CYTOSKELETON-MEMBRANE CONNECTIONS IN THE HUMAN ERYTHROCYTE-MEMBRANE -BAND-4.1 BINDS TO TETRAMERIC BAND-3 PROTEIN

Band 4.1 provides, besides ankyrin, the main linkage between the eryth rocyte membrane and its cytoskeleton. Its predominant binding sites in the membrane are located on the glycophorins. However, the cytoplasmi c domain of band 3 can also bind band 4.1. We have studied which of th e different band 3 oligomers observed (monomers, dimers, tetramers) ca n act as band 4.1 binding sites, by equilibrium sedimentation experime nts on mixtures of purified band 3 and dye-labelled band 4.1 in soluti ons of a nonionic detergent. At low molar ratios of band 4.1 and band 3, the sedimentation equilibrium distributions obtained could all be p erfectly fitted assuming that only two dye-labelled particles were pre sent: uncomplexed band 4.1 and a complex formed between one band 4.1 m olecule and one band 3 tetramer. The presence of small amounts of comp lexes containing band 3 monomers or dimers could not be completely rul ed out but is unlikely. On the other hand, stabilized band 3 dimers ef fectively bound band 4.1. At higher molar band 4.1/band 3 ratio, the b and 3 tetramer apparently could bind up to at least four band 4.1 mole cules. The band 4.1/band 3 tetramer complex was found to be unstable. The results described, together with those reported previously, point at a prominent role of tetrameric band 3 in ligand binding.