Effect Of PCMBS Research Articles

Cysteine 159 delineates a hinge region of the alternating access monocarboxylate transporter1 and is targeted by cysteine-modifying inhibitors.

Monocarboxylate transporter isoforms 1-4, MCT, of the solute carrier SLC16A family facilitate proton-coupled transport of l-lactate. Growth of tumors that exhibit the Warburg effect, that is, high rates of anaerobic glycolysis despite availability of oxygen, relies on swift l-lactate export, whereas oxygenic cancer cells import circulating l-lactate as a fuel. Currently, MCTs are viewed as promising anticancer targets. Small-molecule inhibitors have been found, and, recently, high-resolution protein structures have been obtained. Key questions, however, regarding the exact binding sites of cysteine-modifying inhibitors and the substrate translocation cycle lack a conclusive experimental basis. Here, we report Cys159 of the ubiquitous human MCT1 to reside in a critical hinge region of the alternating access-type transporter. We identified Cys159 as the binding site of the organomercurial pCMBS. The inhibitory effect of pCMBS was proposed to be indirect via modification of the chaperone basigin. We provide evidence that pCMBS locks MCT1 in its outward open conformation in a wedge-like fashion. We corroborated this finding using smaller cysteine-modifying reagents that size-dependently inhibited l-lactate transport. The smallest modifiers targeted additional cysteines as shown by a C159S mutant. We found a Cys399/Cys400 pair to constitute the second hinge of the transporter that tolerated only individual replacement by serine. The hinge cysteines, in particular the selectively addressable Cys159, provide natural anchors for placing probes into MCTs to report, for instance, on the electrostatics or hydration upon binding of the transported l-lactate substrate and the proton cosubstrate.

The Hexose efflux from the peeled grape berry

Aim: The transport of sugars into grape berry mesocarp cells across the plasma and vacuolar membranes after onset of ripening is a complex process. Elements of the sugar transport mechanism may be assessed by exposing the mesocarp cells and investigating sugar movement across the membranes. The purpose of this study was to gain insights into the nature of the transport mechanism by creating conditions conducive to hexose efflux from the peeled berry.Methods and results: The experimental technique employed was a derivate of the ‘berry-cup’ technique. The skin of ripening cv. Shiraz berries was peeled in situ and, after an initial wash, hexose efflux into a collection medium (MES buffer) was monitored. Additionally, during the period of intensive sugar accumulation (one week after veraison) and two weeks later, hexose efflux was assessed following three modifications: (i) using berries excised from the vine, (ii) using MES buffer (2-(N-morpholino)ethanesulfonic acid, pH 5.5) containing PCMBS (p-chloromercuribenzenesulfonic acid, 1mM), and (iii) using cold (10°C) or warm (40°C) MES buffer. Hexose quantities collected into the buffer were dependent on ripening stage and buffer temperature, but they were not dependent on an intact berry-to-cluster connection. The inhibitory effect of PCMBS was observed early in ripening, but not two weeks later.Conclusions: These results lead us to the conclusion that the origin of the collected hexoses was vacuolar as opposed to vascular, and that the hexose efflux mechanism is differently sensitive to PCMBS at the two stages of ripening.Significance and impact of the study: This simple technique was effective at providing insights into hexose transport within the grape berry at the cellular level.

Effect of inhibitors on oxygen permeability of wild type and knockout mouse red blood cells

We now recognize that the paradigm that all gases cross red blood cell (RBC) membranes by dissolving in and diffusing through the lipid phase is not correct. Work on human RBCs genetically deficient in aquaporin‐1 (AQP1) or the Rh complex (mainly RhAG) showed that these two proteins account for ~90% of membrane CO2 permeability (PM,CO2), and that 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonate (DIDS) reduces PM,CO2. We used stopped‐flow (SF) analysis of hemoglobin (Hb) absorbance spectra as we mixed oxygenated RBCs with an extracellular O2 scavenger, and determined the rate constant of Hb deoxygenation (kHbO2). Because kHbO2 depends on Hb kinetics as well as diffusion of O2 to the extracellular space, we used a mathematical model to estimate effects on membrane O2 permeability (PM,O2). We examined the effects of DIDS and chloromercuribenzenesulfonate (pCMBS), agents that decrease PM,CO2. We find that, in RBCs from wild type (WT) mice, pCMBS reduces kHbO2 by 64% (est. PM,O2 by ~84%) and DIDS, by 41% (est. PM,O2 by ~67%). Because pCMBS and DIDS likely act by targeting membrane proteins (rather than lipids), we hypothesized that O2, at least in part, crosses RBC membranes via the same membrane proteins that conduct CO2. Therefore, we examined RBCs from mice genetically deficient in AQP1, RhAG, or both, ± inhibitors. Deletion of AQP1 lowers kHbO2 by 11% (est. PM,O2 by ~27%); RhAG, by 16% (est. PM,O2 by ~35%); the double knockout (dKO), by 33% (est. PM,O2 by ~59%). The combination dKO + pCMBS lowers kHbO2 (vs. WT without inhibitors) by 77% (est. PM,O2 by ~91%), whereas dKO + DIDS lowers kHbO2 by 52% (est. PM,O2 by ~76%). The effects of pCMBS on estimated PM,O2 are very similar when we compare dKO vs. RhAG−/−, or when we compare WT vs. AQP1−/−. Thus, the deletion of AQP1 may have little effect on the sensitivity of PM,O2 to pCMBS—consistent with the hypothesis that AQP1 conducts O2 only via pathways other than the mercury‐sensitive monomeric pores (e.g., via the hydrophobic central pore). In summary, our data suggest that AQP1 and RhAG account for nearly 60% of RBC O2 permeability, and point towards the existence of other O2 channels that account for at least 30%.Support or Funding InformationSupported by ONR N00014‐15‐1‐2060, ONR N00014‐16‐1‐2535 to WFB and NIH K01‐DK107787 to ROThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of Sulfhydryl Modification on Rat Kidney Basolateral Plasma Membrane Transport Function

Transport processes are the hallmark of functioning kidney. Various nephrotoxicants disrupt the transport processes to manifest nephrotoxicity. Of several nephrotoxicants, mercuric chloride (HgCl(2)) depletes the reduced glutathione (GSH) in kidney and has been observed to affect the in vitro p-aminohippurate (PAH) transport by basolateral (BL) membrane vesicles. The role of renal nonprotein sulfhydryls such as, reduced GSH has been demonstrated to affect the PAH transport by BL membrane vesicles. The role of protein sulfhydryls in transport process of PAH by BL membrane is not known. Due to mercury mediated effects on sulfhydryls, the effects of protein-sulfhydryls (-SH) modifying reagents in the current study were investigated on PAH transport by BL membrane. It was observed that modification of -SH by p-chloromercuribenzoate sulphate (pCMBS), and mercuric chloride (HgCl(2)) decreased while recovering the protein -SH with dithiothreitol treatment provided protection against the effects of pCMBS, and HgCl(2) on PAH transport by BL membrane vesicles.

Rigid Motion Near the GABA A Receptor α 1 Subunit First Transmembrane Segment Revealed by pCMBS - Reactivity in Cysteine-Substituted Mutants During Channel Activation

The gamma-aminobutyric receptor (GABAAR) belongs to the pentameric ligand-gated ion channels (pLGIC) family and mediates inhibitory synaptic transmission in the brain. GABA binding in the extracellular domain triggers a conformational change that results channel gating. The molecular details of the events linking the agonist binding site and the gate, distant by ∼60 A, remain uncertain. AChBP crystal structures obtained in the absence and presence of ligand showed that the C-loop undergoes large movement “capping” the agonist in its binding site. The C-loop is directly connected to the first transmembrane segment (M1) of the receptor by the extracellular β10 β-strand. We hypothesize that C-loop movement during gating could be transmitted to M1. We sought to investigate the mobility of the M1 segment of the GABAAR α1 subunit during gating using SCAM. We measured the rates of reaction of the cysteine-specific reagent pCMBS- in various Cys-substituted receptors in the closed and the activated states. The effects of pCMBS- were followed using two electrode voltage clamp of Xenopus oocytes injected with mRNA coding for the Cys-substituted GABAAR. pCMBS- reaction rate is proportional to the solvent accessibility of the substituted residue. The results indicate that the first part of M1 is completely solvent accessible. The most reactive residues are located on one face of the α-helix and the rate of reaction increases significantly with activation. The distribution of rapidly reacting residues does not differ significantly between the closed state and the activated state. This face is directed toward M3 in the adjacent β2 subunit, forming the general anesthetic binding site. We suggest that βM3 moves past αM1 pushing lipid away from the fast reacting face of αM1 thereby increasing the pCMBS reaction rate.

Cysteine-scanning Mutagenesis and Substituted Cysteine Accessibility Analysis of Transmembrane Segment 4 of the Glut1 Glucose Transporter

A low resolution model has been proposed for the exofacial conformation of the Glut1 glucose transporter in which eight transmembrane segments form an inner helical bundle stabilized by four outer helices. The role of transmembrane segment 4, predicted to be an inner helix in this structural model, was investigated by cysteine-scanning mutagenesis in conjunction with the substituted cysteine accessibility method using the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). A functional, cysteine-less, parental Glut1 molecule was used to produce 21 Glut1 point mutants by individually changing each residue along transmembrane helix 4 to a cysteine. The single cysteine mutants were then expressed in Xenopus oocytes, and their expression levels, transport activities, and sensitivities to pCMBS were determined. In striking contrast to all of the other seven predicted inner helices, none of the 21 helix 4 single-cysteine mutants was demonstrably inhibited by pCMBS. However, cysteine substitution within helix 4 resulted in an unusually high number of severely transport-defective mutants. The low absolute transport activities of two of these mutants (G130C and G134C) were due to their extremely low levels of expression, presumably a result of structural instability and consequent degradation in oocytes, suggesting that these two residues play an important role in maintaining the native structure of Glut1. The other two transport-defective mutants (Y143C and E146C) exhibited low specific transport activities, implying that these two residues play an important role in the transport cycle. Based on these data, we conclude that the exoplasmic end of helix 4 lies outside the inner helical bundle in the exofacial configuration of Glut1.

Comparative NMR studies of diffusional water permeability of red blood cells from different species. X. Camel (Camelus dromedarius) and alpaca (Lama pacos)

The diffusional water permeability (P d) of camel and alpaca red blood cells (RBCs) was measured by a doping nuclear magnetic resonance (NMR) technique on control cells and following inhibition withp-chloromercuribenzene sulphonate (PCMBS). The values ofP d were, in the case of alpaca RBC≈4.6×10−3 cm/s at 25°C, 5.4×10−3 cm/s at 30°C, 6.6×10−3 cm/s at 37°C and 7.7×10−3 cm/s at 42°C. In case of camel RBC the values ofP d where ≈4.2×10−3 cm/s and 9.0×10−3 cm/s at 42°C. Systematic studies on the effects of PCMBS on water diffusion in camel RBC indicated that the maximal inhibition was reached in 45 min with 1–2 mm PCMBS. The values of maximal inhibition were around 47% at 25°C and 68% at 30°C for alpaca RBC and around 62% at 25°C and 56% at 37°C for camel RBC. The basal permeability to water of alpaca RBC was estimated at around 2.6×10−3 cm/s at 25°C, 1.7×10−3 cm/s at 30°C and of camel RBC as 1.8×10−3 cm/s at 25°C and 3.0×10−3 cm/s at 37°C. The values of the activation energy of water diffusion (E a, d) were around 23 kJ/mol for camel and 34 kJ/mol for alpaca RBC. This suggests that in addition to the number of transport channels other features of the pathways might be important for defining the temperature dependence of the water permeability.

Effect of PCMBS on CO2 permeability of Xenopus oocytes expressing aquaporin 1 or its C189S mutant.

A recent study on Xenopus oocytes [N. L. Nakhoul, M. F. Romero, B. A. Davis, and W. F. Boron. Am. J. Physiol. 274 (Cell Physiol. 43): C543-548, 1998] injected with carbonic anhydrase showed that expressing aquaporin 1 (AQP1) increases by approximately 40% the rate at which exposing the cell to CO2 causes intracellular pH to fall. This observation is consistent with several interpretations. Overexpressing AQP1 might increase apparent CO2 permeability by 1) allowing CO2 to pass through AQP1, 2) stimulating injected carbonic anhydrase, 3) enhancing the CO2 solubility of the membrane's lipid, or 4) increasing the expression of a native "gas channel." The purpose of the present study was to distinguish among these possibilities. We found that expressing the H2O channel AQP1 in Xenopus oocytes increases the CO2 permeability of oocytes in an expression-dependent fashion, whereas expressing the K+ channel ROMK1 has no effect. The mercury derivative p-chloromercuriphenylsulfonic acid (PCMBS), which inhibits the H2O movement through AQP1, also blocks the AQP1-dependent increase in CO2 permeability. The mercury-insensitive C189S mutant of AQP1 increases the CO2 permeability of the oocyte to the same extent as does the wild-type channel. However, the C189S-dependent increase in CO2 permeability is unaffected by treatment with PCMBS. These data rule out options 2-4 listed above. Thus our results suggest that CO2 passes through the pore of AQP1 and are the first data to demonstrate that a gas can enter a cell by a means other than diffusing through the membrane lipid.

Pathway of phloem loading in the C3tropical orchid hybridOncidiumGoldiana

The apoplast of mature leaves of the tropical orchid Oncidium Goldiana was perfused with 0.5 mM p-chloromercuribenzenesulphonic acid (PCMBS) via the transpiration stream in order to test the mode of phloem loading. The efficacy of introducing PCMBS by perfusion was shown by saffranin O dye movement in the veins and leaf apoplast in control experiments. Photoassimilate export as the result of phloem loading was measured by collection of 14 CO 2 -derived photoassimilates from the basal cut-ends of intact leaves. Phloem loading and translocation of photoassimilates was inhibited by 89% in leaves perfused with PCMBS for 1 h. The effect of PCMBS on leaf photosynthesis was minimal. The amount of radiocarbon fixed by PCMBS-treated leaves averaged 89% of control leaves perfused with distilled water. A negative correlation between the total amount of photoassimilate exuded and the calculated concentration of PCMBS in the leaf apoplast was also observed. The results indicate that phloem loading in Oncidium Goldiana occurs via the apoplastic pathway.

Glycerol permeability of mutant aquaporin 1 and other AQP-MIP proteins: inhibition studies.

In a recent work, we showed that the aquaporins 1 (AQP1) are permeable to certain small solutes such as glycerol. Here, we have further investigated the permeation pathway of glycerol through human AQP1 (hAQP1) by the use of mutants (C189S, H180A, H209A) and inhibitors such as P-chloromercuribenzene sulphonate (pCMBS), CuSO4 or phloretin, in comparison with other AQP-MIP (where MIP denotes major intrinsic protein) proteins: hAQP2, plant water channel gammaTIP and bacterial glycerol permease facilitator, GlpF. Glycerol movements were measured in Xenopus laevis oocytes. Apparent glycerol permeability coefficients (P'gly) were calculated from the rates of oocyte swelling upon exposure to an isoosmotic medium containing an inwardly directed gradient of glycerol and from [3H]glycerol uptake measurements. Similar P'gly values were obtained for hAQP1 and hAQP2 6 to 8 times greater than control indicating that hAQP2 also transports glycerol. P'gly of hAQP2-injected oocytes was pCMBS and CuSO4 sensitive. In contrast, the P'gly value of gammaTIP was close to that of control, indicating that gammaTIP does not transport glycerol. The hAQP1-C189S, -H180A and -H209A mutants gave P'gly values similar to those obtained for wild hAQP1, indicating that these mutations did not affect glycerol movements. However, the H209A mutant has an osmotic water permeability coefficient (Pf) value decreased by 50%. The inhibitory effect pCMBS on P'gly was maintained for the 2 His mutants and, more interestingly, was also conserved for the C189S mutant. CuSO4 significantly inhibited P'gly of oocytes expressing hAQP1, hAQP1-C189S, -H180A, and -H209A mutants and had no effect on P'gly of GlpF-injected oocytes. Phloretin was shown to inhibit by around 80% the glycerol fluxes of wild and mutant hAQP1, hAQP2 and to fully inhibit glycerol uptake in GlpF-injected oocytes.

Comparative nuclear magnetic resonance studies of diffusional water permeability of red blood cells from different species. VIII. Adult and fetal guinea pig (Cavia procellus)

The diffusional water permeability (Pd) of adult, pregnant female and fetal guinea-pig red blood cells (RBCs) was measured by a doping nuclear magnetic resonance (NMR) technique on control cells and following inhibition withp-chloromercuribenzene sulphonate (PCMBS). The values ofPd were around 5.0 × 10−3 cm/s at 15 °C, 5.3 × 10-3 cm/s at 20 °C, 6.6 × 10−3 cm/s at 25 °C, 7.5 × 10−3 cm/s at 30 °C and 8.6 × 10−3 cm/s at 37 °C with no significant differences between adult, pregnant female and fetal RBCs. Systematic studies on the effects of PCMBS on water diffusion indicated that the maximal inhibition was reached in 10 min at 37 °C with 0.1 mm PCMBS. The values of maximal inhibition ranged from 70%–77% at 15–30 °C to 57%–63% at 37 °C in the case of adult and from 64%–67% at 15–30 °C to 51% at 37 °C in the case of fetal RBCs. The basal permeability to water was estimated at 1.1 × 10−3 cm/s at 15 °C ,1.3 × 10−3 cm/s at 20 °C, 1.6 × 10−3 cm/s at 25 °C, 2.2 × 10−3 cm/s at 30 °C and 3.2 × 10−3 cm/s at 37 °C for adult and slightly higher values for fetal guinea pig RBCs as 1.6 × 10−3 cm/s at 15 °C, 2.0 × 10−3 cm/s at 20 °C, 2.4 × 10−3 cm/s at 25 °C, 2.6 × 10−3 cm/s at 30 °C and 4.2 × 10−3 cm/s at 37 °C. The activation energy of water diffusion was around 22 kJ/ mol, with no significant differences between the adult pregnant female and fetal RBCs, and increased to about 40 kJ/mol in the case of adult and pregnant RBCs and 34 kJ/mol for fetal RBCs after incubation with PCMBS in conditions of maximal inhibition of water diffusion. The membrane polypeptide electrophoretic pattern of adult and fetal guinea-pig RBCs was compared with its human counterpart. The guinea-pig membrane contained higher amounts of spectrin (band 1 and 2), whereas the proteins in bands 4.1, 4.2 and 6 were present in lower amounts. Considerable differences in polypeptides migrating in the region of bands 7 and 8 and in front of them were apparent between the two sources of RBC membranes where some bands were present only in the guinea-pig RBC membranes. The adult guinea-pig membranes contained smaller amounts of proteins migrating in band 4.5 and lacked band 8.

Effect of PCMBS on calcium‐ or aluminum‐induced curvature in roots of two watermelon cultivars

Abstract Unilateral application of calcium (Ca) or aluminum (Al) in agar to the primary roots of watermelon [Citrullus lanatus (Thunb.) Matsum. and Nakai] cultivars ‘Dixielee’ and ‘Mirage’ induced root curvature. Root curvature induced by Al was greater than that induced by Ca in both cultivars. PCMBS inhibited Al‐induced root curvature in both cultivars, but had no effect on Ca‐induced curvature. The inhibition of curvature indicated that PCMBS reduced Al uptake. ‘Dixielee’ was more responsive to PCMBS than was ‘Mirage’.

In squid axons the Ca i2+ regulatory site of the Na + /Ca 2+ exchanger is drastically modified by sulfhydryl blocking agents. Evidences that intracellular Ca i2+ regulatory and transport sites are different

We have explored the effect of the sulfhydryl group blocker p-chloromercuryphenylsulfonic acid (PCMBS) on Ca 2+ and Na + interactions with the Na − /Ca 2+ exchanger in squid giant nerve fibers. Steady-state Na o +-dependent Ca 2+ efflux (forward) and Na i +-dependent Ca 2+ influx (reverse) were measured in internally dialyzed, voltage clamped squid axons. External PCMBS (0.5 mM, for 25–35 min) has no effect on the activation of Ca 2+ efflux by Na o +, and Ca o 2+ or on the activatory external monovalent cation site. In contrast, when applied internally it drastically reduces the affinity of the Na + /Ca 2+ exchanger towards Ca i 2+ ions without affecting its maximal rate of transport; in the presence of MgATP the K 0.5 for Ca i 2+ activation of forward Na + /Ca 2+ exchange increases from 1.5 μM to 95 μM; likewise the apparent affinity of the Ca i 2+ stimulation of the reversal exchange decreases 100-fold. Interestingly, no effect of PCMBS was found on the interactions between Na i + and Ca i 2+ ions with the internal transport site(s) (inhibition of Na o + and Ca o 2+-dependent Ca 2+ efflux by Na i +). On the other hand, Na i + ions do not modify the interactions of Ca i 2+ with that site. Two important characteristics of the Ca i 2+ regulatory site are uncover in this work: (i) sulfhydryl groups are important in maintaining the integrity of the Ca 2+ binding domain of the Ca i 2+ regulatory site and (ii) Na i + and Ca i 2+ regulatory, or Na i + and Ca i 2+ transporting sites, are different entities.

Modulation of ionic currents in isolated type I cells of the neonatal rat carotid body by p-chloromercuribenzenesulfonic acid

The sulphyddryl reagent, p-chloromercuribenzenesulfonic acid (PCMB S), irreversible excites the carotid body in vivo. We tested the effects of PCMBS on ionic currents in isolated type I cells, using the whole-cell patch-clamp technique. PCMBS selectively and irreversibly inhibited the Ca 2+-activated K + current (IK ca) in a dose-dependent manner (0.01–1 mM). The same concentrations of PCMBS did not affect the Ca 2+-independent K + current (IK v), but caused a transient enhancement of Ca 2+ current (I ca). The inhibition of IK ca by PCMBS is similar to the previously reported effects of hypoxia, and suggests a central role for the channels underlying this current in chemotransduction.

Involvement of histidine residues and sulfhydryl groups in the function of the biotin transport carrier of rabbit intestinal brush-border membrane

Possible involvement of histidine residues and sulfhydryl groups in the function of the intestinal brush-border membrane (BBM) transporter of biotin was investigated. This was done by examining the effects of pretreatment of BBM vesicle (BBMV) isolated from rabbit intestine with the histidine-specific reagent diethyl pyrocarbonate (DEPC) and the sulfhydryl group-specific reagents p-chloromercuribenzenesulfonic acid ( p-CMBS) and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) on carrier-mediated biotin transport. Pretreatment of BBMV with DEPC caused significant inhibition in the initial rate of biotin transport without affecting the substrate uptake at equilibrium. Addition of biotin plus Na + to vesicle suspensions prior to treatment with DEPC provided significant protection to biotin transport. Treatment of DEPC-pretreated vesicles with the reducing agents dithiothreitol and 2,3-dimercaptopropanol failed to reverse the inhibitory effect of DEPC on biotin transport. The inhibitory effect of DEPC was found to be mediated through a marked decrease in the number of the functional biotin transport carriers with no change in their affinity, as indicated by the severe inhibition in the V max but not the apparent K m of the biotin transport process, respectively. Pretreatment of BBMV with p-CMBS and NBD-Cl also caused significant inhibition in the initial rate of biotin transport without affecting the substrate uptake at equilibrium. Addition of biotin plus Na + to vesicle suspensions prior to treatment with p-CMBS (or NBD-Cl) failed to protect biotin transport from inhibition. On the other hand, treatment of vesicles pretreated with p-CMBS (or NBD-Cl) with the reducing agents dithiothreitol and mercaptoethanol caused significant reversal in the inhibition of biotin transport. The inhibitory effects of p-CMBS (and NBD-Cl) on biotin transport was also found to be mediated through inhibition in the V max, but not the apparent K m, of biotin transport process. These results indicate the involvement of histidine residues and sulfhydryl groups in the normal function of the biotin transport system of rabbit intestinal BBM. Furthermore, the results also suggest that the histidine residues are probably located at (or near) the substrate-binding site while the sulfhydryl groups are located at a site other than the substrate binding region.

Effect of pCMBS on anion transport in human red cell membranes

The kinetics of binding of the mercurial sulfhydryl reagent, pCMVS ( p-chloromercuribenzene sulfonate), to the extracellular site(s) at which pCMBS inhibits water and urea tranport across the human red cell membrane, have previously been characterized. To determine whether pCMBS binding alters Cl − transport, we measured Cl −/NO 3 − exchange by fluorescence enhancement, using the dye SPQ (6-methoxy- N-(3-sulfopropyl)quinolinium). An essentially instantaneous extracellular phase of pCMBS inhibition is followed by a much slower intracellular phase. correlated with pCMBS permeation. We attribute the instantaneous phase to competitive inhibition of Cl − binding to band 3 by the pCMBS anion. The ID 50 of 2.0 ± 0.1 mM agrees with other organic sulfonates, byt is very much greater than that of pCMBS inhibition of urea and water transport, showing that pCMBS reaction with water and urea transport inhibition sites has no effect on anion exchange. The intracelllular inhibition by 1 mM pCMBS (1 h) is apparently non-competitive with K i = 5.5 ± 6.3 mM, presumably an allosteric effect of pCMBS binding to an intracellular band 3-related sulfhydryl group. After N-ethylmaleimide (NEM) treatment to block these band 3 sulfhydryl groups, there is apparent non-competitive inhibition with K i = 2.1 ± 1.2 mM, which suggests that pCMBS reacts with one of the NEM-insensitive sulfhydryl groups on a protein that links band 3 to cytoskeleton, perhaps ankyrin or bands 4.1 and 4.2.

Effect of PCMBS and EPTC on mineral nutrient uptake and translocation in wheat

Abstract Evaluations of the influence of EPTC on mineral ion root uptake and translocation to the foliage were undertaken. Root uptake was measured in wheat (Triticum aestivum L. cv Holley) was grown in 0.5 Hoagland complete mineral nutrient solution. Timed (<4h) accumulation studies were undertaken in the presence of: a) p‐chloromercuribenzenesulfonic acid (PCMBS) which does not penetrate through membranes but does inhibit sulfhydryl groups (SH), and b) dithiothreitol (DTT) which is membrane permeable and protects SH from PCMBS. Mg, Ca, K, Fe, and Mn accumulations were decreased by PCMBS and the partial inhibition was reversed by DTT. Calcium root accumulation data were explicable as two modes of Ca entry into roots and one involved a SH containing enzyme. Ion translocation to the foliage was evaluated in nutrient solutions containing sub‐lethal concentrations of S‐ethyldipropylthiocarbamate (EPTC) (0, 0.625, 0.125, 0.25, or 0.5 mg/L), EPTC did not act as a general sulfhydryl group inhibitor. Shoot dry w...

Sulfhydryl modifying reagents inhibit Q A ? oxidation in reaction centers from Rhodobacter sphaeroides and Capsulatus, but not Rhodopseudomonas viridis

The effects of various sulfhydryl-modifying reagents on reaction centers (RCs) from purple photosynthetic bacteria have been examined, with particular emphasis on the activity of the acceptor quinones, QA and QB, comprising the two electron gate. Mercurial reagents, especially p-chloromercuribenzenesulfonate (pCMBS), were effective in inhibiting QB function in RCs from Rhodobacter sphaeroides and Rb. capsulatus, but not in Rhodopseudomonas viridis. The inhibition was fully reversible by dialysis against dithiothreitol (DTT). The effect on QB function was not an apparent one mediated by an alteration in the redox potential of QA. N-ethylmaleimide (NEM) had no effect on any of the quinone functions, even at very high concentrations. Comparison of the X-ray structures of the RCs from Rb. sphaeroides and Rp. viridis and the known amino acid sequences for all three bacterial RCs suggest that a cysteine residue at position 108 in the L subunit of the Rhodobacter species is the most likely candidate for the site of action of the mercurial reagents. This was strongly supported by the absence of any effect of pCMBS on a site specific mutation of Rb. sphaeroides (L108CS) with residue L108 changed from cysteine to serine. These results imply a long distance (>20 Å) effect on the functioning of QB, perhaps involving a relatively gross structural alteration.

Inhibition of the sodium-dependent uridine co-transporter in bovine renal brush-border membrane vesicles by group-specific reagents

T h e reabsorption of amino acids, glucose and organic anions from the renal filtrate into epithelial cells is coupled to Na' transport down its concentration gradient via specific cotransporters present in the brush-border membrane. Recently, Na + -dependent nucleoside transport has been demonstrated in the proximal tubule of rat kidney 11, 21. In our laboratory, a Na + -dependent uridine transporter has been characterized in rabbit renal brush-border membrane vesicles in terms of substrate specificity, electrogenicity and kinetic properties [3, 41. However, little is known about the molecular nature of this transporter. In the present study, we have attempted to define essential amino acids in the uridine co-transporter of bovine kidney using a variety o f proteinmodifying agents. Fresh bovine kidneys were obtained from a local abattoir and outer renal cortex brush-border membrane vesicles prcpared using a Mgprecipitation technique 1.51. T h e enrichment of the brush-border marker enzyme alkaline phosphatase in the final vesicle suspension was typically 8-14-fold higher than that in the cortical homogenate. T h e initial velocity of uridine uptake into brush-bordcr membrane vesicles was determined by measuring the vesicular content of uridine after incubation periods of 3-5 s at 22°C in the presence of an inwardly directed 100 mM-NaCI gradient, as previously described [3.4]. Initial experiments were performed with the sulphydrylreacting reagents, N-ethylmalcimide ( N E M ) and p chloromecuriphenylsulphonic acid (pCMBS) and the histidyl-modifying reagent, diethylpyrocarbonate (DEP). Table 1 shows that inhibition o f Na'-dependent uridine uptake by N E M occurred only at millimolar concentrations. Similarly, incubation of vesicles with D E P under mild acid conditions also resulted in little inhibition (30/0 inhibition at 20 mM-DEP). In contrast, addition of pCMBS resulted in marked inhibition of uridine influx: 50% inhibition at 30 ,UMpCMBS in the presence of an inwardly directed 100 mMNaCl electrochemical gradient at 22°C. T h e time course for pCMBS inhibition demonstrated that maximal inhibition was obtained after 10 min incubation and remained stable for a further 30 min (data not shown). This result may suggest that the site for pCMBS inhibition is located on the inner surface o f the vesicle. However, no difference in the time course of pCMBS inhibition was observed between incubations performed at 1 and 22°C. Previous studies have established that the transmembrane permeability of pCMBS is significantly reduced at 1°C [6]. Inclusion of equimolar concentrations o f EDTA in the pCMBS incubation mixture had no effect on the inhibition profile, demonstrating that the inhibitory effect of pCMBS is not due to the presence of free Hg?+. Table 1 also shows that pCMBS inhibition was only partially reversed by addition of dithiothreitol (DTT) and Bmercaptoethanol (P-ME). This finding would suggest that, at most, 50% of the inhibition caused by pCMBS is at a reactive thiol group. To investigate whether this possible sulphydryl group was within the active site of the carrier, bovine membrane vesicles were incubated with uridine (5-50 ,UM) in the absence and presence of Na' before treatment with 50 ,UM-

Control of photoassimilate movement in source-leaf tissues of Ipomoea tricolor Cav.

Autoradiographs obtained from discs from mature, peeled Ipomoea tricolor Cav. leaves indicated that a 15-min 2 mM p-chloromercuribenzene sulfonic acid (PCMBS) pretreatment did not prevent the accumulation of (14)CO2-derived (14)C-photosynthate within the minor veins. However, accumulation of (14)C-photosynthate within these veins was almost, or completely prevented by treatment of the leaf discs at pH 9.0. Peeled Ipomoea leaf discs were also used to study the kinetics of exogenously supplied [(14)C]sucrose; influx occurred by the combined operation of a saturable and a first-order kinetic mechanism. Both transport mechanisms were inhibited by a 15-min 2 mM PCMBS pretreatment or by treatment at pH 9.0. An analysis of autoradiographs obtained on leaf discs that had been fed exogenous [(14)C]sucrose, showed that whereas all cells of the leaf took up sucrose at pH 5.0, uptake at pH 9.0 occurred primarily into cells of the minor veins. In contrast with (14)C-photoassimilate experiments, PCMBS pretreatment, at either pH 5.0 or pH 9.0, resulted in a reduction in the radioactivity within the minor veins when the label was supplied exogenously as [(14)C]sucrose. Kinetic experiments conducted with mesophyll cells isolated enzymatically from peeled source-leaf discs indicated that [(14)C]sucrose influx also occurred by a saturable and a first-order kinetic mechanism. Comparative studies between leaf discs and isolated mesophyll cells showed that mesophyll cells could account for 75% of the total uptake occurring into the peeled leaf discs. These results indicate that the effects of PCMBS and alkaline pH on net sucrose uptake by leaf tissues are not exerted solely on phloem tissues, and that some of the effects of these treatments may be attributable to an inhibition of symplasmic transport to the minor veins.