title: High affinity binding of ankyrin induced by volume expansion in skate erythrocytes.
abstract: Volume expansion of little skate (Raja erinacea) erythrocytes increases the affinity of ankyrin binding without altering in the number of binding sites. Potassium iodide-stripped inside-out vesicles (KI-IOV) were used to assess ankyrin binding under volume-expanded conditions. Under isoosmotic conditions, ankyrin binds nearly exclusively to a single class of sites (Bmax, 52 +/- 12 microg/mg; Kd, 150 +/- 28 nM). KI-IOV from volume-expanded cells (either with one-half osmolarity medium or with inclusion of the permeant solute ethylene glycol) demonstrate two ankyrin-binding populations. A high affinity population occurs transiently under volume-expanded conditions. This population has a Bmax of 18 +/- 7 microg/mg and a Kd of 25 +/- 9 nM. Total binding of high and low affinity sites is 57 +/- 17 microg/mg. This change in ankyrin affinity is reversible on volume regulatory decrease. A major target protein in the KI-IOV was identified as the skate homolog of the mammalian red cell anion exchanger band 3. Inclusion of the purified cytoplasmic domain of band 3 competes away more than 80% of the ankyrin binding. To determine whether increased ankyrin affinity is due to band 3 tetramer formation that occurs in volume expansion, cells were treated with pyridoxal 5-phosphate or 4,4'-dinitrostilbene-2,2'-disulfonic acid, two agents that increase tetramer formation under isoosmotic conditions. Both treatments altered the binding affinity with a shift toward higher affinity binding without significant alteration in the number of binding sites.
authors.affiliation: Inflammatory Bowel Disease Center, Department of Medicine, University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: M W
authors.initials: MW
authors.lastname: Musch
authors.forename: L
authors.initials: L
authors.lastname: Goldstein
mesh_terms: D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism; D017487:Ankyrins / Q000378:metabolism*; D001665:Binding Sites; D004911:Erythrocyte Volume; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D012860:Skates, Fish; D013267:Stilbenes / Q000494:pharmacology
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D017487:Ankyrins; D013267:Stilbenes; D011732:Pyridoxal Phosphate; C022306:4,4'-dinitro-2,2'-stilbenedisulfonic acid