4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and 4-4--dibenzamido-2-2--stilbenedisulfonic-acid

Hepatocyte nuclear factor 4 alpha (HNF4α) is a multi-faceted nuclear receptor responsible for governing the development and proper functioning of liver and pancreatic islet cells. Its transcriptional functions encompass the regulation of vital metabolic processes including cholesterol and fatty acid metabolism, and glucose sensing and control. Various genetic mutations and alterations in HNF4α are associated with diabetes, metabolic disorders, and cancers. From a structural perspective, HNF4α is one of the most comprehensively understood nuclear receptors due to its crystallographically observed architecture revealing interconnected DNA binding domains (DBDs) and ligand binding domains (LBDs). This review discusses key properties of HNF4α, including its mode of homodimerization, its binding to fatty acid ligands, the importance of post-translational modifications, and the mechanistic basis for allosteric functions. The surfaces linking HNF4α's DBDs and LBDs create a convergence zone that allows signals originating from any one domain to influence distant domains. The HNF4α-DNA complex serves as a prime illustration of how nuclear receptors utilize individual domains for specific functions, while also integrating these domains to create cohesive higher-order architectures that allow signal responsive functions.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Epithelial Cells; Fatty Acids; Lipid Metabolism

Stilbenedisulfonates are potent competitive inhibitors of the anion exchange (AE) class of transporters. Although these molecules have been extensively used in studies of the anion exchange function, the actual mechanism by which stilbenedisulfonates compete with transported anions has been uncertain. Over the last several years, work in my laboratory has focused on understanding the mechanism of stilbenedisulfonate binding to human erythrocyte band 3 (AE1), with particular emphasis placed on deciding whether stilbenedisulfonates are pure competitive inhibitors, or whether they inhibit transport allosterically. I summarize our results suggesting that stilbenedisulfonates are allosteric inhibitors of band 3 anion exchange. I also summarize results which show that covalent binding of stilbenedisulfonates to one subunit produces allosteric effects which extend to the neighboring subunit in a band 3 dimer. Such allosteric subunit interactions have been observed: a) in divalent anion influx exchange experiments; b) in reversible stilbenedisulfonate binding studies, and c) in thermal unfolding studies of the membrane domain of band 3. In addition, two quaternary conformational states of the band 3 dimer, modulated by ligands of the stilbenedisulfonate site, have been identified in protein crosslinking studies. Finally, new evidence is discussed showing that Southeast Asian ovalocytic band 3 in a heterodimer composed of mutant and wild-type subunits, increases the 4,4'-diisothiocyanodihydro-2,2'-stilbenedisulfonate (H2DIDS) affinity of the wild-type subunit. Taken together, these results challenge the view that band 3 exists as structurally independent monomers. In addition, they suggest that subunit interactions may play a significant role in the transport function.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Allosteric Site; Animals; Anion Exchange Protein 1, Erythrocyte; Binding, Competitive; Erythrocyte Membrane; Humans; Ion Transport; Isothiocyanates; Protein Conformation; Stilbenes

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Allosteric Site; Amino Acid Sequence; Amino Acids; Animals; Anion Exchange Protein 1, Erythrocyte; Anions; Arginine; Binding Sites; Biological Transport; Chemical Phenomena; Chemistry; Chemistry, Physical; Cross-Linking Reagents; Electric Conductivity; Energy Metabolism; Erythrocyte Membrane; Hemoglobins; Humans; Hydrogen-Ion Concentration; Ion Channels; Kinetics; Lipid Bilayers; Macromolecular Substances; Mathematics; Models, Biological; Peptide Fragments; Protein Binding; Protein Conformation; Protein Processing, Post-Translational; Temperature; Thermodynamics

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4-Chloromercuribenzenesulfonate; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Anion Exchange Protein 1, Erythrocyte; Anions; Biological Transport; Cations; Cell Membrane Permeability; Electrolytes; Erythrocyte Membrane; Humans; Hydrogen Bonding; Ion Channels; Membrane Lipids; Models, Molecular; Protein Conformation; Spectrometry, Fluorescence; Sulfhydryl Compounds; Sulfhydryl Reagents; Thermodynamics; Tryptophan; Urea; Water

Band 3 mediates both electroneutral AE (anion exchange) and APCT (anion/proton co-transport). Protons activate APCT and inhibit AE with the same pK (approximately 5.0). SDs (stilbenedisulphonates) bind to a primary, high-affinity site on band 3 and inhibit both AE and APCT functions. In this study, we present fluorescence and kinetic evidence showing that lowering the pH activates a second site on band 3, which binds DBDS (4,4'-dibenzamido-2,2'-stilbenedisulphonate) independently of chloride concentration, and that DBDS binding to the second site inhibits the APCT function of band 3. Activation of the second site correlated with loss of chloride binding to the transport site, thus explaining the lack of competition. The kinetics of DBDS binding at the second site could be simulated by a slow-transition, two-state exclusive binding mechanism (R0<-->T0+D<-->TD<-->RD, where D represents DBDS, R0 and T0 represent alternate conformational states at the second DBDS-binding site, and TD and RD are the same two states with ligand DBDS bound), with a calculated overall Kd of 3.9 microM and a T0+D<-->TD dissociation constant of 55 nM. DBDS binding to the primary SD site inhibited approx. 94% of the proton transport at low pH (KI=68.5+/-11.8 nM). DBDS binding to the second site inhibited approx. 68% of the proton transport (KI=7.27+/-1.27 microM) in a band 3 construct with all primary SD sites blocked through selective cross-linking by bis(sulphosuccinimidyl)suberate. DBDS inhibition of proton transport at the second site could be simulated quantitatively within the context of the slow-transition, two-state exclusive binding mechanism. We conclude that band 3 contains two DBDS-binding sites that can be occupied simultaneously at low pH. The binding kinetic and transport inhibition characteristics of DBDS interaction with the second site suggest that it may be located within a gated access channel leading to the transport site.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Anion Exchange Protein 1, Erythrocyte; Binding Sites; Cell Membrane; Computer Simulation; Erythrocytes; Hydrogen-Ion Concentration; Kinetics; Protein Binding

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 K plus sodium borohydride (i.e., the modification process) exposes a cryptic, conformationally active chloride-binding site which functions to modulate allosterically the conformational state of the band 3 dimer. Chloride binding was determined by measuring the effect of increasing chloride concentration on the rate of DBDS (4,4'-dibenzamido-2,2'-stilbenedisulfonate) release from band 3 using a stopped-flow fluorescence kinetic inhibitor replacement assay with DIDS (4,4'-diisothiocyanato-2,2'-stilbenedisulfonate) as the replacing inhibitor. The time course for DBDS release from unmodified, control band 3 was monophasic and exponential. Chloride binding to the transport site accelerated the rate of DBDS release, with the observed rate constant showing a hyperbolic dependence on chloride concentration, while the total change in reaction fluorescence remained constant. After modification of glutamate 681, DBDS release was monophasic in the absence of chloride, but the rapid addition of chloride at constant ionic strength induced a doubling in the fluorescence quantum yield for the bound DBDS molecules. This was associated with the development of 50:50 biphasic kinetics for DBDS release. Such changes were independent of the degree of modification of the band 3 subunit population between the 66% and 91% levels. Titration of the increase in total reaction fluorescence gave an apparent chloride binding K(d) of between 7 and 10 mM, which is 25-40-fold higher in affinity than chloride binding to the transport site. The dependence of the kinetic constants for both phases of the DBDS release reaction on chloride concentration was nonhyperbolic, which contrasts with unmodified band 3, and is indicative of the presence of two classes of chloride-binding sites on the modified transporter. We have also found that the fraction of subunits capable of binding DBDS reversibly, or DIDS covalently, decreased nonlinearly in the absence of chloride as the level of modification of the band 3 subunit population increased. In contrast, the same DBDS binding correlation plot showed a maximum in the presence of saturating chloride. The observation of such nonlinear

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Allosteric Regulation; Allosteric Site; Anion Exchange Protein 1, Erythrocyte; Borohydrides; Chlorides; Dimerization; Erythrocyte Membrane; Glutamic Acid; Humans; Indicators and Reagents; Ion Transport; Isoxazoles; Kinetics; Models, Chemical; Peptide Fragments; Protein Binding; Protein Conformation; Protein Subunits; Protons; Spectrometry, Fluorescence

Intracellular pH homeostasis and intracellular Cl(-) concentration in cardiac myocytes are regulated by anion exchange mechanisms. In physiological extracellular Cl(-) concentrations, Cl(-)/HCO(3)(-) exchange promotes intracellular acidification and Cl(-) loading sensitive to inhibition by stilbene disulfonates. We investigated the expression of AE anion exchangers in the AT-1 mouse atrial tumor cell line. Cultured AT-1 cells exhibited a substantial basal Na(+)-independent Cl(-)/HCO(3)(-) (but not Cl(-)/OH(-)) exchange activity that was inhibited by DIDS but not by dibenzamidostilbene disulfonic acid (DBDS). AT-1 cell Cl(-)/HCO(3)(-) activity was stimulated two- to threefold by extracellular ATP and ANG II. AE mRNAs detected by RT-PCR in AT-1 cells included brain AE3 (bAE3), cardiac AE3 (cAE3), AE2a, AE2b, AE2c1, AE2c2, and erythroid AE1 (eAE1), but not kidney AE1 (kAE1). Cultured AT-1 cells expressed AE2, cAE3, and bAE3 polypeptides, which were detected by immunoblot and immunocytochemistry. An AE1-like epitope was detected by immunocytochemistry but not by immunoblot. Both bAE3 and cAE3 were present in intact AT-1 tumors. Cultured AT-1 cells provide a useful system for the study of mediators and regulators of Cl(-)/HCO(3)(-) exchange activity in an atrial cell type.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acid-Base Equilibrium; Adenosine Triphosphate; Angiotensin II; Animals; Anion Transport Proteins; Anions; Antiporters; Biological Transport; Chloride-Bicarbonate Antiporters; Extracellular Space; Female; Gene Expression; Heart Atria; Homeostasis; Immunohistochemistry; Membrane Proteins; Mice; Mice, Inbred Strains; Myocardium; Paracrine Communication; RNA, Messenger; SLC4A Proteins; Sodium; Transcription, Genetic; Tumor Cells, Cultured; Vasoconstrictor Agents

The mechanisms involved in 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS)- and 4,4'-dibenzamidostilbene-2, 2'-disulfonic acid (DBDS)- modification of sheep cardiac ryanodine receptor (RyR) channel function have been investigated. DIDS (50-500 microm) exerts at least three effects on single channel function. With Ca2+ as the permeant ion, DIDS increases both channel open probability (Po) and single channel conductance in a similar manner to the effects observed with suramin. Both effects occur immediately and are fully reversible. Similar effects were observed with DBDS (10 microm-2 mm), a compound with the 4,4'-NCS groups of DIDS replaced with NHCOC6H5. DIDS (500 microm) also caused irreversible modification to the fully open channel level in 74% of the channels. This effect was not observed with suramin or DBDS (10 microm-1 mm). Competition studies with DBDS and suramin coupled with the close similarities in the effects of DIDS, DBDS and suramin on gating and conduction suggest that these ligands may all bind to the same sites on RyR. The DIDS-induced irreversible modification to the fully open state may result from the binding of the isothiocyanate groups to positively charged amino acids at or near the suramin binding sites although it is possible that this modification is unrelated to its other effects on channel function.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Calcium; Cations; Cesium; Myocardium; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sheep; Suramin

A novel kinetic approach was used to measure monovalent anion binding to better define the mechanistic basis for competition between stilbenedisulfonates and transportable anions on band 3. An anion-induced acceleration in the release of 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) from its complex with band 3 was measured using monovalent anions of various size and relative affinity for the transport site. The K1/2 values for anion binding were determined and correlated with transport site affinity constants obtained from the literature and the dehydrated radius of each anion. The results show that anions with ionic radii of 120-200 pm fall on a well-defined correlation line where the ranking of the K1/2 values matched the ranking of the transport site affinity constants (thiocyanate < nitrate approximately bromide < chloride < fluoride). The K1/2 values for the anions on this line were about 4-fold larger than expected for anion binding to inhibitor-free band 3. Such a lowered affinity can be explained in terms of allosteric site-site interactions, since the K1/2 values decreased with increasing anionic size. In contrast, iodide, with an ionic radius of about 212 pm, had a 10-fold lower affinity than predicted by the correlation line established by the smaller monovalent anions. These results indicate that smaller monovalent anions have unobstructed access to the transport site within the band 3 / DBDS binary complex, while iodide experiences significant steric hindrance when binding. The observation of steric hindrance in iodide binding to the band 3 / DBDS binary complex, but not in the binding of smaller monovalent anions, suggests that the stilbenedisulfonate binding site is located at the outer surface of an access channel leading to the transport site.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Allosteric Regulation; Anion Exchange Protein 1, Erythrocyte; Anions; Chlorides; Gluconates; Kinetics; Protein Binding

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 removal of Na+o or elevation of K+o. Recovery was unaffected by the stilbene drug DIDS (4,4-diisothiocyanatostilbene-disulphonic acid), but was slowed dose dependently by the stilbene drug DBDS (dibenzamidostilbene-disulphonic acid). 3. In 5 % CO2/HCO3- buffer (pHo 7.40), pHi recovery was faster than in Hepes buffer. It consisted of an initial rapid recovery phase followed by a slow phase. Much of the rapid phase has been attributed to CO2-dependent buffering. The slow phase was inhibited completely by Cl-o removal but not by Na+o removal or K+o elevation. 4. At a test pHi of 7.30 in CO2/HCO3- buffer, the slow phase was inhibited 70 % by DIDS. The mean DIDS-inhibitable acid influx was equivalent in magnitude to the HCO3--stimulated acid influx. Similarly, the DIDS-insensitive influx was equivalent to that estimated in Hepes buffer. 5. We conclude that two independent sarcolemmal acid-loading carriers are stimulated by a rise of pHi and account for the slow phase of recovery from an alkali load. The results are consistent with activation of a DIDS-sensitive Cl--HCO3- anion exchanger (AE) to produce HCO3- efflux, and a DIDS-insensitive Cl--OH- exchanger (CHE) to produce OH- efflux. H+-Cl- co-influx as the alternative configuration for CHE is not, however, excluded. 6. The dual acid-loading system (AE plus CHE), previously shown to be activated by a fall of extracellular pH, is thus activated by a rise of intracellular pH. Activity of the dual-loading system is therefore controlled by pH on both sides of the cardiac sarcolemma.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acetates; Alkalosis; Animals; Benzopyrans; Cells, Cultured; Chlorides; Fluorescent Dyes; Guinea Pigs; Heart; Heart Ventricles; HEPES; Hydrogen-Ion Concentration; Kinetics; Myocardium; Sarcolemma; Sodium

Phenylglyoxalation of the red blood cell membrane leads to three superimposed effects on band 3 protein-mediated anion equilibrium exchange as measured by means of radiosulfate: (1) a shift of the curve relating transport activity to pH towards lower pH values, possibly in combination with an increase of the maximal transport activity. This is accompanied by effect (2), the abolishment of a chloride-stimulated component of anion transport seen at low pH values. Effect (3) consists of inhibition of anion equilibrium exchange. Effect (1) prevails when phenylglyoxalation is performed at low concentrations of PG and low pH, while effect (3) predominates when exposure to PG is executed at high pH and high concentration of PG. Effect (1) is associated with a decrease of the Ki values for inhibition and binding of the reversibly acting stilbene disulfonates DNDS and DBDS. The inhibition observed as a consequence of effect (3) is linearly related to a decrease of the capacity of band 3 to combine with the stilbene disulfonate DBDS. The results are interpreted on the assumption that PG is capable of reacting with two or possibly three distinct binding sites in band 3. Reaction with one of them leads to effect (1) and, perhaps, to effect (2); reaction with the other to effect (3). The latter is possibly due to modification of Arg 730, which is homologous to Arg 748 in mouse band 3. Site-directed mutagenesis of this arginine residue showed that it is required for band 3-mediated anion transport.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Anion Exchange Protein 1, Erythrocyte; Anions; Arginine; Binding Sites; Biological Transport; Chlorides; Erythrocyte Membrane; Humans; Hydrogen-Ion Concentration; Kinetics; Phenylglyoxal; Stilbenes; Sulfates

Band 3 Memphis variant II is a mutant anion-exchange protein associated with the Diego a+ blood group antigen. There are two mutations in this transporter: Lys-56-->Glu within the cytoplasmic domain, and Pro-854-->Leu within the membrane-bound domain. The Pro-854 mutation, which is thought to give rise to the antigenicity, is located within the C-terminal subdomain of the membrane-bound domain. Yet, there is an apparent enhancement in the rate of covalent binding of H2DIDS (4,4'-di-isothiocyanatodihydro-2, 2'-stilbenedisulphonate) to 'lysine A' (Lys-539) in the N-terminal subdomain, suggesting widespread conformational changes. In this report, we have used various kinetic assays which differentiate between conformational changes in the two subdomains, to characterize the stilbenedisulphonate site on band 3 Memphis variant II. We have found a significantly higher H2DIDS (a C-terminal-sensitive inhibitor) affinity for band 3 Memphis variant II, due to a lower H2DIDS 'off' rate constant, but no difference was found between mutant and control when DBDS (4,4'-dibenzamido-2,2'-stilbenedisulphonate) (a C-terminal-insensitive inhibitor) 'off' rates were measured. Furthermore, there were no differences in the rates of covalent binding to lysine A, for either DIDS (4,4'-di-isothiocyanato-2,2'-stilbenedisulphonate) or H2DIDS. However, the rate of covalent intrasubunit cross-linking of Lys-539 and Lys-851 by H2DIDS was abnormally low for band 3 Memphis variant II. These results suggest that the Pro-854-->Leu mutation causes a localized conformational change in the C-terminal subdomain of band 3.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anion Exchange Protein 1, Erythrocyte; Binding Sites; Cross-Linking Reagents; Humans; Kinetics; Mutation; Protein Conformation; Spectrophotometry, Ultraviolet

To determine the mechanism of apparent competitive binding of chloride and stilbenedisulfonates ( S ) to band 3 ( B ), we have compared the binding kinetics of three stilbenedisulfonates [ DIDS, 4,4'-diisothiocyanato-2, 2'-stilbenedisulfonate; H2DIDS 4,4'-diisothiocyanodihydro-2, 2'-stilbenedisulfonate and DBDS, 4,4'-dibenzamido-2, 2'-stilbenedisulfonate ] in the absence and presence of 150 mM sodium chloride at constant ionic strength. Biphasic time courses were observed with the fast phase rate constants following second-order kinetics, and the slow phase rate constants following saturation kinetics according to the mechanism: [formula: see text] The results can be understood in terms of the effect of chloride on each of these reaction steps. Chloride increased k1 by about 2-fold, but decreased k-1 8-fold for H2DIDS. Thus, 150 mM chloride increased the initial affinity of H2DIDS by about 19-fold. There was a 3-fold increase in the initial affinity for DIDS, but little or no effect of chloride on the initial affinity of DBDS. There was no effect of chloride on k2, but, previous "off" rate measurements showed that 150 mM chloride increases k-2 about 16-fold for DBDS and about 12-fold for H2DIDS. Taken together, these results indicate that chloride allosterically competes with stilbenedisulfonates for binding to band 3, predominantly by substantially shifting the second isomerization equilibrium to the left.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Allosteric Regulation; Anion Exchange Protein 1, Erythrocyte; Binding, Competitive; Chlorides; Erythrocyte Membrane; Humans; Kinetics; Protein Binding; Sodium Chloride

4,4'-Diisothiocyanatodihydrostilbene-2,2'-disulfonate and 4,4'-dibenzoylstilbene-2,2'-disulfonate potently inhibit the erythrocyte anion transporter. These inhibitors act by binding, with a 1:1 stoichiometry, to the band 3 transport protein. We have studied, by sedimentation equilibrium analysis in an analytical ultracentrifuge, the effect of the two closely related stilbenedisulfonates on the state of association of band 3 in the nonionic detergent nonaethyleneglycol lauryl ether. It was found that covalent binding of 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate to band 3 did not significantly disturb the monomer/dimer/tetramer association equilibrium shown by the unliganded protein. An entirely different result was obtained after addition of 4,4'-dibenzoylstilbene-2,2'-disulfonate to the protein, at both low and high chloride concentrations. The amount of band 3 dimer in the samples increased with increasing inhibitor concentration c1, and for c1 > or = 15 microM virtually all of the protein was present as dimer. After removal of the inhibitor (by gel filtration or dialysis), the original monomer/dimer/tetramer distribution of the band 3 protein was restored. Our data show that the (noncovalent) binding of 4,4'-dibenzoylstilbene-2,2'-disulfonate drastically changes the coupling between band 3 protomers. In addition, a reversible change in the state of association of band 3 induced by ligand binding is demonstrated.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anion Exchange Protein 1, Erythrocyte; Anions; Hemoglobins; Humans; Ion Transport; Protein Binding

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 ionic strength. The acceleration effect saturated as a function of chloride concentration. This is an indication of specific binding within a ternary complex involving stilbenedisulfonate, chloride, and band 3. To see if stilbenedisulfonates block an access channel to the transport site, we studied the effect of rapidly mixing DBDS-saturated resealed ghosts with chloride at constant ionic strength and osmotic pressure. Once again, we observe a large, uniform acceleration in the rate of DBDS release. These findings are not consistent with molecular models where stilbenedisulfonates are proposed to block access to a deeper transport site. We suggest that the intramonomeric stilbenedisulfonate site is not located on the chloride transport pathway but rather interacts with the transport site though heterotropic allosteric site-site interactions. On the basis of our kinetic evidence for ternary complex formation and on transport inhibition evidence in the literature showing a linear dependence of KI-app on substrate, we suggest that stilbenedisulfonates are linear mixed-type inhibitors of band 3 anion exchange, not pure competitive inhibitors as has been assumed on the basis of analysis of transport inhibition data alone.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Allosteric Regulation; Anion Exchange Protein 1, Erythrocyte; Binding Sites; Chlorides; Erythrocyte Membrane; Flow Injection Analysis; Fluorometry; Humans; Kinetics; Models, Chemical; Stilbenes

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 6.6 mM respectively. Maximal rates of transport were about 5-fold higher in the rat heart cells. 2. 4,4'-Dibenzamidostilbene-2,2'-disulphonate (DBDS), a powerful inhibitor of monocarboxylate transport into erythrocytes [Poole & Halestrap (1991) Biochem. J. 275, 307-312], was found to be a potent but apparently partial inhibitor of lactate and pyruvate transport, with an apparent Ki value at 0.5 mM L-lactate of about 16 microM in both species. Maximal inhibition was 50% and 80% in rat and guinea-pig cells respectively. 3. The maximal extent of inhibition and apparent Ki values were dependent on both the substrate transported and its concentration. Maximum inhibition was less and the Ki was greater at higher substrate concentrations. 4. A variety of other stilbene disulphonates were studied which showed different Ki values and maximal extents of inhibition. 5. Phloretin was a significantly less potent inhibitor of transport into both rat (Ki 25 microM) and guinea-pig (Ki 16 microM) heart cells than into rat erythrocytes (Ki 1.4 microM). In the rat but not the guinea-pig heart cells, inhibition appeared partial (maximal inhibition 84%). 6. We demonstrate that our results can be explained by the presence of two monocarboxylate carriers in heart cells, both with Km values for L-lactate of about 2 mM and inhibited by alpha-cyano-4-hydroxycinnamate, but with different affinities for other substrates and inhibitors. One carrier is sensitive to inhibition by stilbene disulphonates and has lower Km values for pyruvate (0.05-0.10 mM) and D-lactate (5 mM), whereas the other has higher Km values for pyruvate (0.30 mM) and D-lactate (25 mM), and is relatively insensitive to stilbene disulphonates. Rat heart cells possess more of the latter carrier and guinea-pig heart cells more of the former. 7. The significance of these results for the study of lactate transport in the perfused heart is discussed.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Animals; Biological Transport; Carrier Proteins; Erythrocytes; Guinea Pigs; Kinetics; Lactates; Lactic Acid; Myocardium; Phloretin; Pyruvates; Pyruvic Acid; Rats; Stilbenes

We have previously shown that the human red cell glucose transport protein and the anion exchange protein, band 3, are in close enough contact that information can be transmitted from the glucose transport protein to band 3. The present experiments were designed to show whether information could be transferred in the reverse direction, using changes in tryptophan fluorescence to report on the conformation of the glucose transport protein. To see whether tryptophan fluorescence changes could be attributed to the glucose transport protein, we based our experiments on procedures used by Helgerson and Carruthers [Helgerson, A. L., Carruthers, A., (1987) J. Biol. Chem. 262:5464-5475] to displace cytochalasin B (CB), the specific D-glucose transport inhibitor, from its binding site on the inside face of the glucose transport protein, and we showed that these procedures modified tryptophan fluorescence. Addition of 75 mM maltose, a nontransportable disaccharide which also displaces CB, caused a time-dependent biphasic enhancement of tryptophan fluorescence in fresh red cells, which was modulated by the specific anion exchange inhibitor, DBDS (4,4'-dibenzamido-2,2'-stilbene disulfonate). In a study of nine additional disaccharides, we found that both biphasic kinetics and DBDS effects depended upon specific disaccharide conformation, indicating that these two effects could be attributed to a site sensitive to sugar conformation. Long term (800 sec) experiments revealed that maltose binding (+/- DBDS) caused a sustained damped anharmonic oscillation extending over the entire 800 sec observation period. Mathematical analysis of the temperature dependence of these oscillations showed that 2 microM DBDS increased the damping term activation energy, 9.5 +/- 2.8 kcal mol-1 deg-1, by a factor of four to 39.7 +/- 5.1 kcal mol-1 deg-1, providing strong support for the view that signalling between the glucose transport protein and band 3 goes in both directions.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Carbohydrate Metabolism; Carbohydrate Sequence; Disaccharides; Erythrocyte Membrane; Fluorescence; Glucose; Humans; Kinetics; Maltose; Membrane Proteins; Molecular Sequence Data; Monosaccharide Transport Proteins; Protein Conformation; Spectrum Analysis; Tryptophan

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 fully reversible. The most potent reversible inhibitors were 4,4'-dibenzamidostilbene-2,2'-disulphonate (DBDS), DIDS and 4-acetamido-4'isothiocyanostilbene-2,2'-disulphonate (SITS), for which apparent Ki values at 0.5 mM-L-lactate were approx. 36, 53 and 130 microM respectively. 3. DIDS and DBDS were competitive inhibitors with respect to L-lactate, with Ki values of approx 40 microM and 22 microM respectively. 4. After incubation for 1 h at 37 degrees C with DIDS or its dihydro derivative (H2DIDS), which contain the amino-reactive isothiocyanate group, most of the inhibition observed was irreversible. Under these conditions the IC50 value (concn. causing 50% inhibition) for irreversible inhibition by both compounds was approx 100 microM. SITS was much less potent as an irreversible inhibitor of L-lactate transport, approx. 20% inhibition being obtained at 100 microM. 5. The reversible inhibitor DBDS (1 mM) afforded protection against irreversible inhibition by DIDS and H2DIDS (100 microM); protection was 60 and 65% respectively after a 60 min incubation. This indicates that specific binding of the irreversible inhibitors is required before covalent modification can take place. 6. These compounds may be useful high-affinity probes for lactate transport in other tissues and might act as affinity labels for the transport protein(s).

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Carbon Radioisotopes; Erythrocytes; In Vitro Techniques; Kinetics; Lactates; Molecular Structure; Rats; Structure-Activity Relationship

The time course of binding of the fluorescent stilbene anion exchange inhibitor. DBDS (4.4'-dibenzamido-2.2'-stilbene disulfonate), to band 3 can be measured by the stopped-flow method. We have previously used the reaction time constant. tau DBDS, to obtain the kinetic constants for binding and, thus, to report on the conformational state of the band 3 binding site. To validate the method, we have now shown that the ID50 (0.3 +/- 0.1 microM) for H2-DIDS (4.4'-diisothiocyano-2.2'-dihydrostilbene disulfonate) inhibition of tau DBDS is virtually the same as the ID50 (0.47 +/- 0.04 microM) for H2-DIDS inhibition of red cell Cl- flux, thus relating tau DBDS directly to band 3 anion exchange. The specific glucose transport inhibitor, cytochalasin B, causes significant changes in tau DBDS, which can be reversed with intracellular, but not extracellular, D-glucose, ID50 for cytochalasin B modulation of tau DBDS is 0.1 +/- 0.2 microM in good agreement with KD = 0.06 +/- 0.005 microM for cytochalasin B binding to the glucose transport protein. These experiments suggest that the glucose transport protein is either adjacent to band 3, or linked to it through a mechanism, which can transmit conformational information. Ouabain (0.1 microM), the specific inhibitor of red cell Na+,K+-ATPase, increases red cell Cl- exchange flux in red cells by a factor of about two. This interaction indicates that the Na+,K+-ATPase, like the glucose transport protein, is either in contact with, or closely linked to, band 3. These results would be consistent with a transport protein complex, centered on band 3, and responsible for the entire transport process, not only the provision of metabolic energy, but also the actual carriage of the cations and anions themselves.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Anion Transport Proteins; Binding, Competitive; Carrier Proteins; Chlorides; Cytochalasin B; Erythrocyte Membrane; Erythrocytes; Humans; In Vitro Techniques; Kinetics; Monosaccharide Transport Proteins; Ouabain; Spectrometry, Fluorescence; Sulfates

In separated outer medullary collecting duct (MCD) cells, the time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4'-dibenzamido-2,2'-stilbene disulfonate), to the MCD cell analog of band 3, the red blood cell (rbc) anion exchange protein, can be measured by the stopped-flow method and the reaction time constant, tau TDBDS, can be used to report on the conformational state of the band 3 analog. In order to validate the method we have now shown that the ID50D,DBDS,MCD (0.5 +/- 0.1 microM) for the H2-DIDS (4,4'-diisothiocyano-2,2'-dihydrostilbene disulfonate) inhibition of tau DBDS is in agreement with the ID50,Cl-MCD (0.94 +/- 0.07 microM) for H2-DIDS inhibition of MCD cell Cl- flux, thus relating tau DBDS directly to anion exchange. The specific cardiac glycoside cation transport inhibitor, ouabain, not only modulates DBDS binding kinetics, but also increases the time constant for Cl- exchange by a factor of two, from tau Cl- = 0.30 +/- 0.02 sec to 0.56 +/- 0.06 sec (30 mM NaHCO3). The ID50,DBDS,MCD for the ouabain effect on DBDS binding kinetics is 0.003 +/- 0.001 microM, so that binding is about an order of magnitude tighter than that for inhibition of rbc K+ flux (KI,K+,rbc = 0.017 microM). These experiments indicate that the Na+,K+-ATPase, required to maintain cation gradients across the MCD cell membrane, is close enough to the band 3 analog that conformational information can be exchanged. Cytochalasin E (CE), which binds to the spectrin/actin complex in rbc and other cells. modulates DBDS binding kinetics with a physiological ID50,DBDS,MCD (0.076 +/- 0.005 microM); 2 microM CE also more than doubles the Cl- exchange time constant from 0.20 +/- 0.04 sec to 0.50 +/- 0.08 sec (30 mM NaHCO3). These experiments indicate that conformational information can also be exchanged between the MCD cell band 3 analog and the MCD cell cytoskeleton.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Anion Transport Proteins; Carrier Proteins; Chlorides; Cytochalasins; In Vitro Techniques; Kidney Tubules; Kidney Tubules, Collecting; Kinetics; Membrane Proteins; Models, Biological; Ouabain; Protein Binding; Rabbits; Spectrometry, Fluorescence

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 + alpha 4[Cl-]) where alpha 2 and alpha 4 are independent of [Cl-] but dependent on dipyridamole concentration. At high [Cl-] the flux approaches alpha 2/alpha 4, which decreases with increasing dipyridamole concentration. Even when both [Cl-] and dipyridamole concentration assume large values, a small residual flux remains. The equation can be deduced on the assumption that Cl- binding allosterically increases the affinity for dipyridamole binding to band 3 and that the bound dipyridamole produces a non-competitive inhibition of sulfate transport. The mass-law constants for the binding of Cl- and dipyridamole to their respective-binding sites are about 24 mM and 1.5 microM, respectively (pH 6.9, 26 degrees C). Dipyridamole binding leads to a displacement of 4,4'-dibenzoylstilbene-2,2'-disulfonate (DBDS) from the stilbenedisulfonate binding site of band 3. The effect can be predicted quantitatively on the assumption that the Cl- -promoted dipyridamole binding leads to a competitive replacement of the stilbenedisulfonates. For the calculations, the same mass-law constants for binding of Cl- and dipyridamole can be used that were derived from the kinetic studies on Cl- -promoted anion transport inhibition. The newly described Cl- binding site is highly selective with respect to Cl- and other monovalent anion species. There is little competition with SO4(2-), indicating that Cl- binding involves other than purely electrostative forces. The affinity of the binding site to Cl- does not change over the pH range 6.0-7.5. Dipyridamole binds only in its deprotonated state. Binding of the deprotonated dipyridamole is pH-independent over the same range as Cl- binding.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Allosteric Site; Anion Exchange Protein 1, Erythrocyte; Anions; Binding Sites; Binding, Competitive; Biological Transport; Chlorides; Dipyridamole; Erythrocyte Membrane; Humans; Hydrogen-Ion Concentration; Mathematics; Phosphates; Stilbenes; Sulfates

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. Dix and A.K. Solomon, J. Gen. Physiol. 81:421-449, 1983). In the absence of transportable anions, DBDS bound reversibly to a single class of sites on BLMV isolated from rabbit (Kd = 3.8 microM) and rat (3.2 microM); 100 microM dihydro-4,4'-diisothiocyano-2,2'-disulfonic stilbene (H2DIDS) blocked greater than 95% of binding. H2DIDS inhibitable DBDS binding was not detected using rat or rabbit BBMV. In rabbit BLMV, DBDS Kd doubled with 10 mM SO4, 50 mM HCO3 and 100 mM Cl, but was not altered by Na or pH (6-8). In stopped-flow experiments the exponential time constant for DBDS binding slowed with SO4, HCO3 and Cl, but was unaffected by Na. These results are consistent with competitive binding of DBDS and anions at an anion transport site. To relate DBDS binding data to anion transport inhibition we used 35SO4 uptake to characterize several modes of rabbit BLM anion transport: H/SO4 and Na/SO4 cotransport, and Cl/SO4 countertransport. Each transport process was electroneutral and was inhibited by H2DIDS, furosemide, probenecid, chlorothiazide and DBDS. The apparent KI's for DBDS (3-20 microM) were similar to Kd for DBDS binding. These studies define a class of anion transport sites on the proximal tubule basolateral membrane measurable optically by a fluorescent stilbene.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Animals; Anion Transport Proteins; Anions; Carrier Proteins; Diffusion; Erythrocyte Membrane; Fluorescent Dyes; Hydrogen-Ion Concentration; Ion Channels; Kidney Tubules; Kinetics; Membrane Proteins; Rabbits; Rats; Stilbenes

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 both processes consist of a bimolecular association between pCMBS and the membrane site followed by a conformational change. Both processes are very slow and the on rate constant for the water inhibition process is about 10(5) times slower than usual for inhibitor binding to membrane transport proteins. pCMBS binding to the water transport inhibition site can be reversed by cysteine while that to the urea transport inhibition site can not be reversed. The specific stilbene anion exchange inhibitor, DBDS (4,4'-dibenzamidostilbene-2,2'-disulfonate) causes a significant change in the time-course of pCMBS inhibition of water transport, consistent with a linkage between anion exchange and water transport. Consideration of available sulfhydryl groups on band 3 suggests that the urea transport inhibition site is on band 3, but is not a sulfhydryl group, and that, if the water transport inhibition site is a sulfhydryl group, it is located on another protein complexed to band 3, possibly band 4.5.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4-Chloromercuribenzenesulfonate; Anion Exchange Protein 1, Erythrocyte; Body Water; Cell Membrane Permeability; Cysteine; Erythrocyte Membrane; Erythrocytes; Humans; Kinetics; Protein Conformation; Stilbenes; Sulfhydryl Reagents; Urea

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 measured by a fluorescence enhancement technique. Radiation (0-26 Mrad) had no effect on total membrane protein and DBDS binding affinity, whereas DBDS binding stoichiometry decreased exponentially with radiation dose, giving a target size of 59 +/- 4 kDa. H2-4,4'-diisothiocyano-2,2'-disulfonic stilbene (H2-DIDS, 5 microM) blocked greater than 95% of DBDS binding at all radiation doses. pCMBS binding was measured from the time course of tryptophan fluorescence quenching in ghosts treated with the sulfhydryl reagent N-ethylmaleimide (NEM). Radiation did not affect the kinetics of tryptophan quenching, whereas the total amplitude of the fluorescence signal inactivated with radiation with a target size of 31 +/- 6 kDa. These results support the notion that DBDS and pCMBS bind to the transmembrane domain of erythrocyte band 3 in NEM-treated ghosts and demonstrate that radiation inactivation may probe a target significantly smaller than a covalently linked protein subunit. The small target size for the band 3 stilbene binding site may correspond to the intramembrane domain of the band 3 monomer (52 kDa), which is physically distinct from the cytoplasmic domain (42 kDa).

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4-Chloromercuribenzenesulfonate; Anion Exchange Protein 1, Erythrocyte; Anions; Binding Sites; Biological Transport; Erythrocyte Membrane; Ethylmaleimide; Humans; Molecular Weight; Phenylmercury Compounds; Stilbenes; Urea; Water

The effect of chloride on 4,4'-dibenzamido-2,2'-disulfonic stilbene (DBDS) binding to band 3 in unsealed red cell ghost membranes was studied in buffer [NaCl (0 to 500 mM) + Na citrate] at constant ionic strength (160 or 600 mM), pH 7.4, 25 degrees C. In the presence of chloride, DBDS binds to a single class of sites on band 3. At 160 mM ionic strength, the dissociation constant of DBDS increases linearly with chloride concentration in the range [Cl] = 10 to 120 mM; at 600 mM ionic strength, the DBDS dissociation constant saturates hyperbolically with half-saturating [Cl] = 450 mM. The observed rate of DBDS binding to ghost membranes, as measured by fluorescence stopped-flow kinetic experiments, increases with chloride concentration at both 160 and 600 mM ionic strength. The equilibrium and kinetic results have been incorporated into the following model of the DBDS-band 3 interaction: (formula; see text) The equilibrium and rate constants of the model at 600 mM ionic strength are K1 = 0.67 +/- 0.16 microM, k2 = 1.6 +/- 0.7 sec-1, k-2 = 0.17 +/- 0.09 sec-1, K'1 = 6.3 +/- 1.7 microM, k'2 = 9 +/- 4 sec-1 and k'-2 = 7 +/- 3 sec-1. The apparent dissociation constants of chloride from band 3, KCl, are 40 +/- 4 mM (160 mM ionic strength) and 11 +/- 3 mM (600 mM ionic strength). Our results indicate that chloride and DBDS have distinct, interacting binding sites on band 3.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Anion Exchange Protein 1, Erythrocyte; Chlorides; Humans; Kinetics; Mathematics; Models, Biological; Protein Binding; Stilbenes

The binding characteristics of the inhibitor of anion transport in human red cells, 4,4'-dibenzamido-2,2'-disulfonic stilbene (DBDS), to the anion transport protein of red cell ghost membranes in buffer containing 150 mM NaCl have been measured over the temperature range 0-30 degrees C by equilibrium and stopped-flow fluorescence methods. The equilibrium dissociation constant Keq, increased with temperature. No evidence of a 'break' in the ln(Keq) vs. 1/T plot was found. The standard dissociation enthalpy and entropy changes calculated from the temperature dependence are 9.1 +/- 0.9 kcal/mol and 3.2 +/- 0.3 e.u., respectively. Stopped-flow kinetic studies resolve the overall binding into two steps: a bimolecular association of DBDS with the anion transport protein, followed by a unimolecular rearrangement of the DBDS-protein complex. The rate constants for the individual steps in the binding mechanism can be determined from an analysis of the concentration dependence of the binding time course. Arrhenius plots of the rate constants showed no evidence of a break. Activation energies for the individual steps in the binding mechanism are 11.6 +/- 0.9 kcal/mol (bimolecular, forward step), 17 +/- 2 kcal/mol (bimolecular, reverse step), 6.4 +/- 2.3 kcal/mol (unimolecular, forward step), and 10.6 +/- 1.9 kcal/mol (unimolecular, reverse step). Our results indicate that there is an appreciable enthalpic energy barrier for the bimolecular association of DBDS with the transport protein, and appreciable enthalpic and entropic barriers for the unimolecular rearrangement of the DBDS-protein complex.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Anions; Calorimetry; Erythrocyte Membrane; Humans; Kinetics; Mathematics; Stilbenes

The human red cell anion transport protein, band 3, contains six pCMBS (p-chloromercuribenzene sulfonate) reactive SH groups, five of which react with N-ethylmaleimide. We have carried out equilibrium binding experiments using N-ethylmaleimide-treated red cell ghosts and found that the sulfhydryl reactive water transport inhibitor, pCMBS, inhibits the binding to band 3 of the specific anion exchange inhibitor DBDS (4,4'-dibenzoamido-2,2'-disulfonic stilbene) in a non-competitive manner. Stopped-flow kinetic studies, in which DBDS is mixed with ghosts in the presence of pCMBS, show that pCMBS slows the DBDS induced conformational change in band 3. A non-competitive reaction scheme has been developed which incorporates the quantitative results of equilibrium and kinetic studies. The pCMBS effect on DBDS binding and kinetics is reversed with 5 mM cysteine suggesting a sulfhydryl bond is involved in pCMBS binding to band 3. These data suggest that pCMBS has a specific binding site on band 3, consistent with the hypothesis that band 3 mediates red cell water transport.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4-Chloromercuribenzenesulfonate; Anion Exchange Protein 1, Erythrocyte; Binding Sites; Body Water; Erythrocyte Membrane; Ethylmaleimide; Humans; Mathematics; Phenylmercury Compounds; Time Factors

The inhibitor of anion exchange 4,4'-dibenzoamido-2,2'-disulfonic stilbene (DBDS) binds to band 3, the anion transport protein in human red cell ghost membranes, and undergoes a large increase in fluorescence intensity when bound to band 3. Equilibrium binding studies performed in the absence of transportable anions show that DBDS binds to both a class of high-affinity (65 nM) and low-affinity (820 nM) sites with stoichiometry equivalent to 1.6 nmol/mg ghost protein for each site, which is consistent with one DBDS site on each band 3 monomer. The kinetics of DBDS binding were studied both by stopped-flow and temperature-jump experiments. The stopped-flow data indicate that DBDS binding to the apparent high-affinity site involves association with a low-affinity site (3 microM) followed by a slow (4 s-1) conformational change that locks the DBDS molecule in place. A detailed, quantitative fit of the temperature-jump data to several binding mechanisms supports a sequential-binding model, in which a first DBDS molecule binds to one monomer and induces a conformational change. A second DBDS molecule then binds to the second monomer. If the two monomers are assumed to be initially identical, thermodynamic characterization of the binding sites shows that the conformational change induces an interaction between the two monomers that modifies the characteristics of the second DBDS binding site.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Centrifugation; Erythrocyte Membrane; Erythrocytes; Fluorescence; Humans; Kinetics; Membrane Proteins; Models, Biological; Stilbenes; Temperature

Phloretin is an inhibitor of anion exchange and glucose and urea transport in human red cells. Equilibrium binding the kinetic studies indicate that phloretin binds to band 3, a major integral protein of the red cell membrane. Equilibrium phloretin binding has been found to be competitive with the binding of the anion transport inhibitor, 4,4'-dibenzamido-2,2'-disulfonic stilbene (DBDS), which binds specifically to band 3. The apparent binding (dissociation) constant of phloretin to red cell ghost band 3 in 28.5 mM citrate buffer, pH 7.4, 25 degree C, determined from equilibrium binding competition, is 1.8 +/- 0.1 microM. Stopped-flow kinetic studies show that phloretin decreases the rate of DBDS binding to band 3 in a purely competitive manner, with an apparent phloretin constant of 1.6 +/- 0.4 microM. The pH dependence of equilibrium binding studies show that it is the charged, anionic form of phloretin that competes with DBDS binding, with an apparent phloretin inhibition constant of 1.4 microM. The phloretin binding and inhibition constants determined by equilibrium binding, kinetic and pH studies are all similar to the inhibition constant of phloretin for anion exchange. These studies suggest that phloretin inhibits anion exchange in red cells by a specific interaction between phloretin and band 3.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Anion Transport Proteins; Binding, Competitive; Carrier Proteins; Erythrocyte Membrane; Erythrocytes; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Phloretin