Cation-impermeable Inside-out and Right-side-out Vesicles from Human Erythrocyte Membranes

The two surfaces of a membrane can be compared by isolating sealed vesicles which bare one side or the other to impermeable probe molecules. For this purpose, inside-out (IO) and right-side-out (RO) vesicles have been generated from human red blood cell membrane ghosts and partially resolved on dextran density gradients¹. We now report further characterization of this system, showing (a) that vesicles can be made which are impermeable to small solutes, including Na⁺ and K⁺, even though the parent ghosts are quite unsealed; (b) that the dextran gradient fractionation serves to separate sealed vesicles (which happened to be IO and RO, respectively, in the original study¹); (c) that impermeable RO vesicles form instead of IO vesicles if the published protocol is modified by the untimely addition of trace divalent cations; and (d) that the vesicle fractions can and must be accurately monitored for sealing and orientation to avoid misinterpretations such as those encountered in a previous sidedness study².