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H V Westerhoff

Publications and source records attributed to H V Westerhoff.

At least 163 records · Page 9Linked to original sources
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↗
PubMed1988

Kinetics of histone gene expression during early development of Xenopus laevis.

Using literature data for transcriptional and translational rate constants, gene copy numbers, DNA concentrations, and stability constants, we have calculated the expected concentrations of histones and histone mRNA during embryogenesis of Xenopus laevis. The results led us to conclude that: (i) for X. laevis the gene copy number of the histone genes is too low to ensure the synthesis of sufficient histones during very early development, inheritance from the oocyte of either histone protein or histone mRNA (but not necessarily both) is necessary; (ii) from the known storage of histones in the oocyte and the rates of histone synthesis determined by Adamson & Woodland (1977), there would be sufficient histones to structure the newly synthesized DNA up to gastrulation but not thereafter (these empirical rates of histone synthesis may be underestimates); (iii) on the other hand, the amount of H3 mRNA recently observed during early embryogenesis (Koster, 1987, Koster et al., 1988) could direct a higher and sufficient synthesis of H3 protein, also after gastrulation. We present a quantitative model that accounts both for the observed H3 mRNA concentration as a function of time during embryogenesis and for the synthesis of sufficient histones to structure the DNA throughout early embryogenesis. The model suggests that X. laevis exhibits a major (i.e. some 14-fold) reduction in transcription of histone genes approximately 11 hours after fertilization. This reduction could be due to a decrease in the number of transcribed histone genes, a decreased rate constant of transcription with continued transcription of all the histone genes, and/or a reduction in the time during the cell cycle in which histone mRNA synthesis takes place. Alternatively, the histone mRNA stability might decrease approximately 16-fold 11 hours after fertilization.

Animals↗
PubMed1988

Sigmoidal relation between mitochondrial respiration and log ([ATP]/[ADP])out under conditions of extramitochondrial ATP utilization. Implications for the control and thermodynamics of oxidative phosphorylation.

Except for close to state 3, mitochondrial respiration has been observed to vary almost linearly with the extramitochondrial phosphorylation potential. For the understanding of the control, thermodynamics, and stoichiometries of oxidative phosphorylation, it is important if this linearity corresponds to an extension of a near-equilibrium flow-force relationship. Using three methods to determine the extramitochondrial ATP/ADP ratio, we observed that at high ATP/ADP ratios the relationship between respiratory rate and log (ATP/ADP) deviated in a sigmoidal fashion from linearity, if the amount of hexokinase present was modulated. In a titration with uncoupler, the sigmoidicity at high ATP/ADP ratios was absent. This difference between the flow-force relationships of these two experiments suggests that the sigmoidicity in the former case reflects a nonproportional flow-force relationship of the adenine nucleotide translocator. In the latter case, one measures the flow-force relationship of the redox-driven proton pumps alone, which turns out to be virtually linear. We determined the flow-force relation of the adenine nucleotide translocator for two ways of varying the force and confirmed the sigmoidicity in both cases. The implication is that the near-linearity of the flow-force relationships at intermediary respiratory rates does not correspond to an Onsager-type (near equilibrium) linearity. We discuss that this phenomenon requires the application of nonclassical forms of nonequilibrium thermodynamics and may be responsible for some of the control over oxidative phosphorylation that is exerted by the cytosolic ATP consuming processes.

Adenosine Diphosphate↗
PubMed1988

Energization-induced redistribution of charge carriers near membranes.

The electric field arising from proton pumping across a topologically closed biological membrane causes accumulation close to the membrane of ionic charges equivalent to the charge of the pumped protons, positive on the side towards which protons are pumped, negative on the other side. We shall call this the 'active surface charge'. We here use the Poisson-Boltzmann equation to evaluate the effects of zwitterionic buffer molecules and uncharged proteins in the aqueous phase bordering the membrane on the magnitude and ionic composition of the active surface charge. For the positive side of the membrane, the main results are: (1) If the membrane is freely accessible to bulk phase ions, pumped protons exchange with these ions, such that the active surface charge consists of salt cations. (2) If a significant fraction of the ions in bulk solution consists of buffer molecules, then some of the pumped protons will remain close to the membrane and constitute a major fraction of the active surface charge. (3) If a protein layer borders the membrane, a significant part of the transmembrane electric potential difference exists within that protein layer and protons inside this layer dominate the active surface charge. (4) On the negative side of the membrane the corresponding phenomena would occur. (5) All these effects are strictly dependent on the transmembrane electric potential difference arising from proton pumping and would come in addition to the well known effects of buffers and electrically charged proteins on the retention of scalar protons. (6) No additional proton diffusion barrier may be required to account for a deficit in number of protons observed in the aqueous bulk phase upon aeration-induced proton pumping.

Mathematics↗
PubMed1988

Coupling of vectorial proton flow to a biochemical reaction by local electric interactions.

For a transmembrane redox enzyme and a (passive) protonophore, the complete set of rate equations is given. Turnover causes cyclic variation of their electric polarization. This is responsible not only for effects of the electric field on the rate constants but also for the generation of an electric field felt by neighboring molecules. It is calculated that, when the systems are close together at a fixed distance, cycling of the two systems becomes coupled enabling the protonophore to pump protons against their electrochemical gradient. If the electrochemical gradient for protons approaches the input force of the redox reaction, slip (incomplete coupling between the chemical and proton-transport reactions) results. By using different sets of parameters, both kinetically reversible and kinetically irreversible proton pumps can be simulated.

Biological Transport↗
PubMed1987

On the origin of the limited control of mitochondrial respiration by the adenine nucleotide translocator.

A thermodynamic control theory previously developed has been applied to mitochondrial oxidative phosphorylation with emphasis on the role of delta microH and coupling and within the paradigm of delocalized chemiosmotic coupling. The basis for the observed distribution of flux control over the participating enzymes is shown to lie in the relative magnitudes of so-called delta microH elasticity coefficients, i.e., the delta microH dependencies of the different mitochondrial processes. In particular the relatively strong delta microH dependence of mitochondrial respiration is responsible for the significant role of the adenine nucleotide translocator in the control of oxidative phosphorylation. Uncoupling decreases the control exerted by this translocator on respiration but increases that exerted on phosphorylation.

Adenosine Diphosphate↗
PubMed1987

Thermodynamics of the control of metabolism.

A theory is presented, describing the control analysis of metabolic systems in terms of Gibbs free energies, extending earlier work of Kacser and Burns (25), and Heinrich and Rapoport (29). It is shown that relationships exist between flux control coefficients (the degree to which enzymes control steady-state fluxes) and free-energy elasticity coefficients, defined as the fractional change in the rate of a reaction induced by a standard change in one free-energy difference, while all the other free-energy differences are kept constant. Application of this extended control analysis to some biochemical reactions, including proton translocation, demonstrates that 1. Problems arising in the control analysis because of conservation (sum concentration of substrate and product constant) can be circumvented. 2. Although free-energy elasticity coefficients are maximal when the reaction is close to equilibrium, they can also be significant when the reaction is not close to equilibrium. 3. Problems in the control analysis caused by compartmentation can be resolved by defining control parameters that refer to the organelle as a whole. 4. These latter control parameters obey the above-mentioned relationships.

Animals↗
PubMed1987

Variation of efficiency with free-energy dissipation in models of biological energy transduction.

For two models of biological free-energy transducers, it is investigated how free-energy dissipation and efficiency vary as (i) the demand for output free energy, (ii) the input free energy or (iii) the properties of the transducers themselves, are varied. One model is representative of near-equilibrium free-energy transducers in general, the other is a special case of far-from-equilibrium free-energy transduction, reminiscent of proton pumping by bacteriorhodopsin. It turns out that the relationship between efficiency and free-energy dissipation depends strongly on what varies. In some cases, free-energy dissipation increases as the efficiency increases. It is suggested that this is one reason why biological evolution has not resulted in high efficiencies and low rates of free-energy dissipation. For the near-equilibrium free-energy transducer, the free-energy dissipation at the static head steady state is minimal with respect to variations in the output force. For the far-from-equilibrium model (of bacteriorhodopsin), the static head does not correspond to such a minimum, if that free-energy transducer slips.

Bacteriorhodopsins↗
PubMed1987

Can free energy be transduced from electric noise?

Recently, it was shown that free energy can be transduced from a regularly oscillating electric field to do chemical or transport work when coupled through an enzyme with appropriate electrical characteristics. Here we report that randomly pulsed electric fields can also lead to work being done, giving rise to speculation as to whether appropriately designed enzymes can extract and convert free energy from the inherent fluctuations in their environment. The paradox is resolved by showing that equilibrium electrical noise resulting from the environment around an enzyme cannot be completely random but is correlated to the state that the enzyme is in. If the noise has the appropriate reciprocal interaction with the enzyme, its potential to serve as a free-energy source disappears. This is shown by Monte Carlo and other numerical calculations and is proven analytically by use of the diagram method. This method also is used to provide an explicit equation showing that, under a range of conditions, our model enzyme will be induced by uncorrelated ("autonomous") noise to undergo net cyclic flux. That work can be transduced from the "random" noise is demonstrated by using numerical methods.

Electrochemistry↗
PubMed1986

Co-operativity and enzymatic activity in polymer-activated enzymes. A one-dimensional piggy-back binding model and its application to the DNA-dependent ATPase of DNA gyrase.

The binding of a ligand to a one-dimensional lattice in the presence of a second ("rider") ligand, which binds only to the first ligand (piggy-back binding), is studied. A model derived from this study is used to analyze the effects of co-operativity on the reaction rates of enzymes activated by polymeric cofactors that provide multiple binding sites for the enzyme. It is found that in the presence of strong co-operativity, the steady-state reaction rates of polymer-activated enzymes can be very different from the Michaelis-Menten paradigm. By adjusting the co-operativity parameters and the binding constants of the ligands, the model can generate apparent auto-catalytic enhancement by substrates at low substrate concentrations and apparent substrate inhibition at high substrate concentrations. The model is shown to be able to explain the differences in the rates of ATP hydrolysis by DNA gyrase in the presence of long versus short DNA molecules and in the presence of long DNA molecules at different gyrase to DNA ratios.

Adenosine Triphosphatases↗
PubMed1986

How enzymes can capture and transmit free energy from an oscillating electric field.

Recently, it has been demonstrated that free energy from an alternating electric field can drive the active transport of Rb+ by way of the Na+, K+-ATPase. In the present work, it is shown why many transmembrane enzymes can be expected to absorb free energy from an oscillating electric field and transduce that to chemical or transport work. In the theoretical analysis it turned out to be sufficient that (i) the catalytic process be accompanied by either net or cyclic charge translocation across the membrane and (ii) the stability of the enzyme states involved be asymmetric. Calculations based on a four-state model reveal that free-energy transduction occurs with sinusoidal, square-wave, and positive-only oscillating electric fields and for cases that exhibit either linear or exponential field-dependent rate constants. The results suggest that in addition to oscillating electric field-driven transport, the proposed mechanism can also be used to explain, in part, the "missing" free energy term in the cases in which ATP synthesis has been observed with insufficient transmembrane proton electrochemical potential difference.

Adenosine Triphosphatases↗
PubMed1985

A critical appraisal of evidence for localized energy coupling. Kinetic studies on liposomes containing bacteriorhodopsin and ATP synthase.

In intact systems (chloroplasts, mitochondria and bacteria) many experiments have been reported which are indicative of localized coupling between ATP synthase and electron transfer complexes. We have carried out similar experiments with a system in which we may assume that specific interactions between the proton pumps are absent: reconstituted vesicles containing bacteriorhodopsin and yeast mitochondrial ATP synthase. The only experiment that gives results which differ from those previously published for intact systems concerns the effect of uncouplers on the rate of ATP synthesis at different levels of inhibition of the ATP synthase. We propose that this type of experiment may discriminate between localized and delocalized coupling.

ATP Synthetase Complexes↗
PubMed1985

Stochastic free energy transduction.

Theoretical free-energy coupling systems in which the free energy coupling intermediate (e.g., the proton) occurs only in small numbers of molecules per coupling unit are shown to exhibit a number of peculiar properties: (i) the reactions involving the intermediates do not follow conventional kinetic (or nonequilibrium thermodynamic) rate laws in terms of the average concentration or chemical potential of the intermediate, (ii) the variation of the output reaction rate with the average intermediate concentration (or apparent chemical potential) is not unequivocal but depends on whether the input reaction or the leak is varied to alter that concentration, and (iii) when the apparent free energy contained in the average concentration of the intermediate is compared with the average free energy recovered in the output reaction, apparent violations of the second law of thermodynamics can occur. These phenomena are reminiscent of experimental observations in proton-linked free-energy transducing systems that suggest a more direct coupling between electron transfer chains and H+-ATPases than only through a bulk proton gradient, delta muH. Consequently, the chemiosmotic coupling theory can account for those observations if it limits the number of free energy coupling protons per chemiosmotic coupling unit to small values.

Adenosine Triphosphate↗