Rate Of ATP Consumption Research Articles

Prediction of the mechanical response of cardiac alternans by using an electromechanical model of human ventricular myocytes

PurposeAlthough the quantitative analysis of electromechanical alternans is important, previous studies have focused on electrical alternans, and there is a lack quantitative analysis of mechanical alternans at the subcellular level according to various basic cycle lengths (BCLs). Therefore, we used the excitation–contraction (E–C) coupling model of human ventricular cells to quantitatively analyze the mechanical alternans of ventricular cells according to various BCLs.MethodsTo implement E–C coupling, we used calcium transient data, which is the output data of electrical simulation using the electrophysiological model of human ventricular myocytes, as the input data of mechanical simulation using the contractile myofilament dynamics model. Moreover, we applied various loads on ventricular cells for implementation of isotonic and isometric contraction.ResultsAs the BCL was reduced from 1000 to 200 ms at 30 ms increments, mechanical alternans, as well as electrical alternans, were observed. At this time, the myocardial diastolic tension increased, and the contractile ATP consumption rate remained greater than zero even in the resting state. Furthermore, the time of peak tension, equivalent cell length, and contractile ATP consumption rate were all reduced. There are two tendencies that endocardial, mid-myocardial, and epicardial cells have the maximum amplitude of tension and the peak systolic tension begins to appear at a high rate under the isometric condition at a particular BCL.ConclusionsWe observed mechanical alternans of ventricular myocytes as well as electrical alternans, and identified unstable conditions associated with mechanical alternans. We also determined the amount of BCL given to each ventricular cell to generate stable and high tension state in the case of isometric contraction.

Age-Dependent Decrease in Requirement for Glycolysis in Hematopoietic Stem Cells

Metabolic process governs cell cycle and differentiation status of hematopoietic stem cells (HSCs) through regulating the balance between oxidative phosphorylation and glycolysis. Cell cycle and self-renewal capacity dynamically changes during developmental and aging process; in early stage of life, HSCs are highly cycling and self-renew, whereas adult HSCs exhibit quiescent in cell-cycle, and gradually decline in its function as they age. How functional alteration of HSCs is coupled with metabolic program is an important issue for understanding the lifecourse of HSCs. Phosphoglycerate mutase 1 (Pgam1) is an enzyme catalyzing conversion of 3-phosphoglycerate into 2-phosphoglycerate. By deleting Pgam1, ATP production through glycolysis was abrogated while pentose-phosphate pathway (PPP) was spared, allowing us to examine dependence on glycolytic energy production without affecting nucleotide and NADPH production through PPP. Following inducible Pgam1 deletion in mice at various ages, the number of HSCs was measured. At an early life stage (5-week old), Pgam1 deletion resulted in decreased number of HSCs, suggesting an essential role of glycolysis for HSC maintenance. Notably, the number of HSCs in Pgam1-deleted mice restored in an age-dependent manner, with 2-year old mice exhibiting almost comparable number of HSCs with wild type mice. By contrast, upon HSC transplantation, the repopulation capacity of Pgam1-deficient HSCs from any stages of life was uniformly abrogated, indicating that glycolytic energy production is essential for stressed condition in which HSCs are forced to proliferate. Reverse transplantation of wild type HSCs into Pgam1-floxed recipients followed by conditional deletion in non-hematopoietic cells showed no phenotypes in hematopoietic lineages, suggesting a cell-autonomous requirement of Pgam1 for blood cells.To further determine the relevance of glycolysis with maintenance/proliferation balance, we performed in vitro culture utilizing newly-developed quiescence-maintaining condition with or without glucose. The number of HSCs under the quiescence-maintaining condition was irrelevant to the presence or absence of glucose, while cell number under the proliferating condition was markedly decreased in glucose-deprived condition. Furthermore, lymphoid-primed multipotential progenitors (LMPP) with marker phenotype of Flt3+ linage markers- Sca-1+ c-Kit+ cells, which show high cycling status in vivo, were highly dependent on glucose for survival in vitro. Collectively, proliferating cells require glucose irrespective to its cell type, while quiescent HSCs are less dependent on glycolytic energy production in line with age-dependent decrease of requirement for glycolysis in vivo.ATP concentration directly reflects ATP production/consumption rate inside cells. We utilized ATP-biosensor (GO-ATeam2)-transgenic mice to monitor ATP concentration in live HSCs under various metabolic environments in vitro. HSCs cultured in the proliferating condition exhibited gradual decrease in ATP concentration during 7-day culture, whereas in the quiescence-maintaining condition ATP was kept high. In the steady state, cycling multipotent progenitors (MPPs) exhibited lower ATP concentration than quiescent HSCs. These results suggest that quiescent state is characterized by lower ATP production/consumption rate than the proliferating cells. We then compared ATP concentration in young and old HSCs using the same system. The decrease of ATP under the proliferating condition was less prominent in old HSCs, while ATP concentration was comparable in the quiescence-maintaining condition. The result indicates the lower ATP production/consumption rate in old HSCs, presumably explaining lower dependence on glycolytic energy production in the steady state hematopoiesis.Altogether, we concluded that the dependence on glycolysis-driven ATP production derived from glucose decreased with age, underlying mechanism of which is explained by lower ATP consumption rate in old HSCs. DisclosuresOkamoto:Chugai Pharmaceutical Co., Ltd.: Research Funding; Eisai Co.,Ltd.: Research Funding; Bristol-Myers Squibb K.K.: Honoraria, Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Toyama Chemical Co., Ltd.: Research Funding; Alexion Pharmaceuticals, Inc.:: Research Funding; Nippon Shinyaku Co., Ltd: Research Funding; Shionogi & Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Asahi Kasei Pharma Corp.:: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Teijin Pharma Limited: Research Funding; Kyowa Hakko Kirin Co.: Research Funding; Pfizer Inc.: Honoraria, Research Funding; JCR Pharmaceuticals Co., Ltd.: Research Funding.

Faster and more efficient swimming: energy consumption of murine spermatozoa under sperm competition†.

ATP supply is essential for sperm performance and increases in ATP content coevolve with enhanced sperm swimming velocity as a response to sperm competition in rodents. ATP content is the balance between production and consumption but, although ATP production has received much attention, little is known about ATP consumption. The rate of ATP consumption is crucial for the propagation of the flagellar wave, becoming a main determinant of the time and distance sperm could move before exhausting their reserves. A high yield in distance per unit of ATP consumed (efficiency) could provide advantages in sperm competition. We characterized sperm ATP consumption rate in a group of mouse species with different sperm competition levels to understand its impact on swimming velocity, duration, and yield of sperm ATP reserves. Interspecific comparisons revealed that sperm of species with higher sperm competition levels had high ATP consumption rates and faster swimming velocity. Moreover, sperm that consumed ATP at a faster rate swam more efficiently, since they were able to cover more distance per unit of ATP consumed. Our results suggest that by coupling the advantages of higher ATP turnover rates to increased efficiency of ATP expenditure, sperm would respond to increasingly competitive environments while maintaining a positive ATP balance.

Significance of PGR5-dependent cyclic electron flow for optimizing the rate of ATP synthesis and consumption in Arabidopsis chloroplasts.

The proton motive force (PMF) across the chloroplast thylakoid membrane that is generated by electron transport during photosynthesis is the driving force for ATP synthesis in plants. The PMF mainly arises from the oxidation of water in photosystem II and from electron transfer within the cytochrome b6f complex. There are two electron transfer pathways related to PMF formation: linear electron flow and cyclic electron flow. Proton gradient regulation 5 (PGR5) is a major component of the cyclic electron flow pathway, and the Arabidopsis pgr5 mutant shows a substantial reduction in the PMF. How the PGR5-dependent cyclic electron flow contributes to ATP synthesis has not, however, been fully delineated. In this study, we monitored in vivo ATP levels in Arabidopsis chloroplasts in real time using a genetically encoded bioluminescence-based ATP indicator, Nano-lantern(ATP1). The increase in ATP in the chloroplast stroma of pgr5 leaves upon illumination with actinic light was significantly slower than in wild type, and the decrease in ATP levels when this illumination stopped was significantly faster in pgr5 leaves than in wild type. These results indicated that PGR5-dependent cyclic electron flow around photosystem I helps to sustain the rate of ATP synthesis, which is important for growth under fluctuating light conditions.

Understanding start-up problems in yeast glycolysis

Yeast glycolysis has been the focus of research for decades, yet a number of dynamical aspects of yeast glycolysis remain poorly understood at present. If nutrients are scarce, yeast will provide its catabolic and energetic needs with other pathways, but the enzymes catalysing upper glycolytic fluxes are still expressed. We conjecture that this overexpression facilitates the rapid transition to glycolysis in case of a sudden increase in nutrient concentration. However, if starved yeast is presented with abundant glucose, it can enter into an imbalanced state where glycolytic intermediates keep accumulating, leading to arrested growth and cell death. The bistability between regularly functioning and imbalanced phenotypes has been shown to depend on redox balance.We shed new light on these phenomena with a mathematical analysis of an ordinary differential equation model, including NADH to account for the redox balance. In order to gain qualitative insight, most of the analysis is parameter-free, i.e., without assigning a numerical value to any of the parameters.The model has a subtle bifurcation at the switch between an inviable equilibrium state and stable flux through glycolysis. This switch occurs if the ratio between the flux through upper glycolysis and ATP consumption rate of the cell exceeds a fixed threshold. If the enzymes of upper glycolysis would be barely expressed, our model predicts that there will be no glycolytic flux, even if external glucose would be at growth-permissable levels. The existence of the imbalanced state can be found for certain parameter conditions independent of the mentioned bifurcation. The parameter-free analysis proved too complex to directly gain insight into the imbalanced states, but the starting point of a branch of imbalanced states can be shown to exist in detail. Moreover, the analysis offers the key ingredients necessary for successful numerical continuation, which highlight the existence of this bistability and the influence of the redox balance.

Modeling oxygen requirements in ischemic cardiomyocytes

Heart disease remains the leading cause of death globally. Although reperfusion following myocardial ischemia can prevent death by restoring nutrient flow, ischemia/reperfusion injury can cause significant heart damage. The mechanisms that drive ischemia/reperfusion injury are not well understood; currently, few methods can predict the state of the cardiac muscle cell and its metabolic conditions during ischemia. Here, we explored the energetic sustainability of cardiomyocytes, using a model for cellular metabolism to predict the levels of ATP following hypoxia. We modeled glycolytic metabolism with a system of coupled ordinary differential equations describing the individual metabolic reactions within the cardiomyocyte over time. Reduced oxygen levels and ATP consumption rates were simulated to characterize metabolite responses to ischemia. By tracking biochemical species within the cell, our model enables prediction of the cell's condition up to the moment of reperfusion. The simulations revealed a distinct transition between energetically sustainable and unsustainable ATP concentrations for various energetic demands. Our model illustrates how even low oxygen concentrations allow the cell to perform essential functions. We found that the oxygen level required for a sustainable level of ATP increases roughly linearly with the ATP consumption rate. An extracellular O2 concentration of ∼0.007 mm could supply basic energy needs in non-beating cardiomyocytes, suggesting that increased collateral circulation may provide an important source of oxygen to sustain the cardiomyocyte during extended ischemia. Our model provides a time-dependent framework for studying various intervention strategies to change the outcome of reperfusion.

Glycolytic reliance promotes anabolism in photoreceptors.

Vertebrate photoreceptors are among the most metabolically active cells, exhibiting a high rate of ATP consumption. This is coupled with a high anabolic demand, necessitated by the diurnal turnover of a specialized membrane-rich organelle, the outer segment, which is the primary site of phototransduction. How photoreceptors balance their catabolic and anabolic demands is poorly understood. Here, we show that rod photoreceptors in mice rely on glycolysis for their outer segment biogenesis. Genetic perturbations targeting allostery or key regulatory nodes in the glycolytic pathway impacted the size of the outer segments. Fibroblast growth factor signaling was found to regulate glycolysis, with antagonism of this pathway resulting in anabolic deficits. These data demonstrate the cell autonomous role of the glycolytic pathway in outer segment maintenance and provide evidence that aerobic glycolysis is part of a metabolic program that supports the biosynthetic needs of a normal neuronal cell type.

Succinate supplementation improves metabolic performance of mixed glial cell cultures with mitochondrial dysfunction

Mitochondrial dysfunction, the inability to efficiently utilise metabolic fuels and oxygen, contributes to pathological changes following traumatic spinal cord or traumatic brain injury (TBI). In the present study, we tested the hypothesis that succinate supplementation can improve cellular energy state under metabolically stressed conditions in a robust, reductionist in vitro model of mitochondrial dysfunction in which primary mixed glial cultures (astrocytes, microglia and oligodendrocytes) were exposed to the mitochondrial complex I inhibitor rotenone. Cellular response was determined by measuring intracellular ATP, extracellular metabolites (glucose, lactate, pyruvate), and oxygen consumption rate (OCR). Rotenone produced no significant changes in glial ATP levels. However, it induced metabolic deficits as evidenced by lactate/pyruvate ratio (LPR) elevation (a clinically-established biomarker for poor outcome in TBI) and decrease in OCR. Succinate addition partially ameliorated these metabolic deficits. We conclude that succinate can improve glial oxidative metabolism, consistent our previous findings in TBI patients’ brains. The mixed glial cellular model may be useful in developing therapeutic strategies for conditions involving mitochondrial dysfunction, such as TBI.

Quantifying intracellular rates of glycolytic and oxidative ATP production and consumption using extracellular flux measurements

Partitioning of ATP generation between glycolysis and oxidative phosphorylation is central to cellular bioenergetics but cumbersome to measure. We describe here how rates of ATP generation by each pathway can be calculated from simultaneous measurements of extracellular acidification and oxygen consumption. We update theoretical maximum ATP yields by mitochondria and cells catabolizing different substrates. Mitochondrial P/O ratios (mol of ATP generated per mol of [O] consumed) are 2.73 for oxidation of pyruvate plus malate and 1.64 for oxidation of succinate. Complete oxidation of glucose by cells yields up to 33.45 ATP/glucose with a maximum P/O of 2.79. We introduce novel indices to quantify bioenergetic phenotypes. The glycolytic index reports the proportion of ATP production from glycolysis and identifies cells as primarily glycolytic (glycolytic index > 50%) or primarily oxidative. The Warburg effect is a chronic increase in glycolytic index, quantified by the Warburg index. Additional indices quantify the acute flexibility of ATP supply. The Crabtree index and Pasteur index quantify the responses of oxidative and glycolytic ATP production to alterations in glycolysis and oxidative reactions, respectively; the supply flexibility index quantifies overall flexibility of ATP supply; and the bioenergetic capacity quantifies the maximum rate of total ATP production. We illustrate the determination of these indices using C2C12 myoblasts. Measurement of ATP use revealed no significant preference for glycolytic or oxidative ATP by specific ATP consumers. Overall, we demonstrate how extracellular fluxes quantitatively reflect intracellular ATP turnover and cellular bioenergetics. We provide a simple spreadsheet to calculate glycolytic and oxidative ATP production rates from raw extracellular acidification and respiration data.

The effect of electrical conductivity of myocardium on cardiac pumping efficacy: a computational study

Background and aims The existence of non-excitable cells in the myocardium leads to the increasing conduction non-uniformity and decreasing myocardial electrical conductivity. Slowed myocardial conduction velocity (MCV) believed to enhance the probability of cardiac arryhthmia and alter the cardiac mechanical pumping efficacy, even in sinus rhythm. Though several studies on the correlation between MCV and cardiac electrical instabilities exist, there has been no study concerning correlation or causality between MCV and cardiac mechanical pumping efficacy, due to the limitation in clinical methods to document and evaluate cardiac mechanical responses directly. The goal of this study was to examine quantitatively the cardiac pumping efficacy under various MCV conditions using three-dimensional (3D) electromechanical model of canine’s failing ventricle.Methods The electromechanical model used in this study composed of the electrical model coupled with the mechanical contraction model along with a lumped model of the circulatory system. The electrical model consisted of 241,725 nodes and 1,298,751 elements of tetrahedral mesh, whereas the mechanical model consisted of 356 nodes and 172 elements of hexahedral mesh with Hermite basis. First, we performed the electrical simulation for five different MCV conditions, from 30 to 70 cm/s with 10 cm/s interval during sinus pacing. Then, we compared the cardiac electrical and mechanical responses of each MCV condition, such as the electrical activation time (EAT), pressure, volume, and energy consumption of the myocardium. The energy consumption of the myocardium was calculated by integrating ATP consumption rate of each node in myofilament model.ResultsThe result showed that under higher MCV conditions, the EAT, energy consumption, end diastolic and systolic volume are gradually decreased. Meanwhile, the systolic pressure, stroke volume, stroke work, and stroke work to ATP are increased as the MCV values increased. The cardiac functions and performances are more efficient under higher MCV conditions by consuming smaller energy (ATP) while carrying more works.ConclusionIn conclusion, this study reveals that MCV has strong correlation with the cardiac pumping efficacy. The obtained results provide useful information to estimate the effect of MCV on the electro-physiology and hemodynamic responses of the ventricle and can be used for further study about arrhythmogeneis and heart failure.

Efficiency of energy transfer during exercise: what are the limiting factors?

Efficiency of energy transfer during exercise: what are the limiting factors?

Mapping human brain capillary water lifetime: high-resolution metabolic neuroimaging.

Shutter‐speed analysis of dynamic‐contrast‐agent (CA)‐enhanced normal, multiple sclerosis (MS), and glioblastoma (GBM) human brain data gives the mean capillary water molecule lifetime (τ b) and blood volume fraction (v b; capillary density–volume product (ρ † V)) in a high‐resolution 1H2O MRI voxel (40 μL) or ROI. The equilibrium water extravasation rate constant, k po (τ b −1), averages 3.2 and 2.9 s−1 in resting‐state normal white matter (NWM) and gray matter (NGM), respectively (n = 6). The results (italicized) lead to three major conclusions. (A) k po differences are dominated by capillary water permeability (P W †), not size, differences. NWM and NGM voxel k po and vb values are independent. Quantitative analyses of concomitant population‐averaged k po, vb variations in normal and normal‐appearing MS brain ROIs confirm PW † dominance. (B) P W † is dominated (>95%) by a trans(endothelial)cellular pathway, not the P CA † paracellular route. In MS lesions and GBM tumors, PCA † increases but PW † decreases. (C) k po tracks steady‐state ATP production/consumption flux per capillary. In normal, MS, and GBM brain, regional k po correlates with literature MRSI ATP (positively) and Na + (negatively) tissue concentrations. This suggests that the PW † pathway is metabolically active. Excellent agreement of the relative NGM/NWM k po vb product ratio with the literature 31PMRSI‐MT CMRoxphos ratio confirms the flux property. We have previously shown that the cellular water molecule efflux rate constant (k io) is proportional to plasma membrane P‐type ATPase turnover, likely due to active trans‐membrane water cycling. With synaptic proximities and synergistic metabolic cooperativities, polar brain endothelial, neuroglial, and neuronal cells form “gliovascular units.” We hypothesize that a chain of water cycling processes transmits brain metabolic activity to k po, letting it report neurogliovascular unit Na+,K+‐ATPase activity. Cerebral k po maps represent metabolic (functional) neuroimages. The NGM 2.9 s−1 k po means an equilibrium unidirectional water efflux of ~1015 H2O molecules s−1 per capillary (in 1 μL tissue): consistent with the known ATP consumption rate and water co‐transporting membrane symporter stoichiometries. © 2015 The Authors NMR in Biomedicine Published by John Wiley & Sons Ltd.

Redefining the Latch-State in Human Airway Smooth Muscle

Rationale: The latch-state describes the ability of tonic smooth muscle to maintain force at reduced ATP consumption and crossbridge cycling rate. In animal models this is evidenced by reduced unloaded shortening velocity (Vmax), presumably indicative of crossbridge cycling rate, and reduced myosin light chain phosphorylation (pMLC). In this study we aim to assess this phenomenon in human airway smooth muscle (hASM).Methods: hASM tissues were dissected from trachea procured from the International Institute for the Advancement of Medicine. Force-velocity curves and pMLC measurements were performed at several time points in multiple prolonged methacholine 10-6M contractions. Rat ASM strips were tested with the same protocol. A mathematical model was developed to explain the findings.Results: Despite a 40% drop in pMLC between the 1st to the 20th min after the peak contraction, hASM Vmax did not show any change. Conversely, rat ASM Vmax decreased by 50%. A mathematical model of the cross-bridge cycle, combining strain-dependent ADP affinity of myosin with actin regulated binding of dephosphorylated myosin heads, was capable of reproducing our findings.Conclusion: As our results contradict the classical latch-bridge theory, we suggest an alternative model. We propose that Vmax, as calculated from standard force-velocity curves, is not indicative of the crossbridge cycling rate during latch. Instead, Vmax is reduced due to drag caused by dephosphorylated myosin heads binding to actin, with their binding force regulated by actin regulatory proteins and their activation state. The increase in dephosphorylated myosin late in contraction decreases the measured Vmax, unless its binding force is down-regulated by regulatory proteins. Our modelling further shows that force maintenance can be explained by phosphorylated myosin heads remaining attached to actin in a locked configuration, unable to release ADP because of strain dependence of the ADP affinity of myosin.

The compound BTB06584 is an IF1‐dependent selective inhibitor of the mitochondrial F1Fo‐ATPase

Ischaemia compromises mitochondrial respiration. Consequently, the mitochondrial F1 Fo-ATPsynthase reverses and acts as a proton-pumping ATPase, so maintaining the mitochondrial membrane potential (ΔΨm ), while accelerating ATP depletion and cell death. Here we have looked for a molecule that can selectively inhibit this activity without affecting ATP synthesis, preserve ATP and delay ischaemic cell death. We developed a chemoinformatic screen based on the structure of BMS199264, which is reported to selectively inhibit F1 Fo-ATPase activity and which is cardioprotective. Results suggested the molecule BTB06584 (hereafter referred to as BTB). Fluorescence microscopy was used to study its effects on ΔΨm and on the rate of ATP consumption following inhibition of respiration in several cell types. The effect of BTB on oxygen (O2 ) consumption was explored and protective potential determined using ischaemia/reperfusion assays. We also investigated a potential mechanism of action through its interaction with inhibitor protein of F1 subunit (IF1 ), the endogenous inhibitor of the F1 Fo-ATPase. BTB inhibited F1 Fo-ATPase activity with no effect on ΔΨm or O2 consumption. ATP consumption was decreased following inhibition of respiration, and ischaemic cell death was reduced. BTB efficiency was increased by IF1 overexpression and reduced by silencing the protein. In addition, BTB rescued defective haemoglobin synthesis in zebrafish pinotage (pnt) mutants in which expression of the Atpif1a gene is lost. BTB may represent a valuable tool to selectively inhibit mitochondrial F1 Fo-ATPase activity without compromising ATP synthesis and to limit ischaemia-induced injury caused by reversal of the mitochondrial F1 Fo-ATPsynthase.

Simulations of Neck-Linker Modified and One Head Loaded Kinesins

Recently, Czovek et al. established a complete, thermodynamically consistent kinetic model for the two-headed homodimeric motor protein, kinesin. Computational simulations based on the model justified the crucial role of the conformational changes of the neck-linkers (NLs, the peptide chains connecting the two motor domains to the stalk) in the directional movement and force-generation of conventional kinesin. The model was able to reproduce a large number of experimental data (speed, dwell time distribution, randomness, processivity, hydrolysis rate, etc.) astonishingly well under normal as well as under highly unphysiological conditions. Moreover, it enabled a more detailed deconvolution of the mechanochemical cycle than it is experimentally possible. Having such a powerful model, we have applied it to modified versions of the wild-type kinesin, and reproduced (i) the speeds, processivities, and ATP consumption rates of NL modified kinesin; and (ii) the force-velocity relationship of the one-head-pulled kinesin. The good agreement between the simulations and the experiments further justify the legitimacy of the model, which thus provides a detailed understanding of the experimental observations and the basic mechanism of the operation of kinesin.

Measurement of ATP in Single Oocytes: Impact of Maturation and Cumulus Cells on Levels and Consumption

Mitochondria provide the primary source of ATP in the oocyte and early embryo and mitochondrial dysfunction and deficit of mitochondria-derived ATP has been linked to suboptimal developmental competence. We have undertaken a study of ATP in the maturing mouse oocyte using a novel recombinant FRET based probe, AT1.03. We show that AT1.03 can be successfully used to monitor cytosolic ATP levels in single live oocytes over extended time periods. We find that ATP levels undergo dynamic changes associated with specific maturational events and that oocytes display altered rates of ATP consumption at different stages of maturation. Cumulus enclosed oocytes have a higher ATP level during maturation than denuded oocytes and this can be abolished by inhibition of gap junctional communication between the oocyte and cumulus cells. Our work uses a new approach to shed light on regulation of ATP levels and ATP consumption during oocyte maturation.

Oxygen control of intracellular distribution of mitochondria in muscle fibers

Mitochondrial density in skeletal muscle fibers is governed by the demand for aerobic ATP production, but the heterogeneous distribution of these mitochondria appears to be governed by constraints associated with oxygen diffusion. We propose that each muscle fiber has an optimal mitochondrial distribution at which it attains a near maximal rate of ATP consumption (RATPase ) while mitochondria are exposed to a minimal oxygen concentration, thus minimizing reactive oxygen species (ROS) production. We developed a coupled reaction-diffusion/cellular automata (CA) mathematical model of mitochondrial function and considered four fiber types in mouse extensor digitorum longus (EDL) and soleus (SOL) muscle. The developed mathematical model uses a reaction-diffusion analysis of metabolites including oxygen, ATP, ADP, phosphate, and phosphocreatine (PCr) involved in energy metabolism and mitochondrial function. A CA approach governing mitochondrial life cycles in response to the metabolic state of the fiber was superimposed and coupled to the reaction-diffusion approach. The model results show the sensitivity of important model outputs such as the RATPase , effectiveness factor (η) and average oxygen concentration available at each mitochondrion to local oxygen concentration in the fibers through variation in the CA model parameter θdet , which defines the sensitivity of mitochondrial death to the oxygen concentration. The predicted optimal mitochondrial distributions matched previous experimental findings. Deviations from this optimal distribution corresponding to higher CA model parameter values (a more uniform mitochondrial distribution) lead to lower aerobic rates. In contrast, distributions corresponding to lower CA model parameter values (a more asymmetric distribution) lead to an increased exposure of mitochondria to oxygen, usually without substantial increases in aerobic rates, which would presumably result in increased ROS production and thus increased risks of cytotoxicity.

Simulations of Neck Linker Modified and One Head Loaded Kinesin

Recently, Czovek et al. established a complete, thermodynamically consistent kinetic model for the two-headed homodimeric motor protein, kinesin. Computational simulations based on the model justified the crucial role of the conformational changes of the neck-linkers (NLs, the peptide chains connecting the two motor domains to the stalk) in the directional movement and force-generation of conventional kinesin. The model was able to reproduce a large number of experimental data (speed, dwell time distribution, randomness, processivity, hydrolysis rate, etc.) astonishingly well under normal as well as under highly unphysiological conditions. Moreover, it enabled a more detailed deconvolution of the mechanochemical cycle than it is experimentally possible. Having such a powerful model, we have applied it to modified versions of the wild-type kinesin, and reproduced (i) the speeds, processivities, and ATP consumption rates of NL modified kinesin; and (ii) the force-velocity relationship of the one-head-pulled kinesin. The good agreement between the simulations and the experiments further justify the legitimacy of the model, which thus provides a detailed understanding of the experimental observations and the basic mechanism of the operation of kinesin.

Hypoxia associated NMDA receptor 2 subunit composition: developmental comparison between the hypoxia-tolerant subterranean mole-rat, Spalax, and the hypoxia-sensitive rat

Vertebrate brains are sensitive to oxygen depletion, which may lead to cell death. Hypoxia sensitivity originates from the high intrinsic rate of ATP consumption of brain tissue, accompanied by the release of glutamate, leading to the opening of ionotropic glutamate receptors, such as N-methyl-D-aspartate (NMDA) receptors (NMDARs). The relative expression levels of the four NMDAR-2 (NR2) subunits change during mammalian development with higher levels of units NR2B and NR2D observed during early development and correlated with hypoxic tolerance during embryonic and neonatal stages of development. Higher levels of NR2D are also abundant in brains of hypoxia tolerant species such as the crucian carp. The subterranean mole-rat, Spalax spends its life underground in sealed burrows and has developed a wide range of adaptations to this special niche including hypoxia-tolerance. In this study, we compared the in vivo mRNA expression of NR2 subunits in the brains of embryonic, neonatal and adult Spalax and rat. Our results demonstrate that under normoxic conditions, mRNA levels of NR2D are higher in Spalax than in rat at all developmental stages studied and are similar to levels in neonatal rat and in other hypoxia/anoxia tolerant species. Furthermore, under hypoxia Spalax NR2D mRNA levels increase while no response was observed in rat. Similarly, hypoxia induces an increase in mRNA levels of Spalax NR2A, claimed to promote neuronal survival. We suggest that indeed the proportional combinations of NMDAR-2 subunits contribute to the ability of the Spalax brain to cope with hypoxic environments.

The role of the C8 proton of ATP in the catalysis of shikimate kinase and adenylate kinase

BackgroundIt has been demonstrated that the adenyl moiety of ATP plays a direct role in the regulation of ATP binding and/or phosphoryl transfer within a range of kinase and synthetase enzymes. The role of the C8-H of ATP in the binding and/or phosphoryl transfer on the enzyme activity of a number of kinase and synthetase enzymes has been elucidated. The intrinsic catalysis rate mediated by each kinase enzyme is complex, yielding apparent KM values ranging from less than 0.4 μM to more than 1 mM for ATP in the various kinases. Using a combination of ATP deuterated at the C8 position (C8D-ATP) as a molecular probe with site directed mutagenesis (SDM) of conserved amino acid residues in shikimate kinase and adenylate kinase active sites, we have elucidated a mechanism by which the ATP C8-H is induced to be labile in the broader kinase family. We have demonstrated the direct role of the C8-H in the rate of ATP consumption, and the direct role played by conserved Thr residues interacting with the C8-H. The mechanism by which the vast range in KM might be achieved is also suggested by these findings.ResultsWe have demonstrated the mechanism by which the enzyme activities of Group 2 kinases, shikimate kinase (SK) and adenylate kinase 1 (AK1), are controlled by the C8-H of ATP. Mutations of the conserved threonine residues associated with the labile C8-H cause the enzymes to lose their saturation kinetics over the concentration range tested. The relationship between the role C8-H of ATP in the reaction mechanism and the ATP concentration as they influence the saturation kinetics of the enzyme activity is also shown. The SDM clearly identified the amino acid residues involved in both the catalysis and regulation of phosphoryl transfer in SK and AK1 as mediated by C8H-ATP.ConclusionsThe data outlined serves to demonstrate the “push” mechanism associated with the control of the saturation kinetics of Group 2 kinases mediated by ATP C8-H. It is therefore conceivable that kinase enzymes achieve the observed 2,500-fold variation in KM through a combination of the various conserved “push” and “pull” mechanisms associated with the release of C8-H, the proton transfer cascades unique to the class of kinase in question and the resultant/concomitant creation of a pentavalent species from the γ-phosphate group of ATP. Also demonstrated is the interplay between the role of the C8-H of ATP and the ATP concentration in the observed enzyme activity. The lability of the C8-H mediated by active site residues co-ordinated to the purine ring of ATP therefore plays a significant role in explaining the broad KM range associated with kinase steady state enzyme activities.