Studying heterologous associations between membrane proteins by analytical ultracentrifugation: Experience with erythrocyte band 3.

Applying procedures originally suggested for water-soluble proteins (Osborne JC, Powell GM, Brewer HB (1980) Biochim Biophys Acta 619:559-571), our group has studied the association of the intrinsic membrane protein band 3 (from human erythrocytes) with other proteins for which band 3 represents a binding site. In all cases, the association was studied by sedimentation equilibrium analysis on detergent-solubilized band 3 and dye-labeled ligand proteins, under conditions where the proteins show ideal sedimentation behavior. During these studies, a standard step-by-step procedure has developed which allows the determination of the stoichiometry of the predominant heterologous complex and the detection of complexes of different band 3 or ligand content. Application of the procedure to aldolase, erythrocyte band 4.1 protein and hemoglobin as ligand proteins is demonstrated. It is shown that, in all cases studied, the band 3 tetramer represents the predominant or even the exclusive binding site. The number of ligand proteins bound per band 3 tetramer can reach 4 for aldolase and hemoglobin or nearly 8 for band 4.1. [ABSTRACT FROM AUTHOR]