Effects of cross-linking of membrane proteins on vesiculation induced by dimyristoylphosphatidylcholine in human erythrocytes.

To study the effect of cross-linking of membrane proteins on vesiculation of human erythrocytes by dimyristoylphosphatidylcholine (DMPC), red cells were treated with diamide at atmospheric pressure or 100 MPa and then incubated with DMPC in buffers of pH 6.5-8.5. Irrespective of buffer pH, the amount of released vesicles increased upon cross-linking of membrane proteins but approached the control level upon reduction of the cross-linking by dithiothreitol. Similar enhancement of vesicle release was also observed in N-ethylmaleimide-treated red cells. Hemolysis during vesiculation was observed only in red cells treated with diamide at 100 MPa. Furthermore, the composition of membrane proteins in released vesicles was analyzed by SDS-PAGE. Membrane vesicles released from intact red cells or the cells treated with diamide at atmospheric pressure contained band 3 as a major membrane protein. On the other hand, membrane vesicles from red cells treated with diamide at 100 MPa contained protein 4.1 in addition to band 3 and the orientation of these proteins was similar to that in intact cells. These results indicate that the amount and membrane protein composition of DMPC-induced vesicles are much affected by chemical modification of SH-groups in red cell membrane proteins.