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Association state of human red blood cell band 3 and its interaction with
ankyrin
JC Pinder, A Pekrun, AM Maggs, AP Brain and WB Gratzer
Medical Research Council Muscle and Cell Motility Unit, King's College,
London, UK.
We have studied the association state of band 3, the anion channel and
predominant transmembrane protein of the human red blood cell, and the
anomalous stoichiometry and dynamics of its interaction with ankyrin, which
acts as a link to the spectrin of the membrane skeletal network. Band 3
exists in benign nonionic detergent solutions as a dimer. Tetramer is
formed irreversibly in the course of manipulations, particularly in
ion-exchange chromatography. The dimer in solution binds ankyrin without
self-associating. In ankyrin-free inside-out membrane vesicles and when
incorporated into phosphatidylcholine liposomes, only some 10% to 15% of
band 3 chains bind ankyrin at saturation. Moreover, in liposomes this was
independent of protein:lipid ratio between 1:2 and 1:40. The bound fraction
of band 3 remains with the detergent-extracted membrane cytoskeleton, but
is released if the ankyrin has been cleaved with chymotrypsin before
detergent treatment; thus, the attachment to the membrane cytoskeleton is
entirely through ankyrin and not through other constituents such as protein
4.1. The ratio of band 3 to ankyrin in this complex implies that it
consists of two chains of band 3 and one chain of ankyrin, at least after
detergent extraction. The bound and free populations of band 3 exchange
freely in the membrane. In the artificial liposome membrane binding of
ankyrin to band 3 dimers cause association of the band 3 into higher
aggregates, as seen in freeze-fracture electron microscopy. Successive
manipulations of the red blood cell membrane, which are involved in the
preparation of ghosts, of inside-out vesicles, and of inside-out vesicles
stripped of peripheral proteins are accompanied by progressive aggregation
of intramembrane particles, as judged by freeze-fracture electron
microscopy. Thus the intramembrane particles are evidently stabilized in
the intact cell by the peripheral protein network and the cytosolic milieu.
Aggregation may be expected to limit the number of functional ankyrin
binding sites. However, although extraneous ankyrin binds to the unoccupied
binding site on the spectrin tetramers in intact ghost membranes, little or
no ankyrin can bind to the unoccupied band 3 dimers in situ, perhaps by
reason of occlusion of binding sites by the membrane skeletal network.
Volume 85,
Issue 10,
pp. 2951-2961,
05/15/1995
Copyright © 1995 by The American Society of Hematology
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