High affinity binding of ankyrin induced by volume expansion in skate erythrocytes.
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.
Musch
M W
MW
Inflammatory Bowel Disease Center, Department of Medicine, University of Chicago, Chicago, Illinois 60637, USA.
Goldstein
L
L
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
D001457:Anion Exchange Protein 1, Erythrocyte; D017487:Ankyrins; D013267:Stilbenes; D011732:Pyridoxal Phosphate; C022306:4,4'-dinitro-2,2'-stilbenedisulfonic acid