A ROLE FOR BAND-4.2 IN HUMAN ERYTHROCYTE BAND-3 MEDIATED ANION TRANSPORT

Human erythrocyte band 3 was purified essentially free of peripheral proteins, in particular band 4.2, using affinity chromatography. Band 3 protein was then reconstituted into liposomes of lipid type and ratio approximating that of erythrocyte membranes. Stilbenedisulfonate inhibition of band 3 mediated efflux of radiolabeled sulfate from preloaded liposomes was used to test the functionality and correct orientation of the protein. When sulfate efflux, mediated by purified band 3, was compared with partially purified band 3, which contained detectable amounts of bands 4.1 and 4.2, a clear difference in efflux was measured. Sulfate efflux was approximately 30% faster from liposomes containing purified band 3 compared with those containing partially purified protein. In order to investigate further any specific effect of band 4.2 protein on band 3 mediated anion transport, band 4.2 was purified. Increasing amounts of band 4.2 were complexed with purified band 3 and then reconstituted into liposomes. Increasing amounts of band 4.2 complexed with band 3 caused a decrease in band 3 mediated anion transport. The effect of band 4.2 on band 3 mediated anion transport appears to be specific since increasing concentrations of band 4.2 added exogenously to band 3 in reconstituted vesicles (rather than complexed with band 3 before reconstitution) produced no significant changes in sulfate efflux. Further, when increasing amounts of band 4.2 were added to the functionally active transmembrane domain of band 3 and then reconstituted into vesicles, there was also no significant change in sulfate efflux. These results indicate that one of the roles for band 4.2 in situ is possibly as a modulator of band 3 anion transport through its specific interaction with the cytoplasmic domain of band 3.