bongkrekic-acid and carboxyatractyloside

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: 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

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

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 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 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 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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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; Bongkrekic Acid; Cattle; Glycosides; Hydrogen-Ion Concentration; Membranes; Mitochondria, Liver; Mitochondria, Muscle; Myocardium; Oxygen Consumption; Rats; Structure-Activity Relationship