Isolation of an ankyrin-band 3 oligomer from human erythrocyte membranes

A cytoskeleton-associated population of band 3 has been isolated in milligram quantities from human erythrocyte membranes as a stable complex with ankyrin. The major population of band 3 (free band 3) was solubilized from ghosts with 0.1 M KCl/Triton X-100. A detergent-insoluble assembly of proteins (cytoskeletons) contained 10-15% of the band 3, as well as ankyrin, spectrin, band 4.1, actin and other minor polypeptides. The remaining band 3 and ankyrin were extracted in a 1:1 molar ratio from the cytoskeletons with 1 M KCl/Triton X-100, and were copurified with the same 1:1 stoichiometry during DEAE-chromatography, and gel filtration. Free band 3 was isolated by the same procedures, and was clearly resolved from ankyrin-associated band 3 on DEAE-chromatography and gel filtration. Direct evidence that ankyrin and band 3 were associated in a complex was provided by immunoprecipitation with anti-ankyrin IgG of band 3 from the native complex, but not of free band 3 or after denaturation of the complex. Ankyrin-associated band 3 contained a reactive site for H2DIDS (the dihydroanalog of 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid) and thus has an anion transport site. Comparison of 125I-labeled α-chymotryptic peptide fragments of free band 3 and ankyrin-associated band 3 revealed extensive homology with 28 out of 30 identical fragments. The ankyrin-band 3 oligomer is arranged as an αβ dimer with one polypeptide chain of each component, based on the molecular weight calculated from hydrodynamic parameters in dilute solution. Free band 3 behaved under the same conditions as a homodimer. Ankyrin-associated band 3 was capable of band 3 dimerization at concentrations of 1-3 μM, since chemical cross-linking of the oligomer with Cu2+/ophenanthroline produced a 190 000 Mr band 3 dimer on SDS gels. © 1982.

Duke Scholars

Author Vann Bennett Biochemistry