The Effects of Physical States of Phospholipids on the Incorporation and CB Binding Activity of Band 4.5 Protein of human Erythrocyte Membrane in Reconstituted Vesicles

Proteoliposomes were prepared by reconstitution of a solubilized band 4.5 protein of human erythrocyte membrane with a mixture of phosphatidylcholine(PC) and phosphatidylethanolamine(PE) of varying ratios. The incorporation of the band 4.5 protein into the reconstituted vesicles was dependent on the stability of the bilayer of lipid added exogeneously when mixed soybean PE and egg PC were used for reconstitution. The protein/phospholipid ratio was maximal at 25% PC and 75%PE, the composition which give maximal bilayer instability. The specific activity of the cytochalasin B binding of the reconstituted band 4.5 protein increased monotonically as PC content increased in egg PC/soybean PE mixture. These coincide with the results of proteoliposomes reconstituted using crude protein extracts containing band 4.5 protein. Band 4.5 protein, however, changed significantly the phase of the lipid in reconstituted vesicles instead of that protein crude extract modified slightly the phase of the lipid. No lipidic particles (LIP, seen by freeze- fracture electron microscopy) were seen in all lipid mixtures studied. From above findings the fact that pre- existing defects in lipid bilayer promote protein incorporation into the bilayer during reconstitution is confirmed and band 4.5 protein somewhat seems to favor the bilayer structure.