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H V Westerhoff

Publications and source records attributed to H V Westerhoff.

At least 19 recordsLinked to original sources
PubMed2006

Is there an optimal ribosome concentration for maximal protein production?

A core model is presented for protein production in Escherichia coli to address the question whether there is an optimal ribosomal concentration for non-ribosome protein production. Analysing the steady-state solution of the model over a range of mRNA concentrations, indicates that such an optimum ribosomal content exists, and that the optimum shifts to higher ribosomal contents at higher specific growth rates.

Computer Simulation↗
PubMed2006

Summation theorems for flux and concentration control coefficients of dynamic systems.

Metabolic control analysis (MCA) was developed to quantify how system variables are affected by parameter variations in a system. In addition, MCA can express the global properties of a system in terms of the individual catalytic steps, using connectivity and summation theorems to link the control coefficients to the elasticity coefficients. MCA was originally developed for steady-state analysis and not all summation theorems have been derived for dynamic systems. A method to determine time-dependent flux and concentration control coefficients for dynamic systems by expressing the time domain as a function of percentage progression through any arbitrary fixed interval of time is reported. Time-dependent flux and concentration control coefficients of dynamic systems, provided that they are evaluated in this novel way, obey the same summation theorems as steady-state flux and concentration control coefficients, respectively.

Algorithms↗
PubMed2006

Time-dependent hierarchical regulation analysis: deciphering cellular adaptation.

Cells adapt to changes in their environment by the concerted action of many different regulatory mechanisms. Examples of such mechanisms are feedback inhibition by intermediates of metabolism, covalent modification of enzymes and changes in the abundance of mRNAs and proteins. These mechanisms act in parallel at different levels in the cellular hierarchy while regulating a single process. Existing hierarchical regulation analysis determines the relative importance of these mechanisms when the cell regulates a transition from one steady-state to another. Here, the analysis is extended to the regulation of time-dependent phenomena, for which two methods are introduced and illustrated with a kinetic model incorporating transcription and translation of metabolic enzymes.

Adaptation, Physiological↗
PubMed2002

How to distinguish between the vacuum cleaner and flippase mechanisms of the lmrA multi-drug transporter in Lactococcus lactis.

A numerical model of the LmrA multi-drug transport system of Lactococcus lactis is used to explore the possibility of distinguishing experimentally between two putative transport mechanisms, i.e., the vacuum-cleaner and the flippase mechanisms. This comparative model also serves as an example of numerical simulation with the scripting language Python and its scientific add-on Scipy.

Bacterial Proteins↗
PubMed2001

Transcriptome meets metabolome: hierarchical and metabolic regulation of the glycolytic pathway.

The fact that information flows from DNA to RNA to protein to function suggests that regulation is 'hierarchical', i.e. dominated by regulation of gene expression. In the case of dominant regulation at the metabolic level, however, there is no quantitative relationship between mRNA levels and function. We here develop a method to quantitate the relative contributions of metabolic and hierarchical regulation. Applying this method to the glycolytic flux in three species of parasitic protists, we conclude that it is rarely regulated by gene expression alone. This casts strong doubts on whether transcriptome and proteome analysis suffices to assess biological function.

Animals↗
PubMed2001

Building the cellular puzzle: control in multi-level reaction networks.

Quantitative conceptual tools dealing with control and regulation of cellular processes have been mostly developed for and applied to the pathways of intermediary metabolism. Yet, cellular processes are organized in different levels, metabolism forming the lowest level in a cascade of processes. Well-known examples are the DNA-mRNA-enzyme-metabolism cascade and the signal transduction cascades consisting of covalent modification cycles. The reaction network that constitutes each level can be viewed as a "module" in which reactions are linked by mass transfer. Although in principle all of these cellular modules are ultimately linked by mass transfer, in practice they can often be regarded as "isolated" from each other in terms of mass transfer. Here modules can interact with each other only by means of regulatory or catalytic effects-a chemical species in one module may affect the rate of a reaction in another module by binding to an enzyme or transport system or by acting as a catalyst. This paper seeks to answer two questions about the control and regulation of such multi-level reaction networks: (i) How can the control properties of the system as a whole be expressed in terms of the control properties of individual modules and the effects between modules? (ii) How do the control properties of a module in its isolated state change when it is embedded in the whole system through its connections with the other modules? In order to answer these questions a quantitative theoretical framework is developed and applied to systems containing two, three or four fully interacting modules; it is shown how it can be extended in principle to n modules. This newly developed theory therefore makes it possible to quantitatively dissect intermodular, internal and external regulation in multi-level systems.

Animals↗
PubMed2001

Control of glycolytic dynamics by hexose transport in Saccharomyces cerevisiae.

It is becoming accepted that steady-state fluxes are not necessarily controlled by single rate-limiting steps. This leaves open the issue whether cellular dynamics are controlled by single pacemaker enzymes, as has often been proposed. This paper shows that yeast sugar transport has substantial but not complete control of the frequency of glycolytic oscillations. Addition of maltose, a competitive inhibitor of glucose transport, reduced both average glucose consumption flux and frequency of glycolytic oscillations. Assuming a single kinetic component and a symmetrical carrier, a frequency control coefficient of between 0.4 and 0.6 and an average-flux control coefficient of between 0.6 and 0.9 were calculated for hexose transport activity. In a second approach, mannose was used as the carbon and free-energy source, and the dependencies on the extracellular mannose concentration of the transport activity, of the frequency of oscillations, and of the average flux were compared. In this case the frequency control coefficient and the average-flux control coefficient of hexose transport activity amounted to 0.7 and 0.9, respectively. From these results, we conclude that 1) transport is highly important for the dynamics of glycolysis, 2) most but not all control resides in glucose transport, and 3) there should at least be one step other than transport with substantial control.

Biological Transport, Active↗
PubMed2001

The potential role of adenosine in the pathophysiology of the insulin resistance syndrome.

An increased intracellular availability of the co-enzyme A esters of long-chain fatty acids is thought to underlie many aspects of the insulin resistance syndrome. However, the cause of clustering of a hyperdynamic circulation, sympathetic activation, hypertension, hyperuricaemia, and a raised haematocrit in the insulin resistance syndrome remains to be elucidated. We propose a mechanism that expands the etiological role of long-chain fatty acids. By inhibiting adenine nucleotide translocators, elevated intracellular concentrations of the co-enzyme A esters of long-chain fatty acids impair mitochondrial oxidative phosphorylation. This is expected to result in a chronic systemic increase in extracellular adenosine concentrations. As adenosine stimulates the sympathetic nervous system, induces systemic vasodilatation, stimulates erythropoiesis, and induces renal vasoconstriction with renal sodium retention, increased extracellular ADO concentrations may be the common denominator explaining the above-mentioned and still unexplained phenomena associated with the insulin resistance syndrome. Along the same lines, hyperuricaemia can be explained by the fact that adenosine is broken down to urate and because of increased renal urate retention.

Adenosine↗
PubMed2001

A functional genomics strategy that uses metabolome data to reveal the phenotype of silent mutations.

A large proportion of the 6,000 genes present in the genome of Saccharomyces cerevisiae, and of those sequenced in other organisms, encode proteins of unknown function. Many of these genes are "silent, " that is, they show no overt phenotype, in terms of growth rate or other fluxes, when they are deleted from the genome. We demonstrate how the intracellular concentrations of metabolites can reveal phenotypes for proteins active in metabolic regulation. Quantification of the change of several metabolite concentrations relative to the concentration change of one selected metabolite can reveal the site of action, in the metabolic network, of a silent gene. In the same way, comprehensive analyses of metabolite concentrations in mutants, providing "metabolic snapshots," can reveal functions when snapshots from strains deleted for unstudied genes are compared to those deleted for known genes. This approach to functional analysis, using comparative metabolomics, we call FANCY-an abbreviation for functional analysis by co-responses in yeast.

Adenine Nucleotides↗
PubMed2001

Pumping capacity of bacterial reaction centers and backpressure regulation of energy transduction.

Transduction of free-energy by Rhodobacter sphaeroides reaction-center-light-harvesting-complex-1 (RCLH1) was quantified. RCLH1 complexes were reconstituted into liposomal membranes. The capacity of the RCLH1 complex to build up a proton motive force was examined at a range of incident light intensities, and induced proton permeabilities, in the presence of artificial electron donors and acceptors. Experiments were also performed with RCLH1 complexes in which the midpoint potential of the reaction center primary donor was modified over an 85-mV range by replacement of the tyrosine residue at the M210 position of the reaction center protein by histidine, phenylalanine, leucine or tryptophan. The intrinsic driving force with which the reaction center pumped protons tended to decrease as the midpoint potential of the primary donor was increased. This observation is discussed in terms of the control of the energetics of the first steps in light-driven electron transfer on the thermodynamic efficiency of the bacterial photosynthetic process. The light intensity at which half of the maximal proton motive force was generated, increased with increasing proton permeability of the membrane. This presents the first direct evidence for so-called backpressure control exerted by the proton motive force on steady-state cyclic electron transfer through and coupled proton pumping by the bacterial reaction center.

Electron Transport↗
PubMed2001

Regulation of expression of terminal oxidases in Paracoccus denitrificans.

In order to study the induction of terminal oxidases in Paracoccus denitrificans, their promoters were fused to the lacZ reporter gene and analysed in the wild-type strain, in an FnrP-negative mutant, in a cytochrome bc1-negative mutant, and in six single or double oxidase-negative mutant strains. The strains were grown under aerobic, semi-aerobic, and denitrifying conditions. The oxygen-sensing transcriptional-regulatory protein FnrP negatively regulated the activity of the qox promoter, which controls expression of the ba3-type quinol oxidase, while it positively regulated the activity of the cco promoter, which controls expression of the cbb3-type cytochrome c oxidase. The ctaDII and ctaC promoters, which control the expression of the aa3-type cytochrome c oxidase subunits I and II, respectively, were not regulated by FnrP. The activities of the latter two promoters, however, did decrease with decreasing oxygen concentrations in the growth medium, suggesting that an additional oxygen-sensing mechanism exists that regulates transcription of ctaDII and ctaC. Apparently, the intracellular oxygen concentration (as sensed by FnrP) was not the only signal to which the oxidase promoters responded. At given extracellular oxygen status, both the qox and the cco promoters responded to mutations in terminal oxidase genes, whereas the ctaDII and ctaC promoters did not. The change of electron distribution through the respiratory network, resulting from elimination of one or more oxidase genes, may have changed intracellular signals that affect the activities of the qox and cco promoters. On the other hand, the re-routing of electron distribution in the respiratory mutants hardly affected the oxygen consumption rate as compared to that of the wild-type. This suggests that the mutants adapted their respiratory network in such a way that they were able to consume oxygen at a rate similar to that of the wild-type strain.

Base Sequence↗
PubMed2001

Autoamplification of a two-component regulatory system results in "learning" behavior.

We have tested the hypothesis that the autoamplification of two-component regulatory systems results in "learning" behavior, i.e., that bacteria respond faster or more extensively to a signal when a similar signal has been perceived in the past. Indeed, the induction of alkaline phosphatase activity upon phosphate limitation was faster if the cultures had been limited for phosphate previously, and this faster response correlated with the autoamplification of the cognate two-component system.

Acclimatization↗
PubMed2001

Cytochromes c(550), c(552), and c(1) in the electron transport network of Paracoccus denitrificans: redundant or subtly different in function?

Paracoccus denitrificans strains with mutations in the genes encoding the cytochrome c(550), c(552), or c(1) and in combinations of these genes were constructed, and their growth characteristics were determined. Each mutant was able to grow heterotrophically with succinate as the carbon and free-energy source, although their specific growth rates and maximum cell numbers fell variably behind those of the wild type. Maximum cell numbers and rates of growth were also reduced when these strains were grown with methylamine as the sole free-energy source, with the triple cytochrome c mutant failing to grow on this substrate. Under anaerobic conditions in the presence of nitrate, none of the mutant strains lacking the cytochrome bc(1) complex reduced nitrite, which is cytotoxic and accumulated in the medium. The cytochrome c(550)-deficient mutant did denitrify provided copper was present. The cytochrome c(552) mutation had no apparent effect on the denitrifying potential of the mutant cells. The studies show that the cytochromes c have multiple tasks in electron transfer. The cytochrome bc(1) complex is the electron acceptor of the Q-pool and of amicyanin. It is also the electron donor to cytochromes c(550) and c(552) and to the cbb(3)-type oxidase. Cytochrome c(552) is an electron acceptor both of the cytochrome bc(1) complex and of amicyanin, as well as a dedicated electron donor to the aa(3)-type oxidase. Cytochrome c(550) can accept electrons from the cytochrome bc(1) complex and from amicyanin, whereas it is also the electron donor to both cytochrome c oxidases and to at least the nitrite reductase during denitrification. Deletion of the c-type cytochromes also affected the concentrations of remaining cytochromes c, suggesting that the organism is plastic in that it adjusts its infrastructure in response to signals derived from changed electron transfer routes.

Bacterial Proteins↗
PubMed2000

Steady-state cyclic electron transfer through solubilized Rhodobacter sphaeroides reaction centres.

The mechanism, thermodynamics and kinetics of light-induced cyclic electron transfer have been studied in a model energy-transducing system consisting of solubilized Rhodobacter sphaeroides reaction center/light harvesting-1 complexes (so-called core complexes), horse heart cytochrome c and a ubiquinone-0/ubiquinol-0 pool. An analysis of the steady-state kinetics of cytochrome c reduction by ubiquinol-0, after a light-induced steady-state electron flow had been attained, showed that the rate of this reaction is primarily controlled by the one-electron oxidation of the ubiquinol-anion. Re-reduction of the light-oxidized reaction center primary donor by cytochrome c was measured at different reduction levels of the ubiquinone-0/ubiquinol-0 pool. These experiments involved single turnover flash excitation on top of background illumination that elicited steady-state cyclic electron transfer. At low reduction levels of the ubiquinone-0/ubiquinol-0 pool, the total cytochrome c concentration had a major control over the rate of reduction of the primary donor. This control was lost at higher reduction levels of the ubiquinone/ubiquinol-pool, and possible reasons for this behaviour are discussed.

Animals↗
PubMed2000

Glucose and the ATP paradox in yeast.

A sustained decrease in the intracellular ATP concentration has been observed when extra glucose was added to yeast cells growing aerobically under glucose limitation. Because glucose degradation is the main source of ATP-derived free energy, this is a counter-intuitive phenomenon, which cannot be attributed to transient ATP consumption in the initial steps of glycolysis. We present a core model for aerobic growth in which glucose supplies carbon, as well as free energy, for biosynthesis. With Metabolic Control Analysis and numerical simulations, we demonstrate that the decrease in the ATP concentration can be reproduced if the biosynthetic route is more strongly activated by carbon substrates than is the catabolic (ATP-producing) route.

Adenosine Triphosphate↗
PubMed2000

Understanding glucose transport by the bacterial phosphoenolpyruvate:glycose phosphotransferase system on the basis of kinetic measurements in vitro.

The kinetic parameters in vitro of the components of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) in enteric bacteria were collected. To address the issue of whether the behavior in vivo of the PTS can be understood in terms of these enzyme kinetics, a detailed kinetic model was constructed. Each overall phosphotransfer reaction was separated into two elementary reactions, the first entailing association of the phosphoryl donor and acceptor into a complex and the second entailing dissociation of the complex into dephosphorylated donor and phosphorylated acceptor. Literature data on the K(m) values and association constants of PTS proteins for their substrates, as well as equilibrium and rate constants for the overall phosphotransfer reactions, were related to the rate constants of the elementary steps in a set of equations; the rate constants could be calculated by solving these equations simultaneously. No kinetic parameters were fitted. As calculated by the model, the kinetic parameter values in vitro could describe experimental results in vivo when varying each of the PTS protein concentrations individually while keeping the other protein concentrations constant. Using the same kinetic constants, but adjusting the protein concentrations in the model to those present in cell-free extracts, the model could reproduce experiments in vitro analyzing the dependence of the flux on the total PTS protein concentration. For modeling conditions in vivo it was crucial that the PTS protein concentrations be implemented at their high in vivo values. The model suggests a new interpretation of results hitherto not understood; in vivo, the major fraction of the PTS proteins may exist as complexes with other PTS proteins or boundary metabolites, whereas in vitro, the fraction of complexed proteins is much smaller.

Biological Transport↗