Do kinetics of ADP stimulation of mitochondria really change during myocardial maturation?
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Adenosine Diphosphate↗
Biomedical subjects
H V Westerhoff
Publications and source records attributed to H V Westerhoff.
Subtleties in control by metabolic channelling and enzyme organization.
Because of its importance to cell function, the free-energy metabolism of the living cell is subtly and homeostatically controlled. Metabolic control analysis enables a quantitative determination of what controls the relevant fluxes. However, the original metabolic control analysis was developed for idealized metabolic systems, which were assumed to lack enzyme-enzyme association and direct metabolite transfer between enzymes (channelling). We here review the recently developed molecular control analysis, which makes it possible to study non-ideal (channelled, organized) systems quantitatively in terms of what controls the fluxes, concentrations, and transit times. We show that in real, non-ideal pathways, the central control laws, such as the summation theorem for flux control, are richer than in ideal systems: the sum of the control of the enzymes participating in a non-ideal pathway may well exceed one (the number expected in the ideal pathways), but may also drop to values below one. Precise expressions indicate how total control is determined by non-ideal phenomena such as ternary complex formation (two enzymes, one metabolite), and enzyme sequestration. The bacterial phosphotransferase system (PTS), which catalyses the uptake and concomitant phosphorylation of glucose (and also regulates catabolite repression) is analyzed as an experimental example of a non-ideal pathway. Here, the phosphoryl group is channelled between enzymes, which could increase the sum of the enzyme control coefficients to two, whereas the formation of ternary complexes could decrease the sum of the enzyme control coefficients to below one. Experimental studies have recently confirmed this identification, as well as theoretically predicted values for the total control. Macromolecular crowding was shown to be a major candidate for the factor that modulates the non-ideal behaviour of the PTS pathway and the sum of the enzyme control coefficients.
Energy Metabolism↗
The two opposing activities of adenylyl transferase reside in distinct homologous domains, with intramolecular signal transduction.
Adenylyl transferase (ATase) is the bifunctional effector enzyme in the nitrogen assimilation cascade that controls the activity of glutamine synthetase (GS) in Escherichia coli. This study addresses the question of whether the two antagonistic activities of ATase (adenylylation and deadenylylation) occur at the same or at different active sites. The 945 amino acid residue ATase has been truncated in two ways, so as to produce two homologous polypeptides corresponding to amino acids 1-423 (AT-N) and 425-945 (AT-C). We demonstrate that ATase has two active sites; AT-N carries a deadenylylation activity and AT-C carries an adenylylation activity. Glutamine activates the adenylylation reaction of the AT-C domain, whereas alpha-ketoglutarate activates the deadenylylation reaction catalysed by the AT-N domain. With respect to the regulation by the nitrogen status monitor PII, however, the adenylylation domain appears to be dependent on the deadenylylation domain: the deadenylylation activity of AT-N depends on PII-UMP and is inhibited by PII. The adenylylation activity of AT-C is independent of PII (or PII-UMP), whereas in the intact enzyme PII is required for this activity. The implications of this intramolecular signal transduction for the prevention of futile cycling are discussed.
Amino Acid Sequence↗
Quantification of information transfer via cellular signal transduction pathways.
A conceptual framework is developed for the quantitative analysis of signal transfer through cellular signal transduction pathways and networks. This approach is referred to as signal transfer analysis and is based on formalisms that were first developed for the analysis of metabolic networks. Signal transduction is quantified as the sensitivity, known as the response coefficient of a target (e.g. an ion channel or transcription factor) to a signal (e.g. a hormone, growth factor or neurotransmitter). This response coefficient is defined in terms of the fractional change in the activated target brought about by a small fractional change in the signal. Quantifying the signal transduction in this way makes it possible to prove that for an idealized signaling cascade without feedback loops, the total response equals the product of all the local response coefficients, one for each level of the cascade. We show under which conditions merely having more levels in a cascade can boost the sensitivity of a target to a signal. If a signal propagates to a target through two different routes, these routes contribute independently to the total response, provided there is no feedback from the target. This independence makes the behavior of signaling cascades different from that of metabolic pathways, where different branches are connected through Kirchhoffs law. The relations between the total response and the local kinetics at each level are given for a number of network structures, such as branched signaling pathways and pathways with feedback. The formalism introduced here may provide a general approach to quantify cellular information transfer.
Cell Physiological Phenomena↗
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.
ATP-Binding Cassette Transporters↗
Glycolysis in bloodstream form Trypanosoma brucei can be understood in terms of the kinetics of the glycolytic enzymes.
In trypanosomes the first part of glycolysis takes place in specialized microbodies, the glycosomes. Most glycolytic enzymes of Trypanosoma brucei have been purified and characterized kinetically. In this paper a mathematical model of glycolysis in the bloodstream form of this organism is developed on the basis of all available kinetic data. The fluxes and the cytosolic metabolite concentrations as predicted by the model were in accordance with available data as measured in non-growing trypanosomes, both under aerobic and under anaerobic conditions. The model also reproduced the inhibition of anaerobic glycolysis by glycerol, although the amount of glycerol needed to inhibit glycolysis completely was lower than experimentally determined. At low extracellular glucose concentrations the intracellular glucose concentration remained very low, and only at 5 mM of extracellular glucose, free glucose started to accumulate intracellularly, in close agreement with experimental observations. This biphasic relation could be related to the large difference between the affinities of the glucose transporter and hexokinase for intracellular glucose. The calculated intraglycosomal metabolite concentrations demonstrated that enzymes that have been shown to be near-equilibrium in the cytosol must work far from equilibrium in the glycosome in order to maintain the high glycolytic flux in the latter.
Adenosine Diphosphate↗
Simulation of the distribution of parental strains' genomes in RC strains of mice.
Recombinant Congenic strains (RC strains) were developed to facilitate mapping of genes influencing complex traits controlled by multiple genes. They were produced by inbreeding of the progeny derived from a second backcross from a common 'donor' inbred strain to a common 'background' inbred strain. Each RC strain contains a random subset of approximately 12.5% of genes from the donor strain and 87.5% of genes from the background strain. In this way the genetic control of a complex disease may be dissected into its individual components. We simulated the production of the RC strains to study to what extent they have to be characterized in order to obtain sufficient information about the distribution of the parental strains' genomes in these strains and to acquire insight into parameters influencing their effectiveness in mapping quantitative trait loci (QTLs). The donor strain genome in the RC strains is fragmented into many segments. Genetic characterization of these strains with one polymorphic marker per 3.3 centiMorgans (cM) is needed to detect 95% of the donor strain genome. The probability of a donor strain segment being located entirely in between two markers of background strain origin that are 3 cM apart (and hence escaping detection) is 0.003. Although the donor strain genome in the RC strains is split into many segments, the largest part still occurs in relatively long stretches that are mostly concentrated in fewer than 13 autosomes, the median being 9 autosomes. Thus, in mapping QTLs, the use of RC strains facilitates the detection of linkage.
Animals↗
Internal regulation of a modular system: the different faces of internal control.
The living cell houses a multitude of molecular processes that operate simultaneously in a mutually consistent fashion. A certain degree of organization stands out, e.g. in terms of the various metabolic pathways, transcription versus translation, signal transduction versus metabolism. This paper shows that by taking one of the aforementioned organizational principles into account, the complexity of understanding cell function quantitatively may be reduced significantly. To this aim the definition of the corresponding type of organization is refined and the conceptual tools used in the analysis of the control of cell function are adjusted. The approach is elaborated for a theoretical model of cell function, in which the latter depends on a constellation of interdependent but unconnected modules. The organization of a system is reduced to global control within a limited set of partaking modules and the links between them. Information about the systems total internal control and regulability is then drastically reduced to the information specifying global control and the regulability of the pathways that constitute the system. It is shown quantitatively how control at a lower level of organization bears on the control of the cell as a whole. The approach centers on writing the product of control (matrix) and elasticity (matrix) at a number of different levels of aggregation; these products equalling the identity (matrix) under different conditions. We demonstrate that there are at least three ways in which control and regulability of a system can be matched. In one, the true control within and between the modules of the systems is the inverse of the primary regulability (i.e. elasticity plus stoichiometry). In a second, the control internal to a module (but partly determined through the other modules) is matched by the inverse of newly defined 'global' regulabilities for each module separately, which comprise the regulatory impact of the remainder of the system. In the third, the regulabilities are the ones intrinsic to the module and the control is taken equal to the control that would reign in the absence of the regulatory interactions between the units. In making these distinctions, it becomes transparent how much control stems from control within the organizational modules, and how much derives from the regulatory interactions between them. Control through other modules turns out to be equivalent, at steady state, to control within a module. The implications of this type of cellular organization for the location of the steady-state operating point is discussed.
Animals↗
FnrP and NNR of Paracoccus denitrificans are both members of the FNR family of transcriptional activators but have distinct roles in respiratory adaptation in response to oxygen limitation.
The Paracoccus denitrificans fnrP gene encoding a homologue of the Escherichia coli FNR protein was localized upstream of the gene cluster that encodes the high-affinity cbb3-type oxidase. FnrP harbours the invariant cysteine residues that are supposed to be the ligands of the redox-sensitive [4Fe-4S] cluster in FNR. NNR, another FNR-like transcriptional regulator in P. denitrificans, does not. Analysis of FnrP and NNR single and double mutants revealed that the two regulators each exert exclusive control on the expression of a discrete set of target genes. In FnrP mutants, the expression of cytochrome c peroxidase was blocked, that of membrane-bound nitrate reductase and the cbb3-type oxidase was significantly reduced, whilst the activity of the bb3-type quinol oxidase was increased. The amounts of the nitrite and nitric oxide reductases in these FnrP mutants were the same as in the wild type. NNR mutants, on the other hand, were disturbed exclusively in the concentrations of nitrite reductase and nitric oxide reductase. An FnrP.NNR double mutant combined the phenotypes of the single mutant strains. In all three mutants, the concentrations and/or activities of the aa3-type oxidase, cytochrome C550, cytochrome C552, and nitrous oxide reductase equalled those in the wild type. As the FNR boxes in front of the FnrP- and NNR-regulated genes are highly similar to or even identical to each other, the absence of cross-talk between the regulation by FnrP and NNR implies that as yet unidentified factors are important in the control. It is proposed that the redox state of an intracellular redox couple other than the oxygen/water couple is one of the factors that modulates the activity of FnrP.
Amino Acid Sequence↗
Mutational analysis of the nor gene cluster which encodes nitric-oxide reductase from Paracoccus denitrificans.
The genes that encode the hc-type nitric-oxide reductase from Paracoccus denitrificans have been identified. They are part of a cluster of six genes (norCBQDEF) and are found near the gene cluster that encodes the cd1-type nitrite reductase, which was identified earlier [de Boer, A. P. N., Reijnders, W. N. M., Kuenen, J. G., Stouthamer, A. H. & van Spanning, R. J. M. (1994) Isolation, sequencing and mutational analysis of a gene cluster involved in nitrite reduction in Paracoccus denitrificans, Antonie Leeu wenhoek 66, 111-127]. norC and norB encode the cytochrome-c-containing subunit II and cytochrome b-containing subunit I of nitric-oxide reductase (NO reductase), respectively. norQ encodes a protein with an ATP-binding motif and has high similarity to NirQ from Pseudomonas stutzeri and Pseudomonas aeruginosa and CbbQ from Pseudomonas hydrogenothermophila. norE encodes a protein with five putative transmembrane alpha-helices and has similarity to CoxIII, the third subunit of the aa3-type cytochrome-c oxidases. norF encodes a small protein with two putative transmembrane alpha-helices. Mutagenesis of norC, norB, norQ and norD resulted in cells unable to grow anaerobically. Nitrite reductase and NO reductase (with succinate or ascorbate as substrates) and nitrous oxide reductase (with succinate as substrate) activities were not detected in these mutant strains. Nitrite extrusion was detected in the medium, indicating that nitrate reductase was active. The norQ and norD mutant strains retained about 16% and 23% of the wild-type level of NorC, respectively. The norE and norF mutant strains had specific growth rates and NorC contents similar to those of the wild-type strain, but had reduced NOR and NIR activities, indicating that their gene products are involved in regulation of enzyme activity. Mutant strains containing the norCBQDEF region on the broad-host-range vector pEG400 were able to grow anaerobically, although at a lower specific growth rate and with lower NOR activity compared with the wild-type strain.
Amino Acid Sequence↗
Control analysis of glycolytic oscillations.
The principles involved in the control of the frequency of sustained metabolic oscillations are developed in the context of glycolytic oscillations in Saccharomyces cerevisiae. To this purpose, an existing mathematical model that describes the experimentally obtained oscillations was simplified to a core model. Frequency, relative phase, average concentrations and amplitudes of the oscillations were well approximated by writing the two remaining metabolic variables of the core model (representing [ATP] and [hexose]) as harmonic functions of time and by requiring them to fulfill the differential equations. The extent to which an enzyme (-conglomerate) controls the frequency in a sustained oscillation is defined as the log-log derivative of that frequency with respect to enzyme activity. In both the full model and the core model this control of frequency and the control over the average concentrations proved to be distributed over the enzymes. We identified a summation theorem, stating that the sum of such control coefficients over all processes equals unity for frequency and zero for the average concentrations.
Catalysis↗
The signal transduction function for oxidative phosphorylation is at least second order in ADP.
To maintain ATP constant in the cell, mitochondria must sense cellular ATP utilization and transduce this demand to F0-F1-ATPase. In spite of a considerable research effort over the past three decades, no combination of signal(s) and kinetic function has emerged with the power to explain ATP homeostasis in all mammalian cells. We studied this signal transduction problem in intact human muscle using 31P NMR spectroscopy. We find that the apparent kinetic order of the transduction function of the signal cytosolic ADP concentration ([ADP]) is at least second order and not first order as has been assumed. We show that amplified mitochondrial sensitivity to cytosolic [ADP] harmonizes with in vitro kinetics of [ADP] stimulation of respiration and explains ATP homeostasis also in mouse liver and canine heart. This result may well be generalizable to all mammalian cells.
Adenosine Diphosphate↗
Molecular control analysis: control within proteins and molecular processes.
Molecular control analysis is a method for analysing the extent to which the different elementary steps or rate constants within a molecular process limit the steady-state rate (or other variables) of that process. Any process which may be described by a kinetic diagram of transitions between states of the system may be analysed by molecular control analysis, and this approach has previously been used to analyse control within enzymes, transporters, enzyme complexes, channelled pathways, and group-transfer pathways. We outline the theory of molecular control analysis here, and illustrate its use by analysing control within enzymes (three beta-lactamases). Further potential applications include signal-transduction processes, protein folding, and chemical reactions.
Animals↗
What bio technologists knew all along...?
Qualitative, trial-and-error methods designed to increase the flux to desirable biotechnological products have led to new technologies and vast improvements in existing ones. However, these methods now appear in many cases to have approached their limit. In addition, there is a strong feeling in industry that much of the recent boom in academic knowledge of biochemistry and molecular biology passes biotechnology by, simply because one cannot evaluate the implications of molecular kinetics for the functioning of the producer organisms as a whole. New methods, or more rational methods, are called for. One, aimed at increasing only the concentration of a single metabolite by site-directed mutagenesis is developed here.
Animals↗
Synchronized heat flux oscillations in yeast cell populations.
Microcalorimetry was adapted to the study of glycolytic oscillations in suspensions of intact yeast cells. A correction procedure was developed for the distortion of the amplitude and phase of the heat signal, caused by the slow response of the calorimeter. This made it possible to observe oscillations in the heat production rate with a period of less than 1 min, and a relative amplitude of 5-10%. By simultaneously measuring the heat flux and concentrations of glycolytic metabolites, and by comparing acetaldehyde-induced phase shifts of the heat flux oscillations with those of NADH oscillations, the heat flux was found to be 100 degrees out of phase with glucose 6-phosphate, 80 degrees out of phase with fructose 1, 6-bisphosphate, and in phase with NADH. The flux measurement made possible by microcalorimetry allowed the recognition of (i) changes in metabolic capacity that may affect glycolytic dynamics, (ii) implications of glucose carrier kinetics for glycolytic dynamics and (iii) the continued requirement for an acetaldehyde trapping agent for the oscillations.
Calorimetry↗
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'.
Glycolysis↗
Strong control on the transit time in metabolic channelling.
A suite of different characteristic times is used to describe the temporal behavior of a metabolic pathway. Here we focus on the 'transit' time, that is the average time it takes for a molecule, entering the steady-state pathway as a substrate, to exit the pathway as a product. We show that metabolic channelling results in dramatic changes in control exerted by pathway enzymes on the transit time. In an 'ideal' pathway a doubling of the enzyme concentrations halves the transit time. In a dynamic channel such an increase can reduce the transit time by a factor of four or more.
Enzymes↗
Sustained oscillations in free-energy state and hexose phosphates in yeast.
In a population of intact cells of the yeast Saccharomyces cerevisiae the dynamics of glycolytic metabolism were investigated under the condition of sustained oscillations. At 5-s intervals cells were quenched in -40 degrees C methanol, extracted and the intracellular concentrations of glycolytic metabolites, adenine nucleotides and phosphate were analysed. Oscillations were found for the glycolytic intermediates glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-bisphosphate. At variance with earlier reports on transient glycolytic oscillations, some intermediates further down the glycolytic pathway did not oscillate significantly, even though NADH did. In addition, the adenylate energy charge and the free energy of ATP hydrolysis oscillated significantly. Dynamic coupling through the latter may be responsible for this effective compartmentation of glycolytic dynamics.
Adenosine Triphosphate↗