Inside-out plant plasma membrane vesicles of high purity obtained by aqueous two-phase partitioning

AbstractHighly purified plasma membranes obtained from leaves of sugar beet (Beta vulgaris L.) by aqueous two-phase partitioning were separated into two fractions by further phase partition steps. The vesicles partitioning to the interface showed an ATP-dependent H+-uptake (measured using the pH probe acridine orange) and a negligible K+,Mg2+-ATPase latency, while the vesicles partitioning in the upper phase showed only slow H+-uptake and a high ATPase latency on addition of Triton X-100. Based on these results the material at the interface is estimated to contain ∼90% sealed, inside-out vesicles, and the material in the upper phase ∼90% sealed, right-side-out vesicle