A procedure for membrane-protein reconstitution and the functional reconstitution of the anion transport system of the human-erythrocyte membrane

Abstract

A short procedure for the isolation of band-3 protein, the protein responsible for anion exchange in erythrocytes, in a reasonable degree of purity was developed. Using this protein preparation and a novel procedure for membrane-protein reconstitution, vesicles displaying the basic features of the anion-exchange system of the erythrocyte were obtained. The reconstitution procedure is based on slow direct removal of Triton X-100 from aqueous lipid/detergent solutions. According to the composition of the reconstitution medium, either small single-walled or large multi-walled vesicles are obtained. The procedure conserves protein properties well, as is revealed by the similarity of the rates of SO4(2-) exchange in erythrocytes and reconstituted vesicles when corrected for the relevant volumes. A number of functional features of the exchange system were studied and compared with those of the native membrane.