RECONSTITUTION OF A PLASMA-MEMBRANE H-ATPASE INTO BILAYER-LIPID MEMBRANE()

The plasma membrane H+-ATPase of Neurospora has been reconstituted int o planar lipid bilayer membranes by means of the vesicle-fusion techni que described by Finkelstein and his collaborators (Zimmerberg et al., 1980; Cohen et al., 1980, 1984; Akabas et al., 1984). Enzyme was firs t transferred from isolated plasma membrane fragments into asolectin v esicles by a detergent-dialysis procedure (Perlin et al., 1984). After H+-pumping activity had been checked by quenching of acridine orange fluorescence, the vesicles were fused into preformed bilayers. Critica l features of the fusion process include (i) attachment of the vesicle s to the bilayer in the presence of divalent cations (Mg++), and (ii) rapid osmotic swelling, which was enhanced by prior sonication or free ze-thawing of the vesicles, and/or by inclusions of physiologic channe ls. Enough proton pumps could be thus incorporated into bilayers to ac hieve ATP-driven, vanadate-sensitive currents of 0.04-0.4 pA. Aqueous solutions of low ionic strength were used to suppress conductance fluc tuations due to the channels, and when that precaution was taken, we c ould demonstrate the proton pump the work against membrane potentials of at least 50 mV.