Electrogenic transport by the Enterococcus hirae ATPase.

A transport ATPase from Enterococcus hirae was reconstituted in lipid vesicles and its electrogenic action investigated with the fluorescent dye oxonol VI as membrane potential probe. Reconstitution in bacterial and in soybean phospholipid mixtures led to transport-active vesicle preparations. Inside-out oriented ATPase molecules were activated by the addition of ATP to the extravesicular medium, generating in all experiments an intravesicularly positive potential. The extravesicular pH strongly influenced the initial pumping rate and the duration of the pumping activity. At neutral pH, transient pumping activity was observed, lasting for 1-2 min, while at pH 5.6, pumping was continuous. The transport activity was not dependent on the ionic composition of the buffer on either side of the membrane. These findings can be interpreted as the action of a proton ATPase, regulated by the cytoplasmic proton concentration and electrogenically translocating protons from the cytoplasm to the extracellular space.