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
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 Ca2+‐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 ΔpH 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.