title: Restriction by ankyrin of band 3 rotational mobility in human erythrocyte membranes and reconstituted lipid vesicles.
abstract: Rotational diffusion of eosin-5-maleimide-labeled band 3 was measured in erythrocyte membranes at pH 9.4-10.4. Band 3 was found to be more mobile in this pH range than at pH 7.5. Similar results were obtained with spectrin-actin-depleted membranes, where it was further shown that ankyrin is the only detectable protein released from the membrane at pH 10. Further experiments were performed at pH 7.5 to investigate the effects of rebinding purified ankyrin and/or band 4.1 to ghosts stripped of skeletal proteins. Ankyrin was found to reduce band 3 rotational mobility, but band 4.1 had no effect. A fluorescence binding assay revealed that fluorescein isothiocyanate-labeled ankyrin had similar binding parameters to those reported previously using 125I labeling. Finally, the rotational mobility of purified band 3 reconstituted into lipid bilayers was determined before and after ankyrin binding. The results of these reconstitution experiments were globally analyzed, assuming the existence of two populations of band 3 with different correlation times. The faster correlation time is consistent with that expected for either dimers or compact tetramers of band 3. Ankyrin binding reduces the proportion of band 3 contributing to the faster component. This result demonstrates that ankyrin promotes the association of band 3 into more slowly rotating complexes independently of any other components of the erythrocyte membrane. It has been reported that ankyrin contains two binding sites for band 3 [Michaely, P., & Bennett, V. (1995) J. Biol. Chem. 270, 22050-22057]. The results of the present study are thus explained by the ability of ankyrin to cross-link band 3 into larger diameter complexes. Cross-linking by ankyrin in part accounts for the slow components in the anisotropy decays of band 3 in the erythrocyte membrane. Other factors which probably influence band 3 aggregation include the membrane "fluidity" and protein concentration.
authors.affiliation: Department of Biological and Chemical Sciences, Central Campus, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, U.K.
authors.forename: A
authors.initials: A
authors.lastname: Che
authors.forename: I E
authors.initials: IE
authors.lastname: Morrison
authors.forename: R
authors.initials: R
authors.lastname: Pan
authors.forename: R J
authors.initials: RJ
authors.lastname: Cherry
mesh_terms: D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry*; D017487:Ankyrins / Q000737:chemistry*; D003598:Cytoskeletal Proteins; D004058:Diffusion; D004910:Erythrocyte Membrane / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008563:Membrane Lipids / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry; D009479:Neuropeptides; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D017487:Ankyrins; D003598:Cytoskeletal Proteins; D008563:Membrane Lipids; D008565:Membrane Proteins; D009479:Neuropeptides; C028841:erythrocyte membrane band 4.1 protein; C120557:erythrocyte membrane protein band 4.1-like 1