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Theo Wallimann - One of the best experts on this subject based on the ideXlab platform.
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development and performance of an enzyme immunoassay to detect Creatine Kinase isoenzyme mb activity using anti Mitochondrial Creatine Kinase monoclonal antibodies
Scandinavian Journal of Clinical & Laboratory Investigation, 2009Co-Authors: Tadashi Hoshino, Theo Wallimann, Uwe Schlattner, Yasuhiro Sakai, Kazuaki Yamashita, Yasushi Shirahase, Kouji Sakaguchi, Ayumi Asaeda, Kouji Kishi, Mitsuru YanaiAbstract:Objective: The MB fraction of Creatine Kinase (CK-MB) has long been used as a cardiac marker. It is known that the CK-MB immunoinhibition method lacks selectivity and accuracy, because the appearan...
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Progressive decrease of phosphoCreatine, Creatine and Creatine Kinase in skeletal muscle upon transformation to sarcoma.
FEBS Journal, 2008Co-Authors: S. Patra, Uwe Schlattner, Soumen Bera, Soumya Sinharoy, Sarani Ghoshal, Abhimanyu Basu, Theo WallimannAbstract:In vertebrates, phosphoCreatine and ATP are continuously interconverted by the reversible reaction of Creatine Kinase in accordance with cellular energy needs. Sarcoma tissue and its normal counterpart, Creatine-rich skeletal muscle, are good source materials to study the status of Creatine and Creatine Kinase with the progression of malignancy. We experimentally induced sarcoma in mouse leg muscle by injecting either 3-methylcholanthrene or live sarcoma 180 cells into one hind leg. Creatine, phosphoCreatine and Creatine Kinase isoform levels decreased as malignancy progressed and reached very low levels in the final stage of sarcoma development; all these parameters remained unaltered in the unaffected contralateral leg muscle of the same animal. Creatine and Creatine Kinase levels were also reduced significantly in frank malignant portions of human sarcoma and gastric and colonic adenocarcinoma compared with the distal nonmalignant portions of the same samples. In mice, immunoblotting with antibodies against cytosolic muscle-type Creatine Kinase and sarcomeric Mitochondrial Creatine Kinase showed that both of these isoforms decreased as malignancy progressed. Expressions of mRNA of muscle-type Creatine Kinase and sarcomeric Mitochondrial Creatine Kinase were also severely downregulated. In human sarcoma these two isoforms were undetectable also. In human gastric and colonic adenocarcinoma, brain-type Creatine Kinase was found to be downregulated, whereas ubiquitous Mitochondrial Creatine Kinase was upregulated. These significantly decreased levels of Creatine and Creatine Kinase isoforms in sarcoma suggest that: (a) the genuine muscle phenotype is lost during sarcoma progression, and (b) these parameters may be used as diagnostic marker and prognostic indicator of malignancy in this tissue.
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Novel lipid transfer property of two Mitochondrial proteins that bridge the inner and outer membranes.
Biophysical Journal, 2007Co-Authors: Theo Wallimann, Uwe Schlattner, Marie-lise Lacombe, Raquel EpandAbstract:This study provides evidence of a novel function for Mitochondrial Creatine Kinase (MtCK) and nucleoside diphosphate Kinase (NDPK-D). Both are basic peripheral membrane proteins with symmetrical homo-oligomeric structure, which in the case of MtCK was already shown to allow crossbridging of lipid bilayers. Here, different lipid dilution assays clearly demonstrate that both Kinases also facilitate lipid transfer from one bilayer to another. Lipid transfer occurs between liposomes mimicking the lipid composition of Mitochondrial contact sites, containing 30 mol % cardiolipin, but transfer does not occur when cardiolipin is replaced by phosphatidylglycerol. Ubiquitous MtCK, but not NDPK-D, shows some specificity in the nature of the lipids transferred and it is not active with phosphatidylcholine alone. MtCK can undergo reversible oligomerization between dimeric and octameric forms, but only the octamer can bridge membranes and promote lipid transfer. Cytochrome c, another basic Mitochondrial protein known to bind to anionic membranes but not crosslinking them, is also incapable of promoting lipid transfer. The lipid transfer process does not involve vesicle fusion or loss of the internal contents of the liposomes.
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inhibition of the Mitochondrial permeability transition by Creatine Kinase substrates requirement for microcompartmentation
Journal of Biological Chemistry, 2003Co-Authors: Max Dolder, Oliver Speer, Uwe Schlattner, Bernd Walzel, Theo WallimannAbstract:Abstract Mitochondria from transgenic mice, expressing enzymatically active Mitochondrial Creatine Kinase in liver, were analyzed for opening of the permeability transition pore in the absence and presence of Creatine Kinase substrates but with no external adenine nucleotides added. In mitochondria from these transgenic mice, cyclosporin A-inhibited pore opening was delayed by Creatine or cycloCreatine but not by β-guanidinopropionic acid. This observation correlated with the ability of these substrates to stimulate state 3 respiration in the presence of extraMitochondrial ATP. The dependence of transition pore opening on calcium and magnesium concentration was studied in the presence and absence of Creatine. If Mitochondrial Creatine Kinase activity decreased (i.e. by omitting magnesium from the medium), protection of permeability transition pore opening by Creatine or cycloCreatine was no longer seen. Likewise, when Creatine Kinase was added externally to liver mitochondria from wild-type mice that do not express Mitochondrial Creatine Kinase in liver, no protective effect on pore opening by Creatine and its analog was observed. All these findings indicate that Mitochondrial Creatine Kinase activity located within the intermembrane and intercristae space, in conjunction with its tight functional coupling to oxidative phosphorylation, via the adenine nucleotide translocase, can modulate Mitochondrial permeability transition in the presence of Creatine. These results are of relevance for the design of Creatine analogs for cell protection as potential adjuvant therapeutic tools against neurodegenerative diseases.
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Mitochondrial Creatine Kinase and Mitochondrial outer membrane porin show a direct interaction that is modulated by calcium
Journal of Biological Chemistry, 2001Co-Authors: Uwe Schlattner, Max Dolder, Theo Wallimann, Malgorzata TokarskaschlattnerAbstract:Mitochondrial Creatine Kinase (MtCK) co-localizes with Mitochondrial porin (voltage-dependent anion channel) and adenine nucleotide translocator in Mitochondrial contact sites. A specific, direct protein-protein interaction between MtCK and Mitochondrial porin was demonstrated using surface plasmon resonance spectroscopy. This interaction was independent of the immobilized binding partner (porin reconstituted in liposomes or MtCK) or the analyzed isoform (chicken sarcomeric MtCK or human ubiquitous MtCK, human recombinant porin, or purified bovine porin). Increased ionic strength reduced the binding of MtCK to porin, suggesting predominantly ionic interactions. By contrast, micromolar concentrations of Ca(2+) increased the amount of bound MtCK, indicating a physiological regulation of complex formation. No interaction of MtCK with reconstituted adenine nucleotide translocator was detectable in our experimental setup. The relevance of these findings for structure and function of Mitochondrial contact sites is discussed.
Valdur Saks - One of the best experts on this subject based on the ideXlab platform.
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cytoskeleton and regulation of Mitochondrial function the role of beta tubulin ii
Frontiers in Physiology, 2013Co-Authors: Andrey V Kuznetsov, Rita Guzun, Sabzali Javadov, M Grimm, Valdur SaksAbstract:The control of Mitochondrial function is a cardinal issue in the field of cardiac bioenergetics, and the analysis of Mitochondrial regulations is central to basic research and in the diagnosis of many diseases. Interaction between cytoskeletal proteins and mitochondria can actively participate in Mitochondrial regulation. Potential candidates for the key roles in this regulation are the cytoskeletal proteins plectin and tubulin. Analysis of cardiac cells has revealed regular arrangement of β-tubulin II, fully co-localized with mitochondria. β-Tubulin IV demonstrated a characteristic staining of branched network, β-tubulin III was matched with Z-lines, and β-tubulin I was diffusely spotted and fragmentary polymerized. In contrast, HL-1 cells were characterized by the complete absence of β-tubulin II. Comparative analysis of cardiomyocytes and HL-1 cells revealed a dramatic difference in the mechanisms of Mitochondrial regulation. In the heart, colocalization of β-tubulin isotype II with mitochondria suggests that it can participate in the coupling of ATP-ADP translocase (ANT), Mitochondrial Creatine Kinase (MtCK), and VDAC (ANT-MtCK-VDAC). This Mitochondrial supercomplex is responsible for the efficient intracellular energy transfer via the phosphoCreatine pathway. Existing data suggest that cytoskeletal proteins may control the VDAC, contributing to maintenance of Mitochondrial and cellular physiology.
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regulation of respiration in permeabilized muscle cells apparent km for adp shows the Mitochondrial outer membrane permeability
Biophysical Journal, 2013Co-Authors: Rafaela Bagur Quetglas, Valdur Saks, K. Tepp, T. Kaambre, Minna Karuvarikmaa, Madis Metsis, Alexei Grichine, Francois BoucherAbstract:The aim of this work was to study the regulation of respiration and energy fluxes in permeabilized oxidative and glycolytic skeletal muscle fibers. Despite certain similarities of Mitochondrial organization into Intracellular Energetic Units (ICEUs), different muscle types have distinct metabolic pattern with specific regulatory mechanisms of Mitochondrial respiration. In this work we measured ADP fluxes through Mitochondrial outer membrane (MOM) in permeabilized fibers oxygraphically using pyruvate Kinase - phosphoenolpyruvate system for trapping ADP produced in Mitochondrial Creatine Kinase localized behind MOM. These fluxes were high in permeabilized fibers from glycolytic muscles and very low or absent in soleus muscle fibers and cardiac myocytes. These results indicate that the permeability of voltage-dependent anion channel (VDAC) in MOM correlates with the apparent Km for ADP in regulation of respiration. In cardiac cells, the MOM permeability seems to be regulated by the interaction of VDAC with cytoskeletal protein βII tubulin. To ascertain the role of this protein in skeletal muscles we visualized the localization pattern of βII tubulin together with Mitochondrial protein VDAC immunocytochemically. We used the Metabolic Control Analysis to evaluate the Flux Control Coefficients of the respiratory chain complexes (I,III, IV), ANT, ATP synthase, and MtCK in permeabilized soleus muscle fibers under conditions of respiration stimulated by exogenous ADP and by endogenous ADP produced in activated MtCK reaction.
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Systems bioenergetics of Creatine Kinase networks: physiological roles of Creatine and phosphoCreatine in regulation of cardiac cell function
Amino Acids, 2011Co-Authors: Rita Guzun, N. Timohhina, K. Tepp, M. Gonzalez-granillo, I. Shevchuk, V. Chekulayev, A. V. Kuznetsov, T. Kaambre, Valdur SaksAbstract:Physiological role of Creatine (Cr) became first evident in the experiments of Belitzer and Tsybakova in 1939, who showed that oxygen consumption in a well-washed skeletal muscle homogenate increases strongly in the presence of Creatine and with this results in phosphoCreatine (PCr) production with PCr/O_2 ratio of about 5–6. This was the beginning of quantitative analysis in bioenergetics. It was also observed in many physiological experiments that the contractile force changes in parallel with the alteration in the PCr content. On the other hand, it was shown that when heart function is governed by Frank–Starling law, work performance and oxygen consumption rate increase in parallel without any changes in PCr and ATP tissue contents (metabolic homeostasis). Studies of cellular mechanisms of all these important phenomena helped in shaping new approach to bioenergetics, Molecular System Bioenergetics, a part of Systems Biology. This approach takes into consideration intracellular interactions that lead to novel mechanisms of regulation of energy fluxes. In particular, interactions between mitochondria and cytoskeleton resulting in selective restriction of permeability of outer Mitochondrial membrane anion channel (VDAC) for adenine nucleotides and thus their recycling in mitochondria coupled to effective synthesis of PCr by Mitochondrial Creatine Kinase, MtCK. Therefore, Cr concentration and the PCr/Cr ratio became important kinetic parameters in the regulation of respiration and energy fluxes in muscle cells. Decrease in the intracellular contents of Cr and PCr results in a hypodynamic state of muscle and muscle pathology. Many experimental studies have revealed that PCr may play two important roles in the regulation of muscle energetics: first by maintaining local ATP pools via compartmentalized Creatine Kinase reactions, and secondly by stabilizing cellular membranes due to electrostatic interactions with phospholipids. The second mechanism decreases the production of lysophosphoglycerides in hypoxic heart, protects the cardiac cells sarcolemma against ischemic damage, decreases the frequency of arrhythmias and increases the post-ischemic recovery of contractile function. PCr is used as a pharmacological product Neoton in cardiac surgery as one of the components of cardioplegic solutions for protection of the heart against intraoperational injury and injected intravenously in acute myocardial ischemic conditions for improving the hemodynamic response and clinical conditions of patients with heart failure.
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structure function relationships in feedback regulation of energy fluxes in vivo in health and disease Mitochondrial interactosome
Biochimica et Biophysica Acta, 2010Co-Authors: Valdur Saks, Rita Guzun, N. Timohhina, K. Tepp, T. Kaambre, Andrey V Kuznetsov, Minna Varikmaa, Claire Monge, Nathalie Beraud, Lumme KadajaAbstract:The aim of this review is to analyze the results of experimental research of mechanisms of regulation of Mitochondrial respiration in cardiac and skeletal muscle cells in vivo obtained by using the permeabilized cell technique. Such an analysis in the framework of Molecular Systems Bioenergetics shows that the mechanisms of regulation of energy fluxes depend on the structural organization of the cells and interaction of mitochondria with cytoskeletal elements. Two types of cells of cardiac phenotype with very different structures were analyzed: adult cardiomyocytes and continuously dividing cancerous HL-1 cells. In cardiomyocytes mitochondria are arranged very regularly, and show rapid configuration changes of inner membrane but no fusion or fission, diffusion of ADP and ATP is restricted mostly at the level of Mitochondrial outer membrane due to an interaction of heterodimeric tubulin with voltage dependent anion channel, VDAC. VDAC with associated tubulin forms a supercomplex, Mitochondrial Interactosome, with Mitochondrial Creatine Kinase, MtCK, which is structurally and functionally coupled to ATP synthasome. Due to selectively limited permeability of VDAC for adenine nucleotides, Mitochondrial respiration rate depends almost linearly upon the changes of cytoplasmic ADP concentration in their physiological range. Functional coupling of MtCK with ATP synthasome amplifies this signal by recycling adenine nucleotides in mitochondria coupled to effective phosphoCreatine synthesis. In cancerous HL-1 cells this complex is significantly modified: tubulin is replaced by hexoKinase and MtCK is lacking, resulting in direct utilization of Mitochondrial ATP for glycolytic lactate production and in this way contributing in the mechanism of the Warburg effect. Systemic analysis of changes in the integrated system of energy metabolism is also helpful for better understanding of pathogenesis of many other diseases.
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Functional coupling of adenine nucleotide translocase and Mitochondrial Creatine Kinase is enhanced after exercise training in lung transplant skeletal muscle.
AJP - Regulatory Integrative and Comparative Physiology, 2005Co-Authors: Karen Guerrero, Marko Vendelin, Bernard Wuyam, Paulette Mezin, Isabelle Vivodtzev, Jean-christian Borel, Rachid Hacini, Olivier Chavanon, Sandrine Imbeaud, Valdur SaksAbstract:Mechanisms responsible for limitation of exercise capacity in lung transplant recipients (LR) and benefits gained by exercise training were studied. Mitochondrial respiration parameters, energy transfer, and cell structure were assessed in vastus lateralis biopsies using the permeabilized fiber technique with histochemical and morphometric measurements. Twelve male controls (C) and 12 LR performed exercise training over 12 wk. Before exercise training, there were strong correlations between exercise capacity (maximal O(2) consumption and endurance time at 70% maximal power output) and cellular events, as assessed by percentage of type I fibers and apparent K(m) for exogenous ADP. Anticalcineurins were not involved in LR exercise limitation, since there were no differences in maximal Mitochondrial rate of respiration before exercise training and no abnormalities in respiratory chain complexes compared with C. Training resulted in a significant increase in physiological parameters both at the cellular (apparent K(m) for exogenous ADP and stimulating effect of Creatine) and integrated (maximal O(2) consumption, power output at ventilatory threshold, maximal power output, and endurance time at 70% maximal power output) levels in LR and C. After the training period, improvements in maximal O(2) consumption and in maximal Mitochondrial rate of respiration were noted, as well as changes in endurance time and percentage of type I fibers. Because there were no changes in diameters and fiber types, baseline alteration of apparent K(m) for exogenous ADP and its improvement after training might be related to changes within the intracellular energetic units. After the training period, intracellular energetic units exhibited a higher control of Mitochondrial respiration by Creatine linked to a more efficient functional coupling adenine nucleotide translocase-Mitochondrial Creatine Kinase, resulting in better exercise performances in C and LR.
Dieter Brdiczka - One of the best experts on this subject based on the ideXlab platform.
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the function of complexes between the outer Mitochondrial membrane pore vdac and the adenine nucleotide translocase in regulation of energy metabolism and apoptosis
Acta Biochimica Polonica, 2003Co-Authors: Mikhail Yu Vyssokikh, Dieter BrdiczkaAbstract:: The outer Mitochondrial membrane pore (VDAC) changes its structure either voltage-dependently in artificial membranes or physiologically by interaction with the adenine nucleotide translocase (ANT) in the c-conformation. This interaction creates contact sites and leads in addition to a specific organisation of cytochrome c in the VDAC-ANT complexes. The VDAC structure that is specific for contact sites generates a signal at the surface for several proteins in the cytosol to bind with high capacity, such as hexoKinase, glycerol Kinase and Bax. If the VDAC binding site is not occupied by hexoKinase, the VDAC-ANT complex has two critical qualities: firstly, Bax gets access to cytochrome c and secondly the ANT is set in its c-conformation that easily changes conformation into an unspecific channel (uniporter) causing permeability transition. Activity of bound hexoKinase protects against both, it hinders Bax binding and employs the ANT as anti-porter. The octamer of Mitochondrial Creatine Kinase binds to VDAC from the inner surface of the outer membrane. This firstly restrains interaction between VDAC and ANT and secondly changes the VDAC structure into low affinity for hexoKinase and Bax. Cytochrome c in the Creatine Kinase complex will be differently organised, not accessible to Bax and the ANT is run as anti-porter by the active Creatine Kinase octamer. However, when, for example, free radicals cause dissociation of the octamer, VDAC interacts with the ANT with the same results as described above: Bax-dependent cytochrome c release and risk of permeability transition pore opening.
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complexes between porin hexoKinase Mitochondrial Creatine Kinase and adenylate translocator display properties of the permeability transition pore implication for regulation of permeability transition by the Kinases
Biochimica et Biophysica Acta, 1998Co-Authors: Giesela Beutner, Alexander Ruck, Birgit Riede, Dieter BrdiczkaAbstract:Complexes between hexoKinase, outer membrane porin, and the adenylate translocator ANT were recently found to establish properties of the Mitochondrial permeability transition pore in a reconstituted system. The complex was extracted by 0.5% Triton X-100 from rat brain membranes and separated by anion exchanger chromatography. The molecular weight . was approximately 400 kDa suggesting tetramers of hexoKinase monomer 100 kDa . By the same method a porin, Creatine Kinase octamer, ANT complex was isolated and reconstituted in liposomes. Vesicles containing the reconstituted complexes both retained ATP that could be used by either Kinase to phosphorylate external Creatine or glucose. Atractyloside inhibited wx this activity indicating that the ANT was involved in this process and was functionally reconstituted 1 . Exclusively from the hexoKinase complex containing liposome internal malate or ATP was released by addition of Ca 2q in a N-methylVal- 4-cyclosporin sensitive way, suggesting that the hexoKinase porin ANT complex might include the permeability transition . 2q . pore PTP . The Ca dependent opening of the PTP-like structure was inhibited by ADP apparent I ,8 mM and ATP 50 .apparent I ,8 4mM . Also glucose inhibited the PTP-like activity, while glucose-6-phosphate abolished this effect. 50 Although porin and ANT were functionally active in vesicles containing the Creatine Kinase octamer complex ,C a 2q did not induce a release of internal substrates. However, after dissociation of the Creatine Kinase octamer, the complex exhibited PTP-like properties and the vesicles liberated internal metabolites upon addition of Ca 2q . The latter process was also . inhibited by N-methylVal-4-cyclosporin. The activity of peptidyl-prolyl- cis-trans-isomerase representing cyclophilin was followed during complex isolation. Cyp D was co-purified with the hexoKinase complex, while it was absent in the Creatine Kinase complex. The inhibitory effect of N-methylVal-4-cyclosporin on the Creatine Kinase complex may be explained by direct interaction with the Creatine Kinase dimer that appeared to support octamer formation. q 1998 Elsevier Science B.V.
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complexes between porin hexoKinase Mitochondrial Creatine Kinase and adenylate translocator display properties of the permeability transition pore implication for regulation of permeability transition by the Kinases
Biochimica et Biophysica Acta, 1998Co-Authors: Giesela Beutner, Alexander Ruck, Birgit Riede, Dieter BrdiczkaAbstract:Complexes between hexoKinase, outer membrane porin, and the adenylate translocator ANT were recently found to establish properties of the Mitochondrial permeability transition pore in a reconstituted system. The complex was extracted by 0.5% Triton X-100 from rat brain membranes and separated by anion exchanger chromatography. The molecular weight . was approximately 400 kDa suggesting tetramers of hexoKinase monomer 100 kDa . By the same method a porin, Creatine Kinase octamer, ANT complex was isolated and reconstituted in liposomes. Vesicles containing the reconstituted complexes both retained ATP that could be used by either Kinase to phosphorylate external Creatine or glucose. Atractyloside inhibited wx this activity indicating that the ANT was involved in this process and was functionally reconstituted 1 . Exclusively from the hexoKinase complex containing liposome internal malate or ATP was released by addition of Ca 2q in a N-methylVal- 4-cyclosporin sensitive way, suggesting that the hexoKinase porin ANT complex might include the permeability transition . 2q . pore PTP . The Ca dependent opening of the PTP-like structure was inhibited by ADP apparent I ,8 mM and ATP 50 .apparent I ,8 4mM . Also glucose inhibited the PTP-like activity, while glucose-6-phosphate abolished this effect. 50 Although porin and ANT were functionally active in vesicles containing the Creatine Kinase octamer complex ,C a 2q did not induce a release of internal substrates. However, after dissociation of the Creatine Kinase octamer, the complex exhibited PTP-like properties and the vesicles liberated internal metabolites upon addition of Ca 2q . The latter process was also . inhibited by N-methylVal-4-cyclosporin. The activity of peptidyl-prolyl- cis-trans-isomerase representing cyclophilin was followed during complex isolation. Cyp D was co-purified with the hexoKinase complex, while it was absent in the Creatine Kinase complex. The inhibitory effect of N-methylVal-4-cyclosporin on the Creatine Kinase complex may be explained by direct interaction with the Creatine Kinase dimer that appeared to support octamer formation. q 1998 Elsevier Science B.V.
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the role of Creatine Kinase in inhibition of Mitochondrial permeability transition
FEBS Letters, 1997Co-Authors: Eddie Ogorman, Giesela Beutner, Max Dolder, Dieter Brdiczka, Alan P Koretsky, Theo WallimannAbstract:Cyciosporin A sensitive swelling of mitochondria isolated from control mouse livers and from the livers of transgenic mice expressing human ubiquitous Mitochondrial Creatine Kinase occurred in the presence of both 40 gM calcium and 5 gM atractyloside which was accompanied by a 2.5-fold increase over state 4 respiration rates. Creatine and cyclocrea- tine inhibited the latter only in transgenic liver mitochondria. Protein complexes isolated from detergent solubilised rat brain extracts, containing octameric Mitochondrial Creatine Kinase, porin and the adenine nucleotide translocator, were reconstituted into malate loaded lipid vesicles. Dimerisation of Creatine Kinase in the complexes and exposure of the reconstituted complexes to 200 gM calcium induced a cyclosporin A sensitive malate release. No malate release occurred with complexes containing octameric Creatine Kinase under the same conditions. © 1997 Federation of European Biochemical Societies.
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complexes between Kinases Mitochondrial porin and adenylate translocator in rat brain resemble the permeability transition pore
FEBS Letters, 1996Co-Authors: Giesela Beutner, Alexander Ruck, Birgit Riede, Wolfram Welte, Dieter BrdiczkaAbstract:In vitro incubation of isolated hexoKinase isozyme I or isolated dimer of Mitochondrial Creatine Kinase with the outer Mitochondrial membrane pore led to high molecular weight complexes of enzyme oligomers. Similar complexes of hexoKinase and Mitochondrial Creatine Kinase could be extracted by 0.5% Triton X-100 from homogenates of rat brain. HexoKinase and Creatine Kinase complexes could be separated by subsequent chromatography on DEAE anion exchanger. The molecular weight, as determined by gel-permeation chromatography, was approximately 400 kDa for both complexes. The Mr suggested tetramers of hexoKinase (monomer 100 kDa) and Creatine Kinase (active enzyme is a dimer of 80 kDa). The composition of the complexes was further characterised by specific antibodies. Besides either hexoKinase or Creatine Kinase molecules the complexes contained porin and adenylate translocator. It was possible to incorporate the complexes into artificial bilayer membranes and to measure conductance in 1 M KCl. The incorporating channels had a high conductance of 6 nS that was asymmetrically voltage dependent. The complexes were also reconstituted in phospholipid vesicles that were loaded with ATP. Complex containing vesicles retained ATP while vesicles reconstituted with pure porin were leaky. The internal ATP could be used by Creatine Kinase and hexoKinase in the complex to phosphorylate external Creatine or glucose. This process was inhibited by atractyloside. The hexoKinase complex containing vesicles were furthermore loaded with malate or ATP that was gradually released by addition of Ca2+ between 100 and 600 μM. The liberation of malate or ATP by Ca2+ could be inhibited by N-methylVal-4-cyclosporin, suggesting that the porin translocator complex constitutes the permeability transition pore. The results show the physiological existence of Kinase porin translocator complexes at the Mitochondrial surface. It is assumed that such complexes between inner and outer membrane components are the molecular basis of contact sites observed by electron microscopy. Kinase complex formation may serve three regulatory functions, firstly regulation of the Kinase activity, secondly stimulation of oxidative phosphorylation and thirdly regulation of the permeability transition pore.
Uwe Schlattner - One of the best experts on this subject based on the ideXlab platform.
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modelling in vivo Creatine phosphoCreatine in vitro reveals divergent adaptations in human muscle Mitochondrial respiratory control by adp after acute and chronic exercise
The Journal of Physiology, 2016Co-Authors: Mia Ydfors, Uwe Schlattner, Meghan C Hughes, Robert Laham, Jessica Norrbom, Christopher G R PerryAbstract:Key points Mitochondrial respiratory sensitivity to ADP is thought to influence muscle fitness and is partly regulated by cytosolic–Mitochondrial diffusion of ADP or phosphate shuttling via Creatine/phosphoCreatine (Cr/PCr) through Mitochondrial Creatine Kinase (mtCK). Previous measurements of respiration in vitro with Cr (saturate mtCK) or without (ADP/ATP diffusion) show mixed responses of ADP sensitivity following acute exercise vs. less sensitivity after chronic exercise. In human muscle, modelling in vivo ‘exercising’ [Cr:PCr] during in vitro assessments revealed novel responses to exercise that differ from detections with or without Cr (±Cr). Acute exercise increased ADP sensitivity when measured without Cr but had no effect ±Cr or with +Cr:PCr, whereas chronic exercise increased sensitivity ±Cr but lowered sensitivity with +Cr:PCr despite increased markers of Mitochondrial oxidative capacity. Controlling in vivo conditions during in vitro respiratory assessments reveals responses to exercise that differ from typical ±Cr comparisons and challenges our understanding of how exercise improves metabolic control in human muscle. Abstract Mitochondrial respiratory control by ADP (Kmapp) is viewed as a critical regulator of muscle energy homeostasis. However, acute exercise increases, decreases or has no effect on Kmapp in human muscle, whereas chronic exercise surprisingly decreases sensitivity despite greater Mitochondrial content. We hypothesized that modelling in vivo Mitochondrial Creatine Kinase (mtCK)-dependent phosphate-shuttling conditions in vitro would reveal increased sensitivity (lower Kmapp) after acute and chronic exercise. The Kmapp was determined in vitro with 20 mm Cr (+Cr), 0 mm Cr (−Cr) or ‘in vivo exercising’ 20 mm Cr/2.4 mm PCr (Cr:PCr) on vastus lateralis biopsies sampled from 11 men before, immediately after and 3 h after exercise on the first, fifth and ninth sessions over 3 weeks. Dynamic responses to acute exercise occurred throughout training, whereby the first session did not change Kmapp with in vivo Cr:PCr despite increases in −Cr. The fifth session decreased sensitivity with Cr:PCr or +Cr despite no change in −Cr. Chronic exercise increased sensitivity ±Cr in association with increased electron transport chain content (+33–62% complexes I–V), supporting classic proposals that link increased sensitivity to oxidative capacity. However, in vivo Cr:PCr reveals a perplexing decreased sensitivity, contrasting the increases seen ±Cr. Functional responses occurred without changes in fibre type or proteins regulating Mitochondrial–cytosolic energy exchange (mtCK, VDAC and ANT). Despite the dynamic responses seen with ±Cr, modelling in vivo phosphate-shuttling conditions in vitro reveals that ADP sensitivity is unchanged after high-intensity exercise and is decreased after training. These findings challenge our understanding of how exercise regulates skeletal muscle energy homeostasis.
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development and performance of an enzyme immunoassay to detect Creatine Kinase isoenzyme mb activity using anti Mitochondrial Creatine Kinase monoclonal antibodies
Scandinavian Journal of Clinical & Laboratory Investigation, 2009Co-Authors: Tadashi Hoshino, Theo Wallimann, Uwe Schlattner, Yasuhiro Sakai, Kazuaki Yamashita, Yasushi Shirahase, Kouji Sakaguchi, Ayumi Asaeda, Kouji Kishi, Mitsuru YanaiAbstract:Objective: The MB fraction of Creatine Kinase (CK-MB) has long been used as a cardiac marker. It is known that the CK-MB immunoinhibition method lacks selectivity and accuracy, because the appearan...
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Progressive decrease of phosphoCreatine, Creatine and Creatine Kinase in skeletal muscle upon transformation to sarcoma.
FEBS Journal, 2008Co-Authors: S. Patra, Uwe Schlattner, Soumen Bera, Soumya Sinharoy, Sarani Ghoshal, Abhimanyu Basu, Theo WallimannAbstract:In vertebrates, phosphoCreatine and ATP are continuously interconverted by the reversible reaction of Creatine Kinase in accordance with cellular energy needs. Sarcoma tissue and its normal counterpart, Creatine-rich skeletal muscle, are good source materials to study the status of Creatine and Creatine Kinase with the progression of malignancy. We experimentally induced sarcoma in mouse leg muscle by injecting either 3-methylcholanthrene or live sarcoma 180 cells into one hind leg. Creatine, phosphoCreatine and Creatine Kinase isoform levels decreased as malignancy progressed and reached very low levels in the final stage of sarcoma development; all these parameters remained unaltered in the unaffected contralateral leg muscle of the same animal. Creatine and Creatine Kinase levels were also reduced significantly in frank malignant portions of human sarcoma and gastric and colonic adenocarcinoma compared with the distal nonmalignant portions of the same samples. In mice, immunoblotting with antibodies against cytosolic muscle-type Creatine Kinase and sarcomeric Mitochondrial Creatine Kinase showed that both of these isoforms decreased as malignancy progressed. Expressions of mRNA of muscle-type Creatine Kinase and sarcomeric Mitochondrial Creatine Kinase were also severely downregulated. In human sarcoma these two isoforms were undetectable also. In human gastric and colonic adenocarcinoma, brain-type Creatine Kinase was found to be downregulated, whereas ubiquitous Mitochondrial Creatine Kinase was upregulated. These significantly decreased levels of Creatine and Creatine Kinase isoforms in sarcoma suggest that: (a) the genuine muscle phenotype is lost during sarcoma progression, and (b) these parameters may be used as diagnostic marker and prognostic indicator of malignancy in this tissue.
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Novel lipid transfer property of two Mitochondrial proteins that bridge the inner and outer membranes.
Biophysical Journal, 2007Co-Authors: Theo Wallimann, Uwe Schlattner, Marie-lise Lacombe, Raquel EpandAbstract:This study provides evidence of a novel function for Mitochondrial Creatine Kinase (MtCK) and nucleoside diphosphate Kinase (NDPK-D). Both are basic peripheral membrane proteins with symmetrical homo-oligomeric structure, which in the case of MtCK was already shown to allow crossbridging of lipid bilayers. Here, different lipid dilution assays clearly demonstrate that both Kinases also facilitate lipid transfer from one bilayer to another. Lipid transfer occurs between liposomes mimicking the lipid composition of Mitochondrial contact sites, containing 30 mol % cardiolipin, but transfer does not occur when cardiolipin is replaced by phosphatidylglycerol. Ubiquitous MtCK, but not NDPK-D, shows some specificity in the nature of the lipids transferred and it is not active with phosphatidylcholine alone. MtCK can undergo reversible oligomerization between dimeric and octameric forms, but only the octamer can bridge membranes and promote lipid transfer. Cytochrome c, another basic Mitochondrial protein known to bind to anionic membranes but not crosslinking them, is also incapable of promoting lipid transfer. The lipid transfer process does not involve vesicle fusion or loss of the internal contents of the liposomes.
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inhibition of the Mitochondrial permeability transition by Creatine Kinase substrates requirement for microcompartmentation
Journal of Biological Chemistry, 2003Co-Authors: Max Dolder, Oliver Speer, Uwe Schlattner, Bernd Walzel, Theo WallimannAbstract:Abstract Mitochondria from transgenic mice, expressing enzymatically active Mitochondrial Creatine Kinase in liver, were analyzed for opening of the permeability transition pore in the absence and presence of Creatine Kinase substrates but with no external adenine nucleotides added. In mitochondria from these transgenic mice, cyclosporin A-inhibited pore opening was delayed by Creatine or cycloCreatine but not by β-guanidinopropionic acid. This observation correlated with the ability of these substrates to stimulate state 3 respiration in the presence of extraMitochondrial ATP. The dependence of transition pore opening on calcium and magnesium concentration was studied in the presence and absence of Creatine. If Mitochondrial Creatine Kinase activity decreased (i.e. by omitting magnesium from the medium), protection of permeability transition pore opening by Creatine or cycloCreatine was no longer seen. Likewise, when Creatine Kinase was added externally to liver mitochondria from wild-type mice that do not express Mitochondrial Creatine Kinase in liver, no protective effect on pore opening by Creatine and its analog was observed. All these findings indicate that Mitochondrial Creatine Kinase activity located within the intermembrane and intercristae space, in conjunction with its tight functional coupling to oxidative phosphorylation, via the adenine nucleotide translocase, can modulate Mitochondrial permeability transition in the presence of Creatine. These results are of relevance for the design of Creatine analogs for cell protection as potential adjuvant therapeutic tools against neurodegenerative diseases.
Renee Venturaclapier - One of the best experts on this subject based on the ideXlab platform.
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cardiac and skeletal muscle energy metabolism in heart failure beneficial effects of voluntary activity
Cardiovascular Research, 2002Co-Authors: Elvira De Sousa, Patrick Lechene, Dominique Fortin, Benoit Banga Nguessan, Souad Belmadani, Xavier Bigard, Vladimir Veksler, Renee VenturaclapierAbstract:Objective: Mitochondrial function and metabolic profile of slow and fast skeletal muscles and cardiac muscle are altered in chronic heart failure (CHF), suggesting a generalized metabolic myopathy in this disease. The aim of this study was to investigate the potential beneficial effects of voluntary activity on cardiac and skeletal muscle energetics in heart failure. Methods: Heart failure was induced in rats by aortic stenosis. Four months after surgery, part of sham and CHF animals were randomly assigned to activity cages equipped with running wheels for 8 weeks or kept sedentary. Mitochondrial capacity and regulation were measured using saponin skinned fibers in left ventricle, slow and fast skeletal muscles, and metabolic and myosin profiles were established. Results: Despite four times lower performances of CHF rats, alterations in metabolic and myosin parameters (oxidative capacity, Mitochondrial enzymes, cytosolic and Mitochondrial Creatine Kinase, myosin heavy chains) observed in all muscles of CHF animals were almost fully restored in soleus muscle though unchanged in heart and fast skeletal muscles. Conclusions: These results show the powerful beneficial effect of physical activity specifically on active slow oxidative skeletal muscle in CHF, without the worsening of cardiac muscle metabolism.
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metabolic control and metabolic capacity two aspects of Creatine Kinase functioning in the cells
Biochimica et Biophysica Acta, 1996Co-Authors: Valdur Saks, Renee Venturaclapier, Mayis AlievAbstract:Abstract In this short review, the merits and limits of three theoretical concepts explaining the functional role of the Creatine Kinase system in muscle and brain cells are analysed. In addition to the usual concept of an energy buffer system and the recently proposed metabolic capacity theory (Sweeney, H.L. (1994) Med. Sci. Sports Exerc. 26, 30–36), it is proposed that coupled Creatine Kinase systems are involved in effective metabolic regulation of energy fluxes and oxidative phosphorylation, beside their energy transfer function. This aspect of the system is considered on the basis of metabolic control analysis. It is shown by using the results of mathematical modelling that, due to amplification of ADP fluxes from the cytoplasm by the mechanism of metabolic channelling, coupled Mitochondrial Creatine Kinase may exert a flux control coefficient significantly exceeding 1.
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ischaemic metabolic factors high inorganic phosphate and acidosis modulate Mitochondrial Creatine Kinase functional activity in skinned cardiac fibres
Journal of Molecular and Cellular Cardiology, 1994Co-Authors: Vladimir Veksler, Renee VenturaclapierAbstract:Saponin-skinned rat cardiac fibres were used to study the influence of ischaemic factors (high [Pi] and decreased pH) on functional activity of Mitochondrial Creatine Kinase (mi-CK) in situ by evaluation of the stimulation of respiration by Creatine. High (20 mM) [Pi] known to solubilize mi-CK, decreased this stimulation significantly, though mi-CK was still present in the Mitochondrial compartment. Acidosis (pH 6.6) increased the stimulation of respiration by Creatine at low [Pi], and prevented the decrease in mi-CK functional activity at high [Pi]. Thus, these two ischaemic factors act in opposite directions. The data obtained suggest that under physiological or pathological conditions, inorganic phosphate and protons, by changing the functional coupling between Creatine Kinase and translocase, may influence the distribution of energy fluxes through adenylate and Creatine Kinase systems in cardiac tissue. Presence of Creatine Kinase in Mitochondrial compartment is not alone sufficient for functional efficacy of the enzyme.