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D J Morre - One of the best experts on this subject based on the ideXlab platform.
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the sulfonylurea inhibited nadh oxidase activity of hela cell plasma membranes has properties of a protein disulfide Thiol oxidoreductase with protein disulfide Thiol interchange activity
Journal of Bioenergetics and Biomembranes, 1998Co-Authors: Pin Ju Chueh, Juliana Lawler, D J MorreAbstract:Plasma membrane vesicles of HeLa cells are characterized by a drug-responsive oxidation of NADH. The NADH oxidation takes place in an argon or nitrogen atmosphere and in samples purged of oxygen. Direct assay of protein Thiols by reaction with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB; Ellman's Reagent), suggests that protein disulfides may be the natural electron acceptors for NADH oxidation by the plasma membrane vesicles. In the presence of NADH, protein disulfides of the membranes were reduced with a concomitant stoichiometric increase in protein Thiols. The increase in protein Thiols was inhibited in parallel to the inhibition of NADH oxidation by the antitumor sulfonylurea LY181984 with an EC50 of ca. 30 nM. LY181984, with an EC50 of 30 nM, also inhibited a protein disulfide–Thiol interchange activity based on the restoration of activity to inactive (scrambled) RNase and Thiol oxidation. The findings suggest that Thiol oxidation, NADH-dependent disulfide reduction (NADH oxidation), and protein disulfide–Thiol interchange in the absence of NADH all may be manifestations of the same sulfonylurea binding protein of the HeLa plasma membrane. A surface location of the Thiols involved was demonstrated using detergents and the impermeant Thiol Reagent p-chloromercuriphenylsulfonic acid (PCMPS). The surface location precludes a physiological role of the protein in NADH oxidation. Rather, it may carry out some other role more closely related to a function in growth, such as protein disulfide–Thiol interchange coupled to cell enlargement.
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selective inhibition of auxin stimulated nadh oxidase activity and elongation growth of soybean hypocotyls by Thiol Reagents
Plant Physiology, 1995Co-Authors: D J Morre, Andrew O Brightman, A Hidalgo, Placido NavasAbstract:The NADH oxidase activity of isolated vesicles of soybean (Glycine max cv Williams 82) plasma membranes and elongation growth of 1-cm-long hypocotyl segments were stimulated by auxins (indole-3-acetic acid or 2,4-dichlorophenoxyacetic acid [2,4-D]). The auxin-induced stimulations of both NADH oxidase and growth were prevented by the Thiol Reagents N-ethylmaleimide, p-chloromercuribenzoate, 5,5[prime]-dithiobis(2-nitrophenylbenzoic acid), dithiothreitol, and reduced glutathione. These same Reagents largely were without effect on or stimulated slightly the basal levels of NADH oxidase and growth when assayed in the absence of auxins. In the presence of dithiothreitol or reduced glutathione, both 2,4-D and indole-3-acetic acid either failed to stimulate or inhibited the NADH oxidase activity. The rapidity of the response at a given concentration of Thiol Reagent and the degree of inhibition of the 2,4-D-induced NADH oxidase activity were dependent on order of Reagent addition. If the Thiol Reagents were added first, auxin stimulations were prevented. If auxins were added first, the inhibitions by the Thiol Reagents were delayed or higher concentrations of Thiol Reagents were required to achieve inhibition. The results demonstrate a fundamental difference between the auxin-stimulated and the constitutive NADH oxidase activities of soybean plasma membranes that suggest an involvement of active-site Thiols in the auxin-stimulated but not in the constitutive activity.
Roger F. Castilho - One of the best experts on this subject based on the ideXlab platform.
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Ca2+ Induces a Cyclosporin A-Insensitive Permeability Transition Pore in Isolated Potato Tuber Mitochondria Mediated by Reactive Oxygen Species
Journal of Bioenergetics and Biomembranes, 2001Co-Authors: Fabiane Fortes, Roger F. Castilho, Rosana Catisti, Eva G. S. Carnieri, Anibal E. VercesiAbstract:Oxidative damage of mammalian mitochondria induced by Ca^2+ and prooxidants is mediated by the attack of mitochondria-generated reactive oxygen species on membrane protein Thiols promoting oxidation and cross-linkage that leads to the opening of the mitochondrial permeability transition pore (Castilho et al., 1995). In this study, we present evidence that deenergized potato tuber (Solanum tuberosum) mitochondria, which do not possess a Ca^2+ uniport, undergo inner membrane permeabilization when treated with Ca^2+ (>0.2 mM), as indicated by mitochondrial swelling. Similar to rat liver mitochondria, this permeabilization is enhanced by diamide, a Thiol oxidant that creates a condition of oxidative stress by oxidizing pyridine nucleotides. This is inhibited by the antioxidants catalase and dithiothreitol. Potato mitochondrial membrane permeabilization is not inhibited by ADP, cyclosporin A, and ruthenium red, and is partially inhibited by Mg^2+ and acidic pH, well known inhibitors of the mammalian mitochondrial permeability transition. The lack of inhibition of potato mitochondrial permeabilization by cyclosporin A is in contrast to the inhibition of the peptidylprolyl cis–trans isomerase activity, that is related to the cyclosporin A-binding protein cyclophilin. Interestingly, the monofunctional Thiol Reagent mersalyl induces an extensive cyclosporin A-insensitive potato mitochondrial swelling, even in the presence of lower Ca^2+ concentrations (>0.01 mM). In conclusion, we have identified a cyclosporin A-insensitive permeability transition pore in isolated potato mitochondria that is induced by reactive oxygen species.
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Mitochondrial membrane protein Thiol reactivity with N-ethylmaleimide or mersalyl is modified by Ca2+: correlation with mitochondrial permeability transition
Biochimica et biophysica acta, 1997Co-Authors: Alicia J Kowaltowski, Anibal E. Vercesi, Roger F. CastilhoAbstract:Abstract The content of mitochondrial membrane protein Thiol groups accessible to react with the monofunctional Thiol Reagents mersalyl or N-ethylmaleimide (NEM) was determined using Ellman's Reagent. Deenergized mitochondria incubated in the presence of Ca2+ (0–500 μM) undergo a very significant decrease in the content of membrane protein Thiols accessible to NEM, and an increase in the content of Thiols accessible to mersalyl. This process is time-dependent and inhibited by Mg2+, ruthenium red and ADP, but not by cyclosporin A. This suggests that Ca2+ binding to the inner mitochondrial membrane promotes extensive alterations in the conformation of membrane proteins that result in location changes of Thiol groups. The relationship between these alterations and mitochondrial membrane permeability transition was studied through the effect of NEM and mersalyl on mitochondrial swelling induced by Ca2+ plus t-butyl hydroperoxide (t-bOOH) or Ca2+ plus the Thiol cross-linkers 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) or phenylarsine oxide (PhAsO). We observed that the hydrophobic Thiol Reagent NEM inhibits the effects of t-bOOH, DIDS and PhAsO, while the hydrophilic Thiol Reagent mersalyl inhibits only the effect of DIDS. Permeability transition in all the situations studied is accompanied by a significant decrease in the total membrane protein Thiol content. In addition, mitochondrial membrane permeabilization induced by PhAsO is inhibited by EGTA, but not by ruthenium red. This result suggests that PhAsO leads to permeability transition through a mechanism independent of intramitochondrial Ca2+-induced alterations of Thiol group reactivity, but dependent on Ca2+ binding to an extramitochondrial site. This site is sensitive to extramitochondrial Ca2+ concentrations in range of 1–50 μM.
Nilserik L Saris - One of the best experts on this subject based on the ideXlab platform.
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to involvement the conformation of the adenine nucleotide translocase in opening the tl induced permeability transition pore in ca2 loaded rat liver mitochondria
Toxicology in Vitro, 2016Co-Authors: S M Korotkov, Svetlana A Konovalova, Irina V Brailovskaya, Nilserik L SarisAbstract:The conformation of adenine nucleotide translocase (ANT) has a profound impact in opening the mitochondrial permeability transition pore (MPTP) in the inner membrane. Fixing the ANT in 'c' conformation by phenylarsine oxide (PAO), tert-butylhydroperoxide (tBHP), and carboxyatractyloside as well as the interaction of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) with mitochondrial Thiols markedly attenuated the ability of ADP to inhibit the MPTP opening. We earlier found (Korotkov and Saris, 2011) that calcium load of rat liver mitochondria in medium containing TlNO3 and KNO3 stimulated the Tl(+)-induced MPTP opening in the inner mitochondrial membrane. The MPTP opening as well as followed increase in swelling, a drop in membrane potential (ΔΨmito), and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration were visibly enhanced in the presence of PAO, tBHP, DIDS, and carboxyatractyloside. However, these effects were markedly inhibited by ADP and membrane-penetrant hydrophobic Thiol Reagent, N-ethylmaleimide (NEM) which fix the ANT in 'm' conformation. Cyclosporine A additionally potentiated these effects of ADP and NEM. Our data suggest that conformational changes of the ANT may be directly involved in the opening of the Tl(+)-induced MPTP in the inner membrane of Ca(2+)-loaded rat liver mitochondria. Using the Tl(+)-induced MPTP model is discussed in terms finding new transition pore inhibitors and inducers among different chemical and natural compounds.
Anibal E. Vercesi - One of the best experts on this subject based on the ideXlab platform.
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Ca2+ Induces a Cyclosporin A-Insensitive Permeability Transition Pore in Isolated Potato Tuber Mitochondria Mediated by Reactive Oxygen Species
Journal of Bioenergetics and Biomembranes, 2001Co-Authors: Fabiane Fortes, Roger F. Castilho, Rosana Catisti, Eva G. S. Carnieri, Anibal E. VercesiAbstract:Oxidative damage of mammalian mitochondria induced by Ca^2+ and prooxidants is mediated by the attack of mitochondria-generated reactive oxygen species on membrane protein Thiols promoting oxidation and cross-linkage that leads to the opening of the mitochondrial permeability transition pore (Castilho et al., 1995). In this study, we present evidence that deenergized potato tuber (Solanum tuberosum) mitochondria, which do not possess a Ca^2+ uniport, undergo inner membrane permeabilization when treated with Ca^2+ (>0.2 mM), as indicated by mitochondrial swelling. Similar to rat liver mitochondria, this permeabilization is enhanced by diamide, a Thiol oxidant that creates a condition of oxidative stress by oxidizing pyridine nucleotides. This is inhibited by the antioxidants catalase and dithiothreitol. Potato mitochondrial membrane permeabilization is not inhibited by ADP, cyclosporin A, and ruthenium red, and is partially inhibited by Mg^2+ and acidic pH, well known inhibitors of the mammalian mitochondrial permeability transition. The lack of inhibition of potato mitochondrial permeabilization by cyclosporin A is in contrast to the inhibition of the peptidylprolyl cis–trans isomerase activity, that is related to the cyclosporin A-binding protein cyclophilin. Interestingly, the monofunctional Thiol Reagent mersalyl induces an extensive cyclosporin A-insensitive potato mitochondrial swelling, even in the presence of lower Ca^2+ concentrations (>0.01 mM). In conclusion, we have identified a cyclosporin A-insensitive permeability transition pore in isolated potato mitochondria that is induced by reactive oxygen species.
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Mitochondrial membrane protein Thiol reactivity with N-ethylmaleimide or mersalyl is modified by Ca2+: correlation with mitochondrial permeability transition
Biochimica et biophysica acta, 1997Co-Authors: Alicia J Kowaltowski, Anibal E. Vercesi, Roger F. CastilhoAbstract:Abstract The content of mitochondrial membrane protein Thiol groups accessible to react with the monofunctional Thiol Reagents mersalyl or N-ethylmaleimide (NEM) was determined using Ellman's Reagent. Deenergized mitochondria incubated in the presence of Ca2+ (0–500 μM) undergo a very significant decrease in the content of membrane protein Thiols accessible to NEM, and an increase in the content of Thiols accessible to mersalyl. This process is time-dependent and inhibited by Mg2+, ruthenium red and ADP, but not by cyclosporin A. This suggests that Ca2+ binding to the inner mitochondrial membrane promotes extensive alterations in the conformation of membrane proteins that result in location changes of Thiol groups. The relationship between these alterations and mitochondrial membrane permeability transition was studied through the effect of NEM and mersalyl on mitochondrial swelling induced by Ca2+ plus t-butyl hydroperoxide (t-bOOH) or Ca2+ plus the Thiol cross-linkers 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) or phenylarsine oxide (PhAsO). We observed that the hydrophobic Thiol Reagent NEM inhibits the effects of t-bOOH, DIDS and PhAsO, while the hydrophilic Thiol Reagent mersalyl inhibits only the effect of DIDS. Permeability transition in all the situations studied is accompanied by a significant decrease in the total membrane protein Thiol content. In addition, mitochondrial membrane permeabilization induced by PhAsO is inhibited by EGTA, but not by ruthenium red. This result suggests that PhAsO leads to permeability transition through a mechanism independent of intramitochondrial Ca2+-induced alterations of Thiol group reactivity, but dependent on Ca2+ binding to an extramitochondrial site. This site is sensitive to extramitochondrial Ca2+ concentrations in range of 1–50 μM.
S M Korotkov - One of the best experts on this subject based on the ideXlab platform.
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data supporting the involvement of the adenine nucleotide translocase conformation in opening the tl induced permeability transition pore in ca 2 loaded rat liver mitochondria
Data in Brief, 2016Co-Authors: S M KorotkovAbstract:There we made available information about the effects of the adenine nucleotide translocase (ANT) ‘c’ conformation fixers (phenylarsine oxide (PAO), tert-butylhydroperoxide (tBHP), and carboxyatractyloside) as well as Thiol Reagent (4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS)) on isolated rat liver mitochondria. We observed a decrease in A540 (mitochondrial swelling) and respiratory control rates (RCRADP [state 3/state 4] and RCRDNP [2,4-dinitrophenol-uncoupled state/basal state or state 4]), as well as an increase in Ca2+-induced safranin fluorescence (F485/590, arbitrary units), showed a dissipation in the inner membrane potential (ΔΨmito), in experiments with energized rat liver mitochondria, injected into the buffer containing 25–75 mM TlNO3, 125 mM KNO3, and 100 µM Ca2+. The fixers and DIDS, in comparison to Ca2+ alone, greatly increased A540 decline and the rate of Ca2+-induced ΔΨmito dissipation. These Reagents also markedly decreased RCRADP and RCRDNP. The MPTP inhibitors (ADP, cyclosporin A, bongkrekic acid, and N-ethylmaleimide) fixing the ANT in ‘m’ conformation significantly hindered the above-mentioned effects of the fixers and DIDS. A more complete scientific analysis of these findings may be obtained from the manuscript “To involvement the conformation of the adenine nucleotide translocase in opening the Tl+-induced permeability transition pore in Ca2+-loaded rat liver mitochondria” (Korotkov et al., 2016 [1]).
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to involvement the conformation of the adenine nucleotide translocase in opening the tl induced permeability transition pore in ca2 loaded rat liver mitochondria
Toxicology in Vitro, 2016Co-Authors: S M Korotkov, Svetlana A Konovalova, Irina V Brailovskaya, Nilserik L SarisAbstract:The conformation of adenine nucleotide translocase (ANT) has a profound impact in opening the mitochondrial permeability transition pore (MPTP) in the inner membrane. Fixing the ANT in 'c' conformation by phenylarsine oxide (PAO), tert-butylhydroperoxide (tBHP), and carboxyatractyloside as well as the interaction of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) with mitochondrial Thiols markedly attenuated the ability of ADP to inhibit the MPTP opening. We earlier found (Korotkov and Saris, 2011) that calcium load of rat liver mitochondria in medium containing TlNO3 and KNO3 stimulated the Tl(+)-induced MPTP opening in the inner mitochondrial membrane. The MPTP opening as well as followed increase in swelling, a drop in membrane potential (ΔΨmito), and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration were visibly enhanced in the presence of PAO, tBHP, DIDS, and carboxyatractyloside. However, these effects were markedly inhibited by ADP and membrane-penetrant hydrophobic Thiol Reagent, N-ethylmaleimide (NEM) which fix the ANT in 'm' conformation. Cyclosporine A additionally potentiated these effects of ADP and NEM. Our data suggest that conformational changes of the ANT may be directly involved in the opening of the Tl(+)-induced MPTP in the inner membrane of Ca(2+)-loaded rat liver mitochondria. Using the Tl(+)-induced MPTP model is discussed in terms finding new transition pore inhibitors and inducers among different chemical and natural compounds.