atractyloside and carboxyatractyloside

In providing the cell with ATP generated by oxidative phosphorylation, the mitochondrial ADP/ATP carrier plays a central role in aerobic eukaryotic cells. Combining biochemical, genetic, and structural approaches contributes to understanding the molecular mechanism of this essential transport system, the dysfunction of which is implicated in neuromuscular diseases.

Topics: Animals; Atractyloside; Cell Respiration; Gene Expression; Humans; Mitochondria, Muscle; Mitochondrial ADP, ATP Translocases; Neuromuscular Diseases

Atractylis gummifera L. (Asteraceae) is a thistle located in the Mediterranean regions. Despite the plant's well-known toxicity, its ingestion continues to be a common cause of poisoning. The toxicity of Atractylis gummifera resides in atractyloside and carboxyatractyloside, two diterpenoid glucosides capable of inhibiting mitochondrial oxidative phosphorylation. Both constituents interact with a mitochondrial protein, the adenine nucleotide translocator, responsible for the ATP/ADP antiport and involved in mitochondrial membrane permeabilization. Poisoned patients manifest characteristic symptoms such as nausea, vomiting, epigastric and abdominal pain, diarrhoea, anxiety, headache and convulsions, often followed by coma. No specific pharmacological treatment for Atractylis gummifera intoxication is yet available and all the current therapeutic approaches are only symptomatic. In vitro experiments showed that some compounds such as verapamil, or dithiothreitol could protect against the toxic effects of atractyloside, but only if administered before atractyloside exposure. New therapeutic approaches could come from immunotherapy research: some studies have already tried to produce polyclonal Fab fragments against the toxic components of Atractylis gummifera.

Topics: Atractylis; Atractyloside; Child; Child, Preschool; Enzyme Inhibitors; Ethnopharmacology; Humans; Mediterranean Region; Poisoning

In the process of oxidative phosphorylation, the exchange of cytosolic ADP3- against mitochondrial ATP4- across the inner mitochondrial membrane is mediated by a specific carrier protein. Two different conformations for this carrier have been demonstrated on the basis of interactions with specific inhibitors, namely carboxyatractyloside (CATR) and bongkrekic acid (BA). The two conformations, referred to as CATR and BA conformations, are interconvertible, provided that ADP or ATP are present. The functional ADP/ATP carrier is probably organized as a tetramer. In the presence of CATR or BA the tetramer is split into two dimers combined with either of the two inhibitors. The amino acid sequence of the beef heart carrier monomer (297 residues) contains three repeats of about 100 residues each. Experimental results obtained through different approaches, including photolabeling, immunochemistry, and limited proteolysis, can be interpreted on the basis of a model with five or six transmembrane alpha helices per carrier monomer. Two mobile regions involved in the binding of nucleotides and accessible to proteolytic enzymes have been identified. Each of them may be visualized as consisting of two pairs of short amphipathic alpha helices, which can be juxtaposed to form hydrophilic channels facilitating the nucleotide transport. Mutagenesis in yeast is currently being used to detect strategic amino acids in ADP/ATP transport.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Amino Acid Sequence; Animals; Atractyloside; Binding Sites; Bongkrekic Acid; Cattle; Consensus Sequence; Fungal Proteins; Intracellular Membranes; Mitochondria; Mitochondrial ADP, ATP Translocases; Molecular Sequence Data; Oxidative Phosphorylation; Protein Conformation; Saccharomyces cerevisiae; Sequence Alignment; Sequence Homology, Amino Acid

Topics: Animals; Atractyloside; Mathematics; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Oxygen Consumption; Thyroid Hormones

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Adipose Tissue, Brown; Atractyloside; Biological Transport, Active; Bongkrekic Acid; Carrier Proteins; Chemical Phenomena; Chemistry, Physical; Electrochemistry; Guanine Nucleotides; Inosine Nucleotides; Intracellular Membranes; Ion Channels; Macromolecular Substances; Membrane Proteins; Micelles; Mitochondria; Mitochondrial ADP, ATP Translocases; Mitochondrial Proteins; Nucleotidyltransferases; Octoxynol; Polyethylene Glycols; Protein Conformation; Protons; Thermodynamics; Uncoupling Protein 1

The control theory of Kacser and Burns [in: Rate Control of Biological Processes (Davies, D.D. ed) pp. 65-104, Cambridge University Press, London, 1973] and Heinrich and Rapoport [Eur. J. Biochem. (1974) 42, 97-105] has been used to quantify the amount of control exerted by different steps on mitochondrial oxidative phosphorylation in rat-liver mitochondria. Inhibitors were used to manipulate the amount of active enzyme. The control strength of the adenine nucleotide translocator was measured by carrying out titrations with carboxyatractyloside. In state 4, the control strength of the translocator was found to be zero. As the rate of respiration was increased by adding hexokinase, the control strength of the translocator increased to a maximum value of approximately 30% at approximately 80% of state 3 respiration. In state 3, control of respiration is distributed between a number of steps, including the adenine nucleotide translocator, the dicarboxylate carrier and cytochrome c oxidase. The measured values for the distribution of control agree very well with those calculated with the aid of a model for mitochondrial oxidative phosphorylation developed by Bohnensack et al. [Biochim. Biophys. Acta (1982) 680, 271-280].

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Atractyloside; Citrulline; Computers; Glucose-6-Phosphate; Glucosephosphates; Hexokinase; Intracellular Membranes; Kinetics; Membrane Potentials; Mitochondria; Mitochondrial ADP, ATP Translocases; Models, Biological; Oxidative Phosphorylation; Phosphates; Rats; Thermodynamics; Uncoupling Agents

The mitochondrial ADP/ATP carrier (AAC) exports ATP and imports ADP through alternating between cytosol-open (c-) and matrix-open (m-) states. The salt bridge networks near the matrix side (m-gate) and cytosol side (c-gate) are thought to be crucial for state transitions, yet our knowledge on these networks is still limited. In the current work, we focus on more conserved m-gate network in the c-state AAC. All-atom molecular dynamics (MD) simulations on a variety of mutants and the CATR-AAC complex have revealed that: (1) without involvement of other positive residues, the charged residues from the three Px[DE]xx[KR] motifs only are prone to form symmetrical inter-helical network; (2) R235 plays a determinant role for the asymmetry in m-gate network of AAC; (3) R235 significantly strengthens the interactions between H3 and H5; (4) R79 exhibits more significant impact on m-gate than R279; (5) CATR promotes symmetry in m-gate mainly through separating R234 from D231 and fixing R79; (6) vulnerability of the H2-H3 interface near matrix side could be functionally important. Our results provide new insights into the highly conserved yet variable m-gate network in the big mitochondrial carrier family.

Topics: Amino Acid Motifs; Atractyloside; Binding Sites; Mitochondria; Mitochondrial ADP, ATP Translocases; Models, Molecular; Molecular Dynamics Simulation; Mutation; Protein Binding; Protein Conformation

Mitochondrial permeability transition pore (mPTP) closure triggers cardiomyocyte differentiation during development while pathological opening causes cell death during myocardial ischemia-reperfusion and heart failure. Ubiquinone modulates the mPTP; however, little is known about its mechanistic role in health and disease. We previously found excessive proton leak in newborn Fmr1 KO mouse forebrain caused by ubiquinone deficiency and increased open mPTP probability. Because of the physiological differences between the heart and brain during maturation, we hypothesized that developing Fmr1 KO cardiomyocyte mitochondria would demonstrate dissimilar features.. Newborn male Fmr1 KO mice and controls were assessed. Respiratory chain enzyme activity, ubiquinone content, proton leak, and oxygen consumption were measured in cardiomyocyte mitochondria. Cardiac function was evaluated via echocardiography.. In contrast to controls, Fmr1 KO cardiomyocyte mitochondria demonstrated increased ubiquinone content and decreased proton leak. Leak was cyclosporine (CsA)-sensitive in controls and CsA-insensitive in Fmr1 KOs. There was no difference in absolute mitochondrial respiration or cardiac function between strains.. These findings establish the newborn Fmr1 KO mouse as a novel model of excess ubiquinone and closed mPTP in the developing heart. Such a model may help provide insight into the biology of cardiac development and pathophysiology of neonatal heart failure.. Ubiquinone is in excess and the mPTP is closed in the developing FXS heart. Strengthens evidence of open mPTP probability in the normally developing postnatal murine heart and provides new evidence for premature closure of the mPTP in Fmr1 mutants. Establishes a novel model of excess CoQ and a closed pore in the developing heart. Such a model will be a valuable tool used to better understand the role of ubiquinone and the mPTP in the neonatal heart in health and disease.

Topics: Animals; Atractyloside; Cyclosporine; Disease Models, Animal; Electron Transport; Fetal Heart; Fragile X Mental Retardation Protein; Fragile X Syndrome; Guanosine Diphosphate; Male; Mice; Mice, Knockout; Mitochondria, Heart; Mitochondrial Permeability Transition Pore; Myocytes, Cardiac; Oxygen Consumption; Proton-Motive Force; Single-Blind Method; Ubiquinone

Xanthii Fructus is extensively used as an herbal medicine. Ingestion of this herb is associated with severe hepatotoxicity and nephrotoxicity. Atractyloside and carboxyatractyloside are two dominative toxic constituents in Xanthii Fructus. However, their pharmacokinetic study is lacking. In this study, a novel high-performance liquid chromatography-tandem mass spectrometry method was developed to simultaneously quantify the rat plasma concentrations of atractyloside and carboxyatractyloside. After protein precipitation, the analytes were chromatographic separated on a ZORBAX Eclipse Plus column (2.1 × 150 mm id, 5 µm) under gradient elute. In the negative electrospray ionization mode, the transitions at m/z 725.3→645.4 for atractyloside, m/z 769.3→689.4 for carboxyatractyloside, and m/z 479.2→121.1 for paeoniflorin (the internal standard) were acquired by multiple reaction monitoring. This analytical method showed good linearity over 1-500 ng/mL for atractyloside and 2-500 ng/mL for carboxyatractyloside with acceptable precision and accuracy. No matrix effect, instability and carryover occurred in the analysis procedure. The extraction recoveries were greater than 85.0%. This method was applied to a preliminary pharmacokinetic study by orally administering Xanthii Fructus extract (9 g/kg) to rats, which was useful to evaluate the role of these two compounds in Xanthii Fructus-induced toxicity.

Topics: Administration, Oral; Animals; Atractyloside; Chromatography, Liquid; Drugs, Chinese Herbal; Fruit; Male; Medicine, Chinese Traditional; Molecular Conformation; Plant Extracts; Rats; Rats, Sprague-Dawley; Tandem Mass Spectrometry; Xanthium

Topics: Amino Acid Sequence; Atractyloside; Bongkrekic Acid; Cryptosporidium parvum; Cysteine; Lactococcus lactis; Mitochondria; Mitochondrial ADP, ATP Translocases; Models, Molecular; Mutant Proteins; Nucleotides; Phylogeny; Protein Translocation Systems; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Structure-Activity Relationship; Substrate Specificity

As a widely used toxic traditional herbal medicine, the quality of the Fructus Xanthii must be well controlled to ensure the clinical therapeutic efficacy and safety.. A rapid, and sensitive using ultra-high performance liquid chromatography to triple quadrupole tandem mass spectrometry (UPLC-MS/MS) in selected reaction monitoring (SRM) mode was developed and validated for simultaneous quantitation of determination active and toxic ingredients form processed by stir-frying and raw materials of Fructus Xanthii.. Chromatographic separation of all targeted compound was performed on Waters ACQUITY UPLC HSS T3 column (50 mm × 2.1 mm, 1.8 μm). Moreover, the method was successfully applied in thirty-six samples of Fructus Xanthii collected from different sources in China. The processing method was optimized through Box-Behnken statistical design and response surface methodology.. In this work, chemometrics was able to successfully discriminate and classify among samples. The optimal incubation conditions were as follows: under heating in a pot at 295 °C, medicine at 120 °C for 11.0 min with flipping frequently.. Therefore, the established UPLC-QQQ-MS method in combination with chemometric analysis provides a rapid, flexible and reliable method for quality assessment of Fructus Xanthii. Importantly, the optimized experimental value of the processing process provides the basis for future research.

Topics: Atractyloside; Biomarkers; China; Chlorogenic Acid; Chromatography, Liquid; Diterpenes; Drugs, Chinese Herbal; Phenols; Reproducibility of Results; Tandem Mass Spectrometry; Temperature

In order to evaluate the susceptibility of sheep to V. rubricaulis and to establish the clinical signs, serum biochemistry, and pathological findings, eight sheep were fed varying doses of V. rubricaulis. The onset of clinical signs occurred 6-48 h after the ingestion of V. rubricaulis. Clinical courses lasted 6-56 h after the ingestion of the plant. Serum activities of aspartate aminotransferase, gamma-glutamyl transferase, and alkaline phosphatase were highly elevated and glucose blood levels were low in affected sheep. Clinical signs consisted of apathy, anorexia, dry muzzle, respiratory distress, abdominal pain, and mushy feces with streaks of blood and mucus. Two sheep had neurological signs including muscle fasciculation, nystagmus, paddling movements, and blindness. Liver necrosis could be detected antemortem through liver biopsy. Five sheep died and three recovered. The liver was affected in all necropsied sheep; it increased in volume and had marked accentuation of the lobular pattern with red, depressed areas intercalated with a pale yellow network. Ascites and hydropericardium were consistent findings. Microscopically, centrilobular to massive coagulative necrosis was observed. Coagulative necrosis was also observed in a few proximal renal tubules. Microscopic lesions were not found in any other organs. The severity of liver lesions was proportional to the dose. Chemical analysis to detect carboxyatractyloside in V. rubricaulis plant material was negative. It is concluded that V. rubricaulis poisoning in sheep is clinically, biochemically, and pathologically characteristic of an acute hepatoxicosis.

Topics: Alkaline Phosphatase; Animals; Ascites; Aspartate Aminotransferases; Atractyloside; Blood Glucose; gamma-Glutamyltransferase; Liver; Necrosis; Plant Poisoning; Sheep; Sheep Diseases; Vernonia

Non-alcoholic fatty liver disease (NAFLD) is a common metabolic disorder in obese individuals. Adenine nucleotide translocase (ANT) exchanges ADP/ATP through the mitochondrial inner membrane, and Ant2 is the predominant isoform expressed in the liver. Here we demonstrate that targeted disruption of Ant2 in mouse liver enhances uncoupled respiration without damaging mitochondrial integrity and liver functions. Interestingly, liver specific Ant2 knockout mice are leaner and resistant to hepatic steatosis, obesity and insulin resistance under a lipogenic diet. Protection against fatty liver is partially recapitulated by the systemic administration of low-dose carboxyatractyloside, a specific inhibitor of ANT. Targeted manipulation of hepatic mitochondrial metabolism, particularly through inhibition of ANT, may represent an alternative approach in NAFLD and obesity treatment.

Topics: Adenine Nucleotide Translocator 2; Adenosine Triphosphate; Animals; Atractyloside; Diet, High-Fat; Disease Models, Animal; Fatty Liver; Female; Glucose Clamp Technique; Hyperinsulinism; Insulin Resistance; Lipid Metabolism; Lipogenesis; Liver; Male; Mice; Mice, Knockout; Mitochondria, Liver; Mitochondrial Membranes; Non-alcoholic Fatty Liver Disease; Obesity; Protective Agents; Pyruvic Acid

Mitochondrial carriers are proteins that shuttle a variety of metabolites, nucleotides and coenzymes across the inner mitochondrial membrane. The mitochondrial ADP/ATP carriers (AACs) specifically translocate the ATP synthesized within mitochondria to the cytosol in exchange for the cytosolic ADP, playing a key role in energy production, in promoting cell viability and regulating mitochondrial permeability transition pore opening. In Homo sapiens four genes code for AACs with different tissue distribution and expression patterns. Since AACs are dysregulated in several cancer types, the employment of known and new AAC inhibitors might be crucial for inducing mitochondrial-mediated apoptosis in cancer cells. Albeit carboxyatractyloside (CATR) and bongkrekic acid (BKA) are known to be powerful and highly selective AAC inhibitors, able to induce mitochondrial dysfunction at molecular level and poisoning at physiological level, we estimated here for the first time their affinity for the human recombinant AAC2 by in vitro transport assays. We found that the inhibition constants of CATR and BKA are 4 nM and 2.0 μM, respectively. For finding new AAC inhibitors we also performed a docking-based virtual screening of an in-house developed chemical library and we identified about 100 ligands showing high affinity for the AAC2 binding region. By testing 13 commercially available molecules, out of the 100 predicted candidates, we found that 2 of them, namely suramin and chebulinic acid, are competitive AAC2 inhibitors with inhibition constants 0.3 μM and 2.1 μM, respectively. We also demonstrated that chebulinic acid and suramin are "highly selective" AAC2 inhibitors, since they poorly inhibit other human mitochondrial carriers (namely ORC1, APC1 and AGC1).

Topics: Amino Acid Sequence; Atractyloside; B-Lymphocytes; Binding Sites; Bongkrekic Acid; Dose-Response Relationship, Drug; Humans; Mitochondrial ADP, ATP Translocases; Molecular Docking Simulation; Molecular Sequence Data; Protein Transport

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.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Arsenicals; Atractyloside; Calcium; Cyclosporine; Ethylmaleimide; Male; Membrane Potential, Mitochondrial; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Mitochondrial Membrane Transport Proteins; Mitochondrial Membranes; Mitochondrial Permeability Transition Pore; Protein Conformation; Rats, Wistar; Sulfhydryl Compounds; tert-Butylhydroperoxide

The dried ripe fruits of Xanthium sibiricum (Cang'erzi) are used in traditional Chinese medicine for the treatment of nasal congestion, nasal discharge, allergic rhinitis, sinusitis, and wind-cold headaches. Carboxyatractyloside and atractyloside are important constituents of the fruits because these diterpenoid glycosides are responsible for their toxicity. In order to evaluate procedures for reducing the amount of the more toxic carboxyatractyloside, the fruits were dried and heated with different methods. Carboxyatractyloside and atractyloside were analysed by a new reversed-phase high-performance liquid chromatographic method using liquid chromatography-diode array detector-tandem mass spectrometry analysis. The results revealed that temperature and drying methods have a strong influence on the content of carboxyatractyloside and atractyloside. Fruits which were treated at higher temperatures showed a lower content of carboxyatractyloside and an increased content of atractyloside, which is 50 times less toxic. This indicates that the roasting process can reduce toxicity effectively. The microbiological colonisation of Xanthium fruits is also reduced by roasting and by drying above 100 °C. For the safe use of Cang'erzi, the effect of processing should be monitored and analysis of carboxyatractyloside and atractyloside should be obligatory in quality control.

Topics: Atractyloside; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Diterpenes; Drugs, Chinese Herbal; Fruit; Glycosides; Medicine, Chinese Traditional; Molecular Structure; Xanthium

Obesity and diabetes mellitus type 2 (DM2) frequently coexist and increase the propensity of cardiovascular dysfunction by numerous mechanisms. Chief among them are oxidative stress and Ca(2+) dysregulation, and both are inducers of the mitochondrial permeability transition pore (MPTP). Nevertheless, it is unknown whether MPTP formation is triggered in DM2 animals, and thereby contributing to cardiac dysfunction. We assessed MPTP sensitivity and reactive oxygen species production in cardiac mitochondria, as well as cytosolic Ca(2+) handling in ventricular myocytes from rats with DM2.. Male Zucker Fa/fa rats (Fa/fa) 32weeks old presenting DM2, concentric hypertrophy, and diastolic dysfunction were used. MPTP formation was evaluated in isolated mitochondria and Ca(2+) handling in ventricular myocytes, by spectrophotometric and confocal microscope techniques, respectively.. We found that the systolic Ca(2+) transient relaxation was ~40% slower, while mitochondrial H2O2 production increased by ~6-fold. MPTP opening in isolated mitochondria from Fa/fa (mFa/fa) was more sensitive to Ca(2+) than in mitochondria from lean rats (mLean), and correlated with increased thiol group exposure. The mFa/fa showed decreased oxidative phosphorylation capacity. The ATP content decreased in myocytes, while the PCr/ATP ratio remained unchanged and caspase 9 activity largely increased in myocytes from Fa/fa animals.. Our results showed that oxidative stress and diastolic Ca(2+) dysregulation increased MPTP sensitivity leading to mitochondrial dysfunction and apoptosis. Mitochondrial dysfunction could compromise ATP synthesis, and lower ATP could be linked to decreased SERCA2 activity, which might underlie diastolic dysfunction. Prolonged Ca(2+) transients might further exacerbate mitochondrial dysfunction.

Topics: Animals; Atractyloside; Calcium Signaling; Diabetes Mellitus, Type 2; Heart Diseases; Leptin; Lipids; Male; Membrane Potential, Mitochondrial; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Myocytes, Cardiac; Oxidative Stress; Oxygen Consumption; Permeability; Rats; Rats, Zucker; Ultrasonography

Chemical modification of primary amino groups of mitochondrial membrane proteins by the fluorescent probe fluorescamine induces non-specific membrane permeabilisation. Titration of the lysine ϵ-amino group promoted efflux of accumulated Ca(2+), collapse of transmembrane potential and mitochondrial swelling. Ca(2+) release was inhibited by cyclosporin A. Considering the latter, we assumed that fluorescamine induces permeability transition. Carboxyatractyloside also inhibited the reaction. Using a polyclonal antibody for adenine nucleotide translocase, Western blot analysis showed that the carrier appeared labelled with the fluorescent probe. The results point out the importance of the ϵ-amino group of lysine residues, located in the adenine nucleotide carrier, on the modulation of membrane permeability, since its blockage suffices to promote opening of the non-specific nanopore.

Topics: Animals; Atractyloside; Calcium; Cell Membrane Permeability; Fluorescamine; Ion Transport; Lysine; Male; Membrane Potentials; Mitochondria; Mitochondrial ADP, ATP Translocases; Mitochondrial Swelling; Rats; Rats, Wistar

Xanthium strumarium, commonly referred to as "cocklebur," rarely causes poisoning in cattle. When mature, this robust, annual weed bears numerous oval, brownish, spiny burs. Only the seeds in the burs and young seedlings (cotyledonary leaves) contain the toxic principle, carboxyatractyloside. In the Frankfort district of the Free State Province of South Africa, a herd of 150 Bonsmara cows were allowed to graze on the banks of a small river, where mature cocklebur was growing. Four cows died while grazing in this relatively small area. Clinical signs ranged from recumbency, apparent blindness, and hypersensitivity to convulsive seizures. During necropsy, burs completely matted with ingesta were located in the rumen content. The most distinctive microscopic lesions were severe, bridging centrilobular to midzonal hepatocyte necrosis and hemorrhage. Ultrastructurally, periacinar hepatocytes were necrotic, and novel electron-dense cytoplasmic needle-like crystals were observed, often in close association with peroxisomes. Carboxyatractyloside concentrations were determined using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Carboxyatractyloside was present in rumen contents at 2.5 mg/kg; in burs removed from the rumen at 0.17 mg/kg; in liver at 66 ng/g, and was below the limit of quantitation in the kidney sample, estimated at approximately 0.8 ng/g. Based on the presence of the plants on the riverbank, the history of exposure, the clinical findings, the presence of burs in the rumen, and the microscopic and ultrastructural lesions, X. strumarium poisoning in the herd of cattle was confirmed and was supported by LC-HRMS.

Topics: Animals; Atractyloside; Cattle; Cattle Diseases; Female; Gastrointestinal Contents; Kidney; Liver; Necrosis; Plants, Toxic; Rumen; Seeds; South Africa; Xanthium

Ischemic postconditioning (IPoC) modulates the reperfusion maneuver to mitigate ischemia-reperfusion (I/R) injury. This study aims to investigate the effects and protective mechanism of IPoC on intestinal I/R injury.. Intestinal I/R was induced by occluding the superior mesenteric artery for 30 min followed by reperfusion for 60 min on male Wistar rats. IPoC was elicited by three cycles of 30-sec reperfusion and reocclusion of superior mesenteric artery at the initiation of reperfusion. Carboxyatractyloside (CATR), a mitochondrial permeability transition pore (mPTP) opener, and N-methyl-4-isoleucine cyclosporine (NIM811), an mPTP inhibitor, were administered separately in selected groups. The serum and intestinal sections were collected for analysis.. IPoC and the administration of NIM811 significantly diminished the expression of intestinal-type fatty acid-binding protein and lactate dehydrogenase (3427±236.8 U/L for I/R, 1190.5±36.7 U/L for IPoC, 1399.3±295.6 U/L for I/R+NIM811, and 2002±370.9 IU/L for IPoC+CATR) in portal blood, the release of cytosolic cytochrome c, and the cleaved caspase 9 expression in intestinal mucosa after intestinal I/R injury (P<0.05). Histopathologically, IPoC and NIM811 mitigated mucosal damage after I/R as well (Chiu's score, 3.8±0.4 for I/R, 0.2±0.2 for IPoC, 0.4±0.2 for I/R+NIM811, and 4.2±0.2 for IPoC+CATR; apoptotic index, 59.5%±4.6% for I/R, 15.7%±15.7% for I/R+IPoC, 3.5%±3.5% for I/R+NIM811, and 67.1%±9.3% in IPoC+CATR). CATR negated the protection conferred by IPoC.. IPoC and NIM811 attenuate intestinal I/R injury. The addition of CATR negated the effects of IPoC, indicating that the protective mechanism of IPoC was associated with the modulation of mPTP opening.

Topics: Animals; Apoptosis; Atractyloside; Caspase 3; Cyclosporine; Cytochromes c; Disease Models, Animal; Enzyme Activation; Fatty Acid-Binding Proteins; Intestinal Mucosa; Intestine, Small; Ischemic Postconditioning; L-Lactate Dehydrogenase; Ligation; Male; Malondialdehyde; Mesenteric Artery, Superior; Mesenteric Vascular Occlusion; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Oxidative Stress; Rats; Rats, Wistar; Reperfusion Injury; Time Factors

Metabolic response to high altitude remains poorly explored in reptiles. In the present study, the metabolic characteristics of Phrynocephaluserythrurus (Lacertilia: Agamidae), which inhabits high altitudes (4500 m) and Phrynocephalusprzewalskii (Lacertilia: Agamidae), which inhabits low altitudes, were analysed to explore the metabolic regulatory strategies for lizards living at high-altitude environments. The results indicated that the mitochondrial respiratory rates of P. erythrurus were significantly lower than those of P. przewalskii, and that proton leak accounts for 74~79% of state 4 and 7~8% of state3 in P. erythrurus vs. 43~48% of state 4 and 24~26% of state3 in P. przewalskii. Lactate dehydrogenase (LDH) activity in P. erythrurus was lower than in P. przewalskii, indicating that at high altitude the former does not, relatively, have a greater reliance on anaerobic metabolism. A higher activity related to β-hydroxyacyl coenzyme A dehydrogenase (HOAD) and the HOAD/citrate synthase (CS) ratio suggested there was a possible higher utilization of fat in P. erythrurus. The lower expression of PGC-1α and PPAR-γ in P. erythrurus suggested their expression was not influenced by cold and low PO2 at high altitude. These distinct characteristics of P. erythrurus are considered to be necessary strategies in metabolic regulation for living at high altitude and may effectively compensate for the negative influence of cold and low PO2.

Topics: Acclimatization; Altitude; Animals; Atractyloside; Citrate (si)-Synthase; Energy Metabolism; Gene Expression; Guanosine Diphosphate; L-Lactate Dehydrogenase; Liver; Lizards; Male; Mitochondria, Liver; Mitochondria, Muscle; Muscle, Skeletal; Oxygen Consumption; PPAR gamma; Reverse Transcriptase Polymerase Chain Reaction; Species Specificity; Transcription Factors

Atractyloside (ATR) was characterized in 1868 and until now structural studies on diterpenic moiety had been done through the characterization of ATR derivatives; while the glycosidic moiety seemed to be a β-D-glucopyranose a recent crystal structure of the mitochondrial ATP/ADP carrier in complex with CATR showed an α-D-glucopyranose. We decided to re-examine the ATR and CATR structures by crystallographic study of ATR.

Topics: Atractyloside; Crystallography, X-Ray; Models, Molecular; Multiprotein Complexes

To determine carboxyatractyloside and atractyloside in Xanthii Fructus by HPLC.. By HPLC, Agilent ZORBAX SB-phenyl (4.6 mm x 250 mm, 5 microm) column was adopted, with acetonitrile-0.01 mol x L(-1) NaH2PO4 (pH 6) as the mobile phase for gradient elution at the flow rate of 1.0 mL x min(-1). The detection wavelength was 203 nm, and the temperature was set at 35 degrees C.. Carboxyatractyloside showed a good linearity within the range of 0.0972-1.944 microg and atractyloside showed a good linearity within the range of 0.1030-2.060 microg. The recovery rate of carboxyatractyloside was 100. 3% and that of atractyloside was 102.5%. The RSD were 0.67% and 1.4% (n=6).. This method is so simple, practical and highly repeatable that is can be used for quality control of Xanthii Fructus.

Topics: Asteraceae; Atractyloside; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Fruit

Mitochondrial permeability transition is established after massive Ca(2+) accumulation inside the matrix, in addition to an inducer. The closure of the pore can be accomplished by adenosine diphosphate and the immunosuppressant cyclosporin A. Recently, the estrogen antagonist, tamoxifen, has been introduced as an inhibitor of the opening of the permeability transition pore. However, the mechanism by which this drug inhibits pore opening is still under discussion. This work was performed with the purpose of establishing the membrane system involved in tamoxifen-induced pore closure. For this purpose, permeability transition was induced after the addition of carboxyatractyloside, which is a specific reagent that interacts with the adenine nucleotide translocase.. Permeability transition was assessed by analyzing matrix Ca(2+) release, transmembrane electric gradient, and mitochondrial swelling in aged, as well as in freshly prepared mitochondria. Also, cytochrome c content was analyzed in membrane mitochondria as well as in the supernatant.. In freshly prepared mitochondria, tamoxifen, at the concentration of 10 μM, totally inhibited nonspecific membrane permeability induced by 1 μM carboxyatractyloside. In addition, tamoxifen inhibited non-specific permeability in aged mitochondria and diminished membrane fluidity.. Plausibly, the inhibitory effect of tamoxifen on nonspecific membrane permeability, as induced by carboxyatractyloside, should be ascribed to a diminution, of membrane fluidity by this drug.

Topics: Animals; Atractyloside; Calcium; Cytochromes c; Estrogen Antagonists; Mitochondria; Mitochondrial ADP, ATP Translocases; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Rats; Tamoxifen

In liver mitochondria fatty acids act as protonophoric uncouplers mainly with participation of internal membrane protein carriers - ADP/ATP and aspartate/glutamate antiporters. In this study the values of recoupling effects of carboxyatractylate and glutamate (or aspartate) were used to assess the degree of participation of ADP/ATP and aspartate/glutamate antiporters in uncoupling activity of fatty acids. These values were determined from the ability of these recoupling agents to suppress the respiration stimulated by fatty acids and to raise the membrane potential reduced by fatty acids. Increase in palmitic and lauric acid concentration was shown to increase the degree of participation of ADP/ATP antiporter and to decrease the degree of participation of aspartate/glutamate antiporter in uncoupling to the same extent. These data suggest that fatty acids are not only inducers of uncoupling of oxidative phosphorylation, but that they also act the regulators of this process. The linear dependence of carboxyatractylate and glutamate recoupling effects ratio on palmitic and lauric acids concentration was established. Comparison of the effects of fatty acids (palmitic, myristic, lauric, capric, and caprylic having 16, 14, 12, 10, and 8 carbon atoms, respectively) has shown that, as the hydrophobicity of fatty acids decreases, the effectiveness decreases to a greater degree than the respective values of their specific uncoupling activity. The action of fatty acids as regulators of uncoupling is supposed to consist of activation of transport of their anions from the internal to the external monolayer of the internal membrane with participation of ADP/ATP antiporter and, at the same time, in inhibition of this process with the participation of aspartate/glutamate antiporter.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Antiporters; Aspartic Acid; Atractyloside; Cell Respiration; Fatty Acids, Nonesterified; Glutamic Acid; Hydrophobic and Hydrophilic Interactions; Mitochondria, Liver; Oxidative Phosphorylation; Rats; Uncoupling Agents

The fruit of Xanthium strumarium L. (Cang-Er-Zi) is a traditional Chinese medicine that is used in curing nasal diseases and headache according to the Chinese Pharmacopoeia. However, clinical utilization of Xanthium strumarium is relatively limited because of its toxicity. The present investigation was carried out to evaluate the toxic effects on acute liver injury in mice of the two kaurene glycosides (atractyloside and carbxyatractyloside), which are main toxic constituents isolated from Fructus Xanthii on acute liver injury in mice. Histopathological examinations revealed that there were not obviously visible injury in lungs, heart, spleen, and the central nervous system in the mice by intraperitoneal injection of atractyloside (ATR, at the doses 50,125 and 200 mg/kg) and carbxyatractyloside (CATR, at the doses 50,100 and 150 mg/kg) for 5 days. However, it revealed extensive liver injuries compared with the normal group. In the determination of enzyme levels in serum, intraperitoneal injection of ATR and CATR resulted in significantly elevated serum alanine aminotransferase (ALT), asparate aminotransferase (AST), alkaline phosphatase (ALP) activities compared to controls. In the hepatic oxidative stress level, antioxidant-related enzyme activity assays showed that ATR and CATR administration significantly increased hepatic malondialdehyde (MDA) concentration, as well as decreased superoxide dismutase (SOD), catalase (CAT) activities and glutathione (GSH) concentration, and this was in good agreement with the results of serum aminotransferase activity and histopathological examinations. Taken together, our results demonstrate that kaurene glycosides induce hepatotoxicity in mice by way of its induction of oxidative stress as lipid peroxidation in liver, which merited further studies. Therefore, these toxic constituents explain, at least in part, the hepatotoxicity of X. strumarium L. in traditional medicine.

Topics: Alanine Transaminase; Alkaline Phosphatase; Animals; Aspartate Aminotransferases; Atractyloside; Catalase; Chemical and Drug Induced Liver Injury; Diterpenes, Kaurane; Female; Fruit; Glutathione Transferase; Glycosides; Lipid Peroxidation; Liver; Male; Malondialdehyde; Mice; Mice, Inbred ICR; Oxidative Stress; Superoxide Dismutase; Xanthium

The mitochondrial ADP/ATP carrier, or Ancp, is a member of the mitochondrial carrier family responsible for exchanging ADP and ATP across the mitochondrial inner membrane. ADP/ATP transport involves Ancp switching between two conformational states. These can be analyzed using specific inhibitors, carboxyatractyloside (CATR) and bongkrekic acid (BA). The high resolution three-dimensional structure of bovine Anc1p (bAnc1p), as a CATR-carrier complex, has been solved. However, because the structure of the BA-carrier complex has not yet been determined, the detailed mechanism of transport remains unknown. Recently, sample processing for hydrogen/deuterium exchange experiments coupled to mass spectrometry was improved, providing novel insights into bAnc1p conformational transitions due to inhibitor binding. In this work we performed both hydrogen/deuterium exchange-mass spectrometry experiments and genetic manipulations. Because these are very difficult to apply with bovine Anc1p, we used Saccharomyces cerevisiae Anc isoform 2 (ScAnc2p). Significant differences in solvent accessibility were observed throughout the amino acid sequence for ScAnc2p complexed to either CATR or BA. Interestingly, in detergent solution, the conformational dynamics of ScAnc2p were dissimilar to those of bAnc1p, in particular for the upper half of the cavity, toward the intermembrane space, and the m2 loop, which is thought to be easily accessible to the solvent from the matrix in bAnc1p. Our study then focused on the methionyl residues of the Ancp signature sequence, RRRMMM. All our results indicate that the methionine cluster is involved in the ADP/ATP transport mechanism and confirm that the Ancp cavity is a highly dynamic structure.

Topics: Amino Acid Motifs; Anti-Bacterial Agents; Atractyloside; Biological Transport, Active; Bongkrekic Acid; Deuterium Exchange Measurement; Methionine; Mitochondria; Mitochondrial ADP, ATP Translocases; Protein Isoforms; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transcription Factor TFIID

The aim of our study was to analyze a distribution of metabolic flux controls of all mitochondrial complexes of ATP-Synthasome and mitochondrial creatine kinase (MtCK) in situ in permeabilized cardiac cells. For this we used their specific inhibitors to measure flux control coefficients (C(vi)(JATP)) in two different systems: A) direct stimulation of respiration by ADP and B) activation of respiration by coupled MtCK reaction in the presence of MgATP and creatine. In isolated mitochondria the C(vi)(JATP) were for system A: Complex I - 0.19, Complex III - 0.06, Complex IV 0.18, adenine nucleotide translocase (ANT) - 0.11, ATP synthase - 0.01, Pi carrier - 0.20, and the sum of C(vi)(JATP) was 0.75. In the presence of 10mM creatine (system B) the C(vi)(JATP) were 0.38 for ANT and 0.80 for MtCK. In the permeabilized cardiomyocytes inhibitors had to be added in much higher final concentration, and the following values of C(vi)(JATP) were determined for condition A and B, respectively: Complex I - 0.20 and 0.64, Complex III - 0.41 and 0.40, Complex IV - 0.40 and 0.49, ANT - 0.20 and 0.92, ATP synthase - 0.065 and 0.38, Pi carrier - 0.06 and 0.06, MtCK 0.95. The sum of C(vi)(JATP) was 1.33 and 3.84, respectively. Thus, C(vi)(JATP) were specifically increased under conditions B only for steps involved in ADP turnover and for Complex I in permeabilized cardiomyocytes within Mitochondrial Interactosome, a supercomplex consisting of MtCK, ATP-Synthasome, voltage dependent anion channel associated with tubulin βII which restricts permeability of the mitochondrial outer membrane.

Topics: Adenosine Triphosphate; Animals; Antimycin A; Atractyloside; Cell Respiration; Creatine Kinase, Mitochondrial Form; Dinitrofluorobenzene; Energy Metabolism; Enzyme Inhibitors; Male; Mersalyl; Mitochondria; Mitochondrial ADP, ATP Translocases; Mitochondrial Proton-Translocating ATPases; Models, Theoretical; Myocytes, Cardiac; Oxygen Consumption; Rats; Rats, Wistar; Rotenone; Sodium Cyanide; Uncoupling Agents

The adenine nucleotide translocator (ANT) accomplishes the exchange of ATP from the mitochondrial matrix with cytoplasmic ADP. While investigating the biochemical mechanism of retinoic acid (RA) on the ANT via retinoylation, we have found and subsequently demonstrated a positive influence of Coenzyme A (CoA) on the transport of ATP across the membranes of rat liver mitochondria. CoA enhances ANT activity in a dose-dependent manner modifying the V(max) (673.3+/-20.7 nmol ATP/mgprotein/min versus 155.0+/-1.9 nmol ATP/mgprotein/min), the IC(50) for the specific inhibitor carboxyatractyloside (CATR) (0.142+/-0.012 microM versus 0.198+/-0.011 microM) but not the K(m) (22.50+/-0.52 microM versus 22.19+/-0.98 microM). Data suggest a likely enzymatic involvement in the interaction between ANT and CoA. The effect of CoA is observed in mitochondria from several different tissues.

Topics: Adenine Nucleotide Translocator 1; Animals; Atractyloside; Biological Transport; Coenzyme A; Electrophoresis, Gel, Two-Dimensional; Kinetics; Male; Mitochondria, Liver; Models, Molecular; Rats; Rats, Wistar; Time Factors

The lipid peroxidation product 4-hydroxynonenal (HNE) increases the proton conductance of the inner mitochondrial membrane through effects on uncoupling proteins (UCPs) and the adenine nucleotide translocase (ANT); however, the relative contribution of the two carriers to these effects is unclear. To clarify this we isolated mitochondria from skeletal muscle and heart of wild-type and Ucp3 knockout (Ucp3KO) mice. To increase UCP3 expression, some mice were i.p. injected with LPS (12mg/kg body weight). In spite of the increased UCP3 expression levels, basal proton conductance did not change. HNE increased the proton conductance of skeletal muscle and heart mitochondria. In skeletal muscle, this increase was lower in Ucp3KO mice and higher in LPS-treated wild-type mice, and was partially abolished by GDP (UCPs inhibitor) and completely abolished by carboxyatractylate (ANT inhibitor) or addition of both inhibitors. GDP had no effect on HNE-induced conductance in heart mitochondria, but carboxyatractylate or administration of both inhibitors had a partial effect. GDP-mediated inhibition of HNE-activated proton conductance in skeletal muscle mitochondria was not observed in Ucp3KO mice, indicating that GDP is specific for UCP3, at least in muscle. Carboxyatractylate was able to inhibit UCP3, probably through an indirect mechanism. Our results are consistent with the conclusion that, in skeletal muscle, HNE-induced increase in proton conductance is mediated by UCP3 (30%) and ANT, whereas in the heart the increase is mediated by ANT and other carriers, possibly including UCP3.

Topics: Aldehydes; Animals; Atractyloside; Blotting, Western; Cysteine Proteinase Inhibitors; Electron Transport Complex IV; Female; Guanosine Diphosphate; Lipopolysaccharides; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Mitochondria, Heart; Mitochondria, Muscle; Mitochondrial ADP, ATP Translocases; Muscle, Skeletal; Oxygen Consumption; Protons

The mitochondrial ADP/ATP carrier (Ancp) has long been a paradigm for studies of the mitochondrial carrier family due to, among other properties, its natural abundance and the existence of specific inhibitors, namely, carboxyatractyloside (CATR) and bongkrekic acid (BA), which lock the carrier under distinct and stable conformations. Bovine Anc1p isolated in complex with CATR in the presence of an aminoxyde detergent (LAPAO) was crystallized and its 3D structure determined. It is the first mitochondrial carrier structure resolved at high resolution (2.2 A, as reported by Pebay-Peyroula et al. (Nature 426:39-44, 2003)). Analyses revealed a monomer while most of the biochemical studies led to hypothesize Ancp functions as a dimer. To address the structural organization issue, we engineered a mutant of the yeast Ancp that corresponds to a covalent homodimer in view of 3D structure determination. We compare in this chapter the purification yield and quality of the chimera tagged either with six histidines at its C-ter end or nine histidines at its N-ter. We show that, as expected, length and position of the tag are important criteria for qualitative purification. We also discuss the advantages and drawbacks of purifying Ancp either from a natural source or from engineered yeast cells.

Topics: Animals; Atractyloside; Bongkrekic Acid; Cattle; Membrane Proteins; Mitochondrial ADP, ATP Translocases; Recombinant Proteins

The transport of solutes across the inner mitochondrial membrane is highly selective and necessitates membrane proteins mainly from the mitochondrial carrier family (MCF). These carriers are required for the transport of a variety of metabolites implicated in all the important processes occurring within the mitochondrial matrix. Due to its high abundance, the ADP/ATP carrier (AAC) is the member of the family that was studied most. It is the first mitochondrial carrier for which a high-resolution X-ray structure is known. The carrier was crystallized in the presence of a strong inhibitor, the carboxyatractyloside (CATR). The structure gives an insight not only into the overall fold of mitochondrial carriers in general but also into atomic details of the AAC in a conformation that is open toward the intermembrane space (IMS). Molecular dynamics simulations indicate the first events occurring to the carrier after the binding of ADP. A careful analysis of the primary sequences of all the carriers in light with the structure highlights properties of the protein that are related to the substrate.

Topics: Adenosine Diphosphate; Atractyloside; Crystallography, X-Ray; Humans; Membrane Proteins; Mitochondrial ADP, ATP Translocases; Mitochondrial Membranes; Molecular Dynamics Simulation; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary

The mitochondrial adenine nucleotide carrier (Ancp) catalyzes the transport of ADP and ATP across the mitochondrial inner membrane, thus playing an essential role in cellular energy metabolism. During the transport mechanism the carrier switches between two different conformations that can be blocked by two toxins: carboxyatractyloside (CATR) and bongkrekic acid. Therefore, our understanding of the nucleotide transport mechanism can be improved by analyzing structural differences of the individual inhibited states. We have solved the three-dimensional structure of bovine carrier isoform 1 (bAnc1p) in a complex with CATR, but the structure of the carrier-bongkrekic acid complex, and thus, the detailed mechanism of transport remains unknown. Improvements in sample processing in the hydrogen/deuterium exchange technique coupled to mass spectrometry (HDX-MS) have allowed us to gain novel insights into the conformational changes undergone by bAnc1p. This paper describes the first study of bAnc1p using HDX-MS. Results obtained with the CATR-bAnc1p complex were fully in agreement with published results, thus, validating our approach. On the other hand, the HDX kinetics of the two complexes displays marked differences. The bongkrekic acid-bAnc1p complex exhibits greater accessibility to the solvent on the matrix side, whereas the CATR-bAnc1p complex is more accessible on the intermembrane side. These results are discussed with respect to the structural and biochemical data available on Ancp.

Topics: Animals; Atractyloside; Biological Transport; Bongkrekic Acid; Cattle; Deuterium Exchange Measurement; Kinetics; Mass Spectrometry; Mitochondrial ADP, ATP Translocases; Protein Structure, Tertiary

Uncoupling proteins (UCPs) mediate fatty acid-induced proton cycling in mitochondria, which is stimulated by superoxide and inhibited by GDP. Fatty acid anions can also be transported by adenine nucleotide translocase (ANT), thus resulting in the uncoupling of oxidative phosphorylation. In the present work, an attempt was made to distinguish between the protonophoric activity of UCP3 and that of ANT using inhibition analysis. This study was carried out using mitochondria from skeletal muscles of hibernating Yakut ground squirrel, which have a significant level of UCP3 mRNA. We found that millimolar concentrations of GDP, which is considered to be a specific inhibitor of UCPs, slightly recoupled the mitochondrial respiration and restored the membrane potential. Addition of the specific ANT inhibitor CAT (carboxyatractylate), in micromolar concentration, prior to GDP prevented its recoupling effect. Moreover, GDP and ADP exhibited a competitive kinetic behavior with respect to ANT. In brown adipose tissue, CAT did not prevent the UCP1-iduced increase in chloride permeability and the inhibitory effect of GDP, thus confirming the inability of CAT to affect UCP1. These results allow us to conclude that the recoupling effect of purine nucleotides on skeletal muscle mitochondria of hibernating ground squirrels can be explained by interaction of the nucleotides with ANT, whereas UCP3 is not involved in the process.

Topics: Animals; Atractyloside; Enzyme Inhibitors; Hibernation; Ion Channels; Kinetics; Mitochondria, Muscle; Mitochondrial ADP, ATP Translocases; Mitochondrial Proteins; Muscle, Skeletal; Purine Nucleotides; Respiratory Rate; RNA, Messenger; Sciuridae; Uncoupling Protein 3

We studied the effect of mitochondrial ageing on membrane permeability transition. The results obtained indicate that aged mitochondria are neither able to retain Ca2+ nor to maintain a high transmembrane electric gradient. In addition, aged mitochondria undergo a large amplitude swelling. These dysfunctions were circumvented by the addition of cyclosporin A. Furthermore, it is shown that ageing-induced permeability transition causes oxidative damage on the matrix enzyme aconitase. The observed damage in aged mitochondria requires Ca2+ addition; therefore, it was not seen when Sr2+ replaced Ca2+. Two important findings in this work were the fact that despite of the presence of cyclosporin A, carboxyatractyloside was still able to induce permeability transition, and that ageing induced mitochondrial DNA disruption and release of cytochrome c. It is likely that the membrane's increased permeability is due to the effect of fatty acids, since bovine serum albumin makes mitochondria able to retain Ca2+. However, the possibility that the damage might be the result of oxidative stress cannot be discarded.

Topics: Aconitate Hydratase; Adenosine Diphosphate; Animals; Atractyloside; Calcium; Cellular Senescence; Cyclosporine; Cytochromes c; DNA, Mitochondrial; Membrane Potential, Mitochondrial; Mitochondria, Liver; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Mitochondrial Swelling; Oxidative Stress; Rats; Time Factors

This work was undertaken to gain further information on the chemical characteristics of the membrane entity involved in the formation of the nonspecific pore. Mitochondria were subjected to oxidative stress by exposure to UV radiation. The results indicate that ultraviolet C radiation induces structural modifications in the adenine nucleotide translocase that lead to membrane permeability transition. Membrane leakage was assessed by measuring mitochondrial Ca2+ transport, the transmembrane electric gradient, and mitochondrial swelling. UV-irradiated mitochondria were unable to retain matrix Ca2+ or to maintain a high level of membrane potential when Ca2+ was added; furthermore, UV-irradiated mitochondria underwent large amplitude swelling. Release of cytochrome c and formation of malondialdehyde, owing to lipid peroxidation, were also seen. Structural modifications of the translocase were revealed by an increase in the binding of the fluorescent probe eosin-5-maleimide to thiol residues of the ADP/ATP carrier. These modifications, taken together with findings indicating that cyclosporin resulted unable to inhibit carboxyatractyloside-induced permeability transition, prompted us to conclude that the translocase could constitute the nonspecific pore or at least be an important modulator of it.

Topics: Animals; Atractyloside; Cell Membrane Permeability; Cyclosporine; Mass Spectrometry; Rats; Ultraviolet Rays

Berberine [Natural Yellow 18, 5,6-dihydro-9,10-dimethoxybenzo(g)-1,3-benzodioxolo (5,6-a) quinolizinium] is an alkaloid present in plants of the Berberidaceae family and used in traditional Chinese and North American medicine. We have previously demonstrated that berberine causes mitochondrial depolarization and fragmentation, with simultaneous increase in oxidative stress. We also demonstrated that berberine causes an inhibition of mitochondrial respiration and a decrease on calcium loading capacity through induction of the mitochondrial permeability transition (MPT). The objective of the present work is to investigate a common target for both induction of the MPT and inhibition of respiration. The hypothesis is that berberine induces the MPT through interacting with the adenine nucleotide translocator (ANT). By measuring induction of the MPT through increased mitochondrial swelling, membrane depolarization and loss of calcium retention, we observed that the effects of berberine were not inhibited by bongkrekic acid although adenosine diphosphate (ADP)/oligomycin completely prevented the MPT. Also, we observed that berberine increased the depolarization effect of oleic acid on liver mitochondria. The initial depolarization observed when berberine is added to mitochondria was not affected by ANT inhibitors. Taken together, we propose that berberine acts on the ANT, altering the binding of the protein to bongkrekic acid but not to cyclosporin A or ADP. It is also clear that the membrane potential is required for berberine effects, most likely for allowing for its mitochondrial accumulation. Mitochondrial effects of berberine can be relevant not only for its proposed antitumor activity but also for the assessment of its organ toxicity, depending on factors such as tissue accumulation or delivery.

Topics: Adenosine Diphosphate; Animals; Antineoplastic Agents, Phytogenic; Atractyloside; Berberine Alkaloids; Bongkrekic Acid; Calcium; Cell Respiration; Dose-Response Relationship, Drug; In Vitro Techniques; Male; Membrane Potential, Mitochondrial; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Mitochondrial Swelling; Oleic Acid; Oligomycins; Rats; Rats, Wistar; Time Factors; Uncoupling Agents

In this work, we studied the effect of N-ethylmaleimide on permeability transition. The findings indicate that the amine inhibited the effects of carboxyatractyloside and agaric acid. It is known that these reagents interact with the adenine nucleotide carrier through the cytosolic side. When oleate, which interacts through the matrix side, was used it was found that the amine amplified the effects of oleate on permeability transition. The results also show that N-ethylmaleimide strengthened the inhibition induced by carboxyatractyloside, agaric acid, and oleate on ADP exchange. Furthermore, it was also found that oleate improved the binding of eosin-5-maleimide on the adenine nucleotide translocase.

Topics: Adenosine Diphosphate; Atractyloside; Calcium; Citric Acid; Eosine Yellowish-(YS); Ethylmaleimide; Mitochondria; Mitochondrial ADP, ATP Translocases; Mitochondrial Swelling; Oleic Acid; Permeability

Palmitate-induced uncoupling, which involves ADP/ATP and aspartate/glutamate antiporters, has been studied in liver mitochondria of old rats (22-26 months) under conditions of lipid peroxidation and inhibition of oxidative stress by antioxidants--thiourea, Trolox, and ionol. It has been shown that in liver mitochondria of old rats in the absence of antioxidants and under conditions of overproduction of conjugated dienes, the protonophoric uncoupling activity of palmitate is not suppressed by either carboxyatractylate or aspartate used separately. However, the combination of carboxyatractylate and aspartate decreased uncoupling activity of palmitate by 81%. In this case, palmitate-induced uncoupling is limited by a stage insensitive to both carboxyatractylate and aspartate. In the presence of antioxidants, the palmitate-induced protonophoric uncoupling activity is suppressed by either carboxyatractylate or aspartate used separately. Under these conditions, palmitate-induced uncoupling is limited by a stage sensitive to carboxyatractylate (ADP/ATP antiporter) or aspartate (aspartate/glutamate antiporter). In the absence of antioxidants, the uncoupling activity of palmitate is not suppressed by ADP either in the absence or in the presence of aspartate. However, in the presence of thiourea, Trolox, or ionol ADP decreased the uncoupling activity of palmitate by 38%. It is concluded that in liver mitochondria of old rats the development of oxidative stress in the presence of physiological substrates of ADP/ATP and aspartate/glutamate antiporters (ADP and aspartate) results in an increase of the protonophoric uncoupling activity of palmitate.

Topics: Aging; Amino Acid Transport System X-AG; Animals; Antioxidants; Antiporters; Aspartic Acid; Atractyloside; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Respiration; Lipid Peroxidation; Male; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Oxidative Stress; Palmitates; Rats; Uncoupling Agents

The mitochondrial ADP/ATP carrier is a six helix bundle membrane transport protein, which couples the exit of ATP from the mitochondrial matrix to the entry of ADP. Extended (4x20 ns) molecular dynamics simulations of the carrier, in the presence and absence of bound inhibitor (carboxyatractyloside), have been used to explore the conformational dynamics of the protein in a lipid bilayer environment, in the presence and absence of the carboxyatractyloside inhibitor. The dynamic flexibility (measured as conformational drift and fluctuations) of the protein is reduced in the presence of bound inhibitor. Proline residues in transmembrane helices H1, H3 and H5 appear to form dynamic hinges. Fluctuations in inter-helix salt bridges are also observed over the time course of the simulations. Inhibitor-protein and lipid-protein interactions have been characterised in some detail. Overall, the simulations support a transport mechanism in which flexibility about the proline hinges enables a transition between a 'closed' and an 'open' pore-like state of the carrier protein.

Topics: Atractyloside; Computer Simulation; Kinetics; Lipid Bilayers; Mitochondrial ADP, ATP Translocases; Models, Molecular; Pliability; Proline; Protein Binding; Protein Conformation

Leak of protons into the mitochondrial matrix during substrate oxidation partially uncouples electron transport from phosphorylation of ADP, but the functions and source of basal and inducible proton leak in vivo remain controversial. In the present study we describe an endogenous activation of proton conductance in mitochondria isolated from rat and mouse skeletal muscle following addition of respiratory substrate. This endogenous activation increased with time, required a high membrane potential and was diminished by high concentrations of serum albumin. Inhibition of this endogenous activation by GDP [classically considered specific for UCPs (uncoupling proteins)], carboxyatractylate and bongkrekate (considered specific for the adenine nucleotide translocase) was examined in skeletal muscle mitochondria from wild-type and Ucp3-knockout mice. Proton conductance through endogenously activated UCP3 was calculated as the difference in leak between mitochondria from wild-type and Ucp3-knockout mice, and was found to be inhibited by carboxyatractylate and bongkrekate, but not GDP. Proton conductance in mitochondria from Ucp3-knockout mice was strongly inhibited by carboxyatractylate, bongkrekate and partially by GDP. We conclude the following: (i) at high protonmotive force, an endogenously generated activator stimulates proton conductance catalysed partly by UCP3 and partly by the adenine nucleotide translocase; (ii) GDP is not a specific inhibitor of UCP3, but also inhibits proton translocation by the adenine nucleotide translocase; and (iii) the inhibition of UCP3 by carboxyatractylate and bongkrekate is likely to be indirect, acting through the adenine nucleotide translocase.

Topics: Animals; Atractyloside; Bongkrekic Acid; Energy Metabolism; Female; Ion Channels; Male; Malonates; Membrane Potential, Mitochondrial; Mice; Mice, Knockout; Mitochondria, Muscle; Mitochondrial ADP, ATP Translocases; Mitochondrial Proteins; Muscle, Skeletal; Nitrogen Oxides; Palmitates; Proton Pumps; Rats; Rats, Wistar; Serum Albumin, Bovine; Time Factors; Uncoupling Agents; Uncoupling Protein 3

The aim of this work was to characterize the effect of bongkrekic acid (BKA), atractyloside (ATR) and carboxyatractyloside (CAT) on single channel properties of chloride channels from mitochondria. Mitochondrial membranes isolated from a rat heart muscle were incorporated into a bilayer lipid membrane (BLM) and single chloride channel currents were measured in 250/50 mM KCl cis/trans solutions. BKA (1-100 microM), ATR and CAT (5-100 microM) inhibited the chloride channels in dose-dependent manner. The inhibitory effect of the BKA, ATR and CAT was pronounced from the trans side of a BLM and it increased with time and at negative voltages (trans-cis). These compounds did not influence the single channel amplitude, but decreased open dwell time of channels. The inhibitory effect of BKA, ATR and CAT on the mitochondrial chloride channel may help to explain some of their cellular and/or subcellular effects.

Topics: Animals; Atractyloside; Blotting, Western; Bongkrekic Acid; Chloride Channels; Dose-Response Relationship, Drug; Membrane Potential, Mitochondrial; Mitochondrial Membranes; Myocardium; Rats

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.

Topics: Adenosine Diphosphate; Animals; Atractyloside; Calcium Signaling; Copper; Ethylmaleimide; Glutathione; Mersalyl; Mitochondrial ADP, ATP Translocases; Mitochondrial Membranes; Mitochondrial Proteins; Mitochondrial Swelling; Permeability; Rats; Sulfhydryl Compounds; Titrimetry

Genetic expression versus plasmidic overexpression of a functional recombinant fusion protein combining the yeast Saccharomyces cerevisiae mitochondrial ADP/ATP carrier (Anc2p) and the iso-1-cytochrome c (Cyc1p) has been investigated, with the main aim of increasing the polar surface of the carrier to improve its crystallization properties. The gene encoding the his6-tagged fusion protein was expressed in yeast under the control of the regulatory sequences of ScANC2 or under the control of the strong yeast PMA1 promoter. In both cases, the chimeric carrier, Anc2-Cyc1(His6)p, was able to restore growth on a non-fermentable carbon source of a yeast strain devoid of functional ADP/ATP carrier, demonstrating its transport activity. Nevertheless, when the expression vector was used, the level of expression of Anc2-Cyc1(His6)p was no greater than that of the chimeric carrier obtained in yeast mitochondria after homologous recombination. Optimal conditions to extract and to purify Anc2-Cyc1(His6)p were determined. A series of detergents was screened for their ability to extract and to preserve in vitro the chimeric carrier. A rapid, single step purification of Anc2-Cyc1(His6)p was developed, using n-dodecyl-beta-d-maltoside (DoDM) as the best detergent to solubilize the chimeric protein. Carboxyatractyloside- (CATR-) and nucleotide-binding sites were preserved in the purified protein. Moreover, the Cyc1p moiety of Anc2-Cyc1(His6)p-CATR complex solubilized in DoDM was still able to interact in vitro with the cytochrome c oxidase (COX), with the same affinity as yeast Cyc1p. Improved production and purification of Anc2-Cyc1(His6)p-CATR complex opens up new possibilities for the use of this protein in crystallographic approaches to the yeast ADP/ATP carrier. Furthermore, Anc2-Cyc1(His6)p may be an useful molecular tool to investigate in vivo interactions between components of the respiratory chain complexes such as COX and the proteins implicated in ATP biogenesis, such as the ATP/ADP carrier.

Topics: Atractyloside; Blotting, Western; Cytochromes c; Genetic Vectors; Genome, Fungal; Mitochondria; Mitochondrial ADP, ATP Translocases; Oxidation-Reduction; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins

Inner membrane mitochondria undergo a permeability increase elicited after the opening of a nonspecific pore due to supraphysiological matrix Ca2+ load, and the presence of an inducer. Multiple inducers have been used to promote the transition in permeability; among them are carboxyatractyloside (CAT) and reactive oxygen-derived species. In contrast, inhibitors such as ADP and cyclosporin A have been commonly used. In this work, we show that the opening or closure of the nonspecific pore depends on the cationic composition of the incubation medium. It was found that when mitochondria were incubated in either 125 mM KCl or 125 mM LiCl, ADP was essential to maintain selective membrane permeability. Interestingly, the nucleotide was not required when the medium contained 125 mM NaCl. Furthermore, it was established that CAT promotes membrane leakage in K(+)- or Li(+)-incubated mitochondria, while it failed to do so in Na(+)-incubated mitochondria. Evidence is also presented on the ability of Na+ to induce resistance in mitochondria against membrane damage by oxidative stress. Mitochondrial Ca2+ discharge, swelling, and transmembrane electric gradient were analyzed to establish permeability transition. It is concluded that the protection provided by Na+ was accomplished by inducing matrix K+ depletion, which, in turn, diminished the free fraction of matrix Ca2+.

Topics: Adenosine Diphosphate; Animals; Atractyloside; Calcium; Cations, Monovalent; In Vitro Techniques; Kidney Cortex; Lithium; Membrane Potentials; Mitochondria; Mitochondrial Membranes; Mitochondrial Swelling; Oxidative Stress; Permeability; Potassium; Rats; Sodium; Uncoupling Agents

In brown-fat mitochondria, fatty acids induce thermogenic uncoupling through activation of UCP1 (uncoupling protein 1). However, even in brown-fat mitochondria from UCP1-/- mice, fatty-acid-induced uncoupling exists. In the present investigation, we used the inhibitor CAtr (carboxyatractyloside) to examine the involvement of the ANT (adenine nucleotide translocator) in the mediation of this UCP1-independent fatty-acid-induced uncoupling in brown-fat mitochondria. We found that the contribution of ANT to fatty-acid-induced uncoupling in UCP1-/- brown-fat mitochondria was minimal (whereas it was responsible for nearly half the fatty-acid-induced uncoupling in liver mitochondria). As compared with liver mitochondria, brown-fat mitochondria exhibit a relatively high (UCP1-independent) basal respiration ('proton leak'). Unexpectedly, a large fraction of this high basal respiration was sensitive to CAtr, whereas in liver mitochondria, basal respiration was CAtr-insensitive. Total ANT protein levels were similar in brown-fat mitochondria from wild-type mice and in liver mitochondria, but the level was increased in brown-fat mitochondria from UCP1-/- mice. However, in liver, only Ant2 mRNA was found, whereas in brown adipose tissue, Ant1 and Ant2 mRNA levels were equal. The data are therefore compatible with a tentative model in which the ANT2 isoform mediates fatty-acid-induced uncoupling, whereas the ANT1 isoform may mediate a significant part of the high basal proton leak in brown-fat mitochondria.

Topics: Adenine Nucleotide Translocator 1; Adenine Nucleotide Translocator 2; Adipose Tissue, Brown; Animals; Atractyloside; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Respiration; Crosses, Genetic; Fatty Acids; Guanosine Diphosphate; Ion Channels; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Mitochondria, Liver; Mitochondrial Proteins; Models, Biological; Oleic Acid; Organ Specificity; Oxygen Consumption; Palmitates; Protons; Pyruvic Acid; RNA, Messenger; Thermogenesis; Uncoupling Agents; Uncoupling Protein 1

Etiolated early seedlings of durum wheat submitted to moderate and severe salt (NaCl) and osmotic (mannitol) stress showed no relevant increase of both transcript levels of two plant uncoupling protein (pUCP)-related genes and maximal pUCP activity in purified mitochondria (which estimates protein level); contrarily, pUCP functioning due to endogenous free fatty acids strongly increased. These results show that pUCP activation under hyperosmotic stress may be due to modulation of pUCP reaction rather than to an increased protein synthesis. Finally, a properly developed method, based on a single membrane potential measurement, to evaluate both pUCP maximal activity and functioning, is reported.

Topics: Adenosine Triphosphate; Antiporters; Atractyloside; Fatty Acids, Nonesterified; Gene Expression Regulation; Genes, Plant; Hypertonic Solutions; Ion Channels; Membrane Potentials; Mitochondrial Proteins; Osmotic Pressure; Oxidative Phosphorylation; Salts; Seedlings; Transcription, Genetic; Triticum; Uncoupling Protein 1

The mitochondrial adenosine diphosphate/adenosine triphosphate (ADP/ATP) carrier has been recently crystallized in complex with its specific inhibitor carboxyatractyloside (CATR). In the crystal structure, the six-transmembrane helix bundle that defines the nucleotide translocation pathway is closed on the matrix side due to sharp kinks in the odd-numbered helices. The closed conformation is further sealed by the loops protruding into the matrix that interact through an intricate network of charge-pairs. To gain insight into its structural dynamics we performed molecular dynamics (MD) simulation studies of the ADP/ATP carrier with and without its cocrystallized inhibitor. The two trajectories sampled a conformational space around two different configurations characterized by distinct salt-bridge networks with a significant shift from inter- to intrarepeat bonding on the matrix side in the absence of CATR. Analysis of the geometrical parameters defining the transmembrane helices showed that even-numbered helices can undergo a face rotation, whereas odd-numbered helices can undergo a change in the wobble angle with a conserved proline acting as molecular hinge. Our results provide new information on the dynamical properties of the ADP/ATP carrier and for the first time yield a detailed picture of a stable carrier conformation in absence of the inhibitor.

Topics: Animals; Atractyloside; Cattle; Computer Simulation; Mitochondrial ADP, ATP Translocases; Models, Molecular; Molecular Conformation

The mechanism of uncoupling by lauryl sulfate (LS) has been studied. The very fact that uncoupling by low concentration of LS (a strong acid) resembles very much that by fatty acids (weak acids) was used as an argument against the fatty acid cycling scheme of uncoupling where protonated fatty acids operate as a protonophore. We have found that rat liver and heart muscle mitochondria can be uncoupled by low (70 microM) LS concentration in a fashion completely arrested by the ATP/ADP antiporter inhibitor carboxyatractylate (CAtr). On the other hand, uncoupling by two-fold higher LS concentration is not sensitive to CAtr. Addition of oleate desensitizes mitochondria to low LS so that addition of bovine serum albumin becomes necessary to recouple mitochondria. The data are accounted for assuming that low LS releases endogenous fatty acids from some mitochondrial depots, and these fatty acids are responsible for uncoupling. As to high LS, it causes a nonspecific (CAtr-insensitive) damage to the mitochondrial membrane.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Antiporters; Atractyloside; Fatty Acids; Mitochondria, Liver; Mitochondrial Membranes; Rats; Sodium Dodecyl Sulfate; Surface-Active Agents; Uncoupling Agents

Carboxyatractylate (CAT) and atractylate inhibit the mitochondrial adenine nucleotide translocator (ANT) and stimulate the opening of permeability transition pore (PTP). Following pretreatment of mouse liver mitochondria with 5 microM CAT and 75 microM Ca2+, the activity of PTP increased, but addition of 2 mM ADP inhibited the swelling of mitochondria. Extramitochondrial Ca2+ concentration measured with Calcium-Green 5N evidenced that 2 mM ADP did not remarkably decrease the free Ca2+ but the release of Ca2+ from loaded mitochondria was stopped effectively after addition of 2 mM ADP. CAT caused a remarkable decrease of the maximum amount of calcium ions, which can be accumulated by mitochondria. Addition of 2 mM ADP after 5 microM CAT did not change the respiration, but increased the mitochondrial capacity for Ca2+ at more than five times. Bongkrekic acid (BA) had a biphasic effect on PT. In the first minutes 5 microM BA increased the stability of mitochondrial membrane followed by a pronounced opening of PTP too. BA abolished the action about of 1 mM ADP, but was not able to induce swelling of mitochondria in the presence of 2 mM ADP. We conclude that the outer side of inner mitochondrial membrane has a low affinity sensor for ADP, modifying the activity of PTP. The pathophysiological importance of this process could be an endogenous prevention of PT at conditions of energetic depression.

Topics: Adenine Nucleotide Translocator 3; Adenosine Diphosphate; Animals; Atractyloside; Biological Transport; Bongkrekic Acid; Calcium; Intracellular Membranes; Liver; Mice; Mitochondria; Oxygen Consumption; Permeability; Porins; Spectrometry, Fluorescence

A recombinant fusion protein combining the mitochondrial ADP/ATP carrier (Anc2p) and the iso-1-cytochrome c (Cyc1p), both from Saccharomyces cerevisiae, has been genetically elaborated with the aim of increasing the polar surface area of the carrier to facilitate its crystallization. The gene encoding the his-tagged fusion protein was expressed in yeast under the control of the regulatory sequences of ScANC2. The chimeric carrier, Anc2-Cyc1(His6)p, was able to restore growth on a non-fermentable carbon source of a yeast strain devoid of functional ADP/ATP carrier, which demonstrated its transport activity. The kinetic exchange properties of Anc2-Cyc1(His6)p and the wild type his-tagged carrier Anc2(His6)p were very similar. However, Anc2-Cyc1(His6)p restored cell growth less efficiently than Anc2(His6)p which correlates with the lower amount found in mitochondria. Purification of Anc2-Cyc1(His6)p in complex with carboxyatractyloside (CATR), a high affinity inhibitor of ADP/ATP transport, was achieved by combining ion-exchange chromatography and ion-metal affinity chromatography in the presence of LAPAO, an aminoxide detergent. As characterized by absorption in the visible range, heme was found to be present in isolated Anc2-Cyc1(His6)p, giving the protein a red color. Large-scale purification of Anc2-Cyc1(His6)p-CATR complex opens up novel possibilities for the use of crystallographic approaches to the yeast ADP/ATP carrier.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Atractyloside; Chromatography; Cloning, Molecular; Crystallization; Cytochromes c; Escherichia coli; Genetic Engineering; Heme; Histidine; Kinetics; Mitochondrial ADP, ATP Translocases; Molecular Probes; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins

Cocklebur (Xanthium strumarium) is an herbaceous annual plant with worldwide distribution. The seeds contain the glycoside carboxyatractyloside, which is highly toxic to animals. We describe nine cases of carboxyatractyloside poisoning in humans which, to our knowledge, has not previously been reported. The clinical, laboratory and histopathological findings and our therapeutic approach are also discussed.. The patients presented with acute onset abdominal pain, nausea and vomiting, drowsiness, palpitations, sweating and dyspnoea. Three of them developed convulsions followed by loss of consciousness and death.. Laboratory findings showed raised liver enzymes, indicating severe hepatocellular damage. BUN and creatinine levels were raised, especially in the fatal cases who also displayed findings of consumption coagulopathy. CPK-MB values indicative of myocardial injury were also raised, especially in the fatal cases. Three of the patients died within 48 hours of ingesting carboxyatractyloside. Post-mortem histopathology of the liver confirmed centrilobular hepatic necrosis and renal proximal tubular necrosis, secondary changes owing to increased permeability and microvascular haemorrhage in the cerebrum and cerebellum, and leucocytic infiltrates in the muscles and various organs including pancreas, lungs and myocardium.. Carboxyatractyloside poisoning causes multiple organ dysfunction and can be fatal. Coagulation abnormalities, hyponatraemia, marked hypoglycaemia, icterus and hepatic and renal failure are signs of a poor prognosis. No antidote is available and supportive therapy is the mainstay of treatment.

Topics: Adolescent; Adult; Atractyloside; Cardiomyopathies; Child; Child, Preschool; Coma; Disseminated Intravascular Coagulation; Fatal Outcome; Female; Helianthus; Humans; Kidney Diseases; Liver Diseases; Male; Multiple Organ Failure; Plant Poisoning; Seizures; Vomiting; Xanthium

The purpose of this work was to assess the effect of oligomycin on the mitochondrial membrane permeability transition. The antibiotic was found to strengthen cyclosporin A (CSA)-induced protection of non-specific permeability, which is triggered by a matrix Ca2+ load in the absence of ADP. Oligomycin also reinforced the protective effect of CSA on carboxyatractyloside-induced pore opening in the absence of ADP, but failed to do so in mitochondria incubated under anaerobic conditions or after addition of CCCP. Analyzing the efflux of matrix Ca2+, we found that mitochondrial swelling and the collapse of the transmembrane electric gradient coincided with membrane leakage. The effects of the antibiotic were observed in phosphate-containing media but not in the presence of acetate. Furthermore, N-ethylmaleimide hindered the protective effect of oligomycin-CSA. In addition, the matrix phosphate concentration increased concurrently with a diminution in the matrix-free fraction of Ca2+. We concluded that oligomycin increases phosphate uptake by stimulating the phosphate-/OH- exchange reaction.

Topics: Acetates; Animals; Atractyloside; Calcium; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cyclosporine; Drug Synergism; Enzyme Inhibitors; Ethylmaleimide; Intracellular Membranes; Kidney; Membrane Potentials; Mitochondria; Mitochondrial Swelling; Oligomycins; Permeability; Phosphates; Rats; Uncoupling Agents

Pharmacological modulation of the mitochondrial ATP-sensitive K+ channel (mitoKATP) sensitive to diazoxide and 5-hydroxydecanoate (5-HD) represents an attractive strategy to protect cells against ischaemia/reperfusion- and stroke-related injury. To re-evaluate a functional role for the mitoKATP in brain, we used Percoll-gradient-purified brain nonsynaptosomal mitochondria in a light absorbance assay, in radioisotope measurements of matrix volume, and in measurements of respiration, membrane potential (DeltaPsi) and depolarization-induced K+ efflux. The changes in mitochondrial morphology were evaluated by transmission electron microscopy (TEM). Polyclonal antibodies raised against certain fragments of known sulphonylurea receptor subunits, SUR1 and SUR2, and against different epitopes of K+ inward rectifier subunits Kir 6.1 and Kir 6.2 of the ATP-sensitive K+ channel of the plasma membrane (cellKATP), were employed to detect similar subunits in brain mitochondria. A variety of plausible blockers (ATP, 5-hydroxydecanoate, glibenclamide, tetraphenylphosphonium cation) and openers (diazoxide, pinacidil, chromakalim, minoxidil, testosterone) of the putative mitoKATP were applied to show the role of the channel in regulating matrix volume, respiration, and DeltaPsi and K+ fluxes across the inner mitochondrial membrane. None of the pharmacological agents applied to brain mitochondria in the various assays pinpointed processes that could be unequivocally associated with mitoKATP activity. In addition, immunoblotting analysis did not provide explicit evidence for the presence of the mitoKATP, similar to the cellKATP, in brain mitochondria. On the other hand, the depolarization-evoked release of K+ suppressed by ATP could be re-activated by carboxyatractyloside, an inhibitor of the adenine nucleotide translocase (ANT). Moreover, bongkrekic acid, another inhibitor of the ANT, inhibited K+ efflux similarly to ATP. These observations implicate the ANT in ATP-sensitive K+ transport in brain mitochondria.

Topics: Adenosine Triphosphate; Animals; ATP-Binding Cassette Transporters; Atractyloside; Brain; Culture Media; Decanoic Acids; Diazoxide; Hydroxy Acids; In Vitro Techniques; KATP Channels; Male; Mitochondria; Mitochondrial ADP, ATP Translocases; Oxygen Consumption; Potassium; Potassium Channel Blockers; Potassium Channels; Potassium Channels, Inwardly Rectifying; Potassium Chloride; Rats; Rats, Sprague-Dawley; Receptors, Drug; Sulfonylurea Receptors

Ca(2+)-induced mitochondrial depolarization was studied in single isolated rat brain and liver mitochondria. Digital imaging techniques and rhodamine 123 were used for mitochondrial membrane potential measurements. Low Ca(2+) concentrations (about 30--100 nM) initiated oscillations of the membrane potential followed by complete depolarization in brain mitochondria. In contrast, liver mitochondria were less sensitive to Ca(2+); 20 microm Ca(2+) was required to depolarize liver mitochondria. Ca(2+) did not initiate oscillatory depolarizations in liver mitochondria, where each individual mitochondrion depolarized abruptly and irreversibly. Adenine nucleotides dramatically reduced the oscillatory depolarization in brain mitochondria and delayed the onset of the depolarization in liver mitochondria. In both type of mitochondria, the stabilizing effect of adenine nucleotides completely abolished by an inhibition of adenine nucleotide translocator function with carboxyatractyloside, but was not sensitive to bongkrekic acid. Inhibitors of mitochondrial permeability transition cyclosporine A and bongkrekic acid also delayed Ca(2+)-depolarization. We hypothesize that the oscillatory depolarization in brain mitochondria is associated with the transient conformational change of the adenine nucleotide translocator from a specific transporter to a non-specific pore, whereas the non-oscillatory depolarization in liver mitochondria is caused by the irreversible opening of the pore.

Topics: Adenine Nucleotides; Animals; Atractyloside; Brain; Calcium; Cations, Divalent; Cyclosporine; Membrane Potentials; Mitochondria; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Rats

Structure-function relationships of the membrane-embedded Saccharomyces cerevisiae mitochondrial ADP/ATP carrier were investigated through two independent approaches, namely, limited proteolysis and cysteine labeling. Experiments were carried out in the presence of either carboxyatractyloside (CATR) or bongkrekic acid (BA), two specific inhibitors of the ADP/ATP transport that bind to two distinct conformers involved in the translocation process. The proteolysis approach allowed us to demonstrate (i) that N- and C-terminal extremities of ADP/ATP carrier are facing the intermembrane space and (ii) that the central region of the carrier corresponding to the matrix loop m2 is accessible to externally added trypsin in a conformation-sensitive manner, being cleaved at the Lys163-Gly164 and Lys178-Thr179 bonds in the carrier-CATR and the carrier-BA complexes, respectively. The cysteine labeling approach was carried out on the S161C mutant of the ADP/ATP carrier. This variant of the carrier is fully active, displaying nucleotide transport kinetic parameters and inhibitor binding properties similar to that of wild-type carrier. Alkylation experiments, carried out on mitochondria with the nonpermeable reagents eosin-5-maleimide and iodoacetamidyl-3,6-dioxaoctanediamine-biotin, showed that Cys 161 is accessible from the outside in the carrier-CATR complex, whereas it is masked in the carrier-BA complex. Taken together, our results indicate that the matrix loop m2 connecting the transmembrane helices H3 to H4 intrudes to some extent into the inner mitochondrial membrane. Its participation in the translocation of ADP/ATP is strongly suggested, based on the finding that its accessibility to reagents added outside mitochondria is modified according to the conformational state of the carrier.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Atractyloside; Bongkrekic Acid; Mitochondria; Mitochondrial ADP, ATP Translocases; Protein Conformation; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure-Activity Relationship

The existence of a biochemical threshold effect in the metabolic expression of oxidative phosphorylation deficiencies has considerable implications for the understanding of mitochondrial bioenergetics and the study of mitochondrial diseases. However, the molecular bases of this phenomenon remain unclear. We report here a new mechanism to explain this threshold effect, based on a reserve of enzymes not initially participating in the respiratory rate that can be activated either to respond to a flux increase or to compensate for a defect induced by a mutation. We show that this mobilization occurs through 1) the assembly of inactive adenine nucleotide translocator isoform 1 subunits into oligomeric active carriers or 2) conformational changes in the adenine nucleotide translocator isoform 1 in a permeability transition pore-like structure. We discuss how these transitions are sensitive to the steady state of oxidative phosphorylation functioning or tissue and analyze their consequences on the threshold effect.

Topics: Adenine Nucleotide Translocator 1; Adenine Nucleotides; Animals; Atractyloside; Biopsy; Blotting, Western; Electron Transport; Electrophoresis, Polyacrylamide Gel; Humans; Kinetics; Male; Mitochondria; Mitochondrial ADP, ATP Translocases; Mitochondrial Diseases; Models, Biological; Muscles; Mutation; Oxidative Phosphorylation; Oxygen; Oxygen Consumption; Phosphorylation; Protein Conformation; Protein Isoforms; Rats; Rats, Wistar; Tissue Distribution

Mitochondria play a critical role in some forms of apoptosis, and the Ca(2+)-dependent permeability transition (PT) is a key initiator of this process. We quantitatively examined major control mechanisms of PT in rat brain (RBM) and liver (RLM) mitochondria. Compared with RLM, RBM were less sensitive to cyclosporin A (CsA), but the combined action of CsA+ADP was much more pronounced in RBM. Carboxyatractyloside abrogated the effects of all mPTP inhibitors in RBM but not in RLM, where the effects of CsA were not reduced. Estimated H(+)/Ca(2+) ratios were 0.81+/-0.01 for RLM and 0.84-0.93 for RBM, suggesting that Ca(2+) and Pi were sequestered in the matrix as CaHPO(4) and Ca(3)(PO(4))(2) salts, and that RBM sequester more CaPi as the least soluble salt. We conclude that: (1) RBM and RLM differ in their baseline behavior of the PT and in their responses to PT modifiers, and (2) PT modifiers can be functionally divided into those which directly affect the mitochondrial PT pore and are not energy-dependent (CsA, free Ca(2+), ADP(ex), and Mg(2+)), and those which affect the energy-dependent calcium phosphate sequestration process (ADP(mt), CATR, local anesthetics). We also conclude that ANT affects PT by changing mitochondrial capacity for energization.

Topics: Adenosine Diphosphate; Alamethicin; Animals; Atractyloside; Brain; Calcium Phosphates; Cyclosporine; Dibucaine; Hydrogen-Ion Concentration; Intracellular Membranes; Ion Channels; Light; Male; Membrane Potentials; Mitochondria; Mitochondria, Liver; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Oligomycins; Rats; Rats, Inbred Lew; Scattering, Radiation; Titrimetry

It was previously shown that carvedilol, a beta-adrenergic receptor antagonist with antioxidant properties, was able to inhibit the mitochondrial permeability transition (MPT). In the present work, the hypothesis was that the negative impact of carvedilol on the MPT was specifically the result of its antioxidant effect. For the current investigation, we used three different MPT inducers. MPT-associated events were tested to study the protective effect of both carvedilol and cyclosporin-A, the known MPT inhibitor. Carvedilol inhibited mitochondrial swelling with calcium plus phosphate and with calcium plus t-butylhydroperoxide, but not with calcium plus carboxyatractyloside. Carvedilol inhibited the oxidation of thiol groups with calcium plus phosphate (p < 0.01) and with calcium plus t-butylhydroperoxide (p < 0.05), but not with calcium plus carboxyatractyloside--in opposition to the full protection afforded by cyclosporin-A when using calcium and carboxyatractyloside. Our results showed that carvedilol was effective only when the MPT was triggered by a primary oxidative process. This finding implies that the antioxidant properties of carvedilol are crucial for the observed effects and reinforces the advantageous use of carvedilol in cardiac pathologies associated with enhanced cellular oxidative stress.

Topics: Adrenergic beta-Antagonists; Animals; Antioxidants; Atractyloside; Calcium; Carbazoles; Carvedilol; Cyclosporine; Immunosuppressive Agents; In Vitro Techniques; Male; Mitochondria, Heart; Oxidative Stress; Permeability; Propanolamines; Proteins; Rats; Rats, Wistar; Sulfhydryl Compounds; tert-Butylhydroperoxide

To investigate the structural and functional features of the second alpha-helical transmembrane segment (TM2) of the mitochondrial ADP/ATP carrier (AAC), we adopted cysteine scanning mutagenesis analysis. Single-cysteine mutations of yeast AAC were systematically introduced at residues 98-106 in TM2, and the mutants were treated with the fluorescent SH reagent eosin-5-maleimide (EMA). EMA modified different amino acid residues of alpha-helical TM2 between the two distinct carrier conformations, called the m-state and the c-state, in which the substrate recognition site faces the matrix and cytosol, respectively. When amino acids in the helix were projected on a wheel plot, these EMA-modified amino acids were observed at distinct sides of the wheel. Since the SH reagent specifically modified cysteine in the water-accessible environment, these results indicate that distinct helical surfaces of TM2 faced the water-accessible space between the two conformations, possibly as a result of twisting of this helix. In the recently reported crystal structure of bovine AAC, several amino acids faced cocrystallized carboxyatractyloside (CATR), a specific inhibitor of the carrier. These residues correspond to those modified with EMA in the yeast carrier in the c-state. Since the binding site of CATR is known to overlap that of the transport substrate, the water-accessible space was thought to be a substrate transport pathway, and hence, the observed twisting of TM2 between the m-state and the c-state may be involved in the process of substrate translocation. On the basis of the results, the roles of TM2 in the transport function of AAC were discussed.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Atractyloside; Binding Sites; Cattle; Cysteine; Enzyme Inhibitors; Eosine Yellowish-(YS); Intracellular Membranes; Mitochondrial ADP, ATP Translocases; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Conformation; Protein Structure, Secondary; Protein Transport; Saccharomyces cerevisiae Proteins

The mitochondrial permeability transition (MPT) is an event related to severe oxidative stress (for example, during myocardial ischemia and reperfusion) and excessive mitochondrial calcium accumulation, also being implicated in cell death. In this study, we compared the effect of carvedilol on the cardiac MPT induced by calcium and phosphate (Ca/Pi) and calcium/carboxyatractyloside (Ca/Catr). Oxidative stress plays a major role in MPT induction by Ca/Pi, leading to the oxidation of protein thiol groups, in contrast with Ca/Catr, where such oxidation is secondary to MPT induction and is not caused by oxidative stress.. Mitochondria were isolated from rat hearts and parameters related to MPT induction were evaluated (n = 5 for each inducer): mitochondrial swelling and oxidation of protein thiol groups (both measured by spectrophotometry).. Using Ca/Pi, carvedilol protected mitochondria from MPT induction, particularly in its high conductance form. Its effect was demonstrated by analyzing the decrease in mitochondrial swelling amplitude. Simultaneously, we observed inhibition of protein thiol group oxidation (p < 0.001). By contrast, carvedilol did not show any protective effect with Ca/Catr.. Carvedilol was only effective against the MPT when the oxidation of protein thiol groups was the cause and not the consequence of the MPT phenomenon. The results clearly show that during myocardial aggressions (ischemia and reperfusion, for example), the protective effect of carvedilol is primarily due to an antioxidant mechanism, inhibiting the production and effects of reactive oxygen species.

Topics: Animals; Antioxidants; Atractyloside; Calcium; Carbazoles; Carvedilol; Intracellular Membranes; Male; Mitochondria, Heart; Oxidative Stress; Permeability; Phosphates; Propanolamines; Rats; Rats, Wistar; Reactive Oxygen Species

A hallmark of central nervous system (CNS) pathology is reactive astrocyte production of the chronic glial scar that is inhibitory to neuronal regeneration. The reactive astrocyte response is complex; these cells also produce neurotrophic factors and are responsible for removal of extracellular glutamate, the excitatory neurotransmitter that rises to neurotoxic levels in injury and disease. To identify genes expressed by reactive astrocytes, we employed an in vivo model of the glial scar and differential display PCR and found an increase in the level of Ant1, a mitochondrial ATP/ADP exchanger that facilitates the flux of ATP out of the mitochondria. Ant1 expression in reactive astrocytes is regulated by transforming growth factor-beta1, a pluripotent CNS injury-induced cytokine. The significance of increased Ant1 is evident from the observation that glutamate uptake is significantly decreased in astrocytes from Ant1 null mutant mice while a specific Ant inhibitor reduces glutamate uptake in wild-type astrocytes. Thus, the astrocytic response to CNS injury includes an apparent increase in energy mobilization capacity by Ant1 that contributes to neuroprotective, energy-dependent glutamate uptake.

Topics: Adenine Nucleotide Translocator 1; Animals; Astrocytes; Atractyloside; Biological Transport; Brain Injuries; Cells, Cultured; Collodion; Disease Models, Animal; Gene Expression Profiling; Gene Expression Regulation; Genes, Reporter; Gliosis; Glutamic Acid; Implants, Experimental; Male; Mice; Mitochondria; Polymerase Chain Reaction; Rats; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1

We have studied changes in hepatic mitochondrial efficiency induced by 24-h fasting or acclimation at 29 degrees C, two conditions of reduced thermogenesis. Basal and palmitate-induced proton leak, which contribute to mitochondrial efficiency, are not affected after 24-h fasting, when serum free triiodothyronine decreases significantly and serum free fatty acids increase significantly. In rats at 29 degrees C, in which serum free triiodothyronine and fatty acids decrease significantly, basal proton leak increases significantly, while no variation is found in palmitate-induced proton leak. The present results indicate that mitochondrial efficiency in the liver is not related to a physiological decrease in whole body thermogenesis.

Topics: Animals; Atractyloside; Biological Transport; Dose-Response Relationship, Drug; Electron Transport Complex IV; Fasting; Fatty Acids; Hot Temperature; Kinetics; Liver; Male; Membrane Potentials; Mitochondria, Liver; Oxygen Consumption; Palmitic Acid; Protons; Rats; Rats, Wistar; Temperature; Time Factors; Triiodothyronine

KATP channel openers have been shown to protect ischemic-reperfused myocardium by mimicking ischemic preconditioning, although their mechanisms of action have not been fully clarified. In this study we investigated the influence of the adenine nucleotide translocase (ANT) inhibitors--carboxyatractyloside (CAT) and bongkrekic acid (BA)--on the diazoxide- and pinacidil-induced uncoupling of isolated rat heart mitochondria respiring on pyruvate and malate (6 + 6 mM). We found that both CAT (1.3 microM) and BA (20 microM) markedly reduced the uncoupling of mitochondrial oxidative phosphorylation induced by the K(ATP) channel openers. Thus, the uncoupling effect of diazoxide and pinacidil is evident only when ANT is not fixed by inhibitors in neither the C- nor the M-conformation. Moreover, the uncoupling effect of diazoxide and pinacidil was diminished in the presence of ADP or ATP, indicating a competition of K(ATP) channel openers with adenine nucleotides. CAT also abolished K+-dependent mitochondrial respiratory changes. Thus ANT could also be involved in the regulation of K(ATP)-channel-openers-induced K+ flux through the inner mitochondrial membrane.

Topics: Animals; Atractyloside; Bongkrekic Acid; Cells, Cultured; Diazoxide; Dose-Response Relationship, Drug; Extracellular Matrix; Ion Channel Gating; Membrane Potentials; Membrane Proteins; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Pinacidil; Potassium; Potassium Channels; Proton Pumps; Rats; Rats, Wistar

Oxidative stress and mitochondrial dysfunction are associated with disease and aging. Oxidative stress results from overproduction of reactive oxygen species (ROS), often leading to peroxidation of membrane phospholipids and production of reactive aldehydes, particularly 4-hydroxy-2-nonenal. Mild uncoupling of oxidative phosphorylation protects by decreasing mitochondrial ROS production. We find that hydroxynonenal and structurally related compounds (such as trans-retinoic acid, trans-retinal and other 2-alkenals) specifically induce uncoupling of mitochondria through the uncoupling proteins UCP1, UCP2 and UCP3 and the adenine nucleotide translocase (ANT). Hydroxynonenal-induced uncoupling was inhibited by potent inhibitors of ANT (carboxyatractylate and bongkrekate) and UCP (GDP). The GDP-sensitive proton conductance induced by hydroxynonenal correlated with tissue expression of UCPs, appeared in yeast mitochondria expressing UCP1 and was absent in skeletal muscle mitochondria from UCP3 knockout mice. The carboxyatractylate-sensitive hydroxynonenal stimulation correlated with ANT content in mitochondria from Drosophila melanogaster expressing different amounts of ANT. Our findings indicate that hydroxynonenal is not merely toxic, but may be a biological signal to induce uncoupling through UCPs and ANT and thus decrease mitochondrial ROS production.

Topics: Animals; Atractyloside; Bongkrekic Acid; Drosophila melanogaster; Enzyme Inhibitors; Female; Guanosine Diphosphate; Humans; Kidney; Mice; Mice, Knockout; Mitochondria; Mitochondrial ADP, ATP Translocases; Models, Biological; Oxidative Phosphorylation; Protons; Rats; Reactive Oxygen Species; Saccharomyces cerevisiae; Signal Transduction; Structure-Activity Relationship; Tretinoin; Uncoupling Agents

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.

Topics: 2,4-Dinitrophenol; Animals; Aspartic Acid; Atractyloside; Cell Respiration; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glutamic Acid; Laurates; Mersalyl; Mitochondria, Liver; Oxidative Phosphorylation; Palmitates; Phosphates; Rats; Temperature; Thermodynamics; Uncoupling Agents

The intracellular signaling pathway(s) through which second messenger ceramides induce gene expression in human cells has not yet been characterized. In the present study, ceramide-induced expression of intercellular adhesion molecule-1 (ICAM-1), which requires activation of transcription factor activator protein 2 (AP-2), was found to be mediated through a mitochondrial pathway. Inhibitors of mitochondrial electron transport chain (e.g. rotenone, thenoyltrifluoroacetone, and antimycin A) reduced ceramide-induced ICAM-1 expression. Stimulation of human keratinocytes with cell-permeant ceramides at concentrations that did not induce apoptosis (no activation of caspases 3, 8, and 9 and no nucleosomal fragmentation) but caused AP-2 activation and ICAM-1 induction released cytochrome c (cyt c) from mitochondria into the cytoplasm of cells. This cyt c release was an indispensable prerequisite for effective ceramide signaling, because its inhibition by modulating the mitochondrial megachannel with bonkrekic acid or carboxyatractyloside prevented ceramide-induced AP-2 activation and ICAM-1 expression. Analysis of the interaction between cyt c and AP-2 revealed that cyt c oxidized AP-2 and that this redox regulation greatly enhanced the DNA binding capacity of AP-2. Mitochondria thus have a previously unrecognized function in signaling ceramide-induced transcription factor activation and gene regulation.

Topics: Apoptosis; Atractyloside; Blotting, Western; Cell Line; Cell Survival; Cells, Cultured; Ceramides; Coloring Agents; Cytochromes c; Cytoplasm; DNA; DNA-Binding Proteins; Electron Transport; Fibroblasts; Glutathione; HeLa Cells; Humans; Intercellular Adhesion Molecule-1; Keratinocytes; Mitochondria; Oxygen; Precipitin Tests; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Tetrazolium Salts; Thiazoles; Time Factors; Transcription Factor AP-2; Transcription Factors

Two distinct conformations of the mitochondrial ADP/ATP carrier involved in the adenine nucleotide transport are called BA and CATR conformations, as they were distinguished by binding of specific inhibitors bongkrekic acid (BA) and carboxyatractyloside (CATR), respectively. To find out which amino acids are implicated in the transition between these two conformations, which occurs during transport, mutants of the Saccharomyces cerevisiae ADP/ATP carrier Anc2p responsible for resistance of yeast cells to BA were identified and characterized after in vivo chemical or UV mutagenesis. Only four different mutations could be identified in spite of a large number of mutants analyzed. They are located in the Anc2p transmembrane segments I (G30S), II (Y97C), III (L142S), and VI (G298S), and are independently enabling growth of cells in the presence of BA. The variant and wild-type Anc2p were produced practically to the same level in mitochondria, as evidenced by immunochemical analysis and by atractyloside binding experiments. ADP/ATP exchange mediated by Anc2p variants in isolated mitochondria was more efficient than that of the wild-type Anc2p in the presence of BA, confirming that BA resistance of the mutant cells was linked to the functional properties of the modified ADP/ATP carrier. These results suggest that resistance to BA is caused by alternate conformation of Anc2p due to appearance of Ser or Cys at specific positions. Different interactions of these residues with other amino acids and/or BA could prevent formation of stable inactive Anc2p . BA complex.

Topics: Anti-Bacterial Agents; Atractyloside; Bongkrekic Acid; Drug Resistance, Bacterial; Membrane Proteins; Mitochondrial ADP, ATP Translocases; Mitochondrial Proteins; Point Mutation; Protein Conformation; Saccharomyces cerevisiae Proteins

ATP, the principal energy currency of the cell, fuels most biosynthetic reactions in the cytoplasm by its hydrolysis into ADP and inorganic phosphate. Because resynthesis of ATP occurs in the mitochondrial matrix, ATP is exported into the cytoplasm while ADP is imported into the matrix. The exchange is accomplished by a single protein, the ADP/ATP carrier. Here we have solved the bovine carrier structure at a resolution of 2.2 A by X-ray crystallography in complex with an inhibitor, carboxyatractyloside. Six alpha-helices form a compact transmembrane domain, which, at the surface towards the space between inner and outer mitochondrial membranes, reveals a deep depression. At its bottom, a hexapeptide carrying the signature of nucleotide carriers (RRRMMM) is located. Our structure, together with earlier biochemical results, suggests that transport substrates bind to the bottom of the cavity and that translocation results from a transient transition from a 'pit' to a 'channel' conformation.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Amino Acid Sequence; Animals; Atractyloside; Binding Sites; Cattle; Crystallography, X-Ray; Enzyme Inhibitors; Mitochondrial ADP, ATP Translocases; Models, Molecular; Molecular Sequence Data; Protein Conformation; Static Electricity

High plasma level of triglycerides (TGs) is a common feature in atherosclerosis, obesity, diabetes, alcoholism, stress, and infection. Since mitochondria have been implicated in cell death under a variety of metabolic disorders, we examined liver mitochondrial functions in hypertriglyceridemic transgenic mice. Hypertriglyceridemia increased resting respiration and predisposed to mitochondrial permeability transition (MPT). Ciprofibrate therapy reduced plasma TG levels, normalized respiration, and prevented MPT. The higher resting respiration in transgenic mitochondria remained in the presence of the adenine nucleotide carrier inhibitor, carboxyatractyloside, bovine serum albumin, and the uncoupling proteins (UCPs) inhibitor, GDP. UCP2 content was similar in both control and transgenic mitochondria. We propose that faster resting respiration represents a regulated adaptation to oxidize excess free fatty acid in the transgenic mice.

Topics: Animals; Atractyloside; Cell Respiration; Clofibric Acid; Fibric Acids; Guanosine Diphosphate; Hypertriglyceridemia; Hypolipidemic Agents; Ion Channels; Liver; Membrane Potentials; Membrane Transport Proteins; Mice; Mice, Transgenic; Mitochondria; Mitochondrial Proteins; Serum Albumin, Bovine; Triglycerides; Uncoupling Protein 2

The influence of substrates on the role of cyclosporin A, to promote the closure of the permeability transition pore, was studied. It was found that in succinate-oxidizing mitochondria, cyclosporin inhibited pore opening as induced by carboxyatractyloside. The opposite occurred when mitochondrial respiration was supported by malate-glutamate, i.e., cyclosporin A was unable to block pore opening promoted by carboxyatractyloside. We propose that the failure of cyclosporin A to induce pore closure could be due to a low NADH matrix content.

Topics: Adenosine Diphosphate; Animals; Atractyloside; Calcium; Cyclosporine; Immunosuppressive Agents; In Vitro Techniques; Kidney; Male; Membranes; Mitochondria; NAD; Oxidation-Reduction; Permeability; Rats; Succinates

The effect of ethanol on the uncoupling activity of palmitate and recoupling activities of carboxyatractylate and glutamate was studied in liver mitochondria at various Mg2+ concentrations and medium pH values (7.0, 7.4, and 7.8). Ethanol taken at concentration of 0.25 M had no effect on the uncoupling activity of palmitic acid in the presence of 2 mM MgCl2 and decreased the recoupling effects of carboxyatractylate and glutamate added to mitochondria either just before or after the fatty acid. However, ethanol did not modify the overall recoupling effect of carboxyatractylate and glutamate taken in combination. The effect of ethanol decreased as medium pH was decreased to 7.0. Elevated concentration of Mg2+ (up to 8 mM) inhibits the uncoupling effect of palmitate. Ethanol eliminates substantially the recoupling effect of Mg2+ under these conditions, but does not influence the recoupling effects of carboxyatractylate and glutamate. It is inferred that ADP/ATP and aspartate/glutamate antiporters are involved in uncoupling function as single uncoupling complex with the common fatty acid pool. Fatty acid molecules gain the ability to migrate under the action of ethanol: from ADP/ATP antiporter to aspartate/glutamate antiporter on addition of carboxyatractylate and in opposite direction on addition of glutamate. Possible mechanisms of fatty acid translocation from one transporter to another are discussed.

Topics: Animals; Antiporters; Aspartic Acid; Atractyloside; Ethanol; Fatty Acids; Glutamic Acid; Hydrogen-Ion Concentration; Magnesium Chloride; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Oxidative Phosphorylation; Palmitates; Rats; Uncoupling Agents

Effects of the cross-linking catalyst copper-o-phenanthroline [Cu(OP)2] on the bovine heart mitochondrial ADP/ATP carrier solubilized with Triton X-100 were studied under various conditions. Without detergent treatment, Cu(OP)2 specifically catalyzed the formation of intermolecular disulfide bridges in submitochondrial particles between two Cys56 residues in the first loop facing the matrix space of the dimeric carrier [Majima, E., Ikawa, K., Takeda, M., Hashimoto, M., Shinohara, Y., and Terada, H. (1995) J. Biol. Chem. 270, 29548-29554]. However, an intramolecular disulfide bridge between Cys56 and Cys256 in the third loop was formed in the solubilized carrier. Proteolytic digestion of the carrier with lysylendopeptidase showed that it first cleaves the Lys42-Gln43 bond and then the Lys48-Gln49 bond of the first loop in the membrane-bound carrier, but it cleaves both sites almost simultaneously in the solubilized carrier. These features were observed only with the m-state carrier; the c-state carrier was not subject to any cross-linking or proteolytic digestion. It is suggested that the protruding first loop is located close to the third loop, which could be exposed to a certain degree.

Topics: Animals; Atractyloside; Bongkrekic Acid; Cattle; Cross-Linking Reagents; Disulfides; Eosine Yellowish-(YS); Mitochondria; Mitochondrial ADP, ATP Translocases; Myocardium; Octoxynol; Peptide Hydrolases; Phenanthrolines; Sequence Analysis, Protein; Solubility; Staining and Labeling

Effects of cold exposure in vivo and treatment with laurate, carboxyatractylate, atractylate, nucleotides, and BSA in vitro on potato tuber mitochondria have been studied. Cold exposure of tubers for 48-96 h resulted in some uncoupling that could be reversed completely by BSA and partially by ADP, ATP, UDP, carboxyatractylate, and atractylate. UDP was less effective than ADP and ATP, and atractylate was less effective than carboxyatractylate. The recoupling effects of nucleotides were absent when the nucleotides were added after carboxyatractylate. GDP, UDP, and CDP did not recouple mitochondria from either the control or the cold-exposed tubers. This indicates that the cold-induced fatty acid-mediated uncoupling in potato tuber mitochondria is partially due to the operation of the ATP/ADP antiporter. As to the plant uncoupling protein, its contribution to the uncoupling in tuber is negligible or, under the conditions used, somehow desensitized to nucleotides.

Topics: Adaptation, Physiological; Adenosine Diphosphate; Atractyloside; Carrier Proteins; Cold Temperature; Enzyme Inhibitors; Guanosine Diphosphate; Intracellular Membranes; Ion Channels; Lauric Acids; Membrane Potentials; Membrane Proteins; Mitochondria; Mitochondrial Proteins; Oxygen Consumption; Serum Albumin, Bovine; Solanum tuberosum; Uncoupling Protein 1

Fourier transform infrared spectroscopy has been applied to the study of the carboxyatractyloside-inhibited mitochondrial ADP/ATP transporter from the yeast Saccharomyces cerevisiae, either solubilized in dodecyl maltoside or reconstituted in phosphatidylcholine liposomes. Its secondary structure has been estimated by means of Fourier self-deconvolution followed by curve fit. A Voigt function was used to fit the components of the deconvoluted spectrum, aiming to account for any distortions introduced by deconvolution. For any of the states analyzed, reconstituted or solubilized, in solution or in dry films, 60-70% of the amino acids are found to adopt alpha-helix plus unordered structures, coherent with the six transmembrane spanning helix model. Moreover, the problem of structure preservation on drying was addressed, and several observations pointed to a maintenance of the protein structure in dry films. Comparison of reconstituted and solubilized samples indicated the presence of both lipid-induced changes in the protein (decrease of the beta-sheets and increase of unordered structures) and protein-induced changes in the lipids (strong hydrogen bonding of lipid C=O groups). To obtain a better discrimination of alpha-helix and unordered structure contributions for the reconstituted form, H/D exchange experiments were performed. Between 35% and 45% of the amino acids were finally assigned to alpha-helix structures, compatible with the existence of five or six transmembrane spanning helices in the transporter. The level of H/D exchange was determined after 15 h of exposure to D(2)O vapor to be 85%, reflecting a high accessibility of the amide hydrogens even for the carboxyatractyloside-inhibited state.

Topics: Amides; Atractyloside; Detergents; Deuterium Oxide; Enzyme Inhibitors; Glucosides; Histidine; Lipid Bilayers; Micelles; Mitochondria; Mitochondrial ADP, ATP Translocases; Normal Distribution; Phospholipids; Protein Structure, Secondary; Protons; Saccharomyces cerevisiae; Solubility; Spectroscopy, Fourier Transform Infrared

Disulfiram (Ds), a clinically employed alcohol deterrent of the thiuram disulfide (TD) class of compounds, is known to cause hepatitis and neuropathies. Although this drug has been shown to inhibit different thiol-containing enzymes, the actual mechanism of Ds toxicity is not clear. We have previously demonstrated that Ds impairs the permeability of inner mitochondrial membrane (IMM) [Arch. Biochem. Biophys. 356 (1998) 46]. In this report, the effect of Ds and its structural analogue thiram (Th) on mitochondrial functions was studied in detail. We found that mitochondria metabolize TDs in a NAD(P)H- and GSH-dependent manner. At the concentration above characteristic threshold, TDs induced irreversible oxidation of NAD(P)H and glutathione (GSH) pools, collapse of transmembrane potential, and inhibition of oxidative phosphorylation. The presence of Ca(2+) and exhaustion of mitochondrial glutathione (GSH+GSSG) decreased the threshold concentration of TDs. Swelling of the mitochondria and leakage of non-transported fluorescent dye BCECF from the matrix indicated that TDs induced the mitochondrial permeability transition (MPT). Mitochondrial permeabilization by TDs involves two, apparently distinct mechanisms. In the presence of Ca(2+), TDs produced cylosporin A-sensitive swelling of mitochondria, which was inhibited by ADP and accelerated by carboxyatractyloside (CATR) and phosphate. In contrast, the swelling produced by TDs in the absence of Ca(2+) was not sensitive to cyclosporin A (CsA), ADP and CATR but was inhibited by phosphate. Titration with N-ethylmaleimide revealed that these two mechanisms involve different SH-groups and probably different transport proteins on the IMM. Our findings indicate that at pharmacologically relevant concentrations TDs may cause an irreversible mitochondrial injury as a result of induction of the MPT.

Topics: Adenosine Diphosphate; Animals; Atractyloside; Cell Membrane Permeability; Cyclosporine; Disulfiram; Enzyme Inhibitors; Ethylmaleimide; Glutathione; Mitochondria, Liver; Mitochondrial Swelling; NADP; Oxidative Phosphorylation; Phosphates; Rats; Rats, Wistar; Thiram

Apolipoprotein C (apo C) was shown to decrease the Ca2+ capacity and membrane potential of mitochondria isolated from rat liver. The specific ligands of adenine nucleotide carrier, ADP and carboxyatractyloside (CAT), inhibited the effect of apo C on the mitochondrial membrane potential. The effect of ADP and CAT was revealed in the absence of Ca2+. We conclude that in the presence of apo C, adenine nucleosides carrier transforms into a pore, and this causes the decrease in the membrane potential of the mitochondria. ADP and CAT support the primary conformation of the carrier and therefore inhibit the effect of apolipoprotein C.

Topics: Adenosine Diphosphate; Animals; Apolipoproteins C; Atractyloside; Calcium; In Vitro Techniques; Intracellular Membranes; Male; Membrane Potentials; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Rats; Rats, Wistar

Isolated brain mitochondria were examined for their responses to calcium challenges under varying conditions. Mitochondrial membrane potential was monitored by following the distribution of tetraphenylphosphonium ions in the mitochondrial suspension, mitochondrial swelling by observing absorbance changes, calcium accumulation by an external calcium electrode, and oxygen consumption with an oxygen electrode. Both the extent and rate of calcium-induced mitochondrial swelling and depolarization varied greatly depending on the energy source provided to the mitochondria. When energized with succinate plus glutamate, after a calcium challenge, CNS mitochondria depolarized transiently, accumulated substantial calcium, and increased in volume, characteristic of a mitochondrial permeability transition. When energized with 3 mM succinate, CNS mitochondria maintained a sustained calcium-induced depolarization without appreciable swelling and were slow to accumulate calcium. Maximal oxygen consumption was also restricted under these conditions, preventing the electron transport chain from compensating for this increased proton permeability. In 3 mM succinate, cyclosporin A and ADP plus oligomycin restored potential and calcium uptake. This low conductance permeability was not effected by bongkrekic acid or carboxyatractylate, suggesting that the adenine nucleotide translocator was not directly involved. Fura-2FF measurements of [Ca(2+)](i) suggest that in cultured hippocampal neurons glutamate-induced increases reached tens of micromolar levels, approaching those used with mitochondria. We propose that in the restricted substrate environment, Ca(2+) activated a low-conductance permeability pathway responsible for the sustained mitochondrial depolarization.

Topics: Adenosine Diphosphate; Animals; Antineoplastic Agents; Atractyloside; Brain; Calcium; Electric Conductivity; Fluorescent Dyes; Fura-2; Glutamic Acid; Membrane Potentials; Mitochondria; Mitochondrial Swelling; Nerve Degeneration; Oligomycins; Onium Compounds; Organophosphorus Compounds; Oxidation-Reduction; Oxygen Consumption; Rats; Strontium; Succinic Acid; Uncoupling Agents

The roles of mild uncoupling caused by free fatty acids (mediated by plant uncoupling mitochondrial protein (PUMP) and ATP/ADP carrier (AAC)) and non-coupled respiration (alternative oxidase (AO)) on H(2)O(2) formation by plant mitochondria were examined. Both laurate and oleate prevent H(2)O(2) formation dependent on the oxidation of succinate. Conversely, these free fatty acids (FFA) only slightly affect that dependent on malate plus glutamate oxidation. Carboxyatractylate (CAtr), an inhibitor of AAC, completely inhibits oleate- or laurate-stimulated oxygen consumption linked to succinate oxidation, while GDP, an inhibitor of PUMP, caused only a 30% inhibition. In agreement, CAtr completely restores the oleate-inhibited H(2)O(2) formation, while GDP induces only a 30% restoration. Both oleate and laurate cause a mild uncoupling of the electrical potential (generated by succinate), which is then followed by a complete collapse with a sigmoidal kinetic. FFA also inhibit the succinate-dependent reverse electron transfer. Diamide, an inhibitor of AO, favors the malate plus glutamate-dependent H(2)O(2) formation, while pyruvate (a stimulator of AO) inhibits it. These results show that the succinate-dependent H(2)O(2) formation occurs at the level of Complex I by a reverse electron transport. This generation appears to be prevented by mild uncoupling mediated by FFA. The anionic form of FFA appears to be shuttled by AAC rather than PUMP. The malate plus glutamate-dependent H(2)O(2) formation is, conversely, mainly prevented by non-coupled respiration (AO).

Topics: Atractyloside; Carrier Proteins; Glutamic Acid; Guanosine Diphosphate; Hydrogen Peroxide; Ion Channels; Lauric Acids; Malates; Membrane Proteins; Mitochondria; Mitochondrial ADP, ATP Translocases; Mitochondrial Proteins; Oleic Acid; Oxidation-Reduction; Oxidative Phosphorylation; Oxygen Consumption; Pisum sativum; Succinic Acid; Uncoupling Agents; Uncoupling Protein 1

A functional recombinant mitochondrial ADP/ATP carrier from the yeast Saccharomyces cerevisiae that bears a six-histidine tag at the C-terminus, Anc2(His(6))p, has been engineered to allow its purification by immobilized metal-ion affinity chromatography (IMAC). The tagged carrier was expressed at a level similar to that of unmodified Anc2p as determined by immunodetection and titration of the specific atractyloside binding sites. Anc2(His(6))p, enriched by chromatography on hydroxyapatite of detergent extracts of mitochondria, was still contaminated by mitochondrial proteins and a large amount of ergosterol. It was highly purified after adsorption on Ni-NTA resin and elution by imidazole buffer, with a 90-95% overall yield. Anc2(His(6))p interacted differently with immobilized ions depending on whether it was unliganded or bound to carboxyatractyloside (CATR) or bongkrekic acid (BA), two specific inhibitors of the ADP/ATP transport, thus indicating that accessibility of the C-terminus is markedly influenced by the conformational state of the carrier. Fluorometric assays demonstrated that purified unliganded Anc2(His(6))p was in a functional state since it underwent CATR- and BA-sensitive and ADP (or ATP)-induced conformational changes. Large-scale purification of Anc2(His(6))p-CATR and Anc2(His(6))p-BA complexes by IMAC will be of major interest for structural analysis of the ADP/ATP carrier.

Topics: Anti-Bacterial Agents; Atractyloside; Bongkrekic Acid; Chromatography, Affinity; Fluorescence; Fungal Proteins; Histidine; Mitochondria; Mitochondrial ADP, ATP Translocases; Protein Conformation; Protein Isoforms; Saccharomyces cerevisiae

Intermyofibrillar and subsarcolemmal mitochondria were isolated from duckling gastrocnemius muscle. The adenine nucleotide translocase (ANT) content of subsarcolemmal mitochondria was found to be half of that present in intermyofibrillar mitochondria. In addition, cold acclimation resulted in a 1.7-fold increase in subsarcolemmal mitochondrial ANT content, with intermyofibrillar mitochondrial ANT remaining constant. This change in mitochondrial ANT content correlates with the previously reported cold-induced change in the sensitivity of mitochondria to palmitate-inhibited ATP synthesis [Roussel et al. (1998) FEBS Lett. 439, 258-262]. It is suggested that the mitochondrial ANT content enhances or reduces the fatty acid uncoupling activity in tissue, depending on the energetic state of mitochondria.

Topics: Adaptation, Physiological; Adenosine Triphosphate; Animals; Atractyloside; Cell Respiration; Cold Temperature; Ducks; Enzyme Inhibitors; Inhibitory Concentration 50; Male; Mitochondria, Muscle; Mitochondrial ADP, ATP Translocases; Muscle, Skeletal; Oxidative Phosphorylation; Palmitic Acid; Sarcolemma; Titrimetry

Addition of 5 microM copper to rat kidney mitochondria enhances the effect of carboxyatractyloside and oleate on pore opening, in a cyclosporin A-sensitive fashion. The effects of the pair copper-carboxyatractyloside were observed on matrix Ca2+ efflux, mitochondrial swelling and on the transmembrane electric gradient. The effect of Cu2+ emphasizes the importance of membrane thiol groups located, probably, in the ADP/ATP translocase (ANT), on permeability transition. It was also found that Cu2+ does not block the fluorescent label of ANT by eosin 5-maleimide, but abolishes the inhibition by CAT on the labeling. This suggests that the binding of Cu2+ to cysteine residues of ANT promotes a conformational change in the carrier, strengthening the effect of CAT and oleate on membrane leakage.

Topics: Animals; Atractyloside; Copper; Intracellular Membranes; Kidney Cortex; Kinetics; Mitochondria; Mitochondrial ADP, ATP Translocases; Oleic Acid; Permeability; Rats

Mitochondrial proton leak in rat muscle is responsible for approx. 15% of the standard metabolic rate, so its modulation could be important in regulating metabolic efficiency. We report in the present paper that physiological concentrations of AMP (K(0.5)=80 microM) increase the resting respiration rate and double the proton conductance of rat skeletal-muscle mitochondria. This effect is specific for AMP. AMP also doubles proton conductance in skeletal-muscle mitochondria from an ectotherm (the frog Rana temporaria), suggesting that AMP activation is not primarily for thermogenesis. AMP activation in rat muscle mitochondria is unchanged when uncoupling protein-3 is doubled by starvation, indicating that this protein is not involved in the AMP effect. AMP activation is, however, abolished by inhibitors and substrates of the adenine nucleotide translocase (ANT), suggesting that this carrier (possibly the ANT1 isoform) mediates AMP activation. AMP activation of ANT could be important for physiological regulation of metabolic rate.

Topics: Adenosine Monophosphate; Animals; Atractyloside; Carrier Proteins; Dose-Response Relationship, Drug; Electrophysiology; Female; Food Deprivation; Ion Channels; Kinetics; Membrane Potentials; Mitochondria; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Mitochondrial Proteins; Muscle, Skeletal; Oxygen Consumption; Protein Isoforms; Ranidae; Rats; Rats, Wistar; Temperature; Uncoupling Protein 3

Treatment of rat liver mitochondria with aluminum in the presence of Ca2+ results in large amplitude swelling accompanied by loss of endogenous Mg2+ and K+ and oxidation of endogenous pyridine nucleotides. The presence of cyclosporin A, ADP, bongkrekic acid, N-ethylmaleimide and dithioerythritol prevent these effects, indicating that binding of aluminum to the inner mitochondrial membrane, most likely at the level of adenine nucleotide translocase, correlates with the induction of the membrane permeability transition (MPT). Indeed, aluminum binding promotes such a perturbation at the level of ubiquinol-cytochrome c reductase, which favors the production of reactive oxygen species. These metabolites generate an oxidative stress involving two previously defined sites in equilibrium with the glutathione and pyridine nucleotides pools, the levels of which correlate with the increase in MPT induction. Although the above-described phenomena are typical of MPT, they are not paralleled by other events normally observed in response to treatment with inducers of MPT (e.g., phosphate), such as the collapse of the electrochemical gradient and the release of accumulated Ca2+ and oxidized pyridine nucleotides. Biochemical and ultrastructural observations demonstrate that aluminum induces a pore opening having a conformation intermediate between fully open and closed in a subpopulation of mitochondria. While inorganic phosphate enhances the MPT induced by ruthenium red plus a deenergizing agent, aluminum instead inhibits this phenomenon. This finding suggests the presence of a distinct binding site for aluminum differing from that involved in MPT induction.

Topics: Adenosine Diphosphate; Aluminum; Animals; Atractyloside; Biological Transport; Bongkrekic Acid; Calcium; Cations; Cyclosporine; Dose-Response Relationship, Drug; Hydrogen-Ion Concentration; Intracellular Membranes; Membrane Potentials; Microscopy, Electron; Mitochondria, Liver; Mitochondrial Swelling; Nucleotides; Oxidation-Reduction; Permeability; Phosphates; Rats; Sucrose

We show that Ca2+ loading of mitochondria substantially augments the myristate-induced decrease in the transmembrane electric potential difference (deltapsi). Such a Ca2+ action is without effect on the respiration rate and is not accompanied by the high-amplitude swelling when low concentrations of Ca2+ and myristate are used. The myristate-induced deltapsi decrease is prevented and reversed by cyclosporin A (CsA); the decrease is prevented and transiently reversed by nigericin. To explain these effects, we suggest that myristate induces opening of the mitochondrial permeability transition pore at a low-conductance state. Addition of carboxyatractylate (CAtr) after myristate induces the CsA-sensitive uncoupling, but when added after myristate and CsA, CAtr produces a decrease in deltapsi, if the interval between myristate and CsA addition is sufficiently long. The CAtr effect is completely reversed by EGTA and transiently reversed by nigericin. This suggests that the ADP/ATP-antiporter participates in the CsA-sensitive uncoupling when present as a pore complex constituent. ADP/ATP-antiporter that does not take part in the pore complex formation is involved in the CsA-insensitive uncoupling.

Topics: Animals; Atractyloside; Calcium; Cyclosporine; Egtazic Acid; In Vitro Techniques; Membrane Potentials; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Models, Biological; Myristic Acid; Nigericin; Rats; Uncoupling Agents

Mitochondrial permeability transition occurs through a Ca2+-dependent opening of a transmembrane pore, whose identity has been attributed to that of the adenine nucleotide translocase (ANT). In this work, we induced permeability transition by adding 0.5 microM carboxyatractyloside. The process was evaluated analyzing Ca2+ efflux, a drop in transmembrane electric gradient, and swelling. We found that the amphiphyllic cations octylguanidine and octylamine, at the concentration of 100 microM, inhibited, almost completely, nonspecific membrane permeability. Hexylguanidine, hexylamine, as well as guanidine chloride and hydroxylamine failed to do so. The inhibition was reversed after the addition of 40 mM Li+, Na+ K+, Rb+, or Cs+; K+ was the most effective. We propose that the positive charge of the amines interact with negative charges of membrane proteins, more likely the ADP/ATP carrier, while the alkyl chain penetrates into the hydrophobic milieu of the inner membrane, fixing the reagent.

Topics: Adenosine Diphosphate; Amines; Animals; Atractyloside; Calcium; Guanidines; Ion Channels; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Potassium

Nucleotide binding to the cytosolic binding site of the mitochondrial ADP/ATP carrier (AAC) was studied by 1H-nuclear magnetic resonance spectroscopy. Binding (as opposed to translocation) could be identified as a result of the rapid ligand on/off kinetics, using the cytosolic side specific inhibitor carboxyatractyloside (CAT) for the distinction from nonspecific interactions. The off rate constant of the nonhydrolyzable ATP analogue AMP-PCP was more than 3 orders of magnitude larger than the transport rate. The nucleotides adopt an anti conformation in the carrier binding site as shown by measurements of the transferred nuclear overhauser effect (TRNOE). A thermal transition around 14 degreesC that had been previously detected in transport studies [Klingenberg, M., Grebe, K., and Appel, M. (1982) Eur. J. Biochem. 126, 263-269] was reflected by the inhibitor sensitive line broadening, indicating that this transition also affects nucleotide binding. Nucleotide monophosphates were employed to study the relation between nucleotide structure and affinity, using selective excitation, sample spinning with digital suppression of spinning sidebands, and line shape simulation. The binding of purines depends on the distribution of the electrical potential and on the position of ring substituents, while pyrimidines are barely recognized at all by the AAC. It is also shown that the photocleavable "caged" derivatives are more tightly bound than the original nucleotides. A two step model of carrier catalysis will be discussed on the basis of these results.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Atractyloside; Binding, Competitive; Bongkrekic Acid; Cattle; Enzyme Inhibitors; Magnetic Resonance Spectroscopy; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Models, Molecular; Protein Binding; Protein Conformation; Protons; Static Electricity; Surface Properties; Temperature

The effect of 3'-azido-3'-deoxythymidine on nucleoside diphosphate kinase of isolated rat liver mitochondria has been studied. This is done by monitoring the increase in the rate of oxygen uptake by nucleoside diphosphate (TDP, UDP, CDP or GDP) addition to mitochondria in state 4. It is shown that 3'-azido-3'-deoxythymidine inhibits the mitochondrial nucleoside diphosphate kinase in a competitive manner, with a Ki value of about 10 microM as measured for each tested nucleoside diphosphate. It is also shown that high concentrations of GDP prevent 3'-azido-3'-deoxythymidine inhibition of the nucleoside diphosphate kinase.

Topics: Animals; Atractyloside; Binding, Competitive; Cytidine Diphosphate; Enzyme Inhibitors; Guanosine Diphosphate; Kinetics; Mitochondria, Liver; Nucleoside-Diphosphate Kinase; Oligomycins; Oxygen Consumption; Rats; Thymine Nucleotides; Uridine Diphosphate; Zidovudine

Addition of a low concentration of carboxyatractyloside (0.075 microM) renders mitochondria susceptible to the opening of the non-specific pore by 5 microM oleate, in a cyclosporin A-sensitive fashion. Matrix Ca2+ efflux as well as collapse of the transmembrane potential reveal permeability transition. The effect of oleate is reached after the titration, by carboxyatractyloside, of 38 pmol of adenine nucleotide translocase per mg mitochondrial protein. We propose that permeability transition may result from an additive action of carboxyatractyloside plus oleate on the ADP/ATP carrier.

Topics: Animals; Atractyloside; Calcium; Mitochondria; Oleic Acid

The mitochondrial adenine nucleotide translocator (ANT) catalyses the exchange of ATP and ADP between the mitochondria and the cytosol. We have cloned and sequenced the gene encoding the Dictyostelium discoideum ANT (DdANT) and analysed its transcriptional regulation. The single copy D. discoideum ant gene encodes a protein of 309 amino acid residues with a predicted molecular mass of 33,469 Da and a pI of 9.85. These values are comparable to those of ANTs from mammals, insects and fungi. The long N-terminal extension characteristic of plant ANT is absent in DdANT. The protein coding region of the D. discoideum ant gene is interrupted by three introns. Polyclonal antibodies directed against the beef heart mitochondrial ANT or its C-terminal peptide recognized the D. discoideum protein. Northern blot analysis revealed that the expression of the D. discoideum ant gene decreased rapidly during the first hours of multicellular development but the amount of protein remained stable throughout differentiation.

Topics: Animals; Atractyloside; Cell Differentiation; Cell Division; Cloning, Molecular; Dictyostelium; Enzyme Inhibitors; Gene Expression Regulation, Developmental; Mitochondria; Mitochondrial ADP, ATP Translocases; Phylogeny; Protozoan Proteins; RNA, Messenger; Sequence Alignment; Sequence Analysis, DNA

Addition of myristate at low concentration (30-60 nmol/mg protein) to energized rat liver mitochondria resulted in dissipation of the electric membrane potential which, in Ca2+-free media, could be partly reversed by carboxyatractyloside but not by cyclosporin A. In contrast, in mitochondria preloaded with Ca2+ this energy-dissipating effect of fatty acid was partly prevented or reversed by cyclosporin A or ADP. In sucrose media, myristate, but not the protonophore carbonyl cyanide m-chlorophenylhydrazone, induced swelling of Ca2+-loaded mitochondria which was inhibited by cyclosporin A and ADP. We conclude that long-chain fatty acids may induce opening of the mitochondrial permeability transition pore not only because of their protonophoric effect mediated by mitochondrial anion carriers [Skulachev, V.P., FEBS Lett. 294 (1991) 158-162; Wieckowski, M.R. and Wojtczak, L., Biochem. Biophys. Res. Commun. (1997) 232, 414-417] but also by a direct interaction with the pore assembly.

Topics: Adenosine Diphosphate; Animals; Atractyloside; Calcium; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cyclosporine; Fatty Acids; Intracellular Membranes; Ion Channels; Male; Membrane Potentials; Membrane Proteins; Mitochondria, Liver; Mitochondrial Swelling; Myristic Acid; Oxidative Phosphorylation; Permeability; Porins; Rats; Rats, Inbred Strains; Uncoupling Agents; Voltage-Dependent Anion Channels

The present work examined whether the ATP/ADP carrier, other than the plant uncoupling mitochondrial protein, participates in free fatty acid-mediated uncoupling of potato tuber mitochondria. The basal respiration rate of succinate-energized mitochondria was stimulated by a low concentration of palmitate (20 microM). This uncoupling was reversed by 10 microM carboxyatractyloside and by the subsequent addition of 0.1% bovine serum albumin. The decrease in membrane potential caused by palmitate was suppressed by carboxyatractyloside (1 microM) and, to a lesser degree, by bongkrekate (20 microM). GTP could also reversed this decrease via a carboxyatractyloside-independent mechanism. These results indicate that the ATP/ADP carrier, along with the plant uncoupling mitochondrial protein, participates in the protonophoric action of palmitate in potato tuber mitochondria.

Topics: Atractyloside; Carrier Proteins; Fatty Acids, Nonesterified; Intracellular Membranes; Ion Channels; Membrane Potentials; Membrane Proteins; Mitochondria; Mitochondrial Proteins; Oxygen Consumption; Palmitates; Plant Proteins; Receptors, Cytoplasmic and Nuclear; Serum Albumin, Bovine; Solanum tuberosum; Succinates; Uncoupling Protein 1

Earlier it was shown that fatty acid-induced uncoupling in liver mitochondria is suppressed by the substrates of the aspartate/glutamate antiporter (V. N. Samartsev, A. V. Smirnov, I. P. Zeldi, O. V. Markova, E. N. Mokhova, and V. P. Skulachev (1997) Biochim. Biophys. Acta, 1319, 251-257). In this study it is shown that in heart mitochondria aspartate, glutamate, and diethyl pyrocarbonate do not affect oxygen consumption and membrane potential in the presence of laurate at pH 7.4. These compounds have a weak (versus carboxyatractylate) coupling effect at pH 7.0. This effect is manifested only in the presence of carboxyatractylate, magnesium, and phosphate in the incubation medium. It is suggested that these tissue-specific effects are due not only to the specific characteristics of aspartate/glutamate antiporter, but also to the differences in the content of endogenous metabolites in heart mitochondria.

Topics: Animals; Antiporters; Aspartic Acid; Atractyloside; Diethyl Pyrocarbonate; Fatty Acids; Glutamic Acid; Hydrogen-Ion Concentration; In Vitro Techniques; Lauric Acids; Membrane Potentials; Mitochondria, Heart; Oxygen Consumption; Rats; Uncoupling Agents

Membrane permeability was examined in liver mitochondria isolated from hypothyroid rats. It was found that such a thyroid status provides substantial protection from membrane leakiness as induced by Ca2+ loading. Thus, these mitochondria are less prone to undergoing permeability transition than mitochondria from euthyroid rats. The above conclusion was reached on the basis of the following two facts: (1) hypothyroid mitochondria are not strictly dependent on the addition of ADP to retain high matrix Ca2+ concentrations, and (2) carboxyatractyloside, antimycin A or carbonyl cyanide-m-chlorophenyl hydrazone failed to promote Ca2+ efflux. We discuss the possible relevance of the low content of membrane cardiolipin as well as the low expression of the adenine nucleotide translocase as responsible for the resistance to membrane damage.

Topics: Adenosine Diphosphate; Animals; Atractyloside; Calcium; Cyclosporine; Hypothyroidism; Intracellular Membranes; Magnesium; Mitochondria, Liver; Permeability; Rats; Thyroidectomy

We show here that TPP --> TMP conversion can take place in rat liver mitochondria. This occurs via the novel, putative TPP pyrophosphatase localised in the mitochondrial matrix, as shown both by digitonin titration and by an HPLC enzyme assay carried out on the mitochondrial matrix fraction. Certain features of the reaction, including the substrate and pH dependence, are reported. Additional evidence is given that externally added TMP can cross the mitochondrial membrane in a manner consistent with the occurrence of a carrier-mediated process. This can occur both via the TPP translocator and via a novel translocator, inhibited by CAT but different from the ADP/ATP carrier.

Topics: Animals; Atractyloside; Binding, Competitive; Biological Transport; Catalysis; Hydrolysis; Intracellular Membranes; Male; Mitochondria, Liver; Rats; Rats, Wistar; Spectrometry, Fluorescence; Thiamine Monophosphate; Thiamine Pyrophosphate

Possible involvement of the ATP/ADP antiporter and uncoupling protein (UCP) in thermoregulatory uncoupling of oxidative phosphorylation in heart muscle has been studied. To this end, effects of carboxyatractylate (cAtr) and GDP, specific inhibitors of the antiporter and UCP, on the membrane potential of the oligomycin-treated mitochondria from cold-exposed (6 degrees C, 48 h) and control rats have been measured. It is found that cAtr increases the membrane potential level in both cold-exposed and non-exposed groups, the effect being strongly enhanced by cooling. As for GDP, it is effective only in mitochondria from the cold-exposed rats. In these mitochondria, the coupling effect of GDP is smaller than that of cAtr. CDP, which does not interact with UCP, is without any influence on membrane potential. The cold exposure is found to increase the uncoupling efficiency of added natural (palmitate) or artificial (SF6847) uncouplers, the increase being cAtr- and GDP-sensitive in the case of palmitate. The fatty acid-free bovine serum albumin enhances delta psi in both cold-exposed and control groups, the effect being much larger in the former case. It is concluded that in heart muscle mitochondria the ATP/ADP antiporter is responsible for the 'mild uncoupling' under normal conditions and for major portion of the thermoregulatory uncoupling in the cold whereas the rest of thermoregulatory uncoupling is served by UCP (presumably by UCP2 since the UCP2 mRNA level is shown to strongly increase in rat heart muscle under the cold exposure conditions used).

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Atractyloside; Carrier Proteins; Cold Temperature; Guanosine Diphosphate; Ion Channels; Malonates; Membrane Potentials; Membrane Proteins; Mitochondria, Heart; Mitochondrial Proteins; Nitriles; Rats; Serum Albumin, Bovine; Succinic Acid; Tetramethylphenylenediamine; Uncoupling Agents; Uncoupling Protein 1

A fusion protein between cyclophilin-D (CyP-D) and glutathione S-transferase (GST) was shown to bind to purified liver inner mitochondrial membranes (IMMs) in a cyclosporin A (CsA)-sensitive manner. Binding was enhanced by diamide treatment of the IMMs. Immobilized GST-CyP-D avidly bound a single 30 kDa protein present in Triton X-100-solubilized IMMs; immunoblotting showed this to be the adenine nucleotide translocase (ANT). Binding was prevented by pretreatment of the CyP-D with CsA, but not with cyclosporin H. Purified ANT also bound specifically to GST-CyP-D, but porin did not, even in the presence of ANT.

Topics: Animals; Atractyloside; Blotting, Western; Bongkrekic Acid; Chromatography, Affinity; Cyclophilins; Diamide; Glutathione Transferase; Immunoblotting; Immunophilins; Intracellular Membranes; Male; Mitochondria; Mitochondrial ADP, ATP Translocases; Octoxynol; Peptidyl-Prolyl Isomerase F; Permeability; Rats; Rats, Wistar; Recombinant Fusion Proteins; Solubility

Stimulation of the mitochondrial permeability transition (MPT) in de-energized mitochondria by phenylarsine oxide (PheArs) is greater than that by diamide and t-butylhydroperoxide (TBH), yet the increase in CyP binding to the inner mitochondrial membrane (Connern, C. P. and Halestrap, A. P. (1994) Biochem. J. 302, 321-324) is less. From a range of nucleotides tested only ADP, deoxy-ADP, and ATP inhibited the MPT. ADP inhibition involved two sites with Ki values of about 1 and 25 microM which were independent of [Ca2+] and CyP binding. Carboxyatractyloside (CAT) abolished the high affinity site. Following pretreatment of mitochondria with TBH or diamide, the Ki for ADP increased to 50-100 microM, whereas pretreatment with PheArs or eosin maleimide increased the Ki to >500 microM; only one inhibitory site was observed in both cases. Eosin maleimide is known to attack Cys159 of the adenine nucleotide translocase (ANT) in a CAT-sensitive manner (Majima, E., Shinohara, Y., Yamaguchi, N., Hong, Y. M., and Terada, H. (1994) Biochemistry 33, 9530-9536), and here we demonstrate CAT-sensitive binding of the ANT to a PheArs affinity column. In adenine nucleotide-depleted mitochondria, no stimulation of the MPT by uncoupler was observed in the presence or absence of thiol reagents, suggesting that membrane potential may inhibit the MPT by increasing adenine nucleotide binding through an effect on the ANT conformation. We conclude that CsA and ADP inhibit pore opening in distinct ways, CsA by displacing bound CyP and ADP by binding to the ANT. Both mechanisms act to decrease the Ca2+ sensitivity of the pore. Thiol reagents and oxidative stress may modify two thiol groups on the ANT and thus stimulate pore opening by both means.

Topics: Adenosine Diphosphate; Animals; Arsenicals; Atractyloside; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cyclosporine; Diamide; Intracellular Membranes; Membrane Potentials; Mitochondria; Mitochondrial ADP, ATP Translocases; Nucleotides; Oxidative Stress; Permeability; Peroxides; Rats; Reactive Oxygen Species; Sulfhydryl Reagents; tert-Butylhydroperoxide

Recently, we have sequenced a cDNA clone from Arabidopsis thaliana L. encoding a novel putative ATP/ADP translocator (AATP1). Here, we demonstrate that the radioactively labeled AATP1 precursor protein, synthesized in vitro, is targeted to envelope membranes of isolated spinach chloroplasts. Antibodies raised against a synthetic peptide of AATP1 recognized a single polypeptide of about 62 kDa in chloroplast inner envelope preparations. The cDNA coding for the AATP1 protein was functionally expressed in Saccharomyces cerevisiae and Escherichia coli. In both expression systems, increased rates of ATP transport were observed after reconstitution of the extracted protein into proteoliposomes. To our knowledge, this is the first report on the functional expression of an intrinsic plant membrane protein in E. coli. To yield high rates of ATP transport, proteoliposomes had to be preloaded with ADP, indicating a counter-exchange mode of transport. Carboxyatractyloside did not substantially interfere with ATP transport into proteoliposomes containing the plastidic ATP/ADP translocator. An apparent KM for ATP of 28 microM was determined which is similar to values reported for isolated plastids. The data presented here strongly support the conclusion that AATP1 represents a novel eukaryotic adenylate carrier and that it is identical with the so far unknown plastidic ATP/ADP translocator.

Topics: Adenosine Triphosphate; Amino Acid Sequence; Arabidopsis; Arabidopsis Proteins; Atractyloside; Biological Transport; Blotting, Western; Cell Compartmentation; Cell Fractionation; Chloroplasts; Escherichia coli; Intracellular Membranes; Mitochondrial ADP, ATP Translocases; Molecular Sequence Data; Plant Proteins; Proteolipids; Recombinant Proteins; Saccharomyces cerevisiae

The effect of ATP and other nucleotides on the respiration of Saccharomyces cerevisiae mitochondria was investigated. It was observed that ATP induced a stimulation of the respiration rate only in the presence of a salt in mitochondria from the baker's yeast Yeast Foam, whereas an ATP-induced stimulation occurred even in the absence of salt in mitochondria from three different laboratory strains. In both cases, the stimulation was related to a collapse of the transmembrane potential, suggesting the opening of ion- and/or proton-conducting pathways. Not only ATP, but also GTP and CTP, induced these pathways. Moreover, a similar stimulation was obtained with GDP and its analog GDP-beta-S. The fact that, as opposed to NTPs, GDP did not induce any non-specific anion channel, allowed us to use it to demonstrate unambiguously that a proton-conducting pathway was opened through the inner mitochondrial membrane of laboratory strains but not of Yeast Foam. Three additional aspects of this nucleotide-induced permeability were investigated. (i) The proton-conducting pathway was insensitive to Mg2+, whereas the anion-conducting pathway was fully inhibited by 4 mM Mg2-. (ii) The proton-conducting pathway of mitochondria isolated from laboratory strains was opened by the action of nucleotides outside the mitochondrion, since it was fully insensitive to (carboxy)atractyloside, and fully active in mitochondria isolated from op1 and delta anc strains. On the other hand, the cation-conducting pathway of Yeast Foam mitochondria was partly sensitive to (carboxy)atractyloside and insensitive to bongkrekic acid, suggesting a role of the conformational state of ANC in this activity. (iii) Both the proton and cation-conducting pathways were inhibited by very low concentrations of vanadate, under conditions where this oxyanion was polymerized to decavanadate: a competitor to nucleotide-binding sites on some enzymes.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Anions; Atractyloside; Biological Transport; Bongkrekic Acid; Carrier Proteins; Enzyme Inhibitors; Guanosine Diphosphate; Intracellular Membranes; Magnesium; Mitochondria; NAD; Nucleotides; Permeability; Protein Conformation; Protons; Saccharomyces cerevisiae; Salts; Thionucleotides; Valinomycin; Vanadates

Resting state respiration of rat liver mitochondria with succinate or N,N,N',N'-tetrametnyl-p-phenylene-diamine plus ascorbate as substrates was stimulated by myristate. This uncoupling effect was partly reversed not only by carboxyatractyloside and glutamate as reported by others [V. P. Skulachev (1991) FEBS Lett. 294, 158-162; V.N. Samartsev, A.V. Smirnov, I.P. Zeldi, O.V. Markova, E.N. Mokhova, and V.P. Skulachev (1997) Biochim. Biophys. Acta, in press] but also by malonate. Glutamate and malonate also partly restored the mitochondrial membrane potential dissipated by addition of myristate. Myristate inhibited the transport of malonate through the mitochondrial inner membrane, 50% inhibition being reached with 100 nmol fatty acid/mg mitochondrial protein. A similar inhibitory effect was obtained with an azido derivative of long-chain fatty acid, 12-(4-azido-2-nitrophenylamino) dodecanoic acid. This inhibitory effect could be reversed by serum albumin. However, after illumination with ultraviolet light, the inhibition of malonate transport could not be reversed by serum albumin, pointing to photomodification of the dicarboxylate carrier. These results indicate that the dicarboxylate carrier, along with the ADP/ATP carrier and the glutamate carrier, participates in the protonophoric action of fatty acids in mitochondria.

Topics: Animals; Atractyloside; Carrier Proteins; Dicarboxylic Acid Transporters; Dicarboxylic Acids; Fatty Acids; Glutamates; Male; Malonates; Mitochondria, Liver; NADH Dehydrogenase; Oxidative Phosphorylation; Oxygen Consumption; Protons; Rats; Rotenone; Uncoupling Agents

The membrane permeability transition (MPT) induced by Ca2+ and Pi or Asi was studied in rat kidney mitochondria. Membrane potential, Ca2+ transport and swelling were used to monitor the MPT. Asi promoted a faster and more extensive collapse of membrane potential, Ca2+ release and swelling than Pi. The MPT induced by Pi was fully blocked by Mg(2+)+ADP, spermine+ADP, Mg(2+)+ cyclosporin A (CSA), and ADP+CSA. In contrast, the MPT induced by Asi was only prevented, although not completely, by CSA+Mg2+ or ADP+CSA. Asi, but not Pi, was able to cause collapse of membrane potential in the presence of Sr2+. Carboxyatractyloside (CAT) produced collapse of membrane potential at a lower concentration in the presence of Asi+Ca(2+)+ADP than with Pi+Ca(2+)+ADP. The addition of Pi+Ca2+ to [14C]-ADP loaded mitochondria brought about a greater ADP release than Asi+Ca2+. The ADP release was CAT-sensitive with Pi but it was only partially blocked by Asi. The diminution of external pH did not inhibit the MPT induced by Pi or Asi. The results of this study suggest that the adenine nucleotide translocase does not have an essential role in the MPT induced by Asi+Ca2+.

Topics: Adenosine Diphosphate; Animals; Arsenates; Arsenazo III; Atractyloside; Calcium; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cyclosporine; Female; Intracellular Membranes; Kidney; Male; Membrane Potentials; Mitochondria; Oligomycins; Permeability; Phenazines; Phosphates; Rats; Spermine; Trifluoperazine

Substitution of potassium cloride for sucrose in the incubation medium of liver mitochondria or addition of magnesium cloride to the sucrose medium produces a small (if any) effect on the palmitate-induced uncoupling and its sensitivity to carboxyatractylate and aspartate, but it exerts a pronounced increase in the recoupling effect of glutamate on this uncoupling. 20-40 microM cetyltrimethyl ammonium bromide increases the recoupling effect of glutamate and aspartate but decreases that of carboxyatractylate. The data are in line with suggestion that both ATP/ADP antiporter and aspartate/glutamate antiporters are involved in the fatty acid-induced uncoupling.

Topics: Amino Acid Transport Systems, Acidic; Animals; Antiporters; Aspartic Acid; Atractyloside; Carrier Proteins; Cetrimonium; Cetrimonium Compounds; Glutamic Acid; Magnesium Chloride; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Palmitic Acid; Potassium Chloride; Rats; Uncoupling Agents

The pH effect on carboxyatractylate-, glutamate- and aspartate-induced recoupling of palmitate-uncoupled rat liver mitochondria has been studied. Stimulation of respiration by low palmitate concentrations (5-20 microM) in the presence of 3 mM MgCl2 is shown to be pH-independent within the 7.0-7.8 range. The recoupling effect of glutamate (or aspartate) decreases and that of carboxyatractylate increases with increase in pH. The recoupling effect of a combination of carboxyatractylate and glutamate (aspartate) appears to be constant at these pH values, being as high as about 80%. It is concluded that uncoupling by low palmitate in liver mitochondria is mediated mainly by ATP/ADP and aspartate/glutamate antiporter.

Topics: Animals; Antiporters; Aspartic Acid; Atractyloside; Fatty Acids; Glutamic Acid; Hydrogen-Ion Concentration; Magnesium Chloride; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Oxygen Consumption; Palmitic Acid; Rats; Uncoupling Agents

The action of cysteinesulfinate, the substrate of the aspartate/glutamate antiporter on the palmitate-induced uncoupling in rat liver mitochondria and the recoupling effect of glutamate, aspartate, and diethyl pyrocarbonate was studied. In the presence of palmitate and an inhibitor of the ADP/ATP-antiporter carboxyatractylate, cysteinesulfinate exerted a relatively week recoupling effect. However, it significantly decreased the recoupling action of glutamate, aspartate, and diethyl pyrocarbonate. In the presence of cysteinesulfinate, these compounds caused recoupling at higher concentrations. The data show that the recoupling action of glutamate, aspartate, and diethyl pyrocarbonate is due to their interaction with the aspartate/glutamate antiporter. The data also confirm the suggestion that this anion carrier is involved in the uncoupling action of fatty acids.

Topics: Animals; Aspartic Acid; Atractyloside; Cysteine; Diethyl Pyrocarbonate; Fatty Acids; Glutamic Acid; Mitochondria, Liver; Neurotransmitter Agents; Rats

The transport by the mitochondrial ADP/ATP carrier (AAC) has been shown in a preliminary communication to produce electrical capacitive currents on photolysis of caged ATP or ADP with reconstituted AAC liposomes attached to black lipid membranes [Brustovetsky, N., Becker, A., Klingenberg, M., and Bamberg, E. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 664-668]. Here we study the relation of the currents to ADP/ATP fluxes, the interaction of caged ADP and ATP with AAC and other basic facets of this method. Caged ADP and ATP are not transported by the AAC, as shown in mitochondria. Flux measurements with reconstituted AAC show that caged nucleotides are competitive inhibitors (Ki = 5 microM for caged ADP and 1 microM for caged ATP). Caged ATP competes with photolyzed ATP as shown by the dependence of the currents on the caged ATP concentration and on the light intensity. A competition of added ADP with caged ATP on the currents yields Ki = 50 microM for ADP. We conclude that caged ADP and ATP bind tighter to AAC than ADP or ATP, allowing immediate initiation of translocation by in situ photolysis. The caged compounds bind preferentially at the cytosolic side of AAC. With a regenerative hexokinase + glucose system, the currents are stabilized in repetitive flashes and can be used for applying inhibitors etc. during a flash series. The currents are completely inhibited by the combined addition of the AAC inhibitors bongkrekate (BKA) and carboxyatractylate (CAT). The partial inhibition by CAT or BKA is dependent on the number of flash cycles increasing from 60% to 90%, and by replacing chloride with gluconate from only 30% to 90%. The current are increased by a K+ diffusion potential (valinomycin + KCl) and decreased by the permeant anion TPB-. The pH dependence of the currents and of the parallel flux measurements indicates that only the fully charged ATP4- and ADP3- are transported. A strong temperature dependence of the currents with a break at 15 degrees C (EA = 95 and 28 kJ) agrees with former measurements of flux rates in mitochondria. In conclusion, the capacitive currents faithfully reflect AAC transport function and are a powerful tool for investigating the charge transfer in transport.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Atractyloside; Biological Transport; Bongkrekic Acid; Electric Conductivity; Hydrogen-Ion Concentration; Membrane Potentials; Mitochondria, Liver; Photolysis; Rats; Rats, Wistar; Sulfhydryl Reagents; Temperature

In AAC2 from Saccharomyces cerevisiae, nine additional charged residues (six positive, three negative) were neutralized by mutagenesis following the previous mutation of six arginines. Oxidative phosphorylation (OxPhos) in cells and mitochondria, the expression level of AAC protein, and the various transport modes of AAC in the reconstituted system were measured. Mutations are: within the first helix at K38A which is exclusive for AAC; K48A, and R152A, part of a positive triad occurring in the matrix portion of each repeat; two matrix lysines, K179M and K182I, and the negative triad helix-terminating residues, E45G, D149S, D249S. Cellular ATP synthesis (OxPhos) is nearly completely inhibited in K48A, R152A, D149S, and D249S, but still amounts to 10% in K38A and between 30% and 90% in the gly+ mutants K179M, K179I + K182I, and E45G. Comparison of the AAC content measured by ELISA and the binding of [3H]CAT and [3H]BKA reveals discrepancies in K48A, D149S, and D249S mitochondria, which provide evidence that these mutations largely abolish inhibitor binding. Also these mitochondria have undetectable OxPhos. Differently in K38A, CAT and BKA binding are retained at high AAC levels but OxPhos is very low. This reveals a special functional role of K38, different from the more structural role of R152, K48, D149, and D249. Transport activity was measured with reconstituted AAC. The electroneutral ADP/ADP exchange of gly- mutants is largely or fully suppressed in K48A, D149S, and D249S. K38A and R152A are still active at 18% and 30% of wt. The other three exchange modes, ATP/ADP, ADP/ATP, and ATP/ATP, are nearly suppressed in all gly- mutants but remain high in gly+ mutants. ATP-linked modes are higher than the ADP/ADP mode in gly+ but lower in gly- mutants, resulting in an exchange mode inversion (EMI). In the competition for AAC2 transport capacity, the weak ATP exporting modes are suppressed by the much stronger unproductive ADP/ADP mode causing inhibition of OxPhos. Together with previous results all members of three charge triads are now mutagenized, revealing drastic functional rotatory asymmetries within the three repeat domains. In the intrahelical arginine triad the third (R294A), in the positive matrix triad the second (R152A), and in the helix-terminating negative triad the first (E45G) still show high activity.

Topics: Adenosine Triphosphate; Amino Acid Sequence; Anti-Bacterial Agents; Atractyloside; Biological Transport; Bongkrekic Acid; Cell Respiration; Cytochrome c Group; Enzyme-Linked Immunosorbent Assay; Immunoblotting; Liposomes; Membrane Proteins; Mitochondria; Mitochondrial ADP, ATP Translocases; Molecular Sequence Data; Mutagenesis; Oxidative Phosphorylation; Protein Binding; Saccharomyces cerevisiae

The electrophoretic export of ATP against the import of ADP in mitochondria bridges the intra- versus extramitochondrial ATP potential gap. Here we report that the electrical nature of the ADP/ATP exchange by the mitochondrial ADP/ATP carrier (AAC) can be directly studied by measuring the electrical currents via capacitive coupling of AAC-containing vesicles on a planar lipid membrane. The currents were induced by the rapid liberation of ATP or ADP with UV flash photolysis from caged nucleotides. Six different transport modes of the AAC were studied: heteroexchange with either ADP or ATP inside the vesicles, initiated by photolysis of caged ATP or ADP; homoexchange with ADPex/ADPin or ATPex/ATPin; and caged ADP or ATP with unloaded vesicles. The heteroexchange produced the largest currents with the longest duration in line with the electrical charge difference ATP4- versus ADP3-. Surprisingly, also in the homoexchange and with unloaded vesicles, small currents were measured with shorter duration. In all three modes with caged ATP, a negative charge moved into the vesicles and with caged ADP it moved out of the vesicles. All currents were completely inhibited by a mixture of the inhibitors of the AAC, carboxyatractyloside and hongkrekate, which proves that the currents are exclusively due to AAC function. The observed charge movements in the heteroexchange system agree with the prediction from transport studies in mitochondria and reconstituted vesicles. The unexpected charge movements in the homoexchange or unloaded systems are interpreted to reveal transmembrane rearrangements of charged sites in the AAC when occupied with ADP or ATP. The results also indicate that not only ATP4- but also ADP3- contribute, albeit in opposite direction, to the electrical nature of the ADP/ATP exchange, which is at variance with former conclusions from biochemical transport studies. These measurements open up new avenues of studying the electrical interactions of ADP and ATP with the AAC.

Topics: Adenine Nucleotides; Adenosine Triphosphate; Animals; Atractyloside; Binding Sites; Biological Transport; Bongkrekic Acid; Cattle; Electric Conductivity; Guanosine Triphosphate; Membrane Proteins; Membranes, Artificial; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Models, Biological; Ultraviolet Rays

This work shows that 6-ketocholestanol (kCh) inhibits the effect of carbonyl cyanide-m-chlorophenyl hydrazone (CCP) on mitochondrial Ca2+ efflux. Such an effect proved to be caused by diminution of membrane fluidity, therefore, it is affected by the incubation temperature. Furthermore, kCh reversed CCP-induced Ca2+ efflux depending on the accumulation of phosphate. It is also shown that kCh enhances the effect of carboxyatractyloside on membrane permeability transition.

Topics: Animals; Atractyloside; Calcium; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Intracellular Membranes; Ketocholesterols; Kidney; Membrane Fluidity; Mitochondria; Permeability; Rats; Vitamin K

The functional relationship between the adenine nucleotide translocator (ANT) and the mitochondrial multiple conductance channel (MCC) was investigated using patch-clamp techniques. MCC activity with the same conductance, ion selectivity, voltage dependence, and peptide sensitivity could be reconstituted from inner membrane fractions derived from mitochondria of ANT-deficient and wild-type Saccharomyces cerevisiae. In addition, the MCC activity of mouse kidney mitoplasts was unaffected by carboxyatractyloside, a known inhibitor of ANT and inducer of a permeability transition. These results suggest that MCC activity is independent of ANT.

Topics: Amino Acid Sequence; Animals; Atractyloside; Electron Transport Complex IV; Enzyme Inhibitors; Intracellular Membranes; Ion Channels; Kidney; Kinetics; Membrane Potentials; Mice; Mitochondria; Mitochondrial ADP, ATP Translocases; Molecular Sequence Data; Patch-Clamp Techniques; Peptides; Saccharomyces cerevisiae

The exogenous NADH dehydrogenase of heart mitochondria is increasingly reported to mediate cardiomyopathies following adriamycin treatment or reperfusion of ischemic hearts. A great number of studies on the biochemistry and pathobiology exists which indirectly support the existence of this dehydrogenase. Our studies exclude both the rotenone-insensitive NADH dehydrogenase of the outer membrane and the endogenous NADH dehydrogenase of damaged mitochondria as being responsible for external NADH consumption. Reducing equivalents from external NADH were demonstrated to enter complex I of the respiratory chain from the cytosolic phase. Our data support earlier reports on the physical association of the exogenous NADH dehydrogenase with the inner mitochondrial membrane excluding oxidation of external NADH via an enzyme of artefactual origin.

Topics: Animals; Artifacts; Atractyloside; Cytochrome b Group; Cytosol; Kinetics; Male; Mitochondria, Heart; Mitochondria, Liver; Myocardium; NAD; NAD(P)H Dehydrogenase (Quinone); Oligomycins; Oxidation-Reduction; Oxidative Phosphorylation; Oxygen Consumption; Rats; Rats, Sprague-Dawley

In order to gain an understanding of the mechanisms involved in the transfer of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) from the cytosol where it is synthesized to the Golgi lumen where it serves as the universal sulfate donor for sulfate ester formation in higher organisms, we have undertaken a kinetic characterization of the PAPS translocase from rat liver Golgi. Analyzing the PAS translocase activity in both intact Golgi vesicles and in a reconstituted liposome system, we have determined a number of physical and kinetic parameters. Strong competitive inhibition in zero-trans uptake experiments only with beta-methylene PAPS and adenosine 3',5'-biphosphate (PAP) suggest the transporter is highly specific for the 3'-phosphate. The demonstration of trans acceleration as observed by stimulation of transport activity under exchange conditions suggests that the translocase is a carrier with distinct binding sites accessible from both faces of the membrane. The behavior of the PAPS translocase in the presence of equilibrium concentrations of PAP supports the function of an antiport mechanism. Thus the translocase is characterized by its kinetic properties as a specific transporter of PAPS which acts through an antiport mechanism with PAP as the returning ligand. This characterization of the transport activity has proved instrumental in the identification of an approximate 230 kDa Golgi membrane protein as the PAPS translocase protein [Ozeran, J.D., Westley, J., & Schwartz, N.B. (1996) Biochemistry 35, 3695-3703 (accompanying paper)].

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenosine Diphosphate; Animals; Antiporters; Atractyloside; Biological Transport; Cell Membrane; Cytoplasm; Golgi Apparatus; Ion Transport; Kinetics; Liposomes; Liver; Membrane Proteins; Nucleotides; Palmitoyl Coenzyme A; Phosphoadenosine Phosphosulfate; Rats; Sulfates

The role of the adenine nucleotide translocase on Ca2+ homeostasis in mitochondria from brown adipose tissue was examined. It was found that in mitochondria incubated with 50 microM Ca2+, ADP was not needed to retain the cation, but it was required for strengthening the inhibitory effect of cyclosporin on membrane permeability transition as induced by menadione. In addition, carboxyatractyloside was unable to promote matrix Ca2+ release, even though it inhibits the ADP exchange reaction. However, when the Ca2+ concentration was increased to 150 microM carboxyatractyloside did induce Ca2+ release, and ADP favored Ca2+ retention. Determination of cardiolipin content in the inner membrane vesicles showed a greater concentration in brown adipose tissue mitochondria than that found in kidney mitochondria. It suggested that the failure of the adenine nucleotide translocase to influence membrane permeability transition depends on the lipid composition of the inner membrane.

Topics: Adenosine Diphosphate; Adipose Tissue, Brown; Animals; Atractyloside; Calcium; Guanosine Diphosphate; In Vitro Techniques; Ion Transport; Membrane Lipids; Mitochondria; Mitochondrial ADP, ATP Translocases; Rats; Rats, Wistar

Zearalenone (F-2) is a mycotoxin which acts as a protonophoric uncoupler in plant mitochondria [Macri, F. and Vianello, A., (1990) J. Plant Physiol. 136, 754-757]. In the present paper, the mechanism of F-2-induced uncoupling in pea mitochondria was studied. The uncoupling by F-2 was partially reversed by 6-ketocholestanol (kCh) under conditions in which kCh completely reversed the FCCP-induced uncoupling and almost did not affect the palmitate-induced uncoupling. Recoupling effects of carboxyatractylate, ADP and cyclosporin A were small and could not essentially decrease the kCh-insensitive part of F-2-induced uncoupling. It is suggested that a protein, mediating kCh-sensitive uncoupling, is involved in the F-2 effect in plant mitochondria.

Topics: Atractyloside; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Dose-Response Relationship, Drug; Estrogens, Non-Steroidal; Glutamic Acid; Ketocholesterols; Membrane Potentials; Mitochondria; Palmitic Acid; Plants; Uncoupling Agents; Zearalenone

In intact bovine heart mitochondria, cAMP-dependent phosphorylation of 42, 29, 18 and 6.5 kDa proteins was inhibited by carboxyatractyloside. This shows that both mitochondrial cAMP-dependent protein kinase (mtPKA) and its protein substrates are localized at the matrix side of the inner mitochondrial membrane. Proteins of 42, 29, 18, and 6.5 kDa were also bound at the outer surface of mitochondria where they were phosphorylated by the added purified catalytic subunit of PKA. In the cytosol from bovine heart proteins of the above molecular weights were phosphorylated by the cytosolic PKA.

Topics: Animals; Atractyloside; Cattle; Cyclic AMP-Dependent Protein Kinases; Cytosol; Intracellular Membranes; Membrane Proteins; Mitochondria, Heart; Phosphorylation

Peroxidative treatment of rat heart mitochondria results in a gradual increase of the apparent molecular weight of the adenine nucleotide translocase (ANT) by up to 1.2 kDa. ANT isolated from mitochondria treated with 1 mM tert-butyl hydroperoxide and 5-40 microM Cu2+ for 1 h at 37 degrees C exhibited a progressive loss of lysine, cysteine, arginine, and valine residues compared to native ANT. N-Ethylmaleimide, dithiothreitol, and the specific inhibitor of ANT, carboxyatractyloside (CAT), inhibited the peroxidation-induced molecular weight shift without inhibiting lipid peroxidation, which is believed to be the primary cause of the observed ANT modification. Bongkrekic acid, which stabilizes ANT in a conformation different from that brought about by CAT, did not inhibit the ANT molecular weight shift. Dithiothreitol, as well as CAT, was found to protect ANT against most of the losses of amino acid residues, indicating that alteration of sulfhydryl residues is required for chemical modification of, not only cysteine, but also lysine, arginine, and valine. We conclude that the peroxidative modification of ANT is conformation-dependent and involves chemical modification of cysteine as a critical step.

Topics: Animals; Atractyloside; Bongkrekic Acid; Butylated Hydroxytoluene; Copper; Dithiothreitol; Ethylmaleimide; Male; Mannitol; Membrane Proteins; Mitochondria; Mitochondrial ADP, ATP Translocases; Myocardium; Peroxides; Protein Conformation; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; tert-Butylhydroperoxide

Tryptophanyl substitution of the Saccharomyces cerevisiae adenine nucleotide carrier (Anc2p isoform) was not deleterious for the transport activity or the folding of the carrier [preceding paper by Le Saux et al. (1996) Biochemistry 35, 16116-16124]. Conformational changes of the isolated wild-type and Trp-substituted Anc2p variants, induced upon binding of specific substrates [adenosine triphosphate (ATP) or diphosphate (ADP)] or inhibitors [carboxyatractyloside (CATR) or bongkrekic acid (BA)], were studied by measurement of intrinsic fluorescence. Titration of CATR and BA binding sites ended in the same number of sites, namely, 6-7 nmol/mg of wild-type and variant Anc2p. Isolated Anc2p in detergent presented similar emission spectra, suggesting that all tryptophanyl residues were in environments of similar hydrophobicity. Trp87 and Trp126 contributed largely and to a similar extent to the fluorescence enhancement observed in response to ATP binding, while Trp235 contributed negatively and to a small extent to the fluorescence change. Both Trp126 and Trp235, and to a lower extent Trp87, participate in the CATR-induced fluorescence decrease of Anc2p. Responses to BA binding were observed only in the presence of ATP; they consisted of a further fluorescence increase of the Anc2p.ATP complex, which was mainly due to Trp87 and Trp126, Trp235 being much less responsive. The different fluorescence responses of the three Trp residues of Anc2 variants to ATP, CATR, and BA are in agreement with distinct binding sites for these ligands and distinct conformations of the carrier protein recognizing specifically CATR or BA. A mechanistic model is proposed to interpret the transitions between the different conformational states of Anc2p.

Topics: Adenosine Diphosphate; Atractyloside; Binding Sites; Bongkrekic Acid; Kinetics; Mitochondria; Mitochondrial ADP, ATP Translocases; Models, Structural; Mutagenesis, Site-Directed; Point Mutation; Protein Conformation; Protein Structure, Secondary; Recombinant Proteins; Saccharomyces cerevisiae; Spectrometry, Fluorescence; Tryptophan

Mutagenesis of three intrahelical arginines, R96, R204, or R294, and of each member of the arginine triplet R252, R253, R254 into neutral residues had resulted in a strong suppression of oxidative phosphorylation in cells and isolated mitochondria [Müller, V., Basset, G., Nelson, D. R., & Klingenberg, M. (1996) Biochemistry 35, 16132-16143]. Here we determine the transport activity of wild-type and mutant AAC in reconstituted proteoliposomes using a new rapid removal-stop method without relying on the inhibitor stop which can be compromised by mutations. The basic electroneutral ADP/ADP exchange activity is strongly or totally suppressed in six out of seven of these mutations, with the exception of R294A, which retains nearly wild-type activity. Carboxyatractylate (CAT) inhibits the ADP/ATP exchange rate only to 3-10% in wild type and R294A and up to 40% in other mutants, whereas bongkrekic acid (BKA) inhibits 50% (wild type and R294A) and 90% (other mutants). Consequently, AAC is preferentially reconstituted with the matrix surface outside. All these mutations drastically change activity distribution among the four exchange modes ADP/ADP, ADP/ATP, ATP/ADP, and ATP/ATP. Whereas in wild-type AAC the homo ATP/ATP exchange is twice as high as the ADP/ADP exchange, in mutants it is 10 to 15 times lower. Similarly, the hetero ATP/ADP exchange in wild-type AAC is higher than the ADP/ ATP exchange, but in mutants it is several times lower. Thus, these mutations afflict the ATP-linked modes, in particular those linked to external ATP. The inhibition of oxidative phosphorylation is thus explained by the suppression of ATP export versus ADP import mode. The "extra"-inhibition of oxidative phosphorylation in mutant cells is explained by the extreme shift in mutants in favour of ATP import versus ADP export. Besides structural changes, the mutant effects indicate electrostatic interactions of these arginines with the anionic substrates. The loss of one positive charge raises the translocation barrier the more negative the substrate, i.e. more for ATP4- than for ADP3-. In none of these arginine mutants was the binding of CAT or BKA abolished.

Topics: Arginine; Atractyloside; Binding Sites; Kinetics; Liposomes; Mitochondria; Mitochondrial ADP, ATP Translocases; Mutagenesis, Site-Directed; Point Mutation; Recombinant Proteins; Saccharomyces cerevisiae

This study was designed to determine which enzyme activities were first impaired in mitochondria exposed to 2,2'-azobis-(2-amidinopropane) dihydrochloride (AAPH), a known radical initiator. EPR spin-trapping revealed generation of reactive oxygen species although malondialdehyde formation remained very low. With increasing AAPH concentrations, State-3 respiration was progressively depressed with unaltered ADP/O ratios. A top-down approach demonstrated that alterations were located at the phosphorylation level. As shown by inhibitor titrations, ATP/ADP translocase activity was unaffected in the range of AAPH concentrations used. In contrast, AAPH appeared to exert a deleterious effect at the level of F1F0-ATPase, comparable with dicyclohexylcarbodi-imide, which alters Fo proton channel. A comparison of ATP hydrolase activity in uncoupled and broken mitochondria reinforced this finding. In spite of its pro-oxidant properties, AAPH was shown to act as a dose-dependent inhibitor of cyclosporin-sensitive permeability transition initiated by Ca2+, probably as a consequence of its effect on F1F0-ATPase. Resveratrol, a potent antiperoxidant, completely failed to prevent the decrease in State-3 respiration caused by AAPH. The data suggest that AAPH, when used under mild conditions, acted as a radical initiator and was capable of damaging F1F0-ATPase, thereby slowing respiratory chain activity and reducing mitochondrial antioxidant defences.

Topics: Amidines; Animals; Atractyloside; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cyclic N-Oxides; Dicyclohexylcarbodiimide; Electron Spin Resonance Spectroscopy; Female; Free Radicals; Intracellular Membranes; Kinetics; Lipid Peroxidation; Malondialdehyde; Membrane Potentials; Mitochondria, Liver; Oxidative Phosphorylation; Oxygen Consumption; Proton-Translocating ATPases; Rats; Rats, Wistar; Spin Labels; Succinates

Effect of an ADP/ATP antiporter inhibitor carboxyatractylate on permeability of inner mitochondrial membrane was studied. Carboxyatractylate induced two uncoupling systems: Ca(2+)-dependent pore and another system which was sensitive to an inhibitor of calcium uniporter ruthenium red (apparently, Ca(2+)-recycle). Extent of Ca(2+)-recycle induction correlates with a fraction of mitochondrial population which underwent permeabilization; inhibition of permeabilization by cyclosporin A prevents induction of the recycle. Thus, induction of Ca(2+)-dependent pore is direct consequence of carboxyatractylate binding to mitochondria whereas induction of Ca(2+)-recycle is indirect. Both uncoupling systems are induced by carboxyatractylate concentrations which are specific for ADP/ATP antiporter. The data suggest that the antiporter can directly participate in the formation of Ca(2+)-dependent pore in inner mitochondrial membrane.

Topics: Animals; Atractyloside; Calcium; Carrier Proteins; Intracellular Membranes; Ion Channels; Membrane Proteins; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Mitochondrial Proteins; Permeability; Rats; Uncoupling Protein 1

Oxygen consumption and transmembrane electrical potential difference in liver mitochondria from hibernating, arousing and active ground squirrels have been compared. It is found that arousal from hibernation is accompanied by uncoupling which is mediated by two different mechanisms, one sensitive to cyclosporin A and the other suppressed by carboxyatractylate. Both uncoupling effects reach their maxima at 20 - 25 degrees C body temperatures. Involvement of an increase in the free fatty acid concentration in the arousal-induced uncoupling mechanisms is discussed.

Topics: Animals; Arousal; Atractyloside; Cyclosporine; Hibernation; Mitochondria, Liver; Oxygen Consumption; Sciuridae

The effect of calcium on the control exerted by the adenine nucleotide translocator over respiration in isolated heart mitochondria was investigated in order to determine whether calcium directly stimulates the translocator. At respiration rates intermediate between states 3 and 4, Ca2+ is shown to increase the control over 2-oxoglutarate oxidation exerted by the adenine nucleotide translocator in rat heart mitochondria. This did not occur when succinate was the respiratory substrate, even though the control exerted by the translocator was substantial, indicating that Ca2+ does not have a direct effect on the adenine nucleotide translocator. Ca2+ increased the uncoupled oxidation rate of 2-oxoglutarate, but not succinate. Using the summation theorem for flux control, the effect of Ca2+ is explained by a shift of the control over respiration rate toward the adenine nucleotide translocator, from the respiratory chain, presumably as the result of the activation of the 2-oxoglutarate dehydrogenase complex.

Topics: Adenosine Triphosphate; Animals; Atractyloside; Calcium; Dose-Response Relationship, Drug; Ketoglutaric Acids; Male; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Oxidation-Reduction; Oxidative Phosphorylation; Oxygen Consumption; Rats; Rats, Wistar; Succinates; Succinic Acid

The controversial data on the involvement of the ATP/ADP-antiporter in the uncoupling effect of fatty acids in liver mitochondria as well as on the sensitivity of the uncoupling process to the ATP/ADP-antiporter inhibitor, carboxyatractylate, have been analyzed. It has been shown that in liver mitochondria uncoupled by palmitic acid, pyridoxal-5-phosphate, diethyl pyrocarbonate and glutamate produce an additional recoupling action against the background of carboxyatractylate. No such effect is observed during mitochondrial uncoupling by FCCP. Micromolar concentrations of ADP added prior to palmitic acid produce a far more potent coupling action; the subsequent recoupling effect of carboxyatractylate diminishes thereby. This finding suggests that the reduction of endogenous ADP is responsible for the increased sensitivity of the uncoupling action of fatty acids to carboxyatractylate. It is concluded that the ATP/ADP-antiporter and other membrane carriers play a role in the uncoupling action of fatty acids on liver mitochondria.

Topics: Animals; Atractyloside; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Diethyl Pyrocarbonate; Glutamic Acid; Membrane Proteins; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Oxygen; Palmitic Acid; Palmitic Acids; Pyridoxal Phosphate; Rats

We have reconstituted a partially purified extract from rat liver endoplasmic reticulum membrane proteins into phosphatidylcholine liposomes. The resulting proteoliposomes, of an average diameter of 58 nm, transport intact ATP into their lumen in a temperature-dependent manner; transport was saturable (apparent Km = 0.72 microM) and highly specific: CMP-sialic acid and GTP were transported very slowly or not at all. Transport of ATP was inhibited by DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) but not by carboxyatractyloside. Previously, we showed that vesicles derived from rat liver and dog pancreas endoplasmic reticulum translocate ATP into their lumen in vitro but in these studies, following incubations with ATP, most of the phosphate was transferred to proteins because of the many kinases, endogenous acceptors for phosphorylation, and ATP binding proteins present in the vesicle membranes and lumen. This reconstituted system, which yielded a highly functional ATP transporter, can be used for further characterization and purification of this and probably other nucleotide transporters of the endoplasmic reticulum membrane. Previously used reconstitution protocols which were successful for Golgi membrane nucleotide transporters did not yield a functional endoplasmic reticulum ATP transporter.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenosine Triphosphate; Animals; Atractyloside; Biological Transport; Cytidine Monophosphate N-Acetylneuraminic Acid; Dogs; Endoplasmic Reticulum; Guanosine Triphosphate; Kinetics; Liposomes; Liver; Male; Membrane Proteins; Microscopy, Electron; Pancreas; Phosphorylation; Proteolipids; Rats; Rats, Sprague-Dawley; Substrate Specificity

The energy state of rat liver mitochondria on day 10 of cold acclimation, when the body temperature decreases significantly concomitantly with an increase in the content of long-chain acyl-CoAs in the liver, has been studied. State 4 and uncoupling respiration rates increase in parallel; however, the integral potentials of the adenine nucleotide system in the liver diminish. In the presence of oligomycin, ADP (20 and 50 microM) decreases the II(+)-permeability and increases the Ca(2+)-capacity of mitochondria in both control and, in a lesser degree, cold-exposed rats. At 90 microM ADP has the same effect on mitochondria of both groups of rats. Carboxyatractylate abolishes the ADP effect on the mitochondria. In EGTA-containing media carboxyatractylate decreases the respiration rate in oligomycin-treated mitochondria. Palmitoyl-CoA increases the II(+)-permeability of the mitochondrial membrane and decreases the Ca(2+)-capacity of mitochondria. ADP abolished the competitive effects of this long-chain acyl-CoA. Possible involvement of the ATP/ADP antiporter in the thermoregulatory response of liver mitochondria in cold-acclimated rats and the role of fatty acids and long-chain acyl-CoAs in this process are discussed.

Topics: Acyl Coenzyme A; Adaptation, Physiological; Animals; Atractyloside; Body Temperature Regulation; Cold Temperature; Energy Metabolism; Intracellular Membranes; Ion Transport; Male; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Oligomycins; Oxygen; Permeability; Rats; Rats, Wistar

The effect of malate on respiration in liver mitochondria has been studied during oxidation of succinate in the presence of rotenone both in state 3 and after palmitate addition. Malate was shown to stimulate the rate of mitochondrial respiration in the both respiratory states, its effect being increased in the presence of the NAD-dependent substrates of oxidation-glutamate and pyruvate or thiols (cysteine and thiourea) Preincubation of mitochondria for 5 min in the absence of respiratory substrates eliminated the stimulating effect of malate. However, this effect was manifested in the conditions when the NAD-dependent respiratory substrates or thiols were added after preincubation of mitochondria. p-Chloromercuribenzoate eliminated the stimulating effect of malate. Carboxyatractyloside and ATP inhibited mitochondrial respiration in the presence of palmitate. Malate did not influence the action of the first effector but eliminated that of the second effector. It is concluded that malate can regulate oxidative phosphorylation and palmitate-uncoupled respiration by affecting the adenine nucleotide transported. The SH-groups localized outside the mitochondria in the hydrophilic region play an important role in the realization of malate effects.

Topics: Adenosine Triphosphate; Animals; Atractyloside; Electron Transport; Malates; Mitochondria, Liver; Oxidative Phosphorylation; Palmitic Acid; Palmitic Acids; Rats; Sulfhydryl Compounds

Earlier it has been demonstrated that the recoupling action of cyclosporin A on mitochondria deenergized by the opening of the Ca(+)-dependent pore requires higher cyclosporin A concentrations than those, which are necessary for preventing the permeabilization as well as the presence of additional effectors, such as adenine nucleotides and Mg2+. It has been shown that cyclosporin A reseals the pore and prevents its opening when used at the same low concentrations (0.1-0.4 microM), while its higher concentrations (0.5-1.0 microM) produce the recoupling of mitochondria. In contrast with recoupling, resealing of the pore by cyclosporin A does not require Mg2+ or adenine nucleotides. Carboxyatractylate which reverses the cyclosporin A-induced restoration of the membrane potential does not induce repeated opening of the pore. The carboxyatractylate-induced depolarization of the inner mitochondrial membrane is sensitive to ruthenium red. Besides, ruthenium red restores the recoupling action of cyclosporin A in the absence of carboxyatractylate. The data obtained suggest that the loss by cyclosporin A of its recoupling potency may result from the induction of the Ca2+/2H+ antiporter. Induction of the Ca2+/2H+ antiporter in combination with a ruthenium red-sensitive Ca2+ uniporter provides the uncoupling mitochondria even with a closed Ca(2+)-dependent pore. Apparently, Ca2+/2H+ antiporter induction is a result of inhibition of the ADP/ATP antiporter by a natural or exogenous inhibitor. This process seems to require a preliminary release of certain protective factors from the mitochondrial matrix.

Topics: Adenine Nucleotides; Animals; Antiporters; Atractyloside; Calcium; Calcium-Binding Proteins; Cation Transport Proteins; Cyclosporine; Intracellular Membranes; Magnesium; Membrane Potentials; Mitochondria, Liver; Rats; Ruthenium Red

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Adipose Tissue, Brown; Animals; Atractyloside; Carbon Radioisotopes; Carrier Proteins; Cattle; Cell Fractionation; Centrifugation; Chromatography; Chromatography, Gel; Chromatography, Ion Exchange; Cricetinae; Dansyl Compounds; Detergents; Durapatite; Fluorescent Dyes; Indicators and Reagents; Ion Channels; Kinetics; Liposomes; Membrane Proteins; Mesocricetus; Mitochondria; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Mitochondrial Proteins; Octoxynol; Radioisotope Dilution Technique; Solubility; Spectrometry, Fluorescence; Ultracentrifugation; Uncoupling Agents; Uncoupling Protein 1

The present study was designed to establish the characteristics of the Ca2+ fluxes in isolated mitochondria of the protist Euglena gracilis. Uptake of Ca2+ and Sr2+ was supported by succinate and lactate oxidation. Ca2+ influx was slightly inhibited by 5 microM Ruthenium red and completely blocked by La3+ with a half-maximal inhibition attained at 50 microM. The addition of inorganic phosphate induced a 3-fold stimulation of Ca2+ uptake. Ca2+ uptake was inhibited by Mg2+ only in the absence of phosphate. Ca2+ efflux was induced by Na+, Li+ and K+ through a diltiazem-insensitive reaction. Ca2+ release, collapse of membrane potential and swelling were induced by Hg2+ and Cd2+ but not by carboxyatractyloside; cyclosporin A did not prevent the Ca2+ release induced by the heavy metal ions. Ca2+ uptake was achieved in the presence of 3 microM antimycin or 0.1 mM cyanide; this finding indicates that the alternative respiratory chain present in Euglena mitochondria can support this energy-dependent reaction. The data obtained suggest similar pathways, but different regulatory mechanisms, for Ca2+ transport between protist and mammalian mitochondria.

Topics: Animals; Atractyloside; Biological Transport, Active; Cadmium; Calcium; Electron Transport; Euglena gracilis; Ion Transport; Lactates; Lactic Acid; Lanthanum; Lithium; Magnesium; Mercury; Mitochondria; Phosphates; Potassium; Ruthenium Red; Sodium; Strontium; Succinates; Succinic Acid

The effects of various compounds such as the transport substrate ADP and the transport inhibitors carboxyatractyloside (CATR) and bongkrekic acid (BKA) on the labeling of cysteine residues in the ADP/ATP carrier of bovine heart submitochondrial particles by the SH reagent eosin-5-maleimide (EMA) were studied. Of the four cysteine residues in the carrier, the labeling of Cys159 by EMA progressed predominantly and rapidly, and those of Cys56 and Cys256 moderately, but Cys128 was not labeled, as we reported previously [Majima, E., et al. (1993) J. Biol. Chem. 268, 22181-22187]. ADP inhibited the labelings of Cys56, Cys159, and Cys256 by EMA. BKA markedly inhibited the labeling of Cys159 by EMA, and also the labeling of Cys256, but did not affect the labeling of Cys56, suggesting that it binds from the matrix side to a region close to Cys159 in the second loop facing the matrix space. CATR completely inhibited the labeling by EMA when added on the cytosolic side, but had no effect when added on the matrix side. From these results, the conformational changes of the carrier induced by CATR, BKA, and ADP are discussed. Furthermore, a mechanism of adenine nucleotide transport through the ADP/ATP carrier in association with change in its conformation is proposed.

Topics: Adenine Nucleotides; Adenosine Diphosphate; Adenosine Triphosphate; Animals; Atractyloside; Biological Transport; Bongkrekic Acid; Cattle; Cysteine; Eosine Yellowish-(YS); Mitochondria; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Models, Biological; Protein Conformation; Submitochondrial Particles

Iphiona aucheri is responsible for poisoning racing camels (Camelus dromedarius) in the United Arab Emirates U. A. E. Terpenoids, a non-toxic pyrrolizidine alkaloid and two diterpene glycosides, atractyloside and carboxyatractyloside, were isolated and their structures determined by spectroscopic methods. Atractyloside and carboxyatractyloside were identified as the toxic principle of the plant.

Topics: Animals; Atractyloside; Carbohydrate Conformation; Magnetic Resonance Spectroscopy; Mice; Molecular Structure; Plants, Toxic; Spectrometry, Mass, Fast Atom Bombardment

Effects of ADP and Mg2+ on the ability of cyclosporin A to "reseal" mitochondria permeabilized by Ca2+ and P(i) have been studied. Cyclosporin A was completely ineffective, when ADP and Mg2+ were not included into the incubation medium. Both ADP and Mg2+ used at high concentrations potentiated the effect of cyclosporin A and prevented it reversal by carboxyatractylate. Data on the influence of different concentrations of ADP and Mg2+ on the resealing efficiency of cyclosporin A suggest that the true effector modulating the state of the Ca(2+)-dependent pore is the ADP-Mg2+ complex, but not ADP or Mg2+ used separately. The ability of non-hydrolyzable analogs of adenine nucleotides, ADP-S and ATP-S, to potentiate the resealing action of cyclosporin on mitochondria permeabilized by loading of different Ca2+ concentrations to that of ADP was compared. ATP-S was ineffective when the pore was induced by high concentrations of Ca2+. The results obtained are discussed in terms of hypothesis on the direct involvement of the ADP/ATP antiporter in regulation of the inner mitochondrial membrane Ca(2+)-dependent pore state.

Topics: Adenine Nucleotides; Animals; Atractyloside; Calcium; Cyclosporine; Drug Interactions; Hydrolysis; Magnesium; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Protein Conformation; Rats

The effects of carboxyatractylate (CAtr) on delta psi in sunflower hypocotyl and pea stem mitochondria were compared. In sunflower mitochondria, (1) CAtr at higher concentration increased delta psi in the presence of palmitate and delta psi in metabolic state 3; (2) ]1 microM CAtr did not prevent delta psi decrease, induced by ADP addition (in contrast to pea mitochondria); (3) The ATP-generated delta psi was small and was insensitive to 40 microM CAtr. Under the same conditions, in pea mitochondria generation of delta psi by ATP was inhibited by 1 microM CAtr.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Atractyloside; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Fabaceae; Helianthus; Intracellular Membranes; Kinetics; Membrane Potentials; Mitochondria; Oligomycins; Palmitic Acid; Palmitic Acids; Plant Stems; Plants, Medicinal

We studied the relative potencies of cyclosporin A and endogenous effectors (Mg2+ and ADP) to recouple rat liver mitochondria permeabilized by different Ca(2+)-loading in a P(i)-containing medium. Recoupling efficiency of cyclosporin A dramatically decreased at high Ca(2+)-loading (approx. 100 nM of Ca2+/mg protein and more). Mitochondria permeabilized by high Ca2+ were recoupled with approximately equal efficiency by higher cyclosporin A concentrations or by adding 1-5 mM Mg2+ together with low concentrations of cyclosporin A while potentiating effect of ADP on the cyclosporin A recoupling potency was insignificant. Mg2+ ions at concentrations of 3 mM and higher also prevented the carboxyatractylate-induced reversion of cyclosporin A recoupling effect. The data point to competitive relationships between cyclosporin A and/or Mg2+ ions and Ca2+ ions for the site(s) regulating permeability state of the pore.

Topics: 2,4-Dinitrophenol; Adenosine Diphosphate; Animals; Atractyloside; Calcium Chloride; Cyclosporine; Dinitrophenols; Egtazic Acid; Magnesium Sulfate; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Onium Compounds; Organophosphorus Compounds; Permeability; Rats

Carboxyatractyloside partially restored the transmembrane electrical potential difference (delta psi) dissipated by low concentrations of palmitate in pea stem mitochondria. This effect was more marked when mitochondria from sunflower were assayed. It is suggested that the ATP/ADP translocator is involved in the free fatty acid-induced uncoupling of oxidative phosphorylation in plant mitochondria, only when its level is sufficiently high and the concentration of the fatty acid is low to collapse only partially delta psi.

Topics: Atractyloside; Fabaceae; Helianthus; Membrane Potentials; Mitochondria; Mitochondrial ADP, ATP Translocases; NAD; Oxygen Consumption; Palmitic Acid; Palmitic Acids; Plants; Plants, Medicinal; Uncoupling Agents

Topics: Acetylcarnitine; Aging; Animals; Atractyloside; Electron Transport Complex IV; Kinetics; Male; Membrane Lipids; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Oxygen; Phosphorylation; Rats; Rats, Inbred F344

Carboxyatractyloside partially restored the transmembrane electrical potential difference (delta psi) dissipated by low concentrations of palmitate in pea stem mitochondria. This effect was more marked when mitochondria from sunflower were assayed. It is suggested that the ATP/ADP translocator is involved in the free fatty acid-induced uncoupling of oxidative phosphorylation in plant mitochondria, only when its level is sufficiently high and the concentration of the fatty acid is low to collapse only partially the delta psi.

Topics: Atractyloside; Fabaceae; Helianthus; Membrane Potentials; Mitochondria; Mitochondrial ADP, ATP Translocases; NAD; Oxygen Consumption; Palmitic Acid; Palmitic Acids; Plants; Plants, Medicinal; Uncoupling Agents

Oleate (10-60 microM) stimulates oxygen consumption in hepatocytes in the presence of oligomycin. This stimulation of respiration is partially suppressed by 20 microM carboxyatractylate but is insensitive to 0.5 microM cyclosporin A. The results obtained suggest that fatty acids can uncouple oxidative phosphorylation in hepatocytes and that the uncoupling mechanism is the same as that in isolated mitochondria in the presence of EGTA.

Topics: Animals; Atractyloside; Cyclosporine; Drug Synergism; Male; Mitochondria, Liver; Oleic Acid; Oleic Acids; Oligomycins; Oxidative Phosphorylation; Oxygen; Rats; Rats, Sprague-Dawley; Uncoupling Agents

The uncoupling effect of 2-hexadecylmalonic acid (HDMA) and 2-tetradecylmalonic acid (TDMA), the dicarboxylic derivatives of stearic and palmitic acids, have been studied in heart mitochondria. The effects of these compounds and related fatty acids on the induced by ascorbate oxidation with PMS (or TMPD) in the presence of oligomycin, phosphate and rotenone have been compared. It was found that both HDMA and TDMA uncoupled mitochondria with a similar efficiency as did the corresponding fatty acids, while the uncoupling effect of low concentrations of HDMA and TDMA were almost completely suppressed by carboxyatractylate.

Topics: Animals; Atractyloside; Mitochondria, Heart; Oligomycins; Oxidation-Reduction; Palmitic Acids; Phosphates; Rats; Rotenone; Stearic Acids; Uncoupling Agents

It has been shown that the increase of the membrane potential in uncoupled brown fat mitochondria after addition of 1-2 mM GDP to a medium containing potassium acetate is accompanied by swelling of mitochondria which occurs in two phases. The first swelling phase is due to the electrogenic transport of K+ ions, while the second one is activated by carboxyatractylate and carnitine. The swelling is practically completely inhibited by a further rise of the GDP concentration irrespective of high values of the membrane potential. Unlike K+ transport, the potential-dependent transport of Na+ ions occurs more rapidly, is monophasic and insensitive to carboxyatractylate and carnitine. It is suggested that regulation of K+ ion transport in brown fat mitochondria is mediated by the action of nucleotides and fatty acid esters on the uncoupling protein and ADP/ATP antiporter.

Topics: Adipose Tissue, Brown; Animals; Atractyloside; Biological Transport; Guanosine Diphosphate; In Vitro Techniques; Mitochondria; Mitochondrial Swelling; Potassium; Sciuridae

Regulation of the mitochondrial permeability transition pore has been investigated following the release of matrix solutes which normally participate in pore regulation. Under these conditions, neither cyclosporin A nor ADP induces pore closure, as judged by restoration of delta psi, unless Mg2+ is also added. Mg2+ alone is ineffective. In liver mitochondria, the Mg2+ effect is expressed over a 0 to 0.5 mM concentration range with higher concentrations inhibiting repolarization. In heart mitochondria, the inhibitory action of high Mg2+ is not seen and it can be shown that the Mg2+ effect on repolarization increases progressively up to a concentration of 5 mM. In liver mitochondria, when the pore is closed by maximally effective concentrations of Mg2+ plus cyclosporin A or Mg2+ plus ADP, reopening occurs upon the addition of carboxyatractyloside. The latter compound, however, fails to reopen the pore when Mg2+, cyclosporin A, and ADP are present simultaneously. In heart mitochondria, where higher Mg2+ concentrations can be employed, Mg2+ plus cyclosporin A or Mg2+ plus ADP produces pore closure in a carboxyatractyloside insensitive manner. Titration experiments support the adenine nucleotide translocase as the site at which carboxyatractyloside acts to regulate the pore. However, the action of ADP appears to involve a translocase-independent site. In intact mitochondria the action of carboxyatractyloside on pore opening is counteracted by oligomycin, apparently through inhibition of the F1F0 ATP synthase, with a consequent increase in the matrix space ADP/ATP ratio. It is concluded that the permeability transition pore induced by Ca2+ plus P(i) is not formed from the adenine nucleotide translocase although the translocase conformation is one of several factors which regulate the pore. The matrix Mg2+ concentration is also one of these factors. Formation of the pore by a Ca2+ and ADP binding protein is one model which is consistent with the present data.

Topics: Adenosine Diphosphate; Animals; Atractyloside; Binding Sites; Cyclosporine; Intracellular Membranes; Kinetics; Magnesium; Mitochondria, Heart; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Models, Biological; Oligomycins; Permeability; Proton-Translocating ATPases; Rats

The effect of TPP+ on the fatty acid or FCCP-induced uncoupling in rat heart mitochondria was studied. It was found that (a) TPP+ increases the stimulation of oxygen consumption by palmitic acid or FCCP in the presence of oligomycin, (b) TPP+ greatly enhances the palmitic acid or FCCP-induced delta psi decrease. Both effects of TPP+ were strongly suppressed by carboxyatractylate in the case of palmitate but were not in the case of FCCP. The role of ATP/ADP-antiporter in the TPP+ and palmitic acid effects is discussed.

Topics: Animals; Atractyloside; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Fluorescent Dyes; In Vitro Techniques; Mitochondria, Heart; Oligomycins; Onium Compounds; Organophosphorus Compounds; Oxygen Consumption; Palmitic Acid; Palmitic Acids; Phenazines; Rats; Spectrometry, Fluorescence; Uncoupling Agents

The effect of different agents on inner-mitochondrial-membrane permeabilization and lipoperoxidation induced by Ca2+ and the pyridine-nucleotide oxidant t-butylhydroperoxide or inorganic phosphate was investigated. Comparing the protection conferred by ADP, a substrate of the ADP/ATP carrier, dithiothreitol, a disulfide reductant and butylhydroxytoluene, a radical scavenger, it was found that ADP was always the most effective against mitochondrial damage, when present in the incubation medium from the beginning. Moreover, carboxyatractyloside, a specific inhibitor of the ADP/ATP carrier, abolished completely the protective effect of ADP on both the lipoperoxidation and mitochondrial swelling processes. Experiments where deenergized mitochondria were previously incubated with Ca2+ showed a decrease in the content of active ADP/ATP carrier, indicating a direct involvement of this protein in the formation of a non-specific Ca(2+)-dependent pore. Our results also eliminate the possibility of an attack of oxygen radicals on lipids or proteins of the mitochondrial membrane as the primary event triggering the permeability transition of the inner mitochondrial membrane.

Topics: Adenosine Diphosphate; Animals; Atractyloside; Butylated Hydroxytoluene; Calcium; Dithiothreitol; Female; Intracellular Membranes; Lipid Peroxidation; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Mitochondrial Swelling; Oxidants; Permeability; Peroxides; Phosphates; Rats; Rats, Wistar; tert-Butylhydroperoxide

The paper reports a long series of observations on two statistically independent variates which have different nonlinear regressions on the same parameter vector. In our analysis of the data, heteroscedasticity is handled by residual maximum likelihood, REML, but to cope with outliers as well, REML based on a t-specification, called t-REML, is introduced. The data are the results of factorial experiments designed to model the quantitative performance of mitochondria generating aerobic power. The response variates are flux, measured as respiration, and ATP potential as log(ATP/ADP). The background of metabolism and oxidative phosphorylation is briefly sketched.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Aerobiosis; Atractyloside; Hexokinase; Mathematics; Mitochondria; Models, Biological; Models, Statistical; Oxidative Phosphorylation; Oxygen Consumption; Probability; Regression Analysis

Respiring mitochondria drive the electrophoretic uptake of K+ and other cations. In the presence of permeant acids this transport leads to mitochondrial swelling if it is not compensated by electroneutral K+/H+ exchange mediated by the K+/H+ antiporter. The mechanism of influx has yet to be established; however, evidence is accumulating that in addition to leak pathways a specific K+ channel or uniporter may be involved. We examine some of the properties of K+ uniport which are consistent with the existence of a specific ATP-regulated K+ channel. In contrast to the K+/H+ antiporter, K+ uniport shows little dependence on pH. K+ uniport is, however, very sensitive to inhibition by adenine nucleotides. The maximum percent inhibition is increased from 40 to 60% by treatment of mitochondria with N-ethylmaleimide (30 nmol/mg) which stimulates K+ uniport 3.6-fold. N-Ethylmaleimide, however, has no effect on the IC50 values which are 0.5, 2.3, and 8 microM for ADP, ATP, and AMP, respectively. GDP has no effect, while carboxyatractyloside is found to inhibit. The nucleotide analogs Cibacron blue 3GA and erythrosin B exhibit three effects on K+ uniport. Low doses partially inhibit K uniport (I50 = 0.13 microM Cibacron Blue), while higher doses stimulate (EC50 = 13 microM Cibacron Blue). Stimulation is especially apparent in N-ethylmaleimide-treated mitochondria. These analogs also antagonize inhibition by ATP. Since the EC50 values for this antagonism for these two drugs are similar, while the IC50 values for inhibition of ATP transport differ by a factor of five, we suggest that inhibition of K+ uniport by ATP is not mediated via the adenine nucleotide translocase. These data are consistent with the existence of an ATP-regulated K+ channel in the inner mitochondrial membrane.

Topics: Adenine Nucleotides; Adenosine Triphosphate; Animals; Atractyloside; Biological Transport; Erythrosine; Ethylmaleimide; Guanosine Diphosphate; Kinetics; Mitochondria; Mitochondrial Swelling; Oligomycins; Oxygen Consumption; Potassium; Potassium Channels; Proton-Translocating ATPases; Sulfhydryl Reagents; Tetraethylammonium; Tetraethylammonium Compounds; Triazines

Energy coupling parameters were studied in liver mitochondria of ground squirrel during arousal from hibernation. It was found that such mitochondria become uncoupled during incubation with phosphate in a salt medium. The uncoupling was revealed by respiration rate increase and membrane potential decrease in the presence of oligomycin. Both effects were reversed by addition of cyclosporin A. Under the same in vitro conditions, mitochondria from aroused (active) animals showed no uncoupling but could be uncoupled by addition of palmitate in the cyclosporin A-sensitive fashion. It is proposed that formation of cyclosporin A-sensitive pores can be involved in urgent heat production in arousing hibernators.

Topics: Animals; Atractyloside; Cyclosporine; Hibernation; Membrane Potentials; Mitochondria, Liver; Oligomycins; Oxidative Phosphorylation; Oxygen; Sciuridae

This study investigated the cellular regulation of net adenine nucleotide movements between the cytoplasm and mitochondria in intact cells. Such movements are presumed to occur primarily by ATP-Mg exchange with Pi via the mitochondrial ATP-Mg/Pi carrier. Vasopressin, A23187, and thapsigargin all elevate intracellular free [Ca2+] and all caused dose-dependent increases in the mitochondrial adenine nucleotide content (29, 63, and 39%, respectively). Phorbol 12-myristate 13-acetate had no effect. The effect of vasopressin was abolished when cytoplasmic [ATP] was decreased (by 43%) and [Pi] was increased (3-fold) by addition of carboxyatractyloside. The effect of thapsigargin was abolished by addition of xylulose to deplete cytoplasmic [ATP] (by 50%) and [Pi] (> 4-fold). The results indicate that in intact cells Ca2+ activates the mitochondrial ATP-Mg/Pi carrier to enable changes in the subcellular distribution of adenine nucleotides and that the relative [ATP] and [Pi] gradients govern the direction and magnitude of net adenine nucleotide movements between the cytoplasm and mitochondria.

Topics: Adenosine Triphosphate; Animals; Antiporters; Atractyloside; Calcimycin; Calcium-Transporting ATPases; Carrier Proteins; Cell Separation; Cells, Cultured; Cytoplasm; Dose-Response Relationship, Drug; Liver; Male; Mitochondria, Liver; Mitochondrial Proteins; Phosphates; Rats; Rats, Sprague-Dawley; Terpenes; Thapsigargin; Vasopressins

The yeast mitochondrial adenine nucleotide carrier isoform encoded by the ANC2 gene has been specifically expressed in a yeast strain disrupted for the two other genes, ANC1 and ANC3. Isolation of the carrier in a functional form was achieved by utilisation of a mixture of two detergents, dodecylmaltoside and Emulphogen. The intrinsic fluorescence of the Anc2 protein was specifically and rapidly enhanced upon addition of the transportable nucleotides ADP and ATP. Fluorescence enhancement was prevented or reversed by the addition of a stoichiometric amount of CATR. Addition of CATR alone elicited a dose-dependent decrease of fluorescence. The ANC2-specific yeast stain offers the means to study a single ADP/ATP carrier, with a well-defined amino acid sequence, suitable for analysis of substrate- or inhibitor-induced conformational changes.

Topics: Atractyloside; Binding Sites; Fluorescence; Mitochondria; Mitochondrial ADP, ATP Translocases; Saccharomyces cerevisiae

The flux control coefficients of adenine nucleotide translocase, the phosphate transporter, and H(+)-ATPase were determined in rat skeletal muscle mitochondria using glutamate plus malate as substrates and soluble F1-ATPase as load enzyme. It was observed that the flux control coefficients of adenine nucleotide translocase, H(+)-ATPase, and the load enzyme F1-ATPase, at comparable rates of respiration, strongly depend on the phosphate concentration in the incubation medium. So, the flux control exerted by adenine nucleotide translocase, in the intermediate states of mitochondrial respiration (approximately 120 nmol of O2/min/mg) at 10 mM phosphate, was found to be about 0.37. At a phosphate concentration of 1 mM and comparable rates of respiration the flux control coefficient of the translocase decreased to about 0.20. Under these conditions, a sharp increase in the controlling influence of H(+)-ATPase from 0.10 to 0.74 was detected. Furthermore, at this flux rate, the sum of flux control coefficients of adenine nucleotide translocase, H(+)-ATPase, phosphate transporter, and the load enzyme F1-ATPase was noted to be very close to unity. This indicates that under the conditions of intermediate state respiration, all of the other reactions have a negligible controlling influence on oxidative phosphorylation in skeletal muscle mitochondria.

Topics: Animals; Atractyloside; Carrier Proteins; Cattle; Kinetics; Mersalyl; Mitochondria, Heart; Mitochondria, Muscle; Mitochondrial ADP, ATP Translocases; Oligomycins; Oxidative Phosphorylation; Oxygen Consumption; Phosphate-Binding Proteins; Phosphates; Proton-Translocating ATPases; Rats; Regression Analysis

The adenine nucleotide translocator (AdNT) has been isolated from rat heart mitochondria and reconstituted into liposomes containing ATP. Translocator activity was determined using a coupled enzyme system to measure the ADP-induced efflux of ATP from the liposomes. In order to determine specific activity, the number of functional translocators must also be known. Carboxyatractylate (CAT) is a highly selective inhibitor of the AdNT, with Ki < 10 nM, a value sufficiently low relative to the concentration of protein in transport assays to suggest the use of tight-binding inhibitor theory to quantify functional translocators. Ackermann-Potter plots of velocity vs proteoliposome concentration at several different CAT concentrations were used both to demonstrate the occurrence of tight-binding inhibition and to determine the concentration of AdNT catalytic sites in the native orientation. The results obtained agreed well with earlier reports based on [14C]CAT binding; functionally reconstituted AdNT represented 5-10% of the protein added to the system. Specific activities were ca. 7-10 mumol/min mg depending on the lipid composition of the liposomes. Unincorporated protein did not appreciably affect the measurements. This methodology should be readily applicable to any reconstituted systems for which high-affinity inhibitors which bind only to active protein are known.

Topics: Animals; Atractyloside; Binding Sites; Biological Transport, Active; In Vitro Techniques; Kinetics; Liposomes; Male; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Models, Biological; Rats; Rats, Sprague-Dawley

The effect of fatty acids and L-carnitine on Ca2+ retention in rat liver mitochondria have been studied. Ca(2+)-retention was estimated as a sum of consecutive Ca2+ additions which leaded to transient stimulation of respiration coupled with influx of Ca2+ L-carnitine increases the Ca(2+)-retention; such an effect requires ATP. The Ca(2+)-retention was increased in the presence of 50 microM ATP or ADP. In all cases carboxyatractylate prevented the increase in Ca(2+)-retention. Palmitate and FCCP added at concentrations producing similar stimulating effect on respiration inhibit Ca(2+)-retention to about the same degree. The effect of palmitate is strongly diminished by L-carnitine. Again, the L-carnitine effect requires ATP. The data obtained suggest that the protonophoric effect of fatty acid plays a crucial role in Ca(2+)-dependent damage of mitochondria.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Atractyloside; Calcium; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Carnitine; Fatty Acids; Mitochondria, Liver; Rats

This study investigated the hypothesis that changes in the adenine nucleotide (ATP + ADP + AMP) content of kidney mitochondria can occur by a transport mechanism that catalyzes net transfer of adenine nucleotides across the inner mitochondrial membrane. The adenine nucleotide content of isolated kidney mitochondria was 8.23 +/- 0.85 nmol/mg mitochondrial protein. This amount increased or decreased as a function of the external [ATP-Mg] when mitochondria were incubated in phosphate-containing medium. The increases and decreases were inhibited to different extents by 100 microM EGTA (ethylene glycol bis (beta-aminoethyl ether) N,N'-tetraacetic acid) or 5 microM carboxyatractyloside (CAT), suggesting two transport mechanisms. The unidirectional components (influx and efflux) of net flux were examined separately for the CAT-insensitive (EGTA-sensitive) and CAT-sensitive (EGTA-insensitive) mechanisms. CAT-insensitive adenine nucleotide influx and efflux were stimulated by [Ca2+]free up to 2 microM; for ATP influx, Km was 1.7 mM, Vmax was 3.5 nmol/min/mg protein, and Mg2+ was required. Efflux varied as a function of both the external and matrix [ATP] and was completely inhibited by mersalyl. ATP was a better substrate than ADP, and ADP transport did not require Mg2+. The CAT-sensitive mechanism was characterized by studying phosphate-induced adenine nucleotide efflux. Efflux varied with external [Pi] and with matrix [ATP] and was not inhibited by cyclosporin. The amount of CAT required for maximal inhibition was 800 pmol/mg protein. In contrast to CAT-insensitive efflux, this pathway was only partially inhibited by mersalyl and showed no preference for ATP vs ADP. In conclusion, two distinct mechanisms for net adenine nucleotide transport were demonstrated. Both exchange adenine nucleotides (ATP-Mg or ADP) for Pi. One mechanism is identical to the CAT-insensitive ATP-Mg/Pi carrier known in liver mitochondria; the other is a CAT-sensitive mechanism that is not present in liver and may represent a novel function of the ADP/ATP translocase or another CAT-sensitive carrier.

Topics: Adenine Nucleotides; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Atractyloside; Biological Transport; Egtazic Acid; Kidney; Male; Mersalyl; Mitochondria; Phosphates; Rats

The presence of adenine nucleotide translocase (ANT) was found to greatly enhance the partitioning of the ATP analog 2',3'-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP) into reduced Triton X-100 micelles. The protein's effect was studied through the quenching of fluorescence of purified ANT, irreversibly inhibited by carboxyatractyloside (CAT), solubilized in reduced Triton X-100 micelles. The dependence of quenching of the protein's time-resolved tryptophan fluorescence on TNP-ATP concentration was measured and found to follow a Stern-Volmer mechanism. However, the calculated quenching constant was too large to be accounted for by the aqueous TNP-ATP concentration. Experiments were therefore conducted to determine the partitioning of the quencher between the three phases present: aqueous, protein-free micelle, and protein micelle; a system also described by the equation of Omann, G. M., and M. Glaser (1985. Biophys. J. 47:623-627.). By measuring the dependence of the apparent quenching rate constant on the protein concentration and protein/micelle ratios, this equation was used to calculate both the quencher partition coefficient into protein-free micelles (Pm) and into protein-micelles (Ppm), as well as the bimolecular quenching rate constant (kpm) in protein micelles. From the quenching experiments, kpm = 5.0 x 10(8)M-1s-1,Pm = 290 and pyrene quenching experiment to be 325, and by a rapid filtration experiment to be 450. Clearly, the presence of the integral membrane protein ANT-CAT in reduced Triton X-100 micelles greatly increases the partition of TNP-ATP into the micelle. ANT alters the properties and thus, the structure of the detergent micelle, which has direct implications for the use of detergent micelles as a model system for membrane proteins and may indicate that analogous effects occur in the mitochondrial membrane.

Topics: Adenosine Triphosphate; Animals; Atractyloside; Cattle; Chromatography, Gel; Detergents; Energy Transfer; Kinetics; Mathematics; Micelles; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Octoxynol; Polyethylene Glycols; Spectrometry, Fluorescence

Rat myocardial membranes exposed to free radical-generating systems exhibit both lipid peroxidation and protein alterations. The most sensitive protein, a 28-kDa polypeptide, was previously shown to increase slightly in apparent molecular weight before disappearing completely from the protein profile [N. L. Parinandi, C. W. Zwizinski, and H. H. O. Schmid (1991) Arch. Biochem. Biophys. 289, 118-123]. We now report that isolated cardiac mitochondria contain a 28-kDa protein which responds in the same manner to treatment with Cu2+/t-butylhydroperoxide. The protein exhibits several characteristic properties of the mitochondrial adenine nucleotide translocase. This assignment is supported by the finding that carboxyatractyloside, a specific inhibitor of the adenine nucleotide translocase, can prevent the oxidant-induced changes in the 28-kDa protein. Efficient purification schemes for the isolation of milligram quantities of unmodified and oxidatively altered adenine nucleotide translocase from rat heart mitochondria are described.

Topics: Animals; Atractyloside; Chromatography; Copper; Free Radicals; Lipid Peroxidation; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Molecular Weight; Peroxides; Rats; tert-Butylhydroperoxide

The folding of the peptide chain of the beef heart ADP/ATP carrier in the inner mitochondrial membrane was investigated by enzymatic and immunochemical approaches, using specific proteases and polyclonal antibodies directed against the whole protein and specific regions of the carrier. The accessibility of the membrane-bound ADP/ATP carrier to proteases was followed by immunodetection of the cleavage products, using mitochondria devoid of outer membrane (mitoplasts) and inside-out submitochondrial particles (SMP) in the presence of either carboxyatractyloside (CATR) or bongkrekic acid (BA), two specific inhibitors which are able to bind to the outer face or the inner face of the carrier, respectively. Four types of particles were investigated, namely, mitoplasts-CATR, mitoplasts-BA, SMP-CATR, and SMP-BA. Only the ADP/ATP carrier in SMP-BA was cleaved by two specific proteases, namely, trypsin and lysine C endoprotease, at low doses for short periods of time. Two initial cleavage sites were found between Lys-42 and Glu-43, and between Lys-244 and Gly-245. After a longer period of incubation, an additional cleavage site between Lys-146 and Gly-147 could be demonstrated. Despite cleavage of the membrane-embedded carrier, the binding capacity and affinity of SMP for BA were not altered. A number of other proteases tested, including V8 protease, proline C endoprotease, thrombin, alpha-chymotrypsin, and thermolysin had virtually no effect. These results are explained by a dynamic model of the arrangement of the peptide chain of the ADP/ATP carrier.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Animals; Atractyloside; Bongkrekic Acid; Cattle; Endopeptidases; Enzyme-Linked Immunosorbent Assay; Intracellular Membranes; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Molecular Sequence Data; Peptide Fragments; Protein Conformation; Submitochondrial Particles; Trypsin

Maximal rates of O2 uptake by hepatocytes from hypothyroid, euthyroid, and hyperthyroid rats, in the absence of uncoupling agents or ionophores, were achieved by simultaneously stimulating ureogenesis and gluconeogenesis. Rates were increased by pretreatment of the donor animals with thyroid hormone. Only a minor part of the increase could be attributed to stimulation of the activity of plasma membrane (Na+ + K+)-ATPase. No synthesis of glycogen occurred, nor was there any evidence of pyruvate cycling under the conditions used, thus ruling out these processes as potential sources of ATP turnover. Calculation of the O2 uptake necessary to satisfy the energy requirements of gluconeogenesis and ureogenesis, based on anticipated ATP demand and an assumed P:O ratio of 3:1, invariably yielded a theoretical quantity that was less than the experimentally measured increase in O2 uptake. The difference between measured and calculated rates of respiration, possibly representing noncoupled respiration, was related to the thyroid status of the animal, being greatest in cells from hyperthyroid rats. In another experimental approach the O2 uptake associated with ATP synthesis was inhibited by the addition of oligomycin to hepatocytes incubated in the presence of substrates, thus abolishing ATP-coupled respiration. The magnitude of the ATP-coupled as well as the residual respiration was increased in response to thyroid hormone. Thyroid hormone increased the turnover of intramitochondrial ATP. These results imply that a considerable portion of the thermogenic effect of thyroid hormone may be mediated by a stimulatory action on metabolism not directly associated with extramitochondrial ATP-dependent synthetic processes.

Topics: Adenosine Triphosphate; Animals; Atractyloside; Energy Metabolism; Gluconeogenesis; Hyperthyroidism; Hypothyroidism; Liver; Male; Oxygen; Pyruvates; Pyruvic Acid; Rats; Rats, Inbred Strains; Thyroid Hormones

To assess the direct renal toxicity of carboxyatractyloside (CATR), it was administered in relatively low intravenous (i.v.) doses (6.5 and 13.0 mumol/kg) to pentobarbital-anesthetized dogs that were being mechanically ventilated in order to circumvent severe extrarenal effects, such as hypoxemia, that could contribute to its nephrotoxicity. Within 2 h post-CATR, site-specific renal damage was noted in S2 and S3 cells of the proximal tubules; characteristic lesions in both cell types included loss of brush border, condensation of mitochondria and proliferation of small vesicles. Other S2 cells exhibited intense staining and reduced cell height. In 3 of 14 CATR-treated dogs, extrarenal effects were of sufficient magnitude to induce cellular swelling and occlusion of tubular lumina in S3 and thick ascending limb segments. Stevioside (STEV), related to CATR in structure and actions on the mitochondrial ADP/ATP translocase, was totally devoid of acute extrarenal or direct renal effects during the 6-h period following intravenous administration of 2.5 times the higher dose of CATR. The ability of CATR to produce renal toxicity via its renal and extrarenal actions emphasizes the importance of minimizing the latter actions of any toxicant when attempting to ascertain the mechanism by which it adversely affects renal function and ultrastructure.

Topics: Anesthesia; Animals; Atractyloside; Carbohydrate Sequence; Diterpenes; Diterpenes, Kaurane; Dogs; Female; Glucosides; Kidney; Male; Microscopy, Electron; Mitochondria; Mitochondrial ADP, ATP Translocases; Molecular Sequence Data; Pentobarbital; Terpenes

Thermoregulatory uncoupling of oxidative phosphorylation has been studied in heart and skeletal muscle mitochondria of ground squirrels. The respiratory rate of mitochondria in the presence of oligomycin was found to be much higher in winter (in hibernating, arousing, or aroused animals) than in summer. This additional respiration is strongly (arousing animals) or completely (hibernating and aroused animals) inhibited by carboxyatractylate (CAtr) and bovine serum albumin (BSA). The CAtr- and BSA-induced decreases in the rate of respiration are accompanied by membrane potential increases. The rate of the CAtr- and BSA-sensitive respiration is proportional to the content of free fatty acids which, in the heart, decreases in the order: arousing greater than aroused = hibernating greater than summer animals. Maximal respiratory rates observed in the presence of dinitrophenol (arousing greater than aroused greater than summer greater than hibernating animals) do not parallel the fatty acid level. It is assumed that some heat production in the winter animals is due to fatty acid-induced, ATP/ADP-antiporter-mediated uncoupling in heart and skeletal muscle mitochondria. The peak of heat production during arousal after hibernation also includes some other stimulatory effect on mitochondrial respiration.

Topics: Animals; Atractyloside; Body Temperature Regulation; Fatty Acids, Nonesterified; Kinetics; Mitochondria, Heart; Mitochondria, Liver; Mitochondria, Muscle; Oligomycins; Organ Specificity; Oxidative Phosphorylation; Sciuridae; Serum Albumin, Bovine

2-h-old neonatal liver mitochondria, when depleted of adenine nucleotides, showed an 'ohmic' current-voltage relationship and a higher passive proton permeability of the membrane, resembling fetal mitochondrial behaviors for the proton conductance. Incubation of fetal mitochondria with ATP, GDP or carboxyatractyloside promoted a significant reduction in the passive proton permeability of the membrane and the appearance of the characteristic biphasic behavior for the proton conductance. It is concluded that the postnatal increase in intramitochondrial adenine nucleotide concentration promotes, by the interaction of the nucleotides with the adenine nucleotide translocase, the reduction in the passive proton permeability of the mitochondrial membrane, allowing efficient energy conservation in the neonatal liver.

Topics: Adenine Nucleotides; Adenosine Triphosphate; Animals; Animals, Newborn; Atractyloside; Cell Membrane Permeability; Electric Conductivity; Guanosine Diphosphate; Intracellular Membranes; Liver; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Protons; Rats

A protonophore-induced delta psi decrease in a 180-140 mV range causes an increase in the lag-period of Ca(2+)-induced mitochondrial permeabilization but has little effect on the cumene hydroperoxide-induced permeability transition of mitochondria. Suppression of the non-specific permeability induction seems to be mediated by an increase in [ADP] in the mitochondrial matrix. A further decrease in delta psi leads to additional suppression of the non-specific permeability as a result of a partial ruthenium red-sensitive efflux of the previously accumulated Ca2+. On the other hand, complete dissipation of delta psi causes immediate induction of the non-specific permeability. It is concluded that only complete dissipation of delta psi caused by H+ leakages may act as a trigger for non-specific permeability induction.

Topics: Animals; Atractyloside; Benzene Derivatives; Calcium Chloride; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Intracellular Membranes; Kinetics; Membrane Potentials; Mitochondria, Liver; Mitochondrial Swelling; Permeability; Rats; Ruthenium Red; Submitochondrial Particles

The parameters of energy coupling of mitochondria isolated from the livers of hibernating and awakening gophers were studied. The ATP/ADP-antiporter inhibitor carboxyatractylate slowed down the respiration rate, increased delta psi and decreased the ionic conductivity of the inner mitochondrial membrane as measured by the rate of the delta psi decline after addition of cyanide (in the presence of oligomycin and EGTA). A similar effect was produced by BSA, carboxyatractylate being fairly ineffective in the presence of BSA. In hibernating gophers the maximal rate of the uncoupled respiration and the ionic conductivity of the inner mitochondrial membrane were markedly decreased as compared with awakening gophers. The data obtained suggest that in awakening animals fatty acids induce the uncoupling of oxidative phosphorylation by the ATP/ADP-antiporter, this process being simultaneous with the activation of the respiratory chain.

Topics: Animals; Atractyloside; Energy Metabolism; Fatty Acids; Hibernation; Intracellular Membranes; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Oxidative Phosphorylation; Sciuridae; Serum Albumin, Bovine

The respiratory capacities of hepatocytes, derived from hypothyroid, euthyroid and hyperthyroid rats, have been compared by measuring rates of oxygen uptake and by titrating components of the respiratory chain with specific inhibitors. Thyroid hormone increased the maximal rate of substrate-stimulated respiration and also increased the degree of ionophore-stimulated oxygen uptake. In titration experiments, similar concentrations of oligomycin or antimycin were required for maximal inhibition of respiration regardless of thyroid state, suggesting that the changes in respiratory capacity were not the result of variation in the amounts of ATP synthase or cytochrome b. However, less rotenone was required for maximal inhibition of respiration in the hypothyroid state than in cells from euthyroid or hyperthyroid rats, implying that hepatocytes from hypothyroid animals contain less NADH dehydrogenase. The concentration of carboxyatractyloside necessary for maximal inhibition of respiration was 100 microM in hepatocytes from hypothyroid rats, but 200 microM and 300 microM in hepatocytes from euthyroid and hyperthyroid rats, respectively, indicating a possible correlation between levels of thyroid hormone and the amount or activity of adenine nucleotide translocase. The increased capacity for coupled respiration in response to thyroid hormone is not associated with an increase in the components of the electron transport chain or ATP synthase, but correlates with an increased activity of adenine nucleotide translocase.

Topics: Animals; Antimycin A; Atractyloside; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cells, Cultured; Hyperthyroidism; Hypothyroidism; Kinetics; Liver; Male; Oligomycins; Oxygen Consumption; Rats; Rats, Inbred Strains; Reference Values; Rotenone; Thyroid Gland; Triiodothyronine

The role of intramitochondrial K+ content on the increase in membrane permeability to Ca2+, as induced by carboxyatractyloside was studied. In mitochondria containing a high K+ concentration (83 nmol/mg), carboxyatractyloside induced a fast and extensive mitochondrial Ca2+ release, membrane de-energization, and swelling. Conversely, in K(+)-depleted mitochondria (11 nmol/mg), carboxyatractyloside was ineffective. The addition of 40 mM K+ to K(+)-depleted mitochondria restored the capability of atractyloside to induce an increase in membrane permeability to Ca2+ release. The determination of matrix free Ca2+ concentration showed that, at an external free-Ca2+ concentration of 0.8 microM, control mitochondria contained 3.9 microM of free Ca2+ whereas K(+)-depleted mitochondria contained 0.9 microM free Ca2+. It is proposed that intramitochondrial K+ affects the matrix free Ca2+ concentration required to induce a state of high membrane permeability.

Topics: Animals; Atractyloside; Calcium; Cations, Monovalent; Intracellular Membranes; Kidney; Kinetics; Membrane Potentials; Mitochondria; Mitochondrial Swelling; Potassium; Potassium Chloride; Rats

The effect of ATP/ADP-antiporter inhibitors on palmitate-induced uncoupling was studied in heart muscle mitochondria and inside-out submitochondrial particles. In both systems palmitate is found to decrease the respiration-generated membrane potential. In mitochondria, this effect is specifically abolished by carboxyatractylate (CAtr) a non-penetrating inhibitor of antiporter. In submitochondrial particles, CAtr does not abolish the palmitate-induced potential decrease. At the same time, bongkrekic acid, a penetrating inhibitor of the antiporter, suppresses the palmitate effect on the potential both in mitochondria and particles. Palmitoyl-CoA which is known to inhibit the antiporter in mitochondria as well as in particles decreases the palmitate uncoupling efficiency in both these systems. These data are in agreement with the hypothesis that the ATP/ADP-antiporter is involved in the action of free fatty acids as natural uncouplers of oxidative phosphorylation.

Topics: Animals; Atractyloside; Bongkrekic Acid; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Kinetics; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Oxygen Consumption; Palmitic Acid; Palmitic Acids; Rabbits; Sodium Dodecyl Sulfate; Submitochondrial Particles; Uncoupling Agents

The ADP/ATP carrier (AAC) from yeast mitochondria has been reconstituted in phospholipid vesicles essentially according to the procedure described for the reconstitution of AAC from bovine heart mitochondria (Krämer and Heberger (1986) Biochim. Biophys. Acta, 863, 289-296). Liposomes were prepared from the mixed micelles of dodecyl octaoxyethylene ether (C12E8)-solubilized protein and egg yolk phosphatidylcholine by removing the detergent with Amberlite treatment. The micelles were treated with Amberlite either by repeatedly passing through small columns filled with Amberlite XAD-2 beads or by stepwise addition of Amberlite beads to the micelles. All the important variables of the reconstitution components were kept at optimal level and the liposomes obtained by both the methods of Amberlite treatment were analysed for (3H)CAT binding, orientation of AAC and nucleotide exchange activity. Reconstituted AAC showed 80% right side out orientation in the liposomes prepared by either procedure. Lipsomes prepared by the Amberlite column procedure exhibited higher CAT binding but lower ADP exchange activity. Liposome preparation by the stepwise addition of Amberlite is suggested to be the method of choice for functional reconstitution of yeast AAC in view of the higher nucleotide transport activity associated with the liposomes prepared by this method.

Topics: Atractyloside; Detergents; Kinetics; Liposomes; Mitochondria; Mitochondrial ADP, ATP Translocases; Resins, Synthetic; Saccharomyces cerevisiae

The frequency of contacts between the mitochondrial envelope membranes was determined in freeze-fractured samples of isolated mitochondria by means of quantifying the frequency of fracture plane deflections between the two membranes. It was observed that the formation of contacts correlated with the concentration of free ADP despite of inhibition of electron transport by antimycin A. The activity of ATPase partially inhibited by oligomycin or depletion of membrane potential by K+ and valinomycin had no effect on the induction of the contacts by ADP. ATP was ineffective in creating contacts irrespective of the presence or absence of a membrane potential, whereas carboxyatractyloside induced the contacts under all conditions in a manner similar to ADP. These results suggest the involvement of the ATP/ADP translocator in regulation of contact sites. As a consequence, we analyzed its distribution in the inner membrane of kidney and liver mitochondria by binding of [3H]atractyloside to subfractions of this membrane. The experiments demonstrated that the translocator was located in the peripheral part of the inner membrane as well as in the portion which formed the cristae.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Antimycin A; Atractyloside; Electron Transport; Freeze Fracturing; Intracellular Membranes; Membrane Potentials; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Mitochondrial Swelling; Oligomycins; Oxidative Phosphorylation; Proton-Translocating ATPases; Rats

Investigations with dihydrolipoic acid in rat heart mitochondria and mitoplasts reveal an activation of ATP-synthase up to 45%, whereas ATPase activities decrease by 36%. In parallel with an increase in ATP synthesis oligomycin-sensitive mitochondrial -SH groups are activated at 2-4 nmol dihydrolipoic acid/mg protein. ATPase activation by the uncouplers carbonylcyanide-p-trifluoromethoxyphenylhydrazone and oleate is diminished by dihydrolipoic acid, and ATP synthesis depressed by oleate is partially restored. No such efficiency of dihydrolipoic acid is seen with palmitate-induced ATPase activation or decrease of ATP synthesis. This indicates different interference of oleate and palmitate with mitochondria. In addition to its known coenzymatic properties dihydrolipoic acid may act as a substitute for coenzyme A, thereby diminishing the uncoupling efficiency of oleate. Furthermore, dihydrolipoic acid is a very potent antioxidant, shifting the -SH-S-S- equilibrium in mitochondria to the reduced state and improving the energetic state of cells.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Atractyloside; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Female; Kinetics; Mitochondria, Heart; Oleic Acid; Oleic Acids; Oligomycins; Oxidative Phosphorylation; Palmitic Acid; Palmitic Acids; Rats; Rats, Inbred Strains; Sulfhydryl Compounds; Thioctic Acid

Oxygen consumption (QO2) of single isolated axons and their associated glial cell sheath was investigated under a variety of conditions to determine the contribution of each cell type to whole tissue QO2. It was found that the QO2 of the sheath, in the absence of a functional axon, represented approximately 30% of the total tissue QO2. When the axon was injected with carboxyatractyloside, an inhibitor of mitochondrial oxidative phosphorylation that is membrane impermeant, electrophysiological properties of the axon were not affected and glial sheath respiratory activity was stimulated by 1.7 to 2.7 times the untreated control level. These results suggest that glial cell metabolic activity is regulated by the metabolic activity of the axon. Depending on the experimental conditions the glial sheath accounts for 30% to nearly 100% of the QO2 of axon-glial cell tissue. On the basis of these and morphometric measurements we estimate that in a normally functioning axon-glial cell system the glial sheath accounts for 90% of the tissue QO2.

Topics: Animals; Astacoidea; Atractyloside; Axons; Electrophysiology; Microelectrodes; Mitochondria; Neuroglia; Oxygen Consumption; Solutions

Coenzyme A transport was studied by determining [14C]CoA associated with isolated rat heart mitochondria. HPLC analysis of a mitochondrial extract obtained following incubation with [14C] CoA revealed an increase in [14C] CoA. In the presence of pyruvate or alpha-ketoglutarate, [14C]CoA associated with mitochondria was converted to acetyl- or succinyl-[14C]CoA, respectively, demonstrating the intramitochondrial localization of transported CoA. Net uptake of CoA was demonstrated by the findings that the increase in mitochondrial content of CoA following incubation with CoA was equal to the values of CoA uptake obtained from experiments using [14C]CoA. Sequestration of intramitochondrial CoA as metabolically inert derivatives with maleate stimulated CoA uptake, supporting the concept of unidirectional CoA uptake rather than exchange. Altering the membrane electrochemical gradient with valinomycin, nigericin, calcium, phosphate or a combination of phosphate and calcium caused efflux of endogenous CoA. The largest efflux was observed with valinomycin or a combination of Ca2+ and Pi. The Ca2+ and Pi-induced CoA efflux was effectively prevented by succinate or pyruvate. The results suggest that the uptake process, which is dependent on the membrane electrical gradient can be reversed by dissipating the electrical gradient. The relevance of CoA efflux induced by Ca2+ and Pi is discussed with respect to reperfusion injury following ischemic damage. Other factors regulating the maintenance of CoA within the mitochondrial matrix include the matrix pH and the acylation state of CoA.

Topics: Animals; Atractyloside; Biological Transport; Calcium; Carbon Radioisotopes; Chromatography, High Pressure Liquid; Coenzyme A; Ionophores; Ketoglutaric Acids; Male; Maleates; Malonates; Mitochondria, Heart; Phosphates; Rats; Rats, Inbred Strains

Structurally intact and metabolically competent mitochondria isolated from liquid-culture cells of sycamore (Acer pseudoplatanus L.) were shown to incorporate ADPglucose. Employing the double silicone oil layer filtering centrifugation method, we examined the kinetic properties of the uptake of various adenylates as well as the inhibitory effects exerted by carboxyatractyloside, atractyloside and bongkrekic acid, known specific inhibitors of the mitochondrial adenylate translocator. Immunoblot patterns of peptides derived from the partial proteolytic digestion of the mitochondrial and plastid adenylate translocators were shown to be essentially the same. We conclude that the molecular entities engaged in the adenylate transport system operating in two different organelles, mitochondria and amyloplasts, are very similar.

Topics: Adenine Nucleotides; Adenosine Diphosphate Glucose; Atractyloside; Biological Transport; Bongkrekic Acid; Cells, Cultured; Immunoblotting; Kinetics; Mitochondria; Mitochondrial ADP, ATP Translocases; Plants

The addition of oligomycin in the presence of Ca2+ increased the ADP pool in mitochondrial suspension. It is suggested that oligomycin inhibition of Ca(2+)-induced mitochondrial respiratory activation is the function of the increased endogenous ADP pool. Low ADP concentrations (5-20 microM) produce the same inhibitory effect as oligomycin. The increase of ADP levels in the presence of glucose plus hexokinase resulted in the inhibition of Ca(2+)-induced respiration, while the addition of phosphoenol pyruvate plus pyruvate kinase followed by a reduction in ADP levels, reversed the oligomycin inhibitory effect. One of the essential stages of ADP accumulation in mitochondrial suspensions in the presence of oligomycin and Ca2+ is proposed to be the formation of ADP from AMP and ATP, effected by adenylate kinase.

Topics: Adenine Nucleotides; Adenylate Kinase; Animals; Atractyloside; Calcium; In Vitro Techniques; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Oligomycins; Oxygen Consumption; Rats

Cyclosporin A prevents the opening of a nonspecific pore in the inner membrane of liver mitochondria when added prior to Ca2+. In the presence of 10 microM Ca2+ cyclosporin is unable to close the pore and restore the original permeability unless ADP is also added. ADP acts at a high-affinity site (Km 5 microM), corresponding to the adenine nucleotide transporter. This effect of ADP is prevented and reversed by carboxyatractyloside. In the presence of carboxyatractyloside, cyclosporin added with higher concentrations of ADP (Km 70 microM) also can close the pore. This suggests that a lower-affinity ADP-binding component as well as cyclophilin and the adenine nucleotide transporter can modulate the sensitivity of the pore to cyclosporin.

Topics: Adenosine Diphosphate; Animals; Atractyloside; Binding Sites; Cell Membrane Permeability; Cyclosporine; Electrodes; Membrane Potentials; Mitochondria, Liver; Rats

The effects of ATP/ADP-antiporter inhibitors on the uncoupling of oxidative phosphorylation by palmitic acid, detergents and protonophore FCCP in liver mitochondria were studied. The uncoupling activity of these compounds was estimated by their stimulating effect on succinate oxidation and H+ conductivity of the inner mitochondrial membrane in the presence of oligomycin. Carboxyatractylate and pyridoxal 5-phosphate suppressed the uncoupling effects of palmitic acid and anionic detergents but had no effect on the uncoupling action of the nonionic detergent Triton X-100, the cationic detergent CTAB and FCCP. The data obtained are discussed in terms of the putative role of the ATP/ADP-antiporter in the electrophoretic transport of hydrophobic anions from the mitochondria.

Topics: Animals; Atractyloside; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Detergents; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Oxidative Phosphorylation; Oxygen; Palmitic Acid; Palmitic Acids; Pyridoxal Phosphate; Rats

Cockleburs (Xanthium spp.) are herbaceous annuals with worldwide distribution. Toxicoses are usually associated with the consumption of the seedlings in the cotyledon stage, which contain a high concentration of the toxic principle, carboxyatractyloside. The seeds are also known to contain the toxin, but it has long been assumed that the spiny capsule would deter their consumption. Six of 70 yearling calves died while being fed round bale hay composed predominantly of foxtail and mature cocklebur plants with burs. Clinical signs ranged from acute death to hyperexcitability, blindness, tense musculature, and spastic gaits with heads held high and ears erect. Some symptomatic calves would stumble, fall to lateral recumbency, convulse, and later recover. Overall, the herd was very uneasy. Prominent gross lesions were ascites and a firm, pale liver with a mottled hemorrhagic pattern on cut surface. The rumen contained numerous intact burs and well-ruminated grass. Histological examination of the liver revealed marked centrolobular degeneration and necrosis with associated hemorrhage and congestion. Brain lesions were present. Plant and tissue samples were analyzed for carboxyatractyloside with various results. Samples of rumen contents, urine, and burs contained 100-200 ppm, 0.1-0.05 ppm, and 0.1 ppm, respectively. Based on the history, clinical signs, pathological lesions, and chemical analyses, cocklebur toxicosis associated with consumption of mature Xanthium strumarium in hay was confirmed.

Topics: Animal Feed; Animals; Atractyloside; Brain; Cattle; Cattle Diseases; Digestive System; Female; Kidney; Liver; Lung; Male; Plant Poisoning

The ATP synthase capacity of rat heart myocytes can be measured in sonicated cell suspensions and in sonicated preparations of cultured cardiomyocytes. This procedure allows the rapid measurement of mitochondrial function in response to changes in the metabolic status of the cell. In cultured myocytes, transitions in ATP synthase capacity (with no detectable change in cellular ATP concentration) accompany a change to anoxia or electrically stimulated contraction (rise of 70%). These changes are reversed on returning to the original conditions. Exposure of myocytes to low pH has little effect on basal ATP synthase capacity (down to values less than pH 6), but markedly affects cellular ATP levels and the response of the cells to anoxia and reoxygenation, possibly mimicking changes seen in ischaemic heart. Similar effects are seen in suspensions of freshly prepared myocytes, but these preparations are less stable and more pH-sensitive than are cells in culture. It is proposed that mitochondria in vivo are directly regulated at the level of the ATP synthase, and that a regulator protein, the naturally occurring inhibitor protein from mitochondria, may be responsible for this regulation.

Topics: Adenosine Triphosphate; Animals; Atractyloside; Cells, Cultured; Cold Temperature; Electric Stimulation; Energy Metabolism; Hydrogen-Ion Concentration; Hypoxia; In Vitro Techniques; Intracellular Membranes; Ischemia; Male; Mitochondria, Heart; Oligomycins; Ouabain; Proton-Translocating ATPases; Rats; Rats, Inbred Strains; Vanadium

The stimulation of respiration by long-chain fatty acids and FCCP was studied with oligomycin-inhibited mitochondria from rat liver, heart and kidney tissue. By addition of equal amounts of palmitate and oleate, mitochondrial respiration was increased in the order RLM less than RKM less than RHM. Using the classical protonophore FCCP, this difference could not be observed. Inhibition of oleate-stimulated respiration by carboxyatractyloside decreased in the order RHM greater than RKM greater than RLM. As CAT sensitivity of oleate-stimulated respiration and the mitochondrial ANT content were found to be correlated, it is suggested that the weak CAT sensitivity of oleate-stimulated respiration of RLM [(1989) Biochim. Biophys. Acta 977, 266-272] is due to the low content of ANT.

Topics: Animals; Atractyloside; Chloramphenicol O-Acetyltransferase; Fatty Acids; Female; Kidney Cortex; Mitochondria; Mitochondria, Heart; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases; Oleic Acids; Oligomycins; Palmitic Acids; Rats

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Atractyloside; Kinetics; Membrane Potentials; Mitochondria; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases; Oxidative Phosphorylation; Oxygen Consumption; Rats

The action of ATP/ADP-antiporter inhibitors upon the uncoupling effect of palmitate, detergents and 'classical' uncouplers has been studied. The uncoupling effect was estimated by stimulation of succinate oxidation and of H+ permeability of rat liver mitochondria in the presence of oligomycin. It is shown that carboxyatractylate (CAtr) and pyridoxal 5-phosphate (PLP) suppress the uncoupling induced by palmitate and the anionic detergents SDS and cholate, but do not affect that induced by the cationic detergents CTAB, by the non-ionic detergent Triton X-100, as well as by the 'classical' uncouplers FCCP and DNP. The results are discussed in terms of a concept assuming that the ATP/ADP-antiporter facilitates the electrophoretic export of hydrophobic anions from mitochondria.

Topics: Animals; Anions; Atractyloside; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cholic Acid; Cholic Acids; Detergents; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Oligomycins; Oxidation-Reduction; Oxygen Consumption; Palmitic Acid; Palmitic Acids; Permeability; Protons; Pyridoxal Phosphate; Rats; Rotenone; Sodium Dodecyl Sulfate; Succinates; Succinic Acid; Uncoupling Agents

Energy coupling parameters of liver mitochondria from hibernating and arousing ground squirrels have been studied. In the oligomycin-treated mitochondria, carboxyatractylate, an inhibitor of the ATP/ADP-antiporter, is shown to decrease the respiration rate, to increase the membrane potential and to lower the rate of the membrane-potential discharge after the addition of cyanide to liver mitochondria from hibernating and arousing animals. BSA effectively substitutes for carboxyactactylate so that carboxyactactylate, added after BSA, has no effect. In mitochondria from hibernating animals, the maximal respiration rate in the presence of DNP and the rate of the membrane potential discharge in its absence are much lower than in those from arousing animals. It has been concluded that upon arousal of the animals from hibernation, the uncoupling of oxidative phosphorylation, mediated by free fatty acids and ATP/ADP-antiporter, parallels the respiratory chain activation.

Topics: 2,4-Dinitrophenol; Animals; Arousal; Atractyloside; Dinitrophenols; Fatty Acids, Nonesterified; Hibernation; Membrane Potentials; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Oligomycins; Oxidative Phosphorylation; Oxygen Consumption; Palmitic Acid; Palmitic Acids; Sciuridae; Serum Albumin, Bovine

A mathematical model was developed to estimate flux control coefficients (Co) from titration studies with specific non-competitive inhibitors. In contrast to the normally used graphical determination the model pays regard to the dissociation equilibrium (KD) that exists between inhibitor and its binding sites (Eo) as well as to an objective estimation of the initial slope. The model was used for the analysis of titration experiments where the respiration of rat liver mitochondria was inhibited with carboxyatractyloside and antimycin A. It is shown that the graphical estimation of Eo and Co lead to significant overestimation if the ratio KD/Eo is larger than 10(-4) which can be avoided by using our model.

Topics: Animals; Antimycin A; Atractyloside; Enzyme Inhibitors; Enzymes; Kinetics; Mathematics; Mitochondria, Liver; Models, Theoretical; Protein Binding; Rats; Regression Analysis; Rotenone

In mitoplasts, respiratory stimulation by ADP, palmitate, DNP and CCCP and sensitivity of respiration to carboxyatractylate are considerably less pronounced than in mitochondria. Addition of porin-containing preparations (purified outer membranes or solubilized mitochondrial porin) to mitoplasts results in partial restoration of the oxygen consumption and sensitivity to carboxyatractylate (CAT). The uncoupling effect of FCCP in mitoplasts is CAT-resistant and does not depend on added porin. It is suggested that mitochondrial porin may be a natural activator of ADP/ATP antiporter and succinate carrier in mitochondria.

Topics: 2,4-Dinitrophenol; Adenosine Diphosphate; Adenosine Triphosphate; Animals; Anion Transport Proteins; Atractyloside; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Carrier Proteins; Cytochrome c Group; Dinitrophenols; Intracellular Membranes; Ion Channels; Membrane Proteins; Mitochondria, Liver; Oxygen Consumption; Palmitic Acid; Palmitic Acids; Porins; Rats; Succinates; Succinic Acid; Voltage-Dependent Anion Channels

An azido-125I-CoA photolabel was synthesized from N-(3-iodo-[125I]4-azidophenylpropionamido)cysteinyl- 5-(2'thiopyridyl) cysteine ([125I]ACTP) and CoASH, separated by chromatography on a silica gel TLC, and identified by autoradiography. Synthesis of [125I]ACT-CoA from [125I]ACTP and CoA was further confirmed by monitoring the release of one of the products, thiopyridone, at 343 nm and concurrent formation of [125I] ACT-CoA. Beef heart mitochondria were incubated in the presence of the 125I-CoA derivative with or without specific inhibitors and/or substrates of the ADP/ATP carrier, and immediately photolyzed for 5 s. Sodium dodecyl sulfate-gel electrophoresis and autoradiography of the separated proteins revealed exclusive photolabeling of a 30-kDa protein in the absence of inhibitors of the carrier. This specific photolabeling of the 30-kDa protein was prevented in a concentration-dependent manner by either carboxyatractylate or palmitoyl-CoA (0.1-5 microM), two known inhibitors of the ADP/ATP carrier. ADP reduced the extent of photolabeling of the 30-kDa protein, but palmitic acid, free CoASH, and dinitrophenol were ineffective, indicating the specificity of the reaction. CoA photolabels may be useful in probing ligand and/or substrate binding sites and in determining the structure-function relationship of the ADP/ATP carrier.

Topics: Acyl Coenzyme A; Affinity Labels; Animals; Atractyloside; Azides; Cattle; Coenzyme A; In Vitro Techniques; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases; Photochemistry

Methods for isolation of the ADP/ATP carrier (AAC) from yeast (Saccharomyces cerevisiae) are described which allow separation of the carrier from the initially copurified porin which poses a specific problem in yeast. The procedure varies according to whether one wishes to obtain a stable CAT-AAC complex, the free and active AAC for reconstitution, or the SDS-denatured pure AAC peptide. CNBr cleavage of AAC enabled us to differentiate clearly between isogenes AAC-1 and AAC-2 recently found in yeast, due to the exclusive occurrence of a methionine (M-115) residue at the end of the first domain in AAC-2. Thus the AAC isolated from wild-type yeast is primarily or exclusively AAC-2. The isolated AAC is active in ADP/ATP exchange in reconstituted liposomes with a Vmax of 1100 mumol/min per g protein and Km = 15 microM for ADP, and a Vmax of 900 mumol/min per g protein and Km = 9 microM for ATP.

Topics: Atractyloside; Binding Sites; Cyanogen Bromide; Kinetics; Mitochondria; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases; Protein Denaturation; Saccharomyces cerevisiae

The rotational mobility of the mitochondrial ADP/ATP carrier has been studied solubilized in Triton micelles, reincorporated in phospholipid liposomes, and in mitochondria. Spin-labeled analogues of the noncovalent inhibitors carboxyatractyloside and atractyloside were found to be strongly immobilized when bound to the carrier [Munding, A., Beyer, K., & Klingenberg, M. (1983) Biochemistry 22, 1941-1947], such that saturation-transfer electron spin resonance spectroscopy could be used to study the rotational motion of the protein. Spin-labeled maleimide covalently bound to the carrier was found to have independent segmental motion and hence to be unsuitable for studies of protein rotation. The ESR spectra of the carboxyatractyloside and atractyloside spin labels were found to contain a second component from label in the lipid or detergent, necessitating the use of the saturation-transfer ESR integral method [Horváth, L. I., & Marsh, D. (1983) J. Magn. Reson. 54, 363-373] to analyze the rotational motion of the label component bound to the protein. Effective rotational correlation times obtained from integration of the high-field region of the spectrum were lower than those obtained from the total spectral integral, indicating strongly anisotropic rotational diffusion of the carrier in the vesicular and membrane systems, with the spin-label z axis oriented preferentially perpendicular to the rotation axis.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Atractyloside; Cattle; Diffusion; Electron Spin Resonance Spectroscopy; Liposomes; Maleimides; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases; Spin Labels

We have recently synthesized an azido [125I] CoA photolabel, N-(3-iodo-4-azidophenyl propionamide) cysteinyl-5-(2'thiopyridyl cysteine) CoA that specifically labeled the ADP/ATP carrier in beef heart mitochondria. In this study brown adipose tissue mitochondria were photolabeled with the azido [125I] ACT-CoA derivative with or without inhibitors. SDS gel electrophoresis and autoradiography of the separated proteins revealed exclusive photolabeling of two polypeptides corresponding to the ADP/ATP carrier and uncoupling protein. In the presence of carboxyatracytloside only the 32 kD UCP was labeled by [125I] ACT-CoA, whereas preincubation with GDP resulted in exclusive photolabeling of the 30 kD ADP/ATP carrier. Palmitoyl CoA but not palmitic acid inhibited photolabeling of both polypeptides.

Topics: Acyl Coenzyme A; Adipose Tissue, Brown; Affinity Labels; Animals; Atractyloside; Carrier Proteins; Cricetinae; Guanosine Diphosphate; Ion Channels; Membrane Proteins; Mitochondria; Mitochondrial ADP, ATP Translocases; Mitochondrial Proteins; Molecular Weight; Nucleotidyltransferases; Palmitoyl Coenzyme A; Purine Nucleotides; Uncoupling Protein 1

The effect of long-chain fatty acids (LCFA) on respiration and transmembrane potential (delta psi) in the resting state, and the rate of delta psi dissipation [d delta psi/dt)i) was investigated with oligomycin-inhibited rat liver mitochondria using succinate (plus rotenone) as substrate. The results obtained were compared with those of classical protonophores such as 2,4-dinitrophenol (DNP) and 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole (TTFB). The effects of oleate or palmitate and that of DNP or TTFB on respiration and delta psi can be described by a common force-flow relationship. These facts all in all are not compatible with a decoupler-type uncoupling mechanism of LCFA; still, they indicate that the latter are protonophores. Moreover, the oleate-induced increase in the rate of delta psi dissipation closely correlates with that in respiration, suggesting that the uncoupling activity and the protonophoric activity of LCFA are interrelated. Carboxyatractyloside (CAT) exerted only a small inhibitory effect on oleate-induced respiration and delta psi dissipation, indicating that the adenine nucleotide translocase contributes to the uncoupling effect of LCFA to a minor extent only. Proton transport through the lipid region of the membrane as mediated by permeation of the protonated and deprotonated forms of LCFA is interpreted as the main process of the uncoupling of LCFA.

Topics: 2,4-Dinitrophenol; Animals; Atractyloside; Benzimidazoles; Biological Transport; Dinitrophenols; Fatty Acids; Female; Membrane Potentials; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Oleic Acid; Oleic Acids; Oligomycins; Oxidative Phosphorylation; Oxygen Consumption; Palmitic Acid; Palmitic Acids; Protons; Rats; Uncoupling Agents

It was shown that during glutamate+malate oxidation in the presence of creatine, antitumour anthracycline antibiotics strongly inhibit the rate of oxygen uptake by rat heart mitochondria; ADP excess activated the respiration up to the initial level, i.e., that observed after the first addition of ADP. Carboxyatractyloside addition to a system containing creatine (or hexokinase+glucose) results in the stimulation of rubomycin-induced mitochondrial respiration. Substitution of carboxyatractyloside by oligomycin gives very similar results. It is supposed that anthracycline antibiotics exert a manyfold effect on heart mitochondrial membranes which results in impaired compartmentation of enzymatic systems providing for oxidative phosphorylation.

Topics: Adenosine Diphosphate; Animals; Antibiotics, Antineoplastic; Atractyloside; Creatine Kinase; Daunorubicin; Drug Interactions; Mitochondria, Heart; Oxidation-Reduction; Oxygen Consumption; Rats

The catabolism of intramitochondrial guanine nucleotides was examined. During 30 min incubation of rat liver mitochondria at 37 degrees C in the presence of oligomycin and carboxyatractyloside, guanine and xanthine were formed and appeared in the medium. Under these conditions, the direct conversion of GMP to guanine by hypoxanthine-guanine phosphoribosyltransferase is suggested to be the main catabolic route within the organelles. Only very small amounts of guanosine were produced and detected both inside and outside the organelles. [14C]Guanosine and [14C]inosine were taken up by the mitochondria. Therefore, guanosine is suggested to be a precursor of intramitochondrial guanine nucleotides.

Topics: Animals; Atractyloside; Chromatography, High Pressure Liquid; Guanine; Guanine Nucleotides; Guanosine; Inosine; Male; Mitochondria, Liver; Oligomycins; Rats; Rats, Inbred Strains; Subcellular Fractions; Temperature; Xanthine; Xanthines

The degradation of endogenous adenine nucleotides was compared in mitochondria isolated from mouse and rat liver, rat renal cortex and solid hepatoma. Mitochondria were incubated for 30 min at 37 degrees C in the presence of carboxyatractyloside and oligomycin. In rat liver mitochondria ATP, ADP and AMP were degraded by about 25% each, whereas in kidney and hepatoma mitochondria a rapid decline of ATP and ADP but no change in the AMP contents were observed. Main products formed were adenosine, inosine and hypoxanthine in all mitochondria examined. Compartment studies revealed that the main route of intramitochondrial adenine nucleotide catabolism seems to proceed via AMP dephosphorylation and subsequent adenosine deamination.

Topics: Adenine Nucleotides; Animals; Atractyloside; Kidney Cortex; Kinetics; Liver Neoplasms, Experimental; Mice; Mitochondria; Mitochondria, Liver; Oligomycins; Organ Specificity; Rats

The lipophilic triphenylmethylphosphonium cation (TPMP+) has been employed to measure delta psi m, the electrical potential across the inner membrane of the mitochondria of intact hepatocytes. The present studies have examined the validity of this technique in hepatocytes exposed to graded concentrations of inhibitors of mitochondrial energy transduction. Under these conditions, TPMP+ uptake allows a reliable measure of delta psi m in intracellular mitochondria, provided that the ratio [TPMP+]i/[TPMP+]e is greater than 50:1 and that at the end of the incubation more than 80% of the hepatocytes exclude Trypan blue. Hepatocytes, staining with Trypan blue, incubated in the presence of Ca2+, do not concentrate TPMP+. The relationships between delta psi m and two other indicators of cellular energy state, delta GPc and Eh, or between delta psi m and J0, were examined in hepatocytes from fasted rats by titration with graded concentrations of inhibitors of mitochondrial energy transduction. Linear relationships were generally observed between delta psi m and delta GPc, Eh or J0 over the delta psi m range of 120-160 mV, except in the presence of carboxyatractyloside or oligomycin, where delta psi m remained constant. Both the magnitude and the direction of the slope of the observed relationships depended upon the nature of the inhibitor. Hepatocytes from fasted rats synthesized glucose from lactate or fructose, and urea from ammonia, at rates which were generally linear functions of the magnitude of delta psi m, except in the presence of oligomycin or carboxyatractyloside. Linear relationships were also observed between delta psi m and the rate of formation of lactate in cells incubated with fructose and in hepatocytes from fed rats. The linear property of these force-flow relationships is taken as evidence for the operation of thermodynamic regulatory mechanisms within hepatocytes.

Topics: Adenine Nucleotides; Animals; Atractyloside; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Energy Metabolism; Gluconeogenesis; Indicators and Reagents; Intracellular Membranes; Liver; Male; Membrane Potentials; Mitochondria, Liver; Oligomycins; Onium Compounds; Rats; Rats, Inbred Strains; Staining and Labeling; Thermodynamics; Trityl Compounds; Trypan Blue; Urea; Valinomycin

Compounds which induce calcium efflux from calcium-loaded mitochondria generally provoke membrane leakiness. The involvement of the ADP/ATP carrier in modification of mitochondrial membrane properties was studied. The addition of impermeant inhibitors of the ADP/ATP carrier, namely carboxyatractylate, palmitoyl coenzyme A (in the absence of carnitine), and pyridoxal 5-phosphate, to calcium-loaded mitochondria triggered the release of accumulated calcium, the leakage of endogenous ADP, and the swelling of mitochondria. Permeant ligands, such as bongkrekic acid or ADP, showed no damaging effect on membrane permeability; in fact, they impeded the membrane perturbation which was induced by the three impermeant effectors. In addition, both bongkrekic acid and ADP were able to cancel the calcium loss and swelling resulting from the oxidation of intramitochondrial pyridine nucleotides by acetoacetate. In acetoacetate-treated mitochondria, the ADP/ATP carrier was shown to be mainly in a c-state conformation (i.e., the nucleotide binding site had an external orientation). It was concluded that induction of membrane leakiness by calcium ions depends on the conformational state of the adenine nucleotide carrier. The ability of intramitochondrial calcium ions to modify membrane properties is determined by the orientation of the nucleotide binding site. Only the c-state conformation allows membrane destabilization. Consequently, all compounds which stabilize the ADP/ATP carrier in the c-state conformation will have a deleterious effect on calcium-loaded mitochondria.

Topics: Adenosine Diphosphate; Animals; Atractyloside; Binding Sites; Bongkrekic Acid; Calcium; Cell Membrane Permeability; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Mitochondrial Swelling; NAD; Nucleotidyltransferases; Protein Conformation; Rats

The activating anions are found to induce an unexpectedly high (up to 8-fold for sulphite) increase of ATPase activity in intact rat liver mitochondria. This effect is not determined by the observed changes in Km and Ki (ADP) values. The stimulation seems to be caused by dissociation of the inactive complex of ATPase with Mg.ADP. The quantity of this complex formed in the course of ATP hydrolysis is approx. 90% of the total ATPase content in intact mitochondria. The data on toluene-permeabilized mitochondria suggest that the high content of the complex is a result of the stabilizing effect of some matrix macromolecules.

Topics: Adenosine Triphosphate; Animals; Anions; Atractyloside; Azides; Enzyme Activation; Kinetics; Magnesium; Mitochondria, Liver; Oligomycins; Proton-Translocating ATPases; Rats; Sodium Azide; Sulfites

The localization of the mitochondrial elongation activities ('elongases') from Saccharomyces cerevisiae has been investigated. It was shown, using carboxyatractyloside in the incubation mixture, that synthesis of very-long-chain fatty acids probably occurred outside the matrix and, by fractionation experiments, that elongases are membrane-bound enzymes. The solubilization of the outer membrane by digitonin showed that three elongating activities are correlated with a marker of the outer membrane and not with an inner membrane marker. A further partial purification of the outer membrane showed that elongases are present in the outer membrane of mitochondria.

Topics: Acyl-Carrier Protein S-Malonyltransferase; Acyltransferases; Atractyloside; Binding Sites; Centrifugation, Density Gradient; Digitonin; Enzyme Activation; Fatty Acids; Intracellular Membranes; Mitochondria; Oxygen Consumption; Saccharomyces cerevisiae; Sucrose

Atractyloside and carboxyatractyloside partially inhibited nitrogenase activity (acetylene reduction) by isolated vesicles of Frankia strain EAN1pec. Extracts of disrupted vesicles showed nitrogenase activity that was not affected by the inhibitors. The vesicles accumulated ATP by an atractyloside-sensitive mechanism. This inhibition of ATP uptake was reversed when vesicles were permeabilized by detergent. Uptake of ATP was inhibited by excess ATP and ADP, but not AMP or adenosine, and by a calcium-dependent ATPase inhibitor. Uptake was stimulated by calcium ions. Accumulation of ATP was accompanied by release of ADP and AMP from the vesicles. The ATP taken up by vesicles and cells grown with N2 as the nitrogen source was found in the corresponding cell pools only as ATP. The data indicate activity of an ATP-ADP translocase system in vesicles of this organism. The role of ATP translocation in the symbiosis between Frankia strain EAN1pec and plant root nodules is discussed.

Topics: Acetylene; Actinomycetales; Adenosine Diphosphate; Adenosine Triphosphate; Atractyloside; Kinetics; Nitrogenase; Oxidation-Reduction

The ATP/ADP-antiporter inhibitors and ADP decrease the palmitate-induced stimulation of the mitochondrial respiration in the controlled state. The degree of inhibition decreases in the order: carboxyatractylate greater than bongkrekic acid, palmitoyl-CoA, ADP greater than atractylate. GDP is ineffective. The inhibiting concentration of carboxyatractylate coincides with this arresting the state 3 respiration. Carboxyatractylate inhibition decreases when the palmitate concentration increases. Stimulation of controlled respiration by FCCP or gramicidin D at any concentration of these uncouplers is carboxyatractylate-resistant, whereas that by low concentrations of DNP is partially suppressed by carboxyatractylate. These data together with observations that palmitate does not increase H+ conductance in bilayer phospholipid membranes and in cytochrome oxidase-asolectin proteoliposomes indicate that the ATP/ADP-antiporter is somehow involved in the uncoupling by low concentrations of fatty acids (or DNP), whereas that by FCCP and gramicidin D is due to their effect on the phospholipid bilayer. It is suggested that the antiporter facilitates translocation of palmitate anion across the mitochondrial membrane.

Topics: Animals; Atractyloside; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Dinitrophenols; Fatty Acids, Nonesterified; Glycosides; Kinetics; Mitochondria, Liver; Mitochondria, Muscle; Palmitic Acid; Palmitic Acids; Rats; Uncoupling Agents

A regulatory role of adenine nucleotide translocator (ANT) was determined by titration of mitochondrial respiration (state 3) with carboxyatractyloside. It was shown that ANT regulates pyruvate oxidation: the control strength is more pronounced after depletion of endogenous substrates or after the increase in extramitochondrial ATP/ADP. The rate of succinate oxidation is controlled mainly by succinate dehydrogenase, while ANT does not participate in its regulation.

Topics: Adenine Nucleotides; Adenosine Diphosphate; Adenosine Triphosphate; Animals; Atractyloside; Mitochondria, Heart; Oxidative Phosphorylation; Pyruvates; Rabbits; Succinate Dehydrogenase

The regulative role of adenine nucleotide translocators (ANTs) in oxidative phosphorylation has been estimated by the titration of respiration of isolated rabbit heart mitochondria with carboxyatractyloside in the presence of a non-rate limiting creatine phosphokinase ADP-regenerating system. It has been established that the respiration rate is not controlled by ANTs in the two extreme states, state 3 and state 4. On the other hand, at an intermediate respiration rate (30-70% of the state 3 respiration, which roughly corresponds to that under physiological conditions) the ANT control coefficient had a value of 0.62-0.75. Thus, ANTs seem to play a key role in the regulation of oxidative phosphorylation.

Topics: Animals; Atractyloside; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases; Oxidative Phosphorylation; Oxygen Consumption; Phosphocreatine; Rabbits

1. The matrix pyrophosphate (PPi) content of isolated energized rat liver mitochondria incubated in the presence of ATP, Mg2+, Pi and respiratory substrate was about 100 pmol/mg of protein. 2. After incubation with sub-micromolar [Ca2+], this was increased by as much as 300%. There was a correlation between the effects of Ca2+ on PPi and on the increase in matrix volume reported previously [Halestrap, Quinlan, Whipps & Armston (1986) Biochem. J. 236, 779-787]. Half-maximal effects were seen at 0.3 microM-Ca2+. 3. Coincident with these effects, the total adenine nucleotide content increased in a carboxyatractyloside-sensitive manner. 4. Incubation with 0.2-0.5 mM-butyrate induced similar but smaller effects on mitochondrial swelling and matrix PPi and total adenine nucleotide content. Addition of butyrate after Ca2+, or vice versa, caused Ca2+-induced mitochondrial swelling to stop or reverse, while matrix PPi increased 30-fold. 5. Addition of atractyloside or the omission of ATP from incubations greatly enhanced swelling induced by Ca2+ without increasing matrix PPi. 6. Swelling of mitochondria incubated under de-energized conditions in iso-osmotic KSCN was progressively enhanced by the addition of increasing concentrations of PPi (1-20 mM) or valinomycin. 7. In iso-osmotic potassium pyrophosphate swelling was slow initially, but accelerated with time. This acceleration was inhibited by ADP, whereas carboxyatractyloside induced rapid swelling. Swelling in other iso-osmotic PPi salts showed that the rate of entry decreased in the order NH4+ greater than K+ greater than Na+ greater than Li+, whereas choline, tetramethylammonium and Tris did not enter. It is suggested that the adenine nucleotide translocase transports small univalent cations when PPi is bound and that PPi can also be transported when the transporter is in the conformation induced by carboxyatractyloside. 8. It is concluded that Ca2+ and butyrate cause swelling of energized mitochondria through this effect of PPi on K+ permeability of the mitochondrial inner membrane. 9. Freeze-clamped livers from rats treated with glucagon or phenylephrine show 30-50% increases in tissue PPi. It is proposed that Ca2+-mediated increases in mitochondrial PPi are responsible for the increase in matrix volume and total adenine nucleotide content observed after hormone treatment.

Topics: Adenine Nucleotides; Adenosine Triphosphate; Animals; Atractyloside; Butyrates; Butyric Acid; Calcium; Diphosphates; Female; Light; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Mitochondrial Swelling; Rats; Rats, Inbred Strains; Scattering, Radiation

By means of reconstituted systems consisting in rat heart mitochondria and pyruvate kinase it was shown, that the mitochondrial ATP-production is remarkable increased if ADP is produced by enzymes localized in the mitochondrial intermembrane space as creatine kinase or adenylate kinase. This result and different amount of inhibition by atractyloside or carboxyatractyloside of hexokinase- and creatine kinase stimulated respiration further support the concept of dynamic compartmentation of adenine nucleotides in the mitochondrial intermembrane space.

Topics: Adenine Nucleotides; Adenylate Kinase; Animals; Atractyloside; Cell Compartmentation; Creatine Kinase; Cytosol; Hexokinase; In Vitro Techniques; Intracellular Membranes; Mitochondria, Heart; Oxidative Phosphorylation; Oxygen Consumption; Phosphates; Rats

Synthesis of coenzyme A (CoA) in isolated rat heart mitochondria was studied. Mitochondria synthesized CoA from 4'phosphopantetheine (4'PP), a precursor of CoA which is synthesized from pantothenic acid in the cytosol. The synthesis was time dependent and was absolutely dependent on the presence of external ATP under the conditions used. The rate of synthesis was increased either by increasing the pH from 7.4 to 8.5 or by adding 0.01% deoxycholate to the incubation medium. To determine whether the synthesis was intra- or extramitochondrial, mitochondria were separated from the incubation mixture by centrifugation and assayed for CoA. The amount of CoA appearing in the supernatant after 30 mins of incubation increased with increasing concentrations of 4'PP while that in the mitochondrial pellet remained constant over the concentration range studied. Synthesis of CoA from 4'PP was not affected by the uncoupler 2,4-dinitrophenol, or by carboxyatractyloside, or by a combination of the two drugs. The combination was used in an effort to deplete intramitochondrial ATP and to prevent external ATP from entering the mitochondria, thus resulting in mitochondria devoid of matrix ATP. The absolute dependence of synthesis on external ATP, the appearance of newly synthesized CoA in the incubation buffer and the ability of mitochondria lacking matrix-ATP to synthesize CoA suggest that the mitochondrial enzymes responsible for synthesis of CoA from 4'PP are on the outer membrane or on the outer side of the inner membrane.

Topics: Adenosine Triphosphate; Animals; Atractyloside; Coenzyme A; Dinitrophenols; In Vitro Techniques; Mitochondria, Heart; Pantetheine; Rats; Uncoupling Agents

The regulation of oxidative phosphorylation was studied with digitonin-treated epididymal bull spermatozoa in which mitochondria are directly accessible to low molecular compounds in the extracellular medium. Due to the high extramitochondrial ATPase activity in this cell preparation, it was possible to stimulate respiration to a small extent only by added hexokinase in the presence of glucose and adenine nucleotides. Added pyruvate kinase plus phosphoenol pyruvate, however, strongly suppressed the respiration. Under these conditions, the respiration was found to depend on the extramitochondrial [ATP]/[ADP] ratio in the range of 1-100. The contribution of the adenine nucleotide translocator to this dependence was determined by titration with the irreversible inhibitor carboxyatractyloside in the presence of ADP. Using lactate plus malate as substrate, the active state respiration was controlled to about 30% by the translocator, whereas 12 and 4% were determined in the presence of L-glycerol-3-phosphate and malate alone, respectively. In order to compare the results with those for intact cells, the adenine nucleotide patterns were determined in intact and digitonin-treated spermatozoa under conditions of controlled respiration in the presence of vanadate and carboxyatractyloside, respectively. About 21% of total cellular adenine nucleotides were found in digitonin-treated cells representing the mitochondrial compartment. While allowing for the intramitochondrial amount of adenine nucleotides, the cytosolic [ATP]/[ADP] ratio was estimated to be 6-times higher than the mitochondrial ratio in intact cells. It is concluded from the data presented that the principal mechanism by which oxidative phosphorylation in sperm mitochondria is regulated via the extramitochondrial [ATP]/[ADP] ratio is the same as that demonstrated for other isolated mitochondria.

Topics: Adenine Nucleotides; Adenosine Diphosphate; Animals; Atractyloside; Cattle; Cytosol; Digitonin; Epididymis; Hexokinase; Male; Mitochondria; Mitochondrial ADP, ATP Translocases; Oxidative Phosphorylation; Spermatozoa; Succinates

P/2e- stoichiometries in six assay systems spanning different portions of the respiratory chain were estimated by direct determinations of Pi uptake in suspensions of bovine heart mitochondria containing a hexokinase trap. The electron donors were malate + pyruvate, succinate, and ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine, and the electron acceptors were ferricyanide (Site 1, Site 2, and Sites 1 + 2) and O2 (Sites 1 + 2 + 3, Sites 2 + 3, and Site 3). A major objective was to find conditions in which the six systems yield results in sufficiently good agreement to allow confidence as to their reliability. This objective was achieved, and maximum values of 1.1, 0.5, and 1.0 were observed in the Sites 1, 2, and 3 systems, respectively. This required that the energy-conserving reactions be relatively nonlimiting and that the P/2e- ratios be estimated from the slopes of plots of respiration rate versus phosphorylation rate obtained by inhibiting oxidative phosphorylation with respiratory chain inhibitors. The latter requirement allows avoidance of the effect of an apparent endogenous uncoupler and is based on the observation (Tsou, C. S., and Van Dam, K. (1969) Biochim. Biophys. Acta 172, 174-176) that uncoupling agents at low concentrations decrease the rate of phosphorylation nearly as much in absolute amount at low rates of respiration as at high rates. The maximum P/2e- stoichiometry at Site 1 is considered to be 1.0, and the value observed in the Site 1 system is suggested to be higher as a result of H+ ejection at the transhydrogenase level. Respiratory control due to carboxyatractyloside inhibition was examined and found to differ greatly among the systems. It is pointed out that this observation is not consistent with the lack of complete control being due primarily to ion cycling and that, in view of this, the relatively meager control at Site 3 is not consistent with O2 being reduced on the matrix side of the coupling membrane.

Topics: Animals; Ascorbic Acid; Atractyloside; Cattle; Electron Transport; Ferricyanides; Malates; Mitochondria, Heart; Oxidative Phosphorylation; Oxygen Consumption; Phosphates; Pyruvates; Pyruvic Acid; Succinates; Succinic Acid; Tetramethylphenylenediamine; Uncoupling Agents

The ability of isolated rat heart mitochondria to take up coenzyme A (CoA) from the incubation medium was studied. Mitochondria accumulated CoA in a time- and concentration-dependent manner. The accumulation process occurred in two phases. Within the first 30 s of incubation, mitochondrial content of CoA increased, and this phase did not plateau in the concentration range studied. Following this initial increase, a second slower phase of CoA accumulation occurred which plateaued around 50 microM CoA. The initial phase was decreased significantly by ATP or by carboxyatractyloside. In contrast, the presence of ATP or carboxyatractyloside did not affect the second phase. Decreasing the temperature from 30 to 0 degrees C did not affect the initial phase, but the second phase was almost abolished. In the presence of metabolic inhibitors (either 2,4-dinitrophenol or a combination of rotenone and antimycin), the initial "binding" phase was not affected; but the second "uptake" phase was abolished. These results suggest that the first phase of mitochondrial CoA accumulation is probably CoA binding to adenine recognizing sites on the mitochondria while the second phase may represent a specific uptake process for CoA which, although not directly ATP-dependent, is energy-dependent.

Topics: 2,4-Dinitrophenol; Adenosine Triphosphate; Animals; Antimycin A; Atractyloside; Biological Transport, Active; Coenzyme A; Dinitrophenols; Male; Mitochondria, Heart; Rats; Rats, Inbred Strains; Rotenone; Temperature

An improved method for the isolation of rat brain mitochondria is described. The preparation exhibits a respiratory control index (RCI) of 6 or 7.3 in the presence of pyruvate and malate or glutamate and malate, respectively. RCI decreases to 2.5 in the presence of Mg++. When the phosphorylation of extramitochondrially added or formed ADP is suppressed by carboxyatractyloside (CAT) inhibition of the adenine nucleotide translocator, the remaining respiration amounts to 6 nmol O2/min X mg mitochondrial protein. These results and the ratio of 16 to 19 from the quotient of phosphorylating active-state respiration to CAT inhibited respiration refer to a high degree of mitochondrial coupling of respiration. Therefore the remaining respiration in the presence of Mg++ is due to a phosphokinase activity located outside the inner membrane of intact mitochondria or at nonphosphorylating mitochondrial fragments. The following activities were observed: Oligomycin sensitive ATPase, 47 mU/mg protein; hexokinase, 272 mU/mg protein; creatinphosphokinase, 116 mU/mg protein; and a surprisingly low activity of adenylatekinase, 57 mU/mg protein.

Topics: Adenosine Monophosphate; Adenosine Triphosphate; Adenylate Kinase; Animals; Atractyloside; Benzimidazoles; Brain; Brain Chemistry; Creatine Kinase; Hexokinase; Magnesium; Male; Mitochondria; Rats; Rats, Inbred Strains; Substrate Specificity; Uncoupling Agents

The existence of different antigenic determinants in the beef heart adenine nucleotide (AdN) carrier was demonstrated by exploring the reactivity of fragments of the carrier protein with rabbit antisera directed against either the beef heart AdN carrier denatured with sodium dodecyl sulfate (NaDodSO4 carrier) or the beef AdN carrier liganded by the specific inhibitor carboxyatractyloside (CATR-carrier). The antigenic determinants reacting with antiserum to the CATR-carrier appeared to be close to the N- and C-terminal ends of the carrier protein, whereas those reacting with antiserum to the NaDodSO4 carrier were located preferentially in the central region of the protein. The same antisera were used to study the immunogenic specificity of the beef liver AdN carrier, the rat heart AdN carrier, and the rat liver AdN carrier. The beef liver and rat heart AdN carriers were found to react with both antisera whereas the rat liver AdN carrier reacted essentially with the CATR-carrier antiserum.

Topics: Animals; Antigen-Antibody Complex; Atractyloside; Cattle; Epitopes; Immune Sera; Mitochondrial ADP, ATP Translocases; Myocardium; Nucleotidyltransferases; Protein Denaturation; Sodium Dodecyl Sulfate

Interactions between phenylephrine-induced oxygen consumption, lactate and pyruvate output, and urea and glucose production were examined in perfused livers from fed or 48-h-fasted rats. Within 2 min of phenylephrine infusion, oxygen consumption in perfused livers was increased by approximately 40%. Increases in oxygen consumption induced by phenylephrine were essentially abolished in the presence of carboxyatractyloside, whereas those induced by dinitrophenol were still evident. Phenylephrine-induced increases in oxygen consumption were accompanied by enhanced rates of gluconeogenesis and ureogenesis in livers from fed or 48-h-fasted animals. These data indicate that phenylephrine-induced increases in respiration in perfused rat liver may result from an enhanced rate of mitochondrial oxidative phosphorylation in response to an increased cellular energy requirement.

Topics: Adrenergic alpha-Agonists; Animals; Atractyloside; Gluconeogenesis; Glucose; Glycolysis; Lactates; Lactic Acid; Liver; Male; Oxygen Consumption; Perfusion; Phenylephrine; Pyruvates; Pyruvic Acid; Rats; Rats, Inbred Strains; Urea

A fluorescent atractyloside analogue, the 6'-O-dansyl-gamma-aminobutyryl atractyloside (DGA), has been used to probe the binding of the inhibitors carboxyatractyloside (CATR) and bongkrekic acid (BA) and nucleotide substrates to the membrane-bound ADP/ATP carrier protein in beef heart mitochondria. Binding and release of DGA were followed by fluorescence responses. Specifically bound DGA was fully released by CATR alone, or by BA in the presence of micromolar amounts of ADP. In the absence of the inhibitors, ADP increased the rate of the specific binding of DGA. The effect of ADP was shared by transportable nucleotides. Non transportable nucleotides were ineffective. These data are consistent with the previously described CATR and BA conformations of the ADP/ATP carrier that are able to bind CATR and BA respectively, the transition between the two conformations being accelerated by micromolar concentrations of transportable nucleotides.

Topics: Animals; Atractyloside; Binding Sites; Biological Transport, Active; Bongkrekic Acid; Cattle; Dansyl Compounds; Fluorescence; Glycosides; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases; Protein Conformation

Oxidized dialdehyde analogs of ADP or ATP (oADP and oATP) were shown to inhibit irreversibly adenine nucleotide translocator (T) and creatine kinase (CK) in heart mitochondria. Inactivation of T and CK was parallel with carboxyatractyloside - sensitive and (ADP + phosphocreatine) - sensitive incorporation of o[3H]ADP into mitochondria, respectively. o[3H]ADP incorporation sensitive to CAT or ADP+phosphocreatine was used to determine T and CK contents in mitochondria. T content in cardiac mitochondria from rat, rabbit, dog, and chicken was calculated to be 2.6 - 2.9 moles/mole cyt.aa3. The same value of T/cyt.aa3 ratio was found in liver mitochondria with lower cytochrome aa3 content. In all types of cardiac mitochondria CK content was found to be 2.4 - 2.6 moles/mole cyt.aa3. The data show that T and CK are present in molar ratio 1:1 in all types of cardiac mitochondria.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Atractyloside; Carrier Proteins; Chickens; Creatine Kinase; Dogs; Kinetics; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases; Rabbits; Rats

Topics: Animals; Atractyloside; Biological Transport; Carrier Proteins; Cattle; Liposomes; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases; Protein Binding; Radioisotope Dilution Technique; Tritium

Topics: Animals; Anti-Bacterial Agents; Atractyloside; Binding Sites; Bongkrekic Acid; Carbon Radioisotopes; Cattle; Glycosides; Kinetics; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases; Protein Binding; Radioisotope Dilution Technique; Tritium

Determination of the intrinsic or mechanistic P/O ratio of oxidative phosphorylation is difficult because of the unknown magnitude of leak fluxes. Applying a new approach developed to overcome this problem (see our preceding paper in this journal), the relationships between the rate of O2 uptake [( Jo)3], the net rate of phosphorylation (Jp), the P/O ratio, and the respiratory control ratio (RCR) have been determined in rat liver mitochondria when the rate of phosphorylation was systematically varied by three specific means. (a) When phosphorylation is titrated with carboxyatractyloside, linear relationships are observed between Jp and (Jo)3. These data indicate that the upper limit of the mechanistic P/O ratio is 1.80 for succinate and 2.90 for 3-hydroxybutyrate oxidation. (b) Titration with malonate or antimycin yields linear relationships between Jp and (Jo)3. These data give the lower limit of the mechanistic P/O ratio of 1.63 for succinate and 2.66 for 3-hydroxybutyrate oxidation. (c) Titration with a protonophore yields linear relationships between Jp, (Jo)3, and (Jo)4 and between P/O and 1/RCR. Extrapolation of the P/O ratio to 1/RCR = 0 yields P/O ratios of 1.75 for succinate and 2.73 for 3-hydroxybutyrate oxidation which must be equal to or greater than the mechanistic stoichiometry. When published values for the H+/O and H+/ATP ejection ratios are taken into consideration, these measurements suggest that the mechanistic P/O ratio is 1.75 for succinate oxidation and 2.75 for NADH oxidation.

Topics: Adenosine Triphosphate; Animals; Antimycin A; Atractyloside; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; In Vitro Techniques; Mitochondria; NAD; Oxidation-Reduction; Oxidative Phosphorylation; Oxygen Consumption; Protons; Rats; Succinates; Succinic Acid; Thermodynamics

The effects of Reye's plasma, allantoin, and salicylates on mitochondrial structure and Ca2+ transport have been investigated. Measurements of Ca2+ transport showed that when 20-30 microM Ca2+ was added to isolated rat liver mitochondria preincubated with one of these agents, Ca2+ uptake was followed by its spontaneous release into the medium. This was accompanied by large-amplitude swelling; the onset preceded the Ca2+ release. No further Ca2+ release was induced by uncoupler or the Ca2+ ionophore, A23187. The mitochondria continued to swell even after all of the Ca2+ had been released. The time between the addition of Ca2+ and the onset of swelling (or Ca2+ release) depended on the concentration of the agent added and the preincubation time; the extent of swelling did not. These effects were prevented, but not reversed, by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid, ruthenium red, rotenone, or adenine nucleotides. The massive swelling and membrane disruption were confirmed by electron microscopy of the treated vs untreated mitochondria. Similar results concerning swelling and Ca2+ release were also seen with Ca2+ alone, but the time scale was much longer (i.e., greater than 3-4 min), indicating that these agents act by potentiating Ca2+-induced alterations in mitochondrial structure, as suggested by our earlier work (T.Y. Segalman and C.P. Lee (1982) Arch. Biochem. Biophys. 214, 522-530; M.E. Martens and C.P. Lee (1984) Biochem. Pharmacol. 33, 2869-2876). Our data show, therefore, that allantoin, salicylates, and the "toxic" agent in Reye's plasma severely limit the ability of isolated rat liver mitochondria to maintain their structural integrity under conditions of limited Ca2+ loading.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Allantoin; Animals; Atractyloside; Biological Transport; Calcimycin; Calcium; Humans; In Vitro Techniques; Male; Microscopy, Electron; Mitochondria, Liver; Mitochondrial Swelling; Oligomycins; Oxygen Consumption; Rats; Rats, Inbred Strains; Reye Syndrome; Rotenone; Salicylates; Salicylic Acid

Incubation of isolated rat hepatocytes with metabolic inhibitors causes an increase in the -glucose 6-P/+glucose 6-P activity ratio of glycogen synthase after decreasing ATP and increasing AMP levels. Concomitantly, the activity of phosphorylase is increased six-fold by the same treatment. This activation of both enzymes remains after gel filtration of the hepatocyte extracts. Addition of metabolic inhibitors to cells pretreated with an inhibitor of AMP-deaminase results in an accumulation of AMP and, simultaneously, in a further increase in the activation state of glycogen synthase. The correlation coefficient between the intracellular concentration of AMP and glycogen synthase activity is r = 0.93. It is proposed that the covalent activation of glycogen synthase by metabolic inhibitors can be triggered by changes in the level of the intracellular concentrations of adenine nucleotides.

Topics: 2,4-Dinitrophenol; Adenine; Adenine Nucleotides; Animals; Atractyloside; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Dinitrophenols; Enzyme Activation; Glycogen Synthase; Inosine Monophosphate; Liver; Phosphorylases; Rats

Cocklebur poisoning occurred in a herd of cattle in Oklahoma during the month of July. The poisonous dicotyledonary stage of cocklebur plant growth usually occurs during the early spring in Oklahoma. In this instance, dicotyledonary sprouts were in a lowland pasture adjacent to a river. Sprouting and growth occurred after receding water and hot dry weather had created favorable conditions for germination. The diagnosis was made on the basis of the finding of the dicotyledonary stage of plant growth and evidence of the plants having been grazed, as well as the clinical signs, gross necropsy findings, and histopathologic findings in the affected cattle.

Topics: Animals; Atractyloside; Cattle; Cattle Diseases; Glycosides; Plant Poisoning; Plants, Toxic

The rate of oxidative phosphorylation was studied in rat liver mitochondria incubated with free Ca2+ concentrations that range from 10(-9) to 5 X 10(-6) M. The highest rate was observed between 0.5-1.0 microM Ca2+. ATP synthesis was measured by polarographic and spectrophotometric techniques and by uptake of radioactive inorganic phosphate. The concentration of Ca2+ at which maximal rates of ATP synthesis take place is modified by Mg2+ and phosphate. The dependence of oxidative phosphorylation on Ca2+ was observed with alpha-ketoglutarate, glutamate + malate, and succinate, but not with beta-hydroxybutyrate. At 10(-9) M Ca2+ there is a continuous exit of endogenous Ca2+, while with 10(-6) M Ca2+, intramitochondrial Ca2+ levels remained constant throughout time. Apparently the control of the level of internal Ca2+ by external Ca2+ modulates the rate of oxidative phosphorylation. Uncoupler-stimulated respiration also depends on Ca2+ concentration, even though at 10(-9) to 10(-6) M Ca2+ the rate of oxidative phosphorylation is lower than the rate of uncoupled respiration. The contribution of the ADP/ATP carrier and the ATP synthase to the kinetic regulation of ATP synthesis at 10(-9) and 10(-6) M Ca2+ was evaluated by titrations with carboxyatractyloside and oligomycin, respectively. The contribution of the carrier and the synthase to the regulation of the final rate of ATP synthesis was different at the two concentrations of Ca2+; therefore, the concentration of extramitochondrial Ca2+ influences the overall kinetics of oxidative phosphorylation.

Topics: Adenosine Triphosphate; Animals; Atractyloside; Calcium; Female; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Oxidative Phosphorylation; Oxygen Consumption; Proton-Translocating ATPases; Rats; Rats, Inbred Strains

The regulation of the rate of mitochondrial oxidative phosphorylation and arsenylation was studied at two external free Ca2+ concentrations. The rate of arsenate-stimulated respiration in absence of added ADP was not affected by external 10(-9) and 10(-6) M Ca2+ levels or carboxyatractyloside, while state 3 respiration was profoundly modified. In addition, the kinetic analysis showed that the rate of arsenylation in the presence of ADP was more efficient (Vm/Km ratio 3.5 times higher) in the catalytic process than phosphorylation. Therefore, this suggests that the activity of the ATP/ADP carrier is importantly controlled by Ca2+. The evaluation of the control in phosphorylation showed that the flux-control coefficients (Ci) exerted by the ATP/ADP carrier (ranged between 0.23 and 0.48) and the ATP synthase (0.05-0.57) were modified in a reciprocal way by Ca2+ and Pi concentrations. This suggests that these two enzymes are coupling sequentially through a common intermediate, the intramitochondrial ATP/ADP ratio. Other important steps controlling phosphorylation were the b-c1 complex (Ci = 0.30) and the cytochrome oxidase (Ci = 0.23) but they were not modified by Ca2+. It was also found that the main step controlling arsenylation was the ATP synthase (Ci = 0.74). The increment in the inorganic arsenate concentration induced a diminution in the control exerted by the ATP synthase (from 0.73 to 0.56). The results suggest that Ca2+ and Pi (or inorganic arsenate) could be regulated by ATP synthesis through an activating effect on ATP/ADP carrier and/or ATP synthase.

Topics: Adenosine Diphosphate; Animals; Arsenates; Arsenic; Atractyloside; Calcium; Kinetics; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases; Oligomycins; Oxidative Phosphorylation; Proton-Translocating ATPases; Rats

Pea chloroplasts were found to take up actively ATP and ADP and exchange the external nucleotides for internal ones. Using carrier-free [14C]ATP, the rate of nucleotide transport in chloroplasts prepared from 12-14-day-old plants was calculated to be 330 mumol ATP/g chlorophyll/min, and the transport was not affected by light or temperature between 4 and 22 degrees C. Adenine nucleotide uptake was inhibited only slightly by carboxyatractylate, whereas bongkrekic acid was nearly as effective an inhibitor of the translocator in pea chloroplasts as it was in mammalian mitochondria. There was no counter-transport of adenine nucleotides with substrates carried on the phosphate translocator including inorganic phosphate, 3-phosphoglycerate and dihydroxyacetone phosphate. However, internal or external phosphoenolpyruvate, normally considered to be transported on the phosphate carrier in chloroplasts, was able to exchange readily with adenine nucleotides. Furthermore, inorganic pyrophosphate which is not transported by the phosphate carrier initiated efflux of phosphoenolpyruvate as well as ATP from the chloroplast. These findings illustrate some interesting similarities as well as differences between the various plant phosphate and nucleotide transport systems which may relate to their role in photosynthesis.

Topics: Adenine Nucleotides; Anions; Atractyloside; Bongkrekic Acid; Chloroplasts; Kinetics; Light; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases; Temperature

Topics: Anions; Atractyloside; Binding Sites; Biological Transport, Active; Bongkrekic Acid; Intracellular Membranes; Macromolecular Substances; Membrane Potentials; Mitochondria; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases; Phospholipids

Adenine nucleotide efflux from isolated rat heart mitochondria was studied. Inorganic phosphate induced efflux of adenine nucleotides from the mitochondria. This efflux was inhibited by carboxyatractyloside and atractyloside. The rate of efflux showed saturation kinetics with respect to extramitochondrial phosphate (Km, 9.5 mM). Lowering the pH from 7.4 to 6.8 had little or no effect on the rate of efflux. Deenergizing the mitochondria enhanced carboxyatractyloside-insensitive efflux, but it did not affect carboxyatractyloside-sensitive efflux. Extramitochondrial ATP (200 microM) or AMP (200 microM) prevented efflux when the phosphate concentration was 10 mM. AMP (200 microM) did not inhibit efflux when the phosphate concentration was 40 mM. Atractyloside inhibited efflux noncompetitively with respect to inorganic phosphate. Mersalyl (10 nmol/mg protein) did not inhibit efflux. Phenylsuccinate (20 mM) totally inhibited phosphate-induced efflux. The results of this study indicate that under conditions found in the ischemic heart cell (low ATP, high phosphate), adenine nucleotides may be lost from the mitochondria via the adenine nucleotide translocase. Phosphate does not induce this efflux by interacting with the translocase or the phosphate-hydroxyl carrier. The site of action of phosphate may be the dicarboxylate carrier.

Topics: Adenine Nucleotides; Adenosine Triphosphate; Animals; Atractyloside; Coronary Disease; Male; Mersalyl; Mitochondria, Heart; Phosphates; Rats; Rats, Inbred Strains; Succinates

The mechanism of stimulation of hepatic respiration by fatty acids was studied in isolated rat hepatocytes. Stimulation of respiration by fatty acids varied from about 35% to about 105% depending on chain length. The stimulatory effect of octanoate (1 mM) or oleate (0.5 mM) was prevented by oligomycin (2 micrograms/ml). With carboxyatractyloside (100 microM) and ouabain (2 mM) the stimulation of respiration was partially inhibited (by 50-70 and 50-60%, respectively). From these results it can be concluded that the increased rate of respiration after addition of fatty acids is coupled to ATP synthesis. A large part (50-60%) of this ATP is utilized by the (Na+ + K+)-ATPase.

Topics: 2,4-Dinitrophenol; Adenine Nucleotides; Animals; Atractyloside; Caprylates; Dinitrophenols; Energy Metabolism; Fatty Acids; In Vitro Techniques; Liver; Male; Oleic Acid; Oleic Acids; Oligomycins; Ouabain; Oxygen Consumption; Rats; Rats, Inbred Strains

Inhibitor titration experiments carried out with carboxyatractyloside, oligomycin and rotenone show that in the case of heart mitochondria the membrane-bound ATPase and the respiratory chain are the major factors controlling the rate of oxidative phosphorylation whereas the adenine nucleotide carrier exhibits no control strength. As shown by carboxyatractyloside titration curves under different conditions, the relative importance of the adenine nucleotide carrier depends on the mode of regeneration (F1-ATPase or glucose plus hexokinase) of ADP from ATP exported outside mitochondria, on the total concentration of adenine nucleotides present in the medium and on the mode of limitation of the rate of respiration (cyanide, rotenone, oligomycin or mersalyl). Concomitantly with the inhibition of O2 consumption, carboxyatractyloside brings about a rise in membrane potential. The inverse relationship between the two processes is observed for carboxyatractyloside concentrations ranging between 0.7 and 1.5 nmol per mg protein. Carboxyatractyloside concentrations below and above this range increase the membrane potential without affecting significantly the rate of respiration. Titration experiments aimed at comparing the effects of ADP, carboxyatractyloside and the uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, corroborate the conclusion that in heart mitochondria a major limiting factor in oxidative phosphorylation is the capacity of the respiratory chain.

Topics: Adenine Nucleotides; Animals; Atractyloside; Membrane Potentials; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases; Oxidative Phosphorylation; Oxygen Consumption; Rats

Impaired phosphorylation efficiency in liver mitochondria from hypothyroid rats is paralleled by a defect in adenine nucleotide transport. Both of these lesions can be corrected within 15 min by a near-physiological dose of triiodo-L-thyronine. Measurement of the control strength of the translocator shows, however, that this step has a smaller share of the control for oxidative phosphorylation after thyroidectomy and that this is unaltered after 15 min by replacement therapy. Rapid control by triiodothyronine is thus exerted elsewhere than at this transfer and the effects of hormone on the translocator are likely to be indirect.

Topics: Animals; Atractyloside; Hypothyroidism; Kinetics; Male; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases; Oxidative Phosphorylation; Rats; Thyroidectomy; Triiodothyronine

Certain factors that might contribute to the regulation of the rate of glycolysis by rat aorta were investigated. Rat aortic rings were incubated with [14C]glucose, and the release of [14C]lactate was determined. There was good agreement between the lactate production estimated by enzymatic assay and by [14C]lactate release, suggesting that almost all the lactate produced under our experimental conditions was derived from exogenous glucose. When the glucose concentration in the medium was 10 mM or higher, the rate of glucose transport did not limit the rate of lactate production. In most cases studies were done both aerobically and anaerobically. In Hanks' Balanced Salt Solution the aerobic rate of lactate production was 18% of the anaerobic rate. We tested the effects on glycolysis of agents that alter ATP generation by mitochondria or ATP splitting by Na+-K+-ATPase or the mitochondrial ATPase. Under aerobic conditions, ouabain (5 mM) caused a 54% decrease in lactate production, and gramicidin (5 micrograms/ml) caused a 45% increase. Under anaerobic conditions, neither ouabain nor gramicidin affected lactate production. Aerobically dinitrophenol (25 microM) and carboxyatractyloside (0.5 mM) caused substantial increases in lactate production, 72 and 98% respectively. Under anaerobic conditions the effects of dinitrophenol and carboxyatractyloside were much smaller, with dinitrophenol causing a 15% increase and carboxyatractyloside a 12% decrease in lactate production. Increasing the concentration of phosphate in the incubation medium caused marked increases in lactate production. Both aerobically and anaerobically, shifting from 1.3 to 50 mM phosphate in the incubation medium caused a 3.5-fold increase in lactate production.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Triphosphatases; Animals; Aorta; Atractyloside; Biological Transport; Carbon Radioisotopes; Culture Media; Dinitrophenols; Glucose; Glycolysis; Gramicidin; Lactates; Lactic Acid; Male; Mitochondria, Muscle; Ouabain; Oxidative Phosphorylation; Phosphates; Rats; Rats, Inbred Strains; Sodium-Potassium-Exchanging ATPase

Adenosine diphosphatase (ADPase) activity and ATPase activity were assayed in rat liver mitochondria and outer mitochondrial membrane preparations with [beta-32P]ADP and [gamma-32P]ATP as substrates. Inhibition studies were performed with the mitochondrial ATPase inhibitor oligomycin and the adenine nucleotide transport inhibitor, carboxyatractyloside. Kinetic studies were also performed with the nucleotide thiophosphate analogs adenosine 5'-O-thiophosphate, adenosine 5'-O-(2-thiodiphosphate) and adenosine 5'-O-(3-thiotriphosphate) which can act as inhibitors of phosphohydrolases. It is concluded that part of the apparent ADPase activity of intact mitochondria is mediated via ATPase, presumably in conjunction with adenylate kinase. In addition the outer mitochondrial membrane appears to show a distinct ADPase not attributable to contamination by inner membrane ATPase.

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Apyrase; Atractyloside; Female; In Vitro Techniques; Intracellular Membranes; Mitochondria, Liver; Oligomycins; Phosphoric Monoester Hydrolases; Rats; Rats, Inbred Strains; Thionucleotides

At subsaturating concentrations of palmitoyl-CoA, the carnitine-dependent oxidation of the palmitoyl portion by uncoupled rat heart mitochondria was stimulated by ADP or ATP. This effect was traced to the prevention of acyl-CoA binding to adenine nucleotide translocase and the consequent sparing of acyl-CoA for acylcarnitine formation. Palmitoyl-CoA oxidation was stimulated by ITP also although ITP served neither as a transportable substrate nor as an inhibitor of ADP transport. ITP and other nontransportable nucleoside di(tri)phosphates prevented octanoyl-CoA binding to mitochondria. ITP was bound to mitochondria, and this binding was reversed by ADP, octanoyl-CoA, and carboxyatractyloside. Thus, besides a substrate site, there is a site on the translocase that binds nucleoside di(tri)phosphates, CoA and its esters, and atractylosides; inhibition of the translocase results, however, only from the binding of CoA esters of fatty acids and of atractylosides. We suggest that in O2-deficient hearts, when nucleotides decline and fatty acyl-CoA rises, the binding of the latter to the translocase becomes operational to slow fatty acylcarnitine production. By retarding the rise in amphipathic burden, this mechanism could protect heart against irreversible damage during brief periods of ischemia or hypoxia.

Topics: Acetylcarnitine; Acyl Coenzyme A; Adenosine Diphosphate; Adenosine Triphosphate; Animals; Atractyloside; Binding Sites; Fatty Acids; Inosine Triphosphate; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Myocardium; Nucleotidyltransferases; Oxidation-Reduction; Oxygen; Palmitoyl Coenzyme A; Rats

Carboxyatractyloside was labeled with [3H]KBH4 after oxidation of the primary alcohol of the glucose disulfate moiety by dicyclohexylcarbodiimide and P2O5 under anhydrous conditions in a dimethylsulfoxide medium. The 3H-labeled product was purified by DE 52 column chromatography followed by Cellulofine GCL 25 column chromatography. The final 3H-labeled product gave a single spot on a thin-layer chromatogram, and its Rf value was the same as that of authentic carboxyatractyloside. The biological activities (such as inhibition of state 3 respiration and binding to the adenine nucleotide carrier) were also comparable with those of authentic carboxyatractyloside.

Topics: Animals; Atractyloside; Borohydrides; Glycosides; Isotope Labeling; Kinetics; Male; Mitochondria, Liver; Oxygen Consumption; Rats; Rats, Inbred Strains; Tritium

Liver mitochondria provided with an oxidizable substrate, ATP, oxygen, and an ADP-generating system (soluble F1-ATPase) were used to reevaluate the rate-controlling step(s) intrinsic to all of the processes of mitochondrial oxidative phosphorylation. The quantity termed "control strength" (C), previously defined as the fractional change in flux through a (system) induced by a fractional change in the concentration of an individual enzyme in the system, has been used to evaluate rate-influencing steps in this overall process by carefully defining the dimensions of the "system" under analysis. If the system is defined by a suspension of mitochondria provided with substrates, plus an extrinsic ADP-generating process (ATPase), the value of C of the latter for the overall process of phosphorylation-linked respiration is near 1.0 until the capacity of the mitochondria to phosphorylate ADP is approached, after which C for the soluble ATPase becomes zero as the maximum capacity for phosphorylation is attained. Carboxyatractyloside was found only marginally to inhibit respiration stimulated by ATPase, even when a large percentage of adenine nucleotide translocase molecules were immobilized. The relative lack of effect of carboxyatractyloside on phosphorylating respiration is explained by the readjustment of the concentration of one of the substrates (ADP) and an inhibitor (ATP), which results from inhibition of adenine nucleotide translocase. The residual blunted inhibition of respiration is explained by product inhibition of the ADP-regenerating ATPase, and not necessarily to any intrinsically mitochondrial intermediate process. The system being evaluated can be redefined to include only the processes intrinsic to mitochondria. This can be achieved by providing exactly comparable substrate concentrations to the mitochondria under comparable incubation conditions. Under these conditions, the adenine nucleotide translocase is the principal, if not the only, rate-controlling step in the overall process of oxidative phosphorylation until a new rate-limitation is attained (ATP synthesis). These data are consistent with the conclusion that, at intermediate rates of phosphorylation-coupled respiration, the extramitochondrial ATP/ADP ratio regulates this process through its kinetic effects on the catalytic properties of the adenine nucleotide translocase.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Atractyloside; Catalysis; In Vitro Techniques; Kinetics; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Oxidative Phosphorylation; Rats; Rats, Inbred Strains; Substrate Specificity

A Golgi vesicle-enriched preparation from mammary tissue of lactating rats has been used to investigate the phosphorylation of caseins in vitro. Casein kinase, together with its casein substrates, is enclosed within the lumen of Golgi membrane vesicles and has a requirement for Ca2+ and ATP. The permeability characteristics of the Golgi membrane to ATP and Ca2+ therefore have a possible regulatory influence on casein kinase activity. This influence has been investigated by alteration of the permeability characteristics by using several agents having differing degrees of selectivity. The ionophore A23187, which permits loss of Ca2+ from the vesicles, caused a decrease in casein phosphorylation which could be reversed by externally supplied Ca2+. Alamethicin, an ionophore that creates larger transmembrane channels, caused an increase in casein phosphorylation. This increase showed a requirement for divalent metal ions which could be satisfied by either Ca2+ or Mn2+. Under the same conditions, La3+ was inhibitory. Triton X-100 caused loss of intravesicular Ca2+, yet this was accompanied by an increase in phosphate incorporation into the caseins. We conclude from these results that the binding site on casein kinase for ATP is within the Golgi membrane barrier and that they imply the presence of a transmembrane ATP-transport mechanism. Inhibition of casein phosphorylation by atractyloside and carboxyatractyloside lends support to this concept.

Topics: Alamethicin; Animals; Atractyloside; Calcium; Casein Kinases; Caseins; Cations; Female; Golgi Apparatus; In Vitro Techniques; Lactation; Mammary Glands, Animal; Mitochondrial ADP, ATP Translocases; Octoxynol; Phosphorylation; Polyethylene Glycols; Pregnancy; Protein Kinases; Rats; Rats, Inbred Strains

NAD(P)+-stimulated Ca2+ efflux from mitochondria is inhibited by bongkrekate and slightly stimulated by carboxyatractylate. Addition of oxaloacetate, an NAD(P) oxidant, or diamide, a thiol oxidant, to de-energized mitochondria incubated in Ca2+ -free medium induced a small decrease in turbidity of the mitochondrial suspension compatible with small structural changes of mitochondria. Similar to NADP+-stimulated Ca2+ efflux these changes were also inhibited by bongkrekate and slightly stimulated by carboxyatractylate. The similarity between the effects of oxaloacetate and diamide, on both Ca2+ efflux and mitochondrial structure, indicates the existence of a common denominator, possibly the oxidation of specific thiol groups, regarding the mechanism by which these agents stimulate Ca2+ efflux from mitochondria.

Topics: Adenosine Diphosphate; Animals; Atractyloside; Bongkrekic Acid; Calcium; Cell Membrane Permeability; Diamide; Mitochondria, Liver; NADP; Oxaloacetates; Oxidation-Reduction; Rats; Sulfhydryl Compounds

Unidirectional transport (influx and efflux) of adenine nucleotides in rat liver mitochondria was examined using carboxyatractyloside to inhibit rapid exchange of matrix and external adenine nucleotides via the adenine nucleotide translocase. Influx of adenine nucleotides was concentration-dependent. ATP was the preferred substrate with a Km of 2.67 mM and V of the preferred substrate with a Km of 2.67 mM and V of 8.33 nmol/min/mg of protein. For ADP, the Km was 14.7 mM and V was 10.8 nmol/min/mg of protein. Efflux of adenine nucleotides was also concentration-dependent, varying directly as a function of the matrix adenine nucleotide pool size. Any increase in the influx of adenine nucleotides was coupled to an increase in efflux. However, as the external ATP concentration was increased, influx was stimulated to a much greater extent than was efflux. This imbalance suggested that under certain conditions adenine nucleotide movement might be coupled to the movement of an alternate anion such as phosphate. Adenine nucleotide efflux increased as the external phosphate concentration was varied from 0.5 to 4 mM. Also, increasing the external phosphate concentration caused adenine nucleotide influx to decrease, suggesting competition. In the absence of external adenines and phosphate, no efflux occurred. Both adenine nucleotide influx and efflux were depressed if Mg2+ was omitted. Adenine nucleotide efflux in the presence of external phosphate was inhibited much less by lack of Mg2+ than was efflux in the presence of external ATP. This evidence supports a model in which either adenine nucleotides (probably with Mg2+) or phosphate can move across the mitochondrial membrane on a single carrier. Net adenine nucleotide movements can occur when adenine nucleotide movement is coupled to the movement of phosphate in the opposite direction.

Topics: Adenine Nucleotides; Animals; Atractyloside; Glycosides; Kinetics; Magnesium; Male; Mitochondria, Liver; Rats; Rats, Inbred Strains

The effect of palmitoyl coenzyme A, atractylosides, and anion transport inhibitors on translocation into rat liver Golgi vesicles of adenosine 3'-phosphate 5'-phosphosulfate (PAPS), CMP-N-acetylneuraminic acid, and GDP-fucose was studied. Translocation of the above three nucleotide derivatives was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 50% inhibition required 10-20 microM DIDS). The inhibition of translocation of PAPS by DIDS was used to demonstrate that sulfation of macromolecules within Golgi vesicles is preceded by translocation of PAPS into the vesicles. Palmitoyl coenzyme A, at concentrations below its critical micellar concentration, specifically inhibited translocation into Golgi vesicles of PAPS but not CMP-NeuAc and GDP-fucose. Inhibition of PAPS translocation by 50% required 9 microM palmitoyl coenzyme A. Translocation of PAPS but not of CMP-NeuAc or GDP-fucose was also inhibited by atractyloside or carboxyatractyloside with 50% inhibition requiring 15 microM either glycoside. This pattern of inhibition suggests structural similarities between the putative translocator of PAPS in Golgi membranes and the ATP/ADP translocator of mitochondria.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acyl Coenzyme A; Adenine Nucleotides; Animals; Atractyloside; Bongkrekic Acid; Cytidine Monophosphate N-Acetylneuraminic Acid; Glycosides; Golgi Apparatus; Guanosine Diphosphate Fucose; Liver; Nucleoside Diphosphate Sugars; Palmitoyl Coenzyme A; Phosphoadenosine Phosphosulfate; Rats; Sialic Acids

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Atractyloside; Creatine Kinase; Hexokinase; Kinetics; Mitochondria; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases; Oxygen Consumption; Rats; Thermodynamics

The consequence of the complexity of the metabolic network on the amount of control strength of adenine nucleotide translocator was investigated with isolated rat liver mitochondria. Two experimental systems were compared: (i) mitochondria in the presence of yeast hexokinase (hexokinase system) and (ii) the same system plus additional pyruvate kinase (pyruvate kinase system). In both systems the control strength was analysed for the adenine nucleotide translocator by inhibitor titration studies with carboxyatractyloside and for the hexokinase or pyruvate kinase by changing their relative activities. Experimental results were compared with computer simulation of these systems and that of a third one, where the extramitochondrial ATP/ADP ratio was held constant by perifusion (perifusion system). The results demonstrate quite different flux-dependent control strength of the translocator in the three systems. In the hexokinase system the control strength of the translocator on mitochondrial respiration was zero up to respiration rates of about 60 nmol O2/mg protein per min. For higher rates, the control strength increased until the maximum value (0.45) was reached in the fully active state. Here, the same value was also found in the pyruvate kinase system. In all other states of respiration the translocator exerts a higher control strength in the pyruvate kinase system than in the hexokinase system. This different behaviour was attributed to the various changes in the adenine nucleotide pattern caused by partial inhibition of the translocator in the hexokinase and pyruvate kinase system. The data clearly show that the sharing of control strength depends not only on the respiration rate but also on the complexity of the metabolic system.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Atractyloside; Hexokinase; Kinetics; Mathematics; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases; Oxygen Consumption; Pyruvate Kinase; Rats

The spin-label 2,2,5,5-tetramethyl-1-oxy-3-pyrroline-3-carboxylic acid was attached to the inhibitor carboxyatractylate of the mitochondrial ADP/ATP carrier. Being closely linked to the inhibitor, the spin-label should reflect the mobility of the carboxyatractylate. When bound to the carrier in mitochondria, spin-labeled carboxyatractylate reveals a most unusual hyperfine splitting of 72 G. A second spectral component with a hyperfine splitting of 62 G is also mainly due to carrier-bound inhibitor. A similar spectrum with somewhat reduced hyperfine splitting was observed with the detergent-solubilized protein, whereas reincorporation into phospholipid membranes yielded almost the same spectra as in mitochondria. The carrier-bound spin-label is concluded to be highly immobilized. The less immobilized spectral component is discussed in terms of strongly anisotropic label motion. In addition, the unusual splitting is interpreted to indicate the highly polar environment of the nitroxide. The interpretations are supported by the temperature dependence, which indicates a reversible progressive spin-label mobilization up to 50 degrees C. Membrane-impermeable reducing agents showed that the spin-label is easily accessible from the aqueous phase.

Topics: Animals; Atractyloside; Cattle; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Glycosides; Kinetics; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases; Protein Binding; Spin Labels; Temperature

Various modulating influences of negative and positive membrane charges on binding and transport properties of the reconstituted ADP/ATP carrier from mitochondria were investigated. The results are interpreted in terms of functional and structural asymmetries of the adenine nucleotide carrier embedded in the liposomal membrane. The surface potential of liposomes was measured directly either by potential-dependent adsorption of the fluorescent dye 2-p-toluidinylnaphthalene 6-sulfonate (TNS) or by the pK shift of the lipophilic pH indicator pentadecylumbelliferone. These results were correlated with the following observations. (1) Negative surface potentials increase the apparent dissociation constant, Kd, for binding of the negatively charged inhibitor carboxyatractylate to the reconstituted carrier protein. (2) Surface potentials modulate the apparent transport affinity, Km, of the reconstituted adenine nucleotide carrier for ADP and ATP. The interaction of surface charges with the transport function was investigated with carrier proteins oriented both right-side-out and inside-out. Thus the influence of the surface potential on the function of the ADP/ATP carrier could be determined for the internal and external active sites of the translocator on the outer side of the membrane. Large discrepancies were observed not only between the potentials measured directly (fluorescent dyes) and those measured indirectly (binding and transport affinities), but also between the different surface potentials determined from the influence on the alternatively oriented carrier proteins. The effect of surface charges was rather weak on the cytosolic side of the translocator, whereas there was a strong influence of surface charges on the active site at the matrix side. The most obvious explanation, i.e., screening of negative membrane charges by positively charged amino acid residues at the protein surface, could be ruled out. Besides the modulation of binding affinities for substrates and inhibitors, an additional side-specific effect of surface charges on the transport velocity was observed. Again, the influence on the internal active site of the ADP/ATP carrier was found to be much higher than that on the cytosolic site. The observed effects can be explained by a definite structural asymmetry of the carrier embedded in the liposomal membrane. That site which is physiologically exposed to the cytosol is located at a considerable distance from the plane of the membrane, w

Topics: Animals; Atractyloside; Cattle; Cholesterol; Intracellular Membranes; Kinetics; Liposomes; Mathematics; Membrane Lipids; Membrane Potentials; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Models, Biological; Nucleotidyltransferases

The kinetic properties of heart mitochondrial creatine kinase were measured with and without inhibitors of the adenine nucleotide translocator. No significant differences were observed suggesting that mitochondrial creatine kinase is not acting together with the adenine nucleotide translocator as a functional multienzyme complex. Adenine nucleotides from the bulk phase are able to enter the active center of creatine kinase without the necessity of transport via the adenine nucleotide translocator.

Topics: Animals; Atractyloside; Creatine Kinase; Glycosides; Kinetics; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases; Rats

ATP hydrolysis catalysed by the H+-ATPase of intact mitochondria can be induced by addition of ATP in the presence of valinomycin and KCl. This leads to an increase in intramitochondrial Pi and therefore allows investigation of potential Pi efflux pathways in intact mitochondria. Combining this approach with the direct measurement of both internal and external Pi, we have attempted to determine whether Pi efflux occurs via an atractyloside-sensitive transporter, by the classical operation of the Pi/H+ and Pi/dicarboxylate carriers, and/or by other mechanisms. Initial experiments re-examined the evidence that led to the current view that one efflux pathway for Pi is an atractyloside-sensitive ATP/ADP,0.5Pi transporter. No evidence was found in support of this efflux pathway. Rather, atractyloside-sensitivity of the low rate of Pi efflux observed in previous studies (oligomycin present) was accounted for by ATP entry on the well known ATP/ADP transport system followed by hydrolysis of ATP and subsequent Pi efflux. Thus, under these conditions, where ATP hydrolysis is not completely inhibited, Pi efflux becomes atractyloside sensitive most likely because this inhibitor blocks ATP entry, not because it directly inhibits Pi efflux. Substantial efflux of Pi from rat liver mitochondria is observed on generation of high levels of matrix Pi by ATP hydrolysis induced by valinomycin and K+ (oligomycin absent). A portion of this efflux can be inhibited by thiol-specific reagents at concentrations that normally inhibit the Pi/H+ and Pi/dicarboxylate carriers. However, a significant fraction of efflux continues even in the presence of p-chloromercuribenzoate, N-ethylmaleimide plus n-butylmalonate or mersalyl. The mersalyl-insensitive Pi efflux, which is also insensitive to carboxyatractyloside, is a saturable process, thus suggesting carrier mediation. During this efflux the mitochondrial inner membrane retains considerable impermeability to other low-molecular-weight anions (i.e., malate, 2-oxoglutarate). In conclusion, results presented here rule out an atractyloside-sensitive ATP/ADP,0.5Pi transport system as a mechanism for Pi efflux in rat liver mitochondria. Rather Pi efflux appears to occur on the classical Pi/H+ transport system as well as via a mersalyl-insensitive saturable process. The inhibitor-insensitive Pi efflux may occur on a portion of the Pi/H+ carrier molecules that exist in a state different from that normally catalysing Pi influx. Alternatively, a

Topics: Adenosine Triphosphate; Animals; Atractyloside; Chloromercuribenzoates; Male; Mersalyl; Mitochondria, Liver; Oligomycins; p-Chloromercuribenzoic Acid; Phosphates; Potassium; Rats; Sucrose; Valinomycin

Topics: Adenosine Diphosphate; Animals; Atractyloside; Creatine Kinase; Kinetics; Mitochondria, Heart; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases; Oxygen Consumption; Rats

Topics: Animals; Atractyloside; Kinetics; Male; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases; Oxygen Consumption; Rats; Rats, Inbred Strains

Topics: Animals; Atractyloside; Bongkrekic Acid; Cattle; Intracellular Membranes; Mathematics; Micelles; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Molecular Weight; Neutrons; Nucleotidyltransferases; Protein Binding; Scattering, Radiation

The ADP/ATP carrier protein was extracted and purified from beef heart mitochondria, and its binding parameters with respect to 3'-O-naphthoyladenosine 5'-triphosphate (N-ATP), a fluorescent nontransportable analogue of ATP, were studied. The binding of N-ATP to the isolated carrier protein was accompanied by a decrease in fluorescence. Conversely, the release of bound N-ATP upon addition of carboxyatractyloside (CATR) or ATP resulted in a fluorescence increase. The bound N-ATP that was released upon addition of an excess of CATR or ATP was referred to as specifically bound N-ATP, i.e., N-ATP bound to the nucleotide sites of the carrier protein. Two classes of binding sites for N-ATP could be identified; the number of high-affinity sites (Kd less than 10 nM) was equal to the number of low-affinity sites (Kd = 0.45 microM). CATR behaved apparently as a noncompetitive inhibitor of the binding of N-ATP. The amount of N-ATP released increased linearly with the amount of CATR added, indicating an extremely high affinity of the carrier protein for CATR. The number of CATR binding sites was equal to half the total number of N-ATP binding sites (high- and low-affinity sites); at saturating concentrations of N-ATP, the binding of 1 mol of CATR resulted in the release of 2 mol of bound N-ATP, one from the high-affinity site and the other from the low-affinity site, showing unambiguously that each CATR site is interacting with a pair of probably interdependent N-ATP sites. A clear competition between N-ATP and ATP for binding to the carrier protein was demonstrated. The Kd values of the high- and low-affinity sites for ATP were less than 50 nM and 5 microM, respectively. In the presence of high concentrations of ATP, the two classes of N-ATP binding sites became indistinguishable, suggesting interconversion. It is proposed that the asymmetry in affinity for N-ATP binding is induced by the binding step itself, the carrier protein exhibiting a negative cooperativity for N-ATP binding.

Topics: Adenosine Triphosphate; Animals; Atractyloside; Binding Sites; Cattle; Fluorescent Dyes; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases

Topics: Adenosine Triphosphate; Animals; Antibiotics, Antineoplastic; Atractyloside; Binding Sites; Binding, Competitive; Bongkrekic Acid; Cattle; Formycins; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases; Ribonucleotides

Male rats (10 rats/group) were treated with phenobarbital (PB), phenylbutazone (PBZ), stanozolol (3 inducers of cytochrome P450-dependent enzymes), piperonyl butoxide (PBO; a P450 inhibitor), cobaltous chloride (CoCl2; an inhibitor of hemoprotein synthesis), 5,6-benzoflavone (BNF; an inducer of cytochrome P448 dependent enzymes), cysteine [CYS; a glutathione (GSH) precursor], or ethyl maleate (EM; a GSH depletor). The rats were then given a calculated LD50 dosage (13.5 mg/kg of body weight) of carboxyatractyloside (CAT) intraperitoneally. Clinical signs of toxicosis, duration of illness, lethality, gross lesions, and hepatic and renal histopathologic lesions were recorded. Seemingly, (i) CAT toxicosis has independent lethal and cytotoxic components (PBZ decreased lethality and cytotoxicity; CoCl2 decreased cytotoxicity but not lethality; BNF decreased duration of illness, and perhaps lethality, but not cytotoxicity); (ii) CAT cytotoxicity could be partly due to an active metabolite formed by de novo-synthesized, P450-/P448-independent hemoprotein (PBZ and CoCl2 had anticytotoxic effects, but PB, stanozolol, PBO, and BNF did not); (iii) CAT detoxification may occur partly through a hemoprotein-independent, PBZ-inducible enzyme, and partly through a P448-dependent (BNF-inducible) enzyme; and (iv) CAT detoxification apparently is not P450 or GSH-dependent because PB, stanozolol, and CYS had no beneficial effects, and PBO, CoCl2, and EM did not enhance toxicosis. Metabolism of CAT may have a role in its cytotoxic and lethal effects.

Topics: Animals; Atractyloside; Benzoflavones; beta-Naphthoflavone; Cobalt; Cysteine; Glycosides; Kidney Tubules; Lethal Dose 50; Liver; Male; Maleates; Phenobarbital; Phenylbutazone; Piperonyl Butoxide; Plants, Toxic; Rats; Rats, Inbred Strains; Stanozolol

3'-O-(1-Naphthoyl)adenosine 5'-diphosphate (N-ADP), a fluorescent analogue of ADP, was established as a potent inhibitor of adenosine 5'-diphosphate/adenosine 5'-triphosphate (ADP/ATP) transport in mitochondria and inside-out sonic particles; the Ki value was about 5 microM. The inhibition was of a mixed type. On the other hand, N-ADP was not transported in a measurable way in either type of particles. Upon binding to the particles, the fluorescent intensity of N-ADP was decreased; the release of the bound N-ADP upon addition of carboxyatractyloside (CATR) to mitochondria and bongkrekic acid (BA) to sonic particles was reflected by increases of fluorescence. In parallel assays dealing with 14C-labeled N-ADP, specifically bound [14C]N-ADP was equated to [14C]N-ADP released upon addition of either CATR (mitochondria) or BA (sonic particles). The specific binding of N-ADP corresponded to 1.4-1.6 nmol/mg of protein in mitochondria, with a Kd value of 3 microM, and to 1.5-1.6 nmol/mg of protein in sonic particles, with a Kd value of 6 microM. Essentially similar values were obtained for N-ATP binding. These values are at least twice as high as those found for specific ADP or ATP binding, suggesting that N-ADP or N-ATP binds to potential nucleotide binding sites that were not totally occupied by ADP or ATP. Whereas nearly all the specifically bound N-ADP in mitochondria was displaced by an excess of ADP (400 microM) at pH 7.4, only 30% could be removed from sonic particles under the same conditions. Furthermore at pH 6.5, no more than half of the specifically bound N-ADP could be removed by excess ADP in mitochondria and only 10-20% in sonic particles. These results indicate that each ADP/ATP carrier unit contains at least two types of nucleotide sites capable of interacting with N-ADP. Because of the hydrophobic nature of the naphthoyl moiety of N-ADP, the data suggest that difference in N-ADP binding in mitochondria and sonic particles are related to differences in the hydrophobic nature of their sites. Due to the special features of N-ADP (strong specific binding to the ADP/ATP carrier and no competence for transport), this DP analogue was particularly suitable for investigating the sensitivity of the nucleotide binding sites of the carrier to chemical modifiers. Inactivation studies were therefore carried out with mitochondria and sonic particles to compare the sensitivity to UV light and butanedione of the binding of N-ADP, [3H]BA, and [14C]Ac-CATR, a radi

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Atractyloside; Binding Sites; Biological Transport; Bongkrekic Acid; Butylene Glycols; Carrier Proteins; Hydrogen-Ion Concentration; Intracellular Membranes; Kinetics; Mitochondria, Heart

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Atractyloside; Bongkrekic Acid; Carrier Proteins; Kinetics; Ligands; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases; Solutions; Spectrometry, Fluorescence

Cocklebur (Xanthium strumarium) fed to feeder pigs was associated with acute to subacute hepatotoxicosis. Cotyledonary seedings fed at 0.75% to 3% of body weight or ground bur fed at 20% to 30% of the ration caused acute depression, convulsions, and death. Principle gross lesions were marked serofibrinous ascites, edema of the gallbladder wall, and lobular accentuation of the liver. Acute to subacute centrilobular hepatic necrosis was present microscopically. The previously reported toxic principle, hydroquinone, was not recovered from the plant or bur of X. strumarium. Authentic hydroquinone administered orally failed to produce lesions typical of cocklebur intoxication but did produce marked hyperglycemia. Carboxyatractyloside recovered from the aqueous extract of X. strumarium and authentic carboxyatractyloside, when fed to pigs, caused signs and lesions typical of cocklebur intoxication. Marked hypoglycemia and elevated serum glutamic oxaloacetic transaminase and serum isocitric dehydrogenase concentrations occurred in pigs with acute hepatic necrosis that had received either cocklebur seedlings, ground bur or carboxyatractyloside.

Topics: Animals; Atractyloside; Glycosides; Hydroquinones; Liver; Necrosis; Plant Poisoning; Plants, Toxic; Swine; Swine Diseases

Topics: Animals; Atractyloside; Cattle; Mathematics; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Molecular Weight; Nucleotidyltransferases; Polyethylene Glycols; Protein Binding; Protein Conformation

Topics: Animals; Atractyloside; Binding Sites; Carbon Radioisotopes; Cattle; Glycosides; Kinetics; Mitochondria, Heart; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases; Protein Binding; Rats

1. ADP/ATP transport has been reconstituted by incorporation of the purified carrier protein in liposomes filled with ATP. The transport was assayed by uptake of [14C]ADP into the liposomes, and by release of ATP as determined by a luminescence technique. [14C]ADP uptake was strictly dependent on internal ATP. 2. The simplest phospholipid system capable of yielding high rates of ADP/ATP transport was a mixture of phosphatidylethanolamine and cariolipin (92: 8, w/w). 3. ADP/ATP transport in the reconstituted system proceeded by exchange-diffusion with a 1/1 stoichiometry. The specificity for aDP and ATP was absolute. The capacity and the rate of exchange depended on the concentration of ATP present in liposomes. The rate of transport at 20 degrees C, at 20 mM internal ATP, routinely ranged between 300 and 1000 nmol of nucleotide exchanged per min/mg of added carrier protein. The apparent Km value for external ADP was around 10 microM. 4. The ADP/ATP exchange in the reconstituted system was rather stable to ageing. It dropped by only 20% after 1 day of ageing at 20 degrees C. Divalent cations (Mg2+, Mn2+, Ca2+) at concentrations higher than 1 to 2 mM had a deleterious effect on ADP/ATP transport, concomitant with the release of internal ATP and accumulation of multilamellar vesicles. 5. Atractyloside behaved as a competitive inhibitor and carboxyatractyloside as a non-competitive inhibitor. Bongkrekic acid required a slightly acidic pH to be inhibitory. The data concerning atractyloside, carboxyatractyloside and bongkrekic acid were similar to those obtained with whole mitochondria, suggesting that the carrier protein in liposomes has the same asymmetrical arrangement as in the mitochondria. 6. The percentage of competent carrier protein in liposomes was calculated from dose-response data concerning the inhibition of ADP/ATP transport by atractyloside or carboxyatractyloside, and from the amount of bound [3H]-atractyloside removable by ADP. By both methods, 3 to 6% of the added carrier protein was found to be competent in ADP/ATP transport, based on the assumption that the binding of one atractyloside or carboxyatractyloside molecule per 30000 molecular weight carrier unit results in complete inhibition of transport. 7. Freeze-fracture electron microscopy showed that the ADP/ATP carrier protein-lipid preparations are formed by small vesicles, most of which give rise to smooth fracture faces (probably pure lipid vesicles). Only a small percentage of the vesi

Topics: Animals; Atractyloside; Biological Transport; Cattle; Freeze Fracturing; Kinetics; Liposomes; Microscopy, Electron; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Atractyloside; Binding Sites; Bongkrekic Acid; Carrier Proteins; Cattle; Intracellular Membranes; Membrane Proteins; Mitochondria, Heart; Phenylglyoxal

Topics: Adenosine Diphosphate; Animals; Atractyloside; Cattle; Hexokinase; Kinetics; Mitochondria, Heart; Oxidative Phosphorylation; Oxygen Consumption; Phosphates

1. The effects of atractyloside and carboxyatractyloside (between 5 and 40mum) on O(2) uptake, glucose synthesis, urea synthesis, the adenine nucleotide content and the intracellular K(+) concentration were measured in isolated hepatocytes. 2. Urea synthesis was much less inhibited than glucose synthesis by both atractylosides. Measurements of intermediary metabolites of carbohydrate metabolism in freeze-clamped liver after injection of atractyloside into rats indicate that inhibition of gluconeogenesis is due to interference at the cytosolic reactions requiring ATP (phosphoenolpyruvate carboxykinase and 3-phosphoglycerate kinase). 3. The decrease in [ATP]/[ADP]x[P(i)] after addition of atractyloside or carboxyatractyloside was restricted to the cytosol. 4. Dihydroxyacetone can be converted either into glucose with the consumption of 2mol of ATP (per mol of glucose) or into lactate with the production of 2mol of ATP. In the presence of high concentrations of atractyloside and carboxyatractyloside more ATP was produced than was used for the synthesis of glucose from dihydroxyacetone, probably for the maintenance of intracellular [K(+)]. 5. When the rates of respiration were altered by changing substrates, the degrees of inhibition of respiration and translocation by a given concentration of the atractylosides were the same, whereas at a given concentration of HCN the degree of inhibition was high at higher initial rates, and low at lower initial rates. 6. Inhibition of a complex series of reactions by atractyloside does not necessarily indicate that the translocator is a rate-limiting step in that sequence as Th. P. M. Akerboom, H. Bookelman & J. M. Tager [(1977) FEBS. Lett.74, 50-54] assume. This point is discussed.

Topics: Adenine Nucleotides; Adenosine Triphosphate; Animals; Atractyloside; Female; Glucose; Glycosides; In Vitro Techniques; Kinetics; Liver; Oxidative Phosphorylation; Oxygen Consumption; Rats; Urea

The procedure for the isolation from mitochondria of the undenatured ADP, ATP carrier is described. The condition of retaining the nativity are elaborated. 1. As indicator for the ADP, ATP carrier (35S)- or (3H) carboxyatractylate were used. By preloading the mitochondria with carboxyatractylate, a stable carboxyatractylate . protein complex could be retained after solubilization with Triton X-100. Among the polyoxyethylene detergents emulphogen is also solubilizing, whereas Brij and Lubrol fail to solubilize. 2. When unloaded mitochondria are solubilized the capacity for binding carboxyatractylate disappears rapidly, particularly at 20 degrees C. 3. When mitochondria are preloaded with atractylate, the binding after solubilization with Triton X-100 is considerably lower than with carboxyatractylate, indicating that the high affinity of carboxyatractylate is required for effectively protecting the protein. 4. For purification hydroxyapatite is most effective. The carboxyatractylate-protein complex appears in the pass-through whereas the bulk of other mitochondrial proteins are retained such that a 7-fold purification is obtained. The nonadsorptivity to hydroxyapatite is dependent on the undenatured state maintained in the carboxyatractylate . protein complex. 5. Subsequent gel filtration on Sepharose results in a 1.5-fold further enrichment of specific carboxyatractylate binding up to 17 mumol/g protein, corresponding to a 10-fold purification from mitochondria. This value cannot be increased with further measures. 6. At the last purification step, in sodium dodecyl sulfate polyacrylamide gel electrophoresis virtually a single band of 30 000 molecular weight is found, confirming the purity at this stage. A molecular weight of 60 000 is calculated from the carboxyatractylate binding, indicating that the carboxyatractylate protein complex consists of two 30 000 subunits. From this the protein share of the ADP, ATP carrier in beef heart mitochondria can be calculated to amount to 9.5%9 7. The intact carboxyatractylate . protein complex is protected against proteolytic degradation. The release of carboxyatractylate ensues a conformational change of protein as assayed by conformation specific antibodies, concomitant with unmasking of proteolytic site as assayed by tryptic digestion. 8. The amino acid composition indicates hydrophobicity (39% polarity) and a high content of basic amino acid such as lysine and arginine. There is 1.5 mol percent cysteine and a bl

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Amino Acids; Animals; Atractyloside; Cattle; Detergents; Glycosides; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases; Protein Conformation

Topics: Adenosine Diphosphate; Animals; Atractyloside; Bongkrekic Acid; Cattle; Glycosides; Hydrogen-Ion Concentration; Membranes; Mitochondria, Liver; Mitochondria, Muscle; Myocardium; Oxygen Consumption; Rats; Structure-Activity Relationship

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Atractyloside; Biological Transport; Carbon Isotopes; Carboxylic Acids; Chromatography, Paper; Electrophoresis, Paper; Glycosides; Kinetics; Membranes; Mitochondria; Mitochondria, Liver; Models, Structural; Oxidative Phosphorylation; Phenanthrenes; Plant Cells; Plants; Protein Binding; Rats; Receptors, Drug; Sulfur Isotopes