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

Abstract

The mechanism of dissociation of the stable dimer of band 3 was investigated during the incubation of isolated erythrocyte membranes or resealed ghosts 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 correlated with the change in the Stokes radius of band 3. MDB3 integrity was determined as follows: (1) by measuring the fluorescence emission spectrum of 4, 4'-di-isothiocyanostilbene-2,2'-disulphonate (DIDS) bound covalently to MDB3; (2) by measuring the number of DIDS covalent binding sites present after 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 only 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 process involves a conformation change in MDB3. There was a shift in the fluorescence spectrum for DIDS-labelled band 3 and a decrease in the DIDS binding capacity and transport activity of the unlabelled protein. Incubation of membranes at 4 degrees C inhibited the conformational change in MDB3 and the dissociation of dimers. Furthermore, no conformational change in MDB3 was observed when erythrocytes were incubated at 37 degrees C. We suggest that MDB3 unfolding is the molecular event responsible for the subsequent dissociation of stable dimers of band 3 to monomers during the incubation of erythrocyte membranes at 37 degrees C. The monomers so generated are either not functional in anion exchange or they have an attenuated functionality. The absence of a conformational change for band 3 in erythrocytes might imply that haemolysis perturbs the membrane structure and somehow predisposes band 3 to the conformational change that occurs during incubation at 37 degrees C.

Characterization of immunoglobulin binding to isolated human erythrocyte membranes: evidence for selective, temperature-induced binding of naturally occurring autoantibodies to the cytoskeleton

“…Erythrocytes were washed four times in PBS (5 mM sodium phosphate/150 mM NaCl (pH 8)) at 4³C, and the bu¡y coat was removed by aspiration. Stoichiometric labeling of band 3 in intact erythrocytes by DIDS (4,4P-diisothiocyanatostilbene-2,2P-disulfonic acid) was performed as described previously [10]. Samples were incubated for 45 min at 37³C and 50% hematocrit in PBS (hematocrit is de¢ned in this paper as the volume of packed cells or ghosts per total sample volumeU100), or in 25 WM DIDS in PBS.…”

“…Resealed ghosts were prepared essentially as described previously [10], with the following speci¢c modi¢cations. Six milliliters of washed erythrocytes were osmotically hemolyzed 1:5 in ice cold 5P8.…”

“…2.3.1. Unsealed ghosts Fifteen milliliter conical tubes were coated with the protease inhibitors PMSF (phenylmethylsulfonyl £uoride), pepstatin A and leupeptin, by adding methanol solutions containing these inhibitors, and evaporating the methanol as described previously [10]. White unsealed ghosts were placed into these pretreated tubes and brought to 50% hematocrit with 20P8.…”

“…Gel ¢ltration chromatography was performed essentially as described in previous reports from this laboratory [10,15] using Sepharose CL-4B as the matrix. The column bu¡er contained 100 mM NaCl, 5 mM sodium phosphate, 0.1% C 12 E 9 (poly(oxyethylene-9-lauryl ether)) pH 8.…”

Role of Band 3 in the Erythrocyte Membrane Structural Changes Under Isotonic and Hypotonic Conditions

“…The total number of band 3 per single erythrocyte is ∼1 × 10 6 . Thermal fluctuations of the erythrocyte membrane could induce conformational changes of cytoplasmic domain of transmembrane protein band 3 [29]. Such conformational changes result in electrostatic interactions between highly anionic N-terminal domains of band 3 molecules and on that base intensified their short-range self-associative tendency [28].…”