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H V Westerhoff

Publications and source records attributed to H V Westerhoff.

At least 91 records · Page 5Linked to original sources
PubMed1996

How to recognize monofunctional units in a metabolic system.

In intracellular metabolic networks, it is often useful to discern subsystems (modules) of which the metabolites are only produced or consumed by reactions within that subsystem or by a limited number of fluxes crossing the borders of the subsystem. In many cases such subsystems function as units with respect to their effect on the remainder of the system. In this paper we show that the co-response of two metabolic variables outside that subsystem to a perturbation of a subsystem reaction does not depend on which subsystem reaction is perturbed if three conditions are fulfilled: (1) the reactions outside the subsystem are not affected directly by metabolites belonging to the subsystem; (2) there are no conservation relations linking the subsystem to the rest; and (3) the subsystem is linked to the remainder of the system only via one degree of freedom in fluxes. We propose the name "monofunctional units" for subsystems fulfilling these three criteria. Identification of such units greatly simplifies metabolic control analysis. Only one reaction per unit needs to be perturbed to analyse control in the system. Difficulties, such as the inaccessibility of some reactions to experimental perturbation, may be circumvented by perturbing another reaction within the unit that leads to the same co-response coefficients. The analysis can also serve to identify unsuspected regulatory interactions in the metabolic network. The differences in the behaviour between metabolic units and other types of subsystems are illustrated by numerical examples.

Animals↗
PubMed1996

Why and when channelling can decrease pool size at constant net flux in a simple dynamic channel.

Cornish-Bowden and Cárdenas (Cornish-Bowden, A. and Cárdenas M.L. (1993) Eur. J. Biochem. 213, 87-92) have suggested that simulation results peviously published by us (Mendes, P., Kell, D.B. and Westerhoff, H.V. (1992) Eur. J. Biochem. 204, 255-266) which had demonstrated that large reductions of intermediate pool sizes could be accompanied by increasing channel flux in a model metabolic pathway, were an artefact of changes in the pathway's overall flux of the order of 0.0075%, or of inappropriate alterations of enzyme activities. They also asserted to prove that the "channelling of an intermediate cannot affect its free concentration at constant net flux". We consider the co-response of the intermediate metabolite concentration ('pool') and the channel flux to changes in kinetic (or thermodynamic) parameters. Both by analytical proofs and by numerical examples we show that this co-response can be positive, negative or null, depending on the parameter change. In particular, we prove that there is always a number of ways of changing parameters such that the intermediate metabolite concentration decreases with increasing channel flux, whether the total flux varies or is constant. We also show that increased stability of the (dynamic) enzyme-intermediate-enzyme complex, as well as a single parameter change that similarly displays no cross-over effects, can lead to decreased intermediate metabolite concentration and increased channel flux at constant total flux. In general, a non-zero co-response of the intermediate metabolite concentration ('pool') and the channel flux to changes in kinetic (or other) parameters is the rule rather than the exception. More specifically: (i) The algebraic analysis ('general proof') given in Cornish-Bowden and Cárdenas (1993) contains the constraint that the elasticities of various steps to the modulation parameters which were used to vary the channel flux at constant net flux were unity. This is an unfortunate and unnecessary constraint which, when lifted, means that the concentration of the pool in the general case can indeed change at constant net flux. A 'simplified proof' given in Cornish-Bowden and Cárdenas (1993) also fails, due in addition to the consequent failure to include mass conservation relations for some of the enzymes. (ii) In the systems studied by Cornish-Bowden and Cárdenas (1993), flux is properly to be considered as a variable (since it varies during the transition to the steady state), and not a parameter, and as such cannot per se affect the magnitude of other variables in the steady state. (iii) By relaxing the constraint referred to in (i), above, and by making dual modulations (i.e., of more than one parameter at once) which are different from those carried out in Cornish-Bowden and Cárdenas (1993) we find many instances in which channelling (described by a parameter p) does significantly affect the concentration of the pool intermediate C at constant total flux. (iv) In the same pathways, but in which the flux is held constant by setting it via a zero-order flux-generating reaction, the addition of a channel is also able to significantly to modulate the size of the pool at constant total flux. Our results show that the effectiveness of channelling in decreasing a pool, even at constant flux, is very much a reality.

Animals↗
PubMed1996

Reversal of multidrug resistance by valinomycin is overcome by CCCP.

Reversal of P-glycoprotein-mediated multidrug resistance by valinomycin is overcome by the proton ionophore, CCCP. This effect, a complete suppression of the 5- to 10-fold valinomycin-induced reversal ("re-reversal"), exhibits a sharp extracellular potassium concentration ([K+(0)]) dependence. It is observed at [K+(0)] > 2-4 mM and not at [K+(0)] greater than or equal to 2 mM, in the case of the fluorescent substrates rhodamine 123 and daunorubicin. The fact that "re-reversal" is detected only for the combination of CCCP with valinomycin raises the possibility that a direct interaction between these ionophores may explain the phenomenon. We show spectroscopic evidence of such an interaction, with a [K+(0)]-dependence similar to that of the "re-reversal." These data suggest that the reversal of P-glycoprotein activity by valinomycin can be compromised by anionic compounds such as CCCP due to complex formation. More generally, molecular interactions involving P-glycoprotein substrates or reversing agents may significantly affect drug accumulation in multidrug resistant cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
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

Saturable P-glycoprotein kinetics assayed by fluorescence studies of drug efflux from suspended human KB8-5 cells.

This article describes a new and rapid method to determine the pumping rate of P-glycoprotein (P-gp) in intact cells. Multidrug resistant (MDR) human epidermoid carcinoma KB8-5 cells (containing P-gp) were loaded with daunorubicin (DNR) in the absence or in the presence of verapamil, sufficient to inhibit DNR pumping by P-gp. In either case, the cells were resuspended in medium devoid of DNR and the subsequent increase of the DNR fluorescence intensity was measured as a function of time. For cells loaded with the same amount of drug, the free cytosolic drug concentration (Ci(t)) was a unique function of the DNR medium concentration (Co(t)). The cellular drug content in the presence of verapamil decreased nonlinearly with decreasing extracellular drug concentration, indicating that the intracellular drug apparent distribution volume increased with decreasing cellular drug content. At each fluorescence intensity, we calculated the P-gp mediated (verapamil-inhibitable) DNR transport rate from the rate of increase of the DNR fluorescence intensity in the absence of verapamil minus the rate of increase of the DNR fluorescence intensity in the presence of verapamil. When plotted against the intracellular free drug concentration (as calculated from the total cellular drug content and a separately determined relation between the total cellular drug content and the intracellular free drug concentration: the apparent distribution volume), this P-gp mediated DNR transport rate showed saturation of P-gp at higher DNR concentrations. The results imply that P-gp mediated DNR transport is saturable (the value of Km is in the order of 1 microM).

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Changes in the cellular energy state affect the activity of the bacterial phosphotransferase system.

The effect of different cellular free-energy states on the uptake of methyl alpha-D-glucopyranoside, an analogue of glucose, by the Escherichia coli phosphoenolpyruvate:carbohydrate phosphotransferase system was investigated. The intracellular [ATP]/[ADP] ratio was varied by changing the expression of the atp operon, which codes for the H+-ATPase, or by adding an uncoupler of oxidative phosphorylation or an inhibitor of respiration. Corresponding initial phosphotransferase uptake rates were determined using an improved uptake assay that works with growing cells in steady state. The results show that the initial uptake rate was decreased under conditions of lowered intracellular [ATP]/[ADP] ratios, irrespective of which method was used to change the cellular energy state. When either the expression of the atp operon was changed or 2,4-dinitrophenol was added to wild-type cells, the relationship between initial phosphotransferase uptake rate and the logarithm of the [ATP]/[ADP] ratio was approximately linear. These results suggest that the cellular free-energy state, as reflected in the intracellular [ATPI]/[ADP] ratio, plays an important role in regulating the activity of the phosphotransferase system.

2,4-Dinitrophenol↗
PubMed1996

Acetaldehyde mediates the synchronization of sustained glycolytic oscillations in populations of yeast cells.

In the presence of cyanide, populations of yeast cells can exhibit sustained oscillations in the concentration of glycolytic metabolites, NADH and ATP. This study attempts to answer the long-standing question of whether and how oscillations of individual cells are synchronized. It shows that mixing two cell populations that oscillate 180 degrees out of phase only transiently abolishes the macroscopic oscillation. After a few minutes, NADH fluorescence of the mixed population resumes oscillations up to the original amplitude. At low cell densities, addition of acetaldehyde causes transient oscillations. At higher cell densities, where the oscillations are autonomous, 70 microM acetaldehyde causes phase shifts. Extracellular acetaldehyde is shown to oscillate around the 70 microM level. We conclude that acetaldehyde synchronizes the oscillations of the individual cells.

Acetaldehyde↗
PubMed1996

The possible control of chaos in chronic inflammation.

Small differences in inbred animals with a gene for arthritis must be the cause that some of them evolve the disease and others not. These differences are partly stochastic, caused by thermal movement of molecules. It is possible that their effect is amplified by a network of cytokines, known to play an important role in arthritis. Here it is supposed that the cytokines form a chaotic system. Such a system is deterministic but extremely sensitive for small perturbations. However, they can also be used for the control of chaos and this may offer a means of therapy.

Animals↗
PubMed1996

An alternative PII protein in the regulation of glutamine synthetase in Escherichia coli.

The PII protein has been considered pivotal to the dual cascade regulating ammonia assimilation through glutamine synthetase activity. Here we show that PII, encoded by the glnB gene, is not always essential; for instance upon ammonia deprivation of a glnB deletion strain, glutamine synthetase can be deadenylylated as effectively as in the wild-type strain. We describe a new operon, glnK amtB, which encodes a homologue of PII and a putative ammonia transporter. We cloned and overexpressed glnK and found that the expressed protein had almost the same molecular weight as PII, reacted with polyclonal PII antibody, and was 67% identical in terms of amino acid sequence with Escherichia coli PII. Like PII, purified GlnK can activate the adenylylation of glutamine synthetase in vitro, and, in vivo, the GlnK protein is uridylylated in a glnD-dependent fashion. Unlike PII, however, the expression of glnK depends on the presence of UTase, nitrogen regulator I (NRI), and absence of ammonia. Because of a NRI and a sigma N (sigma 54) RNA polymerase-binding consensus sequence upstream from the glnK gene, this suggests that glnK is regulated through the NRI/NRII two-component regulatory system. Indeed, in cells grown in the presence of ammonia, glutamine synthetase deadenylylation upon ammonia depletion depended on PII. Possible regulatory implications of this conditional redundancy of PII are discussed.

Amino Acid Sequence↗
PubMed1996

DNA supercoiling depends on the phosphorylation potential in Escherichia coli.

ATP/ADP ratios were varied in different ways and the degree of negative supercoiling was determined in Escherichia coli. Independent of whether the ATP/ ADP ratio was reduced by a shift to anaerobic conditions, by addition of a protonophore (dinitrophenol) or by potassium cyanide addition, DNA supercoiling decreased similarly with the ATP/ADP ratio. The experiments were performed under well-defined conditions, where oxidative phosphorylation was the dominant route for ATP synthesis, i.e. using a minimal salts medium with succinate as the sole free-energy and carbon source, and in the presence or absence of ammonia as the nitrogen source. The results of the different experiments were consistent with a single linear relationship between the log(ATP/ADP) and the change in linking number. The dependence of DNA supercoiling on the ATP/ADP ratio was not influenced by inhibitors of transcription or translation. Because the ATP/ADP ratio was modulated in different ways, the unique relationship suggests coupling between the phosphorylation potential and DNA supercoiling. This was most probably mediated by the DNA gyrase, independent of topoisomerase I or transcription.

Adenosine Diphosphate↗
PubMed1995

Calcium indirectly increases the control exerted by the adenine nucleotide translocator over 2-oxoglutarate oxidation in rat heart mitochondria.

The effect of calcium on the control exerted by the adenine nucleotide translocator over respiration in isolated heart mitochondria was investigated in order to determine whether calcium directly stimulates the translocator. At respiration rates intermediate between states 3 and 4, Ca2+ is shown to increase the control over 2-oxoglutarate oxidation exerted by the adenine nucleotide translocator in rat heart mitochondria. This did not occur when succinate was the respiratory substrate, even though the control exerted by the translocator was substantial, indicating that Ca2+ does not have a direct effect on the adenine nucleotide translocator. Ca2+ increased the uncoupled oxidation rate of 2-oxoglutarate, but not succinate. Using the summation theorem for flux control, the effect of Ca2+ is explained by a shift of the control over respiration rate toward the adenine nucleotide translocator, from the respiratory chain, presumably as the result of the activation of the 2-oxoglutarate dehydrogenase complex.

Adenosine Triphosphate↗
PubMed1995

An additional PII in Escherichia coli: a new regulatory protein in the glutamine synthetase cascade.

The PII protein in the glutamine synthetase cascade transduces the nitrogen signal, as sensed by uridylyltransferase, both to the NRII/NRI two-component system and to adenylyltransferase, to regulate the activity of glutamine synthetase. Here we describe the amplification of a chromosomal DNA fragment from Escherichia coli which contains the sequence of a PII homologue. The derived amino acid sequence of this DNA fragment is 67% identical to E. coli PII. It contains the conserved tyrosine residue which is known to be the site of uridylylation in PII. E. coli is the first organism in which two different PII proteins have been detected.

Amino Acid Sequence↗
PubMed1995

Structure and partitioning of bacterial DNA: determined by a balance of compaction and expansion forces?

The mechanisms that determine chromosome structure and chromosome partitioning in bacteria are largely unknown. Here we discuss two hypotheses: (i) the structure of the Escherichia coli nucleoid is determined by DNA binding proteins and DNA supercoiling, representing a compaction force on the one hand, and by the coupled transcription/translation/translocation of plasma membrane and cell wall proteins, representing an expansion force on the other hand; (ii) the two forces are important for the partitioning process of chromosomes.

Chromosomes, Bacterial↗
PubMed1995

Reversed electron transfer through the bc1 complex enables a cytochrome c oxidase mutant (delta aa3/cbb3) of Paracoccus denitrificans to grow on methylamine.

In Paracoccus denitrificans four classes of redox proteins are involved in the electron transfer from methylamine to oxygen:methylamine dehydrogenase (MADH), amicyanin, cytochrome c and cytochrome c oxidase. MADH and its electron acceptor amicyanin are indispensable for growth on methylamine. At least three different cytochromes c and two types of cytochrome c oxidase, cytochromes aa3 and cbb3, have previously been proposed to participate in the electron transfer pathways from methylamine to oxygen. In this study, participation of both cytochrome c oxidases and of the quinol oxidase (cytochrome bb3) has indeed been confirmed by analysis of a series of oxidase mutants. Interestingly, a P. denitrificans cytochrome c oxidase mutant (delta aa3/cbb3) retains the capacity to oxidise methylamine. It is demonstrated that the oxidation of the cytochrome c pool in this mutant does not proceed via an alternative cytochrome c oxidase, but rather via an 'uphill' electron transfer through the bc1 complex to ubiquinone, driven by the membrane potential. The subsequent oxidation of ubiquinol proceeds via the only remaining terminal oxidase, the bb3-type quinol oxidase.

Culture Media↗
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

Functional synergism of the magainins PGLa and magainin-2 in Escherichia coli, tumor cells and liposomes.

Xenopus laevis skin secretion contains a mixture of magainins, which are small positively charged oligopeptides with antimicrobial activity. In this study, we show that two of these peptides, i.e. magainin-2 and PGLa, are much more active in biological functions when added together than when added alone. This synergy applies for the antimicrobial activity of these peptides, and for the toxic effects on tumor cells. We show that this peptide combination is also synergistic when permeabilizing protein-free liposomes for glucose, when dissipating the membrane potential in cytochrome oxidase liposomes and Escherichia coli, and, reversibly, when stimulating respiration in the liposomes. The occurrence of synergy in these diverse systems (complex and simple) suggests that the biological synergy results from synergy in the primary activity of the magainin peptides, namely the permeabilization of free-energy transducing membranes, possibly by forming a multimeric transmembrane pore of mixed peptide composition. The antimicrobial activity of X. laevis skin secretions may be greatly enhanced by the application of this binary weapon.

Amino Acid Sequence↗
PubMed1995

Nitrite and nitric oxide reduction in Paracoccus denitrificans is under the control of NNR, a regulatory protein that belongs to the FNR family of transcriptional activators.

The nir and nor genes, which encode nitrite and nitric oxide reductase, lie close together on the DNA of Paracoccus denitrificans. We here identify an adjacent gene, nnr, which is involved in the expression of nir and nor under anaerobic conditions. The corresponding protein of 224 amino acids is homologous with the family of FNR proteins, although it lacks the N-terminal cysteines. A mutation in the nnr gene had a negative effect on the expression of nitrite and nitric oxide reductase. Synthesis of membrane bound nitrate reductase, of nitrous oxide reductase, and of the cbb3-type cytochrome c oxidase were not affected by mutation of this gene. These results suggest that denitrification in P. denitrificans may be governed by a signal transduction network that is similar to that involved in oxygen regulation of nitrogen metabolism in other organisms.

Amino Acid Sequence↗