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H V Westerhoff

Publications and source records attributed to H V Westerhoff.

195 records · Page 11Linked to original sources
PubMed1981

Mosaic nonequilibrium thermodynamics describes biological energy transduction.

A procedure, called "mosaic nonequilibrium thermodynamics," for describing ion movement and energy transduction in biological membranes is tested in a model system: bacteriorhodopsin liposomes. The important steps in the theoretical derivations are summarized; one of the experimental tests of the postulated fundamental flow-force relationships is shown. Furthermore, how the quantitative method, even if used only qualitatively, facilitates analysis and understanding of experimental results (in this case, the effect of medium composition on the development of pH gradient and membrane potential in the bacteriorhodopsin liposomes) is shown. The main advantage of this method lies in its quantitative description of the effect of variation of system parameters on the performance of, in this case, the reconstituted proton pump bacteriorhodopsin. As an example, the method is shown to explain quantitatively the dependence of the steady-state pH gradient on the light intensity. Even in more refined analyses of experiments, the quantitative theoretical description is in full accordance with the experimental results; this is illustrated by considering the effect of valinomycin on the dependence of the initial rate of proton uptake into bacteriorhodopsin liposomes on light intensity. It is concluded that mosaic nonequilibrium thermodynamics describes ion movement and energy transduction in the model system of bacteriorhodopsin liposomes and, therefore, may be applied to any other biological system performing such processes.

Bacteriorhodopsins↗
PubMed1980

Multichannel analysis of intracellular control and intercellular transfer of molecules.

The metabolic regulation and exchanges within intracellular organelles or a cell cluster are studied by multichannel microfluorometry and microinjection of metabolites or tracers. The determination of structure-function relationships relies on the retrieval of cells after microfluorometry, for subsequent morphological evaluation. Rate constants of coenzyme reduction-reoxidation were deduced from a mathematical model of NAD(P) in equilibrium with NAD(P)H transients due to microinjection of metabolites into cultured cells belonging to a variety of normal or malignant lines. Nuclear and cytoplasmic sites operate synchronously or not, depending upon metabolic demand or pathological alterations. Intercellular transit times are determined for tracers and metabolites. Within cell clusters 'communicating territories' are described, which can show metabolically a multicellular integrated state. Microfluorometry in conjunction with ultrastructural and other studies can be used to develop a cybernetic model of the living cell, also yielding dynamic models of cooperative and regulatory interactions between different kinds of specialised cells within a cell cluster.

Animals↗
PubMed1980

New metabolic parameters for the characterization of cells.

Microspectrofluorometric evaluation of coenzyme-linked transient changes in blue fluorescence, triggered by microinjections of metabolic intermediates, allows the definition of dynamic parameters in the characterization of cells. The observed fluorescence transients can be simulated by appropriate equations accounting for NAD(P) reduction-reoxidation, with NAD(P) as rate-limiting or not. From the above, the rate constants K1 and K2 of NAD(P) reduction and reoxidation can be determined. Other useful parameters in the metabolic evaluation of different cell lines, comprising normal and transformed fibroblasts, glia-glioma, melanoma lines, and a mouse embryo clone, can be derived from the relationship between injected dose of substrate and rise or decay rates of NAD(P) in equilibrium or formed from NAD(P)H transients. Reoxidation of NAD(P)H seems to be a useful target for such studies in view of possible impairment in malignant cells and X-irradiated cells. Cells followed by fluorometry are retrieved for subsequent ultrastructural and other analyses. Thus, the metabolic patterns associated with the operation of intracellular pathways or organelle interactions, and their aberrations can be recognized. On this basis eventually a classification of different cell lines according to structure-function should be feasible.

Animals↗
PubMed1979

Bacteriorhodopsin in liposomes. I. A description using irreversible thermodynamics.

A comprehensive description of light-induced ion transport in bacteriorhodopsin liposomes is presented. Linear irreversible thermodynamics and the chemiosmotic theory serve as theoretical bases for the formulation of a limited number of fundamental equations. In these equations mechanistic parameters characterize the dependence of ion movement and flux through the photochemical cycle of bacteriorhodopsin on electrochemical potential differences and a so-called light affinity. By making appropriate steady-state assummptions and carrying out mathematical reduction experimentally testable expressions, still containing the mechanistic parameters, are obtained. In the accompanying article rigid trials to falsify these expressions are shown to be unsuccessful.

Bacteriorhodopsins↗
PubMed1979

Bacteriorhodopsin in liposomes. II. Experimental evidence in support of a theoretical model.

In the preceding article equations describing relevant ion flows in illuminated suspensions of bacteriorhodopsin liposomes have been derived. Here these equations are subjected to experimental tests. Changes in permeability characteristics of the liposomal membrane are brought about by addition of specific ionophores and change of medium composition. Using light-driven proton uptake and electrochemical potential differences for protons across the membrane as observation parameters, ridig attempts to falsify the derived equations are unsuccessful. Agreement between equations and experimental results is established on the point of: (i) the antagonistic effect of valinomycin and nigericin on the two components of the proton-motive force, (ii) the time dependence of the changes in transmembrane electrical and chemical potential differences after the onset of illumination. In three independent experimental systems evidence was obtained for the correctness of the postulated dependence of the turnover rate of the photochemical cycle on back pressure by the transmembrane electrochemical potential difference for protons.

Bacteriorhodopsins↗
PubMedDate not supplied

Quantifying heterogeneity: flow cytometry of bacterial cultures.

Flow cytometry is a technique which permits the characterisation of individual cells in populations, in terms of distributions in their properties such as DNA content, protein content, viability, enzyme activities and so on. We review the technique, and some of its recent applications to microbiological problems. It is concluded that cellular heterogeneity, in both batch and continuous axenic cultures, is far greater than is normally assumed. This has important implications for the quantitative analysis of microbial processes.

Bacteria↗
PubMedDate not supplied

Quantitative approaches to the analysis of the control and regulation of microbial metabolism.

Recently, a number of novel ways of considering the control, regulation and thermodynamics of microbial physiology have been developed and applied. We here present an overview of the new concepts involved, of their limitations and of the most recent attempts to deal with those limitations. We conclude that there no longer exist reasons of principle for vagueness in discussions of the control of microbial physiology and energetics. Further, the novel conceptual methods serve to remove part of the discordance between holistic and reductionistic views of microbial physiology.

Adenosine Monophosphate↗
PubMedDate not supplied

Control theory of metabolic channelling.

Various factors appear to control muscle energetics, often in conjunction. This calls for a quantitative approach of the type provided by Metabolic Control Analysis for intermediary metabolism and mitochondrial oxidative phosphorylation. To the extent that direct transfer of high energy phosphates and spatial organization plays a role in muscle energetics however, the standard Metabolic Control Theory does not apply, neither do its theorems regarding control. This chapter develops the Control Theory that does apply to the muscle system. It shows that direct transfer of high energy phosphates bestows a system with enhanced control: the sum of the control exerted by the participating enzymes on the flux of free energy form the mitochondrial matrix to the actinomyosin may well exceed the 100% mandatory for ideal metabolic pathways. It is also shown how sequestration of high energy phosphates may allow for negative control on pathway flux. The new control theory gives methods functionally to diagnose the extent to which channelling and metabolite sequestration occur.

Animals↗
PubMedDate not supplied

DNA supercoiling by DNA gyrase. A static head analysis.

Using purified DNA gyrase to supercoil circular plasmid pBR322 DNA, we examined how the linking number attained at the steady state ('static head') varies with the concentrations of ATP and ADP, both in the absence and presence of spermidine. In the absence of spermidine at total adenine nucleotide concentrations between 0.35 and 1.4 mM, the static-head linking number was independent of the sum concentration of ATP and ADP, but depended strongly on the ratio of their concentrations. We established that the same linking number was attained independent of the direction from which the steady state was approached. The decrease in linking number at static head is more extensive when spermidine is present in the incubation, but remains a function of the [ATP]-to-[ADP] ratio. These results are discussed in terms of various kinetic schemes for DNA gyrase. We present one kinetic scheme that accounts for the experimental observations. According to this scheme our experimental results imply that there is significant slip in DNA gyrase when spermidine is absent. It is possible that spermidine acts through adjustment of the degree coupling of DNA gyrase.

Adenosine Diphosphate↗
PubMedDate not supplied

Energy coupling and Hill cycles in enzymatic processes.

We review how Hill's work on enzyme catalysis has nurtured our understanding of the mechanism by which enzymes can couple downhill processes to uphill processes. More specifically, we discuss the following questions: (i) Does it make sense to distinguish the chemical potential of the bound ligand from that of the binding enzyme? (ii) To what extent can free-energy transduction be localized at some crucial step in the catalytic cycle? (iii) Need enzymes be optimized so as to even out the profile of basic free energy along the catalytic cycle? (iv) How do continuous models of conformational transitions relate to discrete state diagrams and their kinetic elaborations? We conclude that (1) only in very special cases is it useful to designate a portion of the free energy of the enzyme-ligand complex as the free energy of the bound ligand; (2) only for some mechanisms can free-energy transduction be localized within a part of the catalytic cycle; (3) only in special cases should one expect enzymes to be "optimized" so as to have smooth basic free-energy profiles; and (4) transition rate constants can often be related to conformational diffusion constants, although in certain situations the kinetic description of an enzyme as if jumping between discrete states is impracticable; a diffusion-type description may then be preferable.

Catalysis↗
PubMedDate not supplied

Macromolecular intelligence in microorganisms.

Biochemistry and molecular biology have been focusing on the structural, catalytic, and regulatory properties of individual macromolecules from the perspective of clarifying the mechanisms of metabolism and gene expression. Complete genomes of 'primitive' living organisms seem to be substantially larger than necessary for metabolism and gene expression alone. This is in line with the findings of silent phenotypes for supposedly important genes, apparent redundancy of functions, and variegated networks of signal transduction and transcription factors. Here we propose that evolutionary optimization has been much more intensive than to lead to the bare minima necessary for autonomous life. Much more complex organisms prevail. Much of this complexity arises in the nonlinear interactions between cellular macromolecules and in subtle differences between paralogs (isoenzymes). The complexity can only be understood when analyzed quantitatively, for which quantitative experimentation is needed in living systems that are as simple and manipulatable as possible, yet complex in the above sense. We illustrate this for the glutamine synthetase cascade in Escherichia coli. By reviewing recent molecular findings, we show that this cascade is much more complex than necessary for simple regulation of ammonia assimilation. Simulations suggest that the function of this complexity may lie in quasi-intelligent behavior, including conditioning and learning.

Cell Physiological Phenomena↗
PubMedDate not supplied

[Kinetic modeling of energy metabolism and generation of active forms of oxygen in hepatocyte mitochondria].

Direct nonenzymatic oxidation of semiquinone by oxygen is one of the main sources of superoxide radicals (O2.-) in mitochondria. By using all the known data on hepatocyte mitochondria, we have revealed the correlation between the rate of superoxide generation by the bc1 complex and the transmembrane potential (delta psi). If the main electrogenic stage of the Q cycle is suggested to be the electron transfer between the cytochrome b hemes, then the rate of superoxide generation sharply increases when delta psi grows from 150 mV to 180 mV. However, this interrelation is ambiguous. Indeed, the increase of the generation rate with the growth of the potential can occur faster when succinate dehydrogenase is inhibited by malonate than when external ADP is exhausted. When the potential is changed by adding phosphate or potassium (K+), the rate of O2.- production remains constant, although the comparison of the rate values at the same delta psi reveals the effect of phosphate or potassium. It turned out that the rate of O2.- generation is a function of delta mu H rather than any of its components. Phosphate and K+ have practically no influence on delta mu H, since the change in delta psi is compensated by delta pH. The rate of superoxide generation by the bc1 complex is a multiple function of the electron-transfer activity of enzymes, the processes determining the membrane potential (e.g., loading), and of the oxygen concentration. The kinetic model proposed in this work may serve a tool to understand how the superoxide production is regulated.

Adenosine Diphosphate↗