PHOSPHATE INCORPORATION AND NA, K-ATPASE ACTIVITY IN HUMAN RED BLOOD CELL GHOSTS.

Abstract

AbstractAn 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 Pi32. The PCA‐insoluble fraction contained only Pi32. If the Pi32 contained in the PCA‐insoluble fraction represents an intermediate in the membrane ATPase, this Pi32 should be exchangeable upon restitution of the ATPase activity. However, this Pi32 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.