Biomedical subjects

M Cascante

Publications and source records attributed to M Cascante.

At least 55 records · Page 3Linked to original sources

PubMed1998

Mass isotopomer study of the nonoxidative pathways of the pentose cycle with [1,2-13C2]glucose.

We present a single-tracer method for the study of the pentose phosphate pathway (PPP) using [1,2-13C2]glucose and mass isotopomer analysis. The metabolism of [1,2-13C2]glucose by the glucose-6-phosphate dehydrogenase, transketolase (TK), and transaldolase (TA) reactions results in unique pentose and lactate isotopomers with either one or two 13C substitutions. The distribution of these isotopomers was used to estimate parameters of the PPP using the model of Katz and Rognstad (J. Katz and R. Rognstad. Biochemistry 6: 2227-2247, 1967). Mass and position isotopomers of ribose, and lactate and palmitate (products from triose phosphate) from human hepatoma cells (Hep G2) incubated with 30% enriched [1,2-13C2]glucose were determined using gas chromatography-mass spectrometry. After 24-72 h incubation, 1.9% of lactate molecules in the medium contained one 13C substitution (m1) and 10% contained two 13C substitutions (m2). A similar m1-to-m2 ratio was found in palmitate as expected. Pentose cycle (PC) activity determined from incubation with [1,2-13C2]glucose was 5.73 +/- 0.52% of the glucose flux, which was identical to the value of PC (5.55 +/- 0.73%) determined by separate incubations with [1-13C] and [6-13C]glucose, 13C was found to be distributed in four ribose isotopomers ([1-13C]-, [5-13C]-, [1,2-13C2]-, and [4,5-13C2]ribose). The observed ribose isotopomer distribution was best matched with that provided from simulation by substituting 0.032 for TK and 0.85 for TA activity relative to glucose uptake into the model of Katz and Rognstad. The use of [1,2-13C2]glucose not only permits the determination of PC but also allows estimation of relative rates through the TK and TA reactions.

Carbon Isotopes↗

PubMed1998

Subtleties in control by metabolic channelling and enzyme organization.

Because of its importance to cell function, the free-energy metabolism of the living cell is subtly and homeostatically controlled. Metabolic control analysis enables a quantitative determination of what controls the relevant fluxes. However, the original metabolic control analysis was developed for idealized metabolic systems, which were assumed to lack enzyme-enzyme association and direct metabolite transfer between enzymes (channelling). We here review the recently developed molecular control analysis, which makes it possible to study non-ideal (channelled, organized) systems quantitatively in terms of what controls the fluxes, concentrations, and transit times. We show that in real, non-ideal pathways, the central control laws, such as the summation theorem for flux control, are richer than in ideal systems: the sum of the control of the enzymes participating in a non-ideal pathway may well exceed one (the number expected in the ideal pathways), but may also drop to values below one. Precise expressions indicate how total control is determined by non-ideal phenomena such as ternary complex formation (two enzymes, one metabolite), and enzyme sequestration. The bacterial phosphotransferase system (PTS), which catalyses the uptake and concomitant phosphorylation of glucose (and also regulates catabolite repression) is analyzed as an experimental example of a non-ideal pathway. Here, the phosphoryl group is channelled between enzymes, which could increase the sum of the enzyme control coefficients to two, whereas the formation of ternary complexes could decrease the sum of the enzyme control coefficients to below one. Experimental studies have recently confirmed this identification, as well as theoretically predicted values for the total control. Macromolecular crowding was shown to be a major candidate for the factor that modulates the non-ideal behaviour of the PTS pathway and the sum of the enzyme control coefficients.

Energy Metabolism↗

PubMed1997

Comparison of control analysis data using different approaches: modelling and experiments with muscle extract.

Experimental and model studies have been performed to characterize the control properties of hexokinase and phosphofructokinase in muscle glycolysis and to examine the nature of error associated with experimental flux control coefficient determinations. Different approaches of metabolic control analysis, classical titration, co-response analysis and kinetic modelling indicated that flux control coefficients could be reliably estimated experimentally for the upper part of glycolysis. The kinetic parameters applied to construct the mathematical model were determined in muscle extract under similar conditions used for flux studies. If the kinetic parameters of commercial enzymes are introduced into the model the control analysis data cannot be trusted. Co-response analysis can also be successfully applied to determination of the flux control coefficients of the system. However, the involvement of a rapid-equilibrium enzyme, such as glucose 6-phosphate isomerase, could result in estimation errors for the relevant co-response coefficients that are propagated into the elasticity matrix. If the co-response coefficients related to isomerase activity are replaced by the values obtained by kinetic modelling, the values of elasticities are correct. Our data also suggest that in the upper part of glycolysis hexokinase mainly controls the pathway flux whereas phosphofructokinase exerts dominant control on the turnover of internal metabolite stocks inside the system.

Animals↗

PubMed1997

Oxythiamine and dehydroepiandrosterone inhibit the nonoxidative synthesis of ribose and tumor cell proliferation.

This study investigates the significance of the glucose-6-phosphate dehydrogenase (G6PD) catalyzed oxidative and the transketolase (TK) catalyzed nonoxidative pentose cycle (PC) reactions in the tumor proliferation process by characterizing tumor growth patterns and synthesis of the RNA ribose moiety in the presence of respective inhibitors of G6PD and TK. Mass spectra analysis of 13C-labeled carbons revealed that these PC reactions contribute to over 85% of de novo ribose synthesis in RNA from [1,2-(13)C]glucose in cultured Mia pancreatic adenocarcinoma cells, with the fraction synthesized through the TK pathway predominating (85%). Five days of treatment with the TK inhibitor oxythiamine (OT) and the G6PD inhibitor dehydroepiandrosterone-sulfate (0.5 microM each) exerted a 39 and a 23% maximum inhibitory effect on cell proliferation in culture, which was increased to 60% when the two drugs were administered in combination. In vivo testing of 400 mg/kg OT or dehydroepiandrosterone-sulfate in C57BL/6 mice hosting Ehrlich's ascitic tumor cells revealed a 90.4 and a 46% decrease in the final tumor mass after 3 days of treatment. RNA ribose fractional synthesis through the TK reaction using metabolites directly from glycolysis declined by 9.1 and 23.9% after OT or the combined treatment, respectively. Nonoxidative PC reactions play a central regulating role in the carbon-recruiting process toward de novo nucleic acid ribose synthesis and cell proliferation in vitro and in vivo. Therefore, enzymes or substrates regulating the nonoxidative synthesis of ribose could also be the sites to preferentially target tumor cell proliferation by new anticancer drugs.

Animals↗

PubMed1997

Validation and steady-state analysis of a power-law model of purine metabolism in man.

The paper introduces a model of human purine metabolism in situ. Chosen from among several alternative system descriptions, the model is formulated as a Generalized Mass Action system within Biochemical Systems Theory and validated with analyses of steady-state and dynamic characteristics. Eigenvalue and sensitivity analyses indicate that the model has a stable and robust steady-state. The model quite accurately reproduces numerous biochemical and clinical observations in healthy subjects as well as in patients with disorders of purine metabolism. These results suggest that the model can be used to assess biochemical and clinical aspects of human purine metabolism. It provides a means of exploring effects of enzyme deficiencies and is a potential tool for identifying steps of the pathway that could be the target of therapeutical intervention. Numerous quantitative comparisons with data are given. The model can be used for biomathematical exploration of relationships between enzymic deficiencies and clinically manifested diseases.

Adenine↗

PubMed1997

How did glycogen structure evolve to satisfy the requirement for rapid mobilization of glucose? A problem of physical constraints in structure building.

Optimization of molecular design in cellular metabolism is a necessary condition for guaranteeing a good structure-function relationship. We have studied this feature in the design of glycogen by means of the mathematical model previously presented that describes glycogen structure and its optimization function [Meléndez-Hevia et al. (1993), Biochem J 295: 477-483]. Our results demonstrate that the structure of cellular glycogen is in good agreement with these principles. Because the stored glucose in glycogen must be ready to be used at any phase of its synthesis or degradation, the full optimization of glycogen structure must also imply the optimization of every intermediate stage in its formation. This case can be viewed as a molecular instance of the eye problem, a classical paradigm of natural selection which states that every step in the evolutionary formation of a functional structure must be functional. The glycogen molecule has a highly optimized structure for its metabolic function, but the optimization of the full molecule has meaning and can be understood only by taking into account the optimization of each intermediate stage in its formation.

1,4-alpha-Glucan Branching Enzyme↗

PubMed1996

The metabolic productivity of the cell factory.

It is widely accepted that some performance function has been optimized during the evolution of metabolic pathways. One can study the nature of such a function by analogy with the industrial manufacturing world, in which there have been efforts over recent decades to optimize production chains, and in which it is now accepted that fluxes are not the only important system variables that determine process efficiency, because inventory turnover must also be considered. Inspired by the parallels between living cells and manufacturing factories, we propose that fluxes and transit time may have simultaneously been major targets of natural selection in the optimization of the design, structure and kinetic parameters of metabolic pathways. Accordingly we define the ratio of flux to transit time as a performance index of productivity in metabolic systems: it measures the efficiency with which stocks are administered, and facilitates comparison of a pathway in different steady states or in different tissues or organisms. For a linear chain of two enzymes, at a fixed total equilibrium constant, we have analysed the variation of flux, transit time and productivity index as functions of the equilibrium constants of the two steps. The results show that only the productivity index has a maximum, which represents a good compromise in optimizing flux and transit time. We have extended control analysis to the productivity index and derived the summation theorem that applies to it. For linear chains of different length with maximum productivity index values, the distribution of control coefficients with regard to the three parameters has a characteristic profile independent of the length of the chain. Finally, this control profile changes when other variables are optimized, and we compare the theoretical results with the control profile of the first steps of glycolysis in rat liver.

Animals↗

PubMed1996

Molecular bureaucracy: who controls the delays? Transient times in branched pathways and their control.

Analysis of metabolic control has until now been mainly confined to systems at steady state. This includes studies of the control of "transition time", which is actually a steady-state transit time that does not refer to the transient state. In this paper we examine the control of the transition state of a metabolic pathway in the approach to a stable steady state, showing that the time needed to attain it can be decreased or increased in different branches. Our analysis only applies to branched pathways, and we discuss why similar deviations cannot occur in unbranched pathways. In systems with several branches the acceleration of some branches during the transient phase, so that they reach their steady states more quickly, occurs at the expense of others, which are thus delayed. We present theorems that describe properties of the transient variables and their control.

Animals↗

PubMed1996

Strong control on the transit time in metabolic channelling.

A suite of different characteristic times is used to describe the temporal behavior of a metabolic pathway. Here we focus on the 'transit' time, that is the average time it takes for a molecule, entering the steady-state pathway as a substrate, to exit the pathway as a product. We show that metabolic channelling results in dramatic changes in control exerted by pathway enzymes on the transit time. In an 'ideal' pathway a doubling of the enzyme concentrations halves the transit time. In a dynamic channel such an increase can reduce the transit time by a factor of four or more.

Enzymes↗

PubMed1996

Effect of channelling on the concentration of bulk-phase intermediates as cytosolic proteins become more concentrated.

This paper shows that metabolic channelling can provide a mechanism for decreasing the concentration of metabolites in the cytoplasm when cytosolic proteins become more concentrated. A dynamic complex catalysing the direct transfer of an intermediate is compared with the analogous pathway lacking a channel (an "ideal" pathway). In an ideal pathway a proportional increase in protein content does not result in a change in the steady-state concentration of the bulk-phase intermediate, whereas in a channelling pathway the bulk-phase intermediate either decreases or increases depending on the elemental rate constants within the enzyme mechanisms. When the concentration of the enzymes are equal, the pool size decreases with increasing protein concentration if the elemental step depleting the bulk-phase intermediate exerts more control on its concentration than the step supplying the intermediate. Results are illustrated numerically, and a simplified dynamic channel is analysed in which the concentration of the enzyme-enzyme forms. For such a "hit-and-run" channel it is shown that, when the product-releasing step of the enzyme located upstream is close to equilibrium, the pool size decreases as the concentrations of the enzymes increase in proportion, regardless of the rate, equilibrium constants and concentration ratios of the two sequential enzymes.

Cytosol↗

PubMed1996

The puzzle of the Krebs citric acid cycle: assembling the pieces of chemically feasible reactions, and opportunism in the design of metabolic pathways during evolution.

The evolutionary origin of the Krebs citric acid cycle has been for a long time a model case in the understanding of the origin and evolution of metabolic pathways: How can the emergence of such a complex pathway be explained? A number of speculative studies have been carried out that have reached the conclusion that the Krebs cycle evolved from pathways for amino acid biosynthesis, but many important questions remain open: Why and how did the full pathway emerge from there? Are other alternative routes for the same purpose possible? Are they better or worse? Have they had any opportunity to be developed in cellular metabolism evolution? We have analyzed the Krebs cycle as a problem of chemical design to oxidize acetate yielding reduction equivalents to the respiratory chain to make ATP. Our analysis demonstrates that although there are several different chemical solutions to this problem, the design of this metabolic pathway as it occurs in living cells is the best chemical solution: It has the least possible number of steps and it also has the greatest ATP yielding. Study of the evolutionary possibilities of each one-taking the available material to build new pathways-demonstrates that the emergence of the Krebs cycle has been a typical case of opportunism in molecular evolution. Our analysis proves, therefore, that the role of opportunism in evolution has converted a problem of several possible chemical solutions into a single-solution problem, with the actual Krebs cycle demonstrated to be the best possible chemical design. Our results also allow us to derive the rules under which metabolic pathways emerged during the origin of life.

Acetates↗

PubMed1995

Composite control of cell function: metabolic pathways behaving as single control units.

This paper shows that under some conditions the control exerted by a part of a metabolic network (a pathway) on a flux or concentration in any other part can be described through a single (overall) control coefficient. This has the following implications: (i) the relative contributions of a pathway enzyme to the regulation of the pathway (output) flux and of any flux or concentration outside are identical; therefore, the control analysis of the pathway 'in isolation' allows one to determine the control exerted by any pathway enzyme on the rest of the cell by estimation of the control efficient of just one, arbitrarily chosen enzyme; (ii) the relative control of any two metabolic variables outside the pathway (measured as the ratio of the control coefficients over these two variables outside) is the same for all pathway enzymes. These properties allow one to substitute effectively a pathway by a single (super)reaction and make it possible to consider such a pathway as a metabolic unit within the cellular enzyme network.

Cells↗

PubMed1995

Control analysis of transit time for free and enzyme-bound metabolites: physiological and evolutionary significance of metabolic response times.

Control analysis of transit time, defined as tau = delta/J, has previously been considered with the constraint of low enzyme concentrations compared with free pools of metabolites [Meléndez-Hevia, Torres, Sicilia and Kacser (1990) Biochem. J. 265, 195-202]. One of the conclusions was that the sum of the control coefficients of the transition time with respect to enzyme concentration was -1. Here we demonstrate that, if the enzyme-bound pools are taken into consideration (which would be important at high enzyme concentrations and high affinities), the sum lies between 0 and -1. The transition time between two steady states, which are frequent physiological events, is mainly governed by time constants involved in changing the enzyme concentrations. Some physiological and evolutionary aspects are discussed.

Enzymes↗

PubMed1995

Control theory of metabolic channelling.

Various factors appear to control muscle energetics, often in conjunction. This calls for a quantitative approach of the type provided by Metabolic Control Analysis for intermediary metabolism and mitochondrial oxidative phosphorylation. To the extent that direct transfer of high energy phosphates and spatial organization plays a role in muscle energetics however, the standard Metabolic Control Theory does not apply, neither do its theorems regarding control. This paper develops the Control Theory that does apply to the muscle system. It shows that direct transfer of high energy phosphates bestows a system with enhanced control: the sum of the control exerted by the participating enzymes on the flux of free energy from the mitochondrial matrix to the actinomyosin may well exceed the 100% mandatory for ideal metabolic pathways. It is also shown how sequestration of high energy phosphates may allow for negative control on pathway flux. The new control theory gives method functionally to diagnose the extent to which channelling and metabolite sequestration occur.

Animals↗

PubMed1995

A model of the pentose phosphate pathway in rat liver cells.

A mathematical model based on kinetic data taken from the literature is presented for the pentose phosphate pathway in fasted rat liver steady-state. Since the oxidative and non oxidative pentose phosphate pathway can act independently, the complete (oxidative+non oxidative) and the non oxidative pentose pathway were stimulated. Sensitivity analyses are reported which show that the fluxes are mainly regulated by D-glucose-6-phosphate dehydrogenase (for the oxidative pathway) and by transketolase (for the non oxidative pathway). The most influent metabolites were the group ATP, ADP, P1 and the group NADPH, NADP+ (for the non oxidative pathway).

Animals↗

PubMed1995

Elusive control.

The concept of a single rate-limiting step was proven to be too simplistic for understanding control and regulation of metabolism. Consequently, searches have identified relatively few steps with high control. Here we review a number of such searches and indicate what mechanisms may be responsible for this elusiveness of control. It turns out that this elusiveness of control has itself led to increased understanding of the roles played in metabolic control and regulation of such diverse factors as distributiveness of control, condition dependence, enzyme elasticity, homeostasis, control hierarchies, the input into a pathway, coenzyme sequestration, and redundancy and diversity of control function.

Animals↗

PubMed1995

Comparative characterization of the fermentation pathway of Saccharomyces cerevisiae using biochemical systems theory and metabolic control analysis: model definition and nomenclature.

Mathematical tools that involve the determination of systemic responses to small changes in metabolites or enzymes have demonstrated their utility for analyzing metabolic pathways. The different methodologies based on these ideas allow for modeling and analyzing biochemical pathways focusing on the coordinate behavior of the whole system. However, one must become familiar with the difference in nomenclature and methodology to relate the models and results obtained by applying these techniques and to appreciate their potential for answering fundamental questions about biochemical systems. In the following three papers we show how this can be facilitated by comparing the nomenclature, methodology, and results of the two leading techniques in this area, metabolic control analysis and biochemical systems theory, using a model of the fermentation pathway in Saccharomyces cerevisiae as a reference system. In the present paper we review the nomenclature, technical concepts, and related experimental measurements while creating a practical dictionary for the reference system that makes the relatedness of the two approaches more apparent. In the second paper, subtitled Steady-State Analysis, we show that both approaches give the same picture for many systemic responses of the reference system. In the third paper of this series, subtitled Model Validation and Dynamic Behavior, we show that the quality of the model can be assessed by studying the sensitivity to changes in the system parameters. We hope to illustrate the usefulness of these tools in providing an interpretation of the experimental measurements in a specific metabolic pathway.

Fermentation↗

PubMed1995

Comparative characterization of the fermentation pathway of Saccharomyces cerevisiae using biochemical systems theory and metabolic control analysis: steady-state analysis.

In the preceding paper in this issue, we have shown that metabolic control analysis and biochemical systems theory use the same experimental information to describe a metabolic system. In this paper, we analyze the steady-state properties of this pathway by applying both methods. Our results show the correspondence of the steady-state characterizations and illustrate the relationships between the different nomenclatures used. With both approaches, we identify metabolite pools that are strongly influenced by changes in enzyme concentration when cells are immobilized at pH 5.5. In the final paper of this series, which follows, we discuss the need to assess the quality of a model and the potential difficulties that may arise if the steady-state characterization is accepted without testing its quality. We then validate the different models using parameter sensitivity concepts.

Fermentation↗