1) A highly specific and active Na, K-ATPase preparation was obtained from cerebral microsomes by a slightly modified NaI-treatment. Using the ATPase preparations, the effects of protamine and gangliosides on the binding of rubidium and the activity of Na, K-activated ATPase were examined. 2) The results on the time course of the rubidium-binding by the ATPase preparation indicated that the binding pattern in the presence of ATP and Mg showed a fundamental difference from those in the absence of ATP and Mg. 3) The rubidium-binding by the ATPase preparation was dependent on ATP and Mg. The ATP dependent rubidium-binding was inhibited by ouabain, Ca and K. High concentrations of Na also decreased the binding. 4) The ATP dependent rubidium-binding was apparently inhibited by protamine. The inhibitory effect of protamine was removed by the addition of gangliosides and excess cations. 5) Low concentrations of protamine did not affect the release of bound rubidium from the ATPase preparation. Higher concentrations of protamine caused release of the bound rubidium. 6) The Na, K-activated ATPase activity of the ATPase preparation was selectively inhibited by protamine, though the activity of Mg-ATPase was not influenced. These effects of protamine on the Na, K-activated ATPase activity were antagonized by the addition of gangliosides and cations. 7) The actions of protamine and gangliosides on the binding of rubidium and the Na,K-activated ATPase were discussed on the basis of these results
