Binding of phlorizin to the C-terminal loop 13 of the Na(+)/glucose cotransporter does not depend on the [560-608] disulfide bond

The disulfide bonds of the Na(+)/glucose cotransporter (SGLT1) are believed to participate in the binding of the transport inhibitor phlorizin. Here, we investigated the role of the [560-608] disulfide bond on the phlorizin-binding function of the C-terminal loop 13 of SGLT1 using 3-iodoacetamidophlorizin (3-IAP) as a probe. The reactivity of 3-IAP to the fully reduced loop 13 was competitively inhibited by phlorizin, as evident from the MALDI mass spectra. It indicates that the disulfide bond is not mandatory for phlorizin binding. CD and equilibrium unfolding studies showed that the secondary structure and conformation stability of loop 13 were not affected by removing the disulfide bond. Furthermore, we generated a series of loop 13 mutants to assess the contribution of the disulfide bond to phlorizin binding. A positive correlation between the stability and phlorizin affinity of the mutant proteins was observed, implying that the protein stability, rather than the disulfide bond, is relevant to the phlorizin-binding function of loop 13.     

Hydrogen peroxide involvement in the rhodanese inactivation by dithiothreitol.

The conditions required to obtain rhodanese inactivation in the presence of dithiothreitol indicate the involvement of hydrogen peroxide produced by metal-ion catalyzed oxidation of dithiothreitol. Inhibition of dithiothreitol oxidation by a chelating agent, or by removal of hydrogen peroxide by catalase prevents the enzyme inactivation. The inactivated enzyme contains a disulfide bond resulting from the oxidation of the catalytic sulfhydryl group and another sulfhydryl group close to it. This disulfide might be formed via a sulfenic intermediate.     

Structural state of the Na+ /d-glucose cotransporter in calf kidney brush-border membranes Target size analysis of Na+-dependent phlorizin binding and Na+-dependent d-glucose transport

Target sizes of the renal sodium-d-glucose cotransport system in brush-border membranes of calf kidney cortex were estimated by radiation inactivation. In brush-border vesicles irradiated at ?50°C with 1.5 MeV electron beams, sodium-dependent phlorizin binding, and Na⁺-dependent d-glucose tracer exchange decreased exponentially with increasing doses of radiation (0.4–4.4 Mrad). Inactivation of phlorizin binding was due to a reduction in the number of high-affinity phlorizin binding sites but not in their affinity. The molecular weight of the Na⁺-dependent phlorizin binding unit was estimated to be 230 000 ± 38 000. From the tracer exchange experiments a molecular weight of 345 000 ± 24 500 was calculated for the d-glucose transport unit. The validity of these target size measurements was established by concomitant measurements of two brush-border enzymes, alkaline phosphatase and γ-glutamyltransferase, whose target sizes were found to be 68 570 ± 2670 and 73 500 ± 2270, respectively. These findings provide further evidence for the assumption that the sodium-d-glucose cotransport system is a multimeric structure, in which distinct complexes are responsible for phlorizin binding and d-glucose translocation.     

Diethylpyrocarbonate inhibition of sodium-glucose cotransport in kidney brush-border membrane vesicles

Exposure of kidney brush-border membrane vesicles to the acylating reagent diethylpyrocarbonate resulted in inactivation of the glucose transporter, as demonstrated by inhibition of sodium-coupled D-glucose transport and phlorizin binding. The transport site(s) was protected against inactivation by the simultaneous presence of sodium ions and D-glucose, and were partially protected by phlorizin. Transport activity was not restored by hydroxylamine; this rules out the possibility of diethylpyrocarbonate interaction with histidine, serine or tyrosine transporter residues. Dithiothreitol, a thiol protector, slightly prevented diethylpyrocarbonate inactivation. It is therefore suggested that (an) amino group(s) in the translocation complex is involved, at the level of the sugar transport site and the preferential protection of D-glucose against diethylpyrocarbonate inactivation related to a conformation change caused by the simultaneous binding of sodium and D-glucose to the cotransporter.     

Chemical modification of the neutral amino acid transport system L of Chinese hamster ovary cells with p-chloromercuribenzene sulfonate.

Branched-chain and aromatic neutral amino acids enter mammalian cells predominantly through a Na(+)-independent transport agency called System L. The sulfhydryl specific reagent p-chloromercuribenzene sulfonate (pCMBS) has been shown to be a potent inactivator of System L transport activity in Chinese hamster ovary cells, however, inactivation by pCMBS can be prevented by the presence of System L-specific substrate amino acids during the inactivation reaction. In addition, the presence of amino acids that are not substrates for System L have no effect on pCMBS inactivation of System L. Inactivation of System L activity by pCMBS was sensitive to pH and reversible by incubation with dithiothreitol. These findings suggest that there is a sulfhydryl group in, or very near, the amino acid-binding site of the System L transporter of CHO cells. Substrate protection, however, could be explained by conformational changes in the transporter associated with substrate binding. The presence of a substrate protectable sulfhydryl group on the System L transporter would aid in the attempt to identify this transporter using the technique of differential labeling.     

Identification of human vesicle monoamine transporter (VMAT2) lumenal cysteines that form an intramolecular disulfide bond

The vesicle monoamine transporter (VMAT2) concentrates monoamine neurotransmitter into synaptic vesicles. To obtain structural information regarding this large membrane protein by analysis of disulfide bonds and other intramolecular cross-links, we engineered a strategic thrombin cleavage site into deglycosylated, HA-tagged human VMAT2. Insertion of this protease site did not disrupt ligand binding or serotonin transport. Thrombin cleavage at an engineered site in the predicted cytoplasmic loop between transmembrane (TM) domains 6 and 7 (loop 6/7) was rapid and quantitative in the absence of any detergent. The loop 6/7 thrombin site allowed assessment of an intramolecular disulfide bond between the N- and C-terminal halves of the transporter. Consistent with this hypothesis, after quantitative loop 6/7 thrombin cleavage, in the absence of reducing agent, VMAT2 migrated on SDS-polyacrylamide gels as a full-length transporter. Addition of dithiothreitol resulted in complete conversion from full-length to thrombin-cleaved size, demonstrating a DTT-reversible covalent bond. The identity of the disulfide-bound cysteine pair was suggested by the observation that replacement of Cys 126 or Cys 333 with serine both reduced [(3)H]serotonin transport. Replacement of either Cys 126 or Cys 333 was found to eliminate the DTT-reversible intramolecular covalent bond. We conclude that human VMAT2 Cys 126 in loop 1/2 and Cys 333 in loop 7/8 form a disulfide bond which contributes to efficient monoamine transport.     

Essential disulfide and sulfhydryl groups for organic cation transport in renal brush-border membranes

The disulfide reducing agent, dithiothreitol (DTT) and the sulfhydryl-modifying reagents p-chloromercuribenzenesulfonic acid and N-ethylmaleimide (NEM) were employed to assess the role of disulfide and sulfhydryl groups in organic cation transport. The transport of N1-[3H]methylnicotinamide (NMN), a prototypic organic cation, was examined employing brush-border membrane vesicles isolated from the outer cortex of canine kidneys. DTT inhibited NMN transport reversibly with an IC50 of 250 microM/mg of protein. 5 mM NMN protected against DTT inactivation. The specificity of substrate protection was demonstrated by showing that D-glucose had no effect on the DTT inactivation of NMN transport and conversely that NMN had no effect on the DTT inactivation of D-glucose transport. Disulfide bonds reduced by DTT could be reoxidized by washing with excess buffer or by addition of 0.02% H2O2 thereby restoring NMN transport. p-Chloromercuribenzenesulfonic acid reversibly inactivated NMN transport with an IC50 of 25 microM/mg of protein. 5mM NMN protected against inactivation. NEM irreversibly inactivated transport with an IC50 of 250 microM/mg of protein. The rate of NMN inactivation by NEM followed pseudo-first order reaction kinetics. A replot of the data gave a linear relationship between the apparent rate constants and the NEM concentration with a slope of 1.3. The data are consistent with a simple bimolecular reaction mechanism and imply that one molecule of NEM inactivates 1 sulfhydryl group/active transport unit. The presence of 5 mM NMN affected the rate of NEM (2.5 mM) inactivation: the t1/2 values for inactivation in the presence and absence of substrate were 7.3 and 2.0 min, respectively. The results demonstrate an essential requirement for disulfide and sulfhydryl groups.     

Role of a disulfide bond in the gamma subunit in activation of the ATPase of chloroplast coupling factor 1

The relationship between activation of the latent ATPase activity of isolated chloroplast coupling factor 1 (CF1) and reduction of a disulfide in the gamma subunit has been assessed. The sulfhydryl residues involved in the disulfide bond are distinct from residues normally accessible to maleimide modification during incubation of thylakoids in the dark or the light. Dithiothreitol-induced activation is time dependent, and correlates with reduction of the disulfide. Sulfhydryl residues exposed during activation can be reoxidized to disulfide by incubation with iodosobenzoate , with a concomitant loss of ATPase activity. Activation and deactivation are reversible, but deactivation is prevented by treatment of the reduced enzyme with N-ethylmaleimide. Heat activation does not reduce the disulfide bond unless dithiothreitol is present during activation. Prior heating of CF1, which partially activates the enzyme, renders the disulfide more susceptible to subsequent dithiol reduction. The activity obtained when heat and dithiothreitol are used together is approximately equal to the sum of the partial activations obtained with heat or dithiothreitol alone. Iodosobenzoate has no effect on heat-activated CF1. Enzyme activated by heating in the presence of dithiothreitol can be partially deactivated, consistent with reversal of the activity attributable to the dithiol effect. Fluorescence polarization of anilinonaphthylmaleimide bound to the reduced enzyme indicates that the sulfhydryl residues involved in the disulfide are in a less rigid environment than the other two sulfhydryl residues in the gamma subunit. Polarization of anilinonaphthylmaleimide bound to these sulfhydryls is reduced by heat treatment of CF1. The increased susceptibility of the disulfide to reduction upon heat treatment, and the activation of ATPase activity with or without disulfide bond cleavage are indicative of conformational changes within the gamma subunit that occur during the conversion of CF1 from a latent to an active ATPase. In addition the results are consistent with at least two distinct conformational forms of CF1 that can hydrolyze ATP.     

Inactivation of the renal outer cortical brush-border membrane D-glucose transporter by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline

The inactivation of the renal outer cortical brush-border membrane D-glucose transporter by the covalent carboxyl reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) is studied by monitoring its effects on sodium-dependent phlorizin binding to the active site of the carrier. In the presence of EEDQ, this component of phlorizin binding decreases exponentially and irreversibly with time. The order of this inactivation reaction is very close to 1, indicating that EEDQ modifies the transporter at a single essential site. This site can be partially protected by glucose and by other substrates of the transporter and completely protected by phlorizin, a nontransported competitive inhibitor. By contrast, sodium, a co-transported activator, has no protective effect. The concentration dependence of the protection provided by glucose and phlorizin indicates that the site of action of EEDQ is at or closely related to the substrate binding site on the carrier. The effects of EEDQ on the transporter are mimicked by another carboxyl specific reagent, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate. The rate of inactivation of the transporter by EEDQ increases dramatically with decreasing pH, consistent with the hypothesis that the rate-limiting step in the inactivation process is a reaction with an essential carboxyl group. The properties of this group indicate, however, that it is distinct from the carboxyl group proposed by others as forming (a part of) the sodium binding site of sodium-coupled sugar carriers.     

Thiol/disulfide exchange in the thioredoxin-catalyzed reductive activation of spinach chloroplast fructose-1,6-bisphosphatase. Kinetics and thermodynamics

Two kinetically and thermodynamically distinct thiol/disulfide redox changes are observed during the reversible thioredoxin fb-catalyzed reduction and oxidation of spinach chloroplast fructose-1,6-bisphosphatase by dithiothreitol. The two processes, which occur at different rates and with different equilibrium constants, can be observed independently in either the reduction (activation) or oxidation (inactivation) direction by assaying the enzyme activity at different magnesium and fructose-1,6-bisphosphate concentrations. The two processes, in both the reduction and oxidation directions, are kinetically zero-order in dithiothreitol concentration and first-order in thioredoxin fb concentration. The rate-limiting step in both directions is the reaction of fructose-1,6-bisphosphatase with thioredoxin. The more kinetically and thermodynamically favored reduction of fructose-1,6-bisphosphatase lowers the apparent Km for fructose-1,6-bisphosphate while the less favorable process lowers the Km for magnesium. Both of the thiol/disulfide redox changes reach equilibrium in redox buffers consisting of different ratios of reduced to oxidized dithiothreitol (Ered + DTTox in equilibrium Eox + DTTred). The equilibrium constants (Kox) are 0.12 +/- 0.02 and 0.39 +/- 0.08 for the fast and slow reduction processes at pH 8.0. The equilibrium constants for oxidation of the enzyme by glutathione disulfide (Ered + GSSG in equilibrium Eox + 2 GSH) can be estimated to be approximately 2400 and 7800 M, respectively. Thermodynamically the fructose-1,6-bisphosphatase/thioredoxin fb system is extremely sensitive to oxidation, comparable to disulfide bond formation in extracellular proteins.     

Reduction of the buried intrachain disulfide bond of the constant fragment of the immunoglobulin light chain: global unfolding under physiological conditions

The constant (CL) fragment of the immunoglobulin light chain contains only one intrachain disulfide bond buried in the interior of the molecule. The kinetics of reduction with dithiothreitol of the disulfide bond were studied at various concentrations of guanidine hydrochloride at pH 8.0 and 25 degrees C. It was found that the disulfide bond is reduced even in the absence of guanidine hydrochloride. The results of the reduction kinetics were compared with those of the unfolding and refolding kinetics of the CL fragment previously reported [Goto, Y., & Hamaguchi, K. (1982) J. Mol. Biol. 156, 891-910]. It was shown that the reduction of the disulfide bond proceeds through a species with a conformation very similar to that of the fully unfolded one and that the CL fragment undergoes global unfolding transition even in water.     

Activation of the frog sartorius acetylcholine receptor by a covalently attached group

Summary The frog sartorius motor endplate was treated with the specific disulfide bond reducing agent dithiothreitol and subsequently exposed to a covalently reacting compound (the nitrophenyl ester ofp-carboxyphenyltrimethylammonium iodide, NPTMB) known to activate the dithiothreitol-reduced acetylcholine receptor inElectrophorus electroplax. NPTMB causes a maximum depolarization of about 35 mV when applied to the dithiothreitol-treated sartorius motor endplate. It is ineffective on postjunctional membrane prior to disulfide bond reduction and on extrajunctional regions, reduced or unreduced. High concentrations of a competitive antagonist such as (+)-tubocurarine prevent reaction between NPTMB and the reduced receptor and cause a repolarization of the membrane when applied to the already-depolarized preparation. We conclude that in frog muscle, as in electroplax, the attached activator bridges the acetylcholine binding site of the reduced receptor between a sulfhydryl group, to which it is covalently bound, and a negative subsite, with which it forms a reversible ionic bond.     

Identification of recombinant AtPYL2, an abscisic acid receptor,in E. coli using a substrate-derived bioactive small molecule,a biotin linker with alkyne and amino groups,and a protein cross-linker

Target protein identification of bioactive small molecules is one of the most important research in forward chemical genetics. The affinity chromatography technique to use a resin bound with a small molecule is often used for identification of a target protein of a bioactive small molecule. Here we report a new method to isolate a protein targeted with a bioactive small molecule using a biotin linker with alkyne and amino groups, protein cross-linker containing disulfide bond, and a bioactive small molecule with an azido group (azido probe). After an azido probe is associated with a target protein, the complex of a target protein and azido probe is covalently bound through the biotin linker by azide-alkyne Huisgen cycloaddition and protein cross-linker containing disulfide bond. This ternary complex is immobilized on an affinity matrix with streptavidin, and then the target protein is selectively eluted with a buffer containing a reducing agent for cleavage of disulfide bonds. This method uses a probe having an azido group, which a small functional group, and has the possibility to be a solution strategy to overcome the hindrance of a functional group introduced into the probe that reduces association a target protein. The effectiveness of the method in this study was shown using linker 1, 3′-azidoabscisic acid 3, and protein cross-linker containing a disulfide bond (DTSSP 5).     

On the molecular mechanism of maize phosphoenolpyruvate carboxylase activation by thiol compounds

Incubation of purified phosphoenolpyruvate carboxylase from Zea mays L. leaves with dithiothreitol resulted in an almost 2-fold increase in the enzymic activity. The activated enzyme showed the same affinity for its substrates and the same sensitivity with respect to malate and oxalacetate inhibition. The activation induced by dithiothreitol was reversed by diamide, an oxidant of vicinal dithiols, suggesting that the redox state of disulfide bonds of the enzyme may be important in the expression of the maximal catalytic activity.

Titration of thiol groups before and after activation of maize phosphoenolpyruvate carboxylase by dithiothreitol shows an increase of the accessible groups from 8 to 12 suggesting that the reduction of two disulfide bonds accompanied the activation. The thiols exposed by the treatment with dithiothreitol were available to reagents in nondenatured enzyme and two of them were reoxidized to a disulfide bond by diamide. It is concluded that the mechanism of phosphoenolpyruvate carboxylase activation by dithiothreitol involves the net reduction of two disulfide bonds in the enzyme.

     

Identification of a Disulfide Bridge Important for Transport Function of SNAT4 Neutral Amino Acid Transporter

SNAT4 is a member of system N/A amino acid transport family that primarily expresses in liver and muscles and mediates the transport of L-alanine. However, little is known about the structure and function of the SNAT family of transporters. In this study, we showed a dose-dependent inhibition in transporter activity of SNAT4 with the treatment of reducing agents, dithiothreitol (DTT) and Tris(2-carboxyethyl)phosphine (TCEP), indicating the possible involvement of disulfide bridge(s). Mutation of residue Cys-232, and the two highly conserved residues Cys-249 and Cys-321, compromised the transport function of SNAT4. However, this reduction was not caused by the decrease of SNAT4 on the cell surface since the cysteine-null mutant generated by replacing all five cysteines with alanine was equally capable of being expressed on the cell surface as wild-type SNAT4. Interestingly, by retaining two cysteine residues, 249 and 321, a significant level of L-alanine uptake was restored, indicating the possible formation of disulfide bond between these two conserved residues. Biotinylation crosslinking of free thiol groups with MTSEA-biotin provided direct evidence for the existence of a disulfide bridge between Cys-249 and Cys-321. Moreover, in the presence of DTT or TCEP, transport activity of the mutant retaining Cys-249 and Cys-321 was reduced in a dose-dependent manner and this reduction is gradually recovered with increased concentration of H₂O₂. Disruption of the disulfide bridge also decreased the transport of L-arginine, but to a lesser degree than that of L-alanine. Together, these results suggest that cysteine residues 249 and 321 form a disulfide bridge, which plays an important role in substrate transport but has no effect on trafficking of SNAT4 to the cell surface.     

Characterization of a membrane regulator of insulin receptor affinity

Using the technique of radiation inactivation we have previously shown that the insulin receptor behaves as if it is composed of at least two functional components: a binding component (Mr approximately equal to 100,000) and an affinity regulatory component (Mr approximately equal to 300,000). The interaction between the affinity regulator and binding component results in a decrease in the affinity of the receptor for insulin. To examine in more detail the interaction between this "affinity regulator" and the binding component we have studied the insulin receptor by radiation inactivation under conditions which alter receptor concentration or receptor affinity. Liver membranes of ob/ob mice exhibit a decrease in insulin binding when compared to their lean litter mates which is due to a decrease in receptor concentration. When studied by radiation inactivation, however, there was no detectable change in the interaction or size of the two receptor components. By contrast, under circumstances in which the affinity of the receptor was increased (treatment with high salt, high pH, 1 mM dithiothreitol, 1-5 micrograms/ml of trypsin), the interaction between the regulatory and binding components was either decreased or absent, i.e. there was no increase in binding with irradiation. Conversely, conditions which produce a decrease in receptor affinity resulted in an increase in the interaction between the regulatory and binding components. The changes in receptor affinity and interactions of the two components produced by either high salt or pH were reversible. Partial purification of the solubilized receptor on lectin affinity columns resulted in the apparent removal of the affinity regulator, i.e. receptor affinity was increased. In this state, radiation inactivation studies revealed a monoexponential decay indicating no interaction between binding and regulatory components. Taken together, these results suggest that the affinity regulator is a membrane protein which is both trypsin-sensitive and has disulfide bond(s) essential for its function. The interaction between the affinity regulator and binding component is not via a covalent bond and the two components appear to be separated by lectin chromatography. The interaction between these components appears to be altered in most states associated with altered receptor affinity.     

Sulfhydryl Modification of Two Nicotinic Binding Sites in Mouse Brain

Two distinct binding sites with properties corresponding to those expected for nicotinic cholinergic receptors can be identified in brain by the specific binding of nicotine (or acetylcholine) and alpha-bungarotoxin. The effects of modification of these binding sites by treatment with the disulfide-reducing agent dithiothreitol were examined in tissue prepared from DBA mouse brains. Treatment with dithiothreitol reduced the binding measured with either ligand, and reoxidization of the disulfides fully restored binding. The effects of dithiothreitol treatment appeared to be due to a reduction in the maximal binding of nicotine and to a decrease in the binding affinity for alpha-bungarotoxin. Agonist affinity for the alpha-bungarotoxin binding site was reduced by treatment with low concentrations of dithiothreitol. The nicotine binding sites remaining after disulfide treatment displayed rates of ligand association and dissociation similar to those of unmodified tissue, but treatment of previously unmodified tissue with dithiothreitol accelerated the rate of nicotine dissociation. After reduction, both binding sites could be selectively alkylated with bromoacetylcholine. The results suggest that both putative nicotinic receptors in brain respond similarly to disulfide reduction and that their responses resemble those known for the nicotinic receptor of electric tissue.     

A bacterial glutathione transporter (Escherichia coli CydDC) exports reductant to the periplasm

Glutathione (GSH), a major biological antioxidant, maintains redox balance in prokaryotes and eukaryotic cells and forms exportable conjugates with compounds of pharmacological and agronomic importance. However, no GSH transporter has been characterized in a prokaryote. We show here that a heterodimeric ATP-binding cassette-type transporter, CydDC, mediates GSH transport across the Escherichia coli cytoplasmic membrane. In everted membrane vesicles, GSH is imported via an ATP-driven, protonophore-insensitive, orthovanadate-sensitive mechanism, equating with export to the periplasm in intact cells. GSH transport and cytochrome bd quinol oxidase assembly are abolished in the cydD1 mutant. Glutathione disulfide (GSSG) was not transported in either Cyd(+) or Cyd(-) strains. Exogenous GSH restores defective swarming motility and benzylpenicillin sensitivity in a cydD mutant and also benzylpenicillin sensitivity in a gshA mutant defective in GSH synthesis. Overexpression of the cydDC operon in dsbD mutants defective in disulfide bond formation restores dithiothreitol tolerance and periplasmic cytochrome b assembly, revealing redundant pathways for reductant export to the periplasm. These results identify the first prokaryotic GSH transporter and indicate a key role for GSH in periplasmic redox homeostasis.     

Manipulation of Disulfide Bonds Differentially Affects the Intracellular Transport, Sorting, and Processing of Neuroendocrine Secretory Proteins

Abstract: To investigate if the prevention of disulfide bond formation affects the intracellular transport, sorting, and processing of a distinct set of neuroendocrine proteins in the regulated secretory pathway, we have treated Xenopus intermediate pituitaries with the thiol-reducing agent dithiothreitol. Pulse-chase incubations in combination with immunoprecipitation analysis were used to monitor the fates of the prohormone proopiomelanocortin (POMC), prohormone convertase PC2 and its helper protein 7B2, as well as secretogranin III. Manipulation of the disulfide bonds in POMC and proPC2 blocked their transport to the trans -Golgi network and strongly inhibited their processing. Reduction of the single disulfide bond in 7B2 did not disturb its transport and cleavage, but caused its missorting to the constitutive secretory pathway. Moreover, the liaison between proPC2 and 7B2 was prevented. Dithiothreitol did not affect transport, sorting, and cleavage of secretogranin III, which lacks disulfide bonds. When the reducing agent was washed away, POMC processing, proPC2 maturation, and the association between proPC2 and 7B2 were reestablished. Collectively, our findings indicate that manipulation of disulfide bonds differentially affects the fates of neuroendocrine proteins during their transit through the secretory pathway.