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 pla nt tissue have a right-side-out (cytoplasmic side-in) orientation. Sub sequent purification of plasma membrane vesicles using aqueous two-pha se partitioning leads to a further enrichment in right-side-out vesicl es resulting in preparations with 80-90% of the vesicles in this orien tation. 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 tow ards the cytoplasm, the vesicles have to be inverted.;This is very eff iciently achieved by including 0.05% of the detergent Brij 58 (C(16)E( 20)) in the assay medium, which produces 100% sealed, inside-out (cyto plasmic side-out) vesicles from preparations of 80-90% right-side-out vesicles. This was shown by assaying ATP-dependent H+ pumping using th e Delta pH probe acridine orange and dissipating the H+ gradient with nigericin, and by assaying ATP-dependent Ca2+ transport using Ca-45(2) and dissipating the Ca2+ gradient with the ionophore A23187. The pre sence of intact vesicles was confirmed by electronmicroscopy. The dete rgent 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 (E(20-23)) showed this 'non-detergent behavior', whereas those with smaller head groups (E(8-10)) behaved as normal de tergents and permeabilized the membranes. Thus, a very convenient syst em for studies on ion-pumping activities and other vectorial propertie s of the plasma membrane is obtained by simply including the detergent Brij 58 in the assay medium.