Association between ankyrin and the cytoplasmic domain of band 3 isolated from the human erythrocyte membrane.

Abstract

A 43,000-dalton proteolytic fragment of the cytoplasmic domain of band 3 was purified, and monospecific antibodies against this fragment were prepared.1z61-Labeled 43,000-dalton fragment associated with pure ankyrin in solution, with a KO of 8 X lo"' M and with a stoichiometry of 0.85 mol of fragment/mol of ankyrin (with limiting concentrations of ankyrin).Anti-43,000dalton fragment IgG, which cross-reacted only with band 3, immunoprecipitated ankyrin, and band 4.2, as well as band 3 from detergent extracts of membranes, provided these were not denatured.1261-Labeled ankyrin associated selectively with the cytoplasmic surface of inside-out vesicles depleted of ankyrin as well as spectrin, actin, and bands 4.1,4.2,and 6.The binding of '2sI-labeled ankyrin was decreased over 90% by preincubating this protein above 54"C, or by preincubating the vesicles in 3 M guanidine.Scatchard plots of binding were biphasic, with high affinity (KO = 5 x M) and low affinity (KO = 1.4 X 10" M) components, and extrapolated to 65 to 75 p g of ankyrin/mg of ghost protein, or about 10' sites/erythrocyte.Selective proteolytic cleavage of vesicles and release of the cytoplasmic domain of band 3 abolished the high affinity binding of 12%labeled ankyrin, and decreased the number of low affinity sites by about 50%.Anti-43,OOO-dalton fragment IgG at 12 pg/ml inhibited over 90% of binding of low concentrations of '261-labeled ankyrin, and decreased binding by 60% at high concentrations of ankyri n.The 43,000-dalton fragment blocked binding of '2611-labeled ankyrin to vesicles by a competitive type of inhibition with a ki of 6.6 X 10" M.It is concluded that ankyrin and the cytoplasmic domain of band 3 associate directly without requirement for intermediary proteins, and that the cytoplasmic domain of band 3 participates, perhaps with other protein@), in attachment of ankyrin to the inner surface of the membrane.Human erythrocyte ankyrin (band 2.1') is a polypeptide of M, = 215,000 localized on the cytoplasmic surface of the membrane which has been identified as the high affinity membrane attachment site for spectrin (Bennett and Stenbuck, 1979a; Luna et al., 1979 Yu andGoodman, 1979).Association of spectrin with ankyrin has been demonstrated