Free-Flow Electrophoresis Resolves Plasma Membrane Vesicles Isolated by Aqueous Two Phase Partition into Two Populations of Different Sidedness

Abstract

Transport and other studies with isolated plasma membrane vesicles would be facilitated considerably by the availability of purified preparations of known absolute orientations. In this study, we have used ATPase latency [1] and concanavalin A binding [2] to conclude that vesicles obtained in the upper phase by aqueous two-phase partitioning were mixtures of cytoplasmic side out and cytoplasmic side in vesicles. We used the same approach to demonstrate that free-flow electrophoresis fractions nearest the point of sample injection contained purified plasma membrane of cytoplasmic side out orientation, whereas electrophoretic fractions further from the point of sample injection contained plasma membrane vesicles of the opposite, cytoplasmic side in orientation. Used in series, the two techniques of aqueous two-phase partition and free-flow electrophoresis yielded purified homogeneous fractions of plasma membrane vesicles of opposite orientation.