Molecular mechanisms of band 3 inhibitors. 2. Channel blockers

Band 3 is proposed to contain substrate channels that lead from the aqueous medium to a transport site buried within the membrane, and which can be blocked by inhibitors. The inhibitors 1,2-cyclohexanedione (CHD) and dipyridamole (DP) each inhibit the transport site 35Cl NMR line broadening, but neither competes with Cl- for binding. Thus these inhibitors do not occupy the transport site; instead they slow the migration of Cl- between the transport site and the medium. The simplest explanation for this behavior is that CHD and DP block one or more substrate channels. CHD is an arginine-specific covalent modification reagent, and its effectiveness as a channel blocker indicates that the channel contains arginine positive charges to facilitate the migration of anions through the channel. DP is a noncovalent channel blocker that binds with a stoichiometry of 1 molecule per band 3 dimer. DP binding is unaffected by CHD but is prevented by phenylglyoxal (PG), 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), or niflumic acid. Thus the DP and CHD binding sites are distinct, with DP binding sufficiently close to the transport site to interact with PG and DNDS. It is proposed that substrate channels may be a general feature of transport proteins.     

35Cl nuclear magnetic resonance line broadening shows that eosin-5-maleimide does not block the external anion access channel of band 3.

It has been suggested that Lys-430 of band 3, with which eosin-5-maleimide (EM) reacts, is located in the external channel through which anions gain access to the external transport site, and that EM inhibits anion exchange by blocking this channel. To test this, we have used 35Cl nuclear magnetic resonance (NMR) to measure Cl- binding to the external transport site in control and EM-treated human red blood cells. Intact cells were used rather than ghosts, because in this case all line broadening (LB) results from binding to external sites. In an NMR spectrometer with a 9.4-T magnetic field, red blood cells at 50% concentration (v/v) in 150 mM Cl- medium at 3 degrees C caused 19.0 +/- 1.2 Hz LB. Of this, 7.9 +/- 0.7 Hz was due to Cl- binding to the high affinity band 3 transport sites, because it was prevented by an apparently competitive inhibitor of anion exchange, 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS). The LB was not due to hemoglobin released from the cells, as little LB remained in the supernatant after cells were removed by centrifugation. Saturable Cl- binding remained in EM-treated cells, although the binding was no longer DNDS-sensitive, because EM prevents binding of DNDS. The lower limit for the rate at which Cl- goes from the binding site to the external medium is 2.15 x 10(5) s-1 for control cells and 1.10 x 10(5) s-1 for EM-treated cells, far higher than the Cl- translocation rate at 3 degrees C (about 400 s-1). Thus, EM does not inhibit Cl- exchange by blocking the external access channel. EM may therefore be useful for fixing band 3 in one conformation for studies of Cl- binding to the external transport site.     

Detection of Cl- binding to band 3 by double-quantum-filtered 35Cl nuclear magnetic resonance.

We have applied double-quantum-filtered (DQF) NMR of 35Cl to study binding of Cl- to external sites on intact red blood cells, including the outward-facing anion transport sites of band 3, an integral membrane protein. A DQF 35Cl NMR signal was observed in cell suspensions containing 150 mM KCl, but the DQF signal can be totally eliminated by adding 500 microM 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), an inhibitor that interferes with Cl- binding to the band 3 transport site. Therefore, it seems that only the binding of Cl- to transport sites of band 3 can give rise to a 35Cl DQF signal from red blood cell suspensions. In accordance with this concept, analysis of the single quantum free induction decay (FID) revealed that signals from buffer and DNDS-treated cells were fitted with a single exponential function, whereas the FID signals of untreated control cells were biexponential. The DQF signal remained after the cells were treated with eosin-5-maleimide (EM), a noncompetitive inhibitor of chloride exchange. This result supports previous reports that EM does not block the external chloride binding site. The band 3-dependent DQF signal is shown to be caused at least in part by nonisotropic motions of Cl- in the transport site, resulting in incompletely averaged quadrupolar couplings.     

Evidence for the development of an intermonomeric asymmetry in the covalent binding of 4,4'-diisothiocyanatostilbene-2,2'-disulfonate to human erythrocyte band 3

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) studies have identified two oligomeric forms of band 3 whose proportions on gel profiles were modulated by the particular ligand occupying the intramonomeric stilbenedisulfonate site during intermonomeric cross-linking by BS3 [bis-(sulfosuccinimidyl) suberate] [Salhany et al. (1990) J. Biol. Chem. 265, 17688-17693]. When DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonate) was irreversibly attached to all monomers, BS3 covalent dimers predominated, while with DNDS (4,4'-dinitrostilbene-2,2'-disulfonate) present to protect the intramonomeric stilbenedisulfonate site from attack by BS3, a partially cross-linked band 3 tetramer was observed. In the present study, we investigate the structure of the protected stilbenedisulfonate site within the tetrameric complex by measuring the ability of patent monomers to react irreversibly with DIDS. Our results show two main populations of band 3 monomers present after reaction with DNDS/BS3: (a) inactive monomers resulting from the displacement of reversibly bound DNDS molecules and subsequent irreversible attachment of BS3 to the intramonomeric stilbenedisulfonate site and (b) residual, active monomers. All of the residual activity was fully inhibitable by DIDS under conditions of reversible binding, confirming expectations that all of the monomers responsible for the residual activity have patent stilbenedisulfonate sites. However, within this active population, two subpopulations could be identified: (1) monomers which were irreversibly reactive toward DIDS and (2) monomers which were refractory toward irreversible binding of DIDS at pH 6.9, despite being capable of binding DIDS reversibly. Increasing the pH to 9.5 during treatment of DNDS/BS3-modified cells with 300 microM DIDS did not cause increased irreversible transport inhibition relative to that seen for cells treated at pH 6.9.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional carboxyl groups in the red cell anion exchange protein. Modification with an impermeant carbodiimide

Anion exchange in human red blood cell membranes was inactivated using the impermeant carbodiimide 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide (EAC). The inactivation time course was biphasic: at 30 mM EAC, approximately 50% of the exchange capacity was inactivated within approximately 15 min; this was followed by a phase in which irreversible exchange inactivation was approximately 100-fold slower. The rate and extent of inactivation was enhanced in the presence of the nucleophile tyrosine ethyl ester (TEE), suggesting that the inactivation is the result of carboxyl group modification. Inactivation (to a maximum of 10% residual exchange activity) was also enhanced by the reversible inhibitor of anion exchange 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) at concentrations that were 10(3)-10(4) times higher than those necessary for inhibition of anion exchange. The extracellular binding site for stilbenedisulfonates is essentially intact after carbodiimide modification: the irreversible inhibitor of anion exchange 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) eliminated (most of) the residual exchange activity: DNDS inhibited the residual (DIDS-sensitive) Cl- at concentrations similar to those that inhibit Cl- exchange of unmodified membranes: and Cl- efflux is activated by extracellular Cl-, with half-maximal activation at approximately 3 mM Cl-, which is similar to the value for unmodified membranes. But the residual anion exchange function after maximum inactivation is insensitive to changes of extra- and intracellular pH between pH 5 and 7. The titratable group with a pKa of approximately 5.4, which must be deprotonated for normal function of the native anion exchanger, thus appears to be lost after EAC modification.     

Kinetics of residual chloride transport in human red blood cells after maximum covalent 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid binding

Irreversible inhibition, 99.8% of control values for chloride transport in human red blood cells, was obtained by well-established methods of maximum covalent binding of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The kinetics of the residual chloride transport (0.2%, 106 pmol.cm-2 x s-1) at 38 degrees C, pH 7.2) was studied by means of 36Cl- efflux. The outside apparent affinity, expressed by Ko1/2,c, was 34 mM, as determined by substituting external KCl by sucrose. The residual flux was reversibly inhibited by a reexposure to DIDS, and by 4,4'- dinitrostilbene-2,2'-disulfonate (DNDS), phloretin, salicylate, and alpha-bromo-4-hydroxy-3,5-dinitroacetophenone (Killer III) (Borders, C. L., Jr., D. M. Perez, M. W. Lafferty, A. J. Kondow, J. Brahm, M. B. Fenderson, G. L. Breisford, and V. B. Pett. 1989. Bioorganic Chemistry. 17:96-107), to approximately 0.001% of control cells, which is a flux as low as in lipid bilayers. The reversible DIDS inhibition of the residual chloride flux depended on the extracellular chloride concentration, but was not purely competitive. The half-inhibition concentrations at [Cl(o)] = 150 mM in control cells (Ki,o) and covalently DIDS-treated cells (Ki,c) were: DIDS, Ki,c = 73 nM; DNDS, Ki,o = 6.3 microM, Ki,c = 22 microM; phloretin, Ki,o = 19 microM, Ki,c = 17 microM; salicylate, Ki,o = 4 mM, Ki,c = 8 mM; Killer III, Ki,o = 10 microM, Ki,c = 10 microM.     

Trapping of inhibitor-induced conformational changes in the erythrocyte membrane anion exchanger AE1.

AE1, the chloride/bicarbonate anion exchanger of the erythrocyte plasma membrane, is highly sensitive to inhibition by stilbene disulfonate compounds such as DIDS (4,4'-diisothiocyanostilbene-2, 2'-disulfonate) and DNDS (4,4'-dinitrostilbene-2,2'-disulfonate). Stilbene disulfonates recruit the anion binding site to an outward-facing conformation. We sought to identify the regions of AE1 that undergo conformational changes upon noncovalent binding of DNDS. Since conformational changes induced by stilbene disulfonate binding cause anion transport inhibition, identification of the DNDS binding regions may localize the substrate binding region of the protein. Cysteine residues were introduced into 27 sites in the extracellular loop regions of an otherwise cysteineless form of AE1, called AE1C(-). The ability to label these residues with biotin maleimide [3-(N-maleimidylpropionyl)biocytin] was then measured in the absence and presence of DNDS. DNDS reduced the ability to label residues in the regions around G565, S643-M663, and S731-S742. We interpret these regions either as (i) part of the DNDS binding site or (ii) distal to the binding site but undergoing a conformational change that sequesters the region from accessibility to biotin maleimide. DNDS alters the conformation of residues outside the plane of the bilayer since the S643-M663 region was previously shown to be extramembranous. Upon binding DNDS, AE1 undergoes conformational changes that can be detected in extracellular loops at least 20 residues away from the hydrophobic core of the lipid bilayer. We conclude that the TM7-10 region of AE1 is central to the stilbene disulfonate and substrate binding region of AE1.     

Purification of a stilbene sensitive chloride channel and reconstitution of chloride conductivity into phospholipid vesicles.

A protein conferring passive chloride permeability was isolated from a N-octylglucoside solubilized extract of partially purified H(+)-transporting osteoclast cell membranes. Purification was achieved by binding of solubilized protein to an amine-linked 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) Sepharose 4B column and elution with 50 mM KCl. A major protein, with MR = 60 kD on 10% SDS-PAGE, was obtained, which was further purified to homogeneity by HPLC gel filtration. This protein introduced 36Cl- permeability when reconstituted in phospholipid membranes by equilibrium dialysis. The Cl- transport recovered in reconstituted membranes retained sensitivity to DIDS confirming the identity of the isolated protein as a stilbene-sensitive chloride channel.     

Direct observation of the transmembrane recruitment of band 3 transport sites by competitive inhibitors. A 35Cl NMR study

Numerous models describing anion exchange across the red cell membrane by band 3 have been discussed in literature. These models are readily distinguished from one another by an experiment which tests the ability of band 3 transport sites to be recruited to one side of the membrane. In order to observe directly the transmembrane recruitment of transport sites, we have developed 35Cl NMR techniques that resolve the two transport site populations on opposite sides of the membrane. Using these techniques, we show that the inhibitors 4,4'- dinitrostilbene -2,2'-disulfonate and p- nitrobenzensulfonate each recruit all of the transport sites on both sides of the membrane to the extracellular facing conformation. This result indicates that band 3 has an alternating site transport mechanism: each band 3 transport unit possesses a single functional transport site which is alternately exposed first to one side of the membrane then to the other.     

Location of the stilbenedisulfonate binding site of the human erythrocyte anion-exchange system by resonance energy transfer.

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 fluorescence enhancement upon binding to the inhibitory site on erythrocyte ghosts. The fluorescence properties of all three bound probes indicate a rigid, hydrophobic site with nearby tryptophan residues. The Triton X-100 solublized and purified band 3 protein has similar affinities for DBDS, BADS, and 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) to those observed on intact erythrocytes and erythrocyte ghosts, showing that the anion binding site is not perturbed by the solubilization procedure. The distance between the stilbenedisulfonate binding site and a group of cysteine residues on the 40 000-dalton amino-terminal cytoplasmic domain of band 3 was measured by the fluorescence resonance energy transfer technique. Four different fluorescent sulfhydryl reagents were used as either energy transfer donors or energy transfer acceptors in combination with the stilbenedisulfonates (BIDS, DBDS, BADS, and DNDS). Efficiencies of transfer were measured by sensitized emisssion, donor quenching, and donor lifetime changes. Although these sites are approachable from opposite sides of the membrane by impermeant reagents, they are separated by only 34--42 A, indicating that the anion binding site is located in a protein cleft which extends some distance into the membrane.     

Preparation and characterization of 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and related stilbene disulfonates

4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and other 4,4'-stilbene-2,2'-disulfonate derivatives used as reagents in histochemistry and physiology have been prepared in their E isomeric form, and rearranged to the Z isomers by irradiation with visible light. Infrared, and 1H and 13C nuclear magnetic resonance spectra were recorded for these compounds, and used to establish the chemical structures. In particular, it was shown that the E-isomer of SITS decomposed in aqueous solution by hydrolysis of both the acetamido and isocyano groups yielding a diamine; disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) also decomposed in solution, while disodium 4,4'-dinitrostilbene-2,2'-sulfonate (DNDS) rearranged from the E-isomer to the Z-isomer when solutions were kept unprotected from light. These results indicate that benchworkers should not be surprised when commercial samples of such stilbenes contain large amounts of various types of impurities.     

Characterization of pyridoxal 5'-phosphate affinity labeling of band 3 protein. Evidence for allosterically interacting transport inhibitory subdomains

Pyridoxal 5'-phosphate (PLP) is a substrate of band 3, the erythrocyte anion transport protein. It competitively inhibits anion transport and labels two exofacial chymotryptic domains (the 17-kDa (CH17) and the 35-kDa (CH35) integral fragments). Two mol of PLP are bound/mol of each fragment at saturation. PLP labeling of both domains is competitive with chloride at constant ionic strength. Addition of DNDS (4,4'-dinitrostilbene-2,2'-disulfonate), protects PLP labeling of CH35 but exposes new, nonoverlapping sites on CH17.4,4'-Diisothiocyanostilbene-2,2'-disulfonate reduces PLP labeling to both domains with time, while NAP-taurine (N(-4-azido-2-nitrophenyl)2-aminosulfonate) has no effect on either domain. At low chloride (balance citrate) and high DNDS, we can strongly suppress CH35 labeling and selectively titrate CH17 with PLP. Correlation of fractional transport inhibition with fractional PLP covalent coverage of CH17, quantitatively follows the 1:2 correlation line indicating that full coverage of CH17 sites (which constitute half of the total PLP-labeling sites on band 3) exactly inhibits one-half of transport. PLP labeling of CH35 sites accounts for the other half of inhibition. The inhibition-labeling correlation plots are nonlinear in the absence of DNDS, indicating the presence of allosteric interactions between the domains. We conclude that CH17 and CH35 compose nonoverlapping, functionally equivalent, allosterically linked transport inhibitory subdomains on band 3.     

The relationship between anion exchange and net anion flow across the human red blood cell membrane

The conductive (net) anion permeability of human red blood cells was determined from net KCl or K2SO4 effluxes into low K+ media at high valinomycin concentrations, conditions under which the salt efflux is limited primarily by the net anion permeability. Disulfonic stilbenes, inhibitors of anion exchange, also inhibited KCl or K2SO4 efflux under these conditions, but were less effective at lower valinomycin concentrations where K+ permeability is the primary limiting factor. Various concentrations of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) had similar inhibitory effects on net and exchange sulfate fluxes, both of which were almost completely DIDS sensitive. In the case of Cl-, a high correlation was also found between inhibition of net and exchange fluxes, but in this case about 35% of the net flux was insensitive to DIDS. The net and exchange transport processes differed strikingly in their anion selectivity. Net chloride permeability was only four times as high as net sulfate permeability, whereas chloride exchange is over 10,000 times faster than sulfate exchange. Net OH-permeability, determined by an analogous method, was over four orders of magnitude larger than that of Cl-, but was also sensitive to DIDS. These data and others are discussed in terms of the possibility that a common element may be involved in both net and exchange anion transport.     

Pre-steady state transport by erythrocyte band 3 protein: uphill countertransport induced by the impermeant inhibitor H2DIDS.

Pre-steady state Cl- efflux experiments have been performed to test directly the idea that the transport inhibitor H2DIDS (4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate) binds preferentially to the outward-facing state of the transporter. Cells were equilibrated with a medium consisting of 150 mM sodium phosphate, pH 6.2, N2 atmosphere, and 80-250 microM 36Cl-. Addition of H2DIDS (10-fold molar excess compared with band 3) induces a transient efflux of Cl-, as expected if H2DIDS binds more tightly to outward-facing than to inward-facing states. The size of the H2DIDS-induced efflux depends on the Cl- concentration and is about 700,000 ions per cell at the highest concentrations tested. The size of the transient efflux is larger than would be expected if the catalytic cycle for anion exchange involved one pair of exchanging anions per band 3 dimer. These results are completely consistent with a ping-pong mechanism of anion exchange in which the catalytic cycle consists of one pair of exchanging anions per subunit of the band 3 dimer.     

Reaction mechanism and asymmetric orientation of the reconstituted chloroplast phosphate translocator.

Proteoliposomes loaded with varying levels of internal substrates were used in bisubstrate initial velocity studies to gain insight into the transport mechanism of the reconstituted chloroplast phosphate translocator. The kinetic response to trans substrates clearly indicated that the one-to-one exchange mediated by this translocator proceeds via a ping-pong type, and excluded a sequential type of reaction mechanism. It is also shown that reconstitution of the protein leads to an unidirectional orientation of the protein within the liposomes being orientated right-side-out with respect to chloroplasts. Different transport affinities were observed on either side of the membrane and only the outward-facing transport site of the translocator is able to bind inhibitors i.e. pyridoxal 5'-phosphate (PLP) and 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS).     

Effects of external pH on binding of external sulfate, 4.4-dinitro- stilbene-2,2'-disulfonate (DNDS), and chloride to the band 3 anion exchange protein

A model in which two positively-charged titratable sites enhance the affinity for anionic substrates can explain the increase in external iodide dissociation constant (K(O)(I)) with increasing pH(O) (Liu, S. J., F.-Y. Law, and P.A. Knauf. 1996.f Gen.Physiol. 107:271-291). If sulfate binds to the same external site as I-, this model predicts that the SO(4)= dissociation constant (K(O)(S)) should also increase. The data at pH 0 8.5 to 10 fit this prediction, and the pK for the titration is not significantly different from that (pKc) for the low-pK group that affects K(O)(1). The dissociation constant for the apparently competitive inhibitor, DNDS (4,4-dinitrostilbene-2,2'- disulfonate), also increases greatly as pH(O) increases. Particularly at high pH(O), a noncompetitive inhibition by DNDS is also evident. Increasing pH(O) from 7.2 to 11.2 increases the competitive dissociation constant by 700-fold, but the noncompetitive is only increased 20-fold. The pK values for these effects are similar to pKc for K(O)(1), as expected if DNDS binds near the external transport site, but it seems likely that additional titratable groups also affect DNDS binding. The apparent affinity for external Cl- is also affected by pH(O), in a manner similar to that observed for I-. Pretreatment with the amino-selective reagent, bis-sulfosuccinimidyl suberate (BSSS), decreases the apparent Cl- affinity at pH 8.5, but two titrations are still evident, the first (lower) of which decreases the apparent C- affinity, and the second of which surprisingly increases it. Thus, the BSSS-reactive amino groups (probably Lys-539 and Lys-851) do not seem to be involved in the titrations that affect Cl- affinity. In general, the data support the concept that a positively charged amino group (or groups), together with a guanidino group, plays an important role in the binding of substrates and inhibitors at or near the external transport site.     

Stoichiometry of a half-turnover of band 3, the chloride transport protein of human erythrocytes

The kinetics of human red blood cell Cl transport have been studied under nonequilibrium conditions to determine whether or not an outward Cl gradient can recruit the transport protein from an inward-facing to an outward-facing configuration. Three kinds of evidence are consistent with this outward recruitment. First, the initial net Cl efflux into a Cl-free phosphate medium is independent of the intracellular Cl concentration in the range 20-170 mM. Second, an outward Cl gradient strongly enhances the inhibitory potency of DNDS (4,4'-dinitro-2,2'-stilbene disulfonate), which suggests that DNDS binds primarily to outward-facing states. Finally, we have estimated the number of Cl ions transported during the putative outward recruitment. Resealed red cell ghosts containing only 70 muM 36Cl were resuspended at 0 degrees C in a Cl-free, HCO3-free Na2SO4 medium. In the first 10 s, or approximately 10(6) Cl ions per ghost, followed by a much slower further loss of Cl. The rapid loss of 10(6) Cl ions per ghost, which is abolished by pretreatment with DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonate), appears to represent the Cl that is transported during the first half-turnover of the transport cycle. These data are strong evidence that the influx and efflux events in the catalytic cycle for anion transport do not take place simultaneously, and that the stoichiometry of the transport cycle is close to one pair of anions exchanged per band 3 monomer.     

Effects on water diffusion of inhibitors affecting various transport processes in human red blood cells.

The water permeability of human red blood cells has been monitored by nuclear magnetic resonance (NMR) following exposure to inhibitors of various transport processes across their membranes. No significant inhibition of water diffusion could be detected after the treatment of red blood cells with the anion exchange transport inhibitor dihydro-4,4'-diisothiocyano-stilbene-2,2'-disulfonate (H2DIDS) or the glucose transport inhibitors diallyl-diethyl-stilbestrol (DADES), cytochalasin B, or 30 mM iodoacetamide. It is for the first time that the effects of glucose transport inhibitors has been studied in detail by the NMR approach. A special case proved to be phloretin, an inhibitor of anion, nonelectrolyte and glucose permeability. A small but statistically significant inhibition of water permeability (around 12% at 20 degrees C) was induced by exposure to 2 mM phloretin (for 60 min at 37 degrees C); after a pretreatment of cells with 12 mM N-ethylmaleimide (NEM), for 60 min at 37 degrees C, the degree of inhibition induced by phloretin increased (becoming 17% at 20 degrees C). None of the inhibitors prevented or potentiated the strong inhibitory effect on water diffusion of a mercurial, p-chloromercuribenzene sulfonate (PCMBS). No increase in the activation energy of water diffusion occurred by treatment with the reagents used (exception the effect of PCMBS). The present results clarify some conflicting reports concerning the effects on water permeability of inhibitors of various transport processes in red blood cells and indicate that in addition to the drastic inhibition induced by mercurials other reagents may also have inhibitory effects.     

Preparation and Characterization of 4-Acetamido-4'-Isothiocyanostilbene-2,2'-Disulfonic Acid (Sits) And Related Stilbene Disulfonates

4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonicacid (SITS) and other 4,4'-stilbene-2,2'-disulfonate derivatives used as reagents in histochemistry and physiology have been prepared in their E isomeric form, and rearranged to the Z isomers by irradiation with visible light. Infrared, and 'H and ¹³C nuclear magnetic resonance spectra were recorded for these compounds, and used to establish the chemical structures. In particular, it was shown that the E-isomer of SITS decomposed in aqueous solution by hydrolysis of both the acetamido and isocyano groups yielding a diamine; disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfon-ate (DIDS) also decomposed in solution, while disodium 4,4'-dinilrostilbene-2, 2'-sulfonate (DNDS) rearranged from the E-isomer to the Z-isomer when solutions were kept unprotected from light. These results indicate that benchworkers should not be surprised when commercial samples of such stilbenes contain large amounts of various types of impurities.     

Labeling of human erythrocyte band 3 with maltosylisothiocyanate. Interaction with the anion transporter

Maltosylisothiocyanate (MITC), synthesized as an affinity label for the hexose carrier, has been reported to label a Band 3 or Mr = 100,000 protein in human erythrocytes, in contradistinction to many studies showing the carrier as a Band 4.5 or Mr = 45,000-66,000 protein on gel electrophoresis. In this work the possibility that MITC interacts with the Band 3 anion transporter was studied. In intact human erythrocytes, MITC labeling was largely confined to Band 3 and was decreased by several competitive inhibitors of hexose transport. However, MITC also appeared to react with the anion transport protein, since MITC labeling of Band 3 was irreversibly decreased by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and since MITC also irreversibly inhibited both tritiated dihydro-DIDS labeling of Band 3 and sulfate uptake in intact cells. Although 20 microM DIDS had little effect on hexose transport, the labeling of erythrocyte Band 3 by the dihydro analog was significantly diminished by competitive inhibitors of hexose transport. These data suggest that MITC labels in part the anion transporter as well as other DIDS-reactive sites on Band 3 which appear to be sensitive to competitive inhibitors of hexose transport.