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H V Westerhoff

Publications and source records attributed to H V Westerhoff.

At least 19 recordsLinked to original sources
PubMed1992

Kinetics of daunorubicin transport by P-glycoprotein of intact cancer cells.

Drug permeation across the plasma membrane of multidrug-resistant cells depends on the kinetics of the P-glycoprotein-mediated pump activity as well as on the passive permeation of the drug. We here demonstrate a method to characterize kinetically the pump in intact cells. To this purpose, we examined the membrane-transport properties of daunorubicin in various sensitive cancer cell lines and in their multidrug resistant (MDR) counterparts. First, we determined the passive permeability coefficient for daunorubicin. Then, using a flow-through system, the drug flux into the cell was measured after inhibition of the P-glycoprotein-mediated efflux pump. Combining the two results allowed us to calculate the intracellular free concentration of the drug. In the steady-state, the pump rate must equal the net rate of passive diffusion of the drug and, therefore, the same experiments gave us the pumping rate of daunorubicin. These experiments were then repeated at various extracellular drug concentrations. By plotting the pumping rate versus the intracellular drug concentration, we then characterized the P-glycoprotein kinetically. Four independent methods were used to measure the passive permeability coefficient for the cell line A2780. Similar values were obtained. Maximal pump rates (Vmax) showed a good correlation with the amount of P-glycoprotein in the cell lines used. We obtained saturation curves for the variation of the pump rates with the intracellular daunorubicin concentrations. These curves were typical for positive cooperativity, which provides evidence that at least two binding sites for daunorubicin are present on the active transport system of daunorubicin. The apparent Km values for P-glycoprotein-mediated transport, the intracellular free cytosolic daunorubicin concentrations at half-maximal velocity for the cell lines used, were approximately 1.5 microM. Except for the cell lines with the highest amount of P-glycoprotein, the passive efflux rate of daunorubicin proved to be a substantial part of the total daunorubicin efflux rate for the cell lines used. In cell lines with relatively low levels of P-glycoprotein, passive daunorubicin efflux was even the main route of daunorubicin transport from the cells, determining the intracellular steady-state concentrations of daunorubicin.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Channelling can decrease pool size.

It is widely considered that a possible advantage of metabolite channelling, in which a product of an enzyme is transferred to the next enzyme in a metabolic pathway without being released to the 'bulk' solution, is that channelling can decrease the steady-state concentrations of 'pool' intermediates. This then spares the limited solvent capacity of the cell, and reduces the loss of pathway flux due to leakage or instability of the free intermediate. Recently, however, based on simulations of a particular model of a 'dynamic' channel, Cornish-Bowden ["Failure of channelling to maintain low concentrations of metabolic intermediates" (1991) Eur. J. Biochem. 195, 103-108] has argued that this is not in fact the case; his simulations indicated that the channel was rather ineffective at decreasing the concentration of the pool intermediate, and in some cases actually increased it. However, although his simulations were restricted to very specific thermodynamic and kinetic parameters, he generalised his conclusions, arguing that "channelling has no effect on the free concentration of a channelled intermediate in a pathway". By showing that, for a number of kinetic cases, the concentration of the pool intermediate did decrease substantially with increased channelling, we demonstrate here that the conclusion of Cornish-Bowden is not correct. In particular, if the reaction catalysed by the enzymes forming the channel has an equilibrium constant K higher than 1, and if the enzyme removing the product of the channel reaction is kinetically competent, channelling in the model system studied by Cornish-Bowden (1991) can decrease the steady-state concentration of the pool by a factor of 1000, independently of the mechanism of the terminal reaction and under conditions of essentially constant overall flux. If the channel is a 'static' channel, the decrease in the pool can be to arbitrarily low levels. This conclusion also holds for a system in which other reactions may consume the pool intermediate. Thus, channelling can maintain metabolite concentrations at low levels.

Catalysis↗
PubMed1992

Redox interactions in cytochrome c oxidase: from the "neoclassical" toward "modern" models.

Because of recent experimental data on the redox characteristics of cytochrome c oxidase and renewed interest in the role of cooperativity in energy coupling, the question of redox cooperativity in cytochrome c oxidase is reexamined. Extensive redox cooperativity between more than two redox centers, some of which are spectrally invisible, may be expected for this electron transfer coupled proton pump. Such cooperativity, however, cannot be revealed by the traditional potentiometric experiments based on a difference in absorbance between two wavelengths. Multiwavelength analyses utilizing singular value decomposition and second derivatives of absorbance vs. wavelength have revealed a stronger cooperativity than consistent with the "neoclassical" model, which allowed only for weak negative cooperativity between two equipotential one-electron centers. A thermodynamic analysis of redox cooperativity is developed, which includes the possibilities of strong cooperative redox interactions, the involvement of invisible redox centers, conformational changes, and monomer/dimer equilibrations. The experimental observation of an oxidation of one of the cytochromes (a3) with a decrease in applied redox potential is shown to require both strong negative cooperativity and the participation of more than two one-electron centers. A number of "modern" models are developed using the analytical approaches described in this paper. By testing with experimental data, some of these models are falsified, whereas some are retained with suggestions for further testing.

Binding Sites↗
PubMed1992

METASIM: object-oriented modelling of cell regulation.

Enzymatic processes and substances are modelled as distinct objects, belonging to a limited number of classes. A set of class definitions in C++ is presented that constitutes an object-oriented programming platform. The latter supports 'biological' data types and functions and facilitates simulation of metabolic and regulatory pathways in living cells. To compute the time-evolution, Euler or Runge-Kutta methods are used, though the latter method compromises a strict object-oriented philosophy. As an example, histone gene expression during embryogenesis of Xenopus laevis is modelled. This object-oriented programming system forms a modelling 'language' which is readily understood by both biochemists and programmers. It allows biological problems to be programmed more easily and correctly and brings the program closer to the biological reality, hence making it more meaningful to bioscientists. Moreover, it can readily be extended to new models by class derivation.

Cells↗
PubMed1991

Control theory of regulatory cascades.

We have extended Metabolic Control Theory to include cascades consisting of several modules controlling each other solely via regulatory effects. We derive several theorems that determine how the control properties of a cascade derive from (1) the control properties of each module, taken in isolation and (2) the regulatory interactions between the modules. Two cases are treated explicitly. The first concerns cascades in the absence of feed-back: in this case the internal control behaviour of each module is unaffected by external regulatory interactions. The second includes one feed-back loop and gives a quantitative expression of how feed-back modifies control properties: the internal control matrix within one module can be calculated as if the elasticity matrix of this module was the sum of its intrinsic elasticity matrix and a cyclic regulation matrix. More complex cascades can be analysed recursively by subdividing them into simpler modules, which can be treated individually. The theoretical framework developed here should facilitate quantitative experimental analysis of the control of cell physiology where the latter involves regulatory cascades.

Cells↗
PubMed1991

Magainins affect respiratory control, membrane potential and motility of hamster spermatozoa.

The hypothesis was tested that the magainin peptides, known to compromise bacterial and mitochondrial energetics, are highly active against spermatozoa. A mixture of magainin A and PGLa (1:1) caused a 50% reduction in motility of hamster spermatozoa at 4 micrograms/ml total peptide concentration. All motility was lost at 8 micrograms/ml. At this concentration, respiratory control was released and respiration in the presence of uncoupler was inhibited. Uptake of the lipophilic cation tetraphenyl phosphonium was largely abolished by addition of magainin A and PGLa showed synergism with respect to release of respiratory control.

Amino Acid Sequence↗
PubMed1991

Linear nonequilibrium thermodynamics describes the dynamics of an autocatalytic system.

A model simulating oscillations in glycolysis was formulated in terms of nonequilibrium thermodynamics. In the kinetic rate equations every metabolite concentration was replaced with an exponential function of its chemical potential. This led to nonlinear relations between rates and chemical potentials. Each chemical potential was then expanded around its steady-state value as a Taylor series. The linear (first order) term of the Taylor series sufficed to simulate the dynamic behavior of the system, including the damped and even sustained oscillations at low substrate input or high free-energy load. The glycolytic system is autocatalytic in the first half. Because oscillations were obtained only in the presence of that autocatalytic feed-back loop we conclude that this type of kinetic nonlinearity was sufficient to account for the oscillatory behavior. The matrix of phenomenological coefficients of the system is nonsymmetric. Our results indicate that this is the symmetry property and not the linearity of the flow-force relations in the near equilibrium domain that precludes oscillations. Given autocatalytic properties, a system exhibiting liner flow-force relations and being outside the near equilibrium domain may show bifurcations, leading to self-organized behavior.

Catalysis↗
PubMed1991

Dynamic regulation of yeast glycolytic oscillations by mitochondrial functions.

The control exerted in vivo by mitochondrial functions on the dynamics of glycolysis was investigated in starved yeast cells that were metabolizing glucose semianaerobically. Glycolytic oscillations were triggered after a pulse of glucose by inhibition of mitochondrial respiration with KCN, myxothiazol and antimycin A or in mutants in the bc1 complex (ubiquinol:cytochrome c reductase) that were largely deficient in respiratory capacity. Inhibition of the adenine nucleotide translocator by preincubation with bongkrekic acid also triggered a train of damped sinusoidal oscillations after glucose addition. The oscillations consisted of cycles of reduction and oxidation of the intracellular pool of nicotinamide nucleotides with periods of 45 s to 1 min and amplitudes of 0.8 mM or lower. Preincubation with the uncoupler carbonyl cyamide p-(trifluoromethoxy)phenylhydrazone (FCCP) annihilated cyanide-induced oscillations of NAD(P)H. Evidence for de-energization of mitochondrial membranes in vivo was obtained by mitochondrial staining with dimethylaminostyryl-methyl-pyridiniumiodine (DASPMI) of starved cells. The low rates of NADH reoxidation shown by respiratory mutants and the FCCP-treated X2180 strain open up the possibility that mitochondrial dehydrogenases also control glycolytic oscillations. Low rates of cytosolic NADH reoxidation induced by pyrazole, an inhibitor of alcohol dehydrogenase, were also associated with the disappearance of glycolytic oscillations. From experimental evidence and model calculations we conclude that the modulation of the levels of cytosolic ATP by mitochondrial functions in turn modulates the approach of the dynamic behavior of glycolysis to an oscillatory domain. The mitochondrial NADH dehydrogenase and the glycolytic steps associated with NADH reoxidation downstream from pyruvate appear to provide another control level of glycolysis dynamics in vivo.

Adenosine Triphosphate↗
PubMed1990

Control of DNA structure and gene expression.

In the usual metabolic control theory, the concentrations of enzymes are considered to be parameters rather than variables, i.e., they remain constant as the system relaxes to a new steady state. They can only be reset by interventions. This type of control analysis is useful for understanding principles of metabolic control, and for understanding metabolic changes that are too quick or in too limited a metabolic system to involve changes in gene expression. In actual living systems, metabolic changes are often accompanied by changes in gene expression. In this contribution we shall illustrate how metabolic control analysis is enriched when gene expression is variable. To discuss the new principles emerging in control analysis with variable gene expression, we shall first discuss theoretical model systems. In the first, the number of genes is fixed, but the concentrations of mRNA and enzymes are determined by the activities of RNA polymerase, RNAases, ribosomes and proteases. In a second, there is feedback repression by a metabolite at the level of translation. New coefficients quantifying the strength of regulatory loops will be defined. Also coefficients that indicate to what extent these regulatory strengths themselves are controlled by system parameters, are defined and provided with a summation theorem. The experimental model system we employ, addresses the phenomenon that in prokaryotes, transcription rates are influenced by the extent of supercoiling of the DNA. This includes the transcription of the genes encoding the two enzymes (DNA gyrase and topoisomerase I) involved in the regulation of DNA supercoiling. In vitro the activity of DNA gyrase is influenced by the hydrolytic free energy of ATP. We shall present experimental evidence that the cellular free-energy state influences DNA supercoiling. We shall also discuss experiments inspecting the effect of active transcription on active DNA supercoiling. Also this system will be analyzed in terms of the control analysis with variable gene expression; here the four hierarchical levels (DNA, RNA, enzymes, metabolites) interact, adding complexity to the control analysis.

Computer Simulation↗
PubMed1990

Histones in Xenopus laevis' early development: the race against time.

Using cloned histone gene variants, the expression of three types of histone gene clusters was studied at the RNA level during early development in Xenopus laevis. For each histone class the number of mRNA molecules per embryo strongly decreased upon ovulation and steadily increased during early development, with a slight decrease at the neurula stage. Variation of the stringency of hybridization revealed that none of the histone genes probed is specifically and uniquely expressed at any time point in embryogenesis. The observed variation of histone mRNA content with time after fertilization is consistent with what is known about rate constants for RNA synthesis and degradation and about histone mRNA storage in the oocyte, provided that approximately 11 h after fertilization a regulatory transition is proposed (KOSTER, DESTREE and WESTERHOFF (1988) J. Theor. Biol. 135, 139-167). Similarly, the observed amounts of histone mRNA could well be sufficient to direct the synthesis of the required amount of histone protein. Control Analysis reveals that late in development, the histone to DNA ratio is controlled virtually equally strongly by the rate constants of DNA replication, transcription and translation. The control coefficient for RNA degradation is only a little smaller, whereas that for proteolysis is negligible. Indeed, the deceleration of DNA synthesis around the Mid Blastula Transition (some 8 h after fertilization in our studies) is a regulatory step that is essential in order to allow Xenopus to synthesize sufficient histones to structure its DNA; otherwise the embryo would run out of histone protein and histone mRNA soon thereafter. A model that assumes that the lengthening of the cell cycle around the Mid Blastula Transition it itself a response to the decrease in the concentration of histone protein not complexed to DNA, is shown to account for the dynamics of histone and DNA synthesis during the first 50 h after fertilization.

Animals↗
PubMed1989

A model for fluid secretion in the exocrine pancreas.

Fluid secretion by the isolated rabbit pancreas is strongly dependent on the presence of Na+ in the bathing medium. Substitution of Na+ by another cation such as Li+ or K+ causes an inhibition of fluid secretion rate and a change in the composition of the secreted fluid which is dependent on the nature of the substituent cation. Stimulation of the pancreas by CCK-8 or carbachol increases paracellular ion permeability and, in some cases, also fluid secretion rate. We present a simple, quantitative model for ion and water secretion which accounts for the effects observed upon Na+ substitution and stimulation. The main features are active, Na+-dependent transcellular HCO3- transport and passive, paracellular cation and anion permeation. The activity of the HCO3- pump is dependent on the energy status of the cell and on the Na+ concentration in the bathing medium, and is competitively inhibited by K+. The paracellular ion permeabilities can be modulated by stimulatory agonists. We examine the extent to which, according to the model, fluid secretion is controlled by the various system parameters such as ion permeabilities and ion pump activity, and by external parameters such as the ion concentrations in the bathing medium. In addition, calculation of the effects of changes in these parameters are carried out in order to gain more insight in the mechanisms of secretion.

Animals↗
PubMed1989

Interactions between a new class of eukaryotic antimicrobial agents and isolated rat liver mitochondria.

Members of a newly discovered class of eukaryotic antimicrobial peptides are shown to release respiratory control in isolated rat-liver mitochondria. They also dissipate the membrane potential and inhibit respiration. The uncoupling activity of the peptides decreases with time probably due to the presence of proteases in the mitochondrial preparation. Quinine and Mg2+ reduce the activity of the peptides. The nature of the dependence of the respiratory rate on the concentration of added peptides suggests that they are active in a multimeric form, consistent with the formation of a channel across the inner mitochondrial membrane. The channel allows passage of sucrose.

Animals↗
PubMed1989

Magainin 2 amide and analogues. Antimicrobial activity, membrane depolarization and susceptibility to proteolysis.

We compared the abilities of synthetic magainin 2 amide and its analogues to inhibit the growth of Escherichia coli and to cause membrane depolarization in E. coli cells and cytochrome oxidase liposomes. The analogue, magainin A, was about 40-times more active than magainin 2 amide in inhibiting the growth of E. coli and had a much more sustained effect on the membrane potential. In the liposomal system, however, there was only approx. 20% difference between these two peptides in the reduction of membrane potential and uncoupling of respiration. Studies with pronase digestion suggested that the difference in potency may be due to differential susceptibility to proteolysis in the presence of membranes.

Amides↗
PubMed1989

On the evaluation of data from flow-dialysis experiments.

Flow dialysis can be used to measure (i) ligand binding to macromolecules and (ii) the size of transmembrane ion gradients. Generally an approximate method is used to calculate the binding or gradient parameters from the raw data. Here we present a simple but exact method and evaluate the errors that may arise when the approximate method is used to calculate the magnitude of ion gradients. In addition, equations are presented that allow for a correction for sampling from or additions to the upper compartment of a flow-dialysis vessel during the measurements. Setty and Hendler [(1982) J. Biochem. Biophys. Methods 7, 35-46] have reported artifacts in the measurement of ion-gradients caused by the addition of electron donors to the upper compartment of a flow-dialysis cell. Here we extend their observations and suggest additional methods to prevent such artifacts.

Biopolymers↗
PubMed1989

Control, regulation and thermodynamics of free-energy transduction.

The quantitative formalism called Metabolic Control Theory makes it possible to be precise in discussions of metabolic control. To illustrate this, I will mention 2 experimental systems where free energy is converted from one form to another, i.e., bacteriorhodopsin liposomes and mitochondrial oxidative phosphorylation. More specifically I shall discuss how the distribution of the control of fluxes, concentrations and potentials, among the various enzymes (catalysts) in these systems has been measured and how this distribution can be understood in terms of the enzyme properties. From the outset, Metabolic Control Theory was valid for branched metabolic pathways with non-linear kinetics. Yet, it seemed to be limited to metabolic pathways without enzyme-enzyme interactions and to steady states. It is now clear that these limitations were apparent only and recent extensions to Metabolic Control Theory deal explicitly with enzyme-enzyme interaction and with transient-time analysis. Other limitations are inherent. For instance, Metabolic Control Theory pays for its clarity and exactness by being limited to small modulations. Mosaic Non Equilibrium Thermodynamics and Biochemical System Analysis are formalisms that deal with larger changes, at the cost of accuracy and exactness.

Energy Metabolism↗
PubMed1989

Magainins and the disruption of membrane-linked free-energy transduction.

Magainins, a family of positively charged peptides, are partly if not wholly responsible for antimicrobial activity in skin extracts of Xenopus laevis. We report here that members of the magainin family--i.e., the 21-amino acid peptide PGLa and the 23-amino acid peptide magainin 2 amide (PGSa)--dissipate the electric potential across various energy-transducing membranes and thus uncouple respiration from other free-energy-requiring processes. We propose that this is a likely mechanism for the antimicrobial effects of these compounds.

Animals↗