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1.
ATP synthesis and consumption in respiring cells of the green alga Chlamydomonas reinhardtii were measured with 31P in vivo NMR saturation transfer experiments to determine the intracellular compartmentation of inorganic phosphate. Most of the observed flux towards ATP synthesis was catalyzed by the coupled enzymes glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase (GAPDH/PGK). The attribution of the measured flux to these enzymes is supported by the observation, that (i) the magnetization transfer was strongly reduced by iodoacetate, an irreversible inhibitor of GAPDH and that (ii) the unidirectional flux was much greater than the net flux through the mitochondrial F0F1-ATPase as determined by oxygen consumption measurements. In Chlamydomonas, glycolysis is divided into a chloroplastidic and a cytosolic part with the enzymes GAPDH/PGK being located in the chloroplast stroma (Klein 1986). The 31P-NMR signal of inorganic phosphate must, therefore, originate from the chloroplast. The life time of the magnetic label transferred to Pi by these enzymes is too short for it to be transported to the cytosol via the phosphate translocator of the chloroplast envelope. When the intracellular compartmentation of Pi was taken into consideration the calculated unidirectional ATP synthesis rate was equal to the consumption rate, indicating operation of GAPDH/PGK near equilibrium. The assignment of most of the intracellular Pi to the chloroplast is in contradiction to earlier reports, which attributed the Pi signal to the cytosol. This is of special interest for the use of the chemical shift of the Pi signal as an intracellular pH-marker in plant cells.Abbreviations 3-PGA 3-phosphoglycerate - CW continuous wave - dG6P 2-deoxyglucose-6-phosphate - GAPDH glyceraldehyde-3-phosphate dehydrogenase - MO equilibrium z-magnetization - M0 instantaneous z-magnetization after selective saturation for time t - MDP methylene-diphosphonic acid - PDE phosphodiester - PGK phosphoglycerate kinase - Pi inorganic orthophosphate - polyP polyphosphate - T1 longitudinal relaxation time - 1 longitudinal relaxation time with chemical exchange - TCA cycle tricarboxylic acid cycle Correspondence to: A. Mayer  相似文献   

2.
31P-NMR measurements of saturation transfer have been used to measure phosphate consumption in respiratory competent cells of the yeast Saccharomyces cerevisiae. Measurements of oxygen consumption and maintenance of the cells in a metabolic steady state during the NMR experiments were facilitated by immobilisation of the cells in an agarose gel matrix which could be perfused in the NMR spectrometer. The contribution of glycolysis to the observed rate of phosphate consumption was estimated by simultaneously measuring glucose consumption and ethanol production in the perfusion buffer. The remaining phosphate consumption, which was attributed to flux through the reaction catalysed by the mitochondrial ATP synthase, combined with measurements of oxygen consumption allowed estimation of a P:O ratio (mol ATP synthesised:atoms oxygen consumed) which was close to 3.  相似文献   

3.
The efficiency of (Na+ + K+)-ATPase (i.e. the amount of K+ pumped per ATP hydrolyzed) in intact tumorigenic cells was estimated in this study. This was accomplished by simultaneously measuring the rate of ouabain-sensitive K+ uptake and oxygen consumption in tumorigenic cell suspensions during the reintroduction of K+ to K+-depleted cells. The ATP turnover was then estimated by assuming 5.6–6 ATP/O2 as the stoichiometry of NADH-linked respiration in these cells. In the three cell lines tested (hamster and chick embryo cells transformed with Rous sarcoma virus and Ehrlich ascites cells), the K+/ATP ratio was approximately 2, the same value as that found in normal tissues. Furthermore, only 20% of the total ATP production of these cells was used by (Na+ + K+)-ATPase.  相似文献   

4.
A direct linear relationship between the rate of oxygen consumption and ATP content in starved Zymomonas mobilis cells was observed in the presence of ethanol (0.056–1.12 mM) as the substrate. Both the rate of oxygen consumption and the ATP content were significantly reduced by the exogenously added plant growth substance N6-(2-isopentenyl)adenine (i6Ade), directly proportional to the concentration (0.125–0.5 mM) of i6Ade in the incubation medium. The results obtained are consistent with the current view of ATP synthesis by oxidative phosphorylation in non-growing Z. mobilis cells and gives evidence that i6Ade can be used as a tool to affect in vivo the alcohol dehydrogenase reaction, which provides reducing equivalents for ethanol-dependent aerobic energy generation.  相似文献   

5.
The 31P nuclear magnetic resonance spectrum of cultured human Y-79 retinoblastoma cells was obtained at 121 MHz on intact cells trapped in agarose threads. The spectrum was dominated by monoester peaks, which varied in relative concentration from preparation to preparation. Resonances from phosphocreatine, phosphodiesters and diphosphodiesters also exhibited variability relative to ATP. The main monoester was identified as phosphorylcholine by 31P-NMR of perchloric acid extracts. It was determined that the changes in monoester concentration correlated with feeding pattern. Phosphorus spectra of cells 1,2 and 3 days post feeding showed a 40% decrese in the relative concentration of phosphorylcholine concentration over the 3 day period. Phosphocreatine, phosphodiesters and diphosphodiesters increased relative to ATP during the same period. Growth curve experiments and oxygen consumption measurements indicated that the decrease in phosphorylcholine correlated with a decrease in cellular growth and oxygen consumption. We conclude that monoester concentration may be a useful indicator of nutritional status in these cells and possibly in intact tumors.  相似文献   

6.
5-Iodoacetamidofluorescein (5-IAF) covalently labels dog kidney (Na+ + K+)-ATPase with approximately 2 moles incorporated per mole of enzyme. ATPase and K+-phosphatase activities are fully retained after reaction, and the kinetic parameters for Na+, K+, Mg2+, ATP and p-nitrophenyl phosphate are likewise not significantly affected. The fluorescence of the bound 5-IAF is increased by ATP, Na+, and Mg2+, and decreased by K+. These fluorescence changes likely reflect ligand-induced stabilization of the E1 or E2 states of the enzyme.  相似文献   

7.
The effects of various concentrations of deoxyglucose (DG) on the aerobic metabolism of glucose in glucose-grown repressed Saccharomyces cerevisiae cells were studied at 30°C in a standard pyrophosphate medium containing 4.5 107 cells/ml. 31P-nuclear magnetic resonance (NMR) spectroscopy was used to monitor DG phosphorylation and the formation of polyphosphates. The production of soluble metabolites of glucose was evaluated by 13C- and 1H-NMR and biochemical techniques. The cells were aerobically incubated with 25 mM of glucose and various concentrations of DG (0, 5 and 10 mM) in order to determine the DG concentration leading to optimum of 2-deoxy-d-glucose 6-phosphate (DG6P) formation without over-inhibiting the synthesis of other metabolites. The production of DG6P increased by about 25% when the external DG concentration was doubled (from 5 to 10 mM). The formation of polyphosphates (polyP), on the other hand, was found to be mainly conditioned by the DG concentration. The amount of polyP decreased by a factor of four upon addition of 5 mM DG and became undetectable in the presence of 10 mM DG. The glucose consumption and the production of soluble metabolites of [1-13C]glucose were then evaluated as a function of time in both the absence and presence of 5 mM DG. The effect of DG is to decrease the glucose consumption and the formation of polyphosphates, ethanol, glycerol, trehalose, glutamate, aspartate and succinate while stimulating the formation of arginine and citrate. Upon co-addition of 25 mM glucose and 5 mM DG, the ratio between the initial rates of glucose consumption (0.16 mM/min) and DG6P production (0.027 mM/min) is about (5.9 ± 1.2), not very different from the ratio of the initial concentration of glucose and DG (= 5.0). Therefore, hexokinase can phosphorylate deoxyglucose as well as glucose. However, after 100 min of incubation, the glucose concentration in the external medium decreased by about 64% while only 10% of DG was phosphorylated. DG6P was formed and quickly reached the limiting value about 30 min after co-addition of glucose and DG. Nevertheless, when the maximum quantity of DG6P was obtained, the DG consumption became negligible. By contrast, the glucose consumption and the production of ethanol and glycerol, although substantially reduced by about 42%, varied linearly with time up to 80 min of incubation. Thus even in the presence of an excess of DG, glycolysis is only slowed but not gradually or completely inhibited by DG. The reasons why DG6P cannot accumulate indefinitely in cells are discussed, together with the reasons why the consumption of DG, but not glucose, becomes negligible after 30 min of incubation. In the absence of DG, the amount of polyphosphates (polyP) increased regularly with time as long as glucose was sufficiently present (≥ 5 mM) in the suspension. When glucose was exhausted, long chain polyphosphates disappeared to give rise, at first, to polyP with shorter chains and finally to inorganic phosphate. In the presence of 5 mM DG, the reduction in quantity of polyP can be explained by the fact that ATP, normally used for the polyP synthesis, is now diverted to phosphorylation of DG to DG6P. The presence of 5 mM DG also had significant effects on the glutamate C2, C3 and C4 signal intensity and the production of all aminoacids. The results seem to indicate that the enzymes involved in the Krebs cycle are also affected by the presence of DG.  相似文献   

8.
The disappearance of 2-13C-acetate and the subsequent incorporation of label into cellular metabolites were followed in denitrifying cells of Thiobacillus versutus by 13C NMR spectroscopy. In cells grown under acetate-limitation, the specific rate of consumption was idependent of the density of the cell suspension. An isotopic steady state was reached within 30 min if sufficient substrate was added to the cell suspension. In cells grown under nitrate-limitation, the consumption of 2-13C-acetate proceeded at a significantly lower rate. The decrease and final disappearance of 2-13C-acetate were accompanied by incorporation of 13C into glutamate, glutamine, and by the release of labeled HCO 3 and CO2. The appearance of a broad resonance being the methyl endgroup of poly-3-hydroxybutyrate (PHB) was indicative for PHB mobilization during the incubation. The sequence of label incorporation and the distribution among the various carbon nuclei were consistent with the operation of the tricarboxylic acid cycle.  相似文献   

9.
The role of the hydrolysis products of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and inorganic phosphate (Pi), in the control of myocardial respiration was evaluated in vivo using 31P NMR. These studies were conducted to evaluate whether increases in the ATP hydrolysis products can be detected through the cardiac cycle or during increases in cardiac work. 31P NMR data acquisitions gated to various portions of the cardiac cycle (50 msec time resolution) revealed that cytosolic ATP, ADP and Pi did not change over the course of the cardiac cycle. These metabolites were also monitored during steady-state increases in cardiac work in conjunction with measurements of coronary blood flow and oxygen consumption. No changes were observed during 2 to 3 fold increases in myocardial oxygen consumption induced by various methods. These results demonstrate that the cytosolic ATP, ADP, and Pi concentrations remain relatively constant throughout the cardiac cycle and during physiological increases in cardiac work and oxygen consumption. Furthermore, it is shown that ADP and Pi cannot be solely responsible for the regulation of cardiac respiration in vivo based on the in vitro Km values of these compounds for oxidative phosphorylation. It is concluded that other mechanisms, working in concert with the simple kinetic feedback of ATP hydrolysis products, must be present in the cytosol to provide control of myocardial respiration in vivo.  相似文献   

10.
The proton translocation stoichiometry (H+/ATP ratio) was investigated in membrane vesicles from a Synechocystis 6803 mutant in which the serine at position 37 in the hydrophilic loop of the c-subunit from the wild type was replaced by a negatively charged glutamic acid residue (strain plc37). At this position the c-subunit of chloroplasts and the cyanobacterium Synechococcus 6716 already contains glutamic acid. H+/ATP ratios were determined with active ATP synthase in thermodynamic equilibrium between phosphate potential (G p ) and the proton gradient ( H +) induced by acid–base transition. The mutant displayed a significantly higher H+/ATP ratio than the control strain (wild type with kanamycin resistance) at pH 8 (4.3 vs. 3.3); the higher ratio also being observed in chloroplasts and Synechococcus 6716. Furthermore, the pH dependence of the H+/ATP of strain plc37 resembles that of Synechococcus 6716. When the pH was increased from 7.6 to 8.4, the H+/ATP of the mutant increased from 4.2 to 4.6 whereas in the control strain the ratio decreased from 3.8 to 2.8. Differences in H+/ATP between the mutant and the control strain were confirmed by measuring the light-induced phosphorylation efficiency (P/2e), which changed as expected, i.e., the P/2e ratio in the mutant was significantly less than that in the wild type. The need for more H+ ions used per ATP in the mutant was also reflected by the significantly lower growth rate of the mutant strain. The results are discussed against the background of the present structural and functional models of proton translocation coupled to catalytic activity of the ATP synthase.  相似文献   

11.
The effect of a low phosphate concentration on intracellular adenine nucleotide content, oxygen consumption and poly--hydroxybutyrate deposition was investigated with N-free and NH 4 + batch cultures of Azotobacter vinelandii. When the microorganisms were cultured under low-phosphate concentrations the cells contained much larger amounts of poly--hydroxybutyrate, but displayed lower oxygen consumption activities and energy charge values than did control cells. Also, the ratio ATP to ADP was much higher in control cells and the intracellular levels of ATP were lower in low-phosphate cells.  相似文献   

12.
Chemical composition of the microalga Thalassiosira pseudonana Hasle & Heimdalwas studied with different proton nuclearmagnetic resonance (1H NMR)techniques, and by comparing NMR spectrafrom extraction samples with a spectrumfrom a sample of whole cells we show thathigh-resolution magic angle spinning (HRMAS) 1H NMR can be used for broadrange analysis of metabolic composition inmicroalgal whole cells. Signals fromimportant metabolites such aspolyunsaturated fatty acids (PUFAs)eicosapentaenoic (EPA) and docosahexaenoic(DHA) acids were seen in a 1H NMRspectrum of lipophilic extract, andpossibly also signals from the carotenoidfucoxanthin. In a spectrum of hydrophilicextract we assigned signals to amino acidssuch as glutamine (Gln) and glutamic acid(Glu), carbohydrate and ATP. These findingswere compared to a spectrum of HR MAS1H NMR analysis of whole cells, whereit was possible to find signals coincidentwith the different metabolites seen inspectra of the extraction samples. Sincethe position of resonance peaks in a NMRspectrum depends on the chemicalsurroundings of each atom at the time ofanalysis some peak shift differencesbetween extract and whole cell samplespectra may occur, but signal shifts werenot significantly different between theanalyses here. In addition, application ofHR MAS highly increased spectral resolutionin the complex whole cell sample. Wetherefore suggest that HR MAS 1H NMRanalysis is a suitable analysis tool tostudy metabolic composition directly onwhole cells of microalgae, making itpossible to study a broad range ofmetabolites simultaneously without tediousextraction procedures.  相似文献   

13.
Red cells of hibernating species have a higher relative rate of Na+–K+ pump activity at low temperature than the red cells of a mammal with a typical sensitivity to cold. The kinetics of ATP stimulation of the Na+–K+ pump were determined in guinea pig and ground squirrel red cells at different temperatures between 5 and 37°C by measuring ouabain-sensitive K+ influx at different levels of ATP. In guinea pig cells, elevation of intracellular free Mg2+ to 2 mmol·l-1 by use of the divalent cation ionophore A23187 caused the apparent affinity of the pump for ATP to increase with cooling to 20°C, rather than to decrease, as occurs in cells not loaded with Mg2+. In ground squirrel cells raising intracellular free Mg2+ had little effect on apparent affinity of the pump for ATP at 20°C. ATP affinity rose slightly with cooling both in Mg2+-enriched and in control ground squirrel cells. Increased intracellular free Mg2+ in guinea pig cells stimulated Na+–K+ pump activity so that at 20°C the pump rate was the same in the Mg2+-enriched guinea pig and control ground squirrel cells. Pump activity in Mg2+-enriched guinea pig cells at 5°C was significantly improved but still lower than pump activity in control cells from ground squirrel. Thus, loss of affinity of the Na+–K+ pump for ATP that occurs with cooling in cold-sensitive guinea pig red cells can be, at least partially, prevented by elevating cytoplasmic free Mg2+. Conversely, in ground squirrel red cells natural rise of free Mg2+ may in part account for the preservation of the ATP affinity of their Na+–K+ pump with cooling.Abbreviations K m Michaelis-Menten constant for apparent affinity - MOPS 3-(N-morpholino)-propanesulphonic acid - [Mg2+]i intracellular concentration of free Mg2+ - OD optical density - RBC red blood cell(s) - T b body temperature  相似文献   

14.
Although the sensitivity of the plasma membrane H+-ATPase to vanadate is well known, the metabolic response of plant cells to vanadate is less well characterised in vivo and its use as an inhibitor in whole plant experiments has had mixed success. Experiments with maize (Zea mays, L.) roots and with purified plasma membrane fractions from the same tissues showed that exposure to vanadate caused: (i) a reduction in the capacity for phosphate uptake; (ii) a reduction in the extractable ATPase activity from the tissue; and (iii) a significant increase in the ATP level. The measurements on the extractable ATPase activity and the ATP level showed that the effect of vanadate developed slowly, apparently reflecting the slow accumulation of intracellular vanadate. The marked effect of vanadate on the ATP level-exposure to 500 M vanadate for 5 h doubled the ATP content of the roots tips-indicates that there is no stringent control over the ATP level in the roots and that the plasma membrane H+-ATPase activity is likely to have a significant role in determining the ATP level under normal conditions.  相似文献   

15.
31P NMR spectra were obtained from suspensions of Candida utilis, Saccharomyces cerevisiae and Zygosaccharomyces bailii grown aerobically on glucose. Direct introduction of substrate into the cell suspension, without interruption of the measurements, revealed rapid changes in pH upon addition of the energy source. All 31P NMR spectra of the yeasts studied indicated the presence of two major intracellular inorganic phosphate pools at different pH environments. The pool at the higher pH was assigned to cytoplasmic phosphate from its response to glucose addition and iodoacetate inhibition of glycolysis. After addition of substrate the pH in the compartment containing the second phosphate pool decreased. A parallel response was observed for a significant fraction of the terminal and penultimate phosphates of the polyphosphate observed by 31P NMR. This suggested that the inorganic phosphate fraction at the lower pH and the polyphosphates originated from the same intracellular compartment, most probably the vacuole. In this vacuolar compartment, pH is sensitive to metabolic conditions. In the presence of energy source a pH gradient as large as 0.8 to 1.5 units could be generated across the vacuolar membrane. Under certain conditions net transport of inorganic phosphate across the vacuolar membrane was observed during glycolysis: to the cytoplasm when the cytoplasmic phosphate concentration had become very low due to sugar phosphorylation, and into the vacuole when the former concentration had become high again after glucose exhaustion.Non-Standard Abbreviations NMR nuclear magnetic resonance - ppm parts per million - PP polyphosphate - Pi,c cytoplasmic inorganic phosphate - Pi,v vacuolar inorganic phosphate - pHin,c cytoplasmic pH - pHin,v vacuolar pH - FCCP carbonyl p-trifluoromethoxyphenylhydrazone  相似文献   

16.
A simple device for taking in situ proton NMR measurements in 1H2O is described. This allows aeration of reactions in a 10 mm diameter NMR tube without modifying the magnet or the probe head. With this device, aerobic biotransformations can be monitored in the NMR-tube placed in the spectrometer. It allows in situ analyses of the transformations, separating the aeration period temporally from the measurement time, not unlike traditional Warburg respiratory experiments. Two reactions determining kinetic and stoichieometric parameters: (i) a biotransformation by a growing Pseudomonas putida culture and (ii) l-phenylalanine oxidation catalysed by l-amino acid oxidase [E.C. 1.4.3.2]; both incubations were contained in the magnet.  相似文献   

17.
Whole cells of Desulfobulbus propionicus fermented [1-13C]ethanol to [2-13C] and [3-13C]propionate and [1-13C]-acetate, which indicates the involvement of a randomizing pathway in the formation of propionate. Cell-free extracts prepared from cells grown on lactate (without sulfate) contained high activities of methylmalonyl-CoA: pyruvate transacetylase, acetase kinase and reasonably high activities of NAD(P)-independent L(+)-lactate dehydrogenase NAD(P)-independent pyruvate dehydrogenase, phosphotransacetylase, acetate kinase and reasonably high activity of NAD(P)-independent L(+)-lactate dehydrogenase, fumarate reductase and succinate dehydrogenase. Cell-free extracts catalyzed the conversion of succinate to propionate in the presence of pyruvate, CoA and ATP and the oxaloacetate-dependent conversion of propionate to succinate. After growth on lactate or propionate in the presence of sulfate similar enzyme levels were found except for fumarate reductase which was considerably lower. Fermentative growth on lactate led to higher cytochrome b contents than growth with sulfate as electron acceptor.The labeling studies and the enzyme measurements demonstrate that in Desulfobulbus propionate is formed via a succinate pathway involving a transcarboxylase like in Propionibacterium. The same pathway may be used for the degradation of propionate to acetate in the presence of sulfate.Abbreviations DCPIP 2,6-dichlorophenolindophenol - PEP phosphoenolpyruvate  相似文献   

18.
The controlling effect of ATP, K+ and Na+ on the rate of (Na+ + K+)-ATPase inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is used for the mathematical modelling of the interaction of the effectors with the enzyme under equilibrium conditions.
1. 1. Of a series of conceivable interaction models, designed without conceptual restrictions to describe the effector control of inactivation kinetics, only one fits the experimental data described in a preceding paper.
2. 2. The model is characterized by the coexistence of two binding sites for ATP and the coexistence of two separate binding sites for K+ and Na+ on the enzyme-ATP complex. On the basis of this model, the effector parameters fitting the experimental data most closely are estimated by means of nonlinear least-squares fits.
3. 3. The apparent dissociation constants for ATP of the enzyme-ATP complex or of the enzyme-(ATP)2 complex are computed to lie near 0.0024 mM and 0.34 mM, respectively, irrespective of whether K+ and Na+ were absent or K+ and K+ plus Na+, respectively, were present in the experiments.
4. 4. The origin of the high and the low affinity site for binding of ATP to the (Na+ + K+)-ATPase molecule is traced back to the coexistence of two catalytic centres which, although primarily equivalent as to the reactivity of their thiol groups with NBD-Cl, are induced into anticooperative communication by ATP binding and thus show an induced geometric asymmetry.
Keywords: (Na+ + K+)-ATPase; SH-group alkylation; Inactivation kinetics; Mathematical modelling; Substrate affinity  相似文献   

19.
Summary The regulation of voltage-dependent Ca2+ channels by protein phosphorylation and dephosphorylation was studied using tonoplast-free cells ofNitellopsis. Since the Ca2+-channel activation has a dominant role in the membrane excitation of tonoplast-free cells (T. Shiina and M. Tazawa,J. Membrane Biol. 96:263–276, 1987), it seems to be reasonable to assume that any change of the membrane excitability reflects a modulation of the Ca2+ channel. When agents that enhance phosphoprotein dephosphorylation (protein kinase, inhibitor, phosphoprotein phosphatase-1, -2A) were introduced to the intracellular surface of the plasmalemma (twice-perfused tonoplast-free cells), the membrane potential depolarized and the membrane resistance decreased under current-clamp experiments. By contrast, when cells were challenged with agents that enhance protein phosphorylation (phosphoprotein phosphatase inhibitor-1, -naphthylphosphate), the membrane potential hyperpolarized, and the membrane resistance increased. When phosphoprotein phosphatase-1 or -2A was perfused, the current-voltage (I–V) curve which was obtained under ramp voltage-clamp condition exhibited the so-called N-shaped characteristic, indicating an acceleration of the Ca2+-channel activation. This effect was suppressed by the addition of phosphoprotein phosphatase inhibitors. ATP--S, which is assumed to stimulate protein phosphorylation, decreased the inward current in theI–V curve. The dependence of the Ca2+-channel activation on intracellular ATP was different between the once-perfused and twice-perfused cells. In once-perfused cells, the membrane excitability was reduced by low intracellular ATP concentration. By contrast, in twice-perfused cells, excitability was enhanced by ATP.  相似文献   

20.
Venturicidin behaves as an orthodox energy transfer inhibitor in intact cells of Rhodopseudomonas capsulata as judged by the following criteria. 1. It led to inhibition of respiration. Inhibition was relieved by low concentrations of uncoupling agent. 2. It enhanced light-induced and oxygen dependent H+ efflux. 3. It stimulated light-induced and oxygen dependent carotenoid band shifts. The rate of decay of the band shifts after short flash excitation was decreased in the presence of venturicidin. 4. It stimulated light-induced and oxygen dependent butyltriphenylphosphonium uptake. 5. It inhibited the rise in cellular ATP concentration accompanying either photosynthesis or respiration.  相似文献   

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