Sidedness of plant plasma membrane vesicles altered by conditions of preparation.
Right-side-out vesicles of plasma membrane from soybean (Glycine max Merr.) were isolated by aqueous two-phase partition. Inside-out vesicles were formed when these preparations were diluted or frozen and thawed. Sidedness (orientation) was determined by preparative free-flow electrophoresis, concanavalin A binding, and ATPase latency. Under usual conditions of aqueous two-phase partition, the bulk of the vesicles were strongly reactive with concanavalin A-peroxidase and showed a high level of structure-linked latency as expected of a right-side-out (cytoplasmic-side-in) orientation. The vesicles migrated as a single electrophoretic peak. When frozen and thawed, vesicle diameters were reduced and a second population of vesicles of increased electrophoretic mobility was obtained. This second population of vesicles was weakly reactive with concanavalin A-peroxidase and showed low latency as expected of an inside-out (cytoplasmic-side-out) orientation. If the plasma membrane vesicles were diluted with water, a mixture of right-side-out and inside-out vesicles again was obtained. However, some of the cytoplasmic-side-out vesicles that were concanavalin A-unreactive and had low ATPase latency migrated more slowly as a second, less electronegative peak, upon free-flow electrophoresis. The results suggest that right-side-out and inside-out plasma membrane vesicles differ in electrophoretic mobility but that both the orientation and the absolute electrophoretic mobility of the differently oriented vesicles may be influenced by the preparative conditions.
Brightman
A O
AO
Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, Indiana 47907.
Morré
D J
DJ