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1.
Control of the tricarboxylate cycle and its interactions with glycolysis during acetate utilization in rat heart 总被引:23,自引:18,他引:5
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1. Transient and steady-state changes caused by acetate utilization were studied in perfused rat heart. The transient period occupied 6min and steady-state changes were followed in a further 6min of perfusion. 2. In control perfusions glucose oxidation accounted for 75% of oxygen utilization; the remaining 25% was assumed to represent oxidation of glyceride fatty acids. With acetate in the steady state, acetate oxidation accounted for 80% of oxygen utilization, which increased by 20%; glucose oxidation was almost totally suppressed. The rate of tricarboxylate-cycle turnover increased by 67% with acetate perfusion. The net yield of ATP in the steady state was not altered by acetate. 3. Acetate oxidation increased muscle concentrations of acetyl-CoA, citrate, isocitrate, 2-oxoglutarate, glutamate, alanine, AMP and glucose 6-phosphate, and lowered those of CoA and aspartate; the concentrations of pyruvate, ATP and ADP showed no detectable change. The times for maximum changes were 1min, acetyl-CoA, CoA, alanine and AMP; 6min, citrate, isocitrate, glutamate and aspartate; 2-4min, 2-oxoglutarate. Malate concentration fell in the first minute and rose to a value somewhat greater than in the control by 6min. There was a transient and rapid rise in glucose 6-phosphate concentration in the first minute superimposed on the slower rise over 6min. 4. Acetate perfusion decreased the output of lactate, the muscle concentration of lactate and the [lactate]/[pyruvate] ratio in perfusion medium and muscle in the first minute; these returned to control values by 6min. 5. During the first minute acetate decreased oxygen consumption and lowered the net yield of ATP by 30% without any significant change in muscle ATP or ADP concentrations. 6. The specific radioactivities of cycle metabolites were measured during and after a 1min pulse of [1-(14)C]acetate delivered in the first and twelfth minutes of acetate perfusion. A model based on the known flow rates and concentrations of cycle metabolites was analysed by computer simulation. The model, which assumed single pools of cycle metabolites, fitted the data well with the inclusion of an isotope-exchange reaction between isocitrate and 2-oxoglutarate+bicarbonate. The exchange was verified by perfusions with [(14)C]bicarbonate. There was no evidence for isotope exchange between citrate and acetyl-CoA or between 2-oxoglutarate and malate. There was rapid isotope equilibration between 2-oxoglutarate and glutamate, but relatively poor isotope equilibration between malate and aspartate. 7. It is concluded that the citrate synthase reaction is displaced from equilibrium in rat heart, that isocitrate dehydrogenase and aconitate hydratase may approximate to equilibrium, that alanine aminotransferase is close to equilibrium, but that aspartate transamination is slow for reasons that have yet to be investigated. 8. The slow rise in citrate concentration as compared with the rapid rise in that of acetyl-CoA is attributed to the slow generation of oxaloacetate by aspartate aminotransferase. 9. It is proposed that the tricarboxylate cycle may operate as two spans: acetyl-CoA-->2-oxoglutarate, controlled by citrate synthase, and 2-oxoglutarate-->oxaloacetate, controlled by 2-oxoglutarate dehydrogenase; a scheme for cycle control during acetate oxidation is outlined. The initiating factors are considered to be changes in acetyl-CoA, CoA and AMP concentrations brought about by acetyl-CoA synthetase. 10. Evidence is presented for a transient inhibition of phosphofructokinase during the first minute of acetate perfusion that was not due to a rise in whole-tissue citrate concentration. The probable importance of metabolite compartmentation is stressed. 相似文献
2.
Quantitative analysis of intermediary metabolism in rat hepatocytes incubated in the presence and absence of ethanol with a substrate mixture including ketoleucine.
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Hepatocytes isolated from livers of fed rats were incubated with a mixture of glucose (10 mM), ribose (1.0 mM), acetate (1.25 mM), alanine (3.5 mM), glutamate (2.0 mM), aspartate (2.0 mM), 4-methyl-2-oxovaleric acid (ketoleucine) (3.0 mM), and, in paired flasks, 10 mM-ethanol. One substrate was 14C-radiolabelled in any given incubation. Incorporation of 14C into glucose, glycogen, CO2, lactate, alanine, aspartate, glutamate, acetate, urea, lipid glycerol, fatty acids and the 1- and 2,3,4-positions of ketone bodies was measured after 20 and 40 min of incubation under quasi-steady-state conditions. Data were analysed with the aid of a realistic structural metabolic model. In each of the four conditions examined, there were approx. 77 label incorporation measurements and several measurements of changes in metabolite concentrations. The considerable excess of measurements over the 37 independent flux parameters allowed for a stringent test of the model. A satisfactory fit to these data was obtained for each condition. There were large bidirectional fluxes along the gluconeogenic/glycolytic pathways, with net gluconeogenesis. Rates of ureagenesis, oxygen consumption and ketogenesis were high under all four conditions studied. Oxygen utilization was accurately predicted by three of the four models. There was complete equilibration between mitochondrial and cytosolic pools of acetate and of CO2, but for several of the metabolic conditions, two incompletely equilibrated pools of mitochondrial acetyl-CoA and oxaloacetate were required. Ketoleucine was utilized at a rate comparable to that reported by others in perfused liver and entered the mitochondrial pool of acetyl-CoA directly associated with ketone body formation. Ethanol, which was metabolized at rates comparable to those in vivo, caused relatively few changes in overall flux patterns. Several effects related to the increased NADH/NAD+ ratio were observed. Pyruvate dehydrogenase was completely inhibited and the ratio of acetoacetate to 3-hydroxybutyrate was decreased; flux through glutamate dehydrogenase, the citric acid cycle, and ketoleucine dehydrogenase were, however, only slightly inhibited. Net production of ATP occurred in all conditions studied and was increased by ethanol. Futile cycling was quantified at the glucose/glucose 6-phosphate, glycogen/glucose 6-phosphate, fructose 6-phosphate/fructose 1,6-bis-phosphate, and phosphoenolpyruvate/pyruvate/oxaloacetate substrate cycles. Cycling at these four loci consumed about 22% of cellular ATP production in control hepatocytes and 14% in ethanol-treated cells. 相似文献
3.
The tricarboxylic acid cycle in Dictyostelium discoideum. Metabolite concentrations, oxygen uptake and 14c-labelled amino acid labelling patterns. 总被引:2,自引:1,他引:1
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Some aspects of tricarboxylic acid-cycle activity during differentiation and aging in Dictyostelium discoideum were examined. The concentrations of glutamate, aspartate, alanine, citrate, 2-oxoglutarate, succinate, fumarate, malate, oxaloacetate, pyruvate and acetyl-CoA were determined at four stages over the course of differentiation. The rate of O2 utilization was also determined over differentiation. In addition, experiments are described in which the specific radioactivities of citrate, 2-oxoglutarate, succinate, fumarate and malate were determined during a 30 min labelling of cells from the preculmination stage of development with [14C]glutamate, [14C]aspartate or [14C]alanine. A similar experiment was also performed with cells from the aggregation stage of development using [14C]glutamate. 相似文献
4.
—The time course of changes in glycolytic and citric acid cycle intermediates and in amino acids was studied in acute and steady state hypercapnia. Experiments on unanaesthetized animals exposed to 10% CO2 for 10, 20 and 60s showed that there was a transient decrease in glycogen concentration, progressive increases in glucose-6-phosphate and fructose-6-phosphate and decreases in pyruvate and lactate. During this time the levels of amino acids and Krebs cycle intermediates did not change, except for a small fall in malate at 60s. The results indicate that there was a decrease in glycolytic flux due to an inhibition of the phosphofructokinase reaction. Since the tissue levels of phosphocreatine, ATP, ADP and AMP were unchanged inhibition of phosphofructokinase was probably due to the fall in pH. Anaesthetized animals were exposed to about 5% CO2 (for 2, 5, 15, 30 and 60 min) or to about 45% CO2 (for 5 and 15 min). Except for succinate, which increased, all citric acid cycle metabolites analysed (citrate, α-ketoglutarate, fumarate and malate) decreased with the rise in CO2-tension. The sum of the amino acids analysed (glutamate, glutamine, aspartate, asparagine, alanine and GABA) decreased at extreme hypercapnia. The results suggest that Krebs cycle intermediates and amino acids are partly used as substrates for energy production when there is reduced pyruvate availability due to hypercapnia. It is proposed that amino acid carbon is made available for oxidation via transamination (aspartate aminotransferase reaction) and deamination (glutamate dehydrogenase reaction) and that citric acid cycle intermediates are metabolized following a reversal of reactions usually leading to CO2 fixation. 相似文献
5.
The redistribution of carbon label by the reactions involved in glycolysis, gluconeogenesis and the tricarboxylic acid cycle in rat liver 总被引:4,自引:4,他引:0
D. F. Heath 《The Biochemical journal》1968,110(2):313-335
A scheme is presented that shows how the reactions involved in gluconeogenesis, glycolysis and the tricarboxylic acid cycle are linked in rat liver. Equations are developed that show how label is redistributed in aspartate, glutamate and phosphopyruvate when it is introduced as specifically labelled pyruvate or glucose either at a constant rate (steady-state theory) or at a variable rate (non-steady-state theory). For steady-state theory the fractions of label introduced as specifically labelled pyruvate that are incorporated into glucose and carbon dioxide are also given, and for both theories the specific radioactivities of aspartate and glutamate relative to the specific radioactivity of the substrate. The theories allow for entry of label into the tricarboxylic acid cycle via both oxaloacetate and acetyl-CoA, for (14)CO(2) fixation and for loss of label from the tricarboxylic acid cycle in glutamate, but not for losses in citrate. They also allow for incomplete symmetrization of label in oxaloacetate due to incomplete equilibration with fumarate both in the extramitochondrial part of the cell and in the mitochondrion on entry of oxaloacetate into the tricarboxylic acid cycle. In the latter case failure both of oxaloacetate to equilibrate with malate and of malate to equilibrate with fumarate are considered. 相似文献
6.
The enzyme fumarase catalyzes the reversible hydration of fumarate to malate. The reaction catalyzed by fumarase is critical for cellular energetics as a part of the tricarboxylic acid cycle, which produces reducing equivalents to drive oxidative ATP synthesis. A catalytic mechanism for the fumarase reaction that can account for the kinetic behavior of the enzyme observed in both isotope exchange studies and initial velocity studies has not yet been identified. In the present study, we develop an 11-state kinetic model of the enzyme based on the current consensus on its catalytic mechanism and design a series of experiments to estimate the model parameters and identify the major flux routes through the mechanism. The 11-state mechanism accounts for competitive binding of inhibitors and activation by different anions, including phosphate and fumarate. The model is identified from experimental time courses of the hydration of fumarate to malate obtained over a wide range of buffer and substrate concentrations. Further, the 11-state model is found to effectively reduce to a five-state model by lumping certain successive steps together to yield a mathematically less complex representation that is able to match the data. Analysis suggests the primary reaction route of the catalytic mechanism, with fumarate binding to the free unprotonated enzyme and a proton addition prior to malate release in the fumarate hydration reaction. In the reverse direction (malate dehydration), malate binds the protonated form of the enzyme, and a proton is generated before fumarate is released from the active site. 相似文献
7.
Isidore C Okere Tracy A McElfresh Daniel Z Brunengraber Wenjun Martini Joseph P Sterk Hazel Huang Margaret P Chandler Henri Brunengraber William C Stanley 《Journal of applied physiology》2006,100(1):76-82
In the normal heart, there is loss of citric acid cycle (CAC) intermediates that is matched by the entry of intermediates from outside the cycle, a process termed anaplerosis. Previous in vitro studies suggest that supplementation with anaplerotic substrates improves cardiac function during myocardial ischemia and/or reperfusion. The present investigation assessed whether treatment with the anaplerotic medium-chain fatty acid heptanoate improves contractile function during ischemia and reperfusion. The left anterior descending coronary artery of anesthetized pigs was subjected to 60 min of 60% flow reduction and 30 min of reperfusion. Three treatment groups were studied: saline control, heptanoate (0.4 mM), or hexanoate as a negative control (0.4 mM). Treatment was initiated after 30 min of ischemia and continued through reperfusion. Myocardial CAC intermediate content was not affected by ischemia-reperfusion; however, treatment with heptanoate resulted in a more than twofold increase in fumarate and malate, with no change in citrate and succinate, while treatment with hexanoate did not increase fumarate or malate but increased succinate by 1.8-fold. There were no differences among groups in lactate exchange, glucose oxidation, oxygen consumption, and contractile power. In conclusion, despite a significant increase in the content of carbon-4 CAC intermediates, treatment with heptanoate did not result in improved mechanical function of the heart in this model of reversible ischemia-reperfusion. This suggests that reduced anaplerosis and CAC dysfunction do not play a major role in contractile and metabolic derangements observed with a 60% decrease in coronary flow followed by reperfusion. 相似文献
8.
Son Tran-Dinh Jacqueline A. Hoerter Philippe Mateo Franck Gyppaz Martine Herve 《Biochimie》1998,80(12):1013-1024
A new mathematical model, based on the observation of 13C-NMR spectra of two principal metabolites (glutamate and aspartate), was constructed to determine the citric acid cycle flux in the case of high aspartate transaminase activity leading to the formation of large amounts of labeled aspartate and glutamate. In this model, the labeling of glutamate and aspartate carbons by chemical and isotopic exchange with the citric acid cycle are considered to be interdependent. With [U-13C]Glc or [1,2-13C]acetate as a substrate, all glutamate and aspartate carbons can be labeled. The isotopic transformations of 32 glutamate isotopomers into 16 aspartate isotopomers or vice versa were studied using matrix operations; the results were compiled in two matrices. We showed how the flux constants of the citric acid cycle and the 13C-enrichment of acetyl-CoA can be deduced from 13C-NMR spectra of glutamate and/or aspartate. The citric acid cycle flux in beating Wistar rat hearts, aerobically perfused with [U-13C]glucose in the absence of insulin, was investigated by 13C-NMR spectroscopy. Surprisingly, aspartate instead of glutamate was found to be the most abundantly-labeled metabolite, indicating that aspartate transaminase (which catalyses the reversible reaction: (glutamate + oxaloacetate ↔ 2-oxoglutarate + aspartate) is highly active in the absence of insulin. The amount of aspartate was about two times larger than glutamate. The quantities of glutamate (Go) or aspartate (AO) were approximately the same for all hearts and remained constant during perfusion: G0 = (0.74 ±0.03) μmol/g; A0 = (1.49±0.05) μmol/g. The flux constants, i.e., the fraction of glutamate and aspartate in exchange with the citric acid cycle, were about 1.45 min−1 and 0.72 min−1, respectively; the flux of this cycle is about (1.07±0.02) μmol min-1 g-1. Excellent agreement between the computed and experimental data was obtained, showing that: i) in the absence of insulin, only 41% of acetyl-CoA is formed from glucose while the rest is derived from endogenous substrates; and ii) the exchange between aspartate and oxaloacetate or between glutamate and 2-oxoglutarate is fast in comparison with the biological transformation of intermediate compounds by the citric acid cycle. 相似文献
9.
Carvalho RA Rodrigues TB Zhao P Jeffrey FM Malloy CR Sherry AD 《American journal of physiology. Heart and circulatory physiology》2004,287(2):H889-H895
Rat hearts were perfused with mixtures of [3-(13)C]pyruvate and [3-(13)C]lactate (to alter cytosolic redox) at low (0.5 mM) or high (2.5 mM) Ca(2+) concentrations to alter contractility. Hearts were frozen at various times after exposure to these substrates, were extracted, and were then analyzed by (13)C NMR spectroscopy. The time-dependent multiplets observed in the (13)C NMR resonances of glutamate in all hearts and in malate and aspartate in hearts perfused with high-pyruvate/low-lactate concentrations were analyzed using a kinetic model of the tricarboxylic acid (TCA) cycle. The analysis showed that TCA cycle flux (V(TCA)) and exchange flux (V(X)) that involved cycle intermediates were both sensitive to cell redox and altered Ca(2+) concentration, and the ratio of these fluxes (V(X)/V(TCA)) varied >10-fold. 相似文献
10.
Malonate is an effective inhibitor of succinate dehydrogenase in preparations from brain and other organs. This property was reexamined in isolated rat brain mitochondria during incubation with L-glutamate. The biosynthesis of aspartate was determined by a standard spectrofluorometric method and a radiometric technique. The latter was suitable for aspartate assay after very brief incubations of mitochondria with glutamate. At a concentration of 1 mM or higher, malonate totally inhibited aspartate biosynthesis. At 0.2 mM, the inhibitory effect was still present. It is thus possible that the natural concentration of free malonate in adult rat brain of 192 nmol/g wet weight exerts an effect on citric acid cycle reactions in vivo. The inhibition of glutamate utilization by malonate was readily overcome by the addition of malate which provided oxaloacetate for the transamination of glutamate. The reaction was accompanied by the accumulation of 2-oxoglutarate. The metabolism of glutamate was also blocked by inclusion of arsenite and gamma-vinyl-gamma-aminobutyric acid but again added malate allowed transamination to resume. When arsenite and gamma-vinyl-gamma-aminobutyric acid were present, the role of malonate as an inhibitor of malate entry into the mitochondrial interior could be determined without considering the inhibition of succinate dehydrogenase. The apparent Km and Vmax values for uninhibited malate entry were 0.01 mM and 100 nmol/mg protein/min, respectively. Malonate was a competitive inhibitor of malate transport (Ki = 0.75 mM). 相似文献
11.
Quantification of carbon fluxes through the tricarboxylic acid cycle in early germinating lettuce embryos 总被引:7,自引:0,他引:7
A method involving labeling to isotopic steady state and modeling of the tricarboxylic acid cycle has been used to identify the respiratory substrates in lettuce embryos during the early steps of germination. We have compared the specific radioactivities of aspartate and glutamate and of glutamate C-1 and C-5 after labeling with different substrates. Labeling with [U-14C]acetate and 14CO2 was used to verify the validity of the model for this study; the relative labeling of aspartate and glutamate was that expected from the normal operation of the tricarboxylic acid cycle. After labeling with 14CO2, the label distribution in the glutamate molecule (95% of the label at glutamate C-1) was consistent with an input of carbon via the phosphoenolpyruvate carboxylase reaction, and the relative specific radioactivities of aspartate and glutamate permitted the quantification of the apparent rate of the fumarase reaction. CO2 and intermediates related to the tricarboxylic acid cycle were labeled with [U-14C]acetate, [1-14C] hexanoate, or [U-14C]palmitic acid. The ratios of specific radioactivities of asparate to glutamate and of glutamate C-1 to C-5 indicated that the fatty acids were degraded to acetyl units, suggesting the operation of beta-oxidation, and that the acety-CoA was incorporated directly into citrate. Short-term labeling with [1-14C]hexanoate showed that citrate and glutamate were labeled earlier than malate and aspartate, showing that this fatty acid was metabolized through the tricarboxylic acid cycle rather than the glyoxylate cycle. This was in agreement with the flux into gluconeogenesis compared to efflux as respiratory CO2. The fraction of labeled substrate incorporated into carbohydrates was only about 5% of that converted to CO2; the carbon flux into gluconeogenesis was determined after labeling with 14CO2 and [1-14C]hexanoate from the specific radioactivity of aspartate C-1 and the amount of label incorporated into the carbohydrate fraction. It was only 7.4% of the efflux of respiratory CO2. The labeling of alanine indicates a low activity of either a malic enzyme or the sequence phosphoenolpyruvate carboxykinase/pyruvate kinase. After labeling with [U-14C]glucose, the ratios of specific radioactivities indicated that the labeled carbohydrates contributed less than 10% to the flux of acetyl-CoA. The model indicated that the glycolytic flux is partitioned one-third to pyruvate and two-thirds to oxalacetate and is therefore mainly anaplerotic. The possible role of fatty acids as the main source of acetyl-CoA for respiration is discussed. 相似文献
12.
The interaction of glycolysis, gluconeogenesis and the tricarboxylic acid cycle in rat liver in vivo 总被引:13,自引:12,他引:1
1. The equations derived by Heath (1968) were applied to data from experiments on rats in four metabolic states: fed, post-absorptive, starved and 2hr. after an eventually lethal injury. The data used were: (a) The fractions of label injected as C1-, C2- and C3-pyruvate (where the prefix indicates the position of labelling) that are incorporated into carbon dioxide and glucose in post-absorptive and injured rats (yields). Yields could be corrected to yields on label taken up by the liver. (b) The (C5-label in glutamate)/(total label in glutamate) ratio in the liver after C2-pyruvate in rats in all four states. (c) The distribution of label within glutamate after C2-pyruvate or C2-alanine in the livers of fed, post-absorptive and starved rats. (d) The distribution of label within glucose after C2-lactate or C2-pyruvate in starved rats. (e) The relative specific radioactivities of pyruvate, aspartate, glutamate and (in two states only) of glucose 6-phosphate after injection of [U-(14)C]glucose into rats in all four states. These data were previously published, except those after (e) and some after (b) above, which are given in this paper. 2. In addition the concentrations of pyruvate, citrate, glutamate and aspartate in the livers of post-absorptive and injured rats were found. Injury decreased glutamate and citrate concentrations and to a smaller extent aspartate and pyruvate concentrations. 3. Non-steady-state theory showed that most of the data could be used without serious error in steady-state theory. Steady-state theory correlated all but one observation (the relative yields of (14)CO(2) from C2- and C3-pyruvate) listed after (a)-(e) above within the experimental errors, and gave rough estimates of the rates of pyruvate carboxylation, conversion of pyruvate and fat into acetyl-CoA and utilization of glutamate. The main conclusions were: (a) symmetrization of label in oxaloacetate both in the mitochondrion and in the cytoplasm was far from complete, because oxaloacetate did not equilibrate with fumarate in either. From this and other findings it was deduced: (b) that malate or fumarate or both left the mitochondrion, and not oxaloacetate; (c) that there was a loss from the mitochondrion of a fraction of the malate or fumarate or both formed from succinate, and (d) the resulting deficiency of oxaloacetate for the perpetuation of the tricarboxylic acid cycle was made up from pyruvate in fed and post-absorptive rats, but (e) in the starved rat could only be made up by utilization of glutamate. (f) In the fed rat the tricarboxylic acid cycle ran mostly on pyruvate, but in the post-absorptive and starved rat mostly on fat. (g) In the injured rat the tricarboxylic acid cycle was slowed, label in oxaloacetate was completely symmetrized (cf. conclusion a), and the tricarboxylic acid cycle utilized glutamate. (h) The conclusions were not invalidated by isotopic exchange, i.e. flux of label without net flux of compound, nor by interaction with lipogenic processes. (i) In the kidneys interaction between the tricarboxylic acid cycle and gluconeogenesis was different from in the liver, and was much less. The effects on the theory were roughly assessed, and were small. 4. The experiments and optimum experimental conditions required to check the theory are listed, and several predictions, open to experimental confirmation, are made. 相似文献
13.
M Mourtzakis T E Graham J González-Alonso B Saltin 《Journal of applied physiology》2008,105(2):547-554
Muscle glutamate is central to reactions producing 2-oxoglutarate, a tricarboxylic acid (TCA) cycle intermediate that essentially expands the TCA cycle intermediate pool during exercise. Paradoxically, muscle glutamate drops approximately 40-80% with the onset of exercise and 2-oxoglutarate declines in early exercise. To investigate the physiological relationship between glutamate, oxidative metabolism, and TCA cycle intermediates (i.e., fumarate, malate, 2-oxoglutarate), healthy subjects trained (T) the quadriceps of one thigh on the single-legged knee extensor ergometer (1 h/day at 70% maximum workload for 5 days/wk), while their contralateral quadriceps remained untrained (UT). After 5 wk of training, peak oxygen consumption (VO2peak) in the T thigh was greater than that in the UT thigh (P<0.05); VO2peak was not different between the T and UT thighs with glutamate infusion. Peak exercise under control conditions revealed a greater glutamate uptake in the T thigh compared with rest (7.3+/-3.7 vs. 1.0+/-0.1 micromol.min(-1).kg wet wt(-1), P<0.05) without increase in TCA cycle intermediates. In the UT thigh, peak exercise (vs. rest) induced an increase in fumarate (0.33+/-0.07 vs. 0.02+/-0.01 mmol/kg dry wt (dw), P<0.05) and malate (2.2+/-0.4 vs. 0.5+/-0.03 mmol/kg dw, P<0.05) and a decrease in 2-oxoglutarate (12.2+/-1.6 vs. 32.4+/-6.8 micromol/kg dw, P<0.05). Overall, glutamate infusion increased arterial glutamate (P<0.05) and maintained this increase. Glutamate infusion coincided with elevated fumarate and malate (P<0.05) and decreased 2-oxoglutarate (P<0.05) at peak exercise relative to rest in the T thigh; there were no further changes in the UT thigh. Although glutamate may have a role in the expansion of the TCA cycle, glutamate and TCA cycle intermediates do not directly affect VO2peak in either trained or untrained muscle. 相似文献
14.
Purification, kinetic behavior, and regulation of NAD(P)+ malic enzyme of tumor mitochondria 总被引:1,自引:0,他引:1
The purification and kinetic characterization of an NAD(P)+-malic enzyme from 22aH mouse hepatoma mitochondria are described. The enzyme was purified 328-fold with a final yield of 51% and specific activity of 38.1 units/mg of protein by employing DEAE-cellulose chromatography and an ATP affinity column. Sephadex G-200 chromatography yielded a native Mr = 240,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major subunit with Mr = 61,000, suggesting a tetrameric structure, and also showed that the preparation contained less than 10% polypeptide impurities. Use of the ATP affinity column required the presence of MnCl2 and fumarate (an allosteric activator) in the elution buffers. In the absence of fumarate, the Michaelis constants for malate, NAD+, and NADP+ were 3.6 mM, 55 microM, and 72 microM, respectively; in the presence of fumarate (2 mM), the constants were 0.34 mM, 9 microM, and 13 microM, respectively. ATP was shown to be an allosteric inhibitor, competitive with malate. However, the inhibition by ATP displayed hyperbolic competitive kinetics with a KI (ATP) of 80 microM (minus fumarate) and 0.5 mM (plus 2 mM fumarate). The allosteric properties of the enzyme are integrated into a rationale for its specific role in the pathways of malate and glutamate oxidation in tumor mitochondria. 相似文献
15.
Waagepetersen HS Sonnewald U Larsson OM Schousboe A 《Neurochemistry international》2000,36(4-5):349-358
Cultured neocortical neurons were incubated in medium containing [U-13C]glucose (0.5 mM) and in some cases unlabeled glutamine (0.5 mM). Subsequently the cells were "superfused" for investigation of the effect of depolarization by 55 mM K+. Cell extracts were analyzed by 13C magnetic resonance spectroscopy and gas chromatography/mass spectrometry to determine incorporation of 13C in glutamate, GABA, aspartate and fumarate. The importance of the tricarboxylic acid (TCA) cycle for conversion of the carbon skeleton of glutamine to GABA was evident from the effect of glutamine on the labeling pattern of GABA and glutamate. Moreover, analysis of the labeling patterns of glutamate in particular indicated a depolarization induced increased oxidative metabolism. This effect was only observed in glutamate and not in neurotransmitter GABA. Based on this a hypothesis of mitochondrial compartmentation may be proposed in which mitochondria associated with neurotransmitter synthesis are distinct from those aimed at energy production and influenced by depolarization. The hypothesis of mitochondrial compartmentation was further supported by the finding that the total percent labeling of fumarate and aspartate differed significantly from each other. This can only be explained by the existence of multiple TCA cycles with different turnover rates. 相似文献
16.
The regulation and glutamate metabolism by tricarboxylic acid-cycle activity in rat brain mitochondria 总被引:6,自引:0,他引:6
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1. The interrelationship of metabolism of pyruvate or 3-hydroxybutyrate and glutamate transamination in rat brain mitochondria was studied. 2. If brain mitochondria are incubated in the presence of equimolar concentrations of pyruvate and glutamate and the K(+) concentration is increased from 1 to 20mm, the rate of pyruvate utilization is increased 3-fold, but the rate of production of aspartate and 2-oxoglutarate is decreased by half. 3. Brain mitochondria incubated in the presence of a fixed concentration of glutamate (0.87 or 8.7mm) but different concentrations of pyruvate (0 to 1mm) produce aspartate at rates that decrease as the pyruvate concentration is increased. At 1mm-pyruvate, the rate of aspartate production is decreased to 40% of that when zero pyruvate was present. 4. Brain mitochondria incubated in the presence of glutamate and malate alone produce 2-oxoglutarate at rates stoicheiometric with the rate of aspartate production. Both the 2-oxoglutarate and aspartate accumulate extramitochondrially. 5. Externally added 2-oxoglutarate has little inhibitory effect (K(i) approx. 31mm) on the production of aspartate from glutamate by rat brain mitochondria. 6. It is concluded that the inhibitory effect of increased C(2) flux into the tricarboxylic acid cycle on glutamate transamination is caused by competition for oxaloacetate between the transaminase and citrate synthase. 7. Evidence is provided from a reconstituted malate-aspartate (or Borst) cycle with brain mitochondria that increased C(2) flux into the tricarboxylic acid cycle from pyruvate may inhibit the reoxidation of exogenous NADH. These results are discussed in the light of the relationship between glycolysis and reoxidation of cytosolic NADH by the Borst cycle and the requirement of the brain for a continuous supply of energy. 相似文献
17.
《Biochemical education》1999,27(3):153-154
Some interesting points arose in a discussion on the links between the urea cycle and the TCA cycle. That aspartate is regenerated from fumarate is well known. One of the prime precursors of urea, the bicarbonate ion, is also formed from the CO2, which is generated by the TCA cycle. The flux of acetyl CoA through the TCA cycle can indirectly affect the urea cycle by altering the levels of N-acetyl glutamate. 相似文献
18.
Hypoxia led to a dramatic acceleration of amino acid breakdown together with succinate synthesis in the rat heart. Our data do not confirm the simultaneous conversion of aspartate and glutamate to succinate, which has been repeatedly assumed in the literature (7, 8, 21, 28-30), but rather suggest that different pathways are involved during developing hypoxia and that glutamate is the sole source for anaerobic succinate production from endogenous sources in the glucose-perfused heart. Perfusion of hypoxic rat hearts with 2-oxoglutarate, malate, and fumarate (5 mM each) increased succinate formation three- to fourfold. The beneficial effects of these substances on left ventricular systolic pressure, end diastolic pressure, and time of recovery may be due to the elevated content of ATP in these hearts compared to hypoxic controls with glucose as the sole substrate. However, the maintenance of a high rate of anaerobic glycolysis in hearts perfused with 2-oxoglutarate, malate, and fumarate and not the small stimulation of succinate synthesis is considered to be the most important mechanism of cardiac protection. A proposed pathway assumes that malate, after dehydration to fumarate, may serve as an alternative electron acceptor for cytosolic NADH during conditions of oxygen deficiency, thereby cancelling glycolytic inhibition. 相似文献
19.
In aerobic respiration, the tricarboxylic acid cycle is pivotal to the complete oxidation of carbohydrates, proteins, and lipids to carbon dioxide and water. Plasmodium falciparum, the causative agent of human malaria, lacks a conventional tricarboxylic acid cycle and depends exclusively on glycolysis for ATP production. However, all of the constituent enzymes of the tricarboxylic acid cycle are annotated in the genome of P. falciparum, which implies that the pathway might have important, yet unidentified biosynthetic functions. Here we show that fumarate, a side product of the purine salvage pathway and a metabolic intermediate of the tricarboxylic acid cycle, is not a metabolic waste but is converted to aspartate through malate and oxaloacetate. P. falciparum-infected erythrocytes and free parasites incorporated [2,3-(14)C]fumarate into the nucleic acid and protein fractions. (13)C NMR of parasites incubated with [2,3-(13)C]fumarate showed the formation of malate, pyruvate, lactate, and aspartate but not citrate or succinate. Further, treatment of free parasites with atovaquone inhibited the conversion of fumarate to aspartate, thereby indicating this pathway as an electron transport chain-dependent process. This study, therefore, provides a biosynthetic function for fumarate hydratase, malate quinone oxidoreductase, and aspartate aminotransferase of P. falciparum. 相似文献
20.
Robert Rognstad 《Bulletin of mathematical biology》1995,57(4):557-568
A rather complete model of the gluconeogenic pathway was used, with the known separate pools of mitochondrial and cytosolic
oxalacetate, malate and aspartate. The fumarase, malate dehydrogenase and glutamate oxalacetate transaminase reactions were
assumed to be isotopically actively reversible, but none at isotopic equilibrium. Malate was assumed to exchange actively
between the mitochondrial and cytosol, while aspartate exchange was more limited, in agreement with the known electrogenic
nature of aspartate export from the mitochondria. This model was fit to14C data obtained in hepatocyte studies, and to the whole rat14C data obtained by Heath and Rose (Biochem J.
227, 851–876, 1985). The latter data were easily fit to our model, when a single mitochondrial oxalacetate pool was assumed.
However, invoking two mitochondrial oxalacetate pools, as proposed by Heath and Rose, with the oxalacetate formed via pyruvate
carboxylase preferentially channelled to gluconeogenesis, could not be fit with the known differences in scrambling in glucose
and glutamate produced from L[3-14C]lactate. 相似文献