Effect of pH on the self-association of erythrocyte band 3 in situ.
The human erythrocyte anion exchanger (band 3) contains a cytoplasmic domain (cdb3) that exists in a reversible, pH-dependent structural equilibrium among three native conformations. To understand how this conformational equilibrium might influence the association state of band 3, we have incubated stripped erythrocyte membranes in solutions ranging from pH 6.0 to pH 10.5 and have examined the oligomeric state of the protein by size exclusion high performance liquid chromatography. We demonstrate that incubation of membranes in slightly acidic conditions favors dimer formation, whereas extended incubation at higher pHs (pH>9) leads to irreversible formation of an oligomeric species larger than the tetramer. Since the pH dependence of the conformational state of the cytoplasmic domain exhibits a similar pH profile, we suggest that the conformation of the cytoplasmic domain can modulate the self-association of band 3. Importantly, this modulation would appear to require the structural interactions present within the intact protein, since the isolated membrane-spanning domain does not display any pH dependence of association. The irreversible nature of the alkali-induced aggregation further suggests that a secondary reaction subsequent to band 3 association is required to stabilize the high molecular weight aggregate. Although we were able to eliminate covalent bond formation in this irreversible aggregation process, the exact nature of the secondary reaction remains to be elucidated.
Rettig
M P
MP
Department of Chemistry, 1393 Brown Bldg., Purdue University, West Lafayette, IN 47907, USA.
Orendorff
C J
CJ
Campanella
E
E
Low
P S
PS
D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry*; D017487:Ankyrins / Q000737:chemistry; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D019281:Dimerization; D004910:Erythrocyte Membrane / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation
D001457:Anion Exchange Protein 1, Erythrocyte; D017487:Ankyrins