ASSOCIATION STATE OF HUMAN RED-BLOOD-CELL BAND-3 AND ITS INTERACTION WITH ANKYRIN

We have studied the association state of band 3, the anion channel and predominant transmembrane protein of the human red blood cell, and th e anomalous stoichiometry and dynamics of its interaction with ankyrin , which acts as a link to the spectrin of the membrane skeletal networ k. Band 3 exists in benign nonionic detergent solutions as a dimer. Te tramer is formed irreversibly in the course of manipulations, particul arly in ion-exchange chromatography. The dimer in solution binds ankyr in without self-associating. In ankyrin-free inside-out membrane vesic les and when incorporated into phosphatidylcholine liposomes, only som e 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-extract ed membrane cytoskeleton, but is released if the ankyrin has been clea ved with chymotrypsin before detergent treatment; thus, the attachment to the membrane cytoskeleton is entirely through ankyrin and not thro ugh other constituents such as protein 4.1. The ratio of band 3 to ank yrin in this complex implies that it consists of two chains of band 3 and one chain of ankyrin, at least after detergent extraction, The bou nd and free populations of band 3 exchange freely in the membrane. In the artificial liposome membrane binding of ankyrin to band 3 dimers c ause association of the band 3 into higher aggregates, as seen in free ze-fracture electron microscopy. Successive manipulations of the red b lood cell membrane, which are involved in the preparation of ghosts, o f inside-out vesicles, and of inside-out vesicles stripped of peripher al proteins are accompanied by progressive aggregation of intramembran e particles, as judged by freeze-fracture electron microscopy, Thus th e intramembrane particles are evidently stabilized in the intact cell by the peripheral protein network and the cytosolic milieu. Aggregatio n may be expected to limit the number of functional ankyrin binding si tes. However, although extraneous ankyrin binds to the unoccupied bind ing site on the spectrin tetramers in intact ghost membranes, little o r no ankyrin can bind to the unoccupied band 3 dimers in situ, perhaps by reason of occlusion of binding sites by the membrane skeletal netw ork. (C) 1995 by The American Society of Hematology.