Release of protein 4.1-rich vesicles from diamide-treated erythrocytes under hydrostatic pressure

The effect of cross-linking of membrane proteins on vesiculation under high pressure (2.0 kbar) of human erythrocytes was examined. To get the large molecular weight aggregates characterized by cross-linking of cytoskeletal proteins with integral ones, the erythrocytes were pretreated with diamide under pressure (1.0 kbar) where no vesiculation occurs. Vesicles released at 2.0 kbar from such erythrocytes contained protein 4.1 as major membrane protein. Upon reduction of cross-linking by dithiothreitol prior to vesiculation, the released vesicles contained membrane proteins similar to intact cells. On the other hand, in the erythrocyte pretreated with diamide at atmospheric pressure, no such large molecular weight aggregate was observed and the membrane protein composition of the vesicles released from the cells at 2.0 kbar was also similar to that of intact cells. These results suggest that the membrane protein composition of released vesicles is much affected by the properties of cross-linking of membrane proteins in erythrocytes.