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Volume 271, Number 35, Issue of August 30, 1996 pp. 21221-21225
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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High Affinity Binding of Ankyrin Induced by Volume Expansion in Skate Erythrocytes

(Received for publication, April 26, 1996)

Mark W. Musch ^§ and Leon Goldstein ^§¶

From the  Inflammatory Bowel Disease Center, Department of Medicine, University of Chicago, Chicago, Illinois 60637, the ^§ Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672, and the ^¶ Department of Physiology, Brown University, Providence, Rhode Island 02912

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 (B_max, 52 ± 12 µg/mg; K_d, 150 ± 28 n). 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 B_max of 18 ± 7 µg/mg and a K_d of 25 ± 9 n. Total binding of high and low affinity sites is 57 ± 17 µg/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.

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This article has been cited by other articles:

				M. W. Musch and L. Goldstein Tyrosine kinase inhibition affects skate anion exchanger isoform I alterations after volume expansion Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2005; 288(4): R885 - R890. [Abstract] [Full Text] [PDF]

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