Release of endogenous AChE and in vitro incorporated VSG from human erythrocytes during Ca²⁺-induced vesiculation. [³H]-labeled GPI-anchored molecules were incorporated into erythrocytes as described in Fig 3 and the release of membrane vesicles was induced by loading the cells with calcium using the ionophore A23187. At designated times, erythrocytes were pelleted and the AChE activity (▪) and the radioactivity (○, ◊) were measured in the vesicle-containing supernatants of cells labeled with in vitro incorporated VSG (○) and Pronase-treated VSG (◊). The values represent single determinations from a typical experiment; corresponding results were obtained with in vitro incorporated AChE, procyclin, and Pronase-treated procyclin.