Why cytoplasmic signalling proteins should be recruited to cell membranes.

It has been suggested that localization of signal-transduction proteins close to the cell membrane causes an increase in their rate of encounter after activation. We maintain that such an increase in the first-encounter rate is too small to be responsible for truly enhanced signal transduction. Instead, the function of membrane localization is to increase the number (or average lifetime) of complexes between cognate signal transduction proteins and hence increase the extent of activation of downstream processes. This is achieved by concentrating the proteins in the small volume of the area just below the plasma membrane. The signal-transduction chain is viewed simply as operating at low default intensity because one of its components is present at a low concentration. The steady signalling level of the chain is enhanced 1000-fold by increasing the concentration of that component. This occurs upon 'piggyback' binding to a membrane protein, such as the activated receptor, initiating the signal-transduction chain. For the effect to occur, the protein translocated to the membrane cannot be free but has to remain organized by being piggyback bound to a receptor, membrane lipid(s) or scaffold. We discuss an important structural constraint imposed by this mechanism on signal transduction proteins that might also account for the presence of adaptor proteins.

The macroworld versus the microworld of biochemical regulation and control.

Our understanding of cell physiology has been helped greatly by viewing metabolism as a set of reactions catalysed by independent catalysts (enzymes) in ideal solutions. Yet the differences between this idealized cell and reality have strong implications for biochemical regulation and control. We show here that in the real cell an enzyme controls cell physiology in more than a single way. These different controlling modes in the real 'macroworld' can be related to one another by implementing a new type of control analysis, which is formulated in terms of the 'microworld' of the elemental processes.

The danger of metabolic pathways with turbo design.

Many catabolic pathways begin with an ATP-requiring activation step, after which further metabolism yields a surplus of ATP. Such a 'turbo' principle is useful but also contains an inherent risk. This is illustrated by a detailed kinetic analysis of a paradoxical Saccharomyces cerevisiae mutant; the mutant fails to grow on glucose because of overactive initial enzymes of glycolysis, but is defective only in an enzyme (trehalose 6-phosphate synthase) that appears to have little relevance to glycolysis. The ubiquity of pathways that possess an initial activation step, suggests that there might be many more genes that, when deleted, cause rather paradoxical regulation phenotypes (i.e. growth defects caused by enhanced utilization of growth substrate).

BDI-modelling of complex intracellular dynamics.

A BDI-based continuous-time modelling approach for intracellular dynamics is presented. It is shown how temporalized BDI-models make it possible to model intracellular biochemical processes as decision processes. By abstracting from some of the details of the biochemical pathways, the model achieves understanding in nearly intuitive terms, without losing veracity: classical intentional state properties such as beliefs, desires and intentions are founded in reality through precise biochemical relations. In an extensive example, the complex regulation of Escherichia coli vis-à-vis lactose, glucose and oxygen is simulated as a discrete-state, continuous-time temporal decision manager. Thus a bridge is introduced between two different scientific areas: the area of BDI-modelling and the area of intracellular dynamics.

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.

Systems biology: new paradigms for cell biology and drug design.

In this chapter various facets of and approaches to systems biology will be discussed. This then leads to an illustration of how systems biology may be used in drug target design. We present five new paradigms for drug target research and show how these are based in systems biology.

Signal transduction in bacteria: phospho-neural network(s) in Escherichia coli?

The molecular basis of many forms of signal transfer in living organisms is provided via the transient phosphorylation of regulatory proteins by transfer of phosphoryl groups between these proteins. The dominant form of signal transduction in prokaryotic microorganisms proceeds via so-called two-component regulatory systems. These systems constitute phosphoryl transfer pathways, consisting of two or more components. Most of these pathways are linear, but some converge and some are divergent. The molecular properties of some of the well-characterised representatives of two-component systems comply with the requirements to be put upon the elements of a neural network: they function as logical operators and show the phenomenon of autoamplification. Because there are many phosphoryl transfer pathways in parallel and because there also appears to be cross-talk between these pathways, the total of all two-component regulatory systems in a single prokaryotic cell may show the typical characteristics of a 'phospho-neural network'. This may well lead to signal amplification, associative responses and memory effects, characteristics which are typical for neural networks. One of the main challenges in molecular microbial physiology is to determine the extent of the connectivity of the constituting elements of this presumed 'phospho-neural network', and to outline the extent of intelligence-like behaviour this network can generate. Escherichia coli is the organism of choice for this characterization.

Control theory of one enzyme.

The analogue of metabolic control theory is developed for the control of reactions catalyzed by single enzymes. The control exerted by any of the elemental transitions of enzyme catalytic cycles on reaction rate and on concentrations (probabilities) of enzyme states is quantified in line with the principle of detailed balance. For enzyme reactions with arbitrary kinetic schemes, e.g., with several enzyme cycles, reflecting coupling and slipping of reactions, it is derived what the various sums of the control coefficients are equal to (cycle summation theorems). Total control on flux, state probability and ratios of branch fluxes are 1, 0 and 0, respectively. The general connectivity theorems are derived which indicate how control is determined by the kinetics of the elemental steps. In addition, for enzymes catalyzing single (or completely coupled) processes the control coefficients are expressed in terms of actual and standard free energy differences across the steps. The prevalent qualitative contention that the step with the smallest forward rate constant, or with the largest free energy drop is the step limiting the performance of the enzyme is shown to fail. The new theory should allow subtle analysis of the control of an enzyme catalyzed reaction.

Control of frequency and amplitudes is shared by all enzymes in three models for yeast glycolytic oscillations.

The three main existing models for glycolytic oscillations in yeast were re-examined to investigate how these oscillations are controlled. We implemented the operational definitions provided by metabolic control analysis to quantify the control properties of enzymes with regard to glycolytic oscillations. In all three models, the control of the frequency and that of the amplitudes of the metabolites were distributed among the enzymes. There was no obvious correlation between the control of the average flax and the control of the frequency. Most importantly, the so-called 'oscillophore' of the system, traditionally the enzyme primarily held responsible for the generation of the oscillation, was not the only controlling step. We conclude that just like steady-state flux control is not necessarily limited to a rate-limiting step, oscillations are not dictated by a single 'oscillophore'.

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.

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.

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.

Thermodynamics of growth. Non-equilibrium thermodynamics of bacterial growth. The phenomenological and the mosaic approach.

Microbial growth is analyzed in terms of mosaic and phenomenological non-equilibrium thermodynamics. It turns out that already existing parameters devised to measure bacterial growth, such as YATP, mu, and Q substrate, have as thermodynamic equivalents flow ratio, output flow and input flow. With this characterisation it becomes possible to apply much of the already existing knowledge of phenomenological non-equilibrium thermodynamics to bacterial growth. One of the conclusions is that the frequent observation that YATP is only 50% of its theoretical maximum does not mean that the microbe corresponds to a thermodynamic system that has been optimized for maximal output power, as has been suggested. Rather, at least in some cases, it corresponds to a system that has been optimized towards maximum growth rate. When the degree of reduction of the (single) carbon source is significantly smaller than that of the biomass produced, the efficiency of biomass synthesis has been kept as high (i.e., about 24%) as is consistent with maximization of the growth rate at optimal efficiency. Mosaic thermodynamics allows an analysis of processes which in microbial metabolism may be responsible for any particular growth behaviour. Equations are derived that predict the effect of uncoupling through leaks, futile cycling, or 'slip' on microbial growth. It turns out that uncoupling is expected to affect both the growth rate-independent and the growth rate-dependent 'maintenance coefficient'. The effect on the latter is different when catabolic substrate limits growth than when anabolic substrate limits growth. In the latter case, the growth rate-dependent maintenance coefficient is negative. It is concluded that mosaic non-equilibrium thermodynamics will be a powerful theoretical tool especially in future experimental analyses of the metabolic basis for microbial growth characteristics and growth regulation.

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.

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.

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).

Control analysis of metabolic systems involving quasi-equilibrium reactions.

Reactions for which the rates are extremely sensitive to changes in the concentrations of variable metabolite concentrations contribute little to the control of biochemical reaction networks. Yet they do interfere with the calculation of the system's behaviour, both in terms of numerical integration of the rate equations and in terms of the analysis of metabolic control. We here present a way to solve this problem systematically for systems with time hierarchies. We identify the fast reactions and fast metabolites, group them apart from the other ("slow") reactions and metabolites, and then apply the appropriate quasi-equilibrium condition for the fast subsystem. This then makes it possible to eliminate the fast reactions and their elasticity coefficients from the calculations, allowing the calculation of the control coefficients of the slow reactions in terms of the elasticity coefficients of the slow reactions. As expected, the elasticity coefficients of the fast reactions drop out of the calculations, and they are irrelevant for control at the time resolution of the steady state of the slow reactions. The analysis, when applied iteratively, is expected to be particularly valuable for the control analysis of living cells, where a time hierarchy exists, the fastest being at the level of enzyme kinetics and the slowest at gene expression.

Anthracyclines modulate multidrug resistance protein (MRP) mediated organic anion transport.

We studied the ATP-dependent uptake of dinitrophenyl-glutathione (GS-DNP) into plasma membrane vesicles derived from parental GLC4 cells and from multidrug resistant GLC4/ADR cells. The latter have a high expression of the multidrug resistance protein (MRP). Uptake of GS-DNP into membrane vesicles from GLC4/ADR cells was highly stimulated by the addition of ATP, compared to the uptake into membrane vesicles from GLC4 cells. This ATP-dependent uptake into membrane vesicles from GLC4/ADR cells was saturable with a Km of 1.2 +/- 0.2 microM and a Vmax of 560 +/- 80 pmol/mg prot./min. ATP stimulated GS-DNP uptake with a Km of 187 +/- 4 microM. This uptake was specifically inhibited by a polyclonal serum raised against a fusion protein containing a segment of MRP. The ATP-dependent uptake of GS-DNP was not only inhibited by organic anions, such as oxidized glutathione (GSSG), methotrexate (MTX) and some bile acids, but also by non-anionic natural product drugs, such as anthracyclines, vinca alkaloids and etoposide (VP-16). Uptake of GSSG and MTX into membrane vesicles from GLC4/ADR cells could be stimulated by ATP. The ATP-dependent uptake of GSSG had a Km of 43 +/- 3 microM and a Vmax of 900 +/- 200 nmol/mg protein/min. The ATP-dependent uptake of GS-DNP seemed to be non-competitively inhibited by the anthracycline daunorubicin (DNR), whereas the ATP-dependent GSSG uptake seemed to be competitively inhibited by DNR. A substrate binding site on MRP is proposed that comprises a pocket in which both DNR and GS-DNP or GSSG bind in random order to different, only partly overlapping sites. In this pocket binding of a second compound is influenced by the compound which was bound first.

Relationship between chemiosomotic flows and thermodynamic forces in oxidative phosphorylation.

A set of equations has been derived, describing quantitatively the relationships between flows and thermodynamic forces in the chemiosmotic model of oxidative phosphorylation. Experimental tests of these equations give information on the stoichiometric coupling constants between the different flows.

A minimal hypothesis for membrane-linked free-energy transduction. The role of independent, small coupling units.

Experimental data are reviewed that are not in keeping with the scheme of 'delocalized' protonic coupling in membrane-linked free-energy transduction. It turns out that there are three main types of anomalies: (i) rates of electron transfer and of ATP synthesis do not solely depend on their own driving force and on delta mu H, (ii) the ('static head') ratio of delta Gp to delta mu H varies with delta mu H and (iii) inhibition of either some of the electron-transfer chains or some of the H+-ATPases, does not cause an overcapacity in the other, non-inhibited proton pumps. None of the earlier free-energy coupling schemes, alternative to delocalized protonic coupling, can account for these three anomalies. We propose to add a fifth postulate, namely that of the coupling unit, to the four existing postulates of 'delocalized protonic coupling' and show that, with this postulate, protonic coupling can again account for most experimental observations. We also discuss: (i) how experimental data that might seem to be at odds with the 'coupling unit' hypothesis can be accounted for and (ii) the problem of the spatial arrangement of the electrical field in the different free-energy coupling schemes.