The major polypeptide of the human erythrocyte membrane, band 3, is thought to perform two essential functions. Firstly, band 3 facilitates CO(2) transport to the lungs by catalyzing anion exchange (e.g., bicarbonate for chloride) across the red cell membrane, and secondly, band 3 may serve as a binding site for several peripheral membrane proteins, including the cytoskeleton. The membrane-spanning domain of band 3 is thought to be mainly responsible for the former function, while the cytoplasmic domain has been identified as the site of the latter function. We have cleaved band 3 into the above two domains and have isolated each domain in native form. We propose to examine several structural and functional properties of each domain. We have recently shown that the 40,000-dalton cytoplasmic domain exists in a readily interconvertible, pH dependent equilibrium between two distinct native conformations. We plan to further characterize these two conformations and to determine how those ligands, e.g. Ca++, ADP, which may bind to the cytoplasmic domain influence its conformation. We also intend to examine the influence of pH and the above ligands on the affinity of the cytoplasmic domain for the peripheral proteins with which it interacts, e.g. ankyrin, hemoglobin, glyceraldehyde-3-phosphate dehydrogenase. We ahve observed that the isolated cytoplasmic domain of band 3 reversibly forms a water-insoluble, polymeric structure with hemoglobin. We will investigate the biological significance and behavior of this 1:1 copolymer. We will also explore the properties of the sickle cell hemoglobin-band 3 polymer and determine the conditions under which it forms and dissolves. Out studies of the 53,000-dalton membrane-spanning domain of band 3 indicate that the calorimetric stability of this domain is dependent on the lipids in which it is reconstituted. We plan to determine the lipid properties e.g. head group structure, acyl chain length and degree of saturation, required for reconstituting the native structure and the native function of this anion transport domain. The major methods structure and the native function of this anion transport domain. The major methods of research include differential scanning calorimetry and fluorescence spectroscopy.
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