Mathematical modelling of glycolysis and adenine nucleotide metabolism of human erythrocytes. I. Reaction-kinetic statements, analysis of in vivo state and determination of starting conditions for in vitro experiments

A mathematical model of energy metabolism of human red cells is presented, which includes besides the glycolytic reactions the adenine nucleotide metabolism. The model is based on the network of chemical reactions, the thermodynamic equilibrium constants of fast reversible reactions and on the kinetic equations for irreversible enzyme reactions. The model consists of a system of 16 differential equations and allows the mathematical evaluation of metabolic levels in the steady state of energy metabolism corresponding to the in vivo state erythrocytes with the kinetic data for the enzymes derived from in vitro experiments. The dependence of the levels of metabolites in the steady state on the activity of some enzymes is analysed to characterize the regulatory properties of the system. The comparison of the steady state levels of the model with experimental data makes it possible to estimate values of some controversial enzyme parameters. Estimates of the kinetic parameters of the following intracellular processes are presented: 1) rate constant of AMP-phosphatase, 2) maximum rate of adenylate deaminase, 3) activity of adenine phosphoribosylpyrophosphate transferase and 4) adenosine transport through the cell membrane. The simulation of the preparatory phase before incubation of erythrocytes indicates, that the model also permits to compute the time course of changes of levels of metabolites. To solve the initial problem the stiff differential equation system is integrated numerically by an efficient program without the application of the quasi-steady-state approximation.     

The role of metabolic memory in the ATP paradox and energy homeostasis

In yeast, a sudden transition from glucose limitation to glucose excess leads to a new steady state at increased metabolic fluxes with a sustained decrease in the ATP concentration. Although this behaviour has been rationalized as an adaptive metabolic strategy, the mechanism behind it remains unclear. Nevertheless, it is thought that, on glucose addition, a metabolite derived from glycolysis may up-regulate ATP-consuming reactions. The adenine nucleotides themselves have been ruled out as the signals that mediate this regulation. This is mainly because, in that case, it would be expected that the new steady state at increased fluxes would be accompanied by an increased stationary ATP concentration. In this study, we present a core model consisting of a monocyclic interconvertible enzyme system. Using a supply-demand approach, we demonstrate that this system can account for the empirical observations without involving metabolites other than the adenine nucleotides as effectors. Moreover, memory is an emerging property of such a system, which may allow the cell to sense both the current energy status and the direction of the changes.     

Sucrose is metabolised by sucrose synthase and glycolysis within the phloem complex of Ricinus communis L. seedlings

Metabolites and enzyme activities were measured in the phloem sap exuding from a cut hypocotyl of germinating castor-bean (Ricinus communis L.) seedlings. The sap contained considerable quantities of adenine nucleotides, uridine nucleotides, uridine diphosphoglucose (UDPGlc), all the major phosphorylated metabolites required for glycolysis, fructose-2,6-bisphosphate and pyrophosphate. Supplying 200 mM glucose instead of sucrose to the cotyledons resulted in high concentrations of glucose in the sap, but did not modify the metabolite levels. In contrast, when 200 mM fructose was supplied we found only a low level of fructose but a raised sucrose concentration in the sap, which was accompanied by a three-to fourfold decrease of UDPGlc, and an increase of pyrophosphate, UDP and UTP. The measured levels of metabolites are used to estimate the molar mass action ratios and in-vivo free-energy change associated with the various reactions of sucrose breakdown and glycolysis in the phloem. It is concluded that the reactions catalysed by ATP-dependent phosphofructokinase and pyruvate kinase are removed from equilibrium in the phloem, whereas the reactions catalysed by sucrose synthase, UDPGlc-pyrophosphorylase, phosphoglucose mutase, phosphoglucose isomerase, aldolase, triose-phosphate isomerase, phosphoglycerate mutase and enolase are close to equilibrium within the conducting elements of the phloem. Since the exuded sap contained negligible or undetectable activities of the enzymes, it is concluded, that the responsible proteins are bound, or sequesterd behind plasmodesmata, possibly in the companion cells. It is argued that sucrose mobilisation via a reversible reaction catalysed by sucrose synthase is particularily well suited to allow the rate of sucrose breakdown in the phloem to respond to changes in the metabolic requirement for ATP, and for UDPGlc during callose production. It is also calculated that the transport of nucleotides in the phloem sap implies that there must be a very considerable uptake or de-novo biosynthesis of these cofactors in the phloem.     

Some thoughts on the evolutionary basis for the prominent role of ATP and ADP in cellular energy metabolism

The predominance of the adenosine triphosphate/adenosine diphosphate (ATP/ADP) couple in cellular phosphorylation reactions, including those that form the basis for cellular energy metabolism, cannot be explained on thermodynamic grounds since a variety of "high energy phosphate" compounds (including ADP itself) found in the cell would, based on thermodynamic considerations, be at least as effective as ATP in serving as a phosphoryl donor. How then did present-day organisms come to rely on the ATP/ADP couple as the principal mediator of phosphorylation reactions? The early appearance of adenine compounds in the prebiotic environment is suggested by experiments indicating that, relative to other purine or pyridimine compounds, adenine derivatives are preferentially synthesized under simulated prebiotic conditions (Ponnamperuma et al., 1963). In addition to the roles of adenine nucleotides in phosphorylation reactions, other adenine derivatives (e.g. Coenzyme A, flavin adenine dinucleotide, puridine nucleotides) are employed in a variety of metabolic roles. The principal function of the adenine moiety in these latter cases is in the binding of these derivatives to the relevant enzyme. The capability for binding of the adenine moiety appears to have arisen early in evolution and been exploited in a multitude of contexts, a suggestion consistent with observed similarities between the binding sites of several enzymes employing adenine derivatives as substrate. The early availability of suitable adenine compounds in the biosphere and development of complementary binding sites on cellular proteins, coupled with the expected advantages in having a limited number of metabolites as central mediators of endergonic and exergonic metabolism could readily have led to the observed pre-eminence of adenine nucleotides in cellular energy metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo analysis of metabolic dynamics in Saccharomyces cerevisiae : I. Experimental observations

The goal of this work was to obtain rapid sampling technique to measure transient metabolites in vivo. First, a pulse of glucose was added to a culture of the yeast Saccharomyces cerevisiae growing aerobically under glucose limitation. Next, samples were removed at 2 to 5 s intervals and quenched using methods that depend on the metabolite measured. Extracellular glucose, excreted products, as well as glycolytic intermediates (G6P, F6P, FBP, GAP, 3-PG, PEP, Pyr) and cometabolites (ATP, ADP, AMP, NAD(+), NADH) were measured using enzymatic or HPLC methods. Significant differences between the adenine nucleotide concentrations in the cytoplasm and mitochondria indicated the importance of compartmentation for the regulation of the glycolysis. Changes in the intra- and extracellular levels of metabolites confirmed that glycolysis is regulated on a time scale of seconds. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 305-316, 1997.     

基于文献挖掘的巨大芽胞杆菌代谢网络模型的构建与分析

【目的】通过挖掘实验性文献,建立巨大芽胞杆菌事实型代谢网络模型,以详尽解析生理特性,优化其生理功能。【方法】从PubMed、Derwent Innovations Index、中国知网等公共文献(专利)数据库中获取与巨大芽胞杆菌(Bacillus megaterium)相关的实验性文献建立本地文献数据库。采用文献挖掘工具获取功能基因、酶、代谢物和生化反应等信息,以其为基础构建代谢网络粗模型,进一步借助KEGG等数据库修正以及Matlab程序的模拟得到精细模型(系统生物学标记语言的形式)。【结果】最终的精细模型共有292个生化反应、378个代谢物、220个酶和217个基因。以1.62 mmol/g cell/h的葡萄糖底物吸收速率为限制性条件,模拟的菌体比生长速率为0.089 h-1,略低于实验值0.11 h-1。此外,嘧啶代谢途径的单基因敲除模拟结果表明,准确率为90%。【结论】该代谢网络模型涵盖了中心代谢途径、维生素B12合成途径和氨基酸代谢途径,并在一定程度上反映了营养底物与基因对巨大芽胞杆菌生长性能的影响。     

Estradiol stimulates the biosynthetic pathways of breast cancer cells: detection by metabolic flux analysis

Selective estrogen receptor (ER) modulators are highly successful breast cancer therapies, but they are not effective in patients with ER negative and selective estrogen receptor modulator (SERM)-resistant tumors. Understanding the mechanisms of estrogen-stimulated proliferation may provide a route to design estrogen-independent therapies that would be effective in these patients. In this study, metabolic flux analysis was used to determine the intracellular fluxes that are significantly affected by estradiol stimulation in MCF-7 breast cancer cells. Intracellular fluxes were calculated from nuclear magnetic resonance (NMR)-generated isotope enrichment data and extracellular metabolite fluxes, using a specific flux analysis algorithm. The metabolic pathway model used by the algorithm includes glycolysis, the tricarboxylic acid cycle (TCA cycle), the pentose phosphate pathway, glutamine catabolism, pyruvate carboxylase, and malic enzyme. The pathway model also incorporates mitochondrial compartmentalization and reversible trans-mitochondrial membrane reactions to more accurately describe the role of mitochondria in cancer cell proliferation. Flux results indicate that estradiol significantly increases carbon flow through the pentose phosphate pathway and increases glutamine consumption. In addition, intra-mitochondrial malic enzyme was found to be inactive and the malate-aspartate shuttle (MAS) was only minimally active. The inactivity of these enzymes indicates that glutamine is not oxidized within mitochondria, but is consumed primarily to provide biosynthetic precursors. The excretion of glutamine carbons from the mitochondria has the secondary effect of limiting nicotinamide adenine dinucleotide (NADH) recycle, resulting in NADH buildup in the cytosol and the excretion of lactate. The observed dependence of breast cancer cells on pentose phosphate pathway activity and glutamine consumption for estradiol-stimulated biosynthesis suggests that these pathways may be targets for estrogen-independent breast cancer therapies.     

Changes in intracellular metabolite pools during growth of adherent MDCK cells in two different media

In bioprocess engineering, the growth of continuous cell lines is mainly studied with respect to the changes in cell concentration, the resulting demand for substrates, and the accumulation of extracellular metabolites. The underlying metabolic process rests upon intracellular metabolite pools and their interaction with enzymes in the form of substrates, products, or allosteric effectors. Here, we quantitatively analyze time courses of 29 intracellular metabolites of adherent Madin–Darby canine kidney cells during cultivation in a serum-containing medium and a serum-free medium. The cells, which originated from the same pre-culture, showed similar overall growth behavior and only slight differences in their demand for the substrates glucose (GLC), glutamine (GLN), and glutamate (GLU). Analysis of intracellular metabolites, which mainly cover the glycolytic pathway, the citric acid cycle, and the nucleotide pools, revealed surprisingly similar dynamics for both cultivation conditions. Instead of a strong influence of the medium, we rather observed a growth phase-specific behavior in glycolysis and in the lower citric acid cycle. Furthermore, analysis of the lower part of glycolysis suggests the well-known regulation of pyruvate kinase by fructose 1,6-bisphosphate. The upper citric acid cycle (citrate, cis-aconitate, and isocitrate) is apparently uncoupled from the lower part (α-ketoglutarate, succinate, fumarate, and malate), which is in line with the characteristics of a truncated cycle. Decreased adenosine triphosphate and guanosine triphosphate pools, as well as a relatively low energy charge soon after inoculation of cells, indicate a high demand for cellular energy and the consumption of nucleotides for biosynthesis. We finally conclude that, with sufficient availability of substrates, the dynamics of GLC and GLN/GLU metabolism is influenced mainly by the cellular growth regime and regulatory function of key enzymes.     

Kinetic simulation of anticancer drug effects on metabolic pathway fluxes: Two case studies

This paper presents a brief review of applications of kinetic simulation of multi-enzyme networks to the study of antimetabolite drugs used as anticancer agents. Kinetic models consist of systems of nonlinear differential equations that describe changes in concentrations of cellular metabolites with respect to time. Such models have been used to predict the effect of changes in activity of enzymes, or changes in enzyme kinetic parameters, on sensitivity to inhibition. Kinetic simulation has provided insight into several aspects of the biochemical pharmacology of antimetabolites, including drug sensitivity and resistance, and drug-drug interactions. Two specific studies are described in detail. The first concerns the importance of the ratio of competing enzymes in determining the selectivity of inhibitors of one of the competing enzymes, studied by a simple model. The second case study examines the effect of alternative biosynthetic pathways, thede novo and salvage pathways of pyrimidine nucleotide biosynthesis, on the selectivity of antipyrimidine drugs, as studied by a detailed model of 27 reactions of pyrimidine metabolism.     

Response of the glycolysis of human erythrocytes to the transition from the oxygenated to the deoxygenated state at constant intracellular pH.

The time course of the rate of the glycolysis of human erythrocytes and of some metabolites were determined before and after rapid deoxygenation at constant intracellular pH. For this purpose stripped deoxygenated haemoglobin was used as a rapid oxygen acceptor. Deoxygenation causes an increase of the glycolytic rate by 26%. Glucose 6-phosphate is decreased while the adenine nucleotides and 2,3-bisphosphoglycerate remain constant. Fructose 1,6-bisphosphate and the triose phosphates decrease transiently before rising. The data can be explained by increased binding of phosphocompounds to deoxygenated as compared with oxygenated haemoglobin. Thereby the control enzymes hexokinase and phosphofructokinase are influenced. It is concluded that under physiological conditions changes in the oxygenation state of haemoglobin per se alter the glycolytic rate.     

Response of the glycolysis of human erythrocytes to the transition from the oxygenated to the deoxygenated state at constant intracellular pH

The time course of the rate of the glycolysis of human erythrocytes and of some metabolites were determined before and after rapid deoxygenation at constant intracellular pH. For this purpose stripped deoxygenated haemoglobin was used as a rapid oxgen acceptor.Deoxygenation causes an increase of the glycolytic rate by 26%. Glucose 6-phosphate is decreased while the adenine nucleotides and 2,3-bisphosphoglycerate remain constant. Fructose 1,6-bisphosphate and the triose phosphates decrease transiently before rising.The data can be explained by increased binding of phosphocompounds to deoxygenated as compared with oxygenated haemoglobin. Thereby the control enzymes hexokinase and phosphofructokinase are influenced. It is concluded that under physiological conditions changes in the oxygenation state of haemoglobin per se alter the glycolytic rate.     

Effect of cultural conditions on the concentrations of metabolic intermediates during growth and sporulation of Bacillus licheniformis.

Intracellular concentrations of adenine nucleotides and intermediates of the Embden-Meyerhof pathway and the tricarboxylic acid cycle have been determined during growth and sporulation of Bacillus licheniformis in a variety of different media. The ATP pool was independent of growth rate and nitrogen source, but the use of glucose as a carbon source resulted in a twofold elevation in the ATP pool during exponential growth. The intracellular phosphoenolpyruvate pool was at least twofold higher during gluconeogenesis than during glycolysis. The finding that the use of glutamate as the sole nitrogen source resulted in at least a fivefold elevation of the alpha-ketoglutarate pool suggests a role for alpha-ketoglutarate in the repression of the enzymes of the tricarboxylic acid cycle responsible for alpha-ketoglutarate synthesis. Not one of the metabolites assayed appears to function as a signal of the nutrient deprivation which accompanies the initiation of sporulation.     

The role of stoichiometric analysis in studies of metabolism: an example

Stoichiometric analysis uses matrix algebra to deduce the constraints implicit in metabolic networks. When applied to simple networks, it can often give the impression of being an unnecessarily complicated way of arriving at information that is obvious from inspection, for example, that the sum of the concentrations of the adenine nucleotides is constant. Applied to a more complicated example, that of glycolysis in Trypanosoma brucei, it yields information that is far from obvious and may have importance for developing therapeutic ways of eliminating this parasite. Even in simplified form, the network contains nine reactions or transport steps involving 11 metabolites. This immediately shows that there must be at least two stoichiometric constraints, and indeed two can be recognized by inspection: conservation of adenine nucleotides and conservation of the two forms of NAD. There is, however, a third, which involves eight different phosphorylated intermediates in non-obvious combinations and is very difficult to recognize by inspection. It is also difficult to recognize by inspection that no fourth stoichiometric constraint exists. Gaussian elimination provides a systematic way of analysing a network in such a way that all the stoichiometric relationships that it contains emerge automatically.     

NTPDase and 5' ecto-nucleotidase expression profiles and the pattern of extracellular ATP metabolism in the Walker 256 tumor

In this study, we evaluated the NTPDases and ecto-5'-nucleotidase (CD73) expression profiles and the pattern of adenine nucleotide hydrolysis in rats submitted to the Walker 256 tumor model, 6, 10 and 15 days after the subcutaneous inoculation. Using RT-PCR analysis, we identified mRNA for all of the members of the ecto-nucleoside triphosphate diphosphohydrolase family investigated and a 5'-nucleotidase. By quantitative real-time PCR, Entpd1 (Cd39) and Entpd2 (Cd39L1) and CD73 were identified as the dominant genes expressed by the Walker 256 tumor, at all times studied. Extracellular adenine nucleotide hydrolysis by the Walker 256 tumor was estimated by HPLC analysis. Rapid hydrolysis of extracellular ATP by the tumor cells was observed, leading to the formation of adenosine and inosine in cells obtained from solid tumors at 6 and 10 days after inoculation. Cells obtained from solid tumors at 15 days of growth presented high levels of AMP and presented adenosine as a final product after 90 min of incubation. Results demonstrate that the presence of NTPDases and 5'-nucleotidase enzymes in Walker 256 tumor cells may be important for regulation of the extracellular adenine nucleotides/adenine nucleoside ratio, therefore leading to tumor growth.     

Characterization of glucose transport mutants of Saccharomyces cerevisiae during a nutritional upshift reveals a correlation between metabolite levels and glycolytic flux

Saccharomyces cerevisiae shows a marked preference for glucose and fructose, revealed by the repression of genes whose products are involved in processing other carbon sources. This response seems to be driven by sugar phosphorylation in the first steps of glycolysis rather than by the external sugar concentration. To gain a further insight into the role of the internal sugar signalling mechanisms, were measured the levels of upper intracellular glycolytic metabolites and adenine nucleotides in three mutant strains, HXT1, HXT7 and TM6*, with progressively reduced uptake capacities in comparison with the wild type. Reducing the rate of sugar consumption caused an accumulation of hexose phosphates upstream of the phosphofructokinase (PFK) and a reduction of fructose-1,6-bisphosphate levels. Mathematical modelling showed that these effects may be explained by changes in the kinetics of PFK and phosphoglucose isomerase. Moreover, the model indicated a modified sensitivity of the pyruvate dehydrogenase and the trichloroacetic acid cycle enzymes towards the NAD/NADH in the TM6* strain. The activation of the SNF1 sugar signalling pathway, previously observed in the TM6* strain, does not correlate with a reduction of the ATP : AMP ratio as reported in mammals. The mechanisms that may control the glycolytic rate at reduced sugar transport rates are discussed.     

Dynamic flux cartography of hairy roots primary metabolism

A dynamic model for plant cell and hairy root primary metabolism is presented. The model includes nutrient uptake (Pi, sugars, nitrogen sources), the glycolysis and pentose phosphate pathways, the TCA cycle, amino acid biosynthesis, respiratory chain, biosynthesis of cell building blocks (structural hexoses, organic acids, lipids, and organic phosphated molecules). The energy shuttles (ATP, ADP) and cofactors (NAD/H, NADP/H) are also included. The model describes the kinetics of 44 biochemical reactions (fluxes) of the primary metabolism of plant cells and includes 41 biochemical species (metabolites, nutrients, biomass components). Multiple Michaelis-Menten type kinetics are used to describe biochemical reaction rates. Known regulatory phenomena on metabolic pathways are included using sigmoid switch functions. A visualization framework showing fluxes and metabolite concentrations over time is presented. The visualization of fluxes and metabolites is used to analyze simulation results from Catharanthus roseus hairy root 50 d batch cultures. The visualization of the metabolic system allows analyzing split ratios between pathways and flux time-variations. For carbon metabolism, the cells were observed to have relatively high and stable fluxes for the central carbon metabolism and low and variable fluxes for anabolic pathways. For phosphate metabolism, a very high free intracellular Pi turnover rate was observed with higher flux variations than for the carbon metabolism. Nitrogen metabolism also exhibited large flux variations. The potential uses of the model are also discussed.     

Purine Nucleotide Synthesis in Adrenal Chromaffin Cells

Abstract: The synthesis of purine nucleotides from the salvage precursors adenine and adenosine, and from the de novo precursors formate and glycine, was studied in isolated adrenal chromaffin cells. Both [8-¹⁴C]adenine and [8-¹⁴C]adenosine from extracellular medium are effectively incorporated into intracellular nucleotides. [¹⁴C]Formate and [U-¹⁴C]glycine are also incorporated, but de novo synthesis is clearly lower than synthesis from salvage precursors, although similar to de novo synthesis in liver. The enzymes responsible for adenine and adenosine salvage, adenine phosphoribosyltransferase and adenosine kinase, were purified about 1,500-fold. Both enzymes are mainly cytosolic and exhibit a similar molecular weight of around 42,000. The results suggest that chromaffin cells can replenish their intracellular nucleotides lost during the secretory event by an active synthesis from salvage and de novo precursors.     

A mathematical model of the pyruvate oxidation in liver mitochondria. 1. Regulation of the Krebs cycle by adenine and pyridine nucleotides]

A mathematical model is proposed to describe the behavior of the pyruvate metabolic reactions, Krebs cycle and oxidative phosphorylation over a wide range of changes in the pyruvate influx rate and the activities of ATPase and NADH-reoxidating dehydrogenase. The role of adenine and pyridine nucleotides in various allosteric regulations of the Krebs cycle enzymes is discussed. The accumulation of ATP and NADH has been shown to proceed in definite succession, which makes the allosteric regulation of the Krebs cycle enzymes successive too. First "works" the inhibition by ATP, then by NADH. It has been shown that the properties of the model are in qualitative agreement with the experimental data (Garber A., Hanson R. [1]) on pyruvate oxidation by mitochondria from guinea pig liver, when allosteric regulation of isocitrate dehydrogenase by adenine nucleotides is taken into account.     

Biosynthesis of Caffeine in Flower Buds of Camellia sinensis

The biosynthesis of purine alkaloids in flower buds of tea plantswas investigated. More than 25% of total radioactivity of [8-¹⁴C]adeninetaken up by stamens isolated from tea flower buds was foundto have been incorporated into purine alkaloids, namely, theobromineand caffeine, 24 h after administration of the labelled compound.Pulse-chase experiments indicated that [8-¹⁴C]adenine takenup by the stamens was converted to adenine nucleotides and subsequentlyincorporated into theobromine and caffeine. Since 5 µMcoformycin, an inhibitor of AMP deaminase, inhibited the incorporationof radioactivity into the purine alkaloids, synthesis of caffeinefrom adenine nucleotides seems to be initiated by the reactionof AMP deaminase. Although most of the radioactivity from [8-¹⁴C]inosinewas recovered as CO₂ and ureides, considerable amounts of radioactivitywere recovered as purine alkaloids. The incorporation of radioactivityfrom [8-¹⁴C]inosine into the purine alkaloids was not affectedby coformycin. The five enzymes involved in synthesis of 5-phosphoribosyl-1-pyrophosphatefrom glucose were present in the stamens and petals of tea flowerbuds. From present and previous results, the pathway for thebiosynthesis of caffeine from adenine nucleotides in flowerbuds of tea is discussed.Copyright 1993, 1999 Academic Press Camellia sinensis, tea, stamen, flower, biosynthesis, purine alkaloids, caffeine, theobromine, adenine nucleotides, nucleotide biosynthesis