apparent energies of activation which can be calculated above and below this transition are similar to, but not identical with, those previously reported for activation of the enzyme by cations. 2. Treatment of the enzyme preparation with detergents or lipolysis with phospholipase A eliminates the thermal transition resulting in linear Arrhenius plots. 3. The number of sites available for [3H]ouabain binding is not temperature dependent as the amount of [3H]ouabain bound at equillbrium is not changed between 10 and 37 degrees C. 4. Treatment of the enzyme with phospholipase A results in time-dependent changes in the number of binding sites for [3H]ouabain at equilibrium. 5. Treatment of the membrane enzyme preparations with detergents reveals additional [3H]ouabain binding sites which are extremely sensitive to lipolysis with phospholipase A. 6. There are a number of [3H]ouabain binding sites which remain resistant to lipolysis by phospholipase A in either untreated or detergent-treated membrane preparations. 7. It is suggested that [3H]ouabain binding sites exist in the membrane in at least two different environments, one of which is resistant the other sensitive to attack by phopholipase A.
