ATPase Reaction Research Articles

Unveiling the Metabolic Changes on Muscle Cell Metabolism Underlying p-Phenylenediamine Toxicity.

Rhabdomyolysis is a disorder characterized by acute damage of the sarcolemma of the skeletal muscle leading to release of potentially toxic muscle cell components into the circulation, most notably creatine phosphokinase (CK) and myoglobulin, and is frequently accompanied by myoglobinuria. In the present work, we evaluated the toxicity of p-phenylenediamine (PPD), a main component of hair dyes which is reported to induce rhabdomyolysis. We studied the metabolic effect of this compound in vivo with Wistar rats and in vitro with C2C12 muscle cells. To this aim we have combined multi-omic experimental measurements with computational approaches using model-driven methods. The integrative study presented here has unveiled the metabolic disorders associated to PPD exposure that may underlay the aberrant metabolism observed in rhabdomyolys disease. Animals treated with lower doses of PPD (10 and 20 mg/kg) showed depressed activity and myoglobinuria after 10 h of treatment. We measured the serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatine kinase (CK) in rats after 24, 48, and 72 h of PPD exposure. At all times, treatment with PPD at higher doses (40 and 60 mg/kg) showed an increase of AST and ALT, and also an increase of lactate dehydrogenase (LDH) and CK after 24 h. Blood packed cell volume and hemoglobin levels, as well as organs weight at 48 and 72 h, were also measured. No significant differences were observed in these parameters under any condition. PPD induce cell cycle arrest in S phase and apoptosis (40% or early apoptotic cells) on mus musculus mouse C2C12 cells after 24 h of treatment. Incubation of mus musculus mouse C2C12 cells with [1,2-13C2]-glucose during 24 h, subsequent quantification of 13C isotopologues distribution in key metabolites of glucose metabolic network and a computational fluxomic analysis using in-house developed software (Isodyn) showed that PPD is inhibiting glycolysis, non-oxidative pentose phosphate pathway, glycogen turnover, and ATPAse reaction leading to a reduction in ATP synthesis. These findings unveil the glucose metabolism collapse, which is consistent with a decrease in cell viability observed in PPD-treated C2C12 cells and with the myoglubinuria and other effects observed in Wistar Rats treated with PPD. These findings shed new light on muscle dysfunction associated to PPD exposure, opening new avenues for cost-effective therapies in Rhabdomyolysis disease.

Architecture and mechanism of the central gear in an ancient molecular timer

Molecular clocks are the product of natural selection in organisms from bacteria to human and their appearance early in evolution such as in the prokaryotic cyanobacterium Synechococcus elongatus suggests that these timers served a crucial role in genetic fitness. Thus, a clock allows cyanobacteria relying on photosynthesis and nitrogen fixation to temporally space the two processes and avoid exposure of nitrogenase carrying out fixation to high levels of oxygen produced during photosynthesis. Fascinating properties of molecular clocks are the long time constant, their precision and temperature compensation. Although these are hallmarks of all circadian oscillators, the actual cogs and gears that control clocks vary widely between organisms, indicating that circadian timers evolved convergently multiple times, owing to the selective pressure of an environment with a daily light/dark cycle. In S. elongatus, the three proteins KaiA, KaiB and KaiC in the presence of ATP constitute a so-called post-translational oscillator (PTO). The KaiABC PTO can be reconstituted in an Eppendorf tube and keeps time in a temperature-compensated manner. The ease by which the KaiABC clock can be studied in vitro has made it the best-investigated molecular clock system. Over the last decade, structures of all three Kai proteins and some of their complexes have emerged and mechanistic aspects have been analysed in considerable detail. This review focuses on the central gear of the S. elongatus clock and only enzyme among the three proteins: KaiC. Our determination of the three-dimensional structure of KaiC early in the quest for a better understanding of the inner workings of the cyanobacterial timer revealed its unusual architecture and conformational differences and unique features of the two RecA-like domains constituting KaiC. The structure also pinpointed phosphorylation sites and differential interactions with ATP molecules at subunit interfaces, and helped guide experiments to ferret out mechanistic aspects of the ATPase, auto-phosphorylation and auto-dephosphorylation reactions catalysed by the homo-hexamer. Comparisons between the structure of KaiC and those of nanomachines such as F1-ATPase and CaMKII also exposed shared architectural features (KaiC/ATPase), mechanistic principles (KaiC/CaMKII) and phenomena, such as subunit exchange between hexameric particles critical for function (clock synchronization, KaiABC; memory-storage, CaMKII).

A New and Improved View of Force Production

A central question in muscle contraction is “How is the energy release in ATP hydrolysis converted into mechanical force and work?” With the elucidation of the actomyosin ATPase reaction in the 1970s (1,2) and the fact that a large amount of free energy is produced during Pi release from an actomyosin-ADP-Pi species (3), the Pi release step from actomyosin-ADP-Pi must be associated with the generation of force and shortening. It was also known that increases in the [Pi] concentration in isometrically contracting skinned muscle fibers reduced isometric force (4).

Structural Basis of Backwards Motion in Kinesin-1-Kinesin-14 Chimera: Implication for Kinesin-14 Motility

Kinesin-14 is a unique minus-end-directed microtubule-based motor. A swinging motion of a class-specific N-terminal neck helix has been proposed to produce minus-end directionality. However, it is unclear how swinging of the neck helix is driven by ATP hydrolysis utilizing the highly conserved catalytic core among all kinesins. Here, using a motility assay, we show that in addition to the neck helix, the conserved five residues at the C-terminal region in kinesin-14, namely the neck mimic, are necessary to give kinesin-1 an ability to reverse its directionality toward the minus end of microtubules. Our structural analyses further demonstrate that the C-terminal neck mimic, in cooperation with conformational changes in the catalytic core during ATP binding, forms a kinesin-14 bundle with the N-terminal neck helix to swing toward the minus end of microtubules. Thus, the neck mimic plays a crucial role in coupling the chemical ATPase reaction with the mechanical cycle to produce the minus-end-directed motility of kinesin-14.

ATP binding leads to autophosphorylation of HSV-1 origin binding protein

The tsUL9 gene products exhibit a strongly reduced ATPase function at the nonpermissive temperature, an effect that can be compensated by the addition of unspecific DNA. Here it is demonstrated, that the reduced ATPase activity is caused by altered ATP binding of the tsOBP at the nonpermissive temperature. Furthermore the addition of unspecific DNA to the ATPase reaction not only compensates the ts phenotype at the nonpermissive temperature but also enhances the ATPase activity at the permissive temperature of both wild type and tsOBP. Thereby, the OBP becomes autophosphorylated. Moreover, addition of subgenomic HSV-1 DNA has no further enhancing effect, supporting the inchworm model for the HSV-OBP function.

Restrictions in ATP diffusion within sarcomeres can provoke ATP-depleted zones impairing exercise capacity in chronic obstructive pulmonary disease

BackgroundChronic obstructive pulmonary disease (COPD) is characterized by the inability of patients to sustain a high level of ventilation resulting in perceived exertional discomfort and limited exercise capacity of leg muscles at average intracellular ATP levels sufficient to support contractility. MethodsMyosin ATPase activity in biopsy samples from healthy and COPD individuals was implemented as a local nucleotide sensor to determine ATP diffusion coefficients within myofibrils. Ergometric parameters clinically measured during maximal exercise tests in both groups were used to define the rates of myosin ATPase reaction and aerobic ATP re-synthesis. The obtained parameters in combination with AK- and CK-catalyzed reactions were implemented to compute the kinetic and steady-state spatial ATP distributions within control and COPD sarcomeres. ResultsThe developed reaction–diffusion model of two-dimensional sarcomeric space identified similar, yet extremely low nucleotide diffusion in normal and COPD myofibrils. The corresponding spatio-temporal ATP distributions, constructed during imposed exercise, predicted in COPD sarcomeres a depletion of ATP in the zones of overlap between actin and myosin filaments along the center axis at average cytosolic ATP levels similar to healthy muscles. ConclusionsATP-depleted zones can induce rigor tension foci impairing muscle contraction and increase a risk for sarcomere damages. Thus, intra-sarcomeric diffusion restrictions at limited aerobic ATP re-synthesis can be an additional risk factor contributing to the muscle contractile deficiency experienced by COPD patients. General significanceThis study demonstrates how restricted substrate mobility within a cellular organelle can provoke an energy imbalance state paradoxically occurring at abounding average metabolic resources.

Crystal Structure and Biochemical Characterization of a Mycobacterium smegmatis AAA-Type Nucleoside Triphosphatase Phosphohydrolase (Msm0858).

AAA proteins (ATPases associated with various cellular activities) use the energy of ATP hydrolysis to drive conformational changes in diverse macromolecular targets. Here, we report the biochemical characterization and 2.5-Å crystal structure of a Mycobacterium smegmatis AAA protein Msm0858, the ortholog of Mycobacterium tuberculosis Rv0435c. Msm0858 is a magnesium-dependent ATPase and is active with all nucleoside triphosphates (NTPs) and deoxynucleoside triphosphates (dNTPs) as substrates. The Msm0858 structure comprises (i) an N-terminal domain (amino acids [aa] 17 to 201) composed of two β-barrel modules and (ii) two AAA domains, D1 (aa 212 to 473) and D2 (aa 476 to 744), each of which has ADP in the active site. Msm0858-ADP is a monomer in solution and in crystallized form. Msm0858 domains are structurally homologous to the corresponding modules of mammalian p97. However, the position of the N-domain modules relative to the AAA domains in the Msm0858-ADP tertiary structure is different and would impede the formation of a p97-like hexameric quaternary structure. Mutational analysis of the A-box and B-box motifs indicated that the D1 and D2 AAA domains are both capable of ATP hydrolysis. Simultaneous mutations of the D1 and D2 active-site motifs were required to abolish ATPase activity. ATPase activity was effaced by mutation of the putative D2 arginine finger, suggesting that Msm0858 might oligomerize during the ATPase reaction cycle. A truncated variant Msm0858 (aa 212 to 745) that lacks the N domain was characterized as a catalytically active homodimer. Recent studies have underscored the importance of AAA proteins (ATPases associated with various cellular activities) in the physiology of mycobacteria. This study reports the ATPase activity and crystal structure of a previously uncharacterized mycobacterial AAA protein, Msm0858. Msm0858 consists of an N-terminal β-barrel domain and two AAA domains, each with ADP bound in the active site. Msm0858 is a structural homolog of mammalian p97, with respect to the linear order and tertiary structures of their domains.

The effect of medium viscosity on kinetics of ATP hydrolysis by the chloroplast coupling factor CF1.

The coupling factor CF1 is a catalytic part of chloroplast ATP synthase which is exposed to stroma whose viscosity is many-fold higher than that of reaction mixtures commonly used to measure kinetics of CF1-catalyzed ATP hydrolysis. This study is focused on the effect of medium viscosity modulated by sucrose or bovine serum albumin (BSA) on kinetics of Ca(2+)- and Mg(2+)-dependent ATP hydrolysis by CF1. These agents were shown to reduce the maximal rate of Ca(2+)-dependent ATPase without changing the apparent Michaelis constant (К m), thus supporting the hypothesis on viscosity dependence of CF1 activity. For the sulfite- and ethanol-stimulated Mg(2+)-dependent reaction, the presence of sucrose increased К m without changing the maximal rate that is many-fold as high as that of Ca(2+)-dependent hydrolysis. The hydrolysis reaction was shown to be stimulated by low concentrations of BSA and inhibited by its higher concentrations, with the increasing maximal reaction rate estimated by extrapolation. Sucrose- or BSA-induced inhibition of the Mg(2+)-dependent ATPase reaction is believed to result from diffusion-caused deceleration, while its BSA-induced stimulation is probably caused by optimization of the enzyme structure. Molecular mechanisms of the inhibitory effect of viscosity are discussed. Taking into account high protein concentrations in the chloroplast stroma, it was suggested that kinetic parameters of ATP hydrolysis, and probably those of ATP synthesis in vivo as well, must be quite different from measurements taken at a viscosity level close to that of water.

Abstract P055: Systemic Arterial Hypertension Induces Pulmonary Injury Beyond Protein Degradation and Atrophy of Diaphragm

Background: Systemic arterial hypertension (SAH) is a chronic disease associated with systemic inflammation. Although cardiovascular adaptations resulting from SAH are more evident, little is known about the respiratory alterations. The purinergic receptor P2X7 plays a key role in the immune modulation, beyond to control the vascular tone and the development of inflammation and fibrosis. Aims: Evaluate the effects of SAH on the pulmonary inflammation and remodeling and on the diaphragm, and the involvement of purinergic receptor P2X7 and of ubiquitin-proteasome system (UPS) in this response. Methods: Spontaneously hypertensive rats (SHR) and normotensive Wistar (N) with 18 weeks-of-age were evaluated for: inflammation and remodeling of airways and pulmonary vessels and for P2X7 receptor expression. The morphology and biochemistryin diaphragm muscle for myosin ATPase reaction and ubiquitin-proteasome system (UPS), respectively. Results: The SHR showed a higher wall/lumen of the pulmonary arteries, as well as increased collagen deposition on the wall of these arteries. Increase in P2X7 receptor expression in pulmonary vascular wall (SHR:4.3%±0.7% vs. N:0.3%±0.2%) and bronchial epithelial (SHR:29±0% vs 2.8% N:10.5%±1.3%). The diaphragm was increased cross-sectional area (CSA) of type I fibers (16%) and reduction in CSA of type II (41%), increased the UPS activity and lipid peroxidation. SHR did not change in the analysis of ubiquitinated proteins and misfolded proteins. Conclusion: SAH induces important pulmonary disorders such pulmonary vascular remodeling, with increased expression of the purinergic receptor P2X7 associated with atrophy and protein degradation on diaphragm.

The role of monovalent cations in the ATPase reaction of DNA gyrase.

Four new crystal structures of the ATPase domain of the GyrB subunit of Escherichia coli DNA gyrase have been determined. One of these, solved in the presence of K(+), is the highest resolution structure reported so far for this domain and, in conjunction with the three other structures, reveals new insights into the function of this domain. Evidence is provided for the existence of two monovalent cation-binding sites: site 1, which preferentially binds a K(+) ion that interacts directly with the α-phosphate of ATP, and site 2, which preferentially binds an Na(+) ion and the functional significance of which is not clear. The crystallographic data are corroborated by ATPase data, and the structures are compared with those of homologues to investigate the broader conservation of these sites.

Activity of Na+/K+-activated Mg2+-dependent ATP-hydrolase in the cell-free extracts of the sulfate-reducing bacteria Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9

The aim of our work was to study Na+/K+-activated Mg2+-dependent ATPase activity in cell-free extracts of the sulfate-reducing bacteriaDesulfovibrio pigerVib-7 andDesulfomicrobiumsp. Rod-9 isolated from the human large intestine, and to carry out the kinetic analysis of the enzyme reaction. The maximum ATPase activity for both bacterial strains at +35 ºC was determined. The highest activities of the studied enzyme in the cell-free extracts ofD. pigerVib-7 at pH 7.0 andDesulfomicrobiumsp. Rod-9 at pH 6.5 were measured. Based on experimental data, the analysis of kinetic properties of the ATP-hydrolase reaction by the studied bacteria was carried out. The enzyme activity, initial (instantaneous) reaction rate (V0) and maximum rate of the ATPase reaction (Vmax) was significantly higher inD. pigerVib-7 cells than inDesulfomicrobiumsp. Rod-9. Michaelis constants (Km) of the enzyme reaction for both bacterial strains were determined.

Recurrence of carbamoyl phosphate synthetase 1 (CPS1) deficiency in Turkish patients: Characterization of a founder mutation by use of recombinant CPS1 from insect cells expression

Carbamoyl phosphate synthetase 1 (CPS1) deficiency due to CPS1 mutations is a rare autosomal-recessive urea cycle disorder causing hyperammonemia that can lead to death or severe neurological impairment. CPS1 catalyzes carbamoyl phosphate formation from ammonia, bicarbonate and two molecules of ATP, and requires the allosteric activator N-acetyl-L-glutamate. Clinical mutations occur in the entire CPS1 coding region, but mainly in single families, with little recurrence. We characterized here the only currently known recurrent CPS1 mutation, p.Val1013del, found in eleven unrelated patients of Turkish descent using recombinant His-tagged wild type or mutant CPS1 expressed in baculovirus/insect cell system. The global CPS1 reaction and the ATPase and ATP synthesis partial reactions that reflect, respectively, the bicarbonate and the carbamate phosphorylation steps, were assayed. We found that CPS1 wild type and V1013del mutant showed comparable expression levels and purity but the mutant CPS1 exhibited no significant residual activities. In the CPS1 structural model, V1013 belongs to a highly hydrophobic β-strand at the middle of the central β-sheet of the A subdomain of the carbamate phosphorylation domain and is close to the predicted carbamate tunnel that links both phosphorylation sites. Haplotype studies suggested that p.Val1013del is a founder mutation. In conclusion, the mutation p.V1013del inactivates CPS1 but does not render the enzyme grossly unstable or insoluble. Recurrence of this particular mutation in Turkish patients is likely due to a founder effect, which is consistent with the frequent consanguinity observed in the affected population.

ATP-induced electron transfer by redox-selective partner recognition.

Thermodynamically unfavourable electron transfers are enabled by coupling to an energy-supplying reaction. How the energy is transduced from the exergonic to the endergonic process is largely unknown. Here we provide the structural basis for an energy transduction process in the reductive activation of B12-dependent methyltransferases. The transfer of one electron from an activating enzyme to the cobalamin cofactor is energetically uphill and relies on coupling to an ATPase reaction. Our results demonstrate that the key to coupling is, besides the oxidation state-dependent complex formation, the conformational gating of the electron transfer. Complex formation induces a substitution of the ligand at the electron-accepting Co ion. Addition of ATP initiates electron transfer by provoking conformational changes that destabilize the complex. We show how remodelling of the electron-accepting Co(2+) promotes ATP-dependent electron transfer; an efficient strategy not seen in other electron-transferring ATPases.

USING OF TRADITIONAL AND QUANTITATIVE CYTOCHEMICAL METHODS FOR IDENTIFICATION AND ENZYME CHARACTERIZATION OF SOME E.COLI SEROGROUPS CAUSING ENTERITIS IN BUFFALOES

Enteritis is most fatal disease especially for neonatal and young buffalo calves. Out of 150 fecal samples, 100 from diarrheic and 50 from non diarrheic (apparently healthy) buffalo calves were collected from private farms for isolation and identification of E.coli using morphological, cultural, biochemical and serological examination. Further cytochemical enzyme characterization of the different identified serogroups were carried out through the cytochemical quantitative determination of bacterial mitochondrial Adenosene-tri phosphatase (ATP-ase) and succinic dehydrogenase (SDH-ase) enzyme activities for their possible variations among the isolated and identified E.coli serogroups. Four values to each of E.coli serogroup (2 for each enzyme) as optical densities (OD.) could be obtained : (ATP-ase activity & Actual ATP-ase reaction and SDH-ase activity & Actual SDH-ase reaction) for E.coli-serogroups: E.coli O26, E.coli O55, E.coli O78, E.coli O86 and E.coli O111 as following: (0.595 & 0.285 and 0.560 & 0.210), (0.565 & 0.265 and 0.550 & 0.200), (0.590 & 0.255 and 0.540 & 0.190), (0.590 & 0.205 and 0.490 & 0.180) and (0.600 & 0.160 and 0.560 & 0.150) nm respectively. It was observed that the SDH-ase enzyme activity differentiate between the majority of the studied E.coli-serogroups for the significant (P≤0.05) variations between their enzyme activities (except between the serogroups E.coli O26 and E.coli O111 which showed the same (non-significant) SDH-ase activity, but ATP-ase enzyme activity could not differentiate between the different E.coli serogroups because of its non-significant variations among different serogroups (except with the serogroup E.coli O55 which showed the significant lower ATP-ase enzyme activity and noted to be lower pathogenic than the other E.coli serogroups), so that the actual ATP-ase enzyme activities (the difference between the OD. after and before incubation period) may be used for differentiation between different serogroups. Further cytochemical characterization to other bacterial enzymes and to other E.coli serogroups should be carried out as additional tool for a definite differentiation and characterization of E.coli serogroups causing enteritis in buffalo calves.

Small molecule-mediated refolding and activation of myosin motor function

The small molecule EMD 57033 has been shown to stimulate the actomyosin ATPase activity and contractility of myofilaments. Here, we show that EMD 57033 binds to an allosteric pocket in the myosin motor domain. EMD 57033-binding protects myosin against heat stress and thermal denaturation. In the presence of EMD 57033, ATP hydrolysis, coupling between actin and nucleotide binding sites, and actin affinity in the presence of ATP are increased more than 10-fold. Addition of EMD 57033 to heat-inactivated β-cardiac myosin is followed by refolding and reactivation of ATPase and motile activities. In heat-stressed cardiomyocytes expression of the stress-marker atrial natriuretic peptide is suppressed by EMD 57033. Thus, EMD 57033 displays a much wider spectrum of activities than those previously associated with small, drug-like compounds. Allosteric effectors that mediate refolding and enhance enzymatic function have the potential to improve the treatment of heart failure, myopathies, and protein misfolding diseases. DOI: http://dx.doi.org/10.7554/eLife.01603.001.

Structural Mechanism of Microtubule-Based Motility of Molecular Motor Kinesin

Molecular motor kinesins move processively along microtubules by using energy derived from ATP hydrolysis. Almost all of the intermediate structures of this ATPase reaction cycle have been solved for the monomeric kinesin KIF1A. Based on this structural information, we present here the common atomic mechanisms of kinesin motility, which can be applied not only to the monomeric kinesins but also to the dimeric kinesins. Structural studies have suggested that kinesins accomplish their mission by utilizing the evolutionary conserved strategy among the various ATPases/GTPases/kinases in which they use the energy from the ATP/GTP hydrolysis to attach/detach to/from their effectors.

Sucrose increases the activation energy barrier for actin–myosin strong binding

To determine the mechanism by which sucrose slows in vitro actin sliding velocities, V, we used stopped flow kinetics and a single molecule binding assay, SiMBA. We observed that in the absence of ATP, sucrose (880mM) slowed the rate of actin–myosin (A–M) strong binding by 71±8% with a smaller inhibitory effect observed on spontaneous rigor dissociation (21±3%). Similarly, in the presence of ATP, sucrose slowed strong binding associated with Pi release by 85±9% with a smaller inhibitory effect on ATP-induced A–M dissociation, kT (39±2%). Sucrose had no noticeable effect on any other step in the ATPase reaction. In SiMBA, sucrose had a relatively small effect on the diffusion coefficient for actin fragments (25±2%), and with stopped flow we showed that sucrose increased the activation energy barrier for A–M strong binding by 37±3%, indicating that sucrose inhibits the rate of A–M strong binding by slowing bond formation more than diffusional searching. The inhibitory effects of sucrose on the rate of A–M rigor binding (71%) are comparable in magnitude to sucrose’s effects on both V (79±33% decrease) and maximal actin-activated ATPase, kcat, (81±16% decrease), indicating that the rate of A–M strong bond formation significantly influences both kcat and V.

Conformational Changes Produced by ATP Binding to the Plasma Membrane Calcium Pump

The aim of this work was to study the plasma membrane calcium pump (PMCA) reaction cycle by characterizing conformational changes associated with calcium, ATP, and vanadate binding to purified PMCA. This was accomplished by studying the exposure of PMCA to surrounding phospholipids by measuring the incorporation of the photoactivatable phosphatidylcholine analog 1-O-hexadecanoyl-2-O-[9-[[[2-[(125)I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine to the protein. ATP could bind to the different vanadate-bound states of the enzyme either in the presence or in the absence of Ca(2+) with high apparent affinity. Conformational movements of the ATP binding domain were determined using the fluorescent analog 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate. To assess the conformational behavior of the Ca(2+) binding domain, we also studied the occlusion of Ca(2+), both in the presence and in the absence of ATP and with or without vanadate. Results show the existence of occluded species in the presence of vanadate and/or ATP. This allowed the development of a model that describes the transport of Ca(2+) and its relation with ATP hydrolysis. This is the first approach that uses a conformational study to describe the PMCA P-type ATPase reaction cycle, adding important features to the classical E1-E2 model devised using kinetics methodology only.

Methods for detecting ATP hydrolysis and nucleic acid unwinding of Japanese encephalitis virus NS3 helicase

Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic pathogen that is prevalent in south-east Asia. Because there is no specific antiviral agent, JEV still causes a high rate of neurologic sequelae and mortality in humans. The helicase encoded by the NS3 gene of JEV has emerged recently as a novel antiviral target for treatment. In this study, a soluble recombinant JEV helicase protein was expressed and purified. Methods for detecting the ATP hydrolysis and nucleic acid unwinding activity were developed by luminescence and fluorescence resonance energy transfer (FRET). The concentrations of enzyme, substrate, capture strand, ATP, and divalent ions were optimised in the ATPase and helicase reactions. The feasibility of using these two methods for high-throughput screening of NS3 helicase inhibitors is discussed.

Commentary on: an (un)paralleled process?

As highlighted in the Viewpoint article by Rossiter (2013), studying the adjustment of the energy pathway to cope with ATP demand at the onset of both acute exercise and following exercise training provides a unique window to understand muscle function. Traditionally, it has been thought that the increase in mitochondrial respiration capacity associated with exercise training leads to faster O2 uptake kinetics and smaller relative changes in the cytosolic modulators of oxidative phosphorylation, implying an increased respiration sensitivity of the mitochondria. In this regard, the study by Zoladz et al. (2013) is particularly intriguing and supports previous suggestions that in the early stage of exercise training the sensitivity of mitochondrial respiration can be increased before a change in mitochondrial capacity or mitochondrial density (Green et al. 1991). A potential mechanism contributing to this improved kinetic control may be related to the compartmentalization of energy exchange and the existence of intracellular energetic units. Indeed, it has been suggested that mitochondria, sarcoplasmic reticulum and myofibrils may all be associated functionally in skeletal muscle, such that the ADP produced during intracellular ATPase reactions (myosin- and calcium-ATPases) can be directly channelled to the mitochondria. Considering the substantial, although not significant, reduction in muscle fibre cross-sectional area reported by Zoladz et al. (2013) and the recent documentation of increased ADP sensitivity with short-term training (Layec et al. 2013), one can speculate that the structural arrangement of mitochondria around myofibrils might occur rapidly, after only a few days of exercise training, improving the channelling and regulation of energy within these functional units and metabolic stability.