Electrogenic H+ translocation by the plasma membrane ATPase of Neurospora. Studies on plasma membrane vesicles and reconstituted enzyme

Abstract

Fluorescent probes have been used to measure electrogenic proton pumping by the plasma membrane ATPase of Neurospora. In isolated plasma membrane vesicles, greater than 85% of which are inverted, ATP hydrolysis is accompanied by the formation of an inside acid pH gradient (delta pH) which can be detected by acridine orange fluorescence quenching and an inside positive membrane potential (delta psi) which can be detected by oxonol V fluorescence quenching. Maximal values of delta pH were generated in the presence of a permeant anion (SCN-, NO-3, or Cl-) and maximal delta psi, in the absence of such anions. Cation effects were much less pronounced and can probably be accounted for by non-specific salt effects on the rate of ATP hydrolysis. In addition, a rapid method is described for the reconstitution of the [H+]-ATPase, starting from isolated plasma membranes. When the membranes are solubilized with deoxycholate in the presence of asolectin and detergent is removed by passage through a Bio-Gel P-10 column, vesicles are reformed in which the Mr = 104,000 polypeptide of the ATPase constitutes 35% of the protein. Freeze-fracture electron microscopy of the vesicles has revealed intramembrane particles with a diameter of 116 A, equally distributed between the two fracture faces. Measurements with acridine orange and oxonol V indicate that the reconstituted ATPase retains its transport activity, generating both delta pH and delta psi during the hydrolysis of MgATP.