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PubMed2002

Futile cycles revisited: a markov chain model of simultaneous glycolysis and gluconeogenesis.

We have used a random walk model of glycolysis and gluconeogenesis to investigate the bioenergetic implications of considering the cell cytoplasm to be a uniform well-mixed compartment. Radiotracer studies conducted on hepatocytes harvested from fasted rats and incubated with 40 mM glucose and 10 mM lactate demonstrated simultaneous glycolysis and gluconeogenesis, with net glycolysis. Tracer introduced as glycerol was incorporated both into glucose (via gluconeogenesis) and into pyruvate (via glycolysis). The data allow us to place a lower bound on the energetic cost of futile cycles involving adenosine triphosphate (ATP) hydrolysis in the early phosphorylation steps of glycolysis. Applying the Markov Chain model for glucose undergoing metabolism to pyruvate, the expected number of ATP molecules hydrolysed is not less than 15 ATP molecules per glucose molecule. The data suggest that, in hepatocytes under the circumstances of this experiment, either glycolysis is a net consumer of ATP, or glycolysis and gluconeogenesis are compartmentalized to a greater extent than is generally supposed.

Animals↗
PubMed1991

Modifications of cardiac contractility by redox cycling alkylating and mixed redox cycling/alkylating quinones.

The effects of redox cycling, alkylating and mixed redox cycling/alkylating benzo- and naphthoquinones were examined in electrically driven guinea pig left atria. Cardiac microsomal and mitochondrial NAD(P)H-dependent metabolism of the quinones and consequent generation of superoxide anion (O2.-) were also measured. Mixed redox cycling/alkylating 2-methyl-1,4-naphthoquinone, redox cycling 2,3-dimethoxy-1,4-naphthoquinone and alkylating p-benzoquinone determined concentration-dependent positive inotropic responses, whereas redox cycling 2,3,5,6-tetramethyl-p-benzoquinone had no effect. The positive inotropic effect of 2,3-dimethoxy-1,4-naphthoquinone was completely catecholamine-mediated, that of 2-methyl-1,4-naphthoquinone was approximately 70% adrenergic and 30% direct. p-Benzoquinone acted directly on heart muscle. In time, quinones with alkylating properties caused increases in the resting force of atria, whereas redox cycling quinones did not produce toxic effects. Mitochondrial NADH-oxidoreductase accounted for 90 to 95% of the metabolism of all quinones, whereas the contribution of the microsomal pathway was negligible. Considerable amounts of O2.- were produced by mitochondrial biotransformation of 2-methyl-1,4-naphthoquinone and 2,3-dimethoxy-1,4-naphthoquinone but not of 2,3,5,6-tetramethyl-p-benzoquinone and p-benzoquinone, suggesting a kind of relation between O2.- generation and the release of catecholamines.

Alkylating Agents↗
PubMed2005

Drosophila genome-scale screen for PAN GU kinase substrates identifies Mat89Bb as a cell cycle regulator.

Although traditional organism-based mutational analysis is powerful in identifying genes involved in specific biological processes, limitations include incomplete coverage and time required for gene identification. Biochemical screens using cell transfection or yeast two-hybrid methods are rapid, but they are limited by cDNA library quality. The recent establishment of "uni-gene sets" has made it feasible to biochemically screen an organism's entire genome. Radiolabeled protein pools prepared from the Drosophila Gene Collection were used in a Drosophila in vitro expression cloning ("DIVEC") screen for substrates of PAN GU kinase, which is crucial for S-M embryonic cell cycles. Ablation of one identified substrate, Mat89Bb, by RNAi produces a polyploid phenotype similar to that of pan gu mutants. Xenopus embryos injected with Mat89Bb morpholinos arrest with polyploid nuclei, and Mat89Bb RNAi in HeLa cells gives rise to multinucleated cells. Thus, Mat89Bb plays an evolutionarily conserved role as a crucial regulator of both cell cycle and development.

Animals↗
PubMed2000

Regulation of the futile cycle of fructose phosphate in sea mussel.

Carbohydrate metabolism in mussels shows two phases separated seasonally. During summer and linked to food supply, carbohydrates, mainly glycogen, are accumulated in the mantle tissue. During winter, mantle glycogen decreases concomitantly with an increase in triglyceride synthesis. In spring, after spawning, the animals go in to metabolic rest until the beginning of a new cycle. This cycle is regulated by the futile cycle of fructose phosphate that implicates PFK-1 and FBPase-1 activities. These enzymes and the bifunctional PFK-2/FBPase-2 that regulates the Fru-2,6-P2 levels, are seasonally modulated by covalent phosphorylation/dephosphorylation mechanisms, as a response to unknown factors. The futile cycle of the fructose phosphates also controls the transition from physiological aerobiosis to hypoxia. The process is independent of the phosphorylation state. In this sense, a pH decrease triggers a small Pasteur effect during the first 24 h of aerial exposure. Variations in the concentration of Fru-2,6-P2 and AMP are the sole factor responsible for this effect. Longer periods of hypoxia induce a metabolic depression characterized by a decrease in Fru-2,6-P2 which is hydrolyzed by drop in the pH. In this review, the authors speculate on the two regulation processes.

Animals↗
PubMed2000

Cardiac mechanoenergetics replicated by cross-bridge model.

The aim of this work is to reproduce the experimentally measured linear dependence of cardiac muscle oxygen consumption on stress-strain area using a model, composed of a three-state Huxley-type model for cross-bridge interaction and a phenomenological model of Ca2+-induced activation. By selecting particular cross-bridge cycling rate constants and modifying the cross-bridge activation model, we replicated the linear dependence between oxygen consumption and stress-strain area together with other important mechanical properties of cardiac muscle such as developed stress dependence on the sarcomere length and force-velocity relationship. The model predicts that (1) the amount of the "passenger" cross bridges, i.e., cross bridges that detach without hydrolyzing ATP molecule, is relatively small and (2) ATP consumption rate profile within a beat and the amount of the passenger cross bridges depend on the contraction protocol.

Actomyosin↗
PubMed1995

Gliotoxin inactivates alcohol dehydrogenase by either covalent modification or free radical damage mediated by redox cycling.

The fungal metabolite gliotoxin shows selective toxicity to cells of the immune system and has been implicated in the aetiology of invasive aspergillosis. The related toxin sporidesmin is the causative agent of facial eczema in sheep. The toxicity of these compounds has been related to their ability to redox cycle intracellularly and thus produce damaging free radicals. These toxins are also potentially capable of forming mixed disulphides with thiol groups on proteins by virtue of their bridged disulphide structure. We show here that gliotoxin can inactivate horse liver alcohol dehydrogenase by either oxidative damage or covalent modification of thiol groups on the enzyme. Either Cys-281 or Cys-282 is selectively modified. Neither of these residues are at the active site. Covalent modification occurs in the absence of reducing agents such as dithiothreitol. In the presence of dithiothreitol no protection is observed and the rate of inactivation is enhanced although as expected no covalent modification occurs. Gliotoxin can therefore inhibit alcohol dehydrogenase by either pathway and this will depend on the availability of reducing agents such as glutathione and/or how readily the reactive oxygen species generated are removed.

Alcohol Dehydrogenase↗
PubMed2001

Carbohydrate moiety of time-interval measuring enzyme regulates time measurement through Its interaction with time-holding peptide PIN.

An ATPase called EA4 seems to measure time as a diapause-duration timer in the seasonal cycle of the silkworm, Bombyx mori. A peptide named PIN seems to regulate the time measurement of EA4. We characterize the EA4 as the first step to analyse its interaction with PIN. Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry shows EA4 forms an equimolar complex with PIN. The binding affinity of EA4 for PIN is about 460 nM, as measured by surface plasmon resonance. Western blot analysis of EA4 with a variety of biotinylated lectins suggests that EA4 is a glycoprotein containing N-linked oligosaccharide. On enzymatic cleavage of the glycosyl chain, the carbohydrate is revealed to be essential for the regulation of EA4-time measurement through the interaction with PIN. PIN holds the timer by binding to EA4, and the dissociation of the complex could constitute the cue for the time measurement.

Adenosine Triphosphatases↗
PubMed2004

A mobile loop order-disorder transition modulates the speed of chaperonin cycling.

Molecular machines order and disorder polypeptides as they form and dissolve large intermolecular interfaces, but the biological significance of coupled ordering and binding has been established in few, if any, macromolecular systems. The ordering and binding of GroES co-chaperonin mobile loops accompany an ATP-dependent conformational change in the GroEL chaperonin that promotes client protein folding. Following ATP hydrolysis, disordering of the mobile loops accompanies co-chaperonin dissociation, reversal of the GroEL conformational change, and release of the client protein. "High-affinity" GroEL mutants were identified by their compatibility with "low-affinity" co-chaperonin mutants and incompatibility with high-affinity co-chaperonin mutants. Analysis of binding kinetics using the intrinsic fluorescence of tryptophan-containing co-chaperonin variants revealed that excessive affinity causes the chaperonin to stall in a conformation that forms in the presence of ATP. Destabilizing the beta-hairpins formed by the mobile loops restores the normal rate of dissociation. Thus, the free energy of mobile-loop ordering and disordering acts like the inertia of an engine's flywheel by modulating the speed of chaperonin conformational changes.

Adenosine Triphosphate↗
PubMed1997

MARCKS regulates membrane ruffling and cell spreading.

The dynamic rearrangement of the actin cytoskeleton is fundamental to most biological processes including embryogenesis, morphogenesis, cell movement, wound healing and metastasis [1]. Membrane ruffling and reversible cell-substratum interactions underlie actin-driven cell movement. Protein kinase C (PKC) stimulates membrane ruffling and adhesion [2], but the mechanism by which this occurs is unknown. Myristoylated alaninerich C kinase substrate (MARCKS) is a PKC substrate that cycles on and off membranes by a mechanism termed the myristoyl-electrostatic switch [3-6]. While at the membrane, MARCKS binds to and sequesters acidic phospholipids including phosphatidyl-inositol-4,5-bisphosphate (PIP2) [7]. MARCKS also binds and cross-links filamentous actin, an activity which is regulated by PKC-dependent phosphorylation and calcium-calmodulin [3]. In this report, we demonstrate that expression, in fibroblasts, of MARCKS containing a mutation which abrogates the myristoyl-electrostatic switch prevents cell spreading. The MARCKS mutant arrests the cell during an early stage of spreading, characterized by profuse membrane blebbing, and prevents the formation of membrane ruffles and lamellae usually found at the leading edge of spreading cells. This defect in the regulation of the actin cytoskeleton is accompanied by a decrease in cell-substratum adhesion. Our results provide direct evidence that MARCKS and PKC regulate actin-dependent membrane ruffling and cell adhesion, perhaps via a PIP2-dependent mechanism.

Actins↗
PubMed1986

Transamination of glutamate to tricarboxylic acid-cycle intermediates in cultured neurons correlates with the ability of oxo acids to support neuronal survival in vitro.

Cultures of central-nervous-system neurons at low densities require for their survival exogenous pyruvate, alpha-oxoglutarate or oxaloacetate, even in the presence of high glucose concentrations. Most other alpha-oxo acids support cell survival only in the presence of alpha-amino acids which transaminate to alpha-oxoglutarate, oxaloacetate or pyruvate. The alpha-oxo acids therefore operate as acceptors of amino groups from appropriate donors to generate tricarboxylic acid-cycle-relevant substrates, and these alpha-oxo acids provide for neuronal support only insofar as they make it possible for exogenously supplied alpha-amino acid precursors to generate intracellularly one of the three critical metabolites. To examine more closely the relationship between transamination activity and neuronal survival, we measured 14CO2 production from [14C]glutamate in the presence of appropriate alpha-oxo acid partners by using 8-day-embryonic chick forebrain, dorsal-root-ganglion and ciliary-ganglion neurons. Neuronal survival was measured concurrently in monolayer neuronal cultures maintained with the corresponding amino acid/oxo acid pairs. Forebrain and ganglionic cell suspensions both produced 14CO2 from [14C]glutamate, which accurately correlated with 24 h neuronal survival. Concentrations of glutamate or alpha-oxo acid which provide for maximal neuronal survival also produced maximal amounts of 14CO2. The same ability to generate CO2 from glutamate (in the presence of the appropriate alpha-oxo acids) can ensure neuronal survival in 24 h cultures and therefore must meet energy or other metabolic needs of those neurons which glucose itself is unable to satisfy.

Animals↗
PubMed2001

The localization of human cyclins B1 and B2 determines CDK1 substrate specificity and neither enzyme requires MEK to disassemble the Golgi apparatus.

In this paper, we show that substrate specificity is primarily conferred on human mitotic cyclin-dependent kinases (CDKs) by their subcellular localization. The difference in localization of the B-type cyclin-CDKs underlies the ability of cyclin B1-CDK1 to cause chromosome condensation, reorganization of the microtubules, and disassembly of the nuclear lamina and of the Golgi apparatus, while it restricts cyclin B2-CDK1 to disassembly of the Golgi apparatus. We identify the region of cyclin B2 responsible for its localization and show that this will direct cyclin B1 to the Golgi apparatus and confer upon it the more limited properties of cyclin B2. Equally, directing cyclin B2 to the cytoplasm with the NH(2) terminus of cyclin B1 confers the broader properties of cyclin B1. Furthermore, we show that the disassembly of the Golgi apparatus initiated by either mitotic cyclin-CDK complex does not require mitogen-activated protein kinase kinase (MEK) activity.

Animals↗
PubMed1992

DT diaphorase [NAD(P)H: (quinone acceptor) oxidoreductase] facilitates redox cycling of menadione in channel catfish (Ictalurus punctatus) cytosol.

Characteristics of DT diaphorase (NAD(P)H: (quinone acceptor) oxidoreductase, DTD) activity in Ictalurus punctatus and the effect of DTD activity on menadione (MND)-mediated reduction of acetylated cytochrome c (AcC) were examined. DTD activity in cytosols of four organs followed a distinct gradient in the order stomach greater than gill greater than liver greater than posterior kidney. A similar gradient was observed in organ-specific rates of in vitro AcC reduction in the presence of either NADH or NADPH as reducing equivalent. A greater proportion of the AcC reduction rate was sensitive to inhibition by dicoumarol (DC) in organs with relatively high DTD specific activity (e.g., stomach) than in organs with low DTD activity (e.g., kidney). No such trend was observed in the superoxide dismutase (SOD)-sensitive proportion of AcC reduction rates. DTD was observed to contribute to MND-mediated superoxide production to a greater extent in organs with high DTD activity than in organs with low DTD activity. DC-sensitive (i.e., DTD-mediated) AcC reduction was observed to increase with organ-specific DTD activity, and the majority of the AcC reduction rate was inhibitable by SOD. These findings demonstrate a direct contribution by DTD activity to MND-mediated superoxide production in this in vitro system. The role of I. punctatus DTD as a possible deleterious agent in quinone metabolism and implications regarding the traditional conception of DTD as a detoxifying enzyme are discussed.

Animals↗
PubMed2006

Mitochondrial creatine kinase activity prevents reactive oxygen species generation: antioxidant role of mitochondrial kinase-dependent ADP re-cycling activity.

As recently demonstrated by our group (da-Silva, W. S., Gómez-Puyou, A., Gómez-Puyou, M. T., Moreno-Sanchez, R., De Felice, F. G., de Meis, L., Oliveira, M. F., and Galina, A. (2004) J. Biol. Chem. 279, 39846-39855) mitochondrial hexokinase activity (mt-HK) plays a preventive antioxidant role because of steady-state ADP re-cycling through the inner mitochondrial membrane in rat brain. In the present work we show that ADP re-cycling accomplished by the mitochondrial creatine kinase (mt-CK) regulates reactive oxygen species (ROS) generation, particularly in high glucose concentrations. Activation of mt-CK by creatine (Cr) and ATP or ADP, induced a state 3-like respiration in isolated brain mitochondria and prevention of H(2)O(2) production obeyed the steady-state kinetics of the enzyme to phosphorylate Cr. The extension of the preventive antioxidant role of mt-CK depended on the phosphocreatine (PCr)/Cr ratio. Rat liver mitochondria, which lack mt-CK activity, only reduced state 4-induced H(2)O(2) generation when 1 order of magnitude more exogenous CK activity was added to the medium. Simulation of hyperglycemic conditions, by the inclusion of glucose 6-phosphate in mitochondria performing 2-deoxyglucose phosphorylation via mt-HK, induced H(2)O(2) production in a Cr-sensitive manner. Simulation of hyperglycemia in embryonic rat brain cortical neurons increased both DeltaPsi(m) and ROS production and both parameters were decreased by the previous inclusion of Cr. Taken together, the results presented here indicate that mitochondrial kinase activity performed a key role as a preventive antioxidant against oxidative stress, reducing mitochondrial ROS generation through an ADP-recycling mechanism.

Adenosine Diphosphate↗
PubMed2005

Cyclin specificity in the phosphorylation of cyclin-dependent kinase substrates.

Cell-cycle events are controlled by cyclin-dependent kinases (CDKs), whose periodic activation is driven by cyclins. Different cyclins promote distinct cell-cycle events, but the molecular basis for these differences remains unclear. Here we compare the specificity of two budding yeast cyclins, the S-phase cyclin Clb5 and the M-phase cyclin Clb2, in the phosphorylation of 150 Cdk1 (Cdc28) substrates. About 24% of these proteins were phosphorylated more efficiently by Clb5-Cdk1 than Clb2-Cdk1. The Clb5-specific targets include several proteins (Sld2, Cdc6, Orc6, Mcm3 and Cdh1) involved in early S-phase events. Clb5 specificity depended on an interaction between a hydrophobic patch in Clb5 and a short sequence in the substrate (the RXL or Cy motif). Phosphorylation of Clb5-specific targets during S phase was reduced by replacing Clb5 with Clb2 or by mutating the substrate RXL motif, confirming the importance of Clb5 specificity in vivo. Although we did not identify any highly Clb2-specific substrates, we found that Clb2-Cdk1 possessed higher intrinsic kinase activity than Clb5-Cdk1, enabling efficient phosphorylation of a broad range of mitotic Cdk1 targets. Thus, Clb5 and Clb2 use distinct mechanisms to enhance the phosphorylation of S-phase and M-phase substrates.

Amino Acid Motifs↗
PubMedDate not supplied

Essential motions in a fungal lipase with bound substrate, covalently attached inhibitor and product.

As an aid to understanding the influence of dynamic fluctuations during esterolytic catalysis, we follow protein flexibility at three different steps along the catalytic pathway from substrate binding to product clearance via a covalently attached inhibitor, which represents a transition-state mimic. We have applied a classical approach, using molecular dynamics simulations to monitor protein dynamics in the nanosecond regime. We filter out small amplitude fluctuations and focus on the anharmonic contributions to the overall dynamics. This 'essential dynamics' analysis reveals different modes of response along the pathway suggesting that binding, catalysis and product clearance occur along different energy surfaces. Motions in the enzyme with a covalently attached ligand are more complex and occur along several eigenvectors. The magnitudes of the fluctuations in these individual subspaces are significantly smaller than those observed for the substrate and product molecules, indicating that the energy surface is shallow and that a relatively large number of conformational substates are accessible. On the other hand, substrate binding and product release occur at distinct modes of the protein flexibility suggesting that these processes occur along rough energy surfaces with only a few minima. Detailed energetic analyses along the trajectories indicated that in all cases binding is dominated by van der Waals interactions. The carboxylate form of the product is stabilized by a tight hydrogen bond network involving in particular Ser82, which may be a potential cause of product inhibition. Considerations such as these should aid the understanding of mechanisms of substrate, inhibitor or product recognition and could become of importance in the design of new substrates or inhibitors for enzymes.

Enzyme Inhibitors↗
PubMed1999

Redistribution of metabolic fluxes in Escherichia coli with fermentative lactate dehydrogenase overexpression and deletion.

Under anaerobic conditions, competition for pyruvate between the branch point enzymes pyruvate formate lyase (PFL, Km = 2 mM) and fermentative lactate dehydrogenase (LDH, Km = 7.2 mM) determines the partition of carbon flux. Two Escherichia coli mutant strains, one deficient in ackA, pta, and ldhA and the other overexpressing LDH, were constructed to systematically analyze the effects of these perturbations in the existing pathways on the redistribution of carbon fluxes. Deletion of the lactate and acetate synthesis pathways was detrimental to cell growth. Carbon flux is forced through ethanol and formate production pathways, resulting in a concomitant increase in those fluxes. In addition, overexpression of LDH simultaneously increases the common flux as well as the flux to the competing acetyl-CoA branch. Overexpression of lactate dehydrogenase (ldhA) in the parent strain increases the lactate synthesis rate from 0.19 to 0.40 mmol/g-biomass-h when the LDH activities increases from 1.3 to 15.3 units. Even an increase of more than 10 times in the LDH activity fails to divert a large fraction of the carbon flux to lactate; the majority of the flux still channels through the acetyl-CoA branch. Overexpression of LDH in the parent strain simultaneously increases the common flux as well as the flux through the acetyl-CoA branch. Subsequently, the flux amplification factors (or deviation indices which can be related to the flux control coefficients) are positive for all three fluxes occurring at the pyruvate node.

Acetyltransferases↗
PubMed1999

Classification and sensitivity analysis of a proposed primary metabolic reaction network for Streptomyces lividans.

Constructing a metabolic flux analysis model is in principle fairly straightforward. However, there are a number of mathematical pitfalls. First, dependent reactions are a recurring problem and, second, the choice of reactions to measure may not be straight-forward. A method for systematic identification of dependent reactions and a thorough reactions classification procedure is presented. A well-defined stoichiometric presentation can provide significant insight into metabolic control mechanisms. Two methods for analyzing the impact of perturbations in the measured fluxes on the remaining metabolism and the impact of changes in biomass composition on the calculated metabolic reactions is developed. A metabolic reaction network proposed for Streptomyces lividans is used as an example to demonstrate the outlined analysis. It is concluded that oxygen utilization has the highest influence on the pathway fluxes and that realistic perturbations in the biomass composition do not significantly alter the flux patterns.

Homeostasis↗
PubMed1988

Reversible conversion of coenzyme F420 to the 8-OH-AMP and 8-OH-GMP esters, F390-A and F390-G, on oxygen exposure and reestablishment of anaerobiosis in Methanobacterium thermoautotrophicum.

Intracellular levels of F390 (AMP and GMP adducts of the 5-deazaflavin cofactor F420) in Methanobacterium thermoautotrophicum were analysed after gasing fermenter cultures with several consecutive cycles of substrate gas and gas mixtures containing 5% oxygen. No F390 was detected in growing cells, hydrogen starved cells and CO2 starved cells prior to O2 contamination. Also, no F390 was found in hydrogen depleted cells after O2 treatment. Exposure of exponentially growing cells and CO2 starved cells to oxygen lead to the formation of F390 species; the increase in the detected amount of F390 was coupled to a decrease of the F420 level. As soon as anaerobiosis was reestablished F390 cofactors were degraded and growth proceeded. Independent of the physiological condition of Methanobacterium thermoautotrophicum methanopterin was formed upon O2 exposure. After normal growth conditions were restored the level of detected methanopterin decreased again.

Anaerobiosis↗