Brij 58, a polyoxyethylene acyl ether, creates membrane vesicles of uniform sidedness. A new tool to obtain inside‐out (cytoplasmic side‐out) plasma membrane vesicles

Abstract

Most of the plasma membrane vesicles formed upon homogenization of plant tissue have a right-side-out (cytoplasmic side-in) orientation. Subsequent purification of plasma membrane vesicles using aqueous two-phase partitioning leads to a further enrichment in right-side-out vesicles resulting in preparations with 80-90% of the vesicles in this orientation. Thus, to be able to assay, e.g. the ion-pumping activities of the H(+)-ATPase and the Ca(2+)-ATPase, which expose their active sites towards the cytoplasm, the vesicles have to be inverted. This is very efficiently achieved by including 0.05% of the detergent Brij 58 (C16E20) in the assay medium, which produces 100% sealed, inside-out (cytoplasmic side-out) vesicles from preparations of 80-90% right-side-out vesicles. This was shown by assaying ATP-dependent H+ pumping using the delta pH probe acridine orange and dissipating the H+ gradient with nigericin, and by assaying ATP-dependent Ca2+ transport using 45Ca2+ and dissipating the Ca2+ gradient with the ionophore A23187. The presence of intact vesicles was confirmed by electronmicroscopy. The detergent Brij 58 is a polyoxyethylene acyl ether and a survey among some other members of this series revealed that those with a head group of relatively large size (E20-23) showed this 'non-detergent behavior', whereas those with smaller head groups (E8-10) behaved as normal detergents and permeabilized the membranes. Thus, a very convenient system for studies on ion-pumping activities and other vectorial properties of the plasma membrane is obtained by simply including the detergent Brij 58 in the assay medium.