Phosphate incorporation and Na, K‐ATPase activity in human red blood cell ghosts

Abstract

An attempt is described to demonstrate a phosphorylated intermediate correlated with the membrane ATPase reaction in human red cell ghosts. It was found that ghosts incubated with γ‐labeled ATP³² incorporate 400 to 1600 μM P/kg dry weight and that this incorporation required Mg, could be enhanced by the addition of Na and perhaps of strophanthidin but was inhibited by Ca, cooling or heat denaturation. If the total P³² content of ghosts washed with Tris is taken as the Tris‐insoluble fraction, this fraction can be divided, operationally, into the PCA‐insoluble fraction, and the PCA‐soluble fraction‐B. It was found that the PCA‐soluble fraction‐B was composed primarily of unsplit, intact ATP³² (as shown in parallel experiments with C¹⁴‐labeled ATP) and some P_i³². The PCA‐insoluble fraction contained only P_i³². If the P_i³² contained in the PCA‐insoluble fraction represents an intermediate in the membrane ATPase, this P_i³² should be exchangeable upon restitution of the ATPase activity. However, this P_i³² was found to be stable and nonexchangeable upon reincubation of prelabeled ghosts under conditions which reactivate the ATPase reaction. Two alternative interpretations were considered: (1) that the P³² that is incorporated into an intermediate product of the ATPase reaction is masked by nonspecific binding of P³² and, (2) that the phosphorylated product is resident in the PCA‐soluble fraction‐B. Further experiments are needed to evaluate these possibilities.