In situ cross-linking of human erythrocyte band 3 by bis(sulfosuccinimidyl)suberate. Evidence for ligand modulation of two alternate quaternary forms: covalent band 3 dimers and noncovalent tetramers formed by the association of two covalent dimers.

Salhany, James M.; Sloan, Renee L.; Cordes, Karen A.

doi:10.1016/s0021-9258(18)38218-8

Cited by 48 publications

(26 citation statements)

References 46 publications

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“…In normal cells without BS3, label is mainly in band 3; BS3 cross-links about 80% of the label into the position of the dimer, consistent with the gel scans of unlabeled protein in Figure 2. This result confirms that although stilbenedisulfonates alter the dimer-tetramer associations of band 3 (Salhany et al, 1990(Salhany et al, , 1991, the presence of H2D1DS does not interfere with covalent intermolecular cross-linking by BS3 . In SAO cells the same combined cross-linking protocol gives a different labeling pattern.…”

Section: Resultssupporting

confidence: 76%

“…There is very little higher oligomer. (Salhany et al [1990] showed that BS3 can induce noncovalent tetramers [dimer of dimers] in band 3, but these dissociate under the hightemperature solubilization conditions used here.) We conclude that both normal and SAO band 3 can be cross-linked by BS3 into covalent dimers, with negligible formation of covalent higher oligomers.…”

Section: Resultsmentioning

confidence: 71%

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Anion Exchange Protein in Southeast Asian Ovalocytes: Heterodimer Formation between Normal and Variant Subunits

Jennings

Gosselink²

1995

Biochemistry

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Chemical cross-linking has been used to determine the composition of the erythrocyte band 3 protein dimer in Southeast Asian ovalocytes (SAO). Individuals with SAO are heterozygous for a mutation in which residues 400-408 of band 3 are deleted. Normal and variant protein are present in equal amounts, but the SAO protein does not transport anions or bind stilbenedisulfonates with high affinity. We find that the rate constant for 35SO4(2-) efflux from SAO cells is about 50% that of normal cells, but the time course is a single exponential, indicating that there is no detectable heterogeneity in the distribution of SAO band 3 in the population of cells. Treatment of intact cells with the homobifunctional crosslinker BS3 (bis[sulfosuccinimido]suberate) produces similar amounts of covalent dimer in both normal and SAO cells. In SAO cells, copies of normal band 3 can be distinguished from SAO band 3 by treating with H2DIDS to form a crosslink between major chymotryptic fragments (60 kDa and 35 kDa) within one subunit. Successive treatment of cells with [3H]-4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate ([3H]H2DIDS), BS3, and chymotrypsin gives 3H-labeled products that include homodimer of normal band 3 as well as products of crosslinking normal band 3 with the 60- and 35-kDa fragment of SAO band 3. The results are in semiquantitative agreement with a model in which covalent dimer forms between normal and SAO subunits with the same probability as between two normal subunits. These results indicate that the normal copy of band 3, complexed in a heterodimer with SAO band 3, reacts with H2DIDS as in normal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Resultssupporting

confidence: 76%

Section: Resultsmentioning

confidence: 71%

Anion Exchange Protein in Southeast Asian Ovalocytes: Heterodimer Formation between Normal and Variant Subunits

Jennings

Gosselink²

1995

Biochemistry

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“…Other evidence for tetrameric band 3 is that CuP forms some covalent tetramers in addition to dimers ( 177 , 266 ). Dimers cross-linked by BS 3 can form noncovalent tetramers that are stable in SDS at moderate temperatures, indicating that there are interactions between adjacent dimers ( 267 ). Clusters of band 3 larger than tetramer are associated with a variety of pathophysiological conditions, including oxidative stress, hemoglobin denaturation, and cell senescence ( 268 – 273 ).…”

Section: Associations Of Band 3 Dimersmentioning

confidence: 99%

Cell physiology and molecular mechanism of anion transport by erythrocyte band 3/AE1

Jennings

2021

American Journal of Physiology-Cell Physiology

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The major transmembrane protein of the red blood cell, known as band 3, AE1, and SLC4A1, has two main functions: 1) catalysis of Cl-/HCO3- exchange, one of the steps in CO2 excretion; 2) anchoring the membrane skeleton. This review summarizes the 150 year history of research on red cell anion transport and band 3 as an experimental system for studying membrane protein structure and ion transport mechanisms. Important early findings were that red cell Cl- transport is a tightly coupled 1:1 exchange and band 3 is labeled by stilbenesulfonate derivatives that inhibit anion transport. Biochemical studies showed that the protein is dimeric or tetrameric (paired dimers) and that there is one stilbenedisulfonate binding site per subunit of the dimer. Transport kinetics and inhibitor characteristics supported the idea that the transporter acts by an alternating access mechanism with intrinsic asymmetry. The sequence of band 3 cDNA provided a framework for detailed study of protein topology and amino acid residues important for transport. The identification of genetic variants produced insights into the roles of band 3 in red cell abnormalities and distal renal tubular acidosis. The publication of the membrane domain crystal structure made it possible to propose concrete molecular models of transport. Future research directions include improving our understanding of the transport mechanism at the molecular level and of the integrative relationships among band 3, hemoglobin, carbonic anhydrase, and gradients (both transmembrane and subcellular) of HCO3-, Cl-, O2, CO2, pH, and NO metabolites during pulmonary and systemic capillary gas exchange.

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“…SDS\PAGE analysis of the various preparations was performed with 7.5 % (w\v) polyacrylamide gels ; the gels were stained with Coomassie Blue, as described previously [22].…”

Section: Sds/pagementioning

confidence: 99%

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

2000

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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.

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In situ cross-linking of human erythrocyte band 3 by bis(sulfosuccinimidyl)suberate. Evidence for ligand modulation of two alternate quaternary forms: covalent band 3 dimers and noncovalent tetramers formed by the association of two covalent dimers.

Cited by 48 publications

References 46 publications

Anion Exchange Protein in Southeast Asian Ovalocytes: Heterodimer Formation between Normal and Variant Subunits

Anion Exchange Protein in Southeast Asian Ovalocytes: Heterodimer Formation between Normal and Variant Subunits

Cell physiology and molecular mechanism of anion transport by erythrocyte band 3/AE1

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

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