Freeze-fracture appearance and disposition of band 3 protein from the human erythrocyte membrane in lipid vesicles

Abstract

Single bilayer lipid vesicles were formed by removal of Triton X-100 with Bio Beads SM-2 from a mixture of egg lecithin and a Triton X-100 extract of human erythrocyte ghosts. Upon freeze-fracturing, these vesicles showed intramembrane particles, similar to those seen in the erythrocyte membrane. Similar particles were also observed when a partially purified band 3 preparation was used instead of the crude Triton X-100 extract. In the reconstituted vesicles an equal distribution of the intramembrane particles between the two fracture faces was observed. This is in contrast to the unequal distribution of the particles in the erythrocyte membrane, which did not seem to be altered by removal of the extrinsic proteins. From digestion studies with trypsin and chymotrypsin of vesicles, reconstituted from the crude X-100 extract, it is concluded that band 3 protein in the vesicle bilayer has a similar orientation as in the native membrane.