The plasma membrane (Mg2+)-dependent adenosine triphosphatase from the human erythrocyte is not an ion pump

The plasma membrane (Mg2+)-dependent adenosine triphosphatase [Mg2+)-ATPase) from human erythrocytes has been tested for its ability to transport ions. Using a preparation of inside-out vesicles loaded with the pH-sensitive fluorescence probe 1-hydroxypyrene-3,6,8-trisulfonic acid (HPTS), we have demonstrated the absence of proton movement during (Mg2+)-ATPase activity. From the rate of ATP hydrolysis and the passive proton permeability of these vesicles, an upper limit of 0.03 H+ transported per ATP hydrolyzed was calculated. To verify that proton pumping could be detected in this system, the intravesicular pH was monitored during (Ca2+)-dependent adenosine triphosphatase [Ca2+)-ATPase) activity. Proton efflux associated with (Ca2+)-ATPase activity was observed (in agreement with a recent report of proton pumping by a reconstituted erythrocyte (Ca2+)-ATPase (Niggli, V., Sigel, E., Carafoli, E. (1982) J. Biol. Chem. 257:2350-2356] and was shown to be stimulated by calmodulin. The ability of the (Mg2+)-ATPase to pump 28Mg2+, 35SO2-4 and 86Rb+ was also tested, with the results leading to the conclusion that the human erythrocyte enzyme does not function as an ion transport system.