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The Journal of Biological Chemistry Jan 2024Oxidative phosphorylation, the combined activities of the electron transport chain (ETC) and ATP synthase, has emerged as a valuable target for antibiotics to treat...
Oxidative phosphorylation, the combined activities of the electron transport chain (ETC) and ATP synthase, has emerged as a valuable target for antibiotics to treat infection with Mycobacterium tuberculosis and related pathogens. In oxidative phosphorylation, the ETC establishes a transmembrane electrochemical proton gradient that powers ATP synthesis. Monitoring oxidative phosphorylation with luciferase-based detection of ATP synthesis or measurement of oxygen consumption can be technically challenging and expensive. These limitations reduce the utility of these methods for characterization of mycobacterial oxidative phosphorylation inhibitors. Here, we show that fluorescence-based measurement of acidification of inverted membrane vesicles (IMVs) can detect and distinguish between inhibition of the ETC, inhibition of ATP synthase, and nonspecific membrane uncoupling. In this assay, IMVs from Mycobacterium smegmatis are acidified either through the activity of the ETC or ATP synthase, the latter modified genetically to allow it to serve as an ATP-driven proton pump. Acidification is monitored by fluorescence from 9-amino-6-chloro-2-methoxyacridine, which accumulates and quenches in acidified IMVs. Nonspecific membrane uncouplers prevent both succinate- and ATP-driven IMV acidification. In contrast, the ETC Complex IIIIV inhibitor telacebec (Q203) prevents succinate-driven acidification but not ATP-driven acidification, and the ATP synthase inhibitor bedaquiline prevents ATP-driven acidification but not succinate-driven acidification. We use the assay to show that, as proposed previously, lansoprazole sulfide is an inhibitor of Complex IIIIV, whereas thioridazine uncouples the mycobacterial membrane nonspecifically. Overall, the assay is simple, low cost, and scalable, which will make it useful for identifying and characterizing new mycobacterial oxidative phosphorylation inhibitors.
Topics: Adenosine Triphosphate; Electron Transport Complex III; Mycobacterium tuberculosis; Oxidative Phosphorylation; Anti-Bacterial Agents; Drug Discovery
PubMed: 37992805
DOI: 10.1016/j.jbc.2023.105483 -
PloS One 2015Platelet thrombus formation includes several integrated processes involving aggregation, secretion of granules, release of arachidonic acid and clot retraction, but it...
Platelet thrombus formation includes several integrated processes involving aggregation, secretion of granules, release of arachidonic acid and clot retraction, but it is not clear which metabolic fuels are required to support these events. We hypothesized that there is flexibility in the fuels that can be utilized to serve the energetic and metabolic needs for resting and thrombin-dependent platelet aggregation. Using platelets from healthy human donors, we found that there was a rapid thrombin-dependent increase in oxidative phosphorylation which required both glutamine and fatty acids but not glucose. Inhibition of fatty acid oxidation or glutamine utilization could be compensated for by increased glycolytic flux. No evidence for significant mitochondrial dysfunction was found, and ATP/ADP ratios were maintained following the addition of thrombin, indicating the presence of functional and active mitochondrial oxidative phosphorylation during the early stages of aggregation. Interestingly, inhibition of fatty acid oxidation and glutaminolysis alone or in combination is not sufficient to prevent platelet aggregation, due to compensation from glycolysis, whereas inhibitors of glycolysis inhibited aggregation approximately 50%. The combined effects of inhibitors of glycolysis and oxidative phosphorylation were synergistic in the inhibition of platelet aggregation. In summary, both glycolysis and oxidative phosphorylation contribute to platelet metabolism in the resting and activated state, with fatty acid oxidation and to a smaller extent glutaminolysis contributing to the increased energy demand.
Topics: Adenosine Triphosphate; Adult; Blood Coagulation; Blood Platelets; Energy Metabolism; Female; Glycolysis; Hemostatics; Humans; Male; Middle Aged; Oxidative Phosphorylation; Platelet Aggregation; Rest; Thrombin
PubMed: 25875958
DOI: 10.1371/journal.pone.0123597 -
Genes & Development Mar 2024Metabolic reprogramming of stem cells is a targetable pathway to control regeneration. Activation of stem cells results in down-regulation of oxidative phosphorylation... (Review)
Review
Metabolic reprogramming of stem cells is a targetable pathway to control regeneration. Activation of stem cells results in down-regulation of oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) and turns on glycolysis to provide fuel for proliferation and specific signaling events. How cell type-specific events are regulated is unknown. In this issue of Ciuffoli and colleagues (pp. 151-167) use metabolomic, gene inactivation, and functional approaches to show that phosphoserine aminotransferase (Psat1), an enzyme in serine biosynthesis, is activated in muscle stem cells and contributes to cell expansion and skeletal muscle regeneration via the production of α-ketoglutarate and glutamine.
Topics: Muscle, Skeletal; Oxidative Phosphorylation; Glycolysis; Stem Cells; Satellite Cells, Skeletal Muscle
PubMed: 38485266
DOI: 10.1101/gad.351666.124 -
British Journal of Pharmacology Jan 20031. Tacrolimus and sirolimus are potent immunosuppressors used in transplantation. Tacrolimus has been suspected to alter mitochondrial respiration of different tissues...
1. Tacrolimus and sirolimus are potent immunosuppressors used in transplantation. Tacrolimus has been suspected to alter mitochondrial respiration of different tissues but sirolimus has not been evaluated. 2. We evaluated the in vitro effect of tacrolimus and sirolimus on oxidative phosphorylation of isolated rat kidney mitochondria. 3. Oxygen consumption was measured with a Clark-type electrode. Tacrolimus and sirolimus increased the resting rate (state 4) and had no significant effect on ADP-stimulated respiration (state 3). The decrease of respiratory control ratio was concentration-dependent with a biphasic curve for tacrolimus. The EC(50)s were 3.4 x 10(-11) M and 2.3 x 10(-8) M for tacrolimus and 4.4 x 10(-10) M for sirolimus. The maximal inhibition was 20 and 14% for tacrolimus and sirolimus, respectively. 4. Tacrolimus and sirolimus had an uncoupling effect on oxidative phosphorylation related to a decrease of the inner membrane fluidity. At the opposite of cyclosporin A, no effect on swelling or Ca(2+) fluxes was observed. 5. All events occurred at therapeutic concentrations and then could appear during long-term treatment. Cellular consequences such as chronic nephrotoxicity with tacrolimus are suggested. The risk of cyclosporin A nephrotoxicity potentiation by sirolimus is discussed.
Topics: Animals; Kidney; Male; Mitochondria; Oxidative Phosphorylation; Rats; Rats, Wistar; Sirolimus; Tacrolimus
PubMed: 12540528
DOI: 10.1038/sj.bjp.0705038 -
Proceedings of the National Academy of... Jan 1980Irradiation of the inverted membrane vesicles of Mycobacterium phlei with light at 360 nm inactivated the natural menaquinone [MK(9)(II-H)] and resulted in a loss of...
Irradiation of the inverted membrane vesicles of Mycobacterium phlei with light at 360 nm inactivated the natural menaquinone [MK(9)(II-H)] and resulted in a loss of substrate oxidation, pH gradient, membrane potential, active transport of proline or calcium ions, and oxidative phosphorylation. Restoration of the protonmotive force and active transport occurred on addition of naphthoquinones such as vitamin K(1), menadione, or lapachol to the irradiated membrane vesicles. However, coupled phosphorylation was restored only by vitamin K(1). Menadione and lapachol did not act as uncoupling agents. The magnitude of the pH gradient and membrane potential in the quinone-restored system was a reflection of the rate of oxidation and was correlated with the rate of uptake of proline or Ca(2+). These results are consistent with the chemosmotic hypothesis proposed for the energy transducing mechanism for active transport and further demonstrate that the complete respiratory chain is not required to drive active transport. In contrast, the data suggest that in addition to the driving force (protonmotive force) necessary to establish oxidative phosphorylation, a specific spatial orientation of the respiratory components, such as the naphthaquinones, is essential for the utilization of the proton gradient or membrane potential or both. Bypass of electrons from the respiratory chain with menadione may explain the inability of this quinone to restore oxidative phosphorylation; however, lapachol restores oxidation by the same electron transport pathway as the natural menaquinone but fails to restore phosphorylation. Because all three quinones restore the protonmotive force, other factors that are discussed must be considered in understanding the mechanism of oxidative phosphorylation.
Topics: Biological Transport, Active; Cell Membrane; Light; Membrane Potentials; Mycobacterium; Mycobacterium phlei; Naphthoquinones; Oxidative Phosphorylation; Uncoupling Agents
PubMed: 6928606
DOI: 10.1073/pnas.77.1.102 -
International Journal of Molecular... Nov 2018Melatonin (Mel) is the major biologically active molecule secreted by the pineal gland. Mel and its metabolites, 6-hydroxymelatonin (6(OH)Mel) and 5-methoxytryptamine...
Melatonin (Mel) is the major biologically active molecule secreted by the pineal gland. Mel and its metabolites, 6-hydroxymelatonin (6(OH)Mel) and 5-methoxytryptamine (5-MT), possess a variety of functions, including the scavenging of free radicals and the induction of protective or reparative mechanisms in the cell. Their amphiphilic character allows them to cross cellular membranes and reach subcellular organelles, including the mitochondria. Herein, the action of Mel, 6(OH)Mel, and 5-MT in human MNT-1 melanoma cells against ultraviolet B (UVB) radiation was investigated. The dose of 50 mJ/cm² caused a significant reduction of cell viability up to 48%, while investigated compounds counteracted this deleterious effect. UVB exposure increased catalase activity and led to a simultaneous Ca influx (16%), while tested compounds prevented these disturbances. Additional analysis focused on mitochondrial respiration performed in isolated mitochondria from the liver of BALB/cJ mice where Mel, 6(OH)Mel, and 5-MT significantly enhanced the oxidative phosphorylation at the dose of 10 M with lower effects seen at 10 or 10 M. In conclusion, Mel, 6(OH)Mel and 5-MT protect MNT-1 cells, which express melatonin receptors (MT1 and MT2) against UVB-induced oxidative stress and mitochondrial dysfunction, including the uncoupling of oxidative phosphorylation.
Topics: 5-Methoxytryptamine; Animals; Calcium; Catalase; Cell Line, Tumor; Cell Survival; Humans; Liver; Melanoma; Melatonin; Mice, Inbred BALB C; Mitochondria; Oxidative Phosphorylation; Oxidative Stress; Ultraviolet Rays
PubMed: 30487387
DOI: 10.3390/ijms19123786 -
Journal of Cellular Physiology Dec 2008The combination of laser tweezers, fluorescent imaging, and real-time automated tracking and trapping (RATTS) can measure sperm swimming speed and swimming force... (Comparative Study)
Comparative Study
Comparison of glycolysis and oxidative phosphorylation as energy sources for mammalian sperm motility, using the combination of fluorescence imaging, laser tweezers, and real-time automated tracking and trapping.
The combination of laser tweezers, fluorescent imaging, and real-time automated tracking and trapping (RATTS) can measure sperm swimming speed and swimming force simultaneously with mitochondrial membrane potential (MMP). This approach is used to study the roles of two sources of ATP in sperm motility: oxidative phosphorylation, which occurs in the mitochondria located in the sperm midpiece and glycolysis, which occurs along the length of the sperm tail (flagellum). The relationships between (a) swimming speed and MMP and (b) swimming force and MMP are studied in dog and human sperm. The effects of glucose, oxidative phosphorylation inhibitors and glycolytic inhibitors on human sperm motility are examined. The results indicate that oxidative phosphorylation does contribute some ATP for human sperm motility, but not enough to sustain high motility. The glycolytic pathway is shown to be a primary source of energy for human sperm motility.
Topics: Adenosine Triphosphate; Animals; Antimycin A; Culture Media; Dogs; Glucose; Glycolysis; Humans; Male; Membrane Potential, Mitochondrial; Microscopy, Fluorescence; Optical Tweezers; Oxidative Phosphorylation; Rotenone; Sperm Motility
PubMed: 18683212
DOI: 10.1002/jcp.21549 -
Proceedings of the National Academy of... Jan 1965
Topics: Adenine Nucleotides; Adenosine Triphosphate; Coenzymes; Glycosides; Ion Exchange; Kinetics; Metabolism; Mitochondria; Osmosis; Oxidative Phosphorylation; Phosphates; Research
PubMed: 14283208
DOI: 10.1073/pnas.53.1.73 -
Journal For Immunotherapy of Cancer Mar 2021In response to the recent paper by Chen investigating the triple combination of oxidative phosphorylation inhibition, immunotherapy and radiotherapy, we would like to...
In response to the recent paper by Chen investigating the triple combination of oxidative phosphorylation inhibition, immunotherapy and radiotherapy, we would like to stress that after irradiation, a strong reduction in hypoxia (within 24 hours) can be followed by a strong increase (several days). This is especially the case with larger fraction sizes of radiation therapy, which are often applied in combination with immunotherapy, and is likely to be tumor dependent. All together this may strongly affect the synergistic effect of such a triple combination therapy.
Topics: Humans; Hypoxia; Immunotherapy; Kinetics; Neoplasms; Oxidative Phosphorylation
PubMed: 33707312
DOI: 10.1136/jitc-2020-001793 -
The Journal of Biological Chemistry Apr 1977Catalysis by beef heart submitochondrial particles of the medium Pi in equilibrium HOH, Pi in equilibrium ATP, and the ATP in equilibrium HOH exchanges is strongly...
Catalysis by beef heart submitochondrial particles of the medium Pi in equilibrium HOH, Pi in equilibrium ATP, and the ATP in equilibrium HOH exchanges is strongly inhibited while the ATPase and intermediate Pi in equilibrium HOH exchange are accelerated when medium ADP is removed by pyruvate kinase action. Arsenate readily blocks completely the Pi in equilibrium ATP and medium Pi in equilibrium HOH exchange reactions, but not the ATP in equilibrium HOH exchange reaction. The residual ATP in equilibrium HOH exchange in presence of arsenate is inhibited by 2,4-dinitrophenol. These results and other data are explained by an alternating site model for oxidative phosphorylation. In this model during net oxidative phosphorylation ATP is formed at one site but is transitorily tightly bound and not released until ADP and Pi bind at a second site and the membrane ATPase complex is energized. Under conditions of net ATP hydrolysis, ATP binding at one site is accompanied by hydrolysis of the transitorily tightly bound ATP as a second site. Attractive features are only one site of input for conformational energization of the membrane ATPase, a single conformational transition that accounts for both the promotion of ADP and Pi binding in a competent mode and the release of tightly bound ATP, and a symmetry of catalytic sites. The Pi in equilibrium ATP exchange is not inhibited by increase in MgADP and MgATP at constant ratios, and the energy-linked ADP in equilibrium ATP exchange is not inhibited by increased concentrations of MgATP and Pi at a constant ratio. Such exchange patterns indicate a random binding and release of ADP and Pi.
Topics: Adenosine Triphosphate; Animals; Arsenates; Carrier Proteins; Cattle; Dinitrophenols; Kinetics; Magnesium; Mitochondria, Muscle; Myocardium; Oxidative Phosphorylation; Phosphates
PubMed: 856791
DOI: No ID Found