Analysis of the oligomeric state of Band 3, the anion transport protein of the human erythrocyte membrane, by size exclusion high performance liquid chromatography. Oligomeric stability and origin of heterogeneity |
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Authors: | J R Casey R A Reithmeier |
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Institution: | Department of Medicine, University of Toronto, Ontario, Canada. |
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Abstract: | The oligomeric state of human Band 3 (Mr = 95,000), the erythrocyte membrane anion exchanger, was examined by size exclusion high performance liquid chromatography in solutions containing the nonionic detergent C12E8 (octaethylene glycol n-dodecyl monoether). Band 3 was heterogeneous with respect to oligomeric composition, the predominant (70%) species being a dimer that bound 0.57 mg of C12E8/mg of protein (Stokes radius = 78 A, s20,w = 6.9 S). Variable amounts of larger oligomers were also present; however, no evidence for equilibration between oligomeric species was observed in detergent solution. Analytical and large zone size exclusion chromatography showed that Band 3 could not be dissociated to monomers, other than by protein denaturation. The membrane domain of Band 3 (Mr = 52,000) was also dimeric, but without evidence for higher oligomeric forms, which implies that the interactions responsible for higher associations involve the cytoplasmic domain. Prelabeling of Band 3 with the anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate had no effect upon the oligomeric state of either intact Band 3 or its 52-kDa membrane domain. Band 3 oligomeric state could be reversibly changed in the membrane by altering the pH of the solution. The fraction of Band 3 not associated with the cytoskeleton was almost entirely dimeric. Band 3 purified from erythrocytes separated by density gradient centrifugation revealed that older red cells contained a larger proportion of higher oligomers than did younger cells. We conclude that Band 3, in the membrane and in C12E8 solution, exists as a mixture of dimers and larger oligomers. The higher oligomers interact with the cytoskeleton, increase in amount with cell age, and are held together by interactions of the cytoplasmic domain. |
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