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Roberta F. Colman - One of the best experts on this subject based on the ideXlab platform.
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Monobromobimane occupies a distinct xenobiotic substrate site in glutathione S‐transferase π
Protein Science, 2009Co-Authors: Luis A. Ralat, Roberta F. ColmanAbstract:Monobromobimane (mBBr), functions as a substrate of porcine glutathione S-transferase π (GST π): The enzyme catalyzes the reaction of mBBr with glutathione. S-(Hydroxyethyl)bimane, a nonreactive analog of Monobromobimane, acts as a competitive inhibitor with respect to mBBr as substrate but does not affect the reaction of GST π with another substrate, 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of glutathione, Monobromobimane inactivates GST π at pH 7.0 and 25°C as assayed using mBBr as substrate, with a lesser effect on the enzyme’s use of CDNB as substrate. These results indicate that the sites occupied by CDNB and mBBr are not identical. Inactivation is proportional to the incorporation of 2 moles of bimane/mole of subunit. Modification of GST π with mBBr does not interfere with its binding of 8-anilino-1-naphthalene sulfonate, indicating that this hydrophobic site is not the target of Monobromobimane. S-Methylglutathione and S-(hydroxyethyl)bimane each yield partial protection against inactivation and decrease reagent incorporation, while glutathionyl-bimane protects completely against inactivation. Peptide analysis after trypsin digestion indicates that mBBr modifies Cys45 and Cys99 equally. Modification of Cys45 is reduced in the presence of S-methylglutathione, indicating that this residue is at or near the glutathione binding region. In contrast, modification of Cys99 is reduced in the presence of S-(hydroxyethyl)bimane, suggesting that this residue is at or near the mBBr xenobiotic substrate binding site. Modification of Cys99 can best be understood by reaction with Monobromobimane while it is bound to its xenobiotic substrate site in an alternate orientation. These results support the concept that glutathione S-transferase accomplishes its ability to react with a diversity of substrates in part by harboring distinct xenobiotic substrate sites.
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Monobromobimane occupies a distinct xenobiotic substrate site in glutathione s transferase π
Protein Science, 2009Co-Authors: Luis A. Ralat, Roberta F. ColmanAbstract:Monobromobimane (mBBr), functions as a substrate of porcine glutathione S-transferase π (GST π): The enzyme catalyzes the reaction of mBBr with glutathione. S-(Hydroxyethyl)bimane, a nonreactive analog of Monobromobimane, acts as a competitive inhibitor with respect to mBBr as substrate but does not affect the reaction of GST π with another substrate, 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of glutathione, Monobromobimane inactivates GST π at pH 7.0 and 25°C as assayed using mBBr as substrate, with a lesser effect on the enzyme’s use of CDNB as substrate. These results indicate that the sites occupied by CDNB and mBBr are not identical. Inactivation is proportional to the incorporation of 2 moles of bimane/mole of subunit. Modification of GST π with mBBr does not interfere with its binding of 8-anilino-1-naphthalene sulfonate, indicating that this hydrophobic site is not the target of Monobromobimane. S-Methylglutathione and S-(hydroxyethyl)bimane each yield partial protection against inactivation and decrease reagent incorporation, while glutathionyl-bimane protects completely against inactivation. Peptide analysis after trypsin digestion indicates that mBBr modifies Cys45 and Cys99 equally. Modification of Cys45 is reduced in the presence of S-methylglutathione, indicating that this residue is at or near the glutathione binding region. In contrast, modification of Cys99 is reduced in the presence of S-(hydroxyethyl)bimane, suggesting that this residue is at or near the mBBr xenobiotic substrate binding site. Modification of Cys99 can best be understood by reaction with Monobromobimane while it is bound to its xenobiotic substrate site in an alternate orientation. These results support the concept that glutathione S-transferase accomplishes its ability to react with a diversity of substrates in part by harboring distinct xenobiotic substrate sites.
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Resonance energy transfer between sites in rat liver glutathione S-transferase, 1-1, selectively modified at cysteine-17 and cysteine-111.
Biochemistry, 1997Co-Authors: Roberta F. ColmanAbstract:Monobromobimane (mBBr) can label both Cys111 and Cys17 of rat liver glutathione S-transferase, 1-1 (GST 1-1). However, selective modification of Cys111 was achieved by the maleimide-based sulfhydryl reagents N-ethylmaleimide (NEM) and fluorescein 5-maleimide (NFM). Incubation of GST 1-1 with 5 mM NEM for 30 min at pH 7.5 and 25 °C leads to the formation of modified enzyme with 92% residual activity toward 1-chloro-2,4-dinitrobenzene and completely blocks Cys111 from subsequent reaction with either NFM or mBBr. Reaction of GST 1-1 with 0.2 mM NFM under the same conditions affords a modified enzyme with only 14% residual activity even though NFM and NEM target the same Cys111. The results indicate that when the bulky fluorescein is covalently bound to Cys111, the ligand projects into both the xenobiotic binding site and the glutathione site. After NEM or NFM modification of GST 1-1, the enzyme was further modified by Monobromobimane at Cys17 with loss of activity. Together with the only tryptophan (Trp20), ...
Luis A. Ralat - One of the best experts on this subject based on the ideXlab platform.
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Monobromobimane occupies a distinct xenobiotic substrate site in glutathione S‐transferase π
Protein Science, 2009Co-Authors: Luis A. Ralat, Roberta F. ColmanAbstract:Monobromobimane (mBBr), functions as a substrate of porcine glutathione S-transferase π (GST π): The enzyme catalyzes the reaction of mBBr with glutathione. S-(Hydroxyethyl)bimane, a nonreactive analog of Monobromobimane, acts as a competitive inhibitor with respect to mBBr as substrate but does not affect the reaction of GST π with another substrate, 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of glutathione, Monobromobimane inactivates GST π at pH 7.0 and 25°C as assayed using mBBr as substrate, with a lesser effect on the enzyme’s use of CDNB as substrate. These results indicate that the sites occupied by CDNB and mBBr are not identical. Inactivation is proportional to the incorporation of 2 moles of bimane/mole of subunit. Modification of GST π with mBBr does not interfere with its binding of 8-anilino-1-naphthalene sulfonate, indicating that this hydrophobic site is not the target of Monobromobimane. S-Methylglutathione and S-(hydroxyethyl)bimane each yield partial protection against inactivation and decrease reagent incorporation, while glutathionyl-bimane protects completely against inactivation. Peptide analysis after trypsin digestion indicates that mBBr modifies Cys45 and Cys99 equally. Modification of Cys45 is reduced in the presence of S-methylglutathione, indicating that this residue is at or near the glutathione binding region. In contrast, modification of Cys99 is reduced in the presence of S-(hydroxyethyl)bimane, suggesting that this residue is at or near the mBBr xenobiotic substrate binding site. Modification of Cys99 can best be understood by reaction with Monobromobimane while it is bound to its xenobiotic substrate site in an alternate orientation. These results support the concept that glutathione S-transferase accomplishes its ability to react with a diversity of substrates in part by harboring distinct xenobiotic substrate sites.
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Monobromobimane occupies a distinct xenobiotic substrate site in glutathione s transferase π
Protein Science, 2009Co-Authors: Luis A. Ralat, Roberta F. ColmanAbstract:Monobromobimane (mBBr), functions as a substrate of porcine glutathione S-transferase π (GST π): The enzyme catalyzes the reaction of mBBr with glutathione. S-(Hydroxyethyl)bimane, a nonreactive analog of Monobromobimane, acts as a competitive inhibitor with respect to mBBr as substrate but does not affect the reaction of GST π with another substrate, 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of glutathione, Monobromobimane inactivates GST π at pH 7.0 and 25°C as assayed using mBBr as substrate, with a lesser effect on the enzyme’s use of CDNB as substrate. These results indicate that the sites occupied by CDNB and mBBr are not identical. Inactivation is proportional to the incorporation of 2 moles of bimane/mole of subunit. Modification of GST π with mBBr does not interfere with its binding of 8-anilino-1-naphthalene sulfonate, indicating that this hydrophobic site is not the target of Monobromobimane. S-Methylglutathione and S-(hydroxyethyl)bimane each yield partial protection against inactivation and decrease reagent incorporation, while glutathionyl-bimane protects completely against inactivation. Peptide analysis after trypsin digestion indicates that mBBr modifies Cys45 and Cys99 equally. Modification of Cys45 is reduced in the presence of S-methylglutathione, indicating that this residue is at or near the glutathione binding region. In contrast, modification of Cys99 is reduced in the presence of S-(hydroxyethyl)bimane, suggesting that this residue is at or near the mBBr xenobiotic substrate binding site. Modification of Cys99 can best be understood by reaction with Monobromobimane while it is bound to its xenobiotic substrate site in an alternate orientation. These results support the concept that glutathione S-transferase accomplishes its ability to react with a diversity of substrates in part by harboring distinct xenobiotic substrate sites.
Paolo Bernardi - One of the best experts on this subject based on the ideXlab platform.
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The mitochondrial permeability transition pore is modulated by oxidative agents through both pyridine nucleotides and glutathione at two separate sites.
FEBS Journal, 1996Co-Authors: Boris V. Chernyak, Paolo BernardiAbstract:We studied the modulation of the permeability transition pore (MTP), a cyclosporin-A-sensitive channel, in deenergized mitochondria. Rat liver mitochondria were incubated in a potassium gluconate medium and treated with uncoupler, valinomycin, oligomycin and A23187. Under these conditions the membrane and Donnan potentials are collapsed, and no ion gradients are maintained, allowing the study of the dependence of MTP opening on the Ca2+ concentration under a variety of oxidative conditions without the complexities arising from changes of the membrane potential and matrix pH, and from secondary-ion redistribution. Cross-linking of mitochondrial dithiols with arsenite or phenylarsine oxide, or treatment with tert -butylhydroperoxide leading to complete oxidation of glutathione, increased the sensitivity of MTP opening to Ca2+. This effect could be fully prevented by prior treatment of mitochondria with Monobromobimane and restored by reduction with dithiothreitol. The effect of tert-butylhydroperoxide was not additive with that of AsO, and it was completely blocked by modification of reduced glutathione with 1-chloro 2,4-dinitrobenzene through glutathione-S-transferase, indicating that oxidized glutathione affects the pore through the AsO-reactive and PhAsO-reactive dithiol. Oxidation of mitochondrial pyridine nucleotides by a variety of treatments also increased the sensitivity of MTP opening to Ca2+ under conditions where glutathione was maintained in the reduced state. This effect could be fully prevented when reduced pyridine nucleotides levels were reestablished with 2-hydroxybutyrate but not by treatment with Monobromobimane or dithiothreitol. The effects of dithiol cross-linking or oxidation, and of pyridine nucleotide oxidation on the MTP were additive. The contribution of each of these two oxidative events, when they were induced at the same time, could still be selectively blocked by Monobromobimane and dithiothreitol, or by 2-hydroxybutyrate, respectively. Complete oxidation of pyridine nucleotides did not affect the reactivity of the dithiol towards Monobromobimane, indicating that it remained in the reduced state. After transient opening of the MTP, sensitivity to pyridine nucleotide oxidation was lost while sensitivity to dithiol cross-linking was retained. These data indicate that the dithiol belongs to an MTP regulatory component which is larger than the MTP cutoff of about 1500 Da, or is non-diffusible. Taken together, these findings indicate that the MTP is influenced by oxidation-reduction events at two separate sites that can be distinguished experimentally, and that these sites are not connected by common oxidation-reduction intermediates other than glutathione.
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modulation of the mitochondrial permeability transition pore by pyridine nucleotides and dithiol oxidation at two separate sites
Journal of Biological Chemistry, 1996Co-Authors: Paola Costantini, Valeria Petronilli, B V Chernyak, Paolo BernardiAbstract:Abstract After accumulation of a Ca load, the addition of uncoupler to respiring rat liver mitochondria is followed by opening of the permeability transition pore (MTP), a voltage-dependent channel sensitive to cyclosporin A. The channel's voltage threshold is profoundly affected under conditions of oxidative stress, with a shift to more negative values that may cause MTP opening at physiological membrane potentials. In this paper we further clarify the mechanisms by which oxidative agents affect the apparent voltage dependence of the MTP. We show that two sites can be experimentally distinguished. (i) A first site is in apparent oxidation-reduction equilibrium with the pyridine nucleotide (PN) pool (NADH/NAD + NADPH/NADP); PN oxidation is matched by increased MTP open probability under conditions where the glutathione pool is kept in the fully reduced state; this site can be blocked by N-ethylmaleimide but not by Monobromobimane, a thiol-selective reagent. (ii) A second site coincides with the oxidation-reduction-sensitive dithiol we have recently identified (Petronilli, V., Costantini, P., Scorrano, L., Colonna, R., Passamonti, S., and Bernardi, P.(1994) J. Biol. Chem. 269, 16638-16642); dithiol cross-linking at this site by arsenite or phenylarsine oxide is matched by increased MTP open probability under conditions where the PN pool is kept in the fully reduced state; at variance from the first, this site can be blocked by both N-ethylmaleimide and Monobromobimane and is probably in equilibrium with the glutathione pool. Based on these findings, we reassess the mechanisms by which many oxidative agents affect the MTP and resolve conflicting reports on the relative role of PN and glutathione oxidation in the permeability transition within the framework of MTP (dys)regulation at two separate sites.
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Selective inhibition of the mitochondrial permeability transition pore at the oxidation-reduction sensitive dithiol by Monobromobimane
FEBS Letters, 1995Co-Authors: Paola Costantini, Boris V. Chernyak, Valeria Petronilli, Paolo BernardiAbstract:In this paper we introduce Monobromobimane, a thiol reagent, as a selective blocker of the recently identified dithiol whose oxidation-reduction status modifies voltage sensing by the mitochondrial permeability transition pore, a cyclosporin A-sensitive channel. Monobromobimane does not inhibit the phosphate carrier, nor does it interfere with Ca2+ transport, energy coupling or ATP production and transport. We show that Monobromobimane selectively prevents the shift in pore gating potential caused by some dithiol oxidants or crosslinkers but not by increasing [Ca2+], allowing a clear distinction of the pore agonists which act at this site.
David L. Williams - One of the best experts on this subject based on the ideXlab platform.
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Real-time RT-PCR analysis of glutathione metabolic enzymes in response to various stresses.
2013Co-Authors: Coraline Rigouin, Elyse Nylin, Alexis A. Cogswell, Dirk Schaumlöffel, Dirk Dobritzsch, David L. WilliamsAbstract:Adult worms were exposed to Cd: 100 µM CdCl2; Zn: 100 µM ZnCl2; EA: 10 µM Ethacrynic acid; mBrB: 30 µM Monobromobimane; PZQ: 1 µg/mL praziquantel; H2O2: 100 µM hydrogen peroxide for 6 h. Relative mRNA abundance of PCS (black bar); GST26 (light grey horizontal bar), GST28 (light grey hatched bar), γGT (light grey plain bar); γGCS (dark grey bar); TGR (white bar with black dots) were determined by real-time RT-PCR.
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Real-time RT-PCR analysis of PCS in response to various stresses.
2013Co-Authors: Coraline Rigouin, Elyse Nylin, Alexis A. Cogswell, Dirk Schaumlöffel, Dirk Dobritzsch, David L. WilliamsAbstract:A. Adult worms were exposed to different stressors (Fe: 100 µM FeCl3; Cd: 100 µM CdCl2; PZQ: 1 µg/mL praziquantel; mBrB: 30 µM Monobromobimane; EA: 10 µM ethacrynic acid; H2O2: 100 µM hydrogen peroxide) for 6 h. SmPCS mRNA abundance was determined by real-time RT-PCR. B. Real-time RT-PCR analysis of PCS in response to metal with time. Adult worms were exposed to 100 µM FeCl3 (dark grey) or 100 µM CdCl2 (light grey) during 2, 4 and 6 h. SmPCS mRNA abundance was determined by real-time RT-PCR.
Yves Meyer - One of the best experts on this subject based on the ideXlab platform.
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Cytosolic, Mitochondrial Thioredoxins and Thioredoxin Reductases in Arabidopsis Thaliana
Photosynthesis Research, 2004Co-Authors: Claire Bréhélin, Christophe Laloi, Aaron Setterdahl, David Knaff, Yves MeyerAbstract:Thioredoxins, by reducing disulfide bridges are one of the main participants that regulate cellular redox balance. In plants, the thioredoxin system is particularly complex. The most well-known thioredoxins are the chloroplastic ones, that participate in the regulation of enzymatic activities during the transition between light and dark phases. The mitochondrial system composed of NADPH-dependent thioredoxin reductase and type o thioredoxin has only recently been described. The type h thioredoxin group is better known. Yeast complementation experiments demonstrated that Arabidopsis thaliana thioredoxins h have divergent functions, at least in Saccharomyces cerevisiae. They have diverse affinities for different target proteins, most probably because of structural differences. However, plant thioredoxin h functions still have to be defined. Abbreviations: mBBr-Monobromobimane; NADPH-nicotinamide adenine dinucleotide; NTR-NADPH thioredoxin reductase