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Néstor V. Torres - One of the best experts on this subject based on the ideXlab platform.
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Metabolic Control Analysis of Inhibitory feedback Interaction: Application to Biotechnological Processes
Journal of theoretical biology, 1996Co-Authors: Néstor V. TorresAbstract:Based on a simplified model of a linear metabolic pathway (Kacser & Burns, 1973), and on the principles of Metabolic Control Analysis, an equation useful for predicting the maximum Flux through a system after removing inhibitory feedback interaction is presented. The analysis shows that only when the Flux Control Coefficient of the inhibited enzyme is significant (greater than 0.5) is it worthwhile to act on the regulatory features of the feedback interaction. The approach is then applied to a case study and the magnitude of the Flux increase and the quality of the prediction studied in two different profiles of Flux Control Coefficients.
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Control analysis of rat liver glycolysis under different glucose concentrations. The substrate approach and the role of glucokinase
Molecular and Cellular Biochemistry, 1992Co-Authors: Enrique Meléndez-hevia, Fátima Mateo, Néstor V. TorresAbstract:Control Analysis has been carried out in the first steps of a rat liver glycolytic system. Attention has been focused on the effect of several glucose concentrations on the Control, particularly regarding the role of glucokinase. From kinetic studies of the whole metabolic system we have obtained information on the Flux variation under different glucose concentrations. This information together with the kinetics of glucokinase has allowed us to calculate Flux Control and Elasticity Coefficients for glucokinase and the Response Coefficient of the system with respect to glucose. The changes in of the value of Flux Control Coefficients demonstrates that in conditions of low glucose concentration, glucokinase is the main enzyme in Controlling the Flux through the pathway, but at high glucose concentration the Control moves to phosphofructokinase. Next, we have compared our results with those obtained with the shortening and titration method, previously described (Torres, N.V., Mateo, F., Mélendez-Hevia, E. and Kacser, H., (1986) Biochem. J. 234, 169–174; Torres, N.V. and Meléndez-Hevia, E. 1991. Molec. Cell. Biochem. 101, 1–10). Furthermore, from knowledge of the enzyme kinetics of the system we have been able to build a model of the pathway that allows us computer similation of its behavior and calculation of the Flux Control Coefficient profile at different glucose concentrations. By the three methods the results correlate, supporting the use of the pathway substrate as external modulator of the metabolic system as a tool for practical application of Control Analysis.
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Detailed protocol and critical view for the analysis of Control in metabolic systems by shortening and enzyme titration
Molecular and Cellular Biochemistry, 1991Co-Authors: Néstor V. Torres, Enrique Meléndez-heviaAbstract:In this paper we give a general description of the ‘shortening and enzyme titration method’. This method allows us to determine the Flux Control Coefficients of the different steps of a metabolic pathway in an in vitro experimental system. The system submitted to study is shortened in vitro by means of auxiliary enzymes, and the shortened pathway is titrated with extraneous enzymes. In this way we can modulate the activity of every enzyme of the system and thus every Flux Control Coefficient can be obtained. We criticise its different features in order to comment on the possibilities of its application to different types of systems. Our conclusion is that the method has a general applicability provided: a) that a correct definition of the metabolic pathway is given; b) that the system occurs in only one subcellular fraction and, c) that the dilution of the system by a given factor drives to the same reduction in every enzyme activity of the system.
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Control of glycolysis in rat liver by glucokinase and phosphofructokinase: influence of glucose concentration
Molecular and Cellular Biochemistry, 1990Co-Authors: Néstor V. Torres, Fátima Mateo, José M. Riol-cimas, Enrique Meléndez-heviaAbstract:Control of glucose metabolism in rat liver under different glucose concentrations was studied. Flux Control Coefficients of glucokinase, glucose 6-phosphate isomerase and phosphofructokinase were determined by the ‘shortening and enzyme titration’ method. Results obtained show that glucose concentration in liver can play an important role in Control of liver glycolysis by enhancing the Flux Control Coefficient of phosphofructokinase. Possible physiological significance of this fact is discussed.
Enrique Meléndez-hevia - One of the best experts on this subject based on the ideXlab platform.
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Control analysis of rat liver glycolysis under different glucose concentrations. The substrate approach and the role of glucokinase
Molecular and Cellular Biochemistry, 1992Co-Authors: Enrique Meléndez-hevia, Fátima Mateo, Néstor V. TorresAbstract:Control Analysis has been carried out in the first steps of a rat liver glycolytic system. Attention has been focused on the effect of several glucose concentrations on the Control, particularly regarding the role of glucokinase. From kinetic studies of the whole metabolic system we have obtained information on the Flux variation under different glucose concentrations. This information together with the kinetics of glucokinase has allowed us to calculate Flux Control and Elasticity Coefficients for glucokinase and the Response Coefficient of the system with respect to glucose. The changes in of the value of Flux Control Coefficients demonstrates that in conditions of low glucose concentration, glucokinase is the main enzyme in Controlling the Flux through the pathway, but at high glucose concentration the Control moves to phosphofructokinase. Next, we have compared our results with those obtained with the shortening and titration method, previously described (Torres, N.V., Mateo, F., Mélendez-Hevia, E. and Kacser, H., (1986) Biochem. J. 234, 169–174; Torres, N.V. and Meléndez-Hevia, E. 1991. Molec. Cell. Biochem. 101, 1–10). Furthermore, from knowledge of the enzyme kinetics of the system we have been able to build a model of the pathway that allows us computer similation of its behavior and calculation of the Flux Control Coefficient profile at different glucose concentrations. By the three methods the results correlate, supporting the use of the pathway substrate as external modulator of the metabolic system as a tool for practical application of Control Analysis.
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Detailed protocol and critical view for the analysis of Control in metabolic systems by shortening and enzyme titration
Molecular and Cellular Biochemistry, 1991Co-Authors: Néstor V. Torres, Enrique Meléndez-heviaAbstract:In this paper we give a general description of the ‘shortening and enzyme titration method’. This method allows us to determine the Flux Control Coefficients of the different steps of a metabolic pathway in an in vitro experimental system. The system submitted to study is shortened in vitro by means of auxiliary enzymes, and the shortened pathway is titrated with extraneous enzymes. In this way we can modulate the activity of every enzyme of the system and thus every Flux Control Coefficient can be obtained. We criticise its different features in order to comment on the possibilities of its application to different types of systems. Our conclusion is that the method has a general applicability provided: a) that a correct definition of the metabolic pathway is given; b) that the system occurs in only one subcellular fraction and, c) that the dilution of the system by a given factor drives to the same reduction in every enzyme activity of the system.
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Control of glycolysis in rat liver by glucokinase and phosphofructokinase: influence of glucose concentration
Molecular and Cellular Biochemistry, 1990Co-Authors: Néstor V. Torres, Fátima Mateo, José M. Riol-cimas, Enrique Meléndez-heviaAbstract:Control of glucose metabolism in rat liver under different glucose concentrations was studied. Flux Control Coefficients of glucokinase, glucose 6-phosphate isomerase and phosphofructokinase were determined by the ‘shortening and enzyme titration’ method. Results obtained show that glucose concentration in liver can play an important role in Control of liver glycolysis by enhancing the Flux Control Coefficient of phosphofructokinase. Possible physiological significance of this fact is discussed.
Wolfram S Kunz - One of the best experts on this subject based on the ideXlab platform.
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Differences in Flux Control and reserve capacity of cytochrome c oxidase (COX) in human skeletal muscle and brain suggest different metabolic effects of mild COX deficiencies.
Molecular biology reports, 2002Co-Authors: Alexei Kudin, Stefan Vielhaber, Christian E Elger, Wolfram S KunzAbstract:To evaluate tissue specific Control of oxidative phosphorylation by cytochrome c oxidase (COX) we determined the Flux Control Coefficient and the metabolic reserve capacity of this enzyme in human saponin-permeabilised muscle fibers and digitonin-treated parahippocampal homogenates. In these tissue preparations it is possible to investigate mitochondrial function under conditions which are close to the in vivo situation. In the presence of NAD-dependent substrates we observed, under active state conditions, a Flux Control Coefficient of COX over oxidative phosphorylation of 0.24 +/- 0.07 and a 1.9 +/- 0.2-fold excess capacity in human skeletal muscle fibers. In human parahippocampal gyrus we determined, under similar conditions, a Flux Control Coefficient of COX of 0.12 +/- 0.05 and a 3.9 +/- 0.6-fold excess capacity of the enzyme. The observed difference in metabolic Control can be attributed to activity differences of COX in human brain and muscle mitochondria. Our results predict stronger metabolic effects of mild COX activity deficits in human skeletal muscle than in brain tissue.
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Metabolic consequences of the cytochrome c oxidase deficiency in brain of copper-deficient Mo(vbr) mice.
Journal of neurochemistry, 2001Co-Authors: Wolfram S Kunz, Andrey V. Kuznetsov, Joseph F. Clark, Irene Tracey, Christian E ElgerAbstract:Abstract: Biochemical micromethods were used for the investigation of changes in mitochondrial oxidative phosphorylation associated with cytochrome c oxidase deficiency in brain cortex from Movbr (mottled viable brindled) mice, an animal model of Menkes’ copper deficiency syndrome. Enzymatic analysis of cortex homogenates from Movbr mice showed an approximately twofold decrease in cytochrome c oxidase and a 1.4-fold decrease in NADH:cytochrome c reductase activities as compared with Controls. Assessment of mitochondrial respiratory function was performed using digitonin-treated homogenates of the cortex, which exhibited the main characteristics of isolated brain mitochondria. Despite the substantial changes in respiratory chain enzyme activities, no significant differences were found in maximal pyruvate or succinate oxidation rates of brain cortex homogenates from Movbr and Control mice. Inhibitor titrations were used to determine Flux Control Coefficients of NADH:CoQ oxidoreductase and cytochrome c oxidase on the rate of mitochondrial respiration. Application of amobarbital to titrate the activity of NADH:CoQ oxidoreductase showed very similar Flux Control Coefficients for Control and mutant animals. Alternately, titration of respiration with azide revealed for Movbr mice significantly sharper inhibition curves than for Controls, indicating a more than twofold elevated Flux Control Coefficient of cytochrome c oxidase. Owing to the reserve capacity of respiratory chain enzymes, the reported changes in activities do not seem to affect whole-brain high-energy phosphates, as observed in a previous study using 31P NMR.
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Flux Control of cytochrome c oxidase in human skeletal muscle.
The Journal of biological chemistry, 2000Co-Authors: Wolfram S Kunz, Alexei Kudin, Stefan Vielhaber, Christian E Elger, Giuseppe Attardi, Gaetano VillaniAbstract:In the present work, by titrating cytochrome c oxidase (COX) with the specific inhibitor KCN, the Flux Control Coefficient and the metabolic reserve capacity of COX have been determined in human saponin-permeabilized muscle fibers. In the presence of the substrates glutamate and malate, a 2.3 ± 0.2-fold excess capacity of COX was observed in ADP-stimulated human skeletal muscle fibers. This value was found to be dependent on the mitochondrial substrate supply. In the combined presence of glutamate, malate, and succinate, which supported an approximately 1.4-fold higher rate of respiration, only a 1.4 ± 0.2-fold excess capacity of COX was determined. In agreement with these findings, the Flux Control of COX increased, in the presence of the three substrates, from 0.27 ± 0.03 to 0.36 ± 0.08. These results indicate a tight in vivo Control of respiration by COX in human skeletal muscle. This tight Control may have significant implications for mitochondrial myopathies. In support of this conclusion, the analysis of skeletal muscle fibers from two patients with chronic progressive external ophthalmoplegia, which carried deletions in 11 and 49% of their mitochondrial DNA, revealed a substantially lowered reserve capacity and increased Flux Control Coefficient of COX, indicating severe rate limitations of oxidative phosphorylation by this enzyme.
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Distribution of Flux Control among the enzymes of mitochondrial oxidative phosphorylation in calcium-activated saponin-skinned rat musculus soleus fibers.
European journal of biochemistry, 1995Co-Authors: Elke Wisniewski, Frank N. Gellerich, Wolfram S KunzAbstract:Metabolic Control analysis was applied to describe the Control of mitochondrial oxidative phosphorylation in calcium (approximately 2 microM free calcium) activated saponin-skinned rat musculus soleus fibers oxidizing glutamate and malate. Under these circumstances approximately 80% of mitochondrial active-state respiration was reached due to the activation of ATP turnover by actomyosin ATPase. The Flux Control Coefficients of H(+)-ATPase, adenine-nucleotide translocase, phosphate transporter, NADH:ubiquinone oxidoreductase and cytochrome-c oxidase were determined to be equal to 0.16 +/- 0.08 (n = 6), 0.34 +/- 0.12 (n = 5), 0.08 +/- 0.03 (n = 5), 0.01 +/- 0.006 (n = 4) and 0.09 +/- 0.03 (n = 3) using inhibitor titrations with the specific inhibitors oligomycin, carboxyatractyloside, mersalyl, rotenone and cyanide, respectively, and applying non-linear regression of the entire titration curve. The Flux Control Coefficient of actomyosin ATPase was determined with vanadate to be equal to 0.50 +/- 0.09 (n = 6), measuring independently the vanadate-caused inhibition of fiber respiration and ATP-splitting activity. In contrast to results with isolated rat skeletal muscle mitochondria reconstituted with soluble F1-ATPase the decrease in phosphate concentration from 10 mM to 1 mM only slightly affected the distribution of Flux Control Coefficients. This difference is caused by different kinetic properties of soluble F1-ATPase and actomyosin ATPase. Therefore, phosphate seems to be in skeletal muscle in vivo only a modest modulator of Control of oxidative phosphorylation.
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Phosphate affects the distribution of Flux Control among the enzymes of oxidative phosphorylation in rat skeletal muscle mitochondria.
The Journal of biological chemistry, 1993Co-Authors: E Wisniewski, Wolfram S Kunz, Frank N. GellerichAbstract:Abstract The Flux Control Coefficients of adenine nucleotide translocase, the phosphate transporter, and H(+)-ATPase were determined in rat skeletal muscle mitochondria using glutamate plus malate as substrates and soluble F1-ATPase as load enzyme. It was observed that the Flux Control Coefficients of adenine nucleotide translocase, H(+)-ATPase, and the load enzyme F1-ATPase, at comparable rates of respiration, strongly depend on the phosphate concentration in the incubation medium. So, the Flux Control exerted by adenine nucleotide translocase, in the intermediate states of mitochondrial respiration (approximately 120 nmol of O2/min/mg) at 10 mM phosphate, was found to be about 0.37. At a phosphate concentration of 1 mM and comparable rates of respiration the Flux Control Coefficient of the translocase decreased to about 0.20. Under these conditions, a sharp increase in the Controlling influence of H(+)-ATPase from 0.10 to 0.74 was detected. Furthermore, at this Flux rate, the sum of Flux Control Coefficients of adenine nucleotide translocase, H(+)-ATPase, phosphate transporter, and the load enzyme F1-ATPase was noted to be very close to unity. This indicates that under the conditions of intermediate state respiration, all of the other reactions have a negligible Controlling influence on oxidative phosphorylation in skeletal muscle mitochondria.
Rafael Moreno-sánchez - One of the best experts on this subject based on the ideXlab platform.
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The bifunctional aldehyde-alcohol dehydrogenase Controls ethanol and acetate production in Entamoeba histolytica under aerobic conditions.
FEBS letters, 2012Co-Authors: Erika Pineda, Rusely Encalada, Alfonso Olivos-garcía, Mario Nequiz, Rafael Moreno-sánchez, Emma SaavedraAbstract:By applying metabolic Control analysis and inhibitor titration we determined the degree of Control (Flux Control Coefficient) of pyruvate:ferredoxin oxidoreductase (PFOR) and bifunctional aldehyde–alcohol dehydrogenase (ADHE) over the Fluxes of fermentative glycolysis of Entamoeba histolytica subjected to aerobic conditions. The Flux-Control Coefficients towards ethanol and acetate formation determined for PFOR titrated with diphenyleneiodonium were 0.07 and 0.09, whereas for ADHE titrated with disulfiram were 0.33 and −0.19, respectively. ADHE inhibition induced significant accumulation of glycolytic intermediates and lower ATP content. These results indicate that ADHE exerts significant Flux-Control on the carbon end-product formation of amoebas subjected to aerobic conditions.
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Metabolic Changes Induced by Cold Stress in Rat Liver Mitochondria
Journal of Bioenergetics and Biomembranes, 2001Co-Authors: Concepción Bravo, Martín Vargas-suárez, Sara Rodríguez-enríquez, Herminia Loza-tavera, Rafael Moreno-sánchezAbstract:The mechanisms involved in the metabolic changes induced by cold stress in isolated rat liver mitochondria were studied. Respiration, ATP synthesis, and membrane potential as well as the contents of several metabolites were determined in liver mitochondria from cold-exposed rats. At different times of cold exposure, the force–Flux relationships showed net variation in Flux (enhanced respiration, diminished ATP synthesis) with no associated variation in force (H^+ gradient); this suggested that decoupling rather than classical uncoupling was involved in the effects of cold stress. The Flux Control Coefficient of the H^+ leak on basal respiration was slightly increased by 380 h of cold exposure. Cold stress also induced a diminution in total membrane fatty acids, Zn^2+, Fe^3+, ATP, and ADP/O ratios; the content of cytochromes c + c _1 and b oscillated. The contents of Ca^2+, Na^+, P_i, and cytochromes a + a _3 were not affected, whereas matrix ADP, AMP, K^+, and Mg^2+ were markedly increased. Basal and oleic acid-stimulated respiration of mitochondria from cold-stressed rats was inhibited by GDP, carboxyatractyloside, or albumin. These agents did not affect basal respiration in Control mitochondria. Western blot analysis showed enhanced expression of a protein of about 35 kDa, presumably the uncoupling protein 2, induced by long-term cold exposure. The overall data suggest that cold stress promoted decoupling of oxidative phosphorylation, and hence, changes in several matrix metabolites, by increasing free fatty acids and the UCP2 content.
Frédéric Cadet - One of the best experts on this subject based on the ideXlab platform.
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Identification of Flux checkpoints in a metabolic pathway through white-box, grey-box and black-box modeling approaches
Scientific Reports, 2020Co-Authors: Ophélie Lo-thong, Emma Saavedra, Philippe Charton, Xavier Cadet, Brigitte Grondin-perez, Cédric Damour, Frédéric CadetAbstract:Metabolic pathway modeling plays an increasing role in drug design by allowing better understanding of the underlying regulation and Controlling networks in the metabolism of living organisms. However, despite rapid progress in this area, pathway modeling can become a real nightmare for researchers, notably when few experimental data are available or when the pathway is highly complex. Here, three different approaches were developed to model the second part of glycolysis of E. histolytica as an application example, and have succeeded in predicting the final pathway Flux: one including detailed kinetic information (white-box), another with an added adjustment term (grey-box) and the last one using an artificial neural network method (black-box). Afterwards, each model was used for metabolic Control analysis and Flux Control Coefficient determination. The first two enzymes of this pathway are identified as the key enzymes playing a role in Flux Control. This study revealed the significance of the three methods for building suitable models adjusted to the available data in the field of metabolic pathway modeling, and could be useful to biologists and modelers.