ABSTRACT
It was earlier shown that the calcium load of rat liver mitochondria in medium containing TlNO3 and KNO3 resulted in the Tl+-induced mitochondrial permeability transition pore (MPTP) opening in the inner membrane. This opening was accompanied by an increase in swelling and membrane potential dissipation and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration. This respiratory decrease was markedly leveled by mersalyl (MSL), the phosphate symporter (PiC) inhibitor which poorly stimulated the calcium-induced swelling, but further increased the potential dissipation. All of these effects of Ca2+ and MSL were visibly reduced in the presence of the MPTP inhibitors (ADP, N-ethylmaleimide, and cyclosporine A). High MSL concentrations attenuated the ability of ADP to inhibit the MPTP. Our data suggest that the PiC can participate in the Tl+-induced MPTP opening in the inner membrane of Ca2+-loaded rat liver mitochondria.
Subject(s)
Mersalyl/pharmacology , Mitochondria, Liver/drug effects , Mitochondria, Liver/metabolism , Mitochondrial Membrane Transport Proteins/drug effects , Mitochondrial Membrane Transport Proteins/metabolism , Thallium/pharmacology , Animals , Calcium/metabolism , In Vitro Techniques , Ion Transport/drug effects , Membrane Potential, Mitochondrial/drug effects , Mitochondrial Membranes/drug effects , Mitochondrial Membranes/metabolism , Mitochondrial Permeability Transition Pore , Mitochondrial Swelling/drug effects , Oxygen Consumption/drug effects , Rats , Rats, WistarABSTRACT
To find out whether and how the adenine nucleotide translocator-1 (ANT-1) inhibition due to NH2htau and Aß1-42 is due to an interplay between these two Alzheimer's peptides, ROS and ANT-1 thiols, use was made of mersalyl, a reversible alkylating agent of thiol groups that are oriented toward the external hydrophilic phase, to selectively block and protect, in a reversible manner, the -SH groups of ANT-1. The rate of ATP appearance outside mitochondria was measured as the increase in NADPH absorbance which occurs, following external addition of ADP, when ATP is produced by oxidative phosphorylation and exported from mitochondria in the presence of glucose, hexokinase and glucose-6-phosphate dehydrogenase. We found that the mitochondrial superoxide anions, whose production is induced at the level of Complex I by externally added Aß1-42 and whose release from mitochondria is significantly reduced by the addition of the VDAC inhibitor DIDS, modify the thiol group/s present at the active site of mitochondrial ANT-1, impair ANT-1 in a mersalyl-prevented manner and abrogate the toxic effect of NH2htau on ANT-1 when Aß1-42 is already present. A molecular mechanism is proposed in which the pathological Aß-NH2htau interplay on ANT-1 in Alzheimer's neurons involves the thiol redox state of ANT-1 and the Aß1-42-induced ROS increase. This result represents an important innovation because it suggests the possibility of using various strategies to protect cells at the mitochondrial level, by stabilizing or restoring mitochondrial function or by interfering with the energy metabolism providing a promising tool for treating or preventing AD.
Subject(s)
Adenine Nucleotide Translocator 1/metabolism , Alzheimer Disease/metabolism , Amyloid beta-Peptides/pharmacology , Cerebellum/metabolism , Cytoplasmic Granules/metabolism , Mitochondria/metabolism , Peptide Fragments/pharmacology , tau Proteins/metabolism , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Animals , Cells, Cultured , Cerebellum/cytology , Energy Metabolism , Enzyme Inhibitors/pharmacology , Mersalyl/pharmacology , Models, Neurological , Oxygen Consumption , Polarography , Rats , Rats, Wistar , Reactive Oxygen Species/metabolism , Superoxides/metabolismABSTRACT
The effect of HgCl(2), methylmercury and mersalyl on the glutamine/amino acid (ASCT2) transporter reconstituted in liposomes has been studied. Mercuric compounds externally added to the proteoliposomes, inhibited the glutamine/glutamine antiport catalyzed by the reconstituted transporter. Similar effects were observed by pre-treating the proteoliposomes with the mercurials and then removing unreacted compounds before the transport assay. The inhibition was reversed by DTE, cysteine and N-acetyl-cysteine but not by S-carboxymethyl-cysteine. The data demonstrated that the inhibition was due to covalent reaction of mercuric compounds with Cys residue(s) of the transporter. The IC(50) of the transporter for HgCl(2), methylmercury and mersalyl, were 1.4+/-0.10, 2.4+/-0.16 or 3.1+/-0.19 microM, respectively. Kinetic studies of the inhibition showed that the reagents behaved as non-competitive inhibitor. The presence of glutamine or Na(+) during the incubation of the mercuric compounds with the proteoliposomes did not exerted any protective effect on the inhibition. None of the compounds was transported by the reconstituted transporter. A metal binding motif CXXC has been predicted as possible site of interaction of the mercuric compounds with the transporter on the basis of the homology structural model of ASCT2 obtained using the glutamate transporter homologue from Pyrococcus horikoshii as template.
Subject(s)
Amino Acid Transport System ASC/antagonists & inhibitors , Liposomes/chemistry , Mercuric Chloride/pharmacology , Mercury/pharmacology , Mersalyl/pharmacology , Methylmercury Compounds/pharmacology , Amino Acid Motifs , Amino Acid Transport System ASC/metabolism , Binding Sites , Biological Transport , Glutamine , Minor Histocompatibility Antigens , Models, Molecular , Protein Binding , Protein Conformation , SodiumABSTRACT
The mitochondrial permeability transition (PT) involves the opening of a mitochondrial unselective channel (MUC) resulting in membrane depolarization and increased permeability to ions. PT has been observed in many, but not all eukaryotic species. In some species, PT has been linked to cell death, although other functions, such as matrix ion detoxification or regulation of the rate of oxygen consumption have been considered. The identification of the proteins constituting MUC would help understand the biochemistry and physiology of this channel. It has been suggested that the mitochondrial phosphate carrier is a structural component of MUC and we decided to test this in yeast mitochondria. Mersalyl inhibits the phosphate carrier and it has been reported that it also triggers PT. Mersalyl induced opening of the decavanadate-sensitive Yeast Mitochondrial Unselective Channel (YMUC). In isolated yeast mitochondria from a phosphate carrier-null strain the sensitivity to both phosphate and mersalyl was lost, although the permeability transition was still evoked by ATP in a decavanadate-sensitive fashion. Polyethylene glycol (PEG)-induced mitochondrial contraction results indicated that in mitochondria lacking the phosphate carrier the YMUC is smaller: complete contraction for mitochondria from the wild type and the mutant strains was achieved with 1.45 and 1.1 kDa PEGs, respectively. Also, as expected for a smaller channel titration with 1.1 kDa PEG evidenced a higher sensitivity in mitochondria from the mutant strain. The above data suggest that the phosphate carrier is the phosphate sensor in YMUC and contributes to the structure of this channel.
Subject(s)
Phosphate Transport Proteins/metabolism , Potassium Channels/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Animals , Mersalyl/pharmacology , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondrial Swelling/drug effects , Permeability/drug effects , Phosphate Transport Proteins/antagonists & inhibitors , Phosphates/metabolism , Polyethylene Glycols/pharmacology , Potassium Channels/chemistry , Potassium Channels/deficiency , Potassium Channels/genetics , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Sequence Deletion , Vanadates/pharmacology , Voltage-Dependent Anion Channels/metabolismABSTRACT
To date, the existence of the plant inner membrane anion channel (PIMAC) has been shown only in potato mitochondria, but its physiological role remains unclear. In this study, by means of swelling experiments in K(+) and ammonium salts, we characterize a PIMAC-like anion-conducting pathway in mitochondria from durum wheat (DWM), a monocotyledonous species phylogenetically far from potato. DWM were investigated since they possess a very active potassium channel (PmitoK(ATP)), so implying a very active matching anion uniport pathway and, possibly, a coordinated function. As in potato mitochondria, the electrophoretic uptake of chloride and succinate was inhibited by matrix [H(+)], propranolol, and tributyltin, and was insensitive to Mg(2+), N,N'-dicyclohexylcarbodiimide (DCCD) and mercurials, thus showing PIMAC's existence in DWM. PIMAC actively transports dicarboxylates, oxodicarboxylates, tricarboxylates and Pi. Interestingly, a novel mechanism of swelling in ammonium salts of isolated plant mitochondria is reported, based on electrophoretic anion uptake via PIMAC and ammonium uniport via PmitoK(ATP). PIMAC is inhibited by physiological compounds, such as ATP and free fatty acids, by high electrical membrane potential (Delta Psi), but not by acyl-CoAs or reactive oxygen species. PIMAC was found to cooperate with dicarboxylate carrier by allowing succinate uptake that triggers succinate/malate exchange in isolated DWM. Similar results were obtained using mitochondria from the dicotyledonous species topinambur, so suggesting generalization of results. We propose that PIMAC is normally inactive in vivo due to ATP and Delta Psi inhibition, but activation may occur in mitochondria de-energized by PmitoK(ATP) (or other dissipative systems) to replace or integrate the operation of classical anion carriers.
Subject(s)
Helianthus/metabolism , Ion Channels/metabolism , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Triticum/metabolism , Adenosine Triphosphate/pharmacology , Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone/pharmacology , Fatty Acids/pharmacology , Helianthus/drug effects , Hydrogen Peroxide/pharmacology , Hydrogen-Ion Concentration/drug effects , Linoleic Acid/pharmacology , Malates/metabolism , Membrane Potential, Mitochondrial/drug effects , Mersalyl/pharmacology , Mitochondria/drug effects , Mitochondrial Membranes/drug effects , Mitochondrial Swelling/drug effects , NAD/pharmacology , Osmosis/drug effects , Propranolol/pharmacology , Solutions , Succinic Acid/metabolism , Superoxides/pharmacology , Triticum/drug effectsABSTRACT
The purpose of this work was addressed to provide new information on the effect of thiol reagents on mitochondrial non-specific pore opening, and its response to cyclosporin A (CSA). To meet this proposal phenylarsine oxide (PHA) and mersalyl were employed as tools to induce permeability transition and CSA to inhibit it. PHA-induced mitochondrial dysfunction, characterized by Ca2+ efflux, swelling, and membrane de-energization, was inhibited by N-ethylmaleimide and CSA. Conversely, mersalyl failed to inhibit the inducing effect of phenylarsine oxide, it rather strengthened it. In addition, the effect of mersalyl was associated with cross-linking of membrane proteins. The content of membrane thiol groups accessible to react with PHA, mersalyl, and PHA plus mersalyl was determined. In all situations, permeability transition was accompanied by a significant decrease in the whole free membrane thiol content. Interestingly, it is also shown that mersalyl hinders the protective effect of cyclosporin A on PHA-induced matrix Ca2+ efflux.
Subject(s)
Arsenicals/pharmacology , Cyclosporine/pharmacology , Ion Channel Gating/drug effects , Mersalyl/pharmacology , Mitochondrial Membranes/metabolism , Animals , Calcium/metabolism , Cross-Linking Reagents , Ethylmaleimide/pharmacology , Membrane Potential, Mitochondrial/drug effects , Mitochondria, Liver/metabolism , Mitochondrial ADP, ATP Translocases/metabolism , Mitochondrial Membranes/drug effects , Mitochondrial Swelling/drug effects , Permeability/drug effects , Porins/metabolism , Protein Binding/drug effects , Rats , Sulfhydryl Compounds/chemistry , Sulfhydryl Reagents/chemistry , Sulfhydryl Reagents/pharmacologyABSTRACT
In this work we examined the effect of low concentrations of Cu(2+) on the opening of the mitochondrial non-specific pore. The purpose was addressed to further contribute to the knowledge of the mechanisms that regulate the open/closed cycles of the permeability transition pore. Membrane leakage was established by measuring matrix Ca(2+) efflux and mitochondrial swelling. The experimental results indicate that Cu(2+) at very low concentrations promoted the release of accumulated Ca(2+), as well as mitochondrial swelling, provided 1,10-phenanthroline has been added. Carboxyatractyloside and Cu(2+) exhibited additive effects on these parameters. After Cu(2+) titration of membrane thiols, it might be assumed that the blockage of 5.9nmol of SH/mg protein suffices to open the non-specific pore. Taking into account the reinforcing effect of carboxyatractyloside, the increasing ADP concentrations, and that N-ethylmaleimide inhibited the Cu(2+)-induced Ca(2+) efflux, it is proposed that the target site for Cu(2+) is located in the ADP/ATP carrier.
Subject(s)
Copper/pharmacology , Mitochondrial ADP, ATP Translocases/metabolism , Adenosine Diphosphate/pharmacology , Animals , Atractyloside/analogs & derivatives , Atractyloside/pharmacology , Calcium Signaling/drug effects , Ethylmaleimide/pharmacology , Glutathione/metabolism , Mersalyl/pharmacology , Mitochondrial Membranes/drug effects , Mitochondrial Proteins/isolation & purification , Mitochondrial Swelling/drug effects , Permeability/drug effects , Rats , Sulfhydryl Compounds/metabolism , TitrimetryABSTRACT
H(2)O(2) inactivation of particular GST isoforms has been reported, with no information regarding the overall effect of other ROS on cytosolic GST activity. The present work describes the inactivation of total cytosolic GST activity from liver rats by the oxygen radical-generating system Cu(2+)/ascorbate. We have previously shown that this system may change some enzymatic activities of thiol proteins through two mechanisms: ROS-induced oxidation and non-specific Cu(2+) binding to protein thiol groups. In the present study, we show that nanomolar Cu(2+) in the absence of ascorbate did not modify total cytosolic GST activity; the same concentrations of Cu(2+) in the presence of ascorbate, however, inhibited this activity. Micromolar Cu(2+) in either the absence or presence of ascorbate inhibited cytosolic GST activity. Kinetic studies show that GSH but no 1-chloro-2,4-dinitrobenzene prevent the inhibition on cytosolic GST induced by micromolar Cu(2+) either in the absence or presence of ascorbate. On the other hand, NEM and mersalyl acid, both thiol-alkylating agents, inhibited GST activity with differential reactivity in a dose-dependent manner. Taken together, these results suggest that an inhibitory Cu(2+)-binding effect is likely to be negligible on the overall inhibition of cytosolic GST activity observed by the Cu(2+)/ascorbate system. We discuss how modification of GST-thiol groups is related to the inhibition of cytosolic GST activity.
Subject(s)
Ascorbic Acid/pharmacology , Copper/pharmacology , Cytosol/metabolism , Enzyme Inhibitors/pharmacology , Glutathione Transferase/antagonists & inhibitors , Liver/metabolism , Alkylating Agents/pharmacology , Animals , Binding Sites , Dinitrochlorobenzene/pharmacology , Dose-Response Relationship, Drug , Kinetics , Liver/ultrastructure , Male , Mersalyl/pharmacology , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Sulfhydryl Compounds/chemistry , Sulfhydryl Compounds/metabolismABSTRACT
H(+)-pyrophosphatase (H(+)-PPase) catalyzes pyrophosphate-driven proton transport against the electrochemical potential gradient in various biological membranes. All 50 of the known H(+)-PPase amino acid sequences contain four invariant glutamate residues. In this study, we use site-directed mutagenesis in conjunction with functional studies to determine the roles of the glutamate residues Glu(197), Glu(202), Glu(550), and Glu(649) in the H(+)-PPase of Rhodospirillum rubrum (R-PPase). All residues were replaced with Asp and Ala. The resulting eight variant R-PPases were expressed in Escherichia coli and isolated as inner membrane vesicles. All substitutions, except E202A, generated enzymes capable of PP(i) hydrolysis and PP(i)-energized proton translocation, indicating that the negative charge of Glu(202) is essential for R-PPase function. The hydrolytic activities of all other PPase variants were impaired at low Mg(2+) concentrations but were only slightly affected at high Mg(2+) concentrations, signifying that catalysis proceeds through a three-metal pathway in contrast to wild-type R-PPase, which employs both two- and three-metal pathways. Substitution of Glu(197), Glu(202), and Glu(649) resulted in decreased binding affinity for the substrate analogues aminomethylenediphosphonate and methylenediphosphonate, indicating that these residues are involved in substrate binding as ligands for bridging metal ions. Following the substitutions of Glu(550) and Glu(649), R-PPase was more susceptible to inactivation by the sulfhydryl reagent mersalyl, highlighting a role of these residues in maintaining enzyme tertiary structure. None of the substitutions affected the coupling of PP(i) hydrolysis to proton transport.
Subject(s)
Glutamates/genetics , Glutamates/metabolism , Inorganic Pyrophosphatase/genetics , Inorganic Pyrophosphatase/metabolism , Rhodospirillum rubrum/enzymology , Amino Acid Substitution , Biological Transport , Conserved Sequence , Cytoplasmic Vesicles/metabolism , Diphosphates/metabolism , Diphosphonates/pharmacology , Enzyme Inhibitors/pharmacology , Escherichia coli/cytology , Escherichia coli/metabolism , Hydrolysis , Inorganic Pyrophosphatase/antagonists & inhibitors , Inorganic Pyrophosphatase/chemistry , Kinetics , Magnesium/chemistry , Magnesium/metabolism , Mersalyl/pharmacology , Mutagenesis, Site-Directed , Protons , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Rhodospirillum rubrum/geneticsABSTRACT
The respiration rate of liver mitochondria in the course of succinate oxidation depends on temperature in the presence of palmitate more strongly than in its absence (in state 4). In the Arrhenius plot, the temperature dependence of the palmitate-induced stimulation of respiration has a bend at 22 degrees C which is characterized by transition of the activation energy from 120 to 60 kJ/mol. However, a similar dependence of respiration in state 4 is linear over the whole temperature range and corresponds to the activation energy of 17 kJ/mol. Phosphate partially inhibits the uncoupling effect of palmitate. This effect of phosphate is increased on decrease in temperature. In the presence of phosphate the temperature dependence in the Arrhenius plot also has a bend at 22 degrees C, and the activation energy increases from 128 to 208 kJ/mol in the range from 13 to 22 degrees C and from 56 to 67 kJ/mol in the range from 22 to 37 degrees C. Mersalyl (10 nmol/mg protein), an inhibitor of the phosphate carrier, similarly to phosphate, suppresses the uncoupling effect of laurate, and the effects of mersalyl and phosphate are not additive. The recoupling effects of phosphate and mersalyl seem to show involvement of the phosphate carrier in the uncoupling effect of fatty acids in liver mitochondria. Possible mechanisms of involvement of the phosphate carrier in the uncoupling effect of fatty acids are discussed.
Subject(s)
Atractyloside/analogs & derivatives , Cell Respiration/drug effects , Cell Respiration/physiology , Mitochondria, Liver/drug effects , Mitochondria, Liver/physiology , Palmitates/pharmacology , Phosphates/pharmacology , Uncoupling Agents/pharmacology , 2,4-Dinitrophenol/pharmacology , Animals , Aspartic Acid/pharmacology , Atractyloside/pharmacology , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Glutamic Acid/pharmacology , Laurates/pharmacology , Mersalyl/pharmacology , Mitochondria, Liver/metabolism , Oxidative Phosphorylation/drug effects , Palmitates/metabolism , Phosphates/metabolism , Rats , Temperature , Thermodynamics , Uncoupling Agents/metabolismABSTRACT
UNLABELLED: Plasma membrane (PM) steroid recognition sites are thought to be responsible only for rapid, non-genomic responses without any link to the nuclear receptor-mediated genomic effects of steroids. We focused on a PM "glucocorticoid-importer" (GC-importer) that imports GC into rat liver cells. This site interacts also with particular gestagens (progesterone, P; medroxyprogesterone, MP; ethynodiol, Ethy) and estrogens (ethinylestradiol, EE(2); mestranol), which do not bind to the nuclear GC receptor (GR). To elucidate the role of the GC-importer, we transfected a rat wild-type hepatocyte (CC-1) and a hepatoma cell line, unable to import GC (MH 3924), with a GC<-->GR-responsive luciferase (luc)-reporter gene. Selected steroids were tested for their ability to induce or inhibit luc expression. Corticosterone (B) and dexamethasone (Dex), but also the GC-antagonists cortexolone (Cortex), P and MP, induced luc. Even the PM-impermeable BSA-derivatives of B, Dex and Cortex did so to almost the same extent as the free steroids. MH 3924 cells respond stronger than CC-1 to luc inducing steroids. Luc expression was inhibited by RU 38 486, but also by EE(2) and Ethy. The thiol reactive mesylate-derivatives of B, Dex and Cortex induced to a considerably lesser extent than the free or BSA-steroids. The thiol reagent mersalyl blocks cellular entry of GC and inhibits luc induction in CC-1 cells. Incubation with EE(2) and B of PM-vesicles, isolated from liver cells, resulted in a decrease of the density of two 75 and 52kDa G-proteins reflecting a diminished exchange of GDP by GTP. CONCLUSION: the PM-residing GC-importer, now renamed "Steroid Hormone Recognition and Effector Complex" (SHREC) is an interdependent part of the complete GC signal propagation in which G-proteins are involved. Free SH-groups of SHREC are a prerequisite for genomic GC activity. Specific interactions between SHREC and GC-agonist/-antagonist trigger steroid-dependent signaling. However, import of the ligand into the cell terminates it. Thus, the PM-related non-genomic steroid responses are clearly linked to the GR-related genomic effects.
Subject(s)
Cell Membrane/physiology , Ethynodiol Diacetate/analogs & derivatives , Glucocorticoids/physiology , Receptors, Glucocorticoid/physiology , Animals , Cell Membrane/metabolism , Corticosterone/antagonists & inhibitors , Corticosterone/metabolism , Cortodoxone/metabolism , Dexamethasone/antagonists & inhibitors , Dexamethasone/metabolism , Enzyme Inhibitors/pharmacology , Estrone/metabolism , Ethinyl Estradiol/metabolism , Ethynodiol Diacetate/metabolism , GTP-Binding Proteins/metabolism , Glucocorticoids/antagonists & inhibitors , Glucocorticoids/metabolism , Hepatocytes , Luciferases/genetics , Luciferases/metabolism , Medroxyprogesterone/metabolism , Mersalyl/pharmacology , Progesterone/metabolism , Rats , Receptors, Glucocorticoid/metabolism , Serum Albumin, Bovine/metabolism , Signal Transduction/drug effects , Signal Transduction/physiology , Transfection , Tumor Cells, CulturedABSTRACT
H(+)-translocating pyrophosphatase (H(+)-PPase) of the photosynthetic bacterium Rhodospirillum rubrum was expressed in Escherichia coli C43(DE3) cells. Recombinant H(+)-PPase was observed in inner membrane vesicles, where it catalyzed both PP(i) hydrolysis coupled with H(+) transport into the vesicles and PP(i) synthesis. The hydrolytic activity of H(+)-PPase in E. coli vesicles was eight times greater than that in R. rubrum chromatophores but exhibited similar sensitivity to the H(+)-PPase inhibitor, aminomethylenediphosphonate, and insensitivity to the soluble PPase inhibitor, fluoride. Using this expression system, we showed that substitution of Cys(185), Cys(222), or Cys(573) with aliphatic residues had no effect on the activity of H(+)-PPase but decreased its sensitivity to the sulfhydryl modifying reagent, mersalyl. H(+)-PPase lacking all three Cys residues was completely resistant to the effects of mersalyl. Mg(2+) and MgPP(i) protected Cys(185) and Cys(573) from modification by this agent but not Cys(222). Phylogenetic analyses of 23 nonredundant H(+)-PPase sequences led to classification into two subfamilies. One subfamily invariably contains Cys(222) and includes all known K(+)-independent H(+)-PPases, whereas the other incorporates a conserved Cys(573) but lacks Cys(222) and includes all known K(+)-dependent H(+)-PPases. These data suggest a specific link between the incidence of Cys at positions 222 and 573 and the K(+) dependence of H(+)-PPase.
Subject(s)
Cysteine/chemistry , Enzyme Inhibitors/pharmacology , Escherichia coli/enzymology , Mersalyl/pharmacology , Pyrophosphatases/biosynthesis , Pyrophosphatases/chemistry , Rhodospirillum rubrum/enzymology , Cell Membrane/metabolism , Electrophoresis, Polyacrylamide Gel , Escherichia coli/metabolism , Hydrogen/metabolism , Hydrolysis , Inorganic Pyrophosphatase , Phylogeny , Plasmids/metabolism , Potassium/metabolism , Protein Transport , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Time FactorsABSTRACT
In order to gain a first insight into the effects of reactive oxygen species (ROS) on plant mitochondria, we studied the effect of the ROS producing system consisting of xanthine plus xanthine oxidase on the rate of membrane potential (DeltaPsi) generation due to either succinate or NADH addition to durum wheat mitochondria as monitored by safranin fluorescence. We show that the early ROS production inhibits the succinate-dependent, but not the NADH-dependent, DeltaPsi generation and oxygen uptake. This inhibition appears to depend on the impairment of mitochondrial permeability to succinate. It does not involve mitochondrial thiol groups sensitive to either mersalyl or N-ethylmaleimide and might involve both protein residues and/or membrane lipids, as suggested by the mixed nature. We propose that, during oxidative stress, early generation of ROS can affect plant mitochondria by impairing metabolite transport, thus preventing further substrate oxidation, DeltaPsi generation and consequent large-scale ROS production.
Subject(s)
Mitochondria/metabolism , Reactive Oxygen Species/metabolism , Succinic Acid/metabolism , Triticum/metabolism , Kinetics , Membrane Potentials/drug effects , Mersalyl/pharmacology , Mitochondria/drug effects , Models, Biological , NAD/metabolism , Oxidative Stress , Succinates/pharmacology , Triticum/drug effects , Xanthine/pharmacology , Xanthine Oxidase/pharmacologyABSTRACT
We have examined whether the effects of singlet oxygen (1O2) produced by photodynamic action on the mitochondrial permeability transition (PT) can be modulated by the localization of photosensitizers in irradiated mitochondria. We have previously shown that oxidation due to 1O2 photogenerated in hematoporphyrin (HP)-loaded mitochondria can prevent opening of the PT pores, likely after degradation of some critical histidines (Salet et al, 1997, J. Biol. Chem. 272, 21938-21943). Equally, in the present study we have irradiated mitochondria in the presence of a structurally different photosensitizer producing 1O2, namely 4,5',8-trimethylpsoralen (TMP). Fluorescence studies show that TMP binds to protein sites which differ from those of HP. In sharp contrast with HP, TMP-driven photodynamic action triggers per se pore opening. Interestingly, this inducing effect is inhibited when TMP-treated mitochondria are irradiated after addition of mersalyl, a specific reagent protecting thiol groups of the inner mitochondrial membrane that are oriented toward the external hydrophilic phase. This fact suggests that 1O2-mediated thiol oxidation is responsible for TMP-photoinduced pore opening. Taken together, these findings suggest that 1O2 can activate or inactivate a cellular function such as mitochondrial PT depending on the site where it is produced in the mitochondrial membrane.
Subject(s)
Ion Channels , Membrane Proteins/metabolism , Mitochondria, Liver/metabolism , Oxygen/metabolism , Photosensitizing Agents/metabolism , 1,2-Dipalmitoylphosphatidylcholine/metabolism , Animals , Calcium/metabolism , Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone/pharmacology , Cell Respiration/drug effects , Cell Respiration/radiation effects , Fluorescence Polarization , Hematoporphyrins/metabolism , Intracellular Membranes/drug effects , Intracellular Membranes/metabolism , Intracellular Membranes/radiation effects , Kinetics , Light , Liposomes/chemistry , Liposomes/metabolism , Liposomes/radiation effects , Mersalyl/pharmacology , Mitochondria, Liver/drug effects , Mitochondria, Liver/radiation effects , Mitochondrial Membrane Transport Proteins , Mitochondrial Permeability Transition Pore , Mitochondrial Swelling , Oxidation-Reduction , Oxidative Phosphorylation/drug effects , Oxidative Phosphorylation/radiation effects , Protein Binding , Rats , Scattering, Radiation , Singlet Oxygen , Sulfhydryl Compounds/metabolism , Temperature , Trioxsalen/metabolism , Uncoupling Agents/pharmacologyABSTRACT
An intact alveolar epithelial barrier is thought to be important for alveolar liquid absorption. However, polycations increase alveolar permeability without affecting alveolar liquid absorption (Saumon et al., Am J Physiol 1995: 269:L185-L194). We have reconsidered this issue using polyamines. The polyamine spermine (10(-3) mol/l) produced a large (up to 20-fold), sustained increase in the permeability of the alveolar barrier to mannitol (PAMan) and in alveolar liquid absorption (Jw, twofold) in isolated rat lungs. These increases were inhibited by 5 x 10(-3) mol/l putrescine and 2 x 10(-3) mol/l spermidine. Because spermine is known to affect the phosphoinositide/Ca2+ signalling pathway, we evaluated the effects of thiol reagents known to interfere with this pathway in different ways. Thimerosal, a thiol reagent which sensitizes the inositol 1,4,5-trisphosphate (IP3) receptor, inhibited the spermine-induced increase in PA(Man) and, to a lesser extent, that of Jw. Mersalyl, a thiol reagent which blocks IP3-gated Ca2+ channels, enhanced spermine's effect, whereas N-ethylmaleimide, a non-specific thiol reagent, had no effect. These observations show that large increases in permeability may coexist with increases in Jw. They also suggest that the phosphoinositide/Ca2+ second messenger pathway is involved in modulating the tightness of the alveolar barrier and alveolar liquid absorption.
Subject(s)
Pulmonary Alveoli/drug effects , Pulmonary Alveoli/metabolism , Spermine/pharmacology , Sulfhydryl Compounds/pharmacology , Amiloride/pharmacology , Animals , Biological Transport/drug effects , Biological Transport, Active/drug effects , Calcium/metabolism , Calcium Channel Blockers/pharmacology , Calcium Channels/drug effects , Calcium Channels/physiology , Cell Membrane Permeability/drug effects , Ethylmaleimide/pharmacology , Indicators and Reagents , Inositol 1,4,5-Trisphosphate/pharmacology , Inositol 1,4,5-Trisphosphate Receptors , Ion Channel Gating/drug effects , Mannitol/metabolism , Mersalyl/pharmacology , Phosphatidylinositols/metabolism , Putrescine/pharmacology , Rats , Rats, Wistar , Receptors, Cytoplasmic and Nuclear/drug effects , Receptors, Cytoplasmic and Nuclear/physiology , Second Messenger Systems , Spermidine/pharmacology , Thimerosal/pharmacologyABSTRACT
Heterodisulfide reductase (HDR) is a component of the energy-conserving electron transfer system in methanogens. HDR catalyzes the two-electron reduction of coenzyme B-S-S-coenzyme M (CoB-S-S-CoM), the heterodisulfide product of the methyl-CoM reductase reaction, to free thiols, HS-CoB and HS-CoM. HDR from Methanosarcina thermophila contains two b-hemes and two [Fe(4)S(4)] clusters. The physiological electron donor for HDR appears to be methanophenazine (MPhen), a membrane-bound cofactor, which can be replaced by a water-soluble analog, 2-hydroxyphenazine (HPhen). This report describes the electron transfer pathway from reduced HPhen (HPhenH(2)) to CoB-S-S-CoM. Steady-state kinetic studies indicate a ping-pong mechanism for heterodisulfide reduction by HPhenH(2) with the following values: k(cat) = 74 s(-1) at 25 degrees C, K(m) (HPhenH(2)) = 92 microm, K(m) (CoB-S-S-CoM) = 144 microm. Rapid freeze-quench EPR and stopped-flow kinetic studies and inhibition experiments using CO and diphenylene iodonium indicate that only the low spin heme and the high potential FeS cluster are involved in CoB-S-S-CoM reduction by HPhenH(2). Fe-S cluster disruption by mersalyl acid inhibits heme reduction by HPhenH(2), suggesting that a 4Fe cluster is the initial electron acceptor from HPhenH(2). We propose the following electron transfer pathway: HPhenH(2) to the high potential 4Fe cluster, to the low potential heme, and finally, to CoB-S-S-CoM.
Subject(s)
Iron-Sulfur Proteins/metabolism , Methanosarcina/enzymology , Oxidoreductases/metabolism , Phenazines/metabolism , Phosphothreonine/analogs & derivatives , Carbon Monoxide/pharmacology , Electron Spin Resonance Spectroscopy , Electron Transport , Flow Injection Analysis , Iron-Sulfur Proteins/drug effects , Mersalyl/pharmacology , Mesna/metabolism , Onium Compounds/pharmacology , Oxidoreductases/drug effects , Phosphothreonine/metabolismABSTRACT
We have used site-directed chemical labelling to demonstrate the membrane topology and to identify neighbouring subunits of subunit 8 (Y8) in yeast mitochondrial ATP synthase (mtATPase). Unique cysteine residues were introduced at the N or C-terminus of Y8 by site-directed mutagenesis. Expression and targeting to mitochondria in vivo of each of these variants in a yeast Y8 null mutant was able to restore activity to an otherwise nonfunctional ATP synthase complex. The position of each introduced cysteine relative to the inner mitochondrial membrane was probed with thiol-specific nonpermeant and permeant reagents in both intact and lysed mitochondria. The data indicate that the N-terminus of Y8 is located in the intermembrane space of mitochondria whereas the C-terminus is located within the mitochondrial matrix. The proximity of Y8 to other proteins of mtATPase was tested using heterobifunctional cross-linking reagents, each with one thiol-specific reactive group and one nonspecific, photoactivatible reactive group. These experiments revealed the proximity of the C-terminal domain of Y8 to subunits d and f, and that of the N-terminal domain to subunit f. It is concluded that Y8 possesses a single transmembrane domain which extends across the inner membrane of intact mitochondria. As subunit d is a likely component of the stator stalk of mitochondrial ATP synthase, we propose, on the basis of the observed cross-links, that Y8 may also be part of the stator stalk.
Subject(s)
Cysteine/metabolism , Mitochondria/enzymology , Protein Engineering , Proton-Translocating ATPases/chemistry , Proton-Translocating ATPases/metabolism , Yeasts/enzymology , Amino Acid Sequence , Binding Sites , Blotting, Western , Cell Respiration/drug effects , Cross-Linking Reagents , Cysteine/genetics , Disulfides/metabolism , Enzyme Inhibitors/pharmacology , Fungal Proteins/chemistry , Fungal Proteins/genetics , Fungal Proteins/metabolism , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mersalyl/pharmacology , Mitochondria/drug effects , Mitochondria/metabolism , Models, Molecular , Mutation/genetics , Protein Binding , Protein Structure, Quaternary/drug effects , Protein Subunits , Proton-Translocating ATPases/genetics , Yeasts/cytology , Yeasts/drug effects , Yeasts/metabolismABSTRACT
Mitochondrial transporters, in particular uncoupling proteins and the ADP/ATP carrier, are known to mediate uniport of anionic fatty acids (FAs), allowing FA cycling which is completed by the passive movement of FAs across the membrane in their protonated form. This study investigated the ability of the mitochondrial phosphate carrier to catalyze such a mechanism and, furthermore, how this putative activity is related to the previously observed HgCl(2)-induced uniport mode. The yeast mitochondrial phosphate carrier was expressed in Escherichia coli and then reconstituted into lipid vesicles. The FA-induced H(+) uniport or Cl(-) uniport were monitored fluorometrically after HgCl(2) addition. These transport activities were further characterized by testing various inhibitors of the two different transport modes. The phosphate carrier was found to mediate FA cycling, which led to H(+) efflux in proteoliposomes. This activity was insensitive to ATP, mersalyl or N-ethylmaleimide and was inhibited by methylenediphosphonate and iminodi(methylenephosphonate), which are new inhibitors of mitochondrial phosphate transport. Also, the HgCl(2) induced Cl(-) uniport mediated by the reconstituted yeast PIC, was found to be inhibited by these reagents. Both methylenediphosphonate and iminodi(methylenephosphonate) blocked unidirectional Cl(-) uptake, whereas Cl(-) efflux was inhibited by iminodi(methylenephosphonate) and phosphonoformic acid only. These results suggest that a hydrophobic domain, interacting with FAs, exists in the mitochondrial phosphate carrier, which is distinct from the phosphate transport pathway. This domain allows for FA anion uniport via the phosphate carrier and consequently, FA cycling that should lead to uncoupling in mitochondria. This might be considered as a side function of this carrier.
Subject(s)
Carrier Proteins/metabolism , Fatty Acids/metabolism , Phosphates/metabolism , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/pharmacology , Biological Transport , Carrier Proteins/drug effects , Carrier Proteins/genetics , Chlorides/metabolism , Diphosphonates/metabolism , Diphosphonates/pharmacology , Escherichia coli/genetics , Ethylmaleimide/pharmacology , Foscarnet/pharmacology , Hydrogen/metabolism , Liposomes/metabolism , Mercuric Chloride/metabolism , Mersalyl/pharmacology , Organophosphorus Compounds/pharmacology , Phosphate-Binding ProteinsABSTRACT
The purpose of this study was to investigate the effects exerted by thiol-modifying reagents on themitochondrial sulfonylurea receptor. The thiol-oxidizing agents (timerosal and 5, 5'-dithio-bis(2-nitrobenzoic acid)) were found to produce a large inhibition (70% to 80%) of specific binding of [(3)H]glibenclamide to the beef heart mitochondrial membrane. Similar effects were observed with membrane permeable (N-ethylmaleimide) and non-permeable (mersalyl) thiol modifying agents. Glibenclamide binding was also decreased by oxidizing agents (hydrogen peroxide) but not by reducing agents (reduced gluthatione, dithiothreitol and the 2,3-dihydroxy-1,4-dithiolbutane). The results suggest that intact thiol groups, facing the mitochondrial matrix, are essential for glibenclamide binding to the mitochondrial sulfonylurea receptor.
Subject(s)
ATP-Binding Cassette Transporters , Mitochondria, Heart/drug effects , Potassium Channels, Inwardly Rectifying , Potassium Channels/metabolism , Receptors, Drug/metabolism , Sulfhydryl Reagents/pharmacology , Adenosine Diphosphate/pharmacology , Adenosine Triphosphate/pharmacology , Animals , Binding Sites/drug effects , Cattle , Dithionitrobenzoic Acid/pharmacology , Dose-Response Relationship, Drug , Ethylmaleimide/metabolism , Ethylmaleimide/pharmacology , Glyburide/metabolism , Hydrogen Peroxide/pharmacology , Intracellular Membranes/drug effects , Intracellular Membranes/metabolism , Kinetics , Mersalyl/pharmacology , Mitochondria, Heart/metabolism , Oxidants/pharmacology , Permeability , Reducing Agents/pharmacology , Sulfhydryl Compounds/metabolism , Sulfonylurea ReceptorsABSTRACT
Glutamine transport into rat brain mitochondria (synaptic and non-synaptic) was monitored by the uptake of [3H]glutamine as well as by mitochondrial swelling. The uptake is inversely correlated to medium osmolarity, temperature-dependent, saturable and inhibited by mersalyl, and glutamine is upconcentrated in the mitochondria. These results indicate that glutamine is transported into an osmotically active space by a protein catalyzed mechanism. The uptake is slightly higher in synaptic mitochondria than in non-synaptic ones. It is inhibited both by rotenone and the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, the latter at pH 6.5, showing that the transport is activated by an electrochemical proton gradient. The K+/H+ ionophore nigericin also inhibits the uptake at pH 6.5 in the presence of external K+, which indicates that glutamine, at least in part, is taken up by a proton symport transporter. In addition, glutamine uptake as measured by the swelling technique revealed an additional glutamine transport activity with at least 10 times higher Km value. This uptake is inhibited by valinomycin in the presence of K+ and is thus also activated by the membrane potential. Otherwise, the two methods show similar results. These data indicate that glutamine transport in brain mitochondria cannot be described by merely a simple electroneutral uniport mechanism, but are consistent with the uptake of both the anionic and the zwitterionic glutamine.