Mechanism of band 3 dimer dissociation during incubation of erythrocyte membranes at 37 degrees C

The mechanism of dissociation of the stable dimer of band 3 was investigate d during the incubation of isolated erythrocyte membranes or resealed ghost s at 37 degrees C. The kinetics of changes in the structural and functional integrity of the membrane domain of band 3 (MDB3) were measured and correl ated with the change in the Stokes radius of band 3. MDB3 integrity was det ermined as follows: (1) by measuring the fluorescence emission spectrum of 4,4'-di-isothiocyanostilbene-2,2'-disulphonate (DIDS) bound covalently to M DB3; (2) by measuring the number of DIDS covalent binding sites present aft er incubation of unlabelled resealed ghosts; and (3) by measuring the anion transport V-max by using the same resealed ghosts, Incubation of membranes at 37 degrees C caused the dissociation of band 3 dimers to monomers but o nly after a lag period lasting approx. 50 h, The observation of such a lag implies that dissociation involves a sequence of molecular events beginning with some type of initial process. We have discovered that this initial pr ocess involves a conformation change in MDB3. There was a shift in the fluo rescence spectrum for DIDS-labelled band 3 and a decrease in the DIDS bindi ng capacity and transport activity of the unlabelled protein. Incubation of membranes at 4 degrees C inhibited the conformational change in MDB3 and t he dissociation of dimers. Furthermore, no conformational change in MDB3 wa s observed when erythrocytes were incubated at 37 degrees C, We suggest tha t MDB3 unfolding is the molecular event responsible for the subsequent diss ociation of stable dimers of band 3 to monomers during the incubation of er ythrocyte membranes at 37 degrees C. The monomers so generated are either n ot functional in anion exchange or they have an attenuated functionality. T he absence of a conformational change for band 3 in erythrocytes might impl y that haemolysis perturbs the membrane structure and somehow predisposes b and 3 to the conformational change that occurs during incubation at 37 degr ees C.