Kinetics of conformational changes associated with inhibitor binding to the purified band 3 transporter. Direct observation of allosteric subunit interactions.
AuthorsSalhany JM, Cordes KA, Schopfer LM
JournalBiochemistry
PubMed ID8338838
'Subunit interaction effects were identified for isolated human erythrocyte band 3, the anion exchanger, by observing both static and stopped-flow kinetic protein fluorescence changes associated with inhibitor binding to the intramonomeric stilbenedisulfonate site. We measured the rate of conformational changes associated with reversible binding of H2DIDS (4,4''-diisothiocyanodihydrostilbene-2,2''-disulfonate). The rate of ... More
Interactions between mutant and wild-type band 3 subunits in hereditary Southeast Asian ovalocytic red blood cell membranes.
AuthorsSalhany JM, Schopfer LM
JournalBiochemistry
PubMed ID8555182
'Red cell membranes from individuals with Southeast Asian ovalocytosis (SAO) contain approximately equal proportions of wild-type band 3 and a mutant SAO band 3 which lacks residues 400-408. It is known that the Vmax for anion exchange in SAO cells is reduced by about 50%, that SAO band 3 does ... More
Identification and partial purification of the erythrocyte L-lactate transporter.
AuthorsPoole RC, Halestrap AP
JournalBiochem J
PubMed ID1590773
'1. Intact erythrocytes were incubated with 100 microM-4,4''-di-isothiocyanostilbene-2,2''-disulphonate (DIDS), a concentration sufficient to inhibit lactate transport irreversibly by 65%. DIDS-labelled proteins were detected by immunoblotting of erythrocyte membrane proteins with an anti-DIDS antibody. Labelled polypeptides of 35-45 kDa in rat erythrocytes, and of 40-50 kDa in rabbit and guinea pig ... More
Reversible and irreversible inhibition, by stilbenedisulphonates, of lactate transport into rat erythrocytes. Identification of some new high-affinity inhibitors.
AuthorsPoole RC, Halestrap AP
JournalBiochem J
PubMed ID2025218
'1. Inhibition of L-lactate transport into rat erythrocytes by stilbenedisulphonates was studied under conditions which allowed the contribution of reversible and irreversible inhibition to be assessed. 2. At low temperatures (7 degrees C), 4,4''-di-isothiocyanostilbene-2,2''-disulphonate (DIDS) and other stilbenedisulphonates were found to inhibit lactate transport instantaneously, in a manner which was ... More
Location of the stilbenedisulfonate binding site of the human erythrocyte anion-exchange system by resonance energy transfer.
AuthorsRao A, Martin P, Reithmeier RA, Cantley LC
JournalBiochemistry
PubMed ID497152
'The stilbenedisulfonate inhibitory site of the human erythrocyte anion-exchange system has been characterized by using serveral fluorescent stilbenedisulfonates. The covalent inhibitor 4-benzamido-4''-isothiocyanostilbene-2,2''-disulfonate (BIDS) reacts specifically with the band 3 protein of the plasma membrane when added to intact erythrocytes, and the reversible inhibitors 4,4''-dibenzamidostilbene-2,2''-disulfonate (DBDS) and 4-benzamido-4''-aminostilbene-2,2''-disulfonate (BADS) show a ... More
Characterization of the stilbenedisulfonate binding site on band 3.
AuthorsSchopfer LM, Salhany JM
JournalBiochemistry
PubMed ID7599124
'Stilbenedisulfonates are potent inhibitors of Band 3 mediated anion exchange. They bind tightly to the protein and form a 1-to-1 reversible complex. Those stilbenedisulfonates which contain isothocyanato groups such as DIDS (4,4'-diisothiocyanato-2,2'-stilbenedisulfonate) and H2DIDS (4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate) can also react rapidly with lysine residues within the binding pocket to yield an irreversible ... More
Kinetic evidence for ternary complex formation and allosteric interactions in chloride and stilbenedisulfonate binding to band 3.
AuthorsSalhany JM, Sloan RL, Cordes KA, Schopfer LM
JournalBiochemistry
PubMed ID7918409
'The molecular basis for chloride and stilbenedisulfonate interaction with band 3 was investigated by measuring the kinetics of stilbenedisulfonate release from its complex with the transporter. We found that 150 mM NaCl accelerated the rate of release of DBDS (4,4''-dibenzamidostilbene-2,2''-dibenzamidostilbene-2,2''-disu lfonate) and H2DIDS (4,4''-diisothiocyanodihydrostilbene-2,2''-disulfonate) by more than 10-fold at constant ... More
Electron spin resonance studies on the inorganic-anion-transport system of the human red blood cell. Binding of a disulfonatostilbene spin label (NDS-TEMPO) and inhibition of anion transport.
AuthorsSchnell KF, Elbe W, Käsbauer J, Kaufmann E
JournalBiochim Biophys Acta
PubMed ID6307363
'The disulfonatostilbene spin label, NDS-TEMPO, was synthesized (purity over 96%) and the binding of the spin label to human red-cell ghosts was studied. NDS-TEMPO is readily absorbed to the membrane surface. Both pretreatment of the ghosts with FDNB and DIDS and the presence of DNDS completely prevent the binding of ... More
Sarcolemmal mechanisms for pHi recovery from alkalosis in the guinea-pig ventricular myocyte.
AuthorsLeem CH, Vaughan-Jones RD
JournalJ Physiol
PubMed ID9575297
'1. The mechanism of pHi recovery from an intracellular alkali load (induced by acetate prepulse or by reduction/removal of ambient PCO2) was investigated using intracellular SNARF fluorescence in the guinea-pig ventricular myocyte. 2. In Hepes buffer (pHo 7.40), pHi recovery was inhibited by removal of extracellular Cl-, but not by ... More
Human erythrocyte anion exchange site characterised using a fluorescent probe.
AuthorsDix JA, Verkman AS, Solomon AK, Cantley LC
JournalNature
PubMed ID503233
DIDS modifies the conductance, gating, and inactivation mechanisms of the cardiac ryanodine receptor.
AuthorsHill AP, Sitsapesan R
JournalBiophys J
PubMed ID12023226
The effects of the covalent modifier of amino groups, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) on the single-channel properties of purified sheep cardiac ryanodine receptors (RyR) incorporated into planar phospholipid bilayers were investigated. DIDS increased single-channel conductance and open probability (P(o)) and induced unique modifications to the voltage-dependence of gating. The effects of ... More
Control of red cell urea and water permeability by sulfhydryl reagents.
AuthorsToon MR, Solomon AK
JournalBiochim Biophys Acta
PubMed ID3017418
The binding constant for pCMBS (p-chloromercuribenzenesulfonate) inhibition of human red cell water transport has been determined to be 160 +/- 30 microM and that for urea transport inhibition to be 0.09 +/- 0.06 microM, indicating that there are separate sites for the two inhibition processes. The reaction kinetics show that ... More
Kinetics of the sarcolemmal lactate carrier in single heart cells using BCECF to measure pHi.
AuthorsWang X, Levi AJ, Halestrap AP
JournalAm J Physiol
PubMed ID7977806
The pH-sensitive fluorescent indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was used to measure lactate transport in single cardiac myocytes. Addition of lactate externally caused a rapid fall of intracellular pH (pHi), which was largely inhibited by 5 mM alpha-cyano-4-hydroxycinnamate (CHC), a specific inhibitor of the lactate carrier. Stilbene disulfonates such as 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) ... More
Differential sensitivity of stilbenedisulfonates in their reaction with band 3 HT (Pro-868-->Leu).
AuthorsSalhany JM, Schopfer LM, Kay MM, Gamble DN, Lawrence C
JournalProc Natl Acad Sci U S A
PubMed ID8524861
Band 3 HT (Pro-868-->Leu) is a mutant anion exchange protein which has several phenotypic characteristics, including a 2- to 3-fold larger Vmax, and reduced covalent binding of the anion transport inhibitor 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate (H2DIDS). We have used fluorescence kinetic methods to study inhibitor binding to band 3 to determine if the ... More
Chemical probes for anion transporters of mammalian cell membranes.
AuthorsCabantchik ZI, Greger R
JournalAm J Physiol
PubMed ID1566811
Mammalian cell membranes harbor several types of chloride channels, chloride-cation symporters/cotransporters, and several classes of anion exchangers/antiporters. These transport systems subserve different cellular or organismic functions, depending on the nature of the cell, the spatial organization of transporters, and their functional interplay. Chemical probing has played a central role in ... More
Target molecular weights for red cell band 3 stilbene and mercurial binding sites.
AuthorsVerkman AS, Skorecki KL, Jung CY, Ausiello DA
JournalAm J Physiol
PubMed ID3020989
Radiation inactivation was used to measure the target sizes for binding of disulfonic stilbene anion transport inhibitor 4,4'-dibenzamido-2,2'-disulfonic stilbene (DBDS) and mercurial water transport inhibitor p-chloromercuribenzene sulfonate (pCMBS) to human erythrocytes. The measured target size for erythrocyte ghost acetylcholinesterase was 78 +/- 3 kDa. DBDS binding to ghost membranes was ... More
Interaction of the erythrocyte lactate transporter (monocarboxylate transporter 1) with an integral 70-kDa membrane glycoprotein of the immunoglobulin superfamily.
AuthorsPoole RC, Halestrap AP
JournalJ Biol Chem
PubMed ID9169423
Treatment of intact erythrocytes with 4,4'-diisothiocyanostilbene-2, 2'-disulfonate (DIDS) causes irreversible inhibition and chemical labeling of the lactate transporter, monocarboxylate transporter 1 (MCT1) (Poole, R. C., and Halestrap, A. P. (1992) Biochem. J. 283, 855-862). In rat erythrocytes DIDS also causes cross-linking of MCT1 to another protein in the membrane to ... More
Lactate-proton co-transport and its contribution to interstitial acidification during hypoxia in isolated rat spinal roots.
AuthorsSchneider U, Poole RC, Halestrap AP, Grafe P
JournalNeuroscience
PubMed ID8389429
Exposure of nervous tissue to hypoxia results in interstitial acidification. There is evidence for concomitant decrease in extracellular pH to the increase in tissue lactate. In the present study, we used double-barrelled pH-sensitive microelectrodes to investigate the link between lactate transport and acid-base homeostasis in isolated rat spinal roots. Addition ... More
Regulation of the calcium release channel from skeletal muscle by suramin and the disulfonated stilbene derivatives DIDS, DBDS, and DNDS.
AuthorsO'Neill ER, Sakowska MM, Laver DR
JournalBiophys J
PubMed ID12609870
Activation of skeletal muscle ryanodine receptors (RyRs) by suramin and disulfonic stilbene derivatives (Diisothiocyanostilbene-2',2'-disulfonic acid (DIDS), 4,4'-dibenzamidostilbene-2,2'-disulfonic acid (DBDS),and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS)) was investigated using planar bilayers. One reversible and two nonreversible mechanisms were identified. K(a) for reversible activation (approximately 100 micro M) depended on cytoplasmic [Ca(2+)] and the bilayer ... More
Renal basolateral membrane anion transporter characterized by a fluorescent disulfonic stilbene.
AuthorsChen PY, Verkman AS
JournalJ Membr Biol
PubMed ID2448466
The fluorescence enhancement of 4,4'-dibenzamido-2,2'-disulfonic stilbene (DBDS) upon binding to membranes was used to examine proximal tubule stilbene binding sites. Equilibrium binding studies of DBDS to renal brush border (BBMV) and basolateral membrane vesicles (BLMV) were performed using a fluorescence enhancement technique developed for red blood cells (A.S. Verkman, J.A. ... More
Inhibition of inorganic anion transport across the human red blood cell membrane by chloride-dependent association of dipyridamole with a stilbene disulfonate binding site on the band 3 protein.
AuthorsLegrum B, Passow H
JournalBiochim Biophys Acta
PubMed ID2923878
The inhibition of inorganic anion transport by dipyridamole (2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido[5,4-d] pyrimidine) takes place only in the presence of Cl-, other halides, nitrate or bicarbonate. At any given dipyridamole concentration, the anion flux relative to the flux in the absence of dipyridamole follows the equation: Jrel = (1 + alpha 2[Cl-])/(1 + ... More
Characterization of the inhibition by stilbene disulphonates and phloretin of lactate and pyruvate transport into rat and guinea-pig cardiac myocytes suggests the presence of two kinetically distinct carriers in heart cells.
AuthorsWang X, Poole RC, Halestrap AP, Levi AJ
JournalBiochem J
PubMed ID8439293
1. The kinetics of transport of pyruvate (Km 0.20 mM), L-lactate (Km 2.2 mM) and D-lactate (Ki 10.2 mM) into rat cardiac myocytes were studied and compared with those for guinea-pig heart cells [Poole, Halestrap, Price and Levi (1989) Biochem. J. 264, 409-418] whose equivalent values were 0.07, 2.3 and ... More
The carboxyl side chain of glutamate 681 interacts with a chloride binding modifier site that allosterically modulates the dimeric conformational state of band 3 (AE1). Implications for the mechanism of anion/proton cotransport.
AuthorsSalhany JM, Sloan RL, Cordes KS
JournalBiochemistry
PubMed ID12578372
Glutamate 681 is thought to be located within the transport channel of band 3 (AE1, the chloride/bicarbonate exchanger), where it acts as a proton donor for the anion/proton cotransport function. Here we show that neutralization of the negative charge on glutamate 681 by chemically modifying band 3 with Woodward's reagent ... More
n-Alkanols and halothane inhibit red cell anion transport and increase band 3 conformational change rate.
AuthorsForman SA, Verkman AS, Dix JA, Solomon AK
JournalBiochemistry
PubMed ID4074663
The effects of halothane and n-alkanols on band 3, the anion-exchange protein of the red cell membrane, have been characterized by radioactive sulfate exchange and equilibrium and kinetic binding of a fluorescent anion transport inhibitor, 4,4'-dibenzamido-2,2'-stilbenedisulfonic acid (DBDS), with fluorescence and stopped-flow techniques. Ethanol, butanol, hexanol, heptanol, octanol, and decanol ... More
Mechanism of coupling between Cl- and OH- transport in renal brush-border membranes.
AuthorsIves HE, Chen PY, Verkman AS
JournalBiochim Biophys Acta
PubMed ID3778914
The coupling mechanism for Cl- and H+/OH- transport in renal brush-border vesicles was examined from intravesicular pH changes following imposed H+ and Cl- gradients. Vesicles were loaded with 6-carboxyfluorescein and exposed to H+ gradients and Cl-, gluconate, or sulfate gradients, each with and without a K+/valinomycin voltage clamp. Parallel experiments ... More
The kinetics of intramolecular cross-linking of the band 3 protein in the red blood cell membrane by 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid (H2DIDS).
AuthorsKampmann L, Lepke S, Fasold H, Fritzsch G, Passow H
JournalJ Membr Biol
PubMed ID7186941
The two isothiocyanate groups of the anion transport inhibitor 4,4'-diisothiocyano dihydrostilbene-2-2'-disulfonate (H2DIDS) may react covalently with two lysine residues called a and b that reside on the chymotryptic 60,000 Dalton and 35,000 Dalton segments, respectively, of the band 3 protein of the human erythrocyte membrane. Under suitable conditions, the reaction ... More
Mechanisms of pHi recovery after global ischemia in the perfused heart.
AuthorsVandenberg JI, Metcalfe JC, Grace AA
JournalCirc Res
PubMed ID8386598
A Na(+)-HCO3- coinflux carrier and the Na(+)-H+ antiport have both been shown to contribute to recovery from intracellular acidosis in cardiac tissue. We have investigated the participation of these mechanisms as well as metabolite (lactate and CO2) washout in the recovery of pHi after myocardial ischemia. Isovolumic ferret hearts were ... More