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Sample records for atp iii definition

  1. Inhibition of the Fe(III)-catalyzed dopamine oxidation by ATP and its relevance to oxidative stress in Parkinson's disease.

    PubMed

    Jiang, Dianlu; Shi, Shuyun; Zhang, Lin; Liu, Lin; Ding, Bingrong; Zhao, Bingqing; Yagnik, Gargey; Zhou, Feimeng

    2013-09-18

    Parkinson's disease (PD) is characterized by the progressive degeneration of dopaminergic cells, which implicates a role of dopamine (DA) in the etiology of PD. A possible DA degradation pathway is the Fe(III)-catalyzed oxidation of DA by oxygen, which produces neuronal toxins as side products. We investigated how ATP, an abundant and ubiquitous molecule in cellular milieu, affects the catalytic oxidation reaction of dopamine. For the first time, a unique, highly stable DA-Fe(III)-ATP ternary complex was formed and characterized in vitro. ATP as a ligand shifts the catecholate-Fe(III) ligand metal charge transfer (LMCT) band to a longer wavelength and the redox potentials of both DA and the Fe(III) center in the ternary complex. Remarkably, the additional ligation by ATP was found to significantly reverse the catalytic effect of the Fe(III) center on the DA oxidation. The reversal is attributed to the full occupation of the Fe(III) coordination sites by ATP and DA, which blocks O2 from accessing the Fe(III) center and its further reaction with DA. The biological relevance of this complex is strongly implicated by the identification of the ternary complex in the substantia nigra of rat brain and its attenuation of cytotoxicity of the Fe(III)-DA complex. Since ATP deficiency accompanies PD and neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) induced PD, deficiency of ATP and the resultant impairment toward the inhibition of the Fe(III)-catalyzed DA oxidation may contribute to the pathogenesis of PD. Our finding provides new insight into the pathways of DA oxidation and its relationship with synaptic activity. PMID:23823941

  2. Re-evaluating the kinetics of ATP hydrolysis during initiation of DNA sliding by Type III restriction enzymes

    PubMed Central

    Tóth, Júlia; Bollins, Jack; Szczelkun, Mark D.

    2015-01-01

    DNA cleavage by the Type III restriction enzymes requires long-range protein communication between recognition sites facilitated by thermally-driven 1D diffusion. This ‘DNA sliding’ is initiated by hydrolysis of multiple ATPs catalysed by a helicase-like domain. Two distinct ATPase phases were observed using short oligoduplex substrates; the rapid consumption of ∼10 ATPs coupled to a protein conformation switch followed by a slower phase, the duration of which was dictated by the rate of dissociation from the recognition site. Here, we show that the second ATPase phase is both variable and only observable when DNA ends are proximal to the recognition site. On DNA with sites more distant from the ends, a single ATPase phase coupled to the conformation switch was observed and subsequent site dissociation required little or no further ATP hydrolysis. The overall DNA dissociation kinetics (encompassing site release, DNA sliding and escape via a DNA end) were not influenced by the second phase. Although the data simplifies the ATP hydrolysis scheme for Type III restriction enzymes, questions remain as to why multiple ATPs are hydrolysed to prepare for DNA sliding. PMID:26538601

  3. Inhibition of the Fe(III)-Catalyzed Dopamine Oxidation by ATP and Its Relevance to Oxidative Stress in Parkinson’s Disease

    PubMed Central

    2013-01-01

    Parkinson’s disease (PD) is characterized by the progressive degeneration of dopaminergic cells, which implicates a role of dopamine (DA) in the etiology of PD. A possible DA degradation pathway is the Fe(III)-catalyzed oxidation of DA by oxygen, which produces neuronal toxins as side products. We investigated how ATP, an abundant and ubiquitous molecule in cellular milieu, affects the catalytic oxidation reaction of dopamine. For the first time, a unique, highly stable DA–Fe(III)–ATP ternary complex was formed and characterized in vitro. ATP as a ligand shifts the catecholate–Fe(III) ligand metal charge transfer (LMCT) band to a longer wavelength and the redox potentials of both DA and the Fe(III) center in the ternary complex. Remarkably, the additional ligation by ATP was found to significantly reverse the catalytic effect of the Fe(III) center on the DA oxidation. The reversal is attributed to the full occupation of the Fe(III) coordination sites by ATP and DA, which blocks O2 from accessing the Fe(III) center and its further reaction with DA. The biological relevance of this complex is strongly implicated by the identification of the ternary complex in the substantia nigra of rat brain and its attenuation of cytotoxicity of the Fe(III)–DA complex. Since ATP deficiency accompanies PD and neurotoxin 1-methyl-4-phenylpyridinium (MPP+) induced PD, deficiency of ATP and the resultant impairment toward the inhibition of the Fe(III)-catalyzed DA oxidation may contribute to the pathogenesis of PD. Our finding provides new insight into the pathways of DA oxidation and its relationship with synaptic activity. PMID:23823941

  4. Aspects of Subunit Interactions in the Chloroplast ATP Synthase (I. Isolation of a Chloroplast Coupling Factor 1-Subunit III Complex from Spinach Thylakoids).

    PubMed Central

    Wetzel, C. M.; McCarty, R. E.

    1993-01-01

    A chloroplast ATP synthase complex (CF1 [chloroplast-coupling factor 1]-CF0 [membrane-spanning portion of chloroplast ATP synthase]) depleted of all CF0 subunits except subunit III (also known as the proteolipid subunit) was purified to study the interaction between CF1 and subunit III. Subunit III has a putative role in proton translocation across the thylakoid membrane during photophosphorylation; therefore, an accurate model of subunit inter-actions involving subunit III will be valuable for elucidating the mechanism and regulation of energy coupling. Purification of the complex from a crude CF1-CF0 preparation from spinach (Spinacia oleracea) thylakoids was accomplished by detergent treatment during anion-exchange chromatography. Subunit III in the complex was positively identified by amino acid analysis and N-terminal sequencing. The association of subunit III with CF1 was verified by linear sucrose gradient centrifugation, immunoprecipitation, and incorporation of the complex into asolectin liposomes. After incorporation into liposomes, CF1 was removed from the CF1-III complex by ethylenediaminetetracetate treatment. The subunit III-proteoliposomes were competent to rebind purified CF1. These results indicate that subunit III directly interacts with CF1 in spinach thylakoids. PMID:12231815

  5. Electrophysiological-anatomic correlates of ATP-triggered vagal reflex in the dog. III. Role of cardiac afferents.

    PubMed

    Katchanov, G; Xu, J; Hurt, C M; Pelleg, A

    1996-05-01

    To test the hypothesis that the asymmetry in the afferent traffic of the intra-right atrium (RA) ATP-triggered vagal reflex is due to the stimulation by ATP of extrapulmonary (i.e., cardiac) vagal chemosensitive afferent terminals, ATP, adenosine, and capsaicin were given into the canine RA and the aortic root (AR; n = 12); ATP and adenosine were also administered into the left common carotid artery and the descending aorta (n = 6). The negative chronotropic action [i.e., suppression of sinus node (SN) automaticity] of the test compounds and time to peak effect (tp) were determined. Under baseline conditions, ATP given into the left common carotid artery had a relatively very small effect. ATP given into the descending aorta had no effect. In contrast, intra-RA and intra-AR ATP markedly suppressed SN automaticity, the former less than the latter; the opposite was true for capsaicin. Intra-RA adenosine was much less potent than intra-RA ATP. The tp of intra-RA ATP and intra-RA adenosine were larger than the tp of intra-AR ATP. Pulmonary denervation did not alter the effects of intra-RAATP, intra-ARATP, or intra-AR capsaicin but almost abolished the effect of intra-RA capsaicin. Subsequent bilateral, but not left, cervical vagotomy markedly reduce the effects of ATP and eliminated the difference between the effects of ATP and adenosine. In addition, tp of intra-RA ATP and intra-AR ATP increased substantially and were similar to tp of adenosine. It was concluded that 1) ATP can stimulate vagal afferent terminals not only in the lungs but also in the heart, 2) the latter constitutes the vagal component of the negative chronotropic action of intra-RA or intra-AR ATP on SN automatically, and 3) the asymmetry in the vagal afferent traffic elicited by ATP in the heart (i.e., right vagal dominance) supersedes the symmetrical vagal afferent traffic triggered by intrapulmonary ATP. PMID:8928887

  6. The prevalence of metabolic syndrome according to the Iranian Committee of Obesity and ATP III criteria in Babol, North of Iran.

    PubMed Central

    Mahjoub, Soleiman; Haji Ahmadi, Mahmoud; Faramarzi, Mahbobeh; Ghorbani, Hiva; Moazezi, Zoleika

    2012-01-01

    Background: Metabolic syndrome (MS) is highly significant due to its association to type 2 diabetes and cardiovascular diseases. The purpose of this study was to compare the prevalence of MS according to the report of the Iranian National Committee of Obesity criteria (INCO) versus Adult Treatment Panel III (ATPIII) in Babol, North of Iran. Methods: Data obtained based on criteria ATP III from the Babol Lipid and Glucose Study (from July 2004 to September 2005) and were compared with the new INCO criteria 2010. The data were collected and analyzed. Results: In total, 933 adult males and females were evaluated. According to ATP III criteria, the overall prevalence of metabolic syndrome was 23.7% (95% confidence interval: 21%-26.4%); 28.4% and 9.4% were females and males, respectively; however, the prevalence was 20.5% (95% confidence interval: 17.9%−23.1%) according to the INCO criteria, 22.5% and 15.7% were females and males, respectively. Conclusion: The new INCO criteria for the metabolic syndrome proclaimed by the Iranian Committee of Obesity estimated a lower prevalence of syndrome in comparison with ATP III criteria in Babol. PMID:24358435

  7. In vitro study of accuracy of cervical pedicle screw insertion using an electronic conductivity device (ATPS part III)

    PubMed Central

    Hitzl, Wolfgang; Acosta, Frank; Tauber, Mark; Zenner, Juliane; Resch, Herbert; Yukawa, Yasutsugu; Meier, Oliver; Schmidt, Rene; Mayer, Michael

    2009-01-01

    Reconstruction of the highly unstable, anteriorly decompressed cervical spine poses biomechanical challenges to current stabilization strategies, including circumferential instrumented fusion, to prevent failure. To avoid secondary posterior surgery, particularly in the elderly population, while increasing primary construct rigidity of anterior-only reconstructions, the authors introduced the concept of anterior transpedicular screw (ATPS) fixation and plating. We demonstrated its morphological feasibility, its superior biomechanical pull-out characteristics compared with vertebral body screws and the accuracy of inserting ATPS using a manual fluoroscopically assisted technique. Although accuracy was high, showing non-critical breaches in the axial and sagittal plane in 78 and 96%, further research was indicated refining technique and increasing accuracy. In light of first clinical case series, the authors analyzed the impact of using an electronic conductivity device (ECD, PediGuard) on the accuracy of ATPS insertion. As there exist only experiences in thoracolumbar surgery the versatility of the ECD was also assessed for posterior cervical pedicle screw fixation (pCPS). 30 ATPS and 30 pCPS were inserted alternately into the C3–T1 vertebra of five fresh-frozen specimen. Fluoroscopic assistance was only used for the entry point selection, pedicle tract preparation was done using the ECD. Preoperative CT scans were assessed for sclerosis at the pedicle entrance or core, and vertebrae with dense pedicles were excluded. Pre- and postoperative reconstructed CT scans were analyzed for pedicle screw positions according to a previously established grading system. Statistical analysis revealed an astonishingly high accuracy for the ATPS group with no critical screw position (0%) in axial or sagittal plane. In the pCPS group, 88.9% of screws inserted showed non-critical screw position, while 11.1% showed critical pedicle perforations. The usage of an ECD for posterior and

  8. Structural Features Reminiscent of ATP-Driven Protein Translocases Are Essential for the Function of a Type III Secretion-Associated ATPase

    PubMed Central

    Kato, Junya; Lefebre, Matthew

    2015-01-01

    ABSTRACT Many bacterial pathogens and symbionts utilize type III secretion systems to interact with their hosts. These machines have evolved to deliver bacterial effector proteins into eukaryotic target cells to modulate a variety of cellular functions. One of the most conserved components of these systems is an ATPase, which plays an essential role in the recognition and unfolding of proteins destined for secretion by the type III pathway. Here we show that structural features reminiscent of other ATP-driven protein translocases are essential for the function of InvC, the ATPase associated with a Salmonella enterica serovar Typhimurium type III secretion system. Mutational and functional analyses showed that a two-helix-finger motif and a conserved loop located at the entrance of and within the predicted pore formed by the hexameric ATPase are essential for InvC function. These findings provide mechanistic insight into the function of this highly conserved component of type III secretion machines. IMPORTANCE Type III secretion machines are essential for the virulence or symbiotic relationships of many bacteria. These machines have evolved to deliver bacterial effector proteins into host cells to modulate cellular functions, thus facilitating bacterial colonization and replication. An essential component of these machines is a highly conserved ATPase, which is necessary for the recognition and secretion of proteins destined to be delivered by the type III secretion pathway. Using modeling and structure and function analyses, we have identified structural features of one of these ATPases from Salmonella enterica serovar Typhimurium that help to explain important aspects of its function. PMID:26170413

  9. Radioprotective effectiveness and toxicity of ATP, AET and serotonin applied individually or simultaneously to mice. III. Radioprotective effects of pair combinations.

    PubMed

    Benova, D

    1977-08-01

    The work reported was done as part of an intensive investigation on toxic and radioprotective properties of three substances, ATP, AET and serotonin, administered singly or in combination to mice, with a view to identifying optimal dose ratios for cocktails. Male C57BL mice were exposed to 850 R X-rays (LD100/11) following pretreatment with drug pairs at various dose ratios. Thirty-day survival was scored. For ATP-AET, protection increased with the amount of ATP in the combination; this pair was found to be most effective at an ATP-to-AET ratio of 11:1. A similar trend was observed with ATP-Serotonin, though only up to a certain ATP level beyond which no further increase in protective effect were produced; the most favorable ratio was of 24:1. A maximum synergistic action was displayed by the AET-Serotonin pair as compared to the other two pairs; its best ratio was of 4:1. Using probit analysis, a number of PD50 (protectant dose affording 50% survival in lethal irradiation) values were estimated for the three pairs of protective agents. PMID:898225

  10. ATP synthase.

    PubMed

    Junge, Wolfgang; Nelson, Nathan

    2015-01-01

    Oxygenic photosynthesis is the principal converter of sunlight into chemical energy. Cyanobacteria and plants provide aerobic life with oxygen, food, fuel, fibers, and platform chemicals. Four multisubunit membrane proteins are involved: photosystem I (PSI), photosystem II (PSII), cytochrome b6f (cyt b6f), and ATP synthase (FOF1). ATP synthase is likewise a key enzyme of cell respiration. Over three billion years, the basic machinery of oxygenic photosynthesis and respiration has been perfected to minimize wasteful reactions. The proton-driven ATP synthase is embedded in a proton tight-coupling membrane. It is composed of two rotary motors/generators, FO and F1, which do not slip against each other. The proton-driven FO and the ATP-synthesizing F1 are coupled via elastic torque transmission. Elastic transmission decouples the two motors in kinetic detail but keeps them perfectly coupled in thermodynamic equilibrium and (time-averaged) under steady turnover. Elastic transmission enables operation with different gear ratios in different organisms. PMID:25839341

  11. Assessing the prevalence of the Metabolic Syndrome according to NCEP ATP III in Germany: feasibility and quality aspects of a two step approach in 1550 randomly selected primary health care practices

    PubMed Central

    Moebus, Susanne; Hanisch, Jens Ulrich; Neuhäuser, Markus; Aidelsburger, Pamela; Wasem, Jürgen; Jöckel, Karl-Heinz

    2006-01-01

    Objective: Metabolic Syndrome (MetSyn) describes a cluster of metabolic disorders and is considered a risk factor for development of cardiovascular disease. Although a high prevalence is commonly assumed in Germany data about the degree of its occurrence in the population and in subgroups are still missing. The aim of this study was to assess the prevalence of the MetSyn according to the NCEP ATP-III (National Cholesterol Education Program Adult Treatment Panel III) criteria in persons aged ≥18 years attending a general practitioner in Germany. Here we describe in detail the methods used and the feasibility of determining the MetSyn in a primary health care setting. Research design and methods: The German-wide cross-sectional study was performed during two weeks in October 2005. Blood samples were analyzed in a central laboratory. Waist circumference and blood pressure were assessed, data on smoking, life style, fasting status, socio-demographic characteristics and core information from non-participants collected. Quality control procedures included telephone-monitoring and random on-site visits. In order to achieve a maximal number of fasting blood samples with a minimal need for follow-up appointments a stepwise approach was developed. Basic descriptive statistics were calculated, the Taylor expansion method used to estimate standard errors needed for calculation of confidence intervals for clustered observations. Results: In total, 1511 randomly selected general practices from 397 out of 438 German cities and administrative districts enrolled 35,869 patients (age range: 18-99, women 61.1%). More than 50,000 blood samples were taken. Fasting blood samples were available for 49% of the participants. Of the participating patients 99.3% returned questionnaires to the GP, only 12% were not filled out completely. The overall prevalence of the MetSyn (NCEP/ATP III 2001) was found to be 19.8%, with men showing higher prevalence rates than women (22.7% respective 18

  12. A label-free electrochemiluminescent sensor for ATP detection based on ATP-dependent ligation.

    PubMed

    Zhao, Tingting; Lin, Chunshui; Yao, Qiuhong; Chen, Xi

    2016-07-01

    In this work, we describe a new label-free, sensitive and highly selective strategy for the electrochemiluminescent (ECL) detection of ATP at the picomolar level via ATP-induced ligation. The molecular-beacon like DNA probes (P12 complex) are self-assembled on a gold electrode. The presence of ATP leads to the ligation of P12 complex which blocks the digestion by Exonuclease III (Exo III). The protected P12 complex causes the intercalation of numerous ECL indicators (Ru(phen)3(2+)) into the duplex DNA grooves, resulting in significantly amplified ECL signal output. Since the ligating site of T4 DNA ligase and the nicking site of Exo III are the same, it involves no long time of incubation for conformation change. The proposed strategy combines the amplification power of enzyme and the inherent high sensitivity of the ECL technique and enables picomolar detection of ATP. The developed strategy also shows high selectivity against ATP analogs, which makes our new label-free and highly sensitive ligation-based method a useful addition to the amplified ATP detection arena. PMID:27154705

  13. Electron transfer precedes ATP hydrolysis during nitrogenase catalysis.

    PubMed

    Duval, Simon; Danyal, Karamatullah; Shaw, Sudipta; Lytle, Anna K; Dean, Dennis R; Hoffman, Brian M; Antony, Edwin; Seefeldt, Lance C

    2013-10-01

    The biological reduction of N2 to NH3 catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: kATP = 70 s(-1), 25 °C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (kET = 140 s(-1), 25 °C), (ii) ATP hydrolysis (kATP = 70 s(-1), 25 °C), (iii) Phosphate release (kPi = 16 s(-1), 25 °C), and (iv) Fe protein dissociation from the MoFe protein (kdiss = 6 s(-1), 25 °C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and protein-protein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Fe(ox)(ADP)2 protein and the reduced MoFe protein. PMID:24062462

  14. BIOGENESIS FACTOR REQUIRED FOR ATP SYNTHASE 3 Facilitates Assembly of the Chloroplast ATP Synthase Complex.

    PubMed

    Zhang, Lin; Duan, Zhikun; Zhang, Jiao; Peng, Lianwei

    2016-06-01

    Thylakoid membrane-localized chloroplast ATP synthases use the proton motive force generated by photosynthetic electron transport to produce ATP from ADP. Although it is well known that the chloroplast ATP synthase is composed of more than 20 proteins with α3β3γ1ε1δ1I1II1III14IV1 stoichiometry, its biogenesis process is currently unclear. To unravel the molecular mechanisms underlying the biogenesis of chloroplast ATP synthase, we performed extensive screening for isolating ATP synthase mutants in Arabidopsis (Arabidopsis thaliana). In the recently identified bfa3 (biogenesis factors required for ATP synthase 3) mutant, the levels of chloroplast ATP synthase subunits were reduced to approximately 25% of wild-type levels. In vivo labeling analysis showed that assembly of the CF1 component of chloroplast ATP synthase was less efficient in bfa3 than in the wild type, indicating that BFA3 is required for CF1 assembly. BFA3 encodes a chloroplast stromal protein that is conserved in higher plants, green algae, and a few species of other eukaryotic algae, and specifically interacts with the CF1β subunit. The BFA3 binding site was mapped to a region in the catalytic site of CF1β. Several residues highly conserved in eukaryotic CF1β are crucial for the BFA3-CF1β interaction, suggesting a coevolutionary relationship between BFA3 and CF1β. BFA3 appears to function as a molecular chaperone that transiently associates with unassembled CF1β at its catalytic site and facilitates subsequent association with CF1α during assembly of the CF1 subcomplex of chloroplast ATP synthase. PMID:27208269

  15. The Impact of Extent and Location of Mediastinal Lymph Node Involvement on Survival in Stage III Non-Small Cell Lung Cancer Patients Treated With Definitive Radiotherapy

    SciTech Connect

    Fernandes, Annemarie T.; Mitra, Nandita; Xanthopoulos, Eric; Evans, Tracey; Stevenson, James; Langer, Corey; Kucharczuk, John C.; Lin, Lilie; Rengan, Ramesh

    2012-05-01

    Purpose: Several surgical series have identified subcarinal, contralateral, and multilevel nodal involvement as predictors of poor overall survival in patients with Stage III non-small-cell lung cancer (NSCLC) treated with definitive resection. This retrospective study evaluates the impact of extent and location of mediastinal lymph node (LN) involvement on survival in patients with Stage III NSCLC treated with definitive radiotherapy. Methods and Materials: We analyzed 106 consecutive patients with T1-4 N2-3 Stage III NSCLC treated with definitive radiotherapy at University of Pennsylvania between January 2003 and February 2009. For this analysis, mediastinal LN stations were divided into four mutually exclusive groups: supraclavicular, ipsilateral mediastinum, contralateral mediastinum, and subcarinal. Patients' conditions were then analyzed according to the extent of involvement and location of mediastinal LN stations. Results: The majority (88%) of patients received sequential or concurrent chemotherapy. The median follow-up time for survivors was 32.6 months. By multivariable Cox modeling, chemotherapy use (hazard ratio [HR]: 0.21 [95% confidence interval (CI): 0.07-0.63]) was associated with improved overall survival. Increasing primary tumor [18F]-fluoro-2-deoxy-glucose avidity (HR: 1.11 [CI: 1.06-1.19]), and subcarinal involvement (HR: 2.29 [CI: 1.11-4.73]) were significant negative predictors of overall survival. On univariate analysis, contralateral nodal involvement (HR: 0.70 [CI: 0.33-1.47]), supraclavicular nodal involvement (HR: 0.78 [CI: 0.38-1.67]), multilevel nodal involvement (HR: 0.97 [CI: 0.58-1.61]), and tumor size (HR: 1.04 [CI: 0.94-1.14]) did not predict for overall survival. Patients with subcarinal involvement also had lower rates of 2-year nodal control (51.2% vs. 74.9%, p = 0.047) and 2-year distant control (28.4% vs. 61.2%, p = 0.043). Conclusions: These data suggest that the factors that determine oncologic outcome in Stage III NSCLC

  16. Strategies for Primary Prevention of Coronary Heart Disease Based on Risk Stratification by the ACC/AHA Lipid Guidelines, ATP III Guidelines, Coronary Calcium Scoring, and C-Reactive Protein, and a Global Treat-All Strategy: A Comparative--Effectiveness Modeling Study

    PubMed Central

    Galper, Benjamin Z.; Wang, Y. Claire; Einstein, Andrew J.

    2015-01-01

    Background Several approaches have been proposed for risk-stratification and primary prevention of coronary heart disease (CHD), but their comparative and cost-effectiveness is unknown. Methods We constructed a state-transition microsimulation model to compare multiple approaches to the primary prevention of CHD in a simulated cohort of men aged 45–75 and women 55–75. Risk-stratification strategies included the 2013 American College of Cardiology/American Heart Association (ACC/AHA) guidelines on the treatment of blood cholesterol, the Adult Treatment Panel (ATP) III guidelines, and approaches based on coronary artery calcium (CAC) scoring and C-reactive protein (CRP). Additionally we assessed a treat-all strategy in which all individuals were prescribed either moderate-dose or high-dose statins and all males received low-dose aspirin. Outcome measures included CHD events, costs, medication-related side effects, radiation-attributable cancers, and quality-adjusted-life-years (QALYs) over a 30-year timeframe. Results Treat-all with high-dose statins dominated all other strategies for both men and women, gaining 15.7 million QALYs, preventing 7.3 million myocardial infarctions, and saving over $238 billion, compared to the status quo, far outweighing its associated adverse events including bleeding, hepatitis, myopathy, and new-onset diabetes. ACC/AHA guidelines were more cost-effective than ATP III guidelines for both men and women despite placing 8.7 million more people on statins. For women at low CHD risk, treat-all with high-dose statins was more likely to cause a statin-related adverse event than to prevent a CHD event. Conclusions Despite leading to a greater proportion of the population placed on statin therapy, the ACC/AHA guidelines are more cost-effective than ATP III. Even so, at generic prices, treating all men and women with statins and all men with low-dose aspirin appears to be more cost-effective than all risk-stratification approaches for the

  17. The ATP-binding site of Ca(2+)-ATPase revealed by electron image analysis.

    PubMed Central

    Yonekura, K; Stokes, D L; Sasabe, H; Toyoshima, C

    1997-01-01

    The location of the ATP-binding site of a P-type ion pump, Ca(2+)-ATPase from rabbit sarcoplasmic reticulum, was examined by cryoelectron microscopy. A nonhydrolyzable analog of ATP, beta, gamma-bidentate chromium (III) complex of ATP (CrATP), was used to stabilize the enzyme in the Ca(2+)-occluded state. Tubular crystals were then induced by vanadate in the presence of EGTA, keeping CrATP bound to the enzyme. The three-dimensional structures of the crystals were determined at 14 A resolution by cryoelectron microscopy and helical image analysis. Statistical comparison of the structures with and without CrATP showed clear and significant differences at the groove proposed previously as the ATP-binding pocket. Images FIGURE 3 FIGURE 6 FIGURE 7 PMID:9138598

  18. Optogenetic control of ATP release

    NASA Astrophysics Data System (ADS)

    Lewis, Matthew A.; Joshi, Bipin; Gu, Ling; Feranchak, Andrew; Mohanty, Samarendra K.

    2013-03-01

    Controlled release of ATP can be used for understanding extracellular purinergic signaling. While coarse mechanical forces and hypotonic stimulation have been utilized in the past to initiate ATP release from cells, these methods are neither spatially accurate nor temporally precise. Further, these methods cannot be utilized in a highly effective cell-specific manner. To mitigate the uncertainties regarding cellular-specificity and spatio-temporal release of ATP, we herein demonstrate use of optogenetics for ATP release. ATP release in response to optogenetic stimulation was monitored by Luciferin-Luciferase assay (North American firefly, photinus pyralis) using luminometer as well as mesoscopic bioluminescence imaging. Our result demonstrates repetitive release of ATP subsequent to optogenetic stimulation. It is thus feasible that purinergic signaling can be directly detected via imaging if the stimulus can be confined to single cell or in a spatially-defined group of cells. This study opens up new avenue to interrogate the mechanisms of purinergic signaling.

  19. ATP release through pannexon channels

    PubMed Central

    Dahl, Gerhard

    2015-01-01

    Extracellular adenosine triphosphate (ATP) serves as a signal for diverse physiological functions, including spread of calcium waves between astrocytes, control of vascular oxygen supply and control of ciliary beat in the airways. ATP can be released from cells by various mechanisms. This review focuses on channel-mediated ATP release and its main enabler, Pannexin1 (Panx1). Six subunits of Panx1 form a plasma membrane channel termed ‘pannexon’. Depending on the mode of stimulation, the pannexon has large conductance (500 pS) and unselective permeability to molecules less than 1.5 kD or is a small (50 pS), chloride-selective channel. Most physiological and pathological stimuli induce the large channel conformation, whereas the small conformation so far has only been observed with exclusive voltage activation of the channel. The interaction between pannexons and ATP is intimate. The pannexon is not only the conduit for ATP, permitting ATP efflux from cells down its concentration gradient, but the pannexon is also modulated by ATP. The channel can be activated by ATP through both ionotropic P2X as well as metabotropic P2Y purinergic receptors. In the absence of a control mechanism, this positive feedback loop would lead to cell death owing to the linkage of purinergic receptors with apoptotic processes. A control mechanism preventing excessive activation of the purinergic receptors is provided by ATP binding (with low affinity) to the Panx1 protein and gating the channel shut. PMID:26009770

  20. Structure of ATP-Bound Human ATP:Cobalamin Adenosyltransferase

    SciTech Connect

    Schubert,H.; Hill, C.

    2006-01-01

    Mutations in the gene encoding human ATP:cobalamin adenosyltransferase (hATR) can result in the metabolic disorder known as methylmalonic aciduria (MMA). This enzyme catalyzes the final step in the conversion of cyanocobalamin (vitamin B{sub 12}) to the essential human cofactor adenosylcobalamin. Here we present the 2.5 {angstrom} crystal structure of ATP bound to hATR refined to an R{sub free} value of 25.2%. The enzyme forms a tightly associated trimer, where the monomer comprises a five-helix bundle and the active sites lie on the subunit interfaces. Only two of the three active sites within the trimer contain the bound ATP substrate, thereby providing examples of apo- and substrate-bound-active sites within the same crystal structure. Comparison of the empty and occupied sites indicates that twenty residues at the enzyme's N-terminus become ordered upon binding of ATP to form a novel ATP-binding site and an extended cleft that likely binds cobalamin. The structure explains the role of 20 invariant residues; six are involved in ATP binding, including Arg190, which hydrogen bonds to ATP atoms on both sides of the scissile bond. Ten of the hydrogen bonds are required for structural stability, and four are in positions to interact with cobalamin. The structure also reveals how the point mutations that cause MMA are deficient in these functions.

  1. Synergistic augmentation of ATP-induced interleukin-6 production by arsenite in HaCaT cells.

    PubMed

    Sumi, Daigo; Asao, Masashi; Okada, Hideta; Yogi, Kuniko; Miyataka, Hideki; Himeno, Seiichiro

    2016-06-01

    Chronic arsenic exposure causes cutaneous diseases such as hyperkeratosis and skin cancer. However, little information has been available regarding the molecular mechanisms underlying these symptoms. Because extracellular ATP and interleukin-6 (IL-6) are involved in pathological aspects of cutaneous diseases, we examined whether sodium arsenite (As(III)) affects ATP-induced IL-6 production in human epidermal keratinocyte HaCaT cells. The results showed that the addition of As(III) into the medium of HaCaT cells dose dependently increased the production of IL-6 induced by extracellular ATP, although As(III) alone had no effect on IL-6 production. To elucidate the mechanism of the synergistic effect of As(III) on IL-6 production by extracellular ATP, we next examined the phosphorylation of p38, ERK and epidermal growth factor receptor (EGFR), since we found that these signaling molecules were stimulated by exposure to extracellular ATP. The results indicated that ATP-induced phosphorylation of p38, ERK and EGFR was synergistically enhanced by co-exposure to As(III). To clarify the mechanisms underlying the enhanced phosphorylation of p38, ERK and EGFR by As(III), we explored two possible mechanisms: the inhibition of extracellular ATP degradation and the inhibition of protein tyrosine phosphatases (PTPs) activity by As(III). The degradation of extracellular ATP was not changed by As(III), whereas the activity of PTPs was significantly inhibited by As(III). Our results suggest that As(III) augments ATP-induced IL-6 production in HaCaT cells through enhanced phosphorylation of the EGFR and p38/ERK pathways, which is associated with the inhibition of PTPs activity. PMID:26104857

  2. Androgen suppression adjuvant to definitive radiotherapy in prostate carcinoma-long-term results of phase III RTOG 85-31

    SciTech Connect

    Pilepich, Miljenko V. . E-mail: mpilepich@mednet.ucla.edu; Winter, Kathryn; Lawton, Colleen A.; Krisch, Robert E.; Wolkov, Harvey B.; Movsas, Benjamin; Hug, Eugen B.; Asbell, Sucha O.; Grignon, David

    2005-04-01

    Purpose: Radiation Therapy Oncology Group protocol 85-31 was designed to evaluate the effectiveness of adjuvant androgen suppression, using goserelin, in unfavorable prognosis carcinoma of the prostate treated with definitive radiotherapy (RT). Methods and Materials: Eligible patients were those with palpable primary tumor extending beyond the prostate (clinical Stage T3) or those with regional lymphatic involvement. Patients who had undergone prostatectomy were eligible if penetration through the prostatic capsule to the margin of resection and/or seminal vesicle involvement was documented histologically. Stratification was based on histologic differentiation, nodal status, acid phosphatase status, and prior prostatectomy. The patients were randomized to either RT and adjuvant goserelin (Arm I) or RT alone followed by observation and application of goserelin at relapse (Arm II). In Arm I, the drug was to be started during the last week of RT and was to be continued indefinitely or until signs of progression. Results: Between 1987 and 1992, when the study was closed, 977 patients were entered: 488 to Arm I and 489 to Arm II. As of July 2003, the median follow-up for all patients was 7.6 years and for living patients was 11 years. At 10 years, the absolute survival rate was significantly greater for the adjuvant arm than for the control arm: 49% vs. 39%, respectively (p = 0.002). The 10-year local failure rate for the adjuvant arm was 23% vs. 38% for the control arm (p <0.0001). The corresponding 10-year rates for the incidence of distant metastases and disease-specific mortality was 24% vs. 39% (p <0.001) and 16% vs. 22% (p = 0.0052), respectively, both in favor of the adjuvant arm. Conclusion: In a population of patients with unfavorable prognosis carcinoma of the prostate, androgen suppression applied as an adjuvant after definitive RT was associated not only with a reduction in disease progression but in a statistically significant improvement in absolute

  3. Evaluation of ATP measurements to detect microbial ingress by wastewater and surface water in drinking water.

    PubMed

    Vang, Óluva K; Corfitzen, Charlotte B; Smith, Christian; Albrechtsen, Hans-Jørgen

    2014-11-01

    Fast and reliable methods are required for monitoring of microbial drinking water quality in order to protect public health. Adenosine triphosphate (ATP) was investigated as a potential real-time parameter for detecting microbial ingress in drinking water contaminated with wastewater or surface water. To investigate the ability of the ATP assay in detecting different contamination types, the contaminant was diluted with non-chlorinated drinking water. Wastewater, diluted at 10(4) in drinking water, was detected with the ATP assay, as well as 10(2) to 10(3) times diluted surface water. To improve the performance of the ATP assay in detecting microbial ingress in drinking water, different approaches were investigated, i.e. quantifying microbial ATP or applying reagents of different sensitivities to reduce measurement variations; however, none of these approaches contributed significantly in this respect. Compared to traditional microbiological methods, the ATP assay could detect wastewater and surface water in drinking water to a higher degree than total direct counts (TDCs), while both heterotrophic plate counts (HPC 22 °C and HPC 37 °C) and Colilert-18 (Escherichia coli and coliforms) were more sensitive than the ATP measurements, though with much longer response times. Continuous sampling combined with ATP measurements displays definite monitoring potential for microbial drinking water quality, since microbial ingress in drinking water can be detected in real-time with ATP measurements. The ability of the ATP assay to detect microbial ingress is influenced by both the ATP load from the contaminant itself and the ATP concentration in the specific drinking water. Consequently, a low ATP concentration of the specific drinking water facilitates a better detection of a potential contamination of the water supply with the ATP assay. PMID:25086698

  4. Empirical Derivation to Improve the Definition of the Metabolic Syndrome in the Evaluation of Cardiovascular Disease Risk

    PubMed Central

    Wildman, Rachel P.; McGinn, Aileen P.; Kim, Mimi; Muntner, Paul; Wang, Dan; Cohen, Hillel W.; Ogorodnikova, Alexandra D.; Reynolds, Kristi; Fonseca, Vivian

    2011-01-01

    OBJECTIVE To examine whether a quantitatively derived metabolic syndrome definition predicts incident cardiovascular disease (CVD) events better than do existing definitions. RESEARCH DESIGN AND METHODS Data were pooled from the Atherosclerosis Risk in Communities, Cardiovascular Health, and Framingham Offspring studies (n = 20,581). Incident coronary heart disease and stroke events were ascertained over 9 years. RESULTS The sensitivity for incident CVD events was higher and the specificity lower for the empirically derived versus the Adult Treatment Panel (ATP) III, International Diabetes Federation (IDF), or Harmonized metabolic syndrome definitions (sensitivity/specificity 0.65/0.53 vs. 0.53/0.63, 0.51/0.66, and 0.64/0.56, respectively), resulting in no overall improvement in discrimination. Multivariable-adjusted hazard ratios for incident CVD events were similar across definitions and were 1.7 (95% CI 1.6–1.9) for ATP III, 1.8 (1.6–2.0) for IDF, 1.9 (1.7–2.0) for Harmonized, and 1.7 (1.6–1.9) for the empirically derived definition. CONCLUSIONS Empirical derivation of the metabolic syndrome definition did not improve CVD discrimination or risk prediction. PMID:21285391

  5. Cervical anterior transpedicular screw fixation (ATPS)--Part II. Accuracy of manual insertion and pull-out strength of ATPS.

    PubMed

    Koller, Heiko; Acosta, Frank; Tauber, Mark; Fox, Michael; Martin, Hudelmaier; Forstner, Rosmarie; Augat, Peter; Penzkofer, Rainer; Pirich, Christian; Kässmann, H; Resch, Herbert; Hitzl, Wolfgang

    2008-04-01

    Reconstruction after multilevel decompression of the cervical spine, especially in the weakened osteoporotic, neoplastic or infectious spine often requires circumferential stabilization and fusion. To avoid the additional posterior surgery in these cases while increasing rigidity of anterior-only screw-plate constructs, the authors introduce the concept of anterior transpedicular screw (ATPS) fixation. We demonstrated its morphological feasibility as well as its indications in a previous study in Part I of our project. Consequently, the objectives of the current study were to assess the ex vivo accuracy of placing ATPS into the cervical vertebra as well as the biomechanical performance of ATPS in comparison to traditional vertebral body screws (VBS) in terms of pull-out strength (POS). Twenty-three ATPS were inserted alternately to two screws into the pedicles and vertebral bodies, respectively, of six cadaveric specimens from C3-T1. For insertion of ATPS, a manual fluoroscopically assisted technique was used. Pre- and post insertional CT-scans were used to assess accuracy of ATPS insertion in the axial and sagittal planes. A newly designed grading system and accuracy score were used to delineate accuracy of ATPS insertion. Following insertion of screws, 23 ATPS and 22 VBS were subjected to pull-out testing (POT). The bone mineral density (BMD) of each specimen was assessed prior to POT. Statistical analysis showed that the incidence of correctly placed screws and non-critical pedicles breaches in axial plane was 78.3%, and 95.7% in sagittal plane. Hence, according to our definition of "critical" pedicle breach that exposes neurovascular structures at risk, 21.7% (n = 5) of all ATPS inserted showed a critical pedicle breach in axial plane. Notably, no critical pedicle perforation occurred at the C6 to T1 levels. Pull-out testing of ATPS and VBS revealed that pull-out resistance of ATPS was 2.5-fold that of VBS. Mean POS of 23 ATPS with a mean BMD of 0.566 g/cm(2

  6. Stages III and IV Squamous Cell Carcinoma of the Mouth: Three-Year Experience with Superselective Intraarterial Chemotherapy Using Cisplatin Prior to Definitive Treatment

    SciTech Connect

    Hirai, Toshinori; Korogi, Yukunori; Hamatake, Satoshi; Nishimura, Ryuichi; Baba, Yuji; Takahashi, Mutsumasa; Uji, Yasuyoshi; Taen, Akira

    1999-05-15

    Purpose: This study was designed to assess the 3-year experience with superselective intraarterial chemotherapy prior to definitive treatment for stages III and IV squamous cell carcinomas of the mouth. Methods: Twenty-two patients prospectively received superselective intraarterial chemotherapy using relatively low-dose cisplatin via a transfemoral approach. The locations of the tumors were the tongue (n= 12), gingiva (n= 5), buccal mucosa (n= 2), hard palate (n= 1), floor of the mouth (n= 1), and lip (n= 1). After intraarterial chemotherapy, 21 patients underwent surgery (n= 14), radiation therapy (n= 6), or both (n= 1). The survival rate of 25 patients who underwent surgery with/without radiation therapy until 1992 at Kumamoto University Hospital was also evaluated as a historical control. The survival curve was calculated with the Kaplan-Meier method, and the statistical difference between survival curves was determined with the generalized Wilcoxon test. Results: The overall response rate was 95% [complete response (tumor completely resolved), 24%; partial response (tumor reduction {>=}50%), 71%]. Fifty-two intraarterial infusions were performed without any catheter-related complications. Mild and transient local toxicity such as edema or mucositis of the infused area was relatively common. One patient died of renal failure from cisplatin. After a median follow-up of 20 months (range 2-41 months), the estimated 3-year survival rate for patients who underwent intraarterial chemotherapy plus surgery was 91%. The survival of the patients who underwent intraarterial chemotherapy plus surgery tended to be longer than that of the historical control. Conclusions: Early tumor reduction without delay of subsequent treatments can be obtained by intraarterial chemotherapy while minimizing complications and possibly improving survival. Further investigations of long-term survival with larger series need to be performed.

  7. Phosphate exchange and ATP synthesis by DMSO-pretreated purified bovine mitochondrial ATP synthase.

    PubMed Central

    Beharry, S; Bragg, P D

    2001-01-01

    Purified soluble bovine mitochondrial F(1)F(o)-ATP synthase contained 2 mol of ATP, 2 mol of ADP and 6 mol of P(i)/mol. Incubation of this enzyme with 1 mM [(32)P]P(i) caused the exchange of 2 mol of P(i)/mol of F(1)F(o)-ATP synthase. The labelled phosphates were not displaced by ATP. Transfer of F(1)F(o)-ATP synthase to a buffer containing 30% (v/v) DMSO and 1 mM [(32)P]P(i) resulted in the loss of bound nucleotides with the retention of 1 mol of ATP/mol of F(1)F(o)-ATP synthase. Six molecules of [(32)P]P(i) were incorporated by exchange with the existing bound phosphate. Removal of the DMSO by passage of the enzyme through a centrifuged column of Sephadex G-50 resulted in the exchange of one molecule of bound [(32)P]P(i) into the bound ATP. Azide did not prevent this [(32)P]P(i)<-->ATP exchange reaction. The bound labelled ATP could be displaced from the enzyme by exogenous ATP. Addition of ADP to the DMSO-pretreated F(1)F(o)-ATP synthase in the original DMSO-free buffer resulted in the formation of an additional molecule of bound ATP. It was concluded that following pretreatment with and subsequent removal of DMSO the F(1)F(o)-ATP synthase contained one molecule of ATP at a catalytic site which was competent to carry out a phosphate-ATP exchange reaction using enzyme-bound inorganic radiolabelled phosphate. In the presence of ADP an additional molecule of labelled ATP was formed from enzyme-bound P(i) at a second catalytic site. The bound phosphate-ATP exchange reaction is not readily accommodated by current mechanisms for the ATP synthase. PMID:11139383

  8. Circadian regulation of ATP release in astrocytes.

    PubMed

    Marpegan, Luciano; Swanstrom, Adrienne E; Chung, Kevin; Simon, Tatiana; Haydon, Philip G; Khan, Sanjoy K; Liu, Andrew C; Herzog, Erik D; Beaulé, Christian

    2011-06-01

    Circadian clocks sustain daily oscillations in gene expression, physiology, and behavior, relying on transcription-translation feedback loops of clock genes for rhythm generation. Cultured astrocytes display daily oscillations of extracellular ATP, suggesting that ATP release is a circadian output. We hypothesized that the circadian clock modulates ATP release via mechanisms that regulate acute ATP release from glia. To test the molecular basis for circadian ATP release, we developed methods to measure in real-time ATP release and Bmal1::dLuc circadian reporter expression in cortical astrocyte cultures from mice of different genotypes. Daily rhythms of gene expression required functional Clock and Bmal1, both Per1 and Per2, and both Cry1 and Cry2 genes. Similarly, high-level, circadian ATP release also required a functional clock mechanism. Whereas blocking IP(3) signaling significantly disrupted ATP rhythms with no effect on Bmal1::dLuc cycling, blocking vesicular release did not alter circadian ATP release or gene expression. We conclude that astrocytes depend on circadian clock genes and IP(3) signaling to express daily rhythms in ATP release. PMID:21653839

  9. Circadian regulation of ATP release in astrocytes

    PubMed Central

    Marpegan, Luciano; Swanstrom, Adrienne E.; Chung, Kevin; Simon, Tatiana; Haydon, Philip G.; Khan, Sanjoy K.; Liu, Andrew C.; Herzog, Erik D.; Beaulé, Christian

    2011-01-01

    Circadian clocks sustain daily oscillations in gene expression, physiology and behavior, relying on transcription-translation feedback loops of clock genes for rhythm generation. Cultured astrocytes display daily oscillations of extracellular ATP, suggesting that ATP release is a circadian output. We hypothesized that the circadian clock modulates ATP release via mechanisms that regulate acute ATP release from glia. To test the molecular basis for circadian ATP release, we developed methods to measure in real-time ATP release and Bmal1::dLuc circadian reporter expression in cortical astrocyte cultures from mice of different genotypes. Daily rhythms of gene expression required functional Clock and Bmal1, both Per1 and Per2, and both Cry1 and Cry2 genes. Similarly, high level, circadian ATP release also required a functional clock mechanism. Whereas blocking IP3 signaling significantly disrupted ATP rhythms with no effect on Bmal1::dLuc cycling, blocking vesicular release did not alter circadian ATP release or gene expression. We conclude that astrocytes depend on circadian clock genes and IP3 signaling to express daily rhythms in ATP release. PMID:21653839

  10. On the ATP binding site of the ε subunit from bacterial F-type ATP synthases.

    PubMed

    Krah, Alexander; Takada, Shoji

    2016-04-01

    F-type ATP synthases are reversible machinery that not only synthesize adenosine triphosphate (ATP) using an electrochemical gradient across the membrane, but also can hydrolyze ATP to pump ions under certain conditions. To prevent wasteful ATP hydrolysis, subunit ε in bacterial ATP synthases changes its conformation from the non-inhibitory down- to the inhibitory up-state at a low cellular ATP concentration. Recently, a crystal structure of the ε subunit in complex with ATP was solved in a non-biologically relevant dimeric form. Here, to derive the functional ATP binding site motif, we carried out molecular dynamics simulations and free energy calculations. Our results suggest that the ATP binding site markedly differs from the experimental resolved one; we observe a reorientation of several residues, which bind to ATP in the crystal structure. In addition we find that an Mg(2+) ion is coordinated by ATP, replacing interactions of the second chain in the crystal structure. Thus we demonstrate more generally the influence of crystallization effects on ligand binding sites and their respective binding modes. Furthermore, we propose a role for two highly conserved residues to control the ATP binding/unbinding event, which have not been considered before. Additionally our results provide the basis for the rational development of new biosensors based on subunit ε, as shown previously for novel sensors measuring the ATP concentration in cells. PMID:26780667

  11. Preservation of ATP in Hypersaline Environments

    PubMed Central

    Tuovila, Bruce J.; Dobbs, Fred C.; LaRock, Paul A.; Siegel, B. Z.

    1987-01-01

    High concentrations of particulate ATP were found in the anoxic brines of the Orca Basin and East Flower Garden, Gulf of Mexico. Other measurements indicative of growth and respiration suggested that the microbial community in the brines was inactive, but somehow the ATP associated with the cells persisted. Conceivably, when cells growing just above the interface sank into the brine, the increased osmotic stress could elicit an osmoregulatory response resulting in increased ATP. It was also possible that hydrolytic enzymes were inactivated, resulting in the preservation of ATP. Experiments in which a culture of marine bacteria was suspended in menstrua of different salinities comparable to those found across the Orca Basin interface revealed that as salinity increased, ATP increased three- to sixfold. Within 24 h the ATP fell to its initial level and remained at that concentration for 3 days, at which time the experiment was terminated. In contrast, the control suspensions, at a salinity of 28% (grams per liter) had 1/10th of the initial ATP concentration when the experiment was ended. Cells were also exposed to killing UV irradiation, enabling us to demonstrate with absolute certainty that cellular ATP could be preserved. At the end of the experiment, the viable component of the population was reduced by orders of magnitude by UV irradiation, but the ATP levels of the cells suspended in brine did not decrease. In certain environments it appears that the conventional analytical tools of the microbial ecologist must be interpreted with caution. PMID:16347491

  12. Preservation of ATP in hypersaline environments.

    PubMed

    Tuovila, B J; Dobbs, F C; Larock, P A; Siegel, B Z

    1987-12-01

    High concentrations of particulate ATP were found in the anoxic brines of the Orca Basin and East Flower Garden, Gulf of Mexico. Other measurements indicative of growth and respiration suggested that the microbial community in the brines was inactive, but somehow the ATP associated with the cells persisted. Conceivably, when cells growing just above the interface sank into the brine, the increased osmotic stress could elicit an osmoregulatory response resulting in increased ATP. It was also possible that hydrolytic enzymes were inactivated, resulting in the preservation of ATP. Experiments in which a culture of marine bacteria was suspended in menstrua of different salinities comparable to those found across the Orca Basin interface revealed that as salinity increased, ATP increased three- to sixfold. Within 24 h the ATP fell to its initial level and remained at that concentration for 3 days, at which time the experiment was terminated. In contrast, the control suspensions, at a salinity of 28% (grams per liter) had 1/10th of the initial ATP concentration when the experiment was ended. Cells were also exposed to killing UV irradiation, enabling us to demonstrate with absolute certainty that cellular ATP could be preserved. At the end of the experiment, the viable component of the population was reduced by orders of magnitude by UV irradiation, but the ATP levels of the cells suspended in brine did not decrease. In certain environments it appears that the conventional analytical tools of the microbial ecologist must be interpreted with caution. PMID:16347491

  13. Cervical anterior transpedicular screw fixation (ATPS)—Part II. Accuracy of manual insertion and pull-out strength of ATPS

    PubMed Central

    Acosta, Frank; Tauber, Mark; Fox, Michael; Martin, Hudelmaier; Forstner, Rosmarie; Augat, Peter; Penzkofer, Rainer; Pirich, Christian; Kässmann, H.; Resch, Herbert; Hitzl, Wolfgang

    2008-01-01

    Reconstruction after multilevel decompression of the cervical spine, especially in the weakened osteoporotic, neoplastic or infectious spine often requires circumferential stabilization and fusion. To avoid the additional posterior surgery in these cases while increasing rigidity of anterior-only screw-plate constructs, the authors introduce the concept of anterior transpedicular screw (ATPS) fixation. We demonstrated its morphological feasibility as well as its indications in a previous study in Part I of our project. Consequently, the objectives of the current study were to assess the ex vivo accuracy of placing ATPS into the cervical vertebra as well as the biomechanical performance of ATPS in comparison to traditional vertebral body screws (VBS) in terms of pull-out strength (POS). Twenty-three ATPS were inserted alternately to two screws into the pedicles and vertebral bodies, respectively, of six cadaveric specimens from C3–T1. For insertion of ATPS, a manual fluoroscopically assisted technique was used. Pre- and post insertional CT-scans were used to assess accuracy of ATPS insertion in the axial and sagittal planes. A newly designed grading system and accuracy score were used to delineate accuracy of ATPS insertion. Following insertion of screws, 23 ATPS and 22 VBS were subjected to pull-out testing (POT). The bone mineral density (BMD) of each specimen was assessed prior to POT. Statistical analysis showed that the incidence of correctly placed screws and non-critical pedicles breaches in axial plane was 78.3%, and 95.7% in sagittal plane. Hence, according to our definition of “critical” pedicle breach that exposes neurovascular structures at risk, 21.7% (n = 5) of all ATPS inserted showed a critical pedicle breach in axial plane. Notably, no critical pedicle perforation occurred at the C6 to T1 levels. Pull-out testing of ATPS and VBS revealed that pull-out resistance of ATPS was 2.5-fold that of VBS. Mean POS of 23 ATPS with a mean BMD of 0.566

  14. A Review of the Definition and Measurement of Poverty: Volume I, Summary Review Paper; Volume II, Annotated Bibliography. The Measure of Poverty, Technical Paper III.

    ERIC Educational Resources Information Center

    Oster, Sharon; And Others

    This study reviews the existing literature on a series of issues associated with the defintion and measurement of poverty, and it consists of a summary report covering this research (Volume I), and an annotated bibliography (Volume II). Eleven specific issues were identified and reviewed in this study: (1) the historical definitions of poverty,…

  15. ATP-triggered anticancer drug delivery

    NASA Astrophysics Data System (ADS)

    Mo, Ran; Jiang, Tianyue; Disanto, Rocco; Tai, Wanyi; Gu, Zhen

    2014-03-01

    Stimuli-triggered drug delivery systems have been increasingly used to promote physiological specificity and on-demand therapeutic efficacy of anticancer drugs. Here we utilize adenosine-5'-triphosphate (ATP) as a trigger for the controlled release of anticancer drugs. We demonstrate that polymeric nanocarriers functionalized with an ATP-binding aptamer-incorporated DNA motif can selectively release the intercalating doxorubicin via a conformational switch when in an ATP-rich environment. The half-maximal inhibitory concentration of ATP-responsive nanovehicles is 0.24 μM in MDA-MB-231 cells, a 3.6-fold increase in the cytotoxicity compared with that of non-ATP-responsive nanovehicles. Equipped with an outer shell crosslinked by hyaluronic acid, a specific tumour-targeting ligand, the ATP-responsive nanocarriers present an improvement in the chemotherapeutic inhibition of tumour growth using xenograft MDA-MB-231 tumour-bearing mice. This ATP-triggered drug release system provides a more sophisticated drug delivery system, which can differentiate ATP levels to facilitate the selective release of drugs.

  16. Thermodynamics of proton transport coupled ATP synthesis.

    PubMed

    Turina, Paola; Petersen, Jan; Gräber, Peter

    2016-06-01

    The thermodynamic H(+)/ATP ratio of the H(+)-ATP synthase from chloroplasts was measured in proteoliposomes after energization of the membrane by an acid base transition (Turina et al. 2003 [13], 418-422). The method is discussed, and all published data obtained with this system are combined and analyzed as a single dataset. This meta-analysis led to the following results. 1) At equilibrium, the transmembrane ΔpH is energetically equivalent to the transmembrane electric potential difference. 2) The standard free energy for ATP synthesis (reference reaction) is ΔG°ref=33.8±1.3kJ/mol. 3) The thermodynamic H(+)/ATP ratio, as obtained from the shift of the ATP synthesis equilibrium induced by changing the transmembrane ΔpH (varying either pHin or pHout) is 4.0±0.1. The structural H(+)/ATP ratio, calculated from the ratio of proton binding sites on the c-subunit-ring in F0 to the catalytic nucleotide binding sites on the β-subunits in F1, is c/β=14/3=4.7. We infer that the energy of 0.7 protons per ATP that flow through the enzyme, but do not contribute to shifting the ATP/(ADP·Pi) ratio, is used for additional processes within the enzyme, such as activation, and/or energy dissipation, due e.g. to internal uncoupling. The ratio between the thermodynamic and the structural H(+)/ATP values is 0.85, and we conclude that this value represents the efficiency of the chemiosmotic energy conversion within the chloroplast H(+)-ATP synthase. PMID:26940516

  17. The ADP/ATP Carrier and Its Relationship to Oxidative Phosphorylation in Ancestral Protist Trypanosoma brucei

    PubMed Central

    Gnipová, Anna; Šubrtová, Karolína; Panicucci, Brian; Horváth, Anton; Lukeš, Julius

    2015-01-01

    The highly conserved ADP/ATP carrier (AAC) is a key energetic link between the mitochondrial (mt) and cytosolic compartments of all aerobic eukaryotic cells, as it exchanges the ATP generated inside the organelle for the cytosolic ADP. Trypanosoma brucei, a parasitic protist of medical and veterinary importance, possesses a single functional AAC protein (TbAAC) that is related to the human and yeast ADP/ATP carriers. However, unlike previous studies performed with these model organisms, this study showed that TbAAC is most likely not a stable component of either the respiratory supercomplex III+IV or the ATP synthasome but rather functions as a physically separate entity in this highly diverged eukaryote. Therefore, TbAAC RNA interference (RNAi) ablation in the insect stage of T. brucei does not impair the activity or arrangement of the respiratory chain complexes. Nevertheless, RNAi silencing of TbAAC caused a severe growth defect that coincides with a significant reduction of mt ATP synthesis by both substrate and oxidative phosphorylation. Furthermore, TbAAC downregulation resulted in a decreased level of cytosolic ATP, a higher mt membrane potential, an elevated amount of reactive oxygen species, and a reduced consumption of oxygen in the mitochondria. Interestingly, while TbAAC has previously been demonstrated to serve as the sole ADP/ATP carrier for ADP influx into the mitochondria, our data suggest that a second carrier for ATP influx may be present and active in the T. brucei mitochondrion. Overall, this study provides more insight into the delicate balance of the functional relationship between TbAAC and the oxidative phosphorylation (OXPHOS) pathway in an early diverged eukaryote. PMID:25616281

  18. CrATP as a new inhibitor of ecto-ATPases of trypanosomatids.

    PubMed

    Moreira, O C; Rios, P F; Esteves, F F; Meyer-Fernandes, J R; Barrabin, H

    2009-01-01

    Trypanosomatid protozoa include heteroxenic species some of them pathogenic for men, animals and plants. Parasite membrane contains ecto-enzymes whose active sites face the external medium rather than the cytoplasm. Herpetomonas sp. displayed a Mg2+-dependent ecto-ATPase activity, a Mg-independent ecto-ADPase and an ecto-phosphatase activity. Both, the ecto-ADPase and phosphatase activities were insensitive to CrATP (chromium(III) adenosine 5'-triphosphate complex). Ecto-ATPase activity was reversibly inhibited. At 2 mm ATP the apparent Ki was 4 x 7+/-1 x 0 microm but a fraction of about 40-50% was insensitive to CrATP. Remarkably, at low substrate concentration (0 x 2 mm) more than 90% of the ecto-ATPase was inhibited with Ki=0 x 33+/-0 x 10 microm. These parameter dependences are interpreted as the presence of 2 ecto-ATPases activities, one of them with high ATP apparent affinity and sensitivity to CrATP. DIDS (4,4 diisothiocyanatostilbene 2,2' disulfonic acid), suramin and ADP were also effective as inhibitors. Only ADP presented no additive inhibition with CrATP. The pattern of partial inhibition by CrATP was also observed for the ecto-ATPase activities of Leishmania amazonensis, Trypanosoma cruzi and Trypanosoma rangeli. CrATP emerges as a new inhibitor of ecto-ATPases and as a tool for a better understanding of properties and role of ecto-ATPases in the biology of parasites. PMID:19126268

  19. Epidermal Growth Factor Receptor Mutation Is Associated With Longer Local Control After Definitive Chemoradiotherapy in Patients With Stage III Nonsquamous Non–Small-Cell Lung Cancer

    SciTech Connect

    Yagishita, Shigehiro; Horinouchi, Hidehito; Katsui Taniyama, Tomoko; Nakamichi, Shinji; Kitazono, Satoru; Mizugaki, Hidenori; Kanda, Shintaro; Fujiwara, Yutaka; Nokihara, Hiroshi; Yamamoto, Noboru; Sumi, Minako; Shiraishi, Kouya; Kohno, Takashi; Furuta, Koh; Tsuta, Koji; Tamura, Tomohide

    2015-01-01

    Purpose: To determine the frequency and clinical significance of epidermal growth factor receptor (EGFR) mutations in patients with potentially curable stage III non–small-cell lung cancer (NSCLC) who are eligible for definitive chemoradiotherapy (CRT). Patients and Methods: Between January 2001 and December 2010, we analyzed the EGFR mutational status in consecutive NSCLC patients who were treated by CRT. The response rate, relapse-free survival, 2-year relapse-free rate, initial relapse sites, and overall survival of the patients were investigated. Results: A total of 528 patients received CRT at our hospital during the study period. Of these, 274 were diagnosed as having nonsquamous NSCLC. Sufficient specimens for mutational analyses could be obtained from 198 of these patients. The proportion of patients with EGFR activating mutations was 17%. In addition to the well-known characteristics of patients carrying EGFR mutations (female, adenocarcinoma, and never/light smoker), the proportion of cases with smaller primary lesions (T1/2) was found to be higher in patients with EGFR mutations than in those with wild-type EGFR. Patients with EGFR mutations showed similar response rate, relapse-free survival, and 2-year relapse-free rates as compared to patients with wild-type EGFR. Local relapses as the site of initial relapse occurred significantly less frequently in patients with EGFR mutation (4% vs 21%; P=.045). Patients with EGFR mutations showed longer local control (adjusted hazard ratio 0.49; P=.043). After disease progression, a majority of the patients with EGFR mutations received EGFR tyrosine kinase inhibitors (62%), and these patients showed longer postprogression survival than those with wild-type EGFR. Conclusions: Our study is the first to show radiosensitive biology of EGFR-mutated tumors in definitive CRT with curative intent. This finding could serve as a credible baseline estimate of EGFR-mutated population in stage III nonsquamous NSCLC.

  20. ATP responses in human C nociceptors.

    PubMed

    Hilliges, Marita; Weidner, Christian; Schmelz, Martin; Schmidt, Roland; Ørstavik, Kristin; Torebjörk, Erik; Handwerker, Hermann

    2002-07-01

    Microelectrode recordings of impulse activity in nociceptive C fibres were performed in cutaneous fascicles of the peroneal nerve at the knee level in healthy human subjects. Mechano-heat responsive C units (CMH), mechano-insensitive but heat-responsive (CH) as well as mechano-insensitive and heat-insensitive C units (CM(i)H(i)) were identified. A subgroup of the mechano-insensitive units was readily activated by histamine. We studied the responsiveness of these nociceptor classes to injection of 20 microl 5 mM adenosintriphosphate (ATP) using saline injections as control. Because of mechanical distension during injection, which typically activates mechano-responsive C fibres, interest was focused on responsiveness to ATP after withdrawal of the injection needle. Post-injection responses were observed in 17/27 (63%) mechano-responsive units and in 14/22 (64%) mechano-insensitive units. Excitation by ATP occurred in 9/11 CH units and in 5/11 CM(i)H(i) units. ATP responsive units were found both within the histamine-responsive and the histamine-insensitive group of mechano-insensitive fibres. ATP responses appeared with a delay of 0-180 s after completion of injection; responses were most pronounced during the first 1-3 min of activation, and irregular ongoing activity was observed for up to 10 or even 20 min. ATP responses were dose-dependent, concentrations lower than 5 mM gave weaker responses. No heat or mechanical sensitisation was observed in any of the major fibre classes. In conclusion, we have shown that ATP injections at high concentrations activate C-nociceptors in healthy human skin, without preference for mechano-responsive or mechano-insensitive units. ATP did not sensitise human C fibres for mechanical or heat stimuli. We discuss how various mechanisms might contribute to the observed responses to ATP. PMID:12098617

  1. Metal-Dependent Regulation of ATP7A and ATP7B in Fibroblast Cultures

    PubMed Central

    Lenartowicz, Malgorzata; Moos, Torben; Ogórek, Mateusz; Jensen, Thomas G.; Møller, Lisbeth B.

    2016-01-01

    Deficiency of one of the copper transporters ATP7A and ATP7B leads to the rare X-linked disorder Menkes Disease (MD) or the rare autosomal disorder Wilson disease (WD), respectively. In order to investigate whether the ATP7A and the ATP7B genes may be transcriptionally regulated, we measured the expression level of the two genes at various concentrations of iron, copper, and insulin. Treating fibroblasts from controls or from individuals with MD or WD for 3 and 10 days with iron chelators revealed that iron deficiency led to increased transcript levels of both ATP7A and ATP7B. Copper deficiency obtained by treatment with the copper chelator led to a downregulation of ATP7A in the control fibroblasts, but surprisingly not in the WD fibroblasts. In contrast, the addition of copper led to an increased expression of ATP7A, but a decreased expression of ATP7B. Thus, whereas similar regulation patterns for the two genes were observed in response to iron deficiency, different responses were observed after changes in the access to copper. Mosaic fibroblast cultures from female carriers of MD treated with copper or copper chelator for 6–8 weeks led to clonal selection. Cells that express the normal ATP7A allele had a selective growth advantage at high copper concentrations, whereas more surprisingly, cells that express the mutant ATP7A allele had a selective growth advantage at low copper concentrations. Thus, although the transcription of ATP7A is regulated by copper, clonal growth selection in mosaic cell cultures is affected by the level of copper. Female carriers of MD are rarely affected probably due to a skewed inactivation of the X-chromosome bearing the ATP7A mutation. PMID:27587995

  2. Metal-Dependent Regulation of ATP7A and ATP7B in Fibroblast Cultures.

    PubMed

    Lenartowicz, Malgorzata; Moos, Torben; Ogórek, Mateusz; Jensen, Thomas G; Møller, Lisbeth B

    2016-01-01

    Deficiency of one of the copper transporters ATP7A and ATP7B leads to the rare X-linked disorder Menkes Disease (MD) or the rare autosomal disorder Wilson disease (WD), respectively. In order to investigate whether the ATP7A and the ATP7B genes may be transcriptionally regulated, we measured the expression level of the two genes at various concentrations of iron, copper, and insulin. Treating fibroblasts from controls or from individuals with MD or WD for 3 and 10 days with iron chelators revealed that iron deficiency led to increased transcript levels of both ATP7A and ATP7B. Copper deficiency obtained by treatment with the copper chelator led to a downregulation of ATP7A in the control fibroblasts, but surprisingly not in the WD fibroblasts. In contrast, the addition of copper led to an increased expression of ATP7A, but a decreased expression of ATP7B. Thus, whereas similar regulation patterns for the two genes were observed in response to iron deficiency, different responses were observed after changes in the access to copper. Mosaic fibroblast cultures from female carriers of MD treated with copper or copper chelator for 6-8 weeks led to clonal selection. Cells that express the normal ATP7A allele had a selective growth advantage at high copper concentrations, whereas more surprisingly, cells that express the mutant ATP7A allele had a selective growth advantage at low copper concentrations. Thus, although the transcription of ATP7A is regulated by copper, clonal growth selection in mosaic cell cultures is affected by the level of copper. Female carriers of MD are rarely affected probably due to a skewed inactivation of the X-chromosome bearing the ATP7A mutation. PMID:27587995

  3. Modulation of L-type Ca2+ current by extracellular ATP in ferret isolated right ventricular myocytes.

    PubMed Central

    Qu, Y; Campbell, D L; Strauss, H C

    1993-01-01

    1. The effects of extracellular adenosine triphosphate (ATP) on the basal L-type Ca2+ current (ICa) were investigated in ferret isolated right ventricular myocytes using the gigaohm seal voltage clamp in the whole-cell and cell-attached configurations. 2. Micromolar levels of extracellular ATP reversibly inhibited ICa in a concentration-dependent manner, without any significant changes in the voltage dependence of either the peak ICa I-V relationship or steady-state activation curve. 3. In contrast, micromolar levels of extracellular ATP did significantly alter the inactivation characteristics of ICa. Ten micromolar ATP: (i) increased the degree of steady-state inactivation of ICa; (ii) altered the time constants of ICa inactivation at 0 mV; and (iii) decreased the time constant of ICa recovery from inactivation at -70 mV. 4. The inhibitory effect of ATP on ICa was not blocked by atropine, a muscarinic cholinergic receptor antagonist, or CPDPX (8-cyclopentyl-3,4-dipropylxanthine), an A1 adenosine receptor antagonist. In contrast, the inhibitory effect of 10 microM ATP could be nearly completely antagonized by 100 microM suramin, a purinergic P2 receptor antagonist. 5. The potency order of ATP analogues in inhibiting ICa was 2-methyl-thio-ATP > ATP > alpha,beta-methylene-ATP, indicating involvement of a P2Y-type ATP receptor. 6. Pretreatment of cells with pertussis toxin (PTX) did not prevent the ATP-induced decrease in ICa. However, (i) ATP produced an irreversible decrease of ICa in the presence of intracellular GTP gamma S, and (ii) the inhibitory effect was significantly attenuated in the presence of intracellular GDP beta S, indicating the involvement of a PTX-insensitive G protein in the P2Y receptor-coupling process. 7. Neither (i) replacing extracellular Ca2+ with 1 mM Ba2+, nor (ii) intracellular perfusion of 10 mM BAPTA for at least 30 min attenuated the inhibitory effect of ATP on the current through Ca2+ channels, suggesting that the inhibitory effect

  4. Customized ATP towpreg. [Automated Tow Placement

    NASA Technical Reports Server (NTRS)

    Sandusky, Donald A.; Marchello, Joseph M.; Baucom, Robert M.; Johnston, Norman J.

    1992-01-01

    Automated tow placement (ATP) utilizes robotic technology to lay down adjacent polymer-matrix-impregnated carbon fiber tows on a tool surface. Consolidation and cure during ATP requires that void elimination and polymer matrix adhesion be accomplished in the short period of heating and pressure rolling that follows towpreg ribbon placement from the robot head to the tool. This study examined the key towpreg ribbon properties and dimensions which play a significant role in ATP. Analysis of the heat transfer process window indicates that adequate heating can be achieved at lay down rates as high as 1 m/sec. While heat transfer did not appear to be the limiting factor, resin flow and fiber movement into tow lap gaps could be. Accordingly, consideration was given to towpreg ribbon having uniform yet non-rectangular cross sections. Dimensional integrity of the towpreg ribbon combined with customized ribbon architecture offer great promise for processing advances in ATP of high performance composites.

  5. The Role of ATP in Sleep Regulation

    PubMed Central

    Chikahisa, Sachiko; Séi, Hiroyoshi

    2011-01-01

    One of the functions of sleep is to maintain energy balance in the brain. There are a variety of hypotheses related to how metabolic pathways interact with sleep/wake regulation. A major finding that demonstrates an interaction between sleep and metabolic homeostasis is the involvement of adenosine in sleep homeostasis. An accumulation of adenosine is supplied from ATP, which can act as an energy currency in the cell. Extracellularly, ATP can act as an activity-dependent signaling molecule, especially in regard to communication between neurons and glia, including astrocytes. Furthermore, the intracellular AMP/ATP ratio controls the activity of AMP-activated protein kinase, which is a potent energy regulator and is recently reported to play a role in the regulation of sleep homeostasis. Brain ATP may support multiple functions in the regulation of the sleep/wake cycle and sleep homeostasis. PMID:22207863

  6. Binding of ATP to the progesterone receptor.

    PubMed Central

    Moudgil, V K; Toft, D O

    1975-01-01

    The possible interaction of progesterone--receptor complexes with nucleotides was tested by affinity chromatography. The cytosol progesterone receptor from hen oviduct was partially purified by ammonium sulfate precipitation before use. When progesterone was bound to the receptor, the resulting complex could be selectively adsorbed onto columns of ATP-Sepharose. This interaction was reversible and of an ionic nature since it could be disrupted by high-salt conditions. A competitive binding assay was used to test the specificity of receptor binding to several other nucleotides, including ADP, AMP, and cAMP. A clear specificity for binding ATP was evident from these studies. When ATP was added to receptor preparations, the nucleotide did not affect the sedimentation properties or hormone binding characteristics of the receptor. Although the function of ATP remains unknown, these studies indicate a role of this nucleotide in some aspect of hormone receptor activity. PMID:165493

  7. Cleanup MAC and MBA code ATP

    SciTech Connect

    Russell, V.K.

    1994-10-17

    The K Basins Materials Accounting (MAC) and Material Balance (MBA) database system had some minor code cleanup performed to its code. This ATP describes how the code was to be tested to verify its correctness.

  8. An RNA motif that binds ATP

    NASA Technical Reports Server (NTRS)

    Sassanfar, M.; Szostak, J. W.

    1993-01-01

    RNAs that contain specific high-affinity binding sites for small molecule ligands immobilized on a solid support are present at a frequency of roughly one in 10(10)-10(11) in pools of random sequence RNA molecules. Here we describe a new in vitro selection procedure designed to ensure the isolation of RNAs that bind the ligand of interest in solution as well as on a solid support. We have used this method to isolate a remarkably small RNA motif that binds ATP, a substrate in numerous biological reactions and the universal biological high-energy intermediate. The selected ATP-binding RNAs contain a consensus sequence, embedded in a common secondary structure. The binding properties of ATP analogues and modified RNAs show that the binding interaction is characterized by a large number of close contacts between the ATP and RNA, and by a change in the conformation of the RNA.

  9. ACRIM III

    Atmospheric Science Data Center

    2015-12-30

    ACRIM III Data and Information Active Cavity Radiometer Irradiance ... the ACRIMSAT spacecraft on December 20, 1999. ACRIM III data are reprocessed every 90 days to utilize instrument recalibration.   ... ACRIM III Instrument Team Page ACRIM II Data Sets SCAR-B Block:  SCAR-B Products ...

  10. ATP Synthesis in the Extremely Halophilic Bacteria

    NASA Technical Reports Server (NTRS)

    Hochstein, Lawrence I.; Morrison, David (Technical Monitor)

    1994-01-01

    The proton-translocating ATPases are multimeric enzymes that carry out a multitude of essential functions. Their origin and evolution represent a seminal event in the early evolution of life. Amino acid sequences of the two largest subunits from archaeal ATPases (A-ATPases), vacuolar ATPases (V-ATPases), and FOF1-ATP syntheses (FATPases) suggest these ATPases evolved from an ancestral vacuolar-like ATP syntheses. A necessary consequence of this notion is that the A-ATPases are ATP syntheses. With the possible exception of the A-ATPase from Halobacterium salinarium. no A-ATPase has been demonstrated to synthesize ATP. The evidence for this case is dubious since ATP synthesis occurs only when conditions are distinctively unphysiological. We demonstrated that ATP synthesis in H.saccharovorum is inconsistent with the operation of an A-type ATPase. In order to determine if this phenomenon was unique to H. saccharovorum, ATP synthesis was examined in various extremely halophilic bacteria with the goal of ascertaining if it resembled what occurred in a. saccharovorum, or was consistent with the operation of an A-type ATPase. A-, V-, and F-type ATPases respond singularly to certain inhibitors. Therefore, the effect of these inhibitors on ATP synthesis in several extreme halophiles was determined. Inhibitors that either blocked or collapsed proton-gradients inhibited the steady state synthesis of ATP thus verifying that synthesis took place at the expense of a proton gradient. Azide, an inhibitor of F-ATPases inhibited ATP synthesis. Since the arginine-dependent synthesis of ATP, which occurs by way of substrate-level phosphorylation, was unaffected by azide, it was unlikely that azide acted as an "uncoupler." N -ethylmaleimide and nitrate, which inhibit V- and A-ATPases, either did not inhibit ATP synthesis or resulted in higher steady-state levels of ATP. These results suggest there are two types of proton-motive ATPases in the extreme halophiles (and presumably in other

  11. Rates of various reactions catalyzed by ATP synthase as related to the mechanism of ATP synthesis

    SciTech Connect

    Berkich, D.A.; Williams, G.D.; Masiakos, P.T.; Smith, M.B.; Boyer, P.D.; LaNoue, K.F. )

    1991-01-05

    The forward and reverse rates of the overall reaction catalyzed by the ATP synthase in intact rat heart mitochondria, as measured with 32P, were compared with the rates of two partial steps, as measured with 18O. Such rates have been measured previously, but their relationship to one another has not been determined, nor have the partial reactions been measured in intact mitochondria. The partial steps measured were the rate of medium Pi formation from bound ATP (in state 4 this also equals the rate of medium Pi into bound ATP) and the rate of formation of bound ATP from bound Pi within the catalytic site. The rates of both partial reactions can be measured by 31P NMR analysis of the 18O distribution in Pi and ATP released from the enzyme during incubation of intact mitochondria with highly labeled (18O)Pi. Data were obtained in state 3 and 4 conditions with variation in substrate concentrations, temperature, and mitochondrial membrane electrical potential gradient (delta psi m). Although neither binding nor release of ATP is necessary for phosphate/H2O exchange, in state 4 the rate of incorporation of at least one water oxygen atom into phosphate is approximately twice the rate of the overall reaction rate under a variety of conditions. This can be explained if the release of Pi or ATP at one catalytic site does not occur, unless ATP or Pi is bound at another catalytic site. Such coupling provides strong support for the previously proposed alternating site mechanism. In state 3 slow reversal of ATP synthesis occurs within the mitochondrial matrix and can be detected as incorporation of water oxygen atoms into medium Pi even though medium (32P)ATP does not give rise to 32Pi in state 3. These data can be explained by lack of translocation of ATP from the medium to the mitochondrial matrix.

  12. Compartmentalized ATP synthesis in skeletal muscle triads.

    PubMed

    Han, J W; Thieleczek, R; Varsányi, M; Heilmeyer, L M

    1992-01-21

    Isolated skeletal muscle triads contain a compartmentalized glycolytic reaction sequence catalyzed by aldolase, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase. These enzymes express activity in the structure-associated state leading to synthesis of ATP in the triadic junction upon supply of glyceraldehyde 3-phosphate or fructose 1,6-bisphosphate. ATP formation occurs transiently and appears to be kinetically compartmentalized, i.e., the synthesized ATP is not in equilibrium with the bulk ATP. The apparent rate constants of the aldolase and the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase reaction are significantly increased when fructose 1,6-bisphosphate instead of glyceraldehyde 3-phosphate is employed as substrate. The observations suggest that fructose 1,6-bisphosphate is especially effectively channelled into the junctional gap. The amplitude of the ATP transient is decreasing with increasing free [Ca2+] in the range of 1 nM to 30 microM. In the presence of fluoride, the ATP transient is significantly enhanced and its declining phase is substantially retarded. This observation suggests utilization of endogenously synthesized ATP in part by structure associated protein kinases and phosphatases which is confirmed by the detection of phosphorylated triadic proteins after gel electrophoresis and autoradiography. Endogenous protein kinases phosphorylate proteins of apparent Mr 450,000, 180,000, 160,000, 145,000, 135,000, 90,000, 54,000, 51,000, and 20,000, respectively. Some of these phosphorylated polypeptides are in the Mr range of known phosphoproteins involved in excitation-contraction coupling of skeletal muscle, which might give a first hint at the functional importance of the sequential glycolytic reactions compartmentalized in triads. PMID:1731894

  13. Blockade of Extracellular ATP Effect by Oxidized ATP Effectively Mitigated Induced Mouse Experimental Autoimmune Uveitis (EAU)

    PubMed Central

    Zhao, Ronglan; Liang, Dongchun; Sun, Deming

    2016-01-01

    Various pathological conditions are accompanied by ATP release from the intracellular to the extracellular compartment. Extracellular ATP (eATP) functions as a signaling molecule by activating purinergic P2 purine receptors. The key P2 receptor involved in inflammation was identified as P2X7R. Recent studies have shown that P2X7R signaling is required to trigger the Th1/Th17 immune response, and oxidized ATP (oxATP) effectively blocks P2X7R activation. In this study we investigated the effect of oxATP on mouse experimental autoimmune uveitis (EAU). Our results demonstrated that induced EAU in B6 mice was almost completely abolished by the administration of small doses of oxATP, and the Th17 response, but not the Th1 response, was significantly weakened in the treated mice. Mechanistic studies showed that the therapeutic effects involve the functional change of a number of immune cells, including dendritic cells (DCs), T cells, and regulatory T cells. OxATP not only directly inhibits the T cell response; it also suppresses T cell activation by altering the function of DCs and Foxp3+ T cell. Our results demonstrated that inhibition of P2X7R activation effectively exempts excessive autoimmune inflammation, which may indicate a possible therapeutic use in the treatment of autoimmune diseases. PMID:27196432

  14. Synthetic peptides target ATP translocase of ‘Candidatus Liberibacter asiaticus’ to block ATP uptake

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As an obligate intracellular pathogen, ‘Candidatus Liberibacter asiaticus’ (Las) may act as an “energy parasite” by importing ATP from its host’s cells. We previously demonstrated that the Las translocase NttA (gb|ACX71867.1) is functional in Escherichia coli and enables the direct import of ATP/ADP...

  15. Magnetic field affects enzymatic ATP synthesis.

    PubMed

    Buchachenko, Anatoly L; Kuznetsov, Dmitry A

    2008-10-01

    The rate of ATP synthesis by creatine kinase extracted from V. xanthia venom was shown to depend on the magnetic field. The yield of ATP produced by enzymes with 24Mg2+ and 26Mg2+ ions in catalytic sites increases by 7-8% at 55 mT and then decreases at 80 mT. For enzyme with 25Mg2+ ion in a catalytic site, the ATP yield increases by 50% and 70% in the fields 55 and 80 mT, respectively. In the Earth field the rate of ATP synthesis by enzyme, in which Mg2+ ion has magnetic nucleus 25Mg, is 2.5 times higher than that by enzymes, in which Mg2+ ion has nonmagnetic, spinless nuclei 24Mg or 26Mg. Both magnetic field effect and magnetic isotope effect demonstrate that the ATP synthesis is an ion-radical process, affected by Zeeman interaction and hyperfine coupling in the intermediate ion-radical pair. PMID:18774801

  16. ATP: The crucial component of secretory vesicles.

    PubMed

    Estévez-Herrera, Judith; Domínguez, Natalia; Pardo, Marta R; González-Santana, Ayoze; Westhead, Edward W; Borges, Ricardo; Machado, José David

    2016-07-12

    The colligative properties of ATP and catecholamines demonstrated in vitro are thought to be responsible for the extraordinary accumulation of solutes inside chromaffin cell secretory vesicles, although this has yet to be demonstrated in living cells. Because functional cells cannot be deprived of ATP, we have knocked down the expression of the vesicular nucleotide carrier, the VNUT, to show that a reduction in vesicular ATP is accompanied by a drastic fall in the quantal release of catecholamines. This phenomenon is particularly evident in newly synthesized vesicles, which we show are the first to be released. Surprisingly, we find that inhibiting VNUT expression also reduces the frequency of exocytosis, whereas the overexpression of VNUT drastically increases the quantal size of exocytotic events. To our knowledge, our data provide the first demonstration that ATP, in addition to serving as an energy source and purinergic transmitter, is an essential element in the concentration of catecholamines in secretory vesicles. In this way, cells can use ATP to accumulate neurotransmitters and other secreted substances at high concentrations, supporting quantal transmission. PMID:27342860

  17. ATP synthesis by F-type ATP synthase is obligatorily dependent on the transmembrane voltage.

    PubMed

    Kaim, G; Dimroth, P

    1999-08-01

    ATP synthase is the universal enzyme that manufactures cellular ATP using the energy stored in a transmembrane ion gradient. This energy gradient has two components: the concentration difference (DeltapH or DeltapNa(+)) and the electrical potential difference DeltaPsi, which are thermodynamically equivalent. However, they are not kinetically equivalent, as the mitochondrial and bacterial ATP synthases require a transmembrane potential, DeltaPsi, but the chloroplast enzyme has appeared to operate on DeltapH alone. Here we show that, contrary to the accepted wisdom, the 'acid bath' procedure used to study the chloroplast enzyme develops not only a DeltapH but also a membrane potential, and that this potential is essential for ATP synthesis. Thus, for the chloroplast and other ATP synthases, the membrane potential is the fundamental driving force for their normal operation. We discuss the biochemical reasons for this phenomenon and a model that is consistent with these new experimental facts. PMID:10428951

  18. Pyruvate kinase and the "high ATP syndrome".

    PubMed Central

    Staal, G E; Jansen, G; Roos, D

    1984-01-01

    The erythrocytes of a patient with the so-called "high ATP syndrome" were characterized by a high ATP content and low 2,3-diphosphoglycerate level. The pyruvate kinase activity was specifically increased (about twice the normal level). After separation of the erythrocytes according to age by discontinuous Percoll density centrifugation, the pyruvate kinase activity was found to be increased in all Percoll fractions. Pyruvate kinase of the patient's cells was characterized by a decreased K0.5 for the substrate phosphoenolpyruvate and no inhibition by ATP. The Michaelis constant (Km) value for ADP, the nucleotide specificity, the thermostability, pH optimum, and immunological specific activity were normal. It is concluded that the high pyruvate kinase activity is due to a shift in the R(elaxed) in equilibrium T(ight) equilibrium to the R(elaxed) form. PMID:6736249

  19. Pathway of processive ATP hydrolysis by kinesin

    PubMed Central

    Gilbert, Susan P.; Webb, Martin R.; Brune, Martin; Johnson, Kenneth A.

    2007-01-01

    Direct measurement of the kinetics of kinesin dissociation from microtubules, the release of phosphate and ADP from kinesin, and rebinding of kinesin to the microtubule have defined the mechanism for the kinesin ATPase cycle. The processivity of ATP hydrolysis is ten molecules per site at low salt concentration but is reduced to one ATP per site at higher salt concentration. Kinesin dissociates from the microtubule after ATP hydrolysis. This step is rate-limiting. The subsequent rebinding of kinesin · ADP to the microtubule is fast, so kinesin spends only a small fraction of its duty cycle in the dissociated state. These results provide an explanation for the motility differences between skeletal myosin and kinesin. PMID:7854446

  20. Inhibition of ATP Synthase by Chlorinated Adenosine Analogue

    PubMed Central

    Chen, Lisa S.; Nowak, Billie J.; Ayres, Mary L.; Krett, Nancy L.; Rosen, Steven T.; Zhang, Shuxing; Gandhi, Varsha

    2009-01-01

    8-Chloroadenosine (8-Cl-Ado) is a ribonucleoside analogue that is currently in clinical trial for chronic lymphocytic leukemia. Based on the decline in cellular ATP pool following 8-Cl-Ado treatment, we hypothesized that 8-Cl-ADP and 8-Cl-ATP may interfere with ATP synthase, a key enzyme in ATP production. Mitochondrial ATP synthase is composed of two major parts; FO intermembrane base and F1 domain, containing α and β subunits. Crystal structures of both α and β subunits that bind to the substrate, ADP, are known in tight binding (αdpβdp) and loose binding (αtpβtp) states. Molecular docking demonstrated that 8-Cl-ADP/8-Cl-ATP occupied similar binding modes as ADP/ATP in the tight and loose binding sites of ATP synthase, respectively, suggesting that the chlorinated nucleotide metabolites may be functional substrates and inhibitors of the enzyme. The computational predictions were consistent with our whole cell biochemical results. Oligomycin, an established pharmacological inhibitor of ATP synthase, decreased both ATP and 8-Cl-ATP formation from exogenous substrates, however, did not affect pyrimidine nucleoside analogue triphosphate accumulation. Synthesis of ATP from ADP was inhibited in cells loaded with 8-Cl-ATP. These biochemical studies are in consent with the computational modeling; in the αtpβtp state 8-Cl-ATP occupies similar binding as ANP, a non-hydrolyzable ATP mimic that is a known inhibitor. Similarly, in the substrate binding site (αdpβdp) 8-Cl-ATP occupies a similar position as ATP mimic ADP-BeF3 −. Collectively, our current work suggests that 8-Cl-ADP may serve as a substrate and the 8-Cl-ATP may be an inhibitor of ATP synthase. PMID:19477165

  1. SAGE III

    Atmospheric Science Data Center

    2016-06-15

    SAGE III Data and Information The Stratospheric Aerosol and Gas ... on the spacecraft. SAGE III produced L1 and L2 scientific data from 5/07/2002 until 12/31/2005. The flight of the second instrument is as ... Guide Documents:  Project Guide Data Products User's Guide  (PDF) Relevant Documents:  ...

  2. The structural basis of ATP as an allosteric modulator.

    PubMed

    Lu, Shaoyong; Huang, Wenkang; Wang, Qi; Shen, Qiancheng; Li, Shuai; Nussinov, Ruth; Zhang, Jian

    2014-09-01

    Adenosine-5'-triphosphate (ATP) is generally regarded as a substrate for energy currency and protein modification. Recent findings uncovered the allosteric function of ATP in cellular signal transduction but little is understood about this critical behavior of ATP. Through extensive analysis of ATP in solution and proteins, we found that the free ATP can exist in the compact and extended conformations in solution, and the two different conformational characteristics may be responsible for ATP to exert distinct biological functions: ATP molecules adopt both compact and extended conformations in the allosteric binding sites but conserve extended conformations in the substrate binding sites. Nudged elastic band simulations unveiled the distinct dynamic processes of ATP binding to the corresponding allosteric and substrate binding sites of uridine monophosphate kinase, and suggested that in solution ATP preferentially binds to the substrate binding sites of proteins. When the ATP molecules occupy the allosteric binding sites, the allosteric trigger from ATP to fuel allosteric communication between allosteric and functional sites is stemmed mainly from the triphosphate part of ATP, with a small number from the adenine part of ATP. Taken together, our results provide overall understanding of ATP allosteric functions responsible for regulation in biological systems. PMID:25211773

  3. Distinct Conformation of ATP Molecule in Solution and on Protein

    PubMed Central

    Kobayashi, Eri; Yura, Kei; Nagai, Yoshinori

    2013-01-01

    Adenosine triphosphate (ATP) is a versatile molecule used mainly for energy and a phosphate source. The hydrolysis of γ phosphate initiates the reactions and these reactions almost always start when ATP binds to protein. Therefore, there should be a mechanism to prevent spontaneous hydrolysis reaction and a mechanism to lead ATP to a pure energy source or to a phosphate source. To address these questions, we extensively analyzed the effect of protein to ATP conformation based on the sampling of the ATP solution conformations obtained from molecular dynamics simulation and the sampling of ATP structures bound to protein found in a protein structure database. The comparison revealed mainly the following three points; 1) The ribose ring in ATP molecule, which puckers in many ways in solution, tends to assume either C2′ exo or C2′ endo when it binds to protein. 2) The adenine ring in ATP molecule, which takes open-book motion with the two ring structures, has two distinct structures when ATP binds to protein. 3) The glycosyl-bond and the bond between phosphate and the ribose have unique torsion angles, when ATP binds to protein. The combination of torsion angles found in protein-bound forms is under-represented in ATP molecule in water. These findings suggest that ATP-binding protein exerts forces on ATP molecule to assume a conformation that is rarely found in solution, and that this conformation change should be a trigger for the reactions on ATP molecule. PMID:27493535

  4. The Structural Basis of ATP as an Allosteric Modulator

    PubMed Central

    Wang, Qi; Shen, Qiancheng; Li, Shuai; Nussinov, Ruth; Zhang, Jian

    2014-01-01

    Adenosine-5’-triphosphate (ATP) is generally regarded as a substrate for energy currency and protein modification. Recent findings uncovered the allosteric function of ATP in cellular signal transduction but little is understood about this critical behavior of ATP. Through extensive analysis of ATP in solution and proteins, we found that the free ATP can exist in the compact and extended conformations in solution, and the two different conformational characteristics may be responsible for ATP to exert distinct biological functions: ATP molecules adopt both compact and extended conformations in the allosteric binding sites but conserve extended conformations in the substrate binding sites. Nudged elastic band simulations unveiled the distinct dynamic processes of ATP binding to the corresponding allosteric and substrate binding sites of uridine monophosphate kinase, and suggested that in solution ATP preferentially binds to the substrate binding sites of proteins. When the ATP molecules occupy the allosteric binding sites, the allosteric trigger from ATP to fuel allosteric communication between allosteric and functional sites is stemmed mainly from the triphosphate part of ATP, with a small number from the adenine part of ATP. Taken together, our results provide overall understanding of ATP allosteric functions responsible for regulation in biological systems. PMID:25211773

  5. Mechanisms that match ATP supply to demand in cardiac pacemaker cells during high ATP demand

    PubMed Central

    Yaniv, Yael; Spurgeon, Harold A.; Ziman, Bruce D.; Lyashkov, Alexey E.

    2013-01-01

    The spontaneous action potential (AP) firing rate of sinoatrial node cells (SANCs) involves high-throughput signaling via Ca2+-calmodulin activated adenylyl cyclases (AC), cAMP-mediated protein kinase A (PKA), and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent phosphorylation of SR Ca2+ cycling and surface membrane ion channel proteins. When the throughput of this signaling increases, e.g., in response to β-adrenergic receptor activation, the resultant increase in spontaneous AP firing rate increases the demand for ATP. We hypothesized that an increase of ATP production to match the increased ATP demand is achieved via a direct effect of increased mitochondrial Ca2+ (Ca2+m) and an indirect effect via enhanced Ca2+-cAMP/PKA-CaMKII signaling to mitochondria. To increase ATP demand, single isolated rabbit SANCs were superfused by physiological saline at 35 ± 0.5°C with isoproterenol, or by phosphodiesterase or protein phosphatase inhibition. We measured cytosolic and mitochondrial Ca2+ and flavoprotein fluorescence in single SANC, and we measured cAMP, ATP, and O2 consumption in SANC suspensions. Although the increase in spontaneous AP firing rate was accompanied by an increase in O2 consumption, the ATP level and flavoprotein fluorescence remained constant, indicating that ATP production had increased. Both Ca2+m and cAMP increased concurrently with the increase in AP firing rate. When Ca2+m was reduced by Ru360, the increase in spontaneous AP firing rate in response to isoproterenol was reduced by 25%. Thus, both an increase in Ca2+m and an increase in Ca2+ activated cAMP-PKA-CaMKII signaling regulate the increase in ATP supply to meet ATP demand above the basal level. PMID:23604710

  6. Firefly bioluminescent assay of ATP in the presence of ATP extractant by using liposomes.

    PubMed

    Kamidate, Tamio; Yanashita, Kenji; Tani, Hirofumi; Ishida, Akihiko; Notani, Mizuyo

    2006-01-01

    Liposomes containing phosphatidylcholine (PC) and cholesterol (Chol) were applied to the enhancer for firefly bioluminescence (BL) assay for ATP in the presence of cationic surfactants using as an extractant for the release of ATP from living cells. Benzalkonium chloride (BAC) was used as an ATP extractant. However, BAC seriously inhibited the activity of luciferase, thus resulting in the remarkable decrease in the sensitivity of the BL assay for ATP. On the other hand, we found that BAC was associated with liposomes to form cationic liposomes containing BAC. The association rate of BAC with liposomes was faster than that of BAC with luciferase. As a result, the inhibitory effect of BAC on luciferase was eliminated in the presence of liposomes. In addition, cationic liposomes thus formed enhanced BL emission. BL measurement conditions were optimized in terms of liposome charge type, liposome size, and total concentration of PC and Chol. ATP can be sensitively determined without dilution of analytical samples by using liposomes. The detection limit of ATP with and without liposomes was 100 amol and 25 fmol in aqueous ATP standard solutions containing 0.06% BAC, respectively. The method was applied to the determination of ATP in Escherichia coli extracts. The BL intensity was linear from 4 x 10(4) to 1 x 10(7) cells mL(-1) in the absence of liposomes. On the other hand, the BL intensity was linear from 4 x 10(3) to 4 x 10(6) cells mL(-1) in the presence of liposomes. The detection limit of ATP in E. coli extracts was improved by a factor of 10 via use of liposomes. PMID:16383346

  7. Relationship of tightly bound ADP and ATP to control and catalysis by chloroplast ATP synthase

    SciTech Connect

    Zhou, J.; Xue, Z.; Du, Z.; Melese, T.; Boyer, P.D.

    1988-07-12

    Whether the tightly bound ADP that can cause a pronounced inhibition of ATP hydrolysis by the chloroplast ATP synthase and F/sub 1/ ATPase (CF/sub 1/) is bound at catalytic sites or at noncatalytic regulatory sites or both has been uncertain. The authors have used photolabeling by 2-azido-ATP and 2-azido-ADP to ascertain the location, with Mg/sup 2 +/ activation, of tightly bound ADP (a) that inhibits the hydrolysis of ATP by chloroplast ATP synthase, (b) that can result in an inhibited form of CF/sub 1/ that slowly regains activity during ATP hydrolysis, and (c) that arises when low concentrations of ADP markedly inhibit the hydrolysis of GTP by CF/sub 1/. The data show that in all instances the inhibition is associated with ADP binding without inorganic phosphate (P/sub i/) at catalytic sites. After photophosphorylation of ADP or 2-azido-ADP with (/sup 32/P)P/sub i/, similar amounts of the corresponding triphosphates are present on washed thylakoid membranes. Trials with appropriately labeled substrates show that a small portion of the tightly bound 2-azido-ATP gives rise to covalent labeling with an ATP moiety at noncatalytic sites but that most of the bound 2-azido-ATP gives rise to covalent labeling with an ATP moiety at noncatalytic sites but that most of the bound 2-azido-ATP gives rise to covalent labeling by an ADP moiety at a catalytic site. They also report the occurrence of a 1-2-min delay in the onset of the Mg/sup 2 +/-induced inhibition after addition of CF/sub 1/ to solutions containing Mg/sup 2 +/ and ATP, and that this delay is not associated with the filling of noncatalytic sites. A rapid burst of P/sub i/ formation is followed by a much lower, constant steady-state rate. The burst is not observed with GTP as a substrate or with Ca/sup 2 +/ as the activating cation.

  8. Torque generation mechanism of ATP synthase

    NASA Astrophysics Data System (ADS)

    Miller, John; Maric, Sladjana; Scoppa, M.; Cheung, M.

    2010-03-01

    ATP synthase is a rotary motor that produces adenosine triphosphate (ATP), the chemical currency of life. Our proposed electric field driven torque (EFT) model of FoF1-ATP synthase describes how torque, which scales with the number of c-ring proton binding sites, is generated by the proton motive force (pmf) across the mitochondrial inner membrane. When Fo is coupled to F1, the model predicts a critical pmf to drive ATP production. In order to fully understand how the electric field resulting from the pmf drives the c-ring to rotate, it is important to examine the charge distributions in the protonated c-ring and a-subunit containing the proton channels. Our calculations use a self-consistent field approach based on a refinement of reported structural data. The results reveal changes in pKa for key residues on the a-subunit and c-ring, as well as titration curves and protonation state energy diagrams. Health implications will be briefly discussed.

  9. Calcium and ATP control multiple vital functions

    PubMed Central

    Verkhratsky, Alexei

    2016-01-01

    Life on Planet Earth, as we know it, revolves around adenosine triphosphate (ATP) as a universal energy storing molecule. The metabolism of ATP requires a low cytosolic Ca2+ concentration, and hence tethers these two molecules together. The exceedingly low cytosolic Ca2+ concentration (which in all life forms is kept around 50–100 nM) forms the basis for a universal intracellular signalling system in which Ca2+ acts as a second messenger. Maintenance of transmembrane Ca2+ gradients, in turn, requires ATP-dependent Ca2+ transport, thus further emphasizing the inseparable links between these two substances. Ca2+ signalling controls the most fundamental processes in the living organism, from heartbeat and neurotransmission to cell energetics and secretion. The versatility and plasticity of Ca2+ signalling relies on cell specific Ca2+ signalling toolkits, remodelling of which underlies adaptive cellular responses. Alterations of these Ca2+ signalling toolkits lead to aberrant Ca2+ signalling which is fundamental for the pathophysiology of numerous diseases from acute pancreatitis to neurodegeneration. This paper introduces a theme issue on this topic, which arose from a Royal Society Theo Murphy scientific meeting held in March 2016. This article is part of the themed issue ‘Evolution brings Ca2+ and ATP together to control life and death’. PMID:27377728

  10. Effect of ATP on actin filament stiffness.

    PubMed

    Janmey, P A; Hvidt, S; Oster, G F; Lamb, J; Stossel, T P; Hartwig, J H

    1990-09-01

    Actin is an adenine nucleotide-binding protein and an ATPase. The bound adenine nucleotide stabilizes the protein against denaturation and the ATPase activity, although not required for actin polymerization, affects the kinetics of this assembly Here we provide evidence for another effect of adenine nucleotides. We find that actin filaments made from ATP-containing monomers, the ATPase activity of which hydrolyses ATP to ADP following polymerization, are stiff rods, whereas filaments prepared from ADP-monomers are flexible. ATP exchanges with ADP in such filaments and stiffens them. Because both kinds of actin filaments contain mainly ADP, we suggest the alignment of actin monomers in filaments that have bound and hydrolysed ATP traps them conformationally and stores elastic energy. This energy would be available for release by actin-binding proteins that transduce force or sever actin filaments. These data support earlier proposals that actin is not merely a passive cable, but has an active mechanochemical role in cell function. PMID:2168523

  11. Calcium and ATP control multiple vital functions.

    PubMed

    Petersen, Ole H; Verkhratsky, Alexei

    2016-08-01

    Life on Planet Earth, as we know it, revolves around adenosine triphosphate (ATP) as a universal energy storing molecule. The metabolism of ATP requires a low cytosolic Ca(2+) concentration, and hence tethers these two molecules together. The exceedingly low cytosolic Ca(2+) concentration (which in all life forms is kept around 50-100 nM) forms the basis for a universal intracellular signalling system in which Ca(2+) acts as a second messenger. Maintenance of transmembrane Ca(2+) gradients, in turn, requires ATP-dependent Ca(2+) transport, thus further emphasizing the inseparable links between these two substances. Ca(2+) signalling controls the most fundamental processes in the living organism, from heartbeat and neurotransmission to cell energetics and secretion. The versatility and plasticity of Ca(2+) signalling relies on cell specific Ca(2+) signalling toolkits, remodelling of which underlies adaptive cellular responses. Alterations of these Ca(2+) signalling toolkits lead to aberrant Ca(2+) signalling which is fundamental for the pathophysiology of numerous diseases from acute pancreatitis to neurodegeneration. This paper introduces a theme issue on this topic, which arose from a Royal Society Theo Murphy scientific meeting held in March 2016.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'. PMID:27377728

  12. Electric Field Driven Torque in ATP Synthase

    PubMed Central

    Miller, John H.; Rajapakshe, Kimal I.; Infante, Hans L.; Claycomb, James R.

    2013-01-01

    FO-ATP synthase (FO) is a rotary motor that converts potential energy from ions, usually protons, moving from high- to low-potential sides of a membrane into torque and rotary motion. Here we propose a mechanism whereby electric fields emanating from the proton entry and exit channels act on asymmetric charge distributions in the c-ring, due to protonated and deprotonated sites, and drive it to rotate. The model predicts a scaling between time-averaged torque and proton motive force, which can be hindered by mutations that adversely affect the channels. The torque created by the c-ring of FO drives the γ-subunit to rotate within the ATP-producing complex (F1) overcoming, with the aid of thermal fluctuations, an opposing torque that rises and falls with angular position. Using the analogy with thermal Brownian motion of a particle in a tilted washboard potential, we compute ATP production rates vs. proton motive force. The latter shows a minimum, needed to drive ATP production, which scales inversely with the number of proton binding sites on the c-ring. PMID:24040370

  13. A reusable prepositioned ATP reaction chamber

    NASA Technical Reports Server (NTRS)

    Hoffman, D. G.

    1972-01-01

    Luminescence biometer detects presence of life by means of light-emitting chemical reaction of luciferin and luciferase with adenosine triphosphate (ATP) that occurs in all living cells. Amount of light in reaction chamber is measured to determine presence and extent of life.

  14. Mitochondrial membrane potential and ATP production in primary disorders of ATP synthase.

    PubMed

    Vojtísková, Alena; Jesina, Pavel; Kalous, Martin; Kaplanová, Vilma; Houstek, Josef; Tesarová, Markéta; Fornůsková, Daniela; Zeman, Jirí; Dubot, Audrey; Godinot, Catherine

    2004-01-01

    Studies of fibroblasts with primary defects in mitochondrial ATP synthase (ATPase) due to heteroplasmic mtDNA mutations in the ATP6 gene, affecting protonophoric function or synthesis of subunit a, show that at high mutation loads, mitochondrial membrane potential DeltaPsi(m) at state 4 is normal, but ADP-induced discharge of DeltaPsi(m) is impaired and ATP synthesis at state 3-ADP is decreased. Increased DeltaPsi(m) and low ATP synthesis is also found when the ATPase content is diminished by altered biogenesis of the enzyme complex. Irrespective of the different pathogenic mechanisms, elevated DeltaPsi(m) in primary ATPase disorders could increase mitochondrial production of reactive oxygen species and decrease energy provision. PMID:20021115

  15. Conversion to Paradox 4.02 ATP`s for MAC and mass balance programs

    SciTech Connect

    Russell, V.K.

    1994-10-17

    The K Basins Materials Accounting (MAC) and Material Balance (MBA) database system were converted from Paradox 3.5 to Paradox 4.0. The ATP describes how the code was to be tested to verify its corrections.

  16. Reinterpreting the action of ATP analogs on K(ATP) channels.

    PubMed

    Ortiz, David; Gossack, Lindsay; Quast, Ulrich; Bryan, Joseph

    2013-06-28

    Neuroendocrine-type K(ATP) channels, (SUR1/Kir6.2)4, couple the transmembrane flux of K(+), and thus membrane potential, with cellular metabolism in various cell types including insulin-secreting β-cells. Mutant channels with reduced activity are a cause of congenital hyperinsulinism, whereas hyperactive channels are a cause of neonatal diabetes. A current regulatory model proposes that ATP hydrolysis is required to switch SUR1 into post-hydrolytic conformations able to antagonize the inhibitory action of nucleotide binding at the Kir6.2 pore, thus coupling enzymatic and channel activities. Alterations in SUR1 ATPase activity are proposed to contribute to neonatal diabetes and type 2 diabetes risk. The regulatory model is partly based on the reduced ability of ATP analogs such as adenosine 5'-(β,γ-imino)triphosphate (AMP-PNP) and adenosine 5'-O-(thiotriphosphate) (ATPγS) to stimulate channel activity, presumably by reducing hydrolysis. This study uses a substitution at the catalytic glutamate, SUR(1E1507Q), with a significantly increased affinity for ATP, to probe the action of these ATP analogs on conformational switching. ATPγS, a slowly hydrolyzable analog, switches SUR1 conformations, albeit with reduced affinity. Nonhydrolyzable AMP-PNP and adenosine 5'-(β,γ-methylenetriphosphate) (AMP-PCP) alone fail to switch SUR1, but do reverse ATP-induced switching. AMP-PCP displaces 8-azido-[(32)P]ATP from the noncanonical NBD1 of SUR1. This is consistent with structural data on an asymmetric bacterial ABC protein that shows that AMP-PNP binds selectively to the noncanonical NBD to prevent conformational switching. The results imply that MgAMP-PNP and MgAMP-PCP (AMP-PxP) fail to activate K(ATP) channels because they do not support NBD dimerization and conformational switching, rather than by limiting enzymatic activity. PMID:23665564

  17. Enhanced anticancer efficacy by ATP-mediated liposomal drug delivery.

    PubMed

    Mo, Ran; Jiang, Tianyue; Gu, Zhen

    2014-06-01

    A liposome-based co-delivery system composed of a fusogenic liposome encapsulating ATP-responsive elements with chemotherapeutics and a liposome containing ATP was developed for ATP-mediated drug release triggered by liposomal fusion. The fusogenic liposome had a protein-DNA complex core containing an ATP-responsive DNA scaffold with doxorubicin (DOX) and could release DOX through a conformational change from the duplex to the aptamer/ATP complex in the presence of ATP. A cell-penetrating peptide-modified fusogenic liposomal membrane was coated on the core, which had an acid-triggered fusogenic potential with the ATP-loaded liposomes or endosomes/lysosomes. Directly delivering extrinsic liposomal ATP promoted the drug release from the fusogenic liposome in the acidic intracellular compartments upon a pH-sensitive membrane fusion and anticancer efficacy was enhanced both in vitro and in vivo. PMID:24764317

  18. The c-Ring of the F1FO-ATP Synthase: Facts and Perspectives.

    PubMed

    Nesci, Salvatore; Trombetti, Fabiana; Ventrella, Vittoria; Pagliarani, Alessandra

    2016-04-01

    The F1FO-ATP synthase is the only enzyme in nature endowed with bi-functional catalytic mechanism of synthesis and hydrolysis of ATP. The enzyme functions, not only confined to energy transduction, are tied to three intrinsic features of the annular arrangement of c subunits which constitutes the so-called c-ring, the core of the membrane-embedded FO domain: (i) the c-ring constitution is linked to the number of ions (H(+) or Na(+)) channeled across the membrane during the dissipation of the transmembrane electrochemical gradient, which in turn determines the species-specific bioenergetic cost of ATP, the "molecular currency unit" of energy transfer in all living beings; (ii) the c-ring is increasingly involved in the mitochondrial permeability transition, an event linked to cell death and to most mitochondrial dysfunctions; (iii) the c subunit species-specific amino acid sequence and susceptibility to post-translational modifications can address antibacterial drug design according to the model of enzyme inhibitors which target the c subunits. Therefore, the simple c-ring structure not only allows the F1FO-ATP synthase to perform the two opposite tasks of molecular machine of cell life and death, but it also amplifies the enzyme's potential role as a drug target. PMID:26621635

  19. External Dentin Stimulation Induces ATP Release in Human Teeth.

    PubMed

    Liu, X; Wang, C; Fujita, T; Malmstrom, H S; Nedergaard, M; Ren, Y F; Dirksen, R T

    2015-09-01

    ATP is involved in neurosensory processing, including nociceptive transduction. Thus, ATP signaling may participate in dentin hypersensitivity and dental pain. In this study, we investigated whether pannexins, which can form mechanosensitive ATP-permeable channels, are present in human dental pulp. We also assessed the existence and functional activity of ecto-ATPase for extracellular ATP degradation. We further tested if ATP is released from dental pulp upon dentin mechanical or thermal stimulation that induces dentin hypersensitivity and dental pain and if pannexin or pannexin/gap junction channel blockers reduce stimulation-dependent ATP release. Using immunofluorescence staining, we demonstrated immunoreactivity of pannexin 1 and 2 in odontoblasts and their processes extending into the dentin tubules. Using enzymatic histochemistry staining, we also demonstrated functional ecto-ATPase activity within the odontoblast layer, subodontoblast layer, dental pulp nerve bundles, and blood vessels. Using an ATP bioluminescence assay, we found that mechanical or cold stimulation to the exposed dentin induced ATP release in an in vitro human tooth perfusion model. We further demonstrated that blocking pannexin/gap junction channels with probenecid or carbenoxolone significantly reduced external dentin stimulation-induced ATP release. Our results provide evidence for the existence of functional machinery required for ATP release and degradation in human dental pulp and that pannexin channels are involved in external dentin stimulation-induced ATP release. These findings support a plausible role for ATP signaling in dentin hypersensitivity and dental pain. PMID:26130258

  20. Genomic Analysis of ATP Efflux in Saccharomyces cerevisiae

    PubMed Central

    Peters, Theodore W.; Miller, Aaron W.; Tourette, Cendrine; Agren, Hannah; Hubbard, Alan; Hughes, Robert E.

    2015-01-01

    Adenosine triphosphate (ATP) plays an important role as a primary molecule for the transfer of chemical energy to drive biological processes. ATP also functions as an extracellular signaling molecule in a diverse array of eukaryotic taxa in a conserved process known as purinergic signaling. Given the important roles of extracellular ATP in cell signaling, we sought to comprehensively elucidate the pathways and mechanisms governing ATP efflux from eukaryotic cells. Here, we present results of a genomic analysis of ATP efflux from Saccharomyces cerevisiae by measuring extracellular ATP levels in cultures of 4609 deletion mutants. This screen revealed key cellular processes that regulate extracellular ATP levels, including mitochondrial translation and vesicle sorting in the late endosome, indicating that ATP production and transport through vesicles are required for efflux. We also observed evidence for altered ATP efflux in strains deleted for genes involved in amino acid signaling, and mitochondrial retrograde signaling. Based on these results, we propose a model in which the retrograde signaling pathway potentiates amino acid signaling to promote mitochondrial respiration. This study advances our understanding of the mechanism of ATP secretion in eukaryotes and implicates TOR complex 1 (TORC1) and nutrient signaling pathways in the regulation of ATP efflux. These results will facilitate analysis of ATP efflux mechanisms in higher eukaryotes. PMID:26585826

  1. Energy source of flagellar type III secretion.

    PubMed

    Paul, Koushik; Erhardt, Marc; Hirano, Takanori; Blair, David F; Hughes, Kelly T

    2008-01-24

    Bacterial flagella contain a specialized secretion apparatus that functions to deliver the protein subunits that form the filament and other structures to outside the membrane. This apparatus is related to the injectisome used by many gram-negative pathogens and symbionts to transfer effector proteins into host cells; in both systems this export mechanism is termed 'type III' secretion. The flagellar secretion apparatus comprises a membrane-embedded complex of about five proteins, and soluble factors, which include export-dedicated chaperones and an ATPase, FliI, that was thought to provide the energy for export. Here we show that flagellar secretion in Salmonella enterica requires the proton motive force (PMF) and does not require ATP hydrolysis by FliI. The export of several flagellar export substrates was prevented by treatment with the protonophore CCCP, with no accompanying decrease in cellular ATP levels. Weak swarming motility and rare flagella were observed in a mutant deleted for FliI and for the non-flagellar type-III secretion ATPases InvJ and SsaN. These findings show that the flagellar secretion apparatus functions as a proton-driven protein exporter and that ATP hydrolysis is not essential for type III secretion. PMID:18216859

  2. 25 CFR 291.2 - Definitions

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 25 Indians 1 2010-04-01 2010-04-01 false Definitions 291.2 Section 291.2 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR ECONOMIC ENTERPRISES CLASS III GAMING PROCEDURES § 291.2 Definitions (a) All terms have the same meaning as set forth in the definitional section of IGRA, 25...

  3. Space shuttle (ATP configuration) abort staging investigation

    NASA Technical Reports Server (NTRS)

    Rampy, J. M.; Blackwell, K. L.; Allen, E. C., Jr.; Fossler, I.

    1973-01-01

    A wind tunnel test conducted in a 14-inch trisonic wind tunnel to determine the force and moment characteristics of the ATP Orbiter and modified ATP External Tank/SRB combination during abort staging conditions is discussed. Six component aerodynamic force and moment data were recorded for the orbiter and ET/SRB combination. Pitch polars were obtained for an angle of attack range from minus 10 to plus 10 degrees and orbiter incidence angles (orbiter relative to the ET/SRB combination) of 0 and 2 degrees. A limited amount of yaw data were obtained at 0 degree angle of attack and beta range from minus 10 to plus 10 degrees. In addition, orbiter pitch control effectiveness was determined at several grid points. These force and moment data were obtained for Mach numbers of 0.9, 1.2 and 2.0.

  4. Regulation of mitochondrial translation of the ATP8/ATP6 mRNA by Smt1p

    PubMed Central

    Rak, Malgorzata; Su, Chen Hsien; Xu, Jonathan Tong; Azpiroz, Ricardo; Singh, Angela Mohan; Tzagoloff, Alexander

    2016-01-01

    Expression of the mitochondrially encoded ATP6 and ATP8 genes is translationally regulated by F1 ATPase. We report a translational repressor (Smt1p) of the ATP6/8 mRNA that, when mutated, restores translation of the encoded Atp6p and Atp8p subunits of the ATP synthase. Heterozygous smt1 mutants fail to rescue the translation defect, indicating that the mutations are recessive. Smt1p is an intrinsic inner membrane protein, which, based on its sedimentation, has a native size twice that of the monomer. Affinity purification of tagged Smt1p followed by reverse transcription of the associated RNA and PCR amplification of the resultant cDNA with gene-specific primers demonstrated the presence in mitochondria of Smt1p-ATP8/ATP6 and Smt1p-COB mRNA complexes. These results indicate that Smt1p is likely to be involved in translational regulation of both mRNAs. Applying Occam’s principle, we favor a mechanistic model in which translation of the ATP8/ATP6 bicistronic mRNA is coupled to the availability of F1 for subsequent assembly of the Atp6p and Atp8p products into the ATP synthase. The mechanism of this regulatory pathway is proposed to entail a displacement of the repressor from the translationally mute Smt1-ATP8/ATP6 complex by F1, thereby permitting the Atp22p activator to interact with and promote translation of the mRNA. PMID:26823015

  5. H+/ATP ratio during ATP hydrolysis by mitochondria: modification of the chemiosmotic theory.

    PubMed

    Brand, M D; Lehninger, A L

    1977-05-01

    The stoichiometry of H+ ejection by mitochondria during hydrolysis of a small pulse of ATP (the H+/ATP ratio) has been reexamined in the light of our recent observation that the stoichiometry of H+ ejection during mitochondrial electron transport (the H+/site ratio) was previously underestimated. We show that earlier estimates of the H+/ATP ratio in intact mitochondria were based upon an invalid correction for scaler H+ production and describe a modified method for determination of this ratio which utilizes mersalyl or N-ethylmaleimide to prevent complicating transmembrane movements of phosphate and H+. This method gives a value for the H+/ATP ratio of 2.0 without the need for questionable corrections, compared with a value of 3.0 for the H+/site ratio also obtained by pulse methods. A modified version of the chemiosmotic theory is presented, in which 3 H+ are ejected per pair of electrons traversing each energy-conserving site of the respiratory chain. Of these, 2 H+ return to the matrix through the ATPase to form ATP from ADP and phosphate, and 1 H+ returns through the combined action of the phosphate and adenine nucleotide exchange carriers of the inner membrane to allow the energy-requiring influx of Pi and ADP3- and efflux of ATP4-. Thus, up to one-third of the energy input into synthesis of extramitochondrial ATP may be required for transport work. Since other methods suggest that the H+/site significantly exceeds 3.0, an alternative possibility is that 4 h+ are ejected per site, followed by return of 3 H+ through the ATPase and 1 H+ through the operation of the proton-coupled membrane transport systems. PMID:17116

  6. Nordihydroguaiaretic acid depletes ATP and inhibits a swelling-activated, ATP-sensitive taurine channel.

    PubMed

    Ballatori, N; Wang, W

    1997-05-01

    The mechanism by which nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, prevents swelling-activated organic osmolyte efflux was examined in the human hepatoma cell line Hep G2. When swollen in hypotonic medium, Hep G2 cell exhibited a regulatory volume decrease that was associated with the release of intracellular taurine, an amino acid found at a concentrations of 22.0 +/- 2.5 nmol/mg protein (approximately 5 mM) in these cells. Rate coefficients for swelling-activated [3H]taurine uptake and efflux were unaffected when extracellular taurine was increased from 0.1 to 25 mM, indicating that taurine is released via a channel. Taurine efflux was rapidly activated after cell swelling and immediately inactivated when cells were returned to normal size by restoration of isotonicity. Swelling-activated taurine efflux was not altered by replacement of extracellular Na+ with choline+ or K+ but was inhibited when cellular ATP levels were decreased with a variety of chemical agents, consistent with an ATP-regulated channel previously described in other cell types. NDGA inhibited swelling-activated [3H]taurine efflux in Hep G2 cells at concentrations of 50-150 microM; however, these same concentrations of NDGA also lowered cell ATP levels. Likewise, ketoconazole, an inhibitor of cytochrome P-450 monoxygenases, inhibited [3H]taurine efflux only at concentrations at which cell ATP levels were also lowered. In contrast, other inhibitors of cyclooxygenase (indomethacin, 100 microM) or of lipoxygenases (caffeic acid, 100 microM), as well as arachidonic acid itself (100 microM), had no effect on either taurine efflux or cell ATP. The present findings characterize a swelling-activated, ATP-sensitive osmolyte channel in Hep G2 cells and demonstrate that inactivation of the channel by NDGA is related to the ability of this drug to deplete cellular ATP. PMID:9176131

  7. Continuous measurements of ATP secretion in vivo.

    PubMed

    Smith, J B; Burke, S E; Lefer, A M; Freilich, A

    1984-01-01

    Blood was withdrawn continuously from femoral veins of anesthetized rabbits at a rate of 0.07 ml/min. Sodium citrate was pumped into the blood to prevent coagulation, and luciferin-luciferase reagent was added to permit the continuous detection of extracellular ATP. Subsequently, the red blood cells were lysed and the platelet count was recorded continuously. Injection of platelet activating factor or collagen into rabbit ear veins caused an almost immediate but short-lived increase in extracellular ATP with a simultaneous but more prolonged decrease in the platelet count. Although both the endoperoxide analog 9,11-azo-PGH2 and ADP also decreased the platelet count, little extracellular ATP was detected after the azo-PGH2 and none after ADP. These studies demonstrate that those agents that cause platelet secretion from rabbit platelets in vitro also cause secretion in vivo. The method described should be useful in evaluating the capacity of antithrombotic drugs to modify platelet secretion in vivo. PMID:24277184

  8. ATP synthase: a tentative structural model.

    PubMed

    Engelbrecht, S; Junge, W

    1997-09-15

    Adenosine triphosphate (ATP) synthase produces ATP from ADP and inorganic phosphate at the expense of proton- or sodium-motive force across the respective coupling membrane in Archaea, Bacteria and Eucarya. Cation flow through the intrinsic membrane portion of this enzyme (Fo, subunits ab2c9-12) and substrate turnover in the headpiece (F1, subunits alpha3beta3 gammadeltaepsilon) are mechanically coupled by the rotation of subunit gamma in the center of the catalytic hexagon of subunits (alphabeta)3 in F1. ATP synthase is the smallest rotatory engine in nature. With respect to the headpiece alone, it probably operates with three steps. Partial structures of six out of its at least eight different subunits have been published and a 3-dimensional structure is available for the assembly (alphabeta)3gamma. In this article, we review the available structural data and build a tentative topological model of the holoenzyme. The rotor portion is proposed to consist of a wheel of at least nine copies of subunits c, epsilon and a portion of gamma as a spoke, and another portion of gamma as a crankshaft. The stator is made up from a, the transmembrane portion of b2, delta and the catalytic hexagon of (alphabeta)3. As an educated guess, the model may be of heuristic value for ongoing studies on this fascinating electrochemical-to-mechanical-to-chemical transducer. PMID:9323021

  9. Loss of LRPPRC causes ATP synthase deficiency.

    PubMed

    Mourier, Arnaud; Ruzzenente, Benedetta; Brandt, Tobias; Kühlbrandt, Werner; Larsson, Nils-Göran

    2014-05-15

    Defects of the oxidative phosphorylation system, in particular of cytochrome-c oxidase (COX, respiratory chain complex IV), are common causes of Leigh syndrome (LS), which is a rare neurodegenerative disorder with severe progressive neurological symptoms that usually present during infancy or early childhood. The COX-deficient form of LS is commonly caused by mutations in genes encoding COX assembly factors, e.g. SURF1, SCO1, SCO2 or COX10. However, other mutations affecting genes that encode proteins not directly involved in COX assembly can also cause LS. The leucine-rich pentatricopeptide repeat containing protein (LRPPRC) regulates mRNA stability, polyadenylation and coordinates mitochondrial translation. In humans, mutations in Lrpprc cause the French Canadian type of LS. Despite the finding that LRPPRC deficiency affects the stability of most mitochondrial mRNAs, its pathophysiological effect has mainly been attributed to COX deficiency. Surprisingly, we show here that the impaired mitochondrial respiration and reduced ATP production observed in Lrpprc conditional knockout mouse hearts is caused by an ATP synthase deficiency. Furthermore, the appearance of inactive subassembled ATP synthase complexes causes hyperpolarization and increases mitochondrial reactive oxygen species production. Our findings shed important new light on the bioenergetic consequences of the loss of LRPPRC in cardiac mitochondria. PMID:24399447

  10. Adenosine triphosphate (ATP) as a possible indicator of extraterrestrial biology

    NASA Technical Reports Server (NTRS)

    Chappelle, E. W.; Picciolo, G. L.

    1974-01-01

    The ubiquity of adenosine triphosphate (ATP) in terrestrial organisms provides the basis for proposing the assay of this vital metabolic intermediate for detecting extraterrestrial biological activity. If an organic carbon chemistry is present on the planets, the occurrence of ATP is possible either from biosynthetic or purely chemical reactions. However, ATP's relative complexity minimizes the probability of abiogenic synthesis. A sensitive technique for the quantitative detection of ATP was developed using the firefly bioluminescent reaction. The procedure was used successfully for the determination of the ATP content of soil and bacteria. This technique is also being investigated from the standpoint of its application in clinical medicine.

  11. Suppressors of superoxide production from mitochondrial complex III

    PubMed Central

    Orr, Adam L.; Vargas, Leonardo; Turk, Carolina N.; Baaten, Janine E.; Matzen, Jason T.; Dardov, Victoria J.; Attle, Stephen J.; Li, Jing; Quackenbush, Douglas C.; Goncalves, Renata L. S.; Perevoshchikova, Irina V.; Petrassi, H. Michael; Meeusen, Shelly L.; Ainscow, Edward K.; Brand, Martin D.

    2015-01-01

    Mitochondrial electron transport drives ATP synthesis but also generates reactive oxygen species (ROS), which are both cellular signals and damaging oxidants. Superoxide production by respiratory complex III is implicated in diverse signaling events and pathologies but its role remains controversial. Using high-throughput screening we identified compounds that selectively eliminate superoxide production by complex III without altering oxidative phosphorylation; they modulate retrograde signaling including cellular responses to hypoxic and oxidative stress. PMID:26368590

  12. Suppressors of superoxide production from mitochondrial complex III.

    PubMed

    Orr, Adam L; Vargas, Leonardo; Turk, Carolina N; Baaten, Janine E; Matzen, Jason T; Dardov, Victoria J; Attle, Stephen J; Li, Jing; Quackenbush, Douglas C; Goncalves, Renata L S; Perevoshchikova, Irina V; Petrassi, H Michael; Meeusen, Shelly L; Ainscow, Edward K; Brand, Martin D

    2015-11-01

    Mitochondrial electron transport drives ATP synthesis but also generates reactive oxygen species, which are both cellular signals and damaging oxidants. Superoxide production by respiratory complex III is implicated in diverse signaling events and pathologies, but its role remains controversial. Using high-throughput screening, we identified compounds that selectively eliminate superoxide production by complex III without altering oxidative phosphorylation; they modulate retrograde signaling including cellular responses to hypoxic and oxidative stress. PMID:26368590

  13. Muscle contraction: the step-size distance and the impulse-time per ATP.

    PubMed

    Worthington, C R; Elliott, G F

    1996-02-01

    We derive the step-size distance, and the impulse time per ATP split, from a consideration of Hill's energy rate equation coupled with the enthalpy available per ATP split. This definition of step-size distance is model-independent, and is calculated to have a maximum of 17 A at no load and to reduce to zero at isometric tension, since it will depend on the velocity of shortening. We revisit a derivation of Hill's force-velocity equation depending on impulsive forces working against frictional forces and show that this gives a physical meaning to Hill's constants a and b. This is particularly elegant for Hill's constant b, which is directly related to the impulse time; the value of this impulse time is 1/2 ms. The question that muscle contraction may involve overlapping interactions is considered. However, we find that the step-size distance is not dependent on the possibility of overlapping interactions. PMID:8852761

  14. Welding III.

    ERIC Educational Resources Information Center

    Allegheny County Community Coll., Pittsburgh, PA.

    Instructional objectives and performance requirements are outlined in this course guide for Welding III, an advanced course in arc welding offered at the Community College of Allegheny County to provide students with the proficiency necessary for industrial certification. The course objectives, which are outlined first, specify that students will…

  15. LANDVIEW III

    EPA Science Inventory

    LandView III is a desktop mapping system that includes database extracts from the Environmental Protection Agency, the Bureau of the Census, The U.S. Geological Survey, the Nuclear Regulatory Commission, the Department of Transportation, and the Federal Emergency Management Agenc...

  16. From ATP to PTP and back. A dual function for the mitochondrial ATP synthase

    PubMed Central

    Bernardi, Paolo; Di Lisa, Fabio; Fogolari, Federico; Lippe, Giovanna

    2015-01-01

    Mitochondria play a fundamental role in heart physiology, but are also key effectors of dysfunction and death. This dual role assumes a new meaning following recent advances on the nature and regulation of the permeability transition pore, an inner membrane channel whose opening requires matrix Ca2+ and is modulated by many effectors including reactive oxygen species, matrix cyclophilin D, Pi and matrix pH. The recent demonstration that the F-ATP synthase can reversibly undergo a Ca2+-dependent transition to form a channel that mediates the permeability transition opens new perspectives to the field. These findings demand a reassessment of the modifications of F-ATP synthase that take place in the heart under pathological conditions and of their potential role in determining the transition of F-ATP synthase from and energy-conserving into an energy-dissipating device. PMID:25999424

  17. Bioanalytical Applications of Real-Time ATP Imaging Via Bioluminescence

    SciTech Connect

    Jason Alan Gruenhagen

    2003-12-12

    The research discussed within involves the development of novel applications of real-time imaging of adenosine 5'-triphosphate (ATP). ATP was detected via bioluminescence and the firefly luciferase-catalyzed reaction of ATP and luciferin. The use of a microscope and an imaging detector allowed for spatially resolved quantitation of ATP release. Employing this method, applications in both biological and chemical systems were developed. First, the mechanism by which the compound 48/80 induces release of ATP from human umbilical vein endothelial cells (HUVECs) was investigated. Numerous enzyme activators and inhibitors were utilized to probe the second messenger systems involved in release. Compound 48/80 activated a G{sub q}-type protein to initiate ATP release from HUVECs. Ca{sup 2+} imaging along with ATP imaging revealed that activation of phospholipase C and induction of intracellular Ca{sup 2+} signaling were necessary for release of ATP. Furthermore, activation of protein kinase C inhibited the activity of phospholipase C and thus decreased the magnitude of ATP release. This novel release mechanism was compared to the existing theories of extracellular release of ATP. Bioluminescence imaging was also employed to examine the role of ATP in the field of neuroscience. The central nervous system (CNS) was dissected from the freshwater snail Lymnaea stagnalis. Electrophysiological experiments demonstrated that the neurons of the Lymnaea were not damaged by any of the components of the imaging solution. ATP was continuously released by the ganglia of the CNS for over eight hours and varied from ganglion to ganglion and within individual ganglia. Addition of the neurotransmitters K{sup +} and serotonin increased release of ATP in certain regions of the Lymnaea CNS. Finally, the ATP imaging technique was investigated for the study of drug release systems. MCM-41-type mesoporous nanospheres were loaded with ATP and end-capped with mercaptoethanol functionalized Cd

  18. Mycobacterium tuberculosis Universal Stress Protein Rv2623 Regulates Bacillary Growth by ATP-Binding: Requirement for Establishing Chronic Persistent Infection

    PubMed Central

    Bilder, Patrick; Sun, Meihao; Lim, Jihyeon; Bielefeldt-Ohmann, Helle; Basaraba, Randall; So, Melvin; Zhu, Guofeng; Tufariello, JoAnn M.; Izzo, Angelo A.; Orme, Ian M.; Almo, Steve C.; Leyh, Thomas S.; Chan, John

    2009-01-01

    Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosis universal stress protein (USP) homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant fails to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-Å-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i) M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii) Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii) Rv2623 binds ATP; and iv) the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection. PMID:19478878

  19. Mycobacterium tuberculosis Universal Stress Protein Rv2623 Regulates Bacillary Growth by ATP Binding: Requirement for Establishing Chronic Persistent Infection

    SciTech Connect

    Drumm, J.; Mi, K; Bilder, P; Sun, M; Lim, J; Bielefeldt-Ohmann, H; Basaraba, R; So, M; Zhu, G; et. al.

    2009-01-01

    Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosisuniversal stress protein (USP) homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant fails to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-A-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii Rv2623 binds ATP; and iv the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection.

  20. Release of extracellular ATP by bacteria during growth

    PubMed Central

    2013-01-01

    Background Adenosine triphosphate (ATP) is used as an intracellular energy source by all living organisms. It plays a central role in the respiration and metabolism, and is the most important energy supplier in many enzymatic reactions. Its critical role as the energy storage molecule makes it extremely valuable to all cells. Results We report here the detection of extracellular ATP in the cultures of a variety of bacterial species. The levels of the extracellular ATP in bacterial cultures peaked around the end of the log phase and decreased in the stationary phase of growth. Extracellular ATP levels were dependent on the cellular respiration as bacterial mutants lacking cytochrome bo oxidase displayed lower extracellular ATP levels. We have also shown that Escherichia coli (E. coli) and Salmonella actively depleted extracellular ATP and an ATP supplement in culture media enhanced the stationary survival of E. coli and Salmonella. In addition to E. coli and Salmonella the presence of the extracellular ATP was observed in a variety of bacterial species that contain human pathogens such as Acinetobacter, Pseudomonas, Klebsiella and Staphylococcus. Conclusion Our results indicate that extracellular ATP is produced by many bacterial species during growth and extracellular ATP may serve a role in the bacterial physiology. PMID:24364860

  1. A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif

    PubMed Central

    Schulz, Sarah; Iglesias-Cans, Marina; Krah, Alexander; Yildiz, Özkan; Leone, Vanessa; Matthies, Doreen; Cook, Gregory M.

    2013-01-01

    The anaerobic bacterium Fusobacterium nucleatum uses glutamate decarboxylation to generate a transmembrane gradient of Na+. Here, we demonstrate that this ion-motive force is directly coupled to ATP synthesis, via an F1Fo-ATP synthase with a novel Na+ recognition motif, shared by other human pathogens. Molecular modeling and free-energy simulations of the rotary element of the enzyme, the c-ring, indicate Na+ specificity in physiological settings. Consistently, activity measurements showed Na+ stimulation of the enzyme, either membrane-embedded or isolated, and ATP synthesis was sensitive to the Na+ ionophore monensin. Furthermore, Na+ has a protective effect against inhibitors targeting the ion-binding sites, both in the complete ATP synthase and the isolated c-ring. Definitive evidence of Na+ coupling is provided by two identical crystal structures of the c11 ring, solved by X-ray crystallography at 2.2 and 2.6 Å resolution, at pH 5.3 and 8.7, respectively. Na+ ions occupy all binding sites, each coordinated by four amino acids and a water molecule. Intriguingly, two carboxylates instead of one mediate ion binding. Simulations and experiments demonstrate that this motif implies that a proton is concurrently bound to all sites, although Na+ alone drives the rotary mechanism. The structure thus reveals a new mode of ion coupling in ATP synthases and provides a basis for drug-design efforts against this opportunistic pathogen. PMID:23824040

  2. Fluorescent ATP analog mant-ATP reports dynein activity in the isolated Chlamydomonas axoneme

    NASA Astrophysics Data System (ADS)

    Feofilova, Maria; Howard, Jonathon

    Eukaryotic flagella are long rod-like extensions of cells, which play a fundamental role in single cell movement, as well as in fluid transport. Flagella contain a highly evolutionary conserved mechanical structure called the axoneme. The motion of the flagellum is generated by dynein motor proteins located all along the length of the axoneme. How the force production of motors is controlled spatially and temporally is still an open question. Therefore, monitoring dynein activity in the axonemal structure is expected to provide novel insights in regulation of the beat. We use high sensitivity fluorescence microscopy to monitor the binding and hydrolysis kinetics of the fluorescently labeled ATP analogue mant-ATP (2'(3')-O-(N-methylanthraniloyl) adenosine 5'-triphosphate), which is known to support dynein activity. By studying the kinetics of mant-ATP fluorescence, we identified distinct mant-ATP binding sites in the axoneme. The application of this method to axonemes with reduced amounts of dynein, showed evidence that one of the sites is associated with binding to dynein. In the future, we would like to use this method to find the spatial distribution of dynein activity in the axoneme.

  3. Paradox applications integration ATP`s for MAC and mass balance programs

    SciTech Connect

    Russell, V.K.; Mullaney, J.E.

    1994-10-17

    The K Basins Materials Accounting (MAC) and Material Balance (MBA) database system were set up to run under one common applications program. This Acceptance Test Plan (ATP) describes how the code was to be tested to verify its correctness. The scope of the tests is minimal, since both MAC and MBA have already been tested in detail as stand-alone programs.

  4. Comparison of the H+/ATP ratios of the H+-ATP synthases from yeast and from chloroplast

    PubMed Central

    Petersen, Jan; Förster, Kathrin; Turina, Paola; Gräber, Peter

    2012-01-01

    F0F1-ATP synthases use the free energy derived from a transmembrane proton transport to synthesize ATP from ADP and inorganic phosphate. The number of protons translocated per ATP (H+/ATP ratio) is an important parameter for the mechanism of the enzyme and for energy transduction in cells. Current models of rotational catalysis predict that the H+/ATP ratio is identical to the stoichiometric ratio of c-subunits to β-subunits. We measured in parallel the H+/ATP ratios at equilibrium of purified F0F1s from yeast mitochondria (c/β = 3.3) and from spinach chloroplasts (c/β = 4.7). The isolated enzymes were reconstituted into liposomes and, after energization of the proteoliposomes with acid–base transitions, the initial rates of ATP synthesis and hydrolysis were measured as a function of ΔpH. The equilibrium ΔpH was obtained by interpolation, and from its dependency on the stoichiometric ratio, [ATP]/([ADP]·[Pi]), finally the thermodynamic H+/ATP ratios were obtained: 2.9 ± 0.2 for the mitochondrial enzyme and 3.9 ± 0.3 for the chloroplast enzyme. The data show that the thermodynamic H+/ATP ratio depends on the stoichiometry of the c-subunit, although it is not identical to the c/β ratio. PMID:22733773

  5. ATP-independent contractile proteins from plants

    NASA Astrophysics Data System (ADS)

    Knoblauch, Michael; Noll, Gundula A.; Müller, Torsten; Prüfer, Dirk; Schneider-Hüther, Ingrid; Scharner, Dörte; van Bel, Aart J. E.; Peters, Winfried S.

    2003-09-01

    Emerging technologies are creating increasing interest in smart materials that may serve as actuators in micro- and nanodevices. Mechanically active polymers currently studied include a variety of materials. ATP-driven motor proteins, the actuators of living cells, possess promising characteristics, but their dependence on strictly defined chemical environments can be disadvantagous. Natural proteins that deform reversibly by entropic mechanisms might serve as models for artificial contractile polypeptides with useful functionality, but they are rare. Protein bodies from sieve elements of higher plants provide a novel example. sieve elements form microfluidics systems for pressure-driven transport of photo-assimilates throughout the plant. Unique protein bodies in the sieve elements of legumes act as cellular stopcocks, by undergoing a Ca2+-dependent conformational switch in which they plug the sieve element. In living cells, this reaction is probably controlled by Ca2+-transporters in the cell membrane. Here we report the rapid, reversible, anisotropic and ATP-independent contractility in these protein bodies in vitro. Considering the unique biological function of the legume 'crystalloid' protein bodies and their contractile properties, we suggest to give them the distinctive name forisome ('gate-body'; from the Latin foris, the wing of a gate).

  6. Single molecule thermodynamics of ATP synthesis by F1-ATPase

    NASA Astrophysics Data System (ADS)

    Toyabe, Shoichi; Muneyuki, Eiro

    2015-01-01

    FoF1-ATP synthase is a factory for synthesizing ATP in virtually all cells. Its core machinery is the subcomplex F1-motor (F1-ATPase) and performs the reversible mechanochemical coupling. The isolated F1-motor hydrolyzes ATP, which is accompanied by unidirectional rotation of its central γ -shaft. When a strong opposing torque is imposed, the γ -shaft rotates in the opposite direction and drives the F1-motor to synthesize ATP. This mechanical-to-chemical free-energy transduction is the final and central step of the multistep cellular ATP-synthetic pathway. Here, we determined the amount of mechanical work exploited by the F1-motor to synthesize an ATP molecule during forced rotations using a methodology combining a nonequilibrium theory and single molecule measurements of responses to external torque. We found that the internal dissipation of the motor is negligible even during rotations far from a quasistatic process.

  7. Snapshots of the maltose transporter during ATP hydrolysis

    SciTech Connect

    Oldham, Michael L.; Chen, Jue

    2011-12-05

    ATP-binding cassette transporters are powered by ATP, but the mechanism by which these transporters hydrolyze ATP is unclear. In this study, four crystal structures of the full-length wild-type maltose transporter, stabilized by adenosine 5{prime}-({beta},{gamma}-imido)triphosphate or ADP in conjunction with phosphate analogs BeF{sub 3}{sup -}, VO{sub 4}{sup 3-}, or AlF{sub 4}{sup -}, were determined to 2.2- to 2.4-{angstrom} resolution. These structures led to the assignment of two enzymatic states during ATP hydrolysis and demonstrate specific functional roles of highly conserved residues in the nucleotide-binding domain, suggesting that ATP-binding cassette transporters catalyze ATP hydrolysis via a general base mechanism.

  8. ATP transport in saccular cerebral aneurysms at arterial bends.

    PubMed

    Imai, Yohsuke; Sato, Kodai; Ishikawa, Takuji; Comerford, Andrew; David, Tim; Yamaguchi, Takami

    2010-03-01

    ATP acts as an extracellular signaling molecule in purinergic signaling that regulates vascular tone. ATP binds purinergic P2 nucleotide receptors on endothelial cells. Understanding the mass transport of ATP to endothelial cells by blood flow is thus important to predict functional changes in aneurysmal walls. While some clinical observations indicate a difference of wall pathology between ruptured and unruptured aneurysms, no study has focused on the mass transport in aneurysms. We investigated the characteristics of ATP concentration at aneurysmal wall using a numerical model of ATP transport in aneurysms formed at arterial bends. The magnitude of ATP concentration at the aneurysmal wall was significantly smaller than that at the arterial wall. In particular, significantly low concentration was predicted at the proximal side of the aneurysmal sac. A strong correlation was revealed between the inflow flux at the aneurysmal neck and the resultant concentration at the aneurysmal wall. PMID:20012692

  9. Dimer ribbons of ATP synthase shape the inner mitochondrial membrane

    PubMed Central

    Strauss, Mike; Hofhaus, Götz; Schröder, Rasmus R; Kühlbrandt, Werner

    2008-01-01

    ATP synthase converts the electrochemical potential at the inner mitochondrial membrane into chemical energy, producing the ATP that powers the cell. Using electron cryo-tomography we show that the ATP synthase of mammalian mitochondria is arranged in long ∼1-μm rows of dimeric supercomplexes, located at the apex of cristae membranes. The dimer ribbons enforce a strong local curvature on the membrane with a 17-nm outer radius. Calculations of the electrostatic field strength indicate a significant increase in charge density, and thus in the local pH gradient of ∼0.5 units in regions of high membrane curvature. We conclude that the mitochondrial cristae act as proton traps, and that the proton sink of the ATP synthase at the apex of the compartment favours effective ATP synthesis under proton-limited conditions. We propose that the mitochondrial ATP synthase organises itself into dimer ribbons to optimise its own performance. PMID:18323778

  10. ATP7B detoxifies silver in ciliated airway epithelial cells

    SciTech Connect

    Ibricevic, Aida; Brody, Steven L.; Youngs, Wiley J.; Cannon, Carolyn L.

    2010-03-15

    Silver is a centuries-old antibiotic agent currently used to treat infected burns. The sensitivity of a wide range of drug-resistant microorganisms to silver killing suggests that it may be useful for treating refractory lung infections. Toward this goal, we previously developed a methylated caffeine silver acetate compound, SCC1, that exhibits broad-spectrum antimicrobial activity against clinical strains of bacteria in vitro and when nebulized to lungs in mouse infection models. Preclinical testing of high concentrations of SCC1 in primary culture mouse tracheal epithelial cells (mTEC) showed selective ciliated cell death. Ciliated cell death was induced by both silver- and copper-containing compounds but not by the methylated caffeine portion of SCC1. We hypothesized that copper transporting P-type ATPases, ATP7A and ATP7B, play a role in silver detoxification in the airway. In mTEC, ATP7A was expressed in non-ciliated cells, whereas ATP7B was expressed only in ciliated cells. The exposure of mTEC to SCC1 induced the trafficking of ATP7B, but not ATP7A, suggesting the presence of a cell-specific silver uptake and detoxification mechanisms. Indeed, the expression of the copper uptake protein CTR1 was also restricted to ciliated cells. A role of ATP7B in silver detoxification was further substantiated when treatment of SCC1 significantly increased cell death in ATP7B shRNA-treated HepG2 cells. In addition, mTEC from ATP7B{sup -/-} mice showed enhanced loss of ciliated cells compared to wild type. These studies are the first to demonstrate a cell type-specific expression of the Ag{sup +}/Cu{sup +} transporters ATP7A, ATP7B, and CTR1 in airway epithelial cells and a role for ATP7B in detoxification of these metals in the lung.

  11. ATP release, generation and hydrolysis in exocrine pancreatic duct cells.

    PubMed

    Kowal, J M; Yegutkin, G G; Novak, I

    2015-12-01

    Extracellular adenosine triphosphate (ATP) regulates pancreatic duct function via P2Y and P2X receptors. It is well known that ATP is released from upstream pancreatic acinar cells. The ATP homeostasis in pancreatic ducts, which secrete bicarbonate-rich fluid, has not yet been examined. First, our aim was to reveal whether pancreatic duct cells release ATP locally and whether they enzymatically modify extracellular nucleotides/sides. Second, we wished to explore which physiological and pathophysiological factors may be important in these processes. Using a human pancreatic duct cell line, Capan-1, and online luminescence measurement, we detected fast ATP release in response to pH changes, bile acid, mechanical stress and hypo-osmotic stress. ATP release following hypo-osmotic stress was sensitive to drugs affecting exocytosis, pannexin-1, connexins, maxi-anion channels and transient receptor potential cation channel subfamily V member 4 (TRPV4) channels, and corresponding transcripts were expressed in duct cells. Direct stimulation of intracellular Ca(2+) and cAMP signalling and ethanol application had negligible effects on ATP release. The released ATP was sequentially dephosphorylated through ecto-nucleoside triphosphate diphosphohydrolase (NTPDase2) and ecto-5'-nucleotidase/CD73 reactions, with respective generation of adenosine diphosphate (ADP) and adenosine and their maintenance in the extracellular medium at basal levels. In addition, Capan-1 cells express counteracting adenylate kinase (AK1) and nucleoside diphosphate kinase (NDPK) enzymes (NME1, 2), which contribute to metabolism and regeneration of extracellular ATP and other nucleotides (ADP, uridine diphosphate (UDP) and uridine triphosphate (UTP)). In conclusion, we illustrate a complex regulation of extracellular purine homeostasis in a pancreatic duct cell model involving: ATP release by several mechanisms and subsequent nucleotide breakdown and ATP regeneration via counteracting nucleotide

  12. Binding of ATP by pertussis toxin and isolated toxin subunits

    SciTech Connect

    Hausman, S.Z.; Manclark, C.R.; Burns, D.L. )

    1990-07-03

    The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of ({sup 3}H)ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP > GTP > CTP > TTP for pertussis toxin and ATP > GTP > TTP > CTP for the B oligomer. Phosphate ions inhibited the binding of ({sup 3}H)ATP to pertussis toxin in a competitive manner; however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of ({sup 3}H)ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site.

  13. Diversity and regulation of ATP sulfurylase in photosynthetic organisms

    PubMed Central

    Prioretti, Laura; Gontero, Brigitte; Hell, Ruediger; Giordano, Mario

    2014-01-01

    ATP sulfurylase (ATPS) catalyzes the first committed step in the sulfate assimilation pathway, the activation of sulfate prior to its reduction. ATPS has been studied in only a few model organisms and even in these cases to a much smaller extent than the sulfate reduction and cysteine synthesis enzymes. This is possibly because the latter were considered of greater regulatory importance for sulfate assimilation. Recent evidences (reported in this paper) challenge this view and suggest that ATPS may have a crucial regulatory role in sulfate assimilation, at least in algae. In the ensuing text, we summarize the current knowledge on ATPS, with special attention to the processes that control its activity and gene(s) expression in algae. Special attention is given to algae ATPS proteins. The focus on algae is the consequence of the fact that a comprehensive investigation of ATPS revealed that the algal enzymes, especially those that are most likely involved in the pathway of sulfate reduction to cysteine, possess features that are not present in other organisms. Remarkably, algal ATPS proteins show a great diversity of isoforms and a high content of cysteine residues, whose positions are often conserved. According to the occurrence of cysteine residues, the ATPS of eukaryotic algae is closer to that of marine cyanobacteria of the genera Synechococcus and Prochlorococcus and is more distant from that of freshwater cyanobacteria. These characteristics might have evolved in parallel with the radiation of algae in the oceans and the increase of sulfate concentration in seawater. PMID:25414712

  14. Understanding structure, function, and mutations in the mitochondrial ATP synthase

    PubMed Central

    Xu, Ting; Pagadala, Vijayakanth; Mueller, David M.

    2015-01-01

    The mitochondrial ATP synthase is a multimeric enzyme complex with an overall molecular weight of about 600,000 Da. The ATP synthase is a molecular motor composed of two separable parts: F1 and Fo. The F1 portion contains the catalytic sites for ATP synthesis and protrudes into the mitochondrial matrix. Fo forms a proton turbine that is embedded in the inner membrane and connected to the rotor of F1. The flux of protons flowing down a potential gradient powers the rotation of the rotor driving the synthesis of ATP. Thus, the flow of protons though Fo is coupled to the synthesis of ATP. This review will discuss the structure/function relationship in the ATP synthase as determined by biochemical, crystallographic, and genetic studies. An emphasis will be placed on linking the structure/function relationship with understanding how disease causing mutations or putative single nucleotide polymorphisms (SNPs) in genes encoding the subunits of the ATP synthase, will affect the function of the enzyme and the health of the individual. The review will start by summarizing the current understanding of the subunit composition of the enzyme and the role of the subunits followed by a discussion on known mutations and their effect on the activity of the ATP synthase. The review will conclude with a summary of mutations in genes encoding subunits of the ATP synthase that are known to be responsible for human disease, and a brief discussion on SNPs. PMID:25938092

  15. Application of luciferase assay for ATP to antimicrobial drug susceptibility

    NASA Technical Reports Server (NTRS)

    Chappelle, E. W.; Picciolo, G. L.; Vellend, H.; Tuttle, S. A.; Barza, M. J.; Weinstein, L. (Inventor)

    1977-01-01

    The susceptibility of bacteria, particularly those derived from body fluids, to antimicrobial agents is determined in terms of an ATP index measured by culturing a bacterium in a growth medium. The amount of ATP is assayed in a sample of the cultured bacterium by measuring the amount of luminescent light emitted when the bacterial ATP is reacted with a luciferase-luciferin mixture. The sample of the cultured bacterium is subjected to an antibiotic agent. The amount of bacterial adenosine triphosphate is assayed after treatment with the antibiotic by measuring the luminescent light resulting from the reaction. The ATP index is determined from the values obtained from the assay procedures.

  16. ATP25, a New Nuclear Gene of Saccharomyces cerevisiae Required for Expression and Assembly of the Atp9p Subunit of Mitochondrial ATPase

    PubMed Central

    Zeng, Xiaomei; Barros, Mario H.; Shulman, Theodore

    2008-01-01

    We report a new nuclear gene, designated ATP25 (reading frame YMR098C on chromosome XIII), required for expression of Atp9p (subunit 9) of the Saccharomyces cerevisiae mitochondrial proton translocating ATPase. Mutations in ATP25 elicit a deficit of ATP9 mRNA and of its translation product, thereby preventing assembly of functional F0. Unlike Atp9p, the other mitochondrial gene products, including ATPase subunits Atp6p and Atp8p, are synthesized normally in atp25 mutants. Northern analysis of mitochondrial RNAs in an atp25 temperature-sensitive mutant confirmed that Atp25p is required for stability of the ATP9 mRNA. Atp25p is a mitochondrial inner membrane protein with a predicted mass of 70 kDa. The primary translation product of ATP25 is cleaved in vivo after residue 292 to yield a 35-kDa C-terminal polypeptide. The C-terminal half of Atp25p is sufficient to stabilize the ATP9 mRNA and restore synthesis of Atp9p. Growth on respiratory substrates, however, depends on both halves of Atp25p, indicating that the N-terminal half has another function, which we propose to be oligomerization of Atp9p into a proper size ring structure. PMID:18216280

  17. Evidence for the Synthesis of ATP by an F0F1 ATP Synthase in Membrane Vesicles from Halorubrum Saccharovorum

    NASA Technical Reports Server (NTRS)

    Faguy, David; Lawson, Darion; Hochstein, Lawrence I.; Chang, Sherwood (Technical Monitor)

    1996-01-01

    Vesicles prepared in a buffer containing ADP, Mg(2+) and Pi synthesized ATP at an initial rate of 2 nmols/min/mg protein after acidification of the bulk medium (pH 8 (right arrow) 4). The intravesicular ATP concentration reached a steady state after about 30 seconds and slowly declined thereafter. ATP synthesis was inhibited by low concentrations of dicyclohexylcarbodiimide and m-chlorophenylhydrazone indicating that synthesis took place in response to the proton gradient. NEM and PCMS, which inhibit vacuolar ATPases and the vacuolar-like ATPases of extreme halophiles, did not affect ATP synthesis, and, in fact, produced higher steady state levels of ATP. This suggested that two ATPase activities were present, one which catalyzed ATP synthesis and one that caused its hydrolysis. Azide, a specific inhibitor of F0F1 ATP Synthases, inhibited halobacterial ATP synthesis. The distribution of acridine orange as imposed by a delta pH demonstrated that azide inhibition was not due to the collapse of the proton gradient due to azide acting as a protonophore. Such an effect was observed, but only at azide concentrations higher than those that inhibited ATP synthesis. These results confirm the earler observations with cells of H. saccharovorum and other extreme halophiles that ATP synthesis is inconsistent with the operation of a vacuolar-like ATPase. Therefore, the observation that a vacuolar-like enzyme is responsible for ATP synthesis (and which serves as the basis for imputing ATP synthesis to the vacuolar-like ATPases of the extreme halophiles, and the Archaea in general) should be taken with some degree of caution.

  18. 14 CFR 1259.101 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... definitions shall apply: (a) Field related to space means any academic discipline or field of study (including... activities in the fields related to space: (i) Research; (ii) Training; or (iii) Advisory services. (j)...

  19. 28 CFR 905.1 - Definition.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... QUALIFICATION REQUIREMENTS § 905.1 Definition. “National Fingerprint File” means a database of fingerprints, or... maintained by the FBI to provide positive identification of record subjects indexed in the III System....

  20. ATP synthesis in Halobacterium saccharovorum: evidence that synthesis may be catalysed by an F0F1-ATP synthase

    NASA Technical Reports Server (NTRS)

    Hochstein, L. I.

    1992-01-01

    Halobacterium saccharovorum synthesized ATP in response to a pH shift from 8 to 6.2. Synthesis was inhibited by carbonyl cyanide m-chloro-phenylhydrazone, dicyclohexylcarbodiimide, and azide. Nitrate, an inhibitor of the membrane-bound ATPase previously isolated from this organism, did not inhibit ATP synthesis. N-Ethymaleimide, which also inhibited this ATPase, stimulated the production of ATP. These observations suggested that H. saccharovorum synthesized and hydrolysed ATP using different enzymes and that the vacuolar-like ATPase activity previously described in H. saccharovorum was an ATPase whose function is yet to be identified.

  1. ATP7A trafficking and mechanisms underlying the distal motor neuropathy induced by mutations in ATP7A.

    PubMed

    Yi, Ling; Kaler, Stephen

    2014-05-01

    Diverse mutations in the gene encoding the copper transporter ATP7A lead to X-linked recessive Menkes disease or occipital horn syndrome. Recently, two unique ATP7A missense mutations, T994I and P1386S, were shown to cause isolated adult-onset distal motor neuropathy. These mutations induce subtle defects in ATP7A intracellular trafficking resulting in preferential accumulation at the plasma membrane compared to wild-type ATP7A. Immunoprecipitation assays revealed abnormal interaction between ATP7A(T994I) and p97/VCP, a protein mutated in two autosomal dominant forms of motor neuron disease. Small-interfering RNA knockdown of valosin-containing protein corrected ATP7A(T994I) mislocalization. For ATP7A(P1386S) , flow cytometry documented that nonpermeabilized fibroblasts bound a C-terminal ATP7A antibody, suggesting unstable insertion of the eighth transmembrane segment due to a helix-breaker effect of the amino acid substitution. This could sabotage interaction of ATP7A(P1386S) with adaptor protein complexes. These molecular events appear to selectively disturb normal motor neuron function and lead to neurologic illness that takes years and sometimes decades to develop. PMID:24754450

  2. Carbon and energy metabolism of atp mutants of Escherichia coli.

    PubMed

    Jensen, P R; Michelsen, O

    1992-12-01

    The membrane-bound H(+)-ATPase plays a key role in free-energy transduction of biological systems. We report how the carbon and energy metabolism of Escherichia coli changes in response to deletion of the atp operon that encodes this enzyme. Compared with the isogenic wild-type strain, the growth rate and growth yield were decreased less than expected for a shift from oxidative phosphorylation to glycolysis alone as a source of ATP. Moreover, the respiration rate of a atp deletion strain was increased by 40% compared with the wild-type strain. This result is surprising, since the atp deletion strain is not able to utilize the resulting proton motive force for ATP synthesis. Indeed, the ratio of ATP concentration to ADP concentration was decreased from 19 in the wild type to 7 in the atp mutant, and the membrane potential of the atp deletion strain was increased by 20%, confirming that the respiration rate was not controlled by the magnitude of the opposing membrane potential. The level of type b cytochromes in the mutant cells was 80% higher than the level in the wild-type cells, suggesting that the increased respiration was caused by an increase in the expression of the respiratory genes. The atp deletion strain produced twice as much by-product (acetate) and exhibited increased flow through the tricarboxylic acid cycle and the glycolytic pathway. These three changes all lead to an increase in substrate level phosphorylation; the first two changes also lead to increased production of reducing equivalents. We interpret these data as indicating that E. coli makes use of its ability to respire even if it cannot directly couple this ability to ATP synthesis; by respiring away excess reducing equivalents E. coli enhances substrate level ATP synthesis. PMID:1447134

  3. ATP technology, a tool for monitoring microbes in cooling systems

    SciTech Connect

    Czechowski, M.H.

    1996-11-01

    Rapid and accurate measurement of microbes is important for controlling the formation of troublesome microbial slimes in cooling water systems. One method for accomplishing this involves the measurement of Adenosine Triphosphate (ATP), a compound used to store and transfer energy in microbial cells. Cellular ATP is determined by chemically rupturing cells, which releases ATP that reacts with a luciferase reagent (the firefly enzyme). This reaction produces light which can be detected by a sensitive luminometer/photometer. The amount of light produced is proportional to the amount of ATP in the cell. A quantitative indication of biological activity is obtained in minutes, compared to traditional plating methods which often require days of incubation. The use of ATP for microbial detection has been available for many years; however, industrial usage was limited because the ATP procedure was neither easy to perform nor was it cost effective. Recently, advances in instrument technology, extractant chemistry and enzyme stability have made ATP detection more practical and less expensive. ATP technology can be used for determining microbial content in cooling water systems, predicting biocide effectiveness, and monitoring efficacy of biocides in cooling systems. A good correlation (0.85) was found between microbial ATP values and bacterial Colony Forming Units (CFU) in cooling waters. ATP technology was used to determine the effectiveness of different concentrations of a biocide in a test system within 1 hour after biocide addition. Test results accurately predicted the biocide efficacy in the cooling tower. Effectiveness of other biocides in cooling systems were monitored with results being obtained within minutes after sampling. These findings indicate the potential for ATP technology to be an effective tool in monitoring microbes in cooling water systems.

  4. ATP7B expression confers multidrug resistance through drug sequestration

    PubMed Central

    Moinuddin, F M; Shinsato, Yoshinari; Komatsu, Masaharu; Mitsuo, Ryoichi; Minami, Kentaro; Yamamoto, Masatatsu; Kawahara, Kohich; Hirano, Hirofumi; Arita, Kazunori; Furukawa, Tatsuhiko

    2016-01-01

    We previously reported that ATP7B is involved in cisplatin resistance and ATP7A confers multidrug resistance (MDR) in cancer cells. In this study, we show that ATP7B expressing cells also are resistant to doxorubicin, SN-38, etoposide, and paclitaxel as well as cisplatin. In ATP7B expressing cells, doxorubicin relocated from the nuclei to the late-endosome at 4 hours after doxorubicin exposure. EGFP-ATP7B mainly colocalized with doxorubicin. ATP7B has six metal binding sites (MBSs) in the N-terminal cytoplasmic region. To investigate the role of the MBSs of ATP7B in doxorubicin resistance, we used three mutant ATP7B (Cu0, Cu6 and M6C/S) expressing cells. Cu0 has no MBSs, Cu6 has only the sixth MBS and M6C/S carries CXXC to SXXS mutation in the sixth MBS. Cu6 expressing cells were less resistance to the anticancer agents than wild type ATP7B expressing cells, and had doxorubicin sequestration in the late-endosome. Cu0- and M6C/S-expressing cells were sensitive to doxorubicin. In these cells, doxorubicin did not relocalize to the late-endosome. EGFP-M6C/S mainly localized to the trans-Golgi network (TGN) even in the presence of copper. Thus the cysteine residues in the sixth MBS of ATP7B are essential for MDR phenotype. Finally, we found that ammonium chloride and tamoxifen suppressed late endosomal sequestration of doxorubicin, thereby attenuating drug resistance. These results suggest that the sequestration depends on the acidity of the vesicles partly. We here demonstrate that ATP7B confers MDR by facilitating nuclear drug efflux and late endosomal drug sequestration. PMID:26988911

  5. Carbon and energy metabolism of atp mutants of Escherichia coli.

    PubMed Central

    Jensen, P R; Michelsen, O

    1992-01-01

    The membrane-bound H(+)-ATPase plays a key role in free-energy transduction of biological systems. We report how the carbon and energy metabolism of Escherichia coli changes in response to deletion of the atp operon that encodes this enzyme. Compared with the isogenic wild-type strain, the growth rate and growth yield were decreased less than expected for a shift from oxidative phosphorylation to glycolysis alone as a source of ATP. Moreover, the respiration rate of a atp deletion strain was increased by 40% compared with the wild-type strain. This result is surprising, since the atp deletion strain is not able to utilize the resulting proton motive force for ATP synthesis. Indeed, the ratio of ATP concentration to ADP concentration was decreased from 19 in the wild type to 7 in the atp mutant, and the membrane potential of the atp deletion strain was increased by 20%, confirming that the respiration rate was not controlled by the magnitude of the opposing membrane potential. The level of type b cytochromes in the mutant cells was 80% higher than the level in the wild-type cells, suggesting that the increased respiration was caused by an increase in the expression of the respiratory genes. The atp deletion strain produced twice as much by-product (acetate) and exhibited increased flow through the tricarboxylic acid cycle and the glycolytic pathway. These three changes all lead to an increase in substrate level phosphorylation; the first two changes also lead to increased production of reducing equivalents. We interpret these data as indicating that E. coli makes use of its ability to respire even if it cannot directly couple this ability to ATP synthesis; by respiring away excess reducing equivalents E. coli enhances substrate level ATP synthesis. PMID:1447134

  6. Behavior and stability of adenosine triphosphate (ATP) during chlorine disinfection.

    PubMed

    Nescerecka, Alina; Juhna, Talis; Hammes, Frederik

    2016-09-15

    Adenosine triphosphate (ATP) analysis is a cultivation-independent alternative method for the determination of bacterial viability in both chlorinated and non-chlorinated water. Here we investigated the behavior and stability of ATP during chlorination in detail. Different sodium hypochlorite doses (0-22.4 mg-Cl2 L(-1); 5 min exposure) were applied to an Escherichia coli pure culture suspended in filtered river water. We observed decreasing intracellular ATP with increasing chlorine concentrations, but extracellular ATP concentrations only increased when the chlorine dose exceeded 0.35 mg L(-1). The release of ATP from chlorine-damaged bacteria coincided with severe membrane damage detected with flow cytometry (FCM). The stability of extracellular ATP was subsequently studied in different water matrixes, and we found that extracellular ATP was stable in sterile deionized water and also in chlorinated water until extremely high chlorine doses (≤11.2 mg-Cl2 L(-1); 5 min exposure). In contrast, ATP decreased relatively slowly (k = 0.145 h(-1)) in 0.1 μm filtered river water, presumably due to degradation by either extracellular enzymes or the fraction of bacteria that were able to pass through the filter. Extracellular ATP decreased considerably faster (k = 0.368 h(-1)) during batch growth of a river water bacterial community. A series of growth potential tests showed that extracellular ATP molecules were utilized as a phosphorus source during bacteria proliferation. From the combined data we conclude that ATP released from bacteria at high chlorine doses could promote bacteria regrowth, contributing to biological instability in drinking water distribution systems. PMID:27295623

  7. ATP/P2X7 axis modulates myeloid-derived suppressor cell functions in neuroblastoma microenvironment

    PubMed Central

    Bianchi, G; Vuerich, M; Pellegatti, P; Marimpietri, D; Emionite, L; Marigo, I; Bronte, V; Di Virgilio, F; Pistoia, V; Raffaghello, L

    2014-01-01

    Tumor microenvironment of solid tumors is characterized by a strikingly high concentration of adenosine and ATP. Physiological significance of this biochemical feature is unknown, but it has been suggested that it may affect infiltrating immune cell responses and tumor progression. There is increasing awareness that many of the effects of extracellular ATP on tumor and inflammatory cells are mediated by the P2X7 receptor (P2X7R). Aim of this study was to investigate whether: (i) extracellular ATP is a component of neuroblastoma (NB) microenvironment, (ii) myeloid-derived suppressor cells (MDSCs) express functional P2X7R and (iii) the ATP/P2X7R axis modulates MDSC functions. Our results show that extracellular ATP was detected in NB microenvironment in amounts that increased in parallel with tumor progression. The percentage of CD11b+/Gr-1+ cells was higher in NB-bearing mice compared with healthy animals. Within the CD11b/Gr-1+ population, monocytic MDSCs (M-MDSCs) produced higher levels of reactive oxygen species (ROS), arginase-1 (ARG-1), transforming growth factor-β1 (TGF-β1) and stimulated more potently in vivo tumor growth, as compared with granulocytic MDSCs (G-MDSCs). P2X7R of M-MDSCs was localized at the plasma membrane, coupled to increased functionality, upregulation of ARG-1, TGF-β1 and ROS. Quite surprisingly, the P2X7R in primary MDSCs as well as in the MSC-1 and MSC-2 lines was uncoupled from cytotoxicity. This study describes a novel scenario in which MDSC immunosuppressive functions are modulated by the ATP-enriched tumor microenvironment. PMID:24651438

  8. Renal epithelial cells can release ATP by vesicular fusion

    PubMed Central

    Bjaelde, Randi G.; Arnadottir, Sigrid S.; Overgaard, Morten T.; Leipziger, Jens; Praetorius, Helle A.

    2013-01-01

    Renal epithelial cells have the ability to release nucleotides as paracrine factors. In the intercalated cells of the collecting duct, ATP is released by connexin30 (cx30), which is selectively expressed in this cell type. However, ATP is released by virtually all renal epithelia and the aim of the present study was to identify possible alternative nucleotide release pathways in a renal epithelial cell model. We used MDCK (type1) cells to screen for various potential ATP release pathways. In these cells, inhibition of the vesicular H+-ATPases (bafilomycin) reduced both the spontaneous and hypotonically (80%)-induced nucleotide release. Interference with vesicular fusion using N-ethylamide markedly reduced the spontaneous nucleotide release, as did interference with trafficking from the endoplasmic reticulum to the Golgi apparatus (brefeldin A1) and vesicular transport (nocodazole). These findings were substantiated using a siRNA directed against SNAP-23, which significantly reduced spontaneous ATP release. Inhibition of pannexin and connexins did not affect the spontaneous ATP release in this cell type, which consists of ~90% principal cells. TIRF-microscopy of either fluorescently-labeled ATP (MANT-ATP) or quinacrine-loaded vesicles, revealed that spontaneous release of single vesicles could be promoted by either hypoosmolality (50%) or ionomycin. This vesicular release decreased the overall cellular fluorescence by 5.8 and 7.6% respectively. In summary, this study supports the notion that spontaneous and induced ATP release can occur via exocytosis in renal epithelial cells. PMID:24065923

  9. Role of ATP-conductive anion channel in ATP release from neonatal rat cardiomyocytes in ischaemic or hypoxic conditions

    PubMed Central

    Dutta, Amal K; Sabirov, Ravshan Z; Uramoto, Hiromi; Okada, Yasunobu

    2004-01-01

    It is known that the level of ATP in the interstitial spaces within the heart during ischaemia or hypoxia is elevated due to its release from a number of cell types, including cardiomyocytes. However, the mechanism by which ATP is released from these myocytes is not known. In this study, we examined a possible involvement of the ATP-conductive maxi-anion channel in ATP release from neonatal rat cardiomyocytes in primary culture upon ischaemic, hypoxic or hypotonic stimulation. Using a luciferin–luciferase assay, it was found that ATP was released into the bulk solution when the cells were subjected to chemical ischaemia, hypoxia or hypotonic stress. The swelling-induced ATP release was inhibited by the carboxylate-and stilbene-derivative anion channel blockers, arachidonic acid and Gd3+, but not by glibenclamide. The local concentration of ATP released near the cell surface of a single cardiomyocyte, measured by a biosensor technique, was found to exceed the micromolar level. Patch-clamp studies showed that ischaemia, hypoxia or hypotonic stimulation induced the activation of single-channel events with a large unitary conductance (∼390 pS). The channel was selective to anions and showed significant permeability to ATP4− (PATP/PCl ∼ 0.1) and MgATP2− (PATP/PCl ∼ 0.16). The channel activity exhibited pharmacological properties essentially identical to those of ATP release. These results indicate that neonatal rat cardiomyocytes respond to ischaemia, hypoxia or hypotonic stimulation with ATP release via maxi-anion channels. PMID:15272030

  10. Radioprotective effects of ATP in human blood ex vivo

    SciTech Connect

    Swennen, Els L.R. Dagnelie, Pieter C.; Van den Beucken, Twan; Bast, Aalt

    2008-03-07

    Damage to healthy tissue is a major limitation of radiotherapy treatment of cancer patients, leading to several side effects and complications. Radiation-induced release of pro-inflammatory cytokines is thought to be partially responsible for the radiation-associated complications. The aim of the present study was to investigate the protective effects of extracellular ATP on markers of oxidative stress, radiation-induced inflammation and DNA damage in irradiated blood ex vivo. ATP inhibited radiation-induced TNF-{alpha} release and increased IL-10 release. The inhibitory effect of ATP on TNF- {alpha} release was completely reversed by adenosine 5'-O-thiomonophosphate, indicating a P2Y{sub 11} mediated effect. Furthermore, ATP attenuated radiation-induced DNA damage immediate, 3 and 6 h after irradiation. Our study indicates that ATP administration alleviates radiation-toxicity to blood cells, mainly by inhibiting radiation-induced inflammation and DNA damage.

  11. Amperometric ATP biosensor based on polymer entrapped enzymes.

    PubMed

    Kueng, Angelika; Kranz, Christine; Mizaikoff, Boris

    2004-05-15

    A dual enzyme electrode for the detection of adenosine-5'-triphosphate (ATP) at physiologically relevant pH levels was developed by co-immobilization of the enzymes glucose oxidase (GOD) and hexokinase (HEX) using pH-shift induced deposition of enzyme containing polymer films. Application of a simple electrochemical procedure for the co-immobilization of the enzymes at electrode surfaces exhibits a major improvement of sensitivity, response time, reproducibility, and ease of fabrication of ATP biosensors. Competition between glucose oxidase and hexokinase for the substrate glucose involving ATP as a co-substrate allows the determination of ATP concentrations. Notable control on the immobilization process enables fabrication of micro biosensors with a diameter of 25 microm. The presented concept provides the technological basis for a new generation of fast responding, sensitive, and robust biosensors for the detection of ATP at physiological pH values with a detection limit of 10 nmol l(-1). PMID:15046763

  12. ATP-sulfurylase, sulfur-compounds, and plant stress tolerance.

    PubMed

    Anjum, Naser A; Gill, Ritu; Kaushik, Manjeri; Hasanuzzaman, Mirza; Pereira, Eduarda; Ahmad, Iqbal; Tuteja, Narendra; Gill, Sarvajeet S

    2015-01-01

    Sulfur (S) stands fourth in the list of major plant nutrients after N, P, and K. Sulfate (SO4 (2-)), a form of soil-S taken up by plant roots is metabolically inert. As the first committed step of S-assimilation, ATP-sulfurylase (ATP-S) catalyzes SO4 (2-)-activation and yields activated high-energy compound adenosine-5(')-phosphosulfate that is reduced to sulfide (S(2-)) and incorporated into cysteine (Cys). In turn, Cys acts as a precursor or donor of reduced S for a range of S-compounds such as methionine (Met), glutathione (GSH), homo-GSH (h-GSH), and phytochelatins (PCs). Among S-compounds, GSH, h-GSH, and PCs are known for their involvement in plant tolerance to varied abiotic stresses, Cys is a major component of GSH, h-GSH, and PCs; whereas, several key stress-metabolites such as ethylene, are controlled by Met through its first metabolite S-adenosylmethionine. With the major aim of briefly highlighting S-compound-mediated role of ATP-S in plant stress tolerance, this paper: (a) overviews ATP-S structure/chemistry and occurrence, (b) appraises recent literature available on ATP-S roles and regulations, and underlying mechanisms in plant abiotic and biotic stress tolerance, (c) summarizes ATP-S-intrinsic regulation by major S-compounds, and (d) highlights major open-questions in the present context. Future research in the current direction can be devised based on the discussion outcomes. PMID:25904923

  13. Regulation of ATP supply during muscle contraction: theoretical studies.

    PubMed Central

    Korzeniewski, B

    1998-01-01

    The dynamic computer model of oxidative phosphorylation developed previously and successfully tested for large-scale changes in fluxes and metabolite concentrations was used to study the question of how the rate of ATP production by oxidative phosphorylation is adjusted to meet the energy demand during muscle contraction, which causes a great increase in ATP consumption in relation to the resting state. The changes in the respiration rate and ATP/ADP ratio after the onset of maximal work measured experimentally were compared with simulated changes in the respiration rate and ATP/ADP in several different cases, assuming direct activation of different steps by an external effector. On the basis of the computer simulations performed, it was possible to conclude which enzymes/metabolic blocks should be directly activated to cause the experimentally observable changes in fluxes and metabolite concentrations. The theoretical results obtained suggest that the parallel direct activation of actinomyosin-ATP-ase and oxidative phosphorylation by an external effector (for example calcium ions) is the main mechanism responsible for fitting of ATP production to ATP consumption, while the negative feedback via an increase in ADP concentration (decrease in ATP/ADP), which indirectly activates the ATP supply, plays only a minor role. Additionally, the conclusion is drawn that most of the oxidative phosphorylation steps should be directly activated in order to explain the observed changes in the respiration rate and ATP/ADP ratio (and also in other parameters) during muscle contraction. It is suggested that there should exist a universal external activator/regulatory mechanism which causes a parallel stimulation of different enzymes/processes. A possible nature of such an activator is shortly discussed. PMID:9494084

  14. 12 CFR 26.2 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... million or more; (iii) A senior executive officer as that term is defined in 12 CFR 5.51(c)(3); (iv) A... Definitions. For purposes of this part, the following definitions apply: (a) Affiliate. (1) The term affiliate has the meaning given in section 202 of the Interlocks Act (12 U.S.C. 3201). For purposes of...

  15. Demonstration of a transitory tight binding of ATP and of committed Pi and ADP during ATP synthesis by chloroplasts

    PubMed Central

    Smith, Daniel J.; Boyer, Paul D.

    1976-01-01

    Rapid mixing, quenching, and filtration experiments with chloroplast thylakoid membranes, with energization by acid-base transition, demonstrate that an ATP tightly bound to the isolated membranes is a transient intermediate in the catalytic sequence for ATP synthesis. The experiments also show that most of the Pi and ADP bound at a catalytic site is committed to ATP formation without interchange with medium Pi or ADP. Other results give evidence that upon energization, the tightly bound ADP that is detectable in isolated thylakoid membranes or coupling factor ATPase is rapidly released to the medium from a catalytic site. These findings support an alternating site model in which an energy-requiring conformational transition loosens ATP binding at one site and simultaneously promotes Pi and ADP binding at the other site in a manner favoring ATP formation. PMID:16592374

  16. Multi-allele genotyping platform for the simultaneous detection of mutations in the Wilson disease related ATP7B gene.

    PubMed

    Amvrosiadou, Maria; Petropoulou, Margarita; Poulou, Myrto; Tzetis, Maria; Kanavakis, Emmanuel; Christopoulos, Theodore K; Ioannou, Penelope C

    2015-12-01

    Wilson's disease is an inherited disorder of copper transport in the hepatocytes with a wide range of genotype and phenotype characteristics. Mutations in the ATP7B gene are responsible for the disease. Approximately, over 500 mutations in the ATP7B gene have been described to date. We report a method for the simultaneous detection of the ten most common ATP7B gene mutations in Greek patients. The method comprises 3 simple steps: (i) multiplex PCR amplification of fragments in the ATP7B gene flanking the mutations (ii) multiplex primer extension reaction of the unpurified amplification products using allele-specific primers and (iii) visual detection of the primer extension reaction products within minutes by means of dry-reagent multi-allele dipstick assay using anti-biotin conjugated gold nanoparticles. Optimization studies on the efficiency and specificity of the PEXT reaction were performed. The method was evaluated by genotyping 46 DNA samples of known genotype and 34 blind samples. The results were fully concordant with those obtained by reference methods. The method is simple, rapid, cost-effective and it does not require specialized instrumentation or highly qualified personnel. PMID:26580967

  17. Functional proteomics of synaptic plasma membrane ATP-ases of rat hippocampus: effect of l-acetylcarnitine and relationships with Dementia and Depression pathophysiology.

    PubMed

    Ferrari, Federica; Gorini, Antonella; Villa, Roberto Federico

    2015-06-01

    Synaptic energy state and mitochondrial dysfunction are crucial factors in many brain pathologies. l-acetylcarnitine, a natural derivative of carnitine, improves brain energy metabolism, and has been proposed for the Therapy of many neurological and psychiatric diseases. The effects of the drug on the maximum rate (Vmax) of enzymatic activities related to hippocampal synaptic energy utilization were evaluated, in the perspective of its employment for Dementias and Depression Therapy. Two types of synaptic plasma membranes (SPM1 and SPM2) were isolated from the hippocampus of rats treated with l-acetylcarnitine (30 and 60mg/kg i.p., 28 days, 5 days/week). Acetylcholinesterase (AChE); Na(+), K(+), Mg(2+)-ATP-ase; ouabain-insensitive Mg(2+)-ATP-ase; Na(+), K(+)-ATP-ase; Ca(2+), Mg(2+)-ATP-ase activities were evaluated. In control animals, enzymatic activities were differently expressed in SPM1 , being the evaluated enzymatic activities higher in SPM2. Subchronic treatment with l-acetylcarnitine (i) did not modify AChE on both SPMs; (ii) increased Na(+), K(+), Mg(2+)-ATP-ase, ouabain-insensitive Mg(2+)-ATP-ase and Na(+), K(+)-ATP-ase at the dose of 30 and 60mg/kg on SPM1 and SPM2; (iii) increased Ca(2+), Mg(2+)-ATP-ase activity on both SPMs at the dose of 60mg/kg. These results have been discussed considering the pathophysiology and treatment of Dementias and Depression because, although referred to normal healthy animals, they support the notion that l-acetylcarnitine may have positive effects in these pathologies. PMID:25797282

  18. Bringing Definitions into High Definition

    ERIC Educational Resources Information Center

    Mason, John

    2010-01-01

    Why do definitions play such a central role in mathematics? It may seem obvious that precision about the terms one uses is necessary in order to use those terms reasonably (while reasoning). Definitions are chosen so as to be definite about the terms one uses, but also to make both the statement of, and the reasoning to justify, theorems as…

  19. Authentic role of ATP signaling in micturition reflex

    PubMed Central

    Takezawa, Kentaro; Kondo, Makoto; Kiuchi, Hiroshi; Ueda, Norichika; Soda, Tetsuji; Fukuhara, Shinichiro; Takao, Tetsuya; Miyagawa, Yasushi; Tsujimura, Akira; Matsumoto-Miyai, Kazumasa; Ishida, Yusuke; Negoro, Hiromitsu; Ogawa, Osamu; Nonomura, Norio; Shimada, Shoichi

    2016-01-01

    Adenosine triphosphate (ATP) is a signaling molecule that regulates cellular processes. Based on previous studies of bladder function over the past decade, bladder ATP signaling was thought to have an essential role in the normal micturition reflex. In this study, we performed detailed analyses of bladder function in purinergic receptor-deficient mice using the automated voided stain on paper method and video-urodynamics. Unexpectedly, a lack of P2X2 or P2X3 receptors did not affect bladder function under normal physiological conditions, indicating that bladder ATP signaling is not essential for normal micturition reflex. In contrast, we found that lipopolysaccharide (LPS) induced markedly high levels of ATP release from the urothelium. In addition, LPS-induced rapid bladder hyperactivity was attenuated in P2X2−/− and P2X3−/− mice. Contrary to the previous interpretation, our present findings indicate that bladder ATP signaling has a fundamental role in the micturition reflex, especially in bladder dysfunction, under pathological conditions. Therefore, the bladder ATP signaling pathway might be a highly promising therapeutic target for functional bladder disorders. This study newly defines an authentic role for bladder ATP signaling in the micturition reflex. PMID:26795755

  20. The origin of cytosolic ATP in photosynthetic cells.

    PubMed

    Gardeström, Per; Igamberdiev, Abir U

    2016-07-01

    In photosynthetically active cells, both chloroplasts and mitochondria have the capacity to produce ATP via photophosphorylation and oxidative phosphorylation, respectively. Thus, theoretically, both organelles could provide ATP for the cytosol, but the extent, to which they actually do this, and how the process is regulated, both remain unclear. Most of the evidence discussed comes from experiments with rapid fractionation of isolated protoplasts subjected to different treatments in combination with application of specific inhibitors. The results obtained indicate that, under conditions where ATP demand for photosynthetic CO2 fixation is sufficiently high, the mitochondria supply the bulk of ATP for the cytosol. In contrast, under stress conditions where CO2 fixation is severely limited, ATP will build up in chloroplasts and it can then be exported to the cytosol, by metabolite shuttle mechanisms. Thus, depending on the conditions, either mitochondria or chloroplasts can supply the bulk of ATP for the cytosol. This supply of ATP is discussed in relation to the idea that mitochondrial functions may be tuned to provide an optimal environment for the chloroplast. By balancing cellular redox states, mitochondria can contribute to an optimal photosynthetic capacity. PMID:27087668

  1. ATP and potassium ions: a deadly combination for astrocytes

    NASA Astrophysics Data System (ADS)

    Jackson, David G.; Wang, Junjie; Keane, Robert W.; Scemes, Eliana; Dahl, Gerhard

    2014-04-01

    The ATP release channel Pannexin1 (Panx1) is self-regulated, i.e. the permeant ATP inhibits the channel from the extracellular space. The affinity of the ATP binding site is lower than that of the purinergic P2X7 receptor allowing a transient activation of Panx1 by ATP through P2X7R. Here we show that the inhibition of Panx1 by ATP is abrogated by increased extracellular potassium ion concentration ([K+]o) in a dose-dependent manner. Since increased [K+]o is also a stimulus for Panx1 channels, it can be expected that a combination of ATP and increased [K+]o would be deadly for cells. Indeed, astrocytes did not survive exposure to these combined stimuli. The death mechanism, although involving P2X7R, does not appear to strictly follow a pyroptotic pathway. Instead, caspase-3 was activated, a process inhibited by Panx1 inhibitors. These data suggest that Panx1 plays an early role in the cell death signaling pathway involving ATP and K+ ions. Additionally, Panx1 may play a second role once cells are committed to apoptosis, since Panx1 is also a substrate of caspase-3.

  2. Authentic role of ATP signaling in micturition reflex.

    PubMed

    Takezawa, Kentaro; Kondo, Makoto; Kiuchi, Hiroshi; Ueda, Norichika; Soda, Tetsuji; Fukuhara, Shinichiro; Takao, Tetsuya; Miyagawa, Yasushi; Tsujimura, Akira; Matsumoto-Miyai, Kazumasa; Ishida, Yusuke; Negoro, Hiromitsu; Ogawa, Osamu; Nonomura, Norio; Shimada, Shoichi

    2016-01-01

    Adenosine triphosphate (ATP) is a signaling molecule that regulates cellular processes. Based on previous studies of bladder function over the past decade, bladder ATP signaling was thought to have an essential role in the normal micturition reflex. In this study, we performed detailed analyses of bladder function in purinergic receptor-deficient mice using the automated voided stain on paper method and video-urodynamics. Unexpectedly, a lack of P2X2 or P2X3 receptors did not affect bladder function under normal physiological conditions, indicating that bladder ATP signaling is not essential for normal micturition reflex. In contrast, we found that lipopolysaccharide (LPS) induced markedly high levels of ATP release from the urothelium. In addition, LPS-induced rapid bladder hyperactivity was attenuated in P2X2(-/-) and P2X3(-/-) mice. Contrary to the previous interpretation, our present findings indicate that bladder ATP signaling has a fundamental role in the micturition reflex, especially in bladder dysfunction, under pathological conditions. Therefore, the bladder ATP signaling pathway might be a highly promising therapeutic target for functional bladder disorders. This study newly defines an authentic role for bladder ATP signaling in the micturition reflex. PMID:26795755

  3. Synthesis and fluorescence characteristics of ATP-based FRET probes.

    PubMed

    Hardt, Norman; Hacker, Stephan M; Marx, Andreas

    2013-12-28

    Adenosine triphosphate (ATP) analogues labelled with two dyes suitable for undergoing Förster Resonance Energy Transfer (FRET) have the potential to be valuable tools to continuously study the enzymatic activity of ATP consuming enzymes. Here, we present a synthesis strategy that allows obtaining these ATP analogues in a straight-forward manner. Earlier studies indicate that modifying ATP at the O2'- and the γ-position is a very promising starting point for the design of these probes. We synthesized probes modified with five different combinations of dyes attached to these positions and investigated their fluorescence characteristics in the non-cleaved state as well as after enzymatic hydrolysis. All presented probes largely change their fluorescence characteristics upon cleavage. They include ratiometric FRET probes as well as dark quenched analogues. For typical in vitro applications a combination of the sulfonated polymethine dyes Sulfo-Cy3 and Sulfo-Cy5 seems to be most promising due to their excellent solubility in aqueous buffer and a large change of fluorescence characteristics upon cleavage. For this combination of dyes we also synthesized analogues modified at the γ- and the C2- or the O3'-position, respectively, as these attachment sites are also well accepted by certain ATP consuming enzymes. These analogues show comparably large changes in fluorescence characteristics. Overall, we present new ATP-based FRET probes that have the potential to enable monitoring the enzymatic activity of ATP consuming enzymes. PMID:24173528

  4. Modeling K,ATP-Dependent Excitability in Pancreatic Islets

    PubMed Central

    Silva, Jonathan R.; Cooper, Paige; Nichols, Colin G.

    2014-01-01

    In pancreatic β-cells, K,ATP channels respond to changes in glucose to regulate cell excitability and insulin release. Confirming a high sensitivity of electrical activity to K,ATP activity, mutations that cause gain of K,ATP function cause neonatal diabetes. Our aim was to quantitatively assess the contribution of K,ATP current to the regulation of glucose-dependent bursting by reproducing experimentally observed changes in excitability when K,ATP conductance is altered by genetic manipulation. A recent detailed computational model of single cell pancreatic β-cell excitability reproduces the β-cell response to varying glucose concentrations. However, initial simulations showed that the model underrepresents the significance of K,ATP activity and was unable to reproduce K,ATP conductance-dependent changes in excitability. By altering the ATP and glucose dependence of the L-type Ca2+ channel and the Na-K ATPase to better fit experiment, appropriate dependence of excitability on K,ATP conductance was reproduced. Because experiments were conducted in islets, which contain cell-to-cell variability, we extended the model from a single cell to a three-dimensional model (10×10×10 cell) islet with 1000 cells. For each cell, the conductance of the major currents was allowed to vary as was the gap junction conductance between cells. This showed that single cell glucose-dependent behavior was then highly variable, but was uniform in coupled islets. The study highlights the importance of parameterization of detailed models of β-cell excitability and suggests future experiments that will lead to improved characterization of β-cell excitability and the control of insulin secretion. PMID:25418087

  5. ATP depletion inhibits glucocorticoid-induced thymocyte apoptosis.

    PubMed

    Stefanelli, C; Bonavita, F; Stanic', I; Farruggia, G; Falcieri, E; Robuffo, I; Pignatti, C; Muscari, C; Rossoni, C; Guarnieri, C; Caldarera, C M

    1997-03-15

    In quiescent thymocytes, mitochondrial de-energization was not correlated to apoptotic death. In fact, thymocytes treated with oligomycin, a highly specific inhibitor of ATP synthase, alone or with atractyloside to block ATP translocation from the cytoplasm, were alive, even if their mitochondria were depolarized, as revealed by flow cytometry after Rhodamine 123 staining. Furthermore, oligomycin was a powerful inhibitor of apoptosis induced in rat thymocytes by dexamethasone and, to a lesser extent, by the calcium ionophore A23187 and etoposide, but was without effect when apoptosis was induced by staurosporine, and increased cell death in mitogen-treated thymocytes. The inhibition of apoptosis was confirmed by morphological criteria, inhibition of inter-nucleosomal DNA fragmentation and inhibition of the loss of membrane integrity. The anti-apoptotic effect of oligomycin in cells treated with A23187 or etoposide was correlated to the inhibition of protein synthesis, while inhibition of apoptosis induced by dexamethasone, already evident at an oligomycin concentration of 10 ng/ml, was instead strictly correlated to the effect exerted on the cellular ATP level. Thymocyte apoptosis triggered by dexamethasone was blocked or delayed by inhibitors of respiratory-chain uncouplers, inhibitors of ATP synthase and antioxidants: a lasting protection from dexamethasone-induced apoptosis was always correlated to a drastic and rapid reduction in ATP level (31-35% of control), while a delay in the death process was characterized by a moderate decrease in ATP (73-82% of control). Oligomycin inhibited the specific binding of radioactive corticosteroid to thymocyte nuclei, confirming the inhibitory effect of ATP depletion on glucocorticoid binding and suggesting that ATP depletion is a common mediator of the anti-apoptotic action of different effectors in glucocorticoid-induced apoptosis. In conclusion, the reported data indicate that ATP may act as a cellular modulator of some

  6. Vesicular Nucleotide Transporter-Mediated ATP Release Regulates Insulin Secretion

    PubMed Central

    Geisler, Jessica C.; Corbin, Kathryn L.; Li, Qin; Feranchak, Andrew P.; Nunemaker, Craig S.

    2013-01-01

    Extracellular ATP plays a critical role in regulating insulin secretion in pancreatic β cells. The ATP released from insulin secretory vesicles has been proposed to be a major source of extracellular ATP. Currently, the mechanism by which ATP accumulates into insulin secretory granules remains elusive. In this study, the authors identified the expression of a vesicular nucleotide transporter (VNUT) in mouse pancreas, isolated mouse islets, and MIN6 cells, a mouse β cell line. Immunohistochemistry and immunofluorescence revealed that VNUT colocalized extensively with insulin secretory granules. Functional studies showed that suppressing endogenous VNUT expression in β cells by small hairpin RNA knockdown greatly reduced basal- and glucose-induced ATP release. Importantly, knocking down VNUT expression by VNUT small hairpin RNA in MIN6 cells and isolated mouse islets dramatically suppressed basal insulin release and glucose-stimulated insulin secretion (GSIS). Moreover, acute pharmacologic blockade of VNUT with Evans blue, a VNUT antagonist, greatly attenuated GSIS in a dose-dependent manner. Exogenous ATP treatment effectively reversed the insulin secretion defect induced by both VNUT knockdown and functional inhibition, indicating that VNUT-mediated ATP release is essential for maintaining normal insulin secretion. In contrast to VNUT knockdown, overexpression of VNUT in β cells resulted in excessive ATP release and enhanced basal insulin secretion and GSIS. Elevated insulin secretion induced by VNUT overexpression was reversed by pharmacologic inhibition of P2X but not P2Y purinergic receptors. This study reveals VNUT is expressed in pancreatic β cells and plays an essential and novel role in regulating insulin secretion through vesicular ATP release and extracellular purinergic signaling. PMID:23254199

  7. ATP depletion inhibits glucocorticoid-induced thymocyte apoptosis.

    PubMed Central

    Stefanelli, C; Bonavita, F; Stanic', I; Farruggia, G; Falcieri, E; Robuffo, I; Pignatti, C; Muscari, C; Rossoni, C; Guarnieri, C; Caldarera, C M

    1997-01-01

    In quiescent thymocytes, mitochondrial de-energization was not correlated to apoptotic death. In fact, thymocytes treated with oligomycin, a highly specific inhibitor of ATP synthase, alone or with atractyloside to block ATP translocation from the cytoplasm, were alive, even if their mitochondria were depolarized, as revealed by flow cytometry after Rhodamine 123 staining. Furthermore, oligomycin was a powerful inhibitor of apoptosis induced in rat thymocytes by dexamethasone and, to a lesser extent, by the calcium ionophore A23187 and etoposide, but was without effect when apoptosis was induced by staurosporine, and increased cell death in mitogen-treated thymocytes. The inhibition of apoptosis was confirmed by morphological criteria, inhibition of inter-nucleosomal DNA fragmentation and inhibition of the loss of membrane integrity. The anti-apoptotic effect of oligomycin in cells treated with A23187 or etoposide was correlated to the inhibition of protein synthesis, while inhibition of apoptosis induced by dexamethasone, already evident at an oligomycin concentration of 10 ng/ml, was instead strictly correlated to the effect exerted on the cellular ATP level. Thymocyte apoptosis triggered by dexamethasone was blocked or delayed by inhibitors of respiratory-chain uncouplers, inhibitors of ATP synthase and antioxidants: a lasting protection from dexamethasone-induced apoptosis was always correlated to a drastic and rapid reduction in ATP level (31-35% of control), while a delay in the death process was characterized by a moderate decrease in ATP (73-82% of control). Oligomycin inhibited the specific binding of radioactive corticosteroid to thymocyte nuclei, confirming the inhibitory effect of ATP depletion on glucocorticoid binding and suggesting that ATP depletion is a common mediator of the anti-apoptotic action of different effectors in glucocorticoid-induced apoptosis. In conclusion, the reported data indicate that ATP may act as a cellular modulator of some

  8. ATP stimulates pannexin 1 internalization to endosomal compartments.

    PubMed

    Boyce, Andrew K J; Kim, Michelle S; Wicki-Stordeur, Leigh E; Swayne, Leigh Anne

    2015-09-15

    The ubiquitous pannexin 1 (Panx1) ion- and metabolite-permeable channel mediates the release of ATP, a potent signalling molecule. In the present study, we provide striking evidence that ATP, in turn, stimulates internalization of Panx1 to intracellular membranes. These findings hold important implications for understanding the regulation of Panx1 when extracellular ATP is elevated. In the nervous system, this includes phenomena such as synaptic plasticity, pain, precursor cell development and stroke; outside of the nervous system, this includes things like skeletal and smooth muscle activity and inflammation. Within 15 min, ATP led to significant Panx1-EGFP internalization. In a series of experiments, we determined that hydrolysable ATP is the most potent stimulator of Panx1 internalization. We identified two possible mechanisms for Panx1 internalization, including activation of ionotropic purinergic (P2X) receptors and involvement of a putative ATP-sensitive residue in the first extracellular loop of Panx1 (Trp(74)). Internalization was cholesterol-dependent, but clathrin, caveolin and dynamin independent. Detailed analysis of Panx1 at specific endosome sub-compartments confirmed that Panx1 is expressed in endosome membranes of the classical degradation pathway under basal conditions and that elevation of ATP levels diverts a sub-population to recycling endosomes. This is the first report detailing endosome localization of Panx1 under basal conditions and the potential for ATP regulation of its surface expression. Given the ubiquitous expression profile of Panx1 and the importance of ATP signalling, these findings are of critical importance for understanding the role of Panx1 in health and disease. PMID:26195825

  9. ATP13A2 regulates mitochondrial bioenergetics through macroautophagy

    PubMed Central

    Gusdon, Aaron M.; Zhu, Jianhui; Van Houten, Bennett; Chu, Charleen T.

    2012-01-01

    Mitochondrial dysfunction and autophagy are centrally implicated in Parkinson’s disease (PD). Mutations in ATP13A2, which encodes a lysosomal P-type ATPase of unknown function, cause a rare, autosomal recessive parkinsonian syndrome. Lysosomes are essential for autophagy, and autophagic clearance of dysfunctional mitochondria represents an important element of mitochondrial quality control. In this study, we tested the hypothesis that loss of ATP13A2 function will affect mitochondrial function. Knockdown of ATP13A2 led to an increase in mitochondrial mass in primary mouse cortical neurons and SH-SY5Y cells forced into mitochondrial dependence. ATP13A2-deficient cells exhibited increased oxygen consumption without a significant change in steady-state levels of ATP. Mitochondria in knockdown cells exhibited increased fragmentation and increased production of reactive oxygen species (ROS). Basal levels of the autophagosome marker LC3-II were not significantly changed, however, ATP13A2 knockdown cells exhibited decreased autophagic flux, associated with increased levels of phospho-mTOR, and resistance to autophagy induction by rapamycin. The effects of ATP13A2 siRNA on oxygen consumption, mitochondrial mass and ROS production could be mimicked by inhibiting autophagy induction using siRNA to Atg7. We propose that decreased autophagy associated with ATP13A2 deficiency affects mitochondrial quality control, resulting in increased ROS production. These data are the first to implicate loss of ATP13A2 function in mitochondrial maintenance and oxidative stress, lending further support to converging genetic and environmental evidence for mitochondrial dysregulation in PD pathogenesis. PMID:22198378

  10. Twisting and subunit rotation in single FOF1-ATP synthase

    PubMed Central

    Sielaff, Hendrik; Börsch, Michael

    2013-01-01

    FOF1-ATP synthases are ubiquitous proton- or ion-powered membrane enzymes providing ATP for all kinds of cellular processes. The mechanochemistry of catalysis is driven by two rotary nanomotors coupled within the enzyme. Their different step sizes have been observed by single-molecule microscopy including videomicroscopy of fluctuating nanobeads attached to single enzymes and single-molecule Förster resonance energy transfer. Here we review recent developments of approaches to monitor the step size of subunit rotation and the transient elastic energy storage mechanism in single FOF1-ATP synthases. PMID:23267178

  11. A new system for rapid measurement of ATP.

    PubMed

    Shu, B; Zhou, Y; Ren, S

    1997-01-01

    The paper introduces a new type instrument for rapid measuring ATP. The system consists of a micromodule ATP sensor and an instrument for measuring weak light transmitted by optic fiber. The micromodule ATP sensor mainly is composed of enzyme membrane, a probe and a bundle of optic fiber. The instrument measuring weak light consists of photomultiplier, high voltage power, pulse amplifier and counter. The instrument was characterized by simple structure, small size, rapid response time (< 5s), high sensitivity (10(-12) mol/L), stable performance (measuring the same sample for 50 times, CV < 5%), long enzyme storage time (> 3 months). PMID:9812776

  12. Restoration of intracellular ATP production in banked red blood cells improves inducible ATP export and suppresses RBC-endothelial adhesion

    PubMed Central

    Kirby, Brett S.; Hanna, Gabi; Hendargo, Hansford C.

    2014-01-01

    Transfusion of banked red blood cells (RBCs) has been associated with poor cardiovascular outcomes. Storage-induced alterations in RBC glycolytic flux, attenuated ATP export, and microvascular adhesion of transfused RBCs in vivo could contribute, but the underlying mechanisms have not been tested. We tested the novel hypothesis that improving deoxygenation-induced metabolic flux and the associated intracellular ATP generation in stored RBCs (sRBCs) results in an increased extracellular ATP export and suppresses microvascular adhesion of RBCs to endothelium in vivo following transfusion. We show deficient intracellular ATP production and ATP export by human sRBCs during deoxygenation (impairments ∼42% and 49%, respectively). sRBC pretreatment with a solution containing glycolytic intermediate/purine/phosphate precursors (i.e., “PIPA”) restored deoxygenation-induced intracellular ATP production and promoted extracellular ATP export (improvement ∼120% and 50%, respectively). In a nude mouse model of transfusion, adhesion of human RBCs to the microvasculature in vivo was examined. Only 2% of fresh RBCs (fRBCs) transfused adhered to the vascular wall, compared with 16% of sRBCs transfused. PIPA pretreatment of sRBCs significantly reduced adhesion to just 5%. In hypoxia, adhesion of sRBCs transfused was significantly augmented (up to 21%), but not following transfusion of fRBCs or PIPA-treated sRBCs (3.5% or 6%). Enhancing the capacity for deoxygenation-induced glycolytic flux within sRBCs increases their ability to generate intracellular ATP, improves the inducible export of extracellular anti-adhesive ATP, and consequently suppresses adhesion of stored, transfused RBCs to the vascular wall in vivo. PMID:25305182

  13. Mini review: ATP-dependent proteases in bacteria.

    PubMed

    Bittner, Lisa-Marie; Arends, Jan; Narberhaus, Franz

    2016-08-01

    AAA(+) proteases are universal barrel-like and ATP-fueled machines preventing the accumulation of aberrant proteins and regulating the proteome according to the cellular demand. They are characterized by two separate operating units, the ATPase and peptidase domains. ATP-dependent unfolding and translocation of a substrate into the proteolytic chamber is followed by ATP-independent degradation. This review addresses the structure and function of bacterial AAA(+) proteases with a focus on the ATP-driven mechanisms and the coordinated movements in the complex mainly based on the knowledge of ClpXP. We conclude by discussing strategies how novel protease substrates can be trapped by mutated AAA(+) protease variants. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 505-517, 2016. PMID:26971705

  14. Distinct neurological disorders with ATP1A3 mutations

    PubMed Central

    Heinzen, Erin L.; Arzimanoglou, Alexis; Brashear, Allison; Clapcote, Steven J.; Gurrieri, Fiorella; Goldstein, David B.; Jóhannesson, Sigurður H.; Mikati, Mohamad A.; Neville, Brian; Nicole, Sophie; Ozelius, Laurie J.; Poulsen, Hanne; Schyns, Tsveta; Sweadner, Kathleen J.; van den Maagdenberg, Arn; Vilsen, Bente

    2014-01-01

    Genetic research has shown that mutations that modify the protein-coding sequence of ATP1A3, the gene encoding the α3 subunit of Na+/K+-ATPase, cause both rapid-onset dystonia parkinsonism and alternating hemiplegia of childhood. These discoveries link two clinically distinct neurological diseases to the same gene, however, ATP1A3 mutations are, with one exception, disease-specific. Although the exact mechanism of how these mutations lead to disease is still unknown, much knowledge has been gained about functional consequences of ATP1A3 mutations using a range of in vitro and animal model systems, and the role of Na+/K+-ATPases in the brain. Researchers and clinicians are attempting to further characterise neurological manifestations associated with mutations in ATP1A3, and to build on the existing molecular knowledge to understand how specific mutations can lead to different diseases. PMID:24739246

  15. Distinct neurological disorders with ATP1A3 mutations.

    PubMed

    Heinzen, Erin L; Arzimanoglou, Alexis; Brashear, Allison; Clapcote, Steven J; Gurrieri, Fiorella; Goldstein, David B; Jóhannesson, Sigurður H; Mikati, Mohamad A; Neville, Brian; Nicole, Sophie; Ozelius, Laurie J; Poulsen, Hanne; Schyns, Tsveta; Sweadner, Kathleen J; van den Maagdenberg, Arn; Vilsen, Bente

    2014-05-01

    Genetic research has shown that mutations that modify the protein-coding sequence of ATP1A3, the gene encoding the α3 subunit of Na(+)/K(+)-ATPase, cause both rapid-onset dystonia parkinsonism and alternating hemiplegia of childhood. These discoveries link two clinically distinct neurological diseases to the same gene, however, ATP1A3 mutations are, with one exception, disease-specific. Although the exact mechanism of how these mutations lead to disease is still unknown, much knowledge has been gained about functional consequences of ATP1A3 mutations using a range of in-vitro and animal model systems, and the role of Na(+)/K(+)-ATPases in the brain. Researchers and clinicians are attempting to further characterise neurological manifestations associated with mutations in ATP1A3, and to build on the existing molecular knowledge to understand how specific mutations can lead to different diseases. PMID:24739246

  16. ATP citrate lyase improves mitochondrial function in skeletal muscle.

    PubMed

    Das, Suman; Morvan, Frederic; Jourde, Benjamin; Meier, Viktor; Kahle, Peter; Brebbia, Pascale; Toussaint, Gauthier; Glass, David J; Fornaro, Mara

    2015-06-01

    Mitochondrial dysfunction is associated with skeletal muscle pathology, including cachexia, sarcopenia, and the muscular dystrophies. ATP citrate lyase (ACL) is a cytosolic enzyme that catalyzes mitochondria-derived citrate into oxaloacetate and acetyl-CoA. Here we report that activation of ACL in skeletal muscle results in improved mitochondrial function. IGF1 induces activation of ACL in an AKT-dependent fashion. This results in an increase in cardiolipin, thus increasing critical mitochondrial complexes and supercomplex activity, and a resultant increase in oxygen consumption and cellular ATP levels. Conversely, knockdown of ACL in myotubes not only reduces mitochondrial complex I, IV, and V activity but also blocks IGF1-induced increases in oxygen consumption. In vivo, ACL activity is associated with increased ATP. Activation of this IGF1/ACL/cardiolipin pathway combines anabolic signaling with induction of mechanisms needed to provide required ATP. PMID:26039450

  17. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth

    PubMed Central

    Ahmad, Zulfiqar; Laughlin, Thomas F.; Kady, Ismail O.

    2015-01-01

    We examined the thymoquinone induced inhibition of purified F1 or membrane bound F1FO E. coli ATP synthase. Both purified F1 and membrane bound F1FO were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F1Fo and purified F1 preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null control cells demonstrates that ATP synthase is a molecular target for thymoquinone. This also links the beneficial dietary based antimicrobial and anticancer effects of thymoquinone to its inhibitory action on ATP synthase. PMID:25996607

  18. Control and monitoring the effectiveness of different biocides with the use of free ATP

    SciTech Connect

    Chalut, J.; Small, G.; Payton, J.

    1996-12-01

    The Adenosine Triphosphate (ATP) technology can be used as a measurement of the total living biomass. However, care must be exercised in its application and its interpretation. The use of this technique on samples from cooling water systems clearly indicate that there are three distinct pools of ATP, classified as bacterial ATP, free ATP and total ATP, the latter being the sum of the free ATP and bacterial ATP. The mode of action of certain biocidal agents is by disruption of cell membranes, a process which does not decrease the pool of total ATP, but does move ATP from the bacterial ATP pool to the free ATP pool. As a consequence, it is important that for a realistic interpretation to be made, it is necessary to know which biocidal agents are being used and to fully understand their mode of action.

  19. Inhibition of ATPase activity of the recA protein by ATP ribose-modified analogs.

    PubMed

    Karasaki, Y; Higashi, K

    1984-09-01

    The single-stranded, DNA-dependent ATPase activity of purified recA protein was found to be inhibited competitively by ribose-modified analogs of ATP, 3'-O-anthraniloyl-ATP (Ant-ATP), and 3'-O-(N-methylanthraniloyl)-ATP (Mant-ATP). The Ki values for Ant-ATP and Mant-ATP were around 7 and 3 microM at pH 7.5, respectively. The inhibitions by these analogs were much stronger than that by ADP, which is also a competitive inhibitor for the ATPase activity of the recA protein. The Ki value for ADP is 76 microM. Ant-ATP and Mant-ATP reduced the Hill coefficient for ATP hydrolysis and thus contributed to the cooperative effect of ATP. PMID:6237610

  20. Highly Divergent Mitochondrial ATP Synthase Complexes in Tetrahymena thermophila

    PubMed Central

    Balabaskaran Nina, Praveen; Dudkina, Natalya V.; Kane, Lesley A.; van Eyk, Jennifer E.; Boekema, Egbert J.; Mather, Michael W.; Vaidya, Akhil B.

    2010-01-01

    The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F1 sector catalyzes ATP synthesis, whereas the Fo sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F1 and Fo sectors are highly conserved across prokaryotes and eukaryotes. Therefore, it was a surprise that genes encoding the a and b subunits as well as other components of the Fo sector were undetectable in the sequenced genomes of a variety of apicomplexan parasites. While the parasitic existence of these organisms could explain the apparent incomplete nature of ATP synthase in Apicomplexa, genes for these essential components were absent even in Tetrahymena thermophila, a free-living ciliate belonging to a sister clade of Apicomplexa, which demonstrates robust oxidative phosphorylation. This observation raises the possibility that the entire clade of Alveolata may have invented novel means to operate ATP synthase complexes. To assess this remarkable possibility, we have carried out an investigation of the ATP synthase from T. thermophila. Blue native polyacrylamide gel electrophoresis (BN-PAGE) revealed the ATP synthase to be present as a large complex. Structural study based on single particle electron microscopy analysis suggested the complex to be a dimer with several unique structures including an unusually large domain on the intermembrane side of the ATP synthase and novel domains flanking the c subunit rings. The two monomers were in a parallel configuration rather than the angled configuration previously observed in other organisms. Proteomic analyses of well-resolved ATP synthase complexes from 2-D BN/BN-PAGE identified orthologs of seven canonical ATP synthase subunits, and at least 13 novel proteins that constitute subunits apparently limited to the ciliate lineage. A mitochondrially encoded protein, Ymf66, with predicted eight transmembrane domains could be a substitute for the subunit a

  1. NIF Title III engineering plan

    SciTech Connect

    Deis, G

    1998-06-01

    The purpose of this document is to define the work that must be accomplished by the NIF Project during Title III Engineering. This definition is intended to be sufficiently detailed to provide a framework for yearly planning, to clearly identify the specific deliverables so that the Project teams can focus on them, and to provide a common set of objectives and processes across the Project. This plan has been preceded by similar documents for Title I and Title II design and complements the Site Management Plan, the Project Control Manual, the Quality Assurance Program Plan, the RM Parsons NIF Title III Configuration Control Plan, the Integrated Project Schedule, the Preliminary Safety Analysis Report, the Configuration Management Plan, and the Transition Plan.

  2. Extracellular ATP drives systemic inflammation, tissue damage and mortality

    PubMed Central

    Cauwels, A; Rogge, E; Vandendriessche, B; Shiva, S; Brouckaert, P

    2014-01-01

    Systemic inflammatory response syndromes (SIRS) may be caused by both infectious and sterile insults, such as trauma, ischemia-reperfusion or burns. They are characterized by early excessive inflammatory cytokine production and the endogenous release of several toxic and damaging molecules. These are necessary to fight and resolve the cause of SIRS, but often end up progressively damaging cells and tissues, leading to life-threatening multiple organ dysfunction syndrome (MODS). As inflammasome-dependent cytokines such as interleukin-1β are critically involved in the development of MODS and death in SIRS, and ATP is an essential activator of inflammasomes in vitro, we decided to analyze the ability of ATP removal to prevent excessive tissue damage and mortality in a murine LPS-induced inflammation model. Our results indeed indicate an important pro-inflammatory role for extracellular ATP. However, the effect of ATP is not restricted to inflammasome activation at all. Removing extracellular ATP with systemic apyrase treatment not only prevented IL-1β accumulation but also the production of inflammasome-independent cytokines such as TNF and IL-10. In addition, ATP removal also prevented systemic evidence of cellular disintegration, mitochondrial damage, apoptosis, intestinal barrier disruption and even mortality. Although blocking ATP receptors with the broad-spectrum P2 purinergic receptor antagonist suramin imitated certain beneficial effects of apyrase treatment, it could not prevent morbidity or mortality at all. We conclude that removal of systemic extracellular ATP could be a valuable strategy to dampen systemic inflammatory damage and toxicity in SIRS. PMID:24603330

  3. Characterisation of ATP analogues to cross-link and label P2X receptors

    PubMed Central

    Agboh, Kelvin C.; Powell, Andrew J.; Evans, Richard J.

    2009-01-01

    P2X receptors are a distinct family of ATP-gated ion channels with a number of physiological roles ranging from smooth muscle contractility to the regulation of blood clotting. In this study we determined whether the UV light-reactive ATP analogues 2-azido ATP, ATP azidoanilide (ATP-AA) and 2′,3′-O-(4-benzoylbenzoyl)-ATP (BzATP) can be used to label the ATP binding site of P2X1 receptors. These analogues were agonists, and in patch clamp studies evoked inward currents from HEK293 cells stably expressing the P2X1 receptor. Following irradiation in the presence of these compounds subsequent responses to an EC50 concentration of ATP were reduced by >65%. These effects were partially reversed by co-application of ATP or suramin with the photo-reactive ATP analogue at the time of irradiation. In autoradiographic studies radiolabelled 2-azido [γ32P] ATP and ATP-AA-[γ32P] cross-linked to P2X1 receptors and this binding was reduced by co-incubation with ATP. These studies demonstrate that photo-reactive ATP analogues can be used to label P2X receptor and may prove useful in elucidating the ATP binding site at this novel class of ATP binding proteins. PMID:18599093

  4. Increased Intracellular [dATP] Enhances Cardiac Contraction in Embryonic Chick Cardiomyocytes

    PubMed Central

    Schoffstall, Brenda; Chase, P. Bryant

    2016-01-01

    Although ATP is the physiological substrate for cardiac contraction, cardiac contractility is significantly enhanced in vitro when only 10% of ATP substrate is replaced with 2’-deoxy-ATP (dATP). To determine the functional effects of increased intracellular [dATP] ([dATP]i) within living cardiac cells, we used hypertonic loading with varying exogenous dATP/ATP ratios, but constant total nucleotide concentration, to elevate [dATP]i in contractile monolayers of embryonic chick cardiomyocytes. The increase in [dATP]i was estimated from dilution of dye added in parallel with dATP. Cell viability, average contractile amplitude, rates of contraction/relaxation, spontaneous beat frequency, and Ca2+ transient amplitude and kinetics were examined. At total [dATP]i above ~70 μM, spontaneous contractions ceased, and above ~100 μM [dATP]i, membrane blebbing was also observed, consistent with apoptosis. Interestingly, [dATP]i of ~60 μM (~40% increase over basal [dATP]i levels) enhanced both amplitude of contraction and the rates of contraction and relaxation without affecting beat frequency. With total [dATP]i of ~60 μM or less, we found no significant change in Ca2+ transients. These data indicate that there is an “optimal” concentration of exogenously loaded [dATP]i that under controlled conditions can enhance contractility in living cardiomyocytes without affecting beat frequency or Ca2+ transients. PMID:18452163

  5. Inhibition by nilutamide of the mitochondrial respiratory chain and ATP formation. Possible contribution to the adverse effects of this antiandrogen.

    PubMed

    Berson, A; Schmets, L; Fisch, C; Fau, D; Wolf, C; Fromenty, B; Deschamps, D; Pessayre, D

    1994-07-01

    The effects of nilutamide on the mitochondrial respiratory chain were investigated in rats. In isolated mitochondria, nilutamide (100 microM) inhibited respiration that was supported by substrates feeding electrons into complex I of the respiratory chain but did not inhibit respiration that was supported by substrates donating electrons to complexes II, III or IV. Inhibition of complex I occurred without any lag time. In submitochondrial particles, nilutamide (100 microM) decreased both oxygen consumption mediated by NADH and the oxidation of NADH; addition of superoxide dismutase and catalase did not alleviate inhibition. There was no electron spin resonance evidence for detectable mitochondrial formation of the nilutamide nitro anion free radical by submitochondrial particles or for the formation of iron-nitrosyl complexes with mitochondrial Fe-S clusters in isolated hepatocytes. Severe inhibition of complex I by nilutamide (500 microM) led to upstream inhibition of fatty acid beta-oxidation. Nilutamide (100 microM) decreased the mitochondrial membrane potential and ATP formation in mitochondria energized by malate plus glutamate. In hepatocytes incubated without glucose, nilutamide (500 microM) led to an early (2 hr) drop in cellular ATP and early (4 hr) toxicity. With 5 mM glucose, however, ATP was not decreased and toxicity was mild at these early times. It was concluded that nilutamide itself inhibited the mitochondrial respiratory chain at the level of complex I and decreased ATP in hepatocytes incubated without glucose, which resulted in early toxicity. In the presence of glucose, ATP was not depleted at early times and delayed toxicity was probably the result of an oxidative stress (as previously reported). PMID:8035313

  6. High affinity ATP/ADP analogues as new tools for studying CFTR gating.

    PubMed

    Zhou, Zhen; Wang, Xiaohui; Li, Min; Sohma, Yoshiro; Zou, Xiaoqin; Hwang, Tzyh-Chang

    2005-12-01

    Previous studies using non-hydrolysable ATP analogues and hydrolysis-deficient cystic fibrosis transmembrane conductance regulator (CFTR) mutants have indicated that ATP hydrolysis precedes channel closing. Our recent data suggest that ATP binding is also important in modulating the closing rate. This latter hypothesis predicts that ATP analogues with higher binding affinities should stabilize the open state more than ATP. Here we explore the possibility of using N6-modified ATP/ADP analogues as high-affinity ligands for CFTR gating, since these analogues have been shown to be more potent than native ATP/ADP in other ATP-binding proteins. Among the three N6-modified ATP analogues tested, N6-(2-phenylethyl)-ATP (P-ATP) was the most potent, with a K(1/2) of 1.6 +/- 0.4 microm (>50-fold more potent than ATP). The maximal open probability (P(o)) in the presence of P-ATP was approximately 30% higher than that of ATP, indicating that P-ATP also has a higher efficacy than ATP. Single-channel kinetic analysis showed that as [P-ATP] was increased, the opening rate increased, whereas the closing rate decreased. The fact that these two kinetic parameters have different sensitivities to changes of [P-ATP] suggests an involvement of two different ATP-binding sites, a high-affinity site modulating channel closing and a low affinity site controlling channel opening. The effect of P-ATP on the stability of open states was more evident when ATP hydrolysis was abolished, either by mutating the nucleotide-binding domain 2 (NBD2) Walker B glutamate (i.e. E1371) or by using the non-hydrolysable ATP analogue AMP-PNP. Similar strategies to develop nucleotide analogues with a modified adenine ring could be valuable for future studies of CFTR gating. PMID:16223764

  7. High affinity ATP/ADP analogues as new tools for studying CFTR gating

    PubMed Central

    Zhou, Zhen; Wang, Xiaohui; Li, Min; Sohma, Yoshiro; Zou, Xiaoqin; Hwang, Tzyh-Chang

    2005-01-01

    Previous studies using non-hydrolysable ATP analogues and hydrolysis-deficient cystic fibrosis transmembrane conductance regulator (CFTR) mutants have indicated that ATP hydrolysis precedes channel closing. Our recent data suggest that ATP binding is also important in modulating the closing rate. This latter hypothesis predicts that ATP analogues with higher binding affinities should stabilize the open state more than ATP. Here we explore the possibility of using N6-modified ATP/ADP analogues as high-affinity ligands for CFTR gating, since these analogues have been shown to be more potent than native ATP/ADP in other ATP-binding proteins. Among the three N6-modified ATP analogues tested, N6-(2-phenylethyl)-ATP (P-ATP) was the most potent, with a K½ of 1.6 ± 0.4 μm (>50-fold more potent than ATP). The maximal open probability (Po) in the presence of P-ATP was ∼30% higher than that of ATP, indicating that P-ATP also has a higher efficacy than ATP. Single-channel kinetic analysis showed that as [P-ATP] was increased, the opening rate increased, whereas the closing rate decreased. The fact that these two kinetic parameters have different sensitivities to changes of [P-ATP] suggests an involvement of two different ATP-binding sites, a high-affinity site modulating channel closing and a low affinity site controlling channel opening. The effect of P-ATP on the stability of open states was more evident when ATP hydrolysis was abolished, either by mutating the nucleotide-binding domain 2 (NBD2) Walker B glutamate (i.e. E1371) or by using the non-hydrolysable ATP analogue AMP-PNP. Similar strategies to develop nucleotide analogues with a modified adenine ring could be valuable for future studies of CFTR gating. PMID:16223764

  8. Reaction Dynamics of ATP Hydrolysis Catalyzed by P-Glycoprotein

    PubMed Central

    2015-01-01

    P-glycoprotein (P-gp) is a member of the ABC transporter family that confers drug resistance to many tumors by catalyzing their efflux, and it is a major component of drug–drug interactions. P-gp couples drug efflux with ATP hydrolysis by coordinating conformational changes in the drug binding sites with the hydrolysis of ATP and release of ADP. To understand the relative rates of the chemical step for hydrolysis and the conformational changes that follow it, we exploited isotope exchange methods to determine the extent to which the ATP hydrolysis step is reversible. With γ18O4-labeled ATP, no positional isotope exchange is detectable at the bridging β-phosphorus–O−γ-phosphorus bond. Furthermore, the phosphate derived from hydrolysis includes a constant ratio of three 18O/two 18O/one 18O that reflects the isotopic composition of the starting ATP in multiple experiments. Thus, H2O-exchange with HPO42– (Pi) was negligible, suggesting that a [P-gp·ADP·Pi] is not long-lived. This further demonstrates that the hydrolysis is essentially irreversible in the active site. These mechanistic details of ATP hydrolysis are consistent with a very fast conformational change immediately following, or concomitant with, hydrolysis of the γ-phosphate linkage that ensures a high commitment to catalysis in both drug-free and drug-bound states. PMID:24506763

  9. ATP-dependent degradation of ubiquitin-protein conjugates.

    PubMed Central

    Hershko, A; Leshinsky, E; Ganoth, D; Heller, H

    1984-01-01

    Previous studies have indicated that the ATP-requiring conjugation of ubiquitin with proteins plays a role in the energy-dependent degradation of intracellular proteins. To examine whether such conjugates are indeed intermediates in protein breakdown, conjugates of 125I-labeled lysozyme with ubiquitin were isolated and incubated with a fraction of reticulocyte extract that lacks the enzymes that carry out ubiquitin-protein conjugation. ATP markedly stimulated degradation of the lysozyme moiety of ubiquitin conjugates to products soluble in trichloroacetic acid. By contrast, free 125I-labeled lysozyme was not degraded under these conditions, unless ubiquitin and the three enzymes required for ubiquitin conjugation were supplemented. Mg2+ was absolutely required for conjugate breakdown. Of various nucleotides, only CTP replaced ATP. Nonhydrolyzable analogs of ATP were not effective. In the absence of ATP, free lysozyme is released from ubiquitin-lysozyme conjugates by isopeptidases present in the extract. Thus, ATP is involved in both the formation and the breakdown of ubiquitin-protein conjugates. Images PMID:6324208

  10. Effect of glucose on ATP dephosphorylation in rat spermatids.

    PubMed

    Grootegoed, J A; Jansen, R; van der Molen, H J

    1986-05-01

    Round spermatids were isolated from rat testes and the effects of different energy-yielding substrates on the cellular ATP content were estimated. The ATP content was constant and high (6-8 nmol/10(6) cells) during metabolism of exogenous lactate. During incubation for 30 min in the absence of exogenous lactate, there was a remarkably slow decline of the ATP content, indicating ATP production from other substrates. It was shown that this could reflect beta-oxidation of fatty acids, but not the mobilization of an endogenous pool of acetylcarnitine. Glucose metabolism in the absence of exogenous lactate resulted in a rapid decline of the ATP content. This effect of glucose was correlated with a high fructose 1,6-biphosphate content (6-7 nmol/10(6) cells) and could be prevented by the addition of lactate. It is suggested that metabolism of glucose (and also mannose and fructose, but not galactose) in the absence of exogenous lactate can result in ATP dephosphorylation. PMID:3723480

  11. Catalytic Mechanism of the Maltose Transporter Hydrolyzing ATP.

    PubMed

    Huang, Wenting; Liao, Jie-Lou

    2016-01-12

    We use quantum mechanical and molecular mechanical (QM/MM) simulations to study ATP hydrolysis catalyzed by the maltose transporter. This protein is a prototypical member of a large family that consists of ATP-binding cassette (ABC) transporters. The ABC proteins catalyze ATP hydrolysis to perform a variety of biological functions. Despite extensive research efforts, the precise molecular mechanism of ATP hydrolysis catalyzed by the ABC enzymes remains elusive. In this work, the reaction pathway for ATP hydrolysis in the maltose transporter is evaluated using a QM/MM implementation of the nudged elastic band method without presuming reaction coordinates. The potential of mean force along the reaction pathway is obtained with an activation free energy of 19.2 kcal/mol in agreement with experiments. The results demonstrate that the reaction proceeds via a dissociative-like pathway with a trigonal bipyramidal transition state in which the cleavage of the γ-phosphate P-O bond occurs and the O-H bond of the lytic water molecule is not yet broken. Our calculations clearly show that the Walker B glutamate as well as the switch histidine stabilizes the transition state via electrostatic interactions rather than serving as a catalytic base. The results are consistent with biochemical and structural experiments, providing novel insight into the molecular mechanism of ATP hydrolysis in the ABC proteins. PMID:26666844

  12. Phospho-oligosaccharide dependent phosphorylation of ATP citrate lyase.

    PubMed

    Puerta, J; Mato, J M; Alemany, S

    1990-01-01

    The effect of insulin on ATP citrate lyase phosphorylation has been shown to be mimicked by a phospho-oligosaccharide in intact adipocytes. We demonstrate that the addition of phospho-oligosaccharide to intact adipocytes enhances the phosphorylation of ATP citrate lyase in the same tryptic peptide as insulin does. The addition of phospho-oligosaccharide to an adipocyte extract also results in an increase in ATP citrate lyase phosphorylation but in a different site than that observed in intact cells. The phospho-oligosaccharide-dependent incorporation of phosphate into ATP citrate lyase in intact cells is resistant to isopropanol and acetic acid, but the phosphoenzyme phosphorylated in cell extracts is acid labile. In cell extracts, the addition of phospho-oligosaccharide markedly inhibits ATP hydrolysis, which may explain the effect of this molecule on ATP citrate lyase phosphorylation in broken cells. These results support the hypothesis that this phospho-oligosaccharide mediates some of the effects of insulin on protein phosphorylation. They also indicate that caution should be exercised in interpreting the results obtained by adding phospho-oligosaccharide to broken cell preparations. PMID:2119547

  13. Structural basis for the ATP-induced isomerization of kinesin.

    PubMed

    Chang, Qing; Nitta, Ryo; Inoue, Shigeyuki; Hirokawa, Nobutaka

    2013-06-12

    Kinesin superfamily proteins (KIFs) are microtubule-based molecular motors driven by the energy derived from the hydrolysis of ATP. Previous studies have revealed that the ATP binding step is crucial both for the power stroke to produce motility and for the inter-domain regulation of ATPase activity to guarantee the processive movement of dimeric KIFs. Here, we report the first crystal structure of KIF4 complexed with the non-hydrolyzable ATP analog, AMPPNP (adenylyl imidodiphosphate), at 1.7Å resolution. By combining our structure with previously solved KIF1A structures complexed with two ATP analogs, molecular snapshots during ATP binding reveal that the closure of the nucleotide-binding pocket during ATP binding is achieved by closure of the backdoor. Closure of the backdoor stabilizes two mobile regions, switch I and switch II, to generate the phosphate tube from which hydrolyzed phosphate is released. Through the stabilization of switch II, the local conformational change at the catalytic center is further relayed to the neck-linker element that fully docks to the catalytic core to produce the power stroke. Because the neck linker is a sole element that connects the partner heads in dimeric KIFs, this tight structural coordination between the catalytic center and neck linker enables inter-domain communication between the partner heads. This study also revealed the putative microtubule-binding site of KIF4, thus providing structural insights that describe the specific binding of KIF4 to the microtubule. PMID:23500491

  14. Synthesis and in vitro microbial evaluation of La(III), Ce(III), Sm(III) and Y(III) metal complexes of vitamin B6 drug

    NASA Astrophysics Data System (ADS)

    Refat, Moamen S.; Al-Azab, Fathi M.; Al-Maydama, Hussein M. A.; Amin, Ragab R.; Jamil, Yasmin M. S.

    2014-06-01

    Metal complexes of pyridoxine mono hydrochloride (vitamin B6) are prepared using La(III), Ce(III), Sm(III) and Y(III). The resulting complexes are investigated. Some physical properties, conductivity, analytical data and the composition of the four pyridoxine complexes are discussed. The elemental analysis shows that the formed complexes of La(III), Ce(III), Sm(III) and Y(III) with pyridoxine are of 1:2 (metal:PN) molar ratio. All the synthesized complexes are brown in color and possess high melting points. These complexes are partially soluble in hot methanol, dimethylsulfoxide and dimethylformamide and insoluble in water and some other organic solvents. Elemental analysis data, spectroscopic (IR, UV-vis. and florescence), effective magnetic moment in Bohr magnetons and the proton NMR suggest the structures. However, definite particle size is determined by invoking the X-ray powder diffraction and scanning electron microscopy data. The results obtained suggested that pyridoxine reacted with metal ions as a bidentate ligand through its phenolate oxygen and the oxygen of the adjacent group at the 4‧-position. The molar conductance measurements proved that the pyridoxine complexes are electrolytic in nature. The kinetic and thermodynamic parameters such as: Ea, ΔH*, ΔS* and ΔG* were estimated from the DTG curves. The antibacterial evaluation of the pyridoxine and their complexes were also performed against some gram positive, negative bacteria as well as fungi.

  15. cDNA, genomic sequence cloning and overexpression of giant panda (Ailuropoda melanoleuca) mitochondrial ATP synthase ATP5G1.

    PubMed

    Hou, W-R; Hou, Y-L; Ding, X; Wang, T

    2012-01-01

    The ATP5G1 gene is one of the three genes that encode mitochondrial ATP synthase subunit c of the proton channel. We cloned the cDNA and determined the genomic sequence of the ATP5G1 gene from the giant panda (Ailuropoda melanoleuca) using RT-PCR technology and touchdown-PCR, respectively. The cloned cDNA fragment contains an open reading frame of 411 bp encoding 136 amino acids; the length of the genomic sequence is of 1838 bp, containing three exons and two introns. Alignment analysis revealed that the nucleotide sequence and the deduced protein sequence are highly conserved compared to Homo sapiens, Mus musculus, Rattus norvegicus, Bos taurus, and Sus scrofa. The homologies for nucleotide sequences of the giant panda ATP5G1 to those of these species are 93.92, 92.21, 92.46, 93.67, and 92.46%, respectively, and the homologies for amino acid sequences are 90.44, 95.59, 93.38, 94.12, and 91.91%, respectively. Topology prediction showed that there is one protein kinase C phosphorylation site, one casein kinase II phosphorylation site, five N-myristoylation sites, and one ATP synthase c subunit signature in the ATP5G1 protein of the giant panda. The cDNA of ATP5G1 was transfected into Escherichia coli, and the ATP5G1 fused with the N-terminally GST-tagged protein gave rise to accumulation of an expected 40-kDa polypeptide, which had the characteristics of the predicted protein. PMID:23007995

  16. Molecular mechanism of sulphonylurea block of K(ATP) channels carrying mutations that impair ATP inhibition and cause neonatal diabetes.

    PubMed

    Proks, Peter; de Wet, Heidi; Ashcroft, Frances M

    2013-11-01

    Sulphonylurea drugs are the therapy of choice for treating neonatal diabetes (ND) caused by mutations in the ATP-sensitive K(+) channel (KATP channel). We investigated the interactions between MgATP, MgADP, and the sulphonylurea gliclazide with KATP channels expressed in Xenopus oocytes. In the absence of MgATP, gliclazide block was similar for wild-type channels and those carrying the Kir6.2 ND mutations R210C, G334D, I296L, and V59M. Gliclazide abolished the stimulatory effect of MgATP on all channels. Conversely, high MgATP concentrations reduced the gliclazide concentration, producing a half-maximal block of G334D and R201C channels and suggesting a mutual antagonism between nucleotide and gliclazide binding. The maximal extent of high-affinity gliclazide block of wild-type channels was increased by MgATP, but this effect was smaller for ND channels; channels that were least sensitive to ATP inhibition showed the smallest increase in sulphonylurea block. Consequently, G334D and I296L channels were not fully blocked, even at physiological MgATP concentrations (1 mmol/L). Glibenclamide block was also reduced in β-cells expressing Kir6.2-V59M channels. These data help to explain why patients with some mutations (e.g., G334D, I296L) are insensitive to sulphonylurea therapy, why higher drug concentrations are needed to treat ND than type 2 diabetes, and why patients with severe ND mutations are less prone to drug-induced hypoglycemia. PMID:23835339

  17. [Determination of the optimal proportions as regards toxicity of AET, ATP and serotonin used in combination].

    PubMed

    Benova, D K; Ptev, I Kh

    1985-01-01

    In experiments on mice, a study was made of the quantitative dependence of toxicity of AET, ATP and serotonin applied in combinations. The toxicity decreased when ATP was combined with AET and increased when ATP of AET were combined with serotonin. The toxicity of a combination of all three substances was reduced by introducing high doses of ATP. PMID:3975373

  18. Leishmania donovani: intracellular ATP level regulates apoptosis-like death in luteolin induced dyskinetoplastid cells.

    PubMed

    Sen, Nilkantha; Das, Benu Brata; Ganguly, Agneyo; Banerjee, Bijoylaxhmi; Sen, Tanusree; Majumder, Hemanta K

    2006-11-01

    Leishmaniasis presents a spectrum of diseases ranging from benign cutaneous lesions to the often-fatal visceralizing form. Luteolin, a dietary flavone induces apoptosis-like death in both promastigote and amastigote forms of Leishmania, the causative agent of the diseases. Here, we have elucidated the mechanism of action of luteolin by analyzing the mitochondrial and cytosolic changes associated with apoptosis-like death of leishmanial cells. In Leishmania donovani, treatment with luteolin induces the loss of both maxicircles and minicircles which resulted in the formation of dyskinetoplastid cells. The loss of mitochondrial DNA causes reduction in the activities of complex I, II, III, and IV of electron transport chain. However, the mitochondrial ATPase activity of complex V remains almost unaltered during treatment with luteolin but the sensitivity to oligomycin is lost. The inactivation of ETC complex is associated with decrease in mitochondrial as well as glycolytic ATP production, which is responsible for depolarization of Deltapsi(m) and alteration in mitochondrial structure. This event is followed by the release of cytochrome c from mitochondria in mt-DNA depleted leishmanial cells and causes an activation of caspase like proteases. Collectively our results provide the first insight into the mechanistic pathway of apoptosis-like death where inhibition of glycolytic ATP production is an essential event responsible for depolarization of Deltapsi(m) in mt-DNA depleted cells to propagate apoptosis-like death in leishmanial cells. PMID:16707127

  19. Regulation of yeast acetohydroxyacid synthase by valine and ATP.

    PubMed Central

    Pang, S S; Duggleby, R G

    2001-01-01

    The first step in the common pathway for the biosynthesis of branched-chain amino acids is catalysed by acetohydroxyacid synthase (AHAS; EC 4.1.3.18). The enzyme is found in plants, fungi and bacteria, and is regulated by controls on transcription and translation, and by allosteric modulation of catalytic activity. It has long been known that the bacterial enzyme is composed of two types of subunit, and a similar arrangement has been found recently for the yeast and plant enzymes. One type of subunit contains the catalytic machinery, whereas the other has a regulatory function. Previously, we have shown [Pang and Duggleby (1999) Biochemistry 38, 5222--5231] that yeast AHAS can be reconstituted from its separately purified subunits. The reconstituted enzyme is inhibited by valine, and ATP reverses this inhibition. In the present work, we further characterize the structure and the regulatory properties of reconstituted yeast AHAS. High phosphate concentrations are required for reconstitution and it is shown that these conditions are necessary for physical association between the catalytic and regulatory subunits. It is demonstrated by CD spectral changes that ATP binds to the regulatory subunit alone, most probably as MgATP. Neither valine nor MgATP causes dissociation of the regulatory subunit from the catalytic subunit. The specificity of valine inhibition and MgATP activation are examined and it is found that the only effective analogue of either regulator of those tested is the non-hydrolysable ATP mimic, adenosine 5'-[beta,gamma-imido]triphosphate. The kinetics of regulation are studied in detail and it is shown that the activation by MgATP depends on the valine concentration in a complex manner that is consistent with a proposed quantitative model. PMID:11463345

  20. Protease La from Escherichia coli Hydrolyzes ATP and Proteins in a Linked Fashion

    NASA Astrophysics Data System (ADS)

    Waxman, Lloyd; Goldberg, Alfred L.

    1982-08-01

    The energy requirement for protein breakdown in Escherichia coli results from an ATP requirement for the function of protease La, the product of the lon gene. This novel serine protease contains an ATPase activity that is essential for proteolysis. ATP and protein hydrolysis show the same Km for ATP (30-40 μ M) and are affected similarly by various inhibitors, activators, and ATP analogs. Vanadate inhibited ATP cleavage and caused a proportionate reduction in casein hydrolysis, and inhibitors of serine proteases reduced ATP cleavage. Thus, ATP and protein hydrolysis appear to be linked stoichiometrically. Furthermore, ATP hydrolysis is stimulated two- to threefold by polypeptides that are substrates for the protease (casein, glucagon) but not by nonhydrolyzed polypeptides (insulin, RNase). Unlike hemoglobin or native albumin, globin and denatured albumin stimulated ATP hydrolysis and were substrates for proteolysis. It is suggested that the stimulation of ATP hydrolysis by potential substrates triggers activation of the proteolytic function.

  1. Structural Mechanism of Allosteric Activity Regulation in a Ribonucleotide Reductase with Double ATP Cones.

    PubMed

    Johansson, Renzo; Jonna, Venkateswara Rao; Kumar, Rohit; Nayeri, Niloofar; Lundin, Daniel; Sjöberg, Britt-Marie; Hofer, Anders; Logan, Derek T

    2016-06-01

    Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides. Their overall activity is stimulated by ATP and downregulated by dATP via a genetically mobile ATP cone domain mediating the formation of oligomeric complexes with varying quaternary structures. The crystal structure and solution X-ray scattering data of a novel dATP-induced homotetramer of the Pseudomonas aeruginosa class I RNR reveal the structural bases for its unique properties, namely one ATP cone that binds two dATP molecules and a second one that is non-functional, binding no nucleotides. Mutations in the observed tetramer interface ablate oligomerization and dATP-induced inhibition but not the ability to bind dATP. Sequence analysis shows that the novel type of ATP cone may be widespread in RNRs. The present study supports a scenario in which diverse mechanisms for allosteric activity regulation are gained and lost through acquisition and evolutionary erosion of different types of ATP cone. PMID:27133024

  2. CFTR mediates noradrenaline-induced ATP efflux from DRG neurons

    PubMed Central

    2011-01-01

    In our earlier study, noradrenaline (NA) stimulated ATP release from dorsal root ganglion (DRG) neurons as mediated via β3 adrenoceptors linked to Gs protein involving protein kinase A (PKA) activation, to cause allodynia. The present study was conducted to understand how ATP is released from DRG neurons. In an outside-out patch-clamp configuration from acutely dissociated rat DRG neurons, single-channel currents, sensitive to the P2X receptor inhibitor PPADS, were evoked by approaching the patch-electrode tip close to a neuron, indicating that ATP is released from DRG neurons, to activate P2X receptor. NA increased the frequency of the single-channel events, but such NA effect was not found for DRG neurons transfected with the siRNA to silence the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In the immunocytochemical study using acutely dissociated rat DRG cells, CFTR was expressed in neurons alone, but not satellite cells, fibroblasts, or Schwann cells. It is concluded from these results that CFTR mediates NA-induced ATP efflux from DRG neurons as an ATP channel. PMID:21943397

  3. ATP Hydrolyzing Salivary Enzymes of Caterpillars Suppress Plant Defenses

    PubMed Central

    Wu, Shuang; Peiffer, Michelle; Luthe, Dawn S.; Felton, Gary W.

    2012-01-01

    The oral secretions of herbivores are important recognition cues that can be used by plants to mediate induced defenses. In this study, a degradation of adenosine-5′-triphosphate (ATP) in tomato leaves was detected after treatment with Helicoverpa zea saliva. Correspondingly, a high level of ATPase activity in saliva was detected and three ATP hydrolyzing enzymes: apyrase, ATP synthase and ATPase 13A1 were identified in salivary glands. To determine the functions of these proteins in mediating defenses, they were cloned from H. zea and expressed in Escherichia coli. By applying the purified expressed apyrase, ATP synthase or ATPase 13A1 to wounded tomato leaves, it was determined that these ATP hydrolyzing enzymes suppressed the defensive genes regulated by the jasmonic acid and ethylene pathways in tomato plant. Suppression of glandular trichome production was also observed after treatment. Blood-feeding arthropods employ 5′-nucleotidase family of apyrases to circumvent host responses and the H. zea apyrase, is also a member of this family. The comparatively high degree of sequence similarity of the H. zea salivary apyrase with mosquito apyrases suggests a broader evolutionary role for salivary apyrases than previously envisioned. PMID:22848670

  4. ATP P2X3 receptors and neuronal sensitization

    PubMed Central

    Fabbretti, Elsa

    2013-01-01

    Increasing evidence indicates the importance of extracellular adenosine triphosphate (ATP) in the modulation of neuronal function. In particular, fine control of ATP release and the selective and discrete ATP receptor operation are crucial elements of the crosstalk between neuronal and non-neuronal cells in the peripheral and central nervous systems. In peripheral neurons, ATP signaling gives an important contribution to neuronal sensitization, especially that involved in neuropathic pain. Among other subtypes, P2X3 receptors expressed on sensory neurons are sensitive even to nanomolar concentrations of extracellular ATP, and therefore are important transducers of pain stimuli. P2X3 receptor function is highly sensitive to soluble factors like neuropeptides and neurotrophins, and is controlled by transduction mechanisms, protein-protein interactions and discrete membrane compartmentalization. More recent findings have demonstrated that P2X3 receptors interact with the synaptic scaffold protein calcium/calmodulin-dependent serine protein kinase (CASK) in a state dependent fashion, indicating that CASK plays a crucial role in the modulation of P2X3 receptor stability and efficiency. Activation of P2X3 receptors within CASK/P2X3 complex has important consequences for neuronal plasticity and possibly for the release of neuromodulators and neurotransmitters. Better understanding of the interactome machinery of P2X3 receptors and their integration with other receptors and channels on neuronal surface membranes, is proposed to be essential to unveil the process of neuronal sensitization and related, abnormal pain signaling. PMID:24363643

  5. Cardiac Metabolism in Heart Failure - Implications beyond ATP production

    PubMed Central

    Doenst, Torsten; Nguyen, T. Dung; Abel, E. Dale

    2013-01-01

    The heart has a high rate of ATP production and turnover which is required to maintain its continuous mechanical work. Perturbations in ATP generating processes may therefore affect contractile function directly. Characterizing cardiac metabolism in heart failure revealed several metabolic alterations termed metabolic remodeling, ranging from changes in substrate utilization to mitochondrial dysfunction, ultimately resulting in ATP deficiency and impaired contractility. However, ATP depletion is not the only relevant consequence of metabolic remodeling during heart failure. By providing cellular building blocks and signaling molecules, metabolic pathways control essential processes such as cell growth and regeneration. Thus, alterations in cardiac metabolism may also affect the progression to heart failure by mechanisms beyond ATP supply. Our aim is therefore to highlight that metabolic remodeling in heart failure not only results in impaired cardiac energetics, but also induces other processes implicated in the development of heart failure such as structural remodeling and oxidative stress. Accordingly, modulating cardiac metabolism in heart failure may have significant therapeutic relevance that goes beyond the energetic aspect. PMID:23989714

  6. Regulation of CFTR ion channel gating by MgATP.

    PubMed

    Aleksandrov, A A; Riordan, J R

    1998-07-10

    Single channel currents of wild-type CFTR reconstituted in lipid bilayers were recorded to study the temperature dependence of channel gating between +20 degrees C and +40 degrees C. The opening of the channel was highly temperature dependent and required an activation energy of about 100 kJ/mol. Closing of the channel was only weakly temperature dependent with an activation energy close to that of diffusion in water. We found no significant difference in the free energy between the open and closed states. Most of the excess energy needed to activate channel opening is used to diminish the entropy of the open state. This structural reorganization is initiated by ATP binding followed by interconversion to the open channel structure as the CFTR-ATP-Mg complex passes to the transition state for hydrolysis. The energy of the CFTR-ATP-Mg interaction in the transition state is responsible for the CFTR ion channel opening rather than the energy of ATP hydrolysis. Channel closing is a diffusion limited process and does not require additional ATP binding. PMID:9684873

  7. ATP synthase: from single molecule to human bioenergetics

    PubMed Central

    KAGAWA, Yasuo

    2010-01-01

    ATP synthase (FoF1) consists of an ATP-driven motor (F1) and a H+-driven motor (Fo), which rotate in opposite directions. FoF1 reconstituted into a lipid membrane is capable of ATP synthesis driven by H+ flux. As the basic structures of F1 (α3β3γδε) and Fo (ab2c10) are ubiquitous, stable thermophilic FoF1 (TFoF1) has been used to elucidate molecular mechanisms, while human F1Fo (HF1Fo) has been used to study biomedical significance. Among F1s, only thermophilic F1 (TF1) can be analyzed simultaneously by reconstitution, crystallography, mutagenesis and nanotechnology for torque-driven ATP synthesis using elastic coupling mechanisms. In contrast to the single operon of TFoF1, HFoF1 is encoded by both nuclear DNA with introns and mitochondrial DNA. The regulatory mechanism, tissue specificity and physiopathology of HFoF1 were elucidated by proteomics, RNA interference, cytoplasts and transgenic mice. The ATP synthesized daily by HFoF1 is in the order of tens of kilograms, and is primarily controlled by the brain in response to fluctuations in activity. PMID:20689227

  8. Rotation and structure of FoF1-ATP synthase.

    PubMed

    Okuno, Daichi; Iino, Ryota; Noji, Hiroyuki

    2011-06-01

    F(o)F(1)-ATP synthase is one of the most ubiquitous enzymes; it is found widely in the biological world, including the plasma membrane of bacteria, inner membrane of mitochondria and thylakoid membrane of chloroplasts. However, this enzyme has a unique mechanism of action: it is composed of two mechanical rotary motors, each driven by ATP hydrolysis or proton flux down the membrane potential of protons. The two molecular motors interconvert the chemical energy of ATP hydrolysis and proton electrochemical potential via the mechanical rotation of the rotary shaft. This unique energy transmission mechanism is not found in other biological systems. Although there are other similar man-made systems like hydroelectric generators, F(o)F(1)-ATP synthase operates on the nanometre scale and works with extremely high efficiency. Therefore, this enzyme has attracted significant attention in a wide variety of fields from bioenergetics and biophysics to chemistry, physics and nanoscience. This review summarizes the latest findings about the two motors of F(o)F(1)-ATP synthase as well as a brief historical background. PMID:21524994

  9. ATP can be dispensable for prespliceosome formation in yeast

    PubMed Central

    Perriman, Rhonda; Ares, Manuel

    2000-01-01

    The first ATP-dependent step in pre-mRNA splicing involves the stable binding of U2 snRNP to form the prespliceosome. We show that a prespliceosome-like complex forms in the absence of ATP in yeast extracts lacking the U2 suppressor protein CUS2. These complexes display the same pre-mRNA and U snRNA requirements as authentic prespliceosomes and can be chased through the splicing pathway, indicating that they are a functional intermediate in the spliceosome assembly pathway. ATP-independent prespliceosome-like complexes are also observed in extracts containing a mutant U2 snRNA. Loss of CUS2 does not bypass the role of PRP5, an RNA helicase family member required for ATP-dependent prespliceosome formation. Genetic interactions between CUS2 and a heat-sensitive prp5 allele parallel those observed between CUS2 and U2, and suggest that CUS2 mediates functional interactions between U2 RNA and PRP5. We propose that CUS2 enforces ATP dependence during formation of the prespliceosome by brokering an interaction between PRP5 and the U2 snRNP that depends on correct U2 RNA structure. PMID:10640279

  10. Neuroretinitis -- definition

    MedlinePlus

    ... page: //medlineplus.gov/ency/article/007624.htm Neuroretinitis - definition To use the sharing features on this page, ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

  11. Timely management of developing class III malocclusion.

    PubMed

    Yelampalli, M R; Rachala, M R

    2012-01-01

    Timing of orthodontic treatment, especially for children with developing class III malocclusions, has always been somewhat controversial, and definitive treatment tends to be delayed for severe class III cases. Developing class III patients with moderate to severe anterior crossbite and deep bite may need early intervention in some selected cases. Class III malocclusion may develop in children as a result of an inherent growth abnormality, i.e. true class III malocclusion, or as a result of premature occlusal contacts causing forward functional shift of the mandible, which is known as pseudo class III malocclusion. These cases, if not treated at the initial stage of development, interfere with normal growth of the jaw bases and may result in severe facial deformities. The treatment should be carried out as early as possible for permitting normal growth of the skeletal bases. This paper deals with the selection of an appropriate appliance from the various current options available for early intervention in developing class III malocclusion through two case reports. PMID:22565523

  12. Pyrophosphate-Dependent ATP Formation from Acetyl Coenzyme A in Syntrophus aciditrophicus , a New Twist on ATP Formation

    DOE PAGESBeta

    James, Kimberly L.; Ríos-Hernández, Luis A.; Wofford, Neil Q.; Mouttaki, Housna; Sieber, Jessica R.; Sheik, Cody S.; Nguyen, Hong H.; Yang, Yanan; Xie, Yongming; Erde, Jonathan; et al

    2016-08-16

    Syntrophus aciditrophicusis a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation byS. aciditrophicus. However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome ofS. aciditrophicusleaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA) and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show thatS. aciditrophicususes AMP-forming, acetyl-CoA synthetase (Acs1)more » for ATP synthesis from acetyl-CoA.acs1mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, ofS. aciditrophicusgrown in pure culture and coculture. Cell extracts ofS. aciditrophicushad low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified fromS. aciditrophicusand recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4) inS. aciditrophicuscells support the operation of Acs1 in the acetate-forming direction. Thus,S. aciditrophicushas a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase. We find bacteria use two enzymes, phosphate acetyltransferase and acetate kinase, to make ATP from acetyl-CoA, while acetate-forming archaea use a single enzyme, an ADP-forming, acetyl-CoA synthetase, to synthesize ATP and acetate from acetyl-CoA.Syntrophus aciditrophicusapparently relies on a different approach to conserve energy during acetyl-CoA metabolism, as

  13. ATP level and caffeine efficiency on cytokinesis inhibition in plants.

    PubMed

    López-Sáez, J F; Mingo, R; González-Fernández, A

    1982-06-01

    Plant cytokinesis appears to be a topographically organized process of exocytosis. Golgi vesicles which contain cell wall precursors are translocated during telophase, by interzonal microtubules, to the equatorial region of the mitotic apparatus where they fuse with each other giving rise to the new cell wall. Caffeine inhibits cytokinesis by hindering Golgi vesicle coalescence. The present results demonstrate that treatments which increase the cellular ATP level (adenosine, cycloheximide and anisomycin) counteract caffein-induced cytokinesis inhibition in meristem cells of onion root tips (Allium cepa L.), while treatments which decrease ATP level potentiate this caffeine effect (dinitrophenol, fluoroacetate, low oxygen tensions, etc.). We postulate that caffeine, in competition with the cellular ATP level, blocks cell plate formation by inhibiting a certain ATPase activity required for membrane fusion of Golgi vesicles. PMID:7117265

  14. Rotary catalysis of FoF1-ATP synthase

    PubMed Central

    Watanabe, Rikiya

    2013-01-01

    The synthesis of ATP, the key reaction of biological energy metabolism, is accomplished by the rotary motor protein; FoF1-ATP synthase (FoF1). In vivo, FoF1, located on the cell membrane, carries out ATP synthesis by using the proton motive force. This heterologous energy conversion is supposed to be mediated by the mechanical rotation of FoF1; however, it still remained unclear. Recently, we developed the novel experimental setup to reproduce the proton motive force in vitro and succeeded in directly observing the proton-driven rotation of FoF1. In this review, we describe the interesting working principles determined so far for FoF1 and then introduce results from our recent study. PMID:27493540

  15. Structural Basis for Substrate Binding and the Catalytic Mechanism of Type III Pantothenate Kinase

    SciTech Connect

    Yang, Kun; Strauss, Erick; Huerta, Carlos; Zhang, Hong

    2008-07-15

    Pantothenate kinase (PanK) catalyzes the first step of the universal five-step coenzyme A (CoA) biosynthetic pathway. The recently characterized type III PanK (PanK-III, encoded by the coaX gene) is distinct in sequence, structure and enzymatic properties from both the long-known bacterial type I PanK (PanK-I, exemplified by the Escherichia coli CoaA protein) and the predominantly eukaryotic type II PanK (PanK-II). PanK-III enzymes have an unusually high K{sub m} for ATP, are resistant to feedback inhibition by CoA, and are unable to utilize the N-alkylpantothenamide family of pantothenate analogues as alternative substrates, thus making type III PanK ineffective in generating CoA analogues as antimetabolites in vivo. Previously, we reported the crystal structure of the PanK-III from Thermotoga maritima and identified it as a member of the 'acetate and sugar kinase/heat shock protein 70/actin' (ASKHA) superfamily. Here we report the crystal structures of the same PanK-III in complex with one of its substrates (pantothenate), its product (phosphopantothenate) as well as a ternary complex structure of PanK-III with pantothenate and ADP. These results are combined with isothermal titration calorimetry experiments to present a detailed structural and thermodynamic characterization of the interactions between PanK-III and its substrates ATP and pantothenate. Comparison of substrate binding and catalytic sites of PanK-III with that of eukaryotic PanK-II revealed drastic differences in the binding modes for both ATP and pantothenate substrates, and suggests that these differences may be exploited in the development of new inhibitors specifically targeting PanK-III.

  16. The alpha-subunit of Leishmania F1 ATP synthase hydrolyzes ATP in presence of tRNA.

    PubMed

    Goswami, Srikanta; Adhya, Samit

    2006-07-14

    Import of tRNAs into the mitochondria of the kinetoplastid protozoon Leishmania requires the tRNA-dependent hydrolysis of ATP leading to the generation of membrane potential through the pumping of protons. Subunit RIC1 of the inner membrane RNA import complex is a bi-functional protein that is identical to the alpha-subunit of F1F0 ATP synthase and specifically binds to a subset (Type I) of importable tRNAs. We show that recombinant, purified RIC1 is a Type I tRNA-dependent ATP hydrolase. The activity was insensitive to oligomycin, sensitive to mutations within the import signal of the tRNA, and required the cooperative interaction between the ATP-binding and C-terminal domains of RIC1. The ATPase activity of the intact complex was inhibited by anti-RIC1 antibody, while knockdown of RIC1 in Leishmania tropica resulted in deficiency of the tRNA-dependent ATPase activity of the mitochondrial inner membrane. Moreover, RIC1 knockdown extracts failed to generate a membrane potential across reconstituted proteoliposomes, as shown by a rhodamine 123 uptake assay, but activity was restored by adding back purified RIC1. These observations identify RIC1 as a novel form of the F1 ATP synthase alpha-subunit that acts as the major energy transducer for tRNA import. PMID:16735512

  17. Comparing the catalytic strategy of ATP hydrolysis in biomolecular motors.

    PubMed

    Kiani, Farooq Ahmad; Fischer, Stefan

    2016-07-27

    ATP-driven biomolecular motors utilize the chemical energy obtained from the ATP hydrolysis to perform vital tasks in living cells. Understanding the mechanism of enzyme-catalyzed ATP hydrolysis reaction has substantially progressed lately thanks to combined quantum/classical molecular mechanics (QM/MM) simulations. Here, we present a comparative summary of the most recent QM/MM results for myosin, kinesin and F1-ATPase motors. These completely different motors achieve the acceleration of ATP hydrolysis through a very similar catalytic mechanism. ATP hydrolysis has high activation energy because it involves the breaking of two strong bonds, namely the Pγ-Oβγ bond of ATP and the H-O bond of lytic water. The key to the four-fold decrease in the activation barrier by the three enzymes is that the breaking of the Pγ-Oβγ bond precedes the deprotonation of the lytic water molecule, generating a metaphosphate hydrate complex. The resulting singly charged trigonal planar PγO3(-) metaphosphate is a better electrophilic target for attack by an OaH(-) hydroxyl group. The formation of this OaH(-) is promoted by a strong polarization of the lytic water: in all three proteins, this water is forming a hydrogen-bond with a backbone carbonyl group and interacts with the carboxylate group of glutamate (either directly or via an intercalated water molecule). This favors the shedding of one proton by the attacking water. The abstracted proton is transferred to the γ-phosphate via various proton wires, resulting in a H2PγO4(-)/ADP(3-) product state. This catalytic strategy is so effective that most other nucleotide hydrolyzing enzymes adopt a similar approach, as suggested by their very similar triphosphate binding sites. PMID:27296627

  18. Molecular structure of the Menkes disease gene (ATP7A)

    SciTech Connect

    Dierick, H.A.; Glover, T.W.; Ambrosini, L.

    1995-08-10

    We report a detailed molecular analysis of the genomic structure of the Menkes disease gene (MNK; ATP7A). There are 23 exons in ATP7A covering a genomic region of approximately 140 kb. The size of the individual coding exons varies between 77 and 726 bp, and introns vary in size between 196 bp and approximately 60 kb. All of the splice sites obey the consensus GT-AG rule except the splice donor of intron 9, which is GC instead of GT. The exon following this rare splice donor variant is alternatively spliced. A PGAM pseudogene and two highly polymorphic CA repeats map to introns within the gene. The structure is very similar to that of the closely related Wilson disease gene (WND; ATP7B). From exon 5 (exon 3 in ATP7B) to the end, all of the splice sites occur at exactly the same nucleotide positions as in the WND gene, except for the boundary between exons 17 and 18 (exons 15 and 16 in ATP7B) and a single codon difference at the boundary between exons 4 and 5 of the MNK gene (exons 2 and 3 in ATP7B). In contrast to the WND gene, in which the first four of six metal binding domains are contained in 1 exon, metal binding domains 1 to 4 are divided over 3 exons. The striking similarity of the MNK and WND genes at the genomic level is consistent with their relatively recent divergence from a common ancestral gene. 39 refs., 4 figs., 1 tab.

  19. Diverse Functional Properties of Wilson Disease ATP7B Variants

    PubMed Central

    Huster, Dominik; Kühne, Angelika; Bhattacharjee, Ashima; Raines, Lily; Jantsch, Vanessa; Noe, Johannes; Schirrmeister, Wiebke; Sommerer, Ines; Sabri, Osama; Berr, Frieder; Mössner, Joachim; Stieger, Bruno; Caca, Karel; Lutsenko, Svetlana

    2012-01-01

    BACKGROUND & AIMS Wilson disease is a severe disorder of copper metabolism caused by mutations in ATP7B, which encodes a copper-transporting adenosine triphosphatase. The disease presents with a variable phenotype that complicates the diagnostic process and treatment. Little is known about the mechanisms that contribute to the different phenotypes of the disease. METHODS We analyzed 28 variants of ATP7B from patients with Wilson disease that affected different functional domains; the gene products were expressed using the baculovirus expression system in Sf9 cells. Protein function was analyzed by measuring catalytic activity and copper (64Cu) transport into vesicles. We studied intracellular localization of variants of ATP7B that had measurable transport activities and were tagged with green fluorescent protein in mammalian cells using confocal laser scanning microscopy. RESULTS Properties of ATP7B variants with pathogenic amino-acid substitution varied greatly even if substitutions were in the same functional domain. Some variants had complete loss of catalytic and transport activity, whereas others lost transport activity but retained phosphor-intermediate formation or had partial losses of activity. In mammalian cells, transport-competent variants differed in stability and subcellular localization. CONCLUSIONS Variants in ATP7B associated with Wilson disease disrupt the protein’s transport activity, result in its mislocalization, and reduce its stability. Single assays are insufficient to accurately predict the effects of ATP7B variants the function of its product and development of Wilson disease. These findings will contribute to our understanding of genotype–phenotype correlation and mechanisms of disease pathogenesis. PMID:22240481

  20. ATP and AMP Mutually Influence Their Interaction with the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) at Separate Binding Sites*

    PubMed Central

    Randak, Christoph O.; Dong, Qian; Ver Heul, Amanda R.; Elcock, Adrian H.; Welsh, Michael J.

    2013-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP ⇆ 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, also have adenylate kinase activity. All three ABC adenylate kinases bind and hydrolyze ATP in the absence of other nucleotides. However, little is known about how an ABC adenylate kinase interacts with ATP and AMP when both are present. Based on data from non-ABC adenylate kinases, we hypothesized that ATP and AMP mutually influence their interaction with CFTR at separate binding sites. We further hypothesized that only one of the two CFTR ATP-binding sites is involved in the adenylate kinase reaction. We found that 8-azidoadenosine 5′-triphosphate (8-N3-ATP) and 8-azidoadenosine 5′-monophosphate (8-N3-AMP) photolabeled separate sites in CFTR. Labeling of the AMP-binding site with 8-N3-AMP required the presence of ATP. Conversely, AMP enhanced photolabeling with 8-N3-ATP at ATP-binding site 2. The adenylate kinase active center probe P1,P5-di(adenosine-5′) pentaphosphate interacted simultaneously with an AMP-binding site and ATP-binding site 2. These results show that ATP and AMP interact with separate binding sites but mutually influence their interaction with the ABC adenylate kinase CFTR. They further indicate that the active center of the adenylate kinase comprises ATP-binding site 2. PMID:23921386

  1. ATP and AMP mutually influence their interaction with the ATP-binding cassette (ABC) adenylate kinase cystic fibrosis transmembrane conductance regulator (CFTR) at separate binding sites.

    PubMed

    Randak, Christoph O; Dong, Qian; Ver Heul, Amanda R; Elcock, Adrian H; Welsh, Michael J

    2013-09-20

    Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP &lrarr2; 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, also have adenylate kinase activity. All three ABC adenylate kinases bind and hydrolyze ATP in the absence of other nucleotides. However, little is known about how an ABC adenylate kinase interacts with ATP and AMP when both are present. Based on data from non-ABC adenylate kinases, we hypothesized that ATP and AMP mutually influence their interaction with CFTR at separate binding sites. We further hypothesized that only one of the two CFTR ATP-binding sites is involved in the adenylate kinase reaction. We found that 8-azidoadenosine 5'-triphosphate (8-N3-ATP) and 8-azidoadenosine 5'-monophosphate (8-N3-AMP) photolabeled separate sites in CFTR. Labeling of the AMP-binding site with 8-N3-AMP required the presence of ATP. Conversely, AMP enhanced photolabeling with 8-N3-ATP at ATP-binding site 2. The adenylate kinase active center probe P(1),P(5)-di(adenosine-5') pentaphosphate interacted simultaneously with an AMP-binding site and ATP-binding site 2. These results show that ATP and AMP interact with separate binding sites but mutually influence their interaction with the ABC adenylate kinase CFTR. They further indicate that the active center of the adenylate kinase comprises ATP-binding site 2. PMID:23921386

  2. ATP Exhibits Antimicrobial Action by Inhibiting Bacterial Utilization of Ferric Ions

    PubMed Central

    Tatano, Yutaka; Kanehiro, Yuichi; Sano, Chiaki; Shimizu, Toshiaki; Tomioka, Haruaki

    2015-01-01

    ATP up-regulates macrophage antimycobacterial activity in a P2X7-dependent manner, but little is known about whether ATP directly exhibits antimicrobial effects against intracellular mycobacteria. In this study, we found that ATP inhibited the growth of various bacteria, including Staphylococcus, Pseudomonas, and mycobacteria, without damaging bacterial surface structures. Using gene technology, we newly established an enterobactin-deficient (entB−) mutant from ATP-resistant Klebsiella pneumoniae, and found the recovery of ATP susceptibility in the enterobactin-deleted mutant. Therefore, ATP's antibacterial activity is attributable to its iron-chelating ability. Since ATP distributed in the cytosol of macrophages at high concentrations, ATP appears to augment macrophage's antimicrobial activity by directly attacking intracytosolic and intra-autophagosomal pathogens. Furthermore, ATP exhibited combined effects with some antimicrobials against methicillin-resistant S. aureus (MRSA) and M. intracellulare, suggesting its usefulness as an adjunctive drug in the chemotherapy of certain intractable infections. PMID:25712807

  3. Cloning, expression and bioinformatics analysis of ATP sulfurylase from Acidithiobacillus ferrooxidans ATCC 23270 in Escherichia coli

    PubMed Central

    Jaramillo, Michael L; Abanto, Michel; Quispe, Ruth L; Calderón, Julio; del Valle, Luís J; Talledo, Miguel; Ramírez, Pablo

    2012-01-01

    Molecular studies of enzymes involved in sulfite oxidation in Acidithiobacillus ferrooxidans have not yet been developed, especially in the ATP sulfurylase (ATPS) of these acidophilus tiobacilli that have importance in biomining. This enzyme synthesizes ATP and sulfate from adenosine phosphosulfate (APS) and pyrophosphate (PPi), final stage of the sulfite oxidation by these organisms in order to obtain energy. The atpS gene (1674 bp) encoding the ATPS from Acidithiobacillus ferrooxidans ATCC 23270 was amplified using PCR, cloned in the pET101-TOPO plasmid, sequenced and expressed in Escherichia coli obtaining a 63.5 kDa ATPS recombinant protein according to SDS-PAGE analysis. The bioinformatics and phylogenetic analyses determined that the ATPS from A. ferrooxidans presents ATP sulfurylase (ATS) and APS kinase (ASK) domains similar to ATPS of Aquifex aeolicus, probably of a more ancestral origin. Enzyme activity towards ATP formation was determined by quantification of ATP formed from E. coli cell extracts, using a bioluminescence assay based on light emission by the luciferase enzyme. Our results demonstrate that the recombinant ATP sulfurylase from A. ferrooxidans presents an enzymatic activity for the formation of ATP and sulfate, and possibly is a bifunctional enzyme due to its high homology to the ASK domain from A. aeolicus and true kinases. PMID:23055613

  4. Cloning, expression and bioinformatics analysis of ATP sulfurylase from Acidithiobacillus ferrooxidans ATCC 23270 in Escherichia coli.

    PubMed

    Jaramillo, Michael L; Abanto, Michel; Quispe, Ruth L; Calderón, Julio; Del Valle, Luís J; Talledo, Miguel; Ramírez, Pablo

    2012-01-01

    Molecular studies of enzymes involved in sulfite oxidation in Acidithiobacillus ferrooxidans have not yet been developed, especially in the ATP sulfurylase (ATPS) of these acidophilus tiobacilli that have importance in biomining. This enzyme synthesizes ATP and sulfate from adenosine phosphosulfate (APS) and pyrophosphate (PPi), final stage of the sulfite oxidation by these organisms in order to obtain energy. The atpS gene (1674 bp) encoding the ATPS from Acidithiobacillus ferrooxidans ATCC 23270 was amplified using PCR, cloned in the pET101-TOPO plasmid, sequenced and expressed in Escherichia coli obtaining a 63.5 kDa ATPS recombinant protein according to SDS-PAGE analysis. The bioinformatics and phylogenetic analyses determined that the ATPS from A. ferrooxidans presents ATP sulfurylase (ATS) and APS kinase (ASK) domains similar to ATPS of Aquifex aeolicus, probably of a more ancestral origin. Enzyme activity towards ATP formation was determined by quantification of ATP formed from E. coli cell extracts, using a bioluminescence assay based on light emission by the luciferase enzyme. Our results demonstrate that the recombinant ATP sulfurylase from A. ferrooxidans presents an enzymatic activity for the formation of ATP and sulfate, and possibly is a bifunctional enzyme due to its high homology to the ASK domain from A. aeolicus and true kinases. PMID:23055613

  5. Sequence analysis and mapping of a novel human mitochondrial ATP synthase subunit 9 cDNA (ATP5G3)

    SciTech Connect

    Yan, W.L.; Gusella, J.F. |; Haines, J.L. |

    1994-11-15

    The authors describe the cloning, sequence analysis, and chromosomal mapping of a novel mitochondrial ATP synthase subunit 9 cDNA, P3. Subunit 9 transports protons across the inner mitochondrial membrane to the F{sub 1}-ATPase protruding on the matrix side, resulting in the generation of ATP. Sequence analysis of the P3 cDNA reveals only 80% identity with the human subunit 9 genes P1 and P2 in the DNA sequence encoding the mature protein identical to P1 and P2. The predicted sequence of the P3 leader peptide differs from the P1 and P2 leaders, but retains the {open_quotes}RFS{close_quotes} motif critical for mitochondrial import and maturation. The P3 gene (ATP5G3) maps to chromosome 2. 8 refs., 1 fig., 1 tab.

  6. Natural variation in the ATPS1 isoform of ATP sulfurylase contributes to the control of sulfate levels in Arabidopsis.

    PubMed

    Koprivova, Anna; Giovannetti, Marco; Baraniecka, Patrycja; Lee, Bok-Rye; Grondin, Cécile; Loudet, Olivier; Kopriva, Stanislav

    2013-11-01

    Sulfur is an essential macronutrient for all living organisms. Plants take up inorganic sulfate from the soil, reduce it, and assimilate it into bioorganic compounds, but part of this sulfate is stored in the vacuoles. In our first attempt to identify genes involved in the control of sulfate content in the leaves, we reported that a quantitative trait locus (QTL) for sulfate content in Arabidopsis (Arabidopsis thaliana) was underlain by the APR2 isoform of the key enzyme of sulfate assimilation, adenosine 5'-phosphosulfate reductase. To increase the knowledge of the control of this trait, we cloned a second QTL from the same analysis. Surprisingly, the gene underlying this QTL encodes the ATPS1 isoform of the enzyme ATP sulfurylase, which precedes adenosine 5'-phosphosulfate reductase in the sulfate assimilation pathway. Plants with the Bay allele of ATPS1 accumulate lower steady-state levels of ATPS1 transcript than those with the Sha allele, which leads to lower enzyme activity and, ultimately, the accumulation of sulfate. Our results show that the transcript variation is controlled in cis. Examination of ATPS1 sequences of Bay-0 and Shahdara identified two deletions in the first intron and immediately downstream the gene in Bay-0 shared with multiple other Arabidopsis accessions. The average ATPS1 transcript levels are lower in these accessions than in those without the deletions, while sulfate levels are significantly higher. Thus, sulfate content in Arabidopsis is controlled by two genes encoding subsequent enzymes in the sulfate assimilation pathway but using different mechanisms, variation in amino acid sequence and variation in expression levels. PMID:24027241

  7. Pyrophosphate-Dependent ATP Formation from Acetyl Coenzyme A in Syntrophus aciditrophicus, a New Twist on ATP Formation

    PubMed Central

    James, Kimberly L.; Ríos-Hernández, Luis A.; Wofford, Neil Q.; Mouttaki, Housna; Sieber, Jessica R.; Sheik, Cody S.; Nguyen, Hong H.; Yang, Yanan; Xie, Yongming; Erde, Jonathan; Rohlin, Lars; Karr, Elizabeth A.; Loo, Joseph A.; Ogorzalek Loo, Rachel R.; Hurst, Gregory B.; Gunsalus, Robert P.; Szweda, Luke I.

    2016-01-01

    ABSTRACT Syntrophus aciditrophicus is a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation by S. aciditrophicus. However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome of S. aciditrophicus leaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA) and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show that S. aciditrophicus uses AMP-forming, acetyl-CoA synthetase (Acs1) for ATP synthesis from acetyl-CoA. acs1 mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, of S. aciditrophicus grown in pure culture and coculture. Cell extracts of S. aciditrophicus had low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified from S. aciditrophicus and recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4) in S. aciditrophicus cells support the operation of Acs1 in the acetate-forming direction. Thus, S. aciditrophicus has a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase. PMID:27531911

  8. The Q Motif Is Involved in DNA Binding but Not ATP Binding in ChlR1 Helicase

    PubMed Central

    Ding, Hao; Guo, Manhong; Vidhyasagar, Venkatasubramanian; Talwar, Tanu; Wu, Yuliang

    2015-01-01

    Helicases are molecular motors that couple the energy of ATP hydrolysis to the unwinding of structured DNA or RNA and chromatin remodeling. The conversion of energy derived from ATP hydrolysis into unwinding and remodeling is coordinated by seven sequence motifs (I, Ia, II, III, IV, V, and VI). The Q motif, consisting of nine amino acids (GFXXPXPIQ) with an invariant glutamine (Q) residue, has been identified in some, but not all helicases. Compared to the seven well-recognized conserved helicase motifs, the role of the Q motif is less acknowledged. Mutations in the human ChlR1 (DDX11) gene are associated with a unique genetic disorder known as Warsaw Breakage Syndrome, which is characterized by cellular defects in genome maintenance. To examine the roles of the Q motif in ChlR1 helicase, we performed site directed mutagenesis of glutamine to alanine at residue 23 in the Q motif of ChlR1. ChlR1 recombinant protein was overexpressed and purified from HEK293T cells. ChlR1-Q23A mutant abolished the helicase activity of ChlR1 and displayed reduced DNA binding ability. The mutant showed impaired ATPase activity but normal ATP binding. A thermal shift assay revealed that ChlR1-Q23A has a melting point value similar to ChlR1-WT. Partial proteolysis mapping demonstrated that ChlR1-WT and Q23A have a similar globular structure, although some subtle conformational differences in these two proteins are evident. Finally, we found ChlR1 exists and functions as a monomer in solution, which is different from FANCJ, in which the Q motif is involved in protein dimerization. Taken together, our results suggest that the Q motif is involved in DNA binding but not ATP binding in ChlR1 helicase. PMID:26474416

  9. Calcium-induced conformational changes in the regulatory domain of the human mitochondrial ATP-Mg/Pi carrier.

    PubMed

    Harborne, Steven P D; Ruprecht, Jonathan J; Kunji, Edmund R S

    2015-10-01

    The mitochondrial ATP-Mg/Pi carrier imports adenine nucleotides from the cytosol into the mitochondrial matrix and exports phosphate. The carrier is regulated by the concentration of cytosolic calcium, altering the size of the adenine nucleotide pool in the mitochondrial matrix in response to energetic demands. The protein consists of three domains; (i) the N-terminal regulatory domain, which is formed of two pairs of fused calcium-binding EF-hands, (ii) the C-terminal mitochondrial carrier domain, which is involved in transport, and (iii) a linker region with an amphipathic α-helix of unknown function. The mechanism by which calcium binding to the regulatory domain modulates substrate transport in the carrier domain has not been resolved. Here, we present two new crystal structures of the regulatory domain of the human isoform 1. Careful analysis by SEC confirmed that although the regulatory domain crystallised as dimers, full-length ATP-Mg/Pi carrier is monomeric. Therefore, the ATP-Mg/Pi carrier must have a different mechanism of calcium regulation than the architecturally related aspartate/glutamate carrier, which is dimeric. The structure showed that an amphipathic α-helix is bound to the regulatory domain in a hydrophobic cleft of EF-hand 3/4. Detailed bioinformatics analyses of different EF-hand states indicate that upon release of calcium, EF-hands close, meaning that the regulatory domain would release the amphipathic α-helix. We propose a mechanism for ATP-Mg/Pi carriers in which the amphipathic α-helix becomes mobile upon release of calcium and could block the transport of substrates across the mitochondrial inner membrane. PMID:26164100

  10. Calcium-induced conformational changes in the regulatory domain of the human mitochondrial ATP-Mg/Pi carrier

    PubMed Central

    Harborne, Steven P.D.; Ruprecht, Jonathan J.; Kunji, Edmund R.S.

    2015-01-01

    The mitochondrial ATP-Mg/Pi carrier imports adenine nucleotides from the cytosol into the mitochondrial matrix and exports phosphate. The carrier is regulated by the concentration of cytosolic calcium, altering the size of the adenine nucleotide pool in the mitochondrial matrix in response to energetic demands. The protein consists of three domains; (i) the N-terminal regulatory domain, which is formed of two pairs of fused calcium-binding EF-hands, (ii) the C-terminal mitochondrial carrier domain, which is involved in transport, and (iii) a linker region with an amphipathic α-helix of unknown function. The mechanism by which calcium binding to the regulatory domain modulates substrate transport in the carrier domain has not been resolved. Here, we present two new crystal structures of the regulatory domain of the human isoform 1. Careful analysis by SEC confirmed that although the regulatory domain crystallised as dimers, full-length ATP-Mg/Pi carrier is monomeric. Therefore, the ATP-Mg/Pi carrier must have a different mechanism of calcium regulation than the architecturally related aspartate/glutamate carrier, which is dimeric. The structure showed that an amphipathic α-helix is bound to the regulatory domain in a hydrophobic cleft of EF-hand 3/4. Detailed bioinformatics analyses of different EF-hand states indicate that upon release of calcium, EF-hands close, meaning that the regulatory domain would release the amphipathic α-helix. We propose a mechanism for ATP-Mg/Pi carriers in which the amphipathic α-helix becomes mobile upon release of calcium and could block the transport of substrates across the mitochondrial inner membrane. PMID:26164100

  11. Time to Definitive Health-Related Quality of Life Score Deterioration in Patients with Resectable Metastatic Colorectal Cancer Treated with FOLFOX4 versus Sequential Dose-Dense FOLFOX7 followed by FOLFIRI: The MIROX Randomized Phase III Trial

    PubMed Central

    Hamidou, Zeinab; Chibaudel, Benoist; Hebbar, Mohamed; Hug de Larauze, Marine; André, Thierry; Louvet, Christophe; Brusquant, David; Garcia-Larnicol, Marie-Line; de Gramont, Aimery; Bonnetain, Franck

    2016-01-01

    Purpose We previously showed that a sequential chemotherapy with dose-dense oxaliplatin (FOLFOX7) and irinotecan (FOLFIRI; irinotecan plus 5-fluorouracil/leucovorin) is not superior to FOLFOX4 in patients at advanced stage of colorectal cancer with liver metastases. Here we aimed to determine whether time to health-related quality of life (HRQoL) score definitive deterioration (TUDD) differs by study arm. Methods HRQoL was evaluated using the European Organization for Research and Treatment of Cancer (EORTC) QLQ-C30 at baseline and every 4 cycles until the end of the study or death. Functional scale, symptom scale, global health status, and financial difficulties were analyzed. The TUDD was defined as the time interval between randomization and the first decrease in HRQoL score ≥ 5-point with no further improvement in HRQoL score ≥ 5 points or any further HRQoL data. TUDD was estimated using the Kaplan-Meier method and the long-rank test. Cox regression analyses were used to identify HRQoL items influencing TUDD. Sensitivity analyses were done using a multiple imputation method and different definitions of TUDD. Results Of the 284 patients, 171 (60.2%) completed HRQoL questionnaires. Cox multivariate analysis showed no statistically significant difference in TUDD for most of the QLQ-C30 scales between treatments. Patients with dyspnea and those without symptoms at baseline had a significantly longer TUDD when there was a delay >12 months between diagnosis of the primary tumor and metastases (HR 0.48 [0.26–0.89]) and when there was diarrhea (HR 0.59 [0.36–0.96]), respectively. Conclusion This study shows that TUDD does not differ significantly according to type of treatment. The TUDD method produces meaningful longitudinal HRQoL results that may facilitate effective clinical decision making in patients with mCRC. Trial Registration ClinicalTrials.gov NCT00268398 PMID:27310205

  12. The Na(+)-F(1)F(0)-ATPase operon from Acetobacterium woodii. Operon structure and presence of multiple copies of atpE which encode proteolipids of 8- and 18-kda.

    PubMed

    Rahlfs, S; Aufurth, S; Müller, V

    1999-11-26

    Eight genes (atpI, atpB, atpE(1), atpE(2), atpE(3), atpF, atpH, and atpA) upstream of and contiguous with the previously described genes atpG, atpD, and atpC were cloned from chromosomal DNA of Acetobacterium woodii. Northern blot analysis revealed that the eleven atp genes are transcribed as a polycistronic message. The atp operon encodes the Na(+)-F(1)F(0)-ATPase of A. woodii, as evident from a comparison of the biochemically derived N termini of the subunits with the amino acid sequences deduced from the DNA sequences. The molecular analysis revealed that all of the F(1)F(0)-encoding genes from Escherichia coli have homologs in the Na(+)-F(1)F(0)-ATPase operon from A. woodii, despite the fact that only six subunits were found in previous preparations of the enzyme from A. woodii. These results unequivocally prove that the Na(+)-ATPase from A. woodii is an enzyme of the F(1)F(0) class. Most interestingly, the gene encoding the proteolipid underwent quadruplication. Two gene copies (atpE(2) and atpE(3)) encode identical 8-kDa proteolipids. Two additional gene copies were fused to form the atpE(1) gene. Heterologous expression experiments as well as immunolabeling studies with native membranes revealed that atpE(1) encodes a duplicated 18-kDa proteolipid. This is the first demonstration of multiplication and fusion of proteolipid-encoding genes in F(1)F(0)-ATPase operons. Furthermore, AtpE(1) is the first duplicated proteolipid ever found to be encoded by an F(1)F(0)-ATPase operon. PMID:10567365

  13. ATP stimulates calcium influx in primary astrocyte cultures

    SciTech Connect

    Neary, J.T.; van Breemen, C.; Forster, E.; Norenberg, L.O.; Norenberg, M.D.

    1988-12-30

    The effect of ATP and other purines on /sup 45/Ca uptake was studied in primary cultures of rat astrocytes. Treatment of the cells with ATP for 1 to 30 min brought about an increase in cellular /sup 45/Ca. Stimulation of calcium influx by ATP was investigated using a 90 sec exposure to /sup 45/Ca and over a concentration range of 0.1 nM to 3 mM; a biphasic dose-response curve was obtained with EC50 values of 0.3 nM and 9 uM, indicating the presence of low and high affinity purinergic binding sites. Similar levels of /sup 45/Ca influx at 90 sec were observed with ATP, ADP and adenosine (all at 100 uM). Prior treatment of the cultures with LaCl3 blocked the purine-induced /sup 45/Ca influx. These findings indicate that one pathway for calcium entry in astrocytes involves purinergic receptor-operated, calcium channels.

  14. ATP-induced noncooperative thermal unfolding of hen lysozyme

    SciTech Connect

    Liu, Honglin; Yin, Peidong; He, Shengnan; Sun, Zhihu; Tao, Ye; Huang, Yan; Zhuang, Hao; Zhang, Guobin; Wei, Shiqiang

    2010-07-02

    To understand the role of ATP underlying the enhanced amyloidosis of hen egg white lysozyme (HEWL), the synchrotron radiation circular dichroism, combined with tryptophan fluorescence, dynamic light-scattering, and differential scanning calorimetry, is used to examine the alterations of the conformation and thermal unfolding pathway of the HEWL in the presence of ATP, Mg{sup 2+}-ATP, ADP, AMP, etc. It is revealed that the binding of ATP to HEWL through strong electrostatic interaction changes the secondary structures of HEWL and makes the exposed residue W62 move into hydrophobic environments. This alteration of W62 decreases the {beta}-domain stability of HEWL, induces a noncooperative unfolding of the secondary structures, and produces a partially unfolded intermediate. This intermediate containing relatively rich {alpha}-helix and less {beta}-sheet structures has a great tendency to aggregate. The results imply that the ease of aggregating of HEWL is related to the extent of denaturation of the amyloidogenic region, rather than the electrostatic neutralizing effect or monomeric {beta}-sheet enriched intermediate.

  15. Abiogenic photophosphorylation of ADP to ATP sensitized by flavoproteinoid microspheres.

    PubMed

    Kolesnikov, Michael P; Telegina, Taisiya A; Lyudnikova, Tamara A; Kritsky, Mikhail S

    2008-06-01

    A model for abiogenic photophosphorylation of ADP by orthophosphate to yield ATP was studied. The model is based on the photochemical activity of flavoproteinoid microspheres that are formed by aggregation in an aqueous medium of products of thermal condensation of a glutamic acid, glycine and lysine mixture (8:3:1) and contain, along with amino acid polymers (proteinoids), abiogenic isoalloxazine (flavin) pigments. Irradiation of aqueous suspensions of microspheres with blue visible light or ultraviolet in the presence of ADP and orthophosphate resulted in ATP formation. The yield of ATP in aerated suspensions was 10-20% per one mol of starting ADP. Deaeration reduced the photophosphorylating activity of microspheres five to 10 times. Treatment of aerated microsphere suspensions with superoxide dismutase during irradiation partially suppressed ATP formation. Deaerated microspheres restored completely their photophosphorylating activity after addition of hydrogen peroxide to the suspension. The photophosphorylating activity of deaerated suspensions of flavoproteinoid microspheres was also recovered by introduction of Fe3+-cytochrome c, an electron acceptor alternative to oxygen. On the basis of the results obtained, a chemical mechanism of phosphorylation is proposed in which the free radical form of reduced flavin sensitizer (F1H*) and ADP are involved. PMID:18386156

  16. ATP-enhanced peroxidase-like activity of gold nanoparticles.

    PubMed

    Shah, Juhi; Purohit, Rahul; Singh, Ragini; Karakoti, Ajay Singh; Singh, Sanjay

    2015-10-15

    Gold nanoparticles (AuNPs) are known to possess intrinsic biological peroxidase-like activity that has applications in development of numerous biosensors. The reactivity of the Au atoms at the surface of AuNPs is critical to the performance of such biosensors, yet little is known about the effect of biomolecules and ions on the peroxidase-like activity. In this work, the effect of ATP and other biologically relevant molecules and ions over peroxidase-like activity of AuNPs are described. Contrary to the expectation that nanoparticles exposed to biomolecules may lose the catalytic property, ATP and ADP addition enhanced the peroxidase-like activity of AuNPs. The catalytic activity was unaltered by the addition of free phosphate, sulphate and carbonate anions however, addition of ascorbic acid to the reaction mixture diminished the intrinsic peroxidase-like activity of AuNPs, even in the presence of ATP and ADP. In contrast to AuNPs, ATP did not synergize and improve the peroxidase activity of the natural peroxidase enzyme, horseradish peroxidase. PMID:26111515

  17. Detection of ATP and NADH: A Bioluminescent Experience.

    ERIC Educational Resources Information Center

    Selig, Ted C.; And Others

    1984-01-01

    Described is a bioluminescent assay for adenosine triphosphate (ATP) and reduced nicotineamide-adenine dinucleotide (NADH) that meets the requirements of an undergraduate biochemistry laboratory course. The 3-hour experiment provides students with experience in bioluminescence and analytical biochemistry yet requires limited instrumentation,…

  18. Animation Model to Conceptualize ATP Generation: A Mitochondrial Oxidative Phosphorylation

    ERIC Educational Resources Information Center

    Jena, Ananta Kumar

    2015-01-01

    Adenosine triphosphate (ATP) is the molecular unit of intracellular energy and it is the product of oxidative phosphorylation of cellular respiration uses in cellular processes. The study explores the growth of the misconception levels amongst the learners and evaluates the effectiveness of animation model over traditional methods. The data…

  19. ATP synthesis during exogenous NADH oxidation. A reappraisal.

    PubMed

    Bernardi, P; Azzone, G F

    1982-01-20

    This paper reports a reinvestigation on the pathway for mitochondrial oxidation of exogenous NADH and on the related ATP synthesis, first reported 30 years ago (Lehninger, A.L. (1951) J. Biol. Chem. 190, 345-359). NADH oxidation, both in intact and in water-treated mitochondria, is 90% inhibited by mersalyl, an inhibitor of the outer membrane NADH-cytochrome b5 reductase, and 10% inhibited by rotenone. The mersalyl-sensitive, but not the rotenone-sensitive, portion of NADH oxidation is stimulated by exogenous cytochrome c. Part of ATP synthesis is independent of exogenous NADH and cytochrome c, and is inhibited by rotenone and antimycin A, and is therefore due to oxidation of endogenous substrates. Another part of ATP synthesis is dependent on exogenous NADH and cytochrome c, is insensitive to rotenone and antimycin A, and is due to operation of cytochrome oxidase. It is concluded that (i) oxidation of exogenous NADH in the presence of cytochrome c proceeds mostly through NADH-cytochrome b5 reductase and cytochrome b5 on the outer membrane and then through cytochrome oxidase via the cytochrome c shuttle, and (ii) ATP synthesis during oxidation of exogenous NADH is partly due to oxidation of endogenous substrates and partly to operation of cytochrome oxidase receiving electrons from the outer membrane via cytochrome c. PMID:6275889

  20. ATP Binding Turns Plant Cryptochrome Into an Efficient Natural Photoswitch

    NASA Astrophysics Data System (ADS)

    Müller, Pavel; Bouly, Jean-Pierre; Hitomi, Kenichi; Balland, Véronique; Getzoff, Elizabeth D.; Ritz, Thorsten; Brettel, Klaus

    2014-06-01

    Cryptochromes are flavoproteins that drive diverse developmental light-responses in plants and participate in the circadian clock in animals. Plant cryptochromes have found application as photoswitches in optogenetics. We have studied effects of pH and ATP on the functionally relevant photoreduction of the oxidized FAD cofactor to the semi-reduced FADH. radical in isolated Arabidopsis cryptochrome 1 by transient absorption spectroscopy on nanosecond to millisecond timescales. In the absence of ATP, the yield of light-induced radicals strongly decreased with increasing pH from 6.5 to 8.5. With ATP present, these yields were significantly higher and virtually pH-independent up to pH 9. Analysis of our data in light of the crystallographic structure suggests that ATP-binding shifts the pKa of aspartic acid D396, the putative proton donor to FAD.-, from ~7.4 to >9, and favours a reaction pathway yielding long-lived aspartate D396-. Its negative charge could trigger conformational changes necessary for signal transduction.

  1. ATP drives direct photosynthetic production of 1-butanol in cyanobacteria

    PubMed Central

    Lan, Ethan I.; Liao, James C.

    2012-01-01

    While conservation of ATP is often a desirable trait for microbial production of chemicals, we demonstrate that additional consumption of ATP may be beneficial to drive product formation in a nonnatural pathway. Although production of 1-butanol by the fermentative coenzyme A (CoA)-dependent pathway using the reversal of β-oxidation exists in nature and has been demonstrated in various organisms, the first step of the pathway, condensation of two molecules of acetyl-CoA to acetoacetyl-CoA, is thermodynamically unfavorable. Here, we show that artificially engineered ATP consumption through a pathway modification can drive this reaction forward and enables for the first time the direct photosynthetic production of 1-butanol from cyanobacteria Synechococcus elongatus PCC 7942. We further demonstrated that substitution of bifunctional aldehyde/alcohol dehydrogenase (AdhE2) with separate butyraldehyde dehydrogenase (Bldh) and NADPH-dependent alcohol dehydrogenase (YqhD) increased 1-butanol production by 4-fold. These results demonstrated the importance of ATP and cofactor driving forces as a design principle to alter metabolic flux. PMID:22474341

  2. Interaction between ATP, metal ions, glycine, and several minerals

    NASA Technical Reports Server (NTRS)

    Rishpon, J.; Ohara, P. J.; Lawless, J. G.; Lahav, N.

    1982-01-01

    Interactions between ATP, glycine and montmorillonite and kaolinite clay minerals in the presence of various metal cations are investigated. The adsorption of adenine nucleotides on clays and Al(OH)3 was measured as a function of pH, and glycine condensation was followed in the presence of ATP, ZnCl2, MgCl2 and either kaolinite or montmorillonite. The amounts of ATP and ADP adsorbed are found to decrease with increasing Ph, and to be considerably enhanced in experiments with Mg(2+)- and Zn(2+)-montmorillonite with respect to Na(+)-montmorillonite. The effects of divalent cations are less marked in kaolinite. Results for Al(OH)3 show the importance of adsorption at clay platelet edges at high pH. The decomposition of ATP during drying at high temperature is observed to be inhibited by small amounts of clay, vacuum, or Mg(2+) or Zn(2+) ions, and to be accompanied by peptide formation in the presence of glycine. Results suggest the importance of Zn(2+) and Mg(2+) in chemical evolution.

  3. ATP binding turns plant cryptochrome into an efficient natural photoswitch.

    PubMed

    Müller, Pavel; Bouly, Jean-Pierre; Hitomi, Kenichi; Balland, Véronique; Getzoff, Elizabeth D; Ritz, Thorsten; Brettel, Klaus

    2014-01-01

    Cryptochromes are flavoproteins that drive diverse developmental light-responses in plants and participate in the circadian clock in animals. Plant cryptochromes have found application as photoswitches in optogenetics. We have studied effects of pH and ATP on the functionally relevant photoreduction of the oxidized FAD cofactor to the semi-reduced FADH(·) radical in isolated Arabidopsis cryptochrome 1 by transient absorption spectroscopy on nanosecond to millisecond timescales. In the absence of ATP, the yield of light-induced radicals strongly decreased with increasing pH from 6.5 to 8.5. With ATP present, these yields were significantly higher and virtually pH-independent up to pH 9. Analysis of our data in light of the crystallographic structure suggests that ATP-binding shifts the pKa of aspartic acid D396, the putative proton donor to FAD·(-), from ~7.4 to >9, and favours a reaction pathway yielding long-lived aspartate D396(-). Its negative charge could trigger conformational changes necessary for signal transduction. PMID:24898692

  4. [Suggested mitochondrial ancestry of non-mitochondrial ATP/ADP].

    PubMed

    Emel'ianov, V V

    2007-01-01

    One of the major evolutionary events that transformed endosymbiotic bacterium into mitochondrion was an acquisition of ATP/ADP carrier in order to supply the host with respiration-derived ATP. Along with mitochondrial carrier, unrelated carrier is known which is characteristic of intracellular chlamydiae, plastids, parasitic intracellular eukaryote Encephalitozoon cuniculi, and the genus Rickettsia of obligate endosymbiotic alpha-Proteobacteria. This non-mitochondrial ATP/ADP carrier was recently described in rickettsia-like endosymbionts - a group of obligate intracellular bacteria, classified with the order Rickettsiales, which have diverged after free-living alpha-Proteobacteria but before sister groups of the Rickettsiaceae assemblage (true rickettsiae) and mitochondria. Published controversial phylogenetic data on the non-mitochondrial carrier were reanalysed in the present work using both DNA and protein sequences, and various methods including Bayesian analysis. The data presented are consistent with classic endosymbiont theory for the origin of mitochondria and also suggest that even last but one common ancestor of rickettsiae and organelles may have been an endosymbiotic bacterium in which ATP/ADP carrier has first originated. PMID:17380892

  5. Cyclodextrin-based microcapsules as bioreactors for ATP biosynthesis.

    PubMed

    Li, Jian-Hu; Wang, Yi-Fu; Ha, Wei; Liu, Yan; Ding, Li-Sheng; Li, Bang-Jing; Zhang, Sheng

    2013-09-01

    A biomimetic energy converter was fabricated via the assembly of CF0F1-ATPase on lipid-coated hollow nanocapsules composed of α-cyclodextrins/chitosan-graft-poly(ethylene glycol) methacrylate. Upon entrapped GOD into these capsules, the addition of glucose could trigger proton-motive force and then drive the rotation of ATPase to synthesize ATP. PMID:23962233

  6. Rapid and precise determination of ATP using a modified photometer

    USGS Publications Warehouse

    Shultz, David J.; Stephens, Doyle W.

    1980-01-01

    An inexpensive delay timer was designed to modify a commercially available ATP photometer which allows a disposable tip pipette to be used for injecting either enzyme or sample into the reaction cuvette. The disposable tip pipette is as precise and accurate as a fixed-needle syringe but eliminates the problem of sample contamination and decreases analytical time. (USGS)

  7. Teacher Development Program for ATP 2000. Project Report.

    ERIC Educational Resources Information Center

    Sutphin, Dean; And Others

    Agri Tech Prep 2000 (ATP 2000) is a 4-year tech prep program linking high school and postsecondary curricula designed to prepare New York students for careers in agriculture or acceptance into a college program in agriculture. Because teacher development was designated an integral project component for fiscal year 1991-1992, a weeklong teacher…

  8. Activated sludge optimization using ATP in pulp and paper industry.

    PubMed

    Bäckman, Göran; Gytel, Ulla

    2015-01-01

    The activated sludge process is an old technology, but still the most commonly used one for treatment of wastewater. Despite the wide spread usage the technology still suffers from instability (Tandoi et al. 2006) and high operating cost. Activated sludge processes often carry a large solids inventory. Managing the total inventory without interference is the key component of the optimization process described in this paper. Use of nutrients is common in pulp and paper effluent treatment. Feeding enough nutrients to support the biomass growth is a delicate balance. Overfeeding or underfeeding of nutrients can result in higher costs. Detrimental substances and toxic components in effluents entering a biological treatment system can cause severe, long lasting disturbances (Hynninen & Ingman 1998; Bergeron & Pelletier 2004). A LumiKem test kit is used to measure biological activity with adenosine triphosphate (ATP) in a pulp and paper mill. ATP data are integrated with other standardized mill parameters. Measurements of active volatile suspended solids based on ATP can be used to quantify the living biomass in the activated sludge process and to ensure that sufficient biomass is present in order to degrade the wastewater constituents entering the process. Information about active biomass will assist in optimizing sludge inventories and feeding of nutrients allowing the living biomass to re-populate to create optimal efficiency. ATP measurements can also be used to alert operators if any components toxic to bacteria are present in wastewater. The bio stress index represents the stress level experienced by the microbiological population. This parameter is very useful in monitoring toxicity in and around bioreactors. Results from the wastewater process optimization and ATP measurements showed that treatment cost could be reduced by approximately 20-30% with fewer disturbances and sustained biological activity compared to the reference period. This was mainly achieved by

  9. ATP Is an Allosteric Inhibitor of Coxsackievirus B3 Polymerase.

    PubMed

    Karr, Jonathan P; Peersen, Olve B

    2016-07-19

    The RNA-dependent RNA polymerases from positive-strand RNA viruses, such as picornaviruses and flaviviruses, close their active sites for catalysis via a unique NTP-induced conformational change in the palm domain. Combined with a fully prepositioned templating nucleotide, this mechanism is error-prone and results in a distribution of random mutations in the viral progeny often described as a quasi-species. Here we examine the extent to which noncognate NTPs competitively inhibit single-cycle elongation by coxsackievirus B3 3D(pol), a polymerase that generates three to four mutations per 10 kb of RNA synthesized during viral infection. Using an RNA with a templating guanosine combined with 2-aminopurine fluorescence as a reporter for elongation, we find that the cognate CTP has a Km of 24 μM and the three noncognate nucleotides competitively inhibit the reaction with Kic values of 500 μM for GTP, 1300 μM for ATP, and 3000 μM for UTP. Unexpectedly, ATP also acted as an uncompetitive inhibitor with a Kiu of 1800 μM, resulting in allosteric modulation of 3D(pol) that slowed the polymerase elongation rate ≈4-fold. ATP uncompetitive inhibition required the β- and γ-phosphates, and its extent was significantly diminished in two previously characterized low-fidelity polymerases. This led to further mutational analysis and the identification of a putative allosteric binding site below the NTP entry channel at the interface of conserved motifs A and D, although cocrystallization failed to reveal any density for bound ATP in this pocket. The potential role of an ATP allosteric effect during the virus life cycle is discussed. PMID:27319576

  10. Regulation of CFTR Cl- channel gating by ATP binding and hydrolysis.

    PubMed

    Ikuma, M; Welsh, M J

    2000-07-18

    Opening and closing of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is regulated by the interaction of ATP with its two cytoplasmic nucleotide-binding domains (NBD). Although ATP hydrolysis by the NBDs is required for normal gating, the influence of ATP binding versus hydrolysis on specific steps in the gating cycle remains uncertain. Earlier work showed that the absence of Mg(2+) prevents hydrolysis. We found that even in the absence of Mg(2+), ATP could support channel activity, albeit at a reduced level compared with the presence of Mg(2+). Application of ATP with a divalent cation, including the poorly hydrolyzed CaATP complex, increased the rate of opening. Moreover, in CFTR variants with mutations that disrupt hydrolysis, ATP alone opened the channel and Mg(2+) further enhanced ATP-dependent opening. These data suggest that ATP alone can open the channel and that divalent cations increase ATP binding. Consistent with this conclusion, when we mutated an aspartate thought to bind Mg(2+), divalent cations failed to increase activity compared with ATP alone. Two observations suggested that divalent cations also stabilize the open state. In wild-type CFTR, CaATP generated a long duration open state, whereas ATP alone did not. With a CFTR variant in which hydrolysis was disrupted, MgATP, but not ATP alone, produced long openings. These results suggest a gating cycle for CFTR in which ATP binding opens the channel and either hydrolysis or dissociation leads to channel closure. In addition, the data suggest that ATP binding and hydrolysis by either NBD can gate the channel. PMID:10880569

  11. Regulation of CFTR Cl− channel gating by ATP binding and hydrolysis

    PubMed Central

    Ikuma, Mutsuhiro; Welsh, Michael J.

    2000-01-01

    Opening and closing of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is regulated by the interaction of ATP with its two cytoplasmic nucleotide-binding domains (NBD). Although ATP hydrolysis by the NBDs is required for normal gating, the influence of ATP binding versus hydrolysis on specific steps in the gating cycle remains uncertain. Earlier work showed that the absence of Mg2+ prevents hydrolysis. We found that even in the absence of Mg2+, ATP could support channel activity, albeit at a reduced level compared with the presence of Mg2+. Application of ATP with a divalent cation, including the poorly hydrolyzed CaATP complex, increased the rate of opening. Moreover, in CFTR variants with mutations that disrupt hydrolysis, ATP alone opened the channel and Mg2+ further enhanced ATP-dependent opening. These data suggest that ATP alone can open the channel and that divalent cations increase ATP binding. Consistent with this conclusion, when we mutated an aspartate thought to bind Mg2+, divalent cations failed to increase activity compared with ATP alone. Two observations suggested that divalent cations also stabilize the open state. In wild-type CFTR, CaATP generated a long duration open state, whereas ATP alone did not. With a CFTR variant in which hydrolysis was disrupted, MgATP, but not ATP alone, produced long openings. These results suggest a gating cycle for CFTR in which ATP binding opens the channel and either hydrolysis or dissociation leads to channel closure. In addition, the data suggest that ATP binding and hydrolysis by either NBD can gate the channel. PMID:10880569

  12. Ectopic ATP synthase in endothelial cells: a novel cardiovascular therapeutic target.

    PubMed

    Fu, Yi; Zhu, Yi

    2010-01-01

    Adenosine triphosphate (ATP) synthase produces ATP in cells and is found on the inner membrane of mitochondria or the cell plasma membrane (ectopic ATP synthase). Here, we summarize the functions of ectopic ATP synthase in vascular endothelial cells (ECs). Ectopic ATP synthase is involved in adenosine metabolism on the cell surface through its ATP generation or hydrolysis activity. The ATP/ADP generated by the enzyme on the plasma membrane can bind to P2X/P2Y receptors and activate the related signalling pathways to regulate endothelial function. The β-chain of ectopic ATP synthase on the EC surface can recruit inflammatory cells and activate cytotoxic activity to damage ECs and induce vascular inflammation. Angiostatin and other angiogenesis inhibitors can have anti-angiogenic functions by inhibiting ectopic ATP synthase on ECs. Moreover, ectopic ATP synthase on ECs is a receptor for apoA-I, the acceptor of cholesterol efflux, which implies that endothelial ectopic ATP synthase is involved in cholesterol metabolism. Coupling factor 6 (CF6), a part of ectopic ATP synthase, is released from ECs and can inhibit prostacyclin synthesis and promote nitric oxide (NO) degradation to enhance NO bioactivity. Because ATP/ADP generated by ectopic ATP synthase can induce NO production, substances such as CF6 can inhibit NO generation by inhibiting surface ATP/ADP production. Thus, the components of ectopic ATP synthase are associated with regulation of vascular tone. Through these functions, ectopic ATP synthase on ECs is considered a potential and novel therapeutic target for atherosclerosis, hypertension and lipid disorders. PMID:21247400

  13. Evidence for Extracellular ATP as a Stress Signal in a Single-Celled Organism.

    PubMed

    Sivaramakrishnan, Venketesh; Fountain, Samuel J

    2015-08-01

    ATP is omnipresent in biology and acts as an extracellular signaling molecule in mammals. Information regarding the signaling function of extracellular ATP in single-celled eukaryotes is lacking. Here, we explore the role of extracellular ATP in cell volume recovery during osmotic swelling in the amoeba Dictyostelium. Release of micromolar ATP could be detected during cell swelling and regulatory cell volume decrease (RVD) phases during hypotonic challenge. Scavenging ATP with apyrase caused profound cell swelling and loss of RVD. Apyrase-induced swelling could be rescued by 100 μM βγ-imidoATP. N-Ethylmalemide (NEM), an inhibitor of vesicular exocytosis, caused heightened cell swelling, loss of RVD, and inhibition of ATP release. Amoebas with impaired contractile vacuole (CV) fusion (drainin knockout [KO] cells) displayed increased swelling but intact ATP release. One hundred micromolar Gd(3+) caused cell swelling while blocking any recovery by βγ-imidoATP. ATP release was 4-fold higher in the presence of Gd(3+). Cell swelling was associated with an increase in intracellular nitric oxide (NO), with NO-scavenging agents causing cell swelling. Swelling-induced NO production was inhibited by both apyrase and Gd(3+), while NO donors rescued apyrase- and Gd(3+)-induced swelling. These data suggest extracellular ATP released during cell swelling is an important signal that elicits RVD. Though the cell surface receptor for ATP in Dictyostelium remains elusive, we suggest ATP operates through a Gd(3+)-sensitive receptor that is coupled with intracellular NO production. PMID:26048010

  14. Extracellular ATP inhibits root gravitropism at concentrations that inhibit polar auxin transport

    NASA Technical Reports Server (NTRS)

    Tang, Wenqiang; Brady, Shari R.; Sun, Yu; Muday, Gloria K.; Roux, Stanley J.

    2003-01-01

    Raising the level of extracellular ATP to mM concentrations similar to those found inside cells can block gravitropism of Arabidopsis roots. When plants are grown in Murashige and Skoog medium supplied with 1 mM ATP, their roots grow horizontally instead of growing straight down. Medium with 2 mM ATP induces root curling, and 3 mM ATP stimulates lateral root growth. When plants are transferred to medium containing exogenous ATP, the gravity response is reduced or in some cases completely blocked by ATP. Equivalent concentrations of ADP or inorganic phosphate have slight but usually statistically insignificant effects, suggesting the specificity of ATP in these responses. The ATP effects may be attributable to the disturbance of auxin distribution in roots by exogenously applied ATP, because extracellular ATP can alter the pattern of auxin-induced gene expression in DR5-beta-glucuronidase transgenic plants and increase the response sensitivity of plant roots to exogenously added auxin. The presence of extracellular ATP also decreases basipetal auxin transport in a dose-dependent fashion in both maize (Zea mays) and Arabidopsis roots and increases the retention of [(3)H]indole-3-acetic acid in root tips of maize. Taken together, these results suggest that the inhibitory effects of extracellular ATP on auxin distribution may happen at the level of auxin export. The potential role of the trans-plasma membrane ATP gradient in auxin export and plant root gravitropism is discussed.

  15. Potential therapeutic target for malignant paragangliomas: ATP synthase on the surface of paraganglioma cells

    PubMed Central

    Fliedner, Stephanie MJ; Yang, Chunzhang; Thompson, Eli; Abu-Asab, Mones; Hsu, Chang-Mei; Lampert, Gary; Eiden, Lee; Tischler, Arthur S; Wesley, Robert; Zhuang, Zhengping; Lehnert, Hendrik; Pacak, Karel

    2015-01-01

    F1FoATP synthase (ATP synthase) is a ubiquitous enzyme complex in eukaryotes. In general it is localized to the mitochondrial inner membrane and serves as the last step in the mitochondrial oxidative phosphorylation of ADP to ATP, utilizing a proton gradient across the inner mitochondrial membrane built by the complexes of the electron transfer chain. However some cell types, including tumors, carry ATP synthase on the cell surface. It was suggested that cell surface ATP synthase helps tumor cells thriving on glycolysis to survive their high acid generation. Angiostatin, aurovertin, resveratrol, and antibodies against the α and β subunits of ATP synthase were shown to bind and selectively inhibit cell surface ATP synthase, promoting tumor cell death. Here we show that ATP synthase β (ATP5B) is present on the cell surface of mouse pheochromocytoma cells as well as tumor cells of human SDHB-derived paragangliomas (PGLs), while being virtually absent on chromaffin primary cells from bovine adrenal medulla by confocal microscopy. The cell surface location of ATP5B was verified in the tissue of an SDHB-derived PGL by immunoelectron microscopy. Treatment of mouse pheochromocytoma cells with resveratrol as well as ATP5B antibody led to statistically significant proliferation inhibition. Our data suggest that PGLs carry ATP synthase on their surface that promotes cell survival or proliferation. Thus, cell surface ATP synthase may present a novel therapeutic target in treating metastatic or inoperable PGLs. PMID:26101719

  16. Mitochondrial ATP synthase activity is impaired by suppressed O-GlcNAcylation in Alzheimer's disease.

    PubMed

    Cha, Moon-Yong; Cho, Hyun Jin; Kim, Chaeyoung; Jung, Yang Ouk; Kang, Min Jueng; Murray, Melissa E; Hong, Hyun Seok; Choi, Young-Joo; Choi, Heesun; Kim, Dong Kyu; Choi, Hyunjung; Kim, Jisoo; Dickson, Dennis W; Song, Hyun Kyu; Cho, Jin Won; Yi, Eugene C; Kim, Jungsu; Jin, Seok Min; Mook-Jung, Inhee

    2015-11-15

    Glycosylation with O-linked β-N-acetylglucosamine (O-GlcNAc) is one of the protein glycosylations affecting various intracellular events. However, the role of O-GlcNAcylation in neurodegenerative diseases such as Alzheimer's disease (AD) is poorly understood. Mitochondrial adenosine 5'-triphosphate (ATP) synthase is a multiprotein complex that synthesizes ATP from ADP and Pi. Here, we found that ATP synthase subunit α (ATP5A) was O-GlcNAcylated at Thr432 and ATP5A O-GlcNAcylation was decreased in the brains of AD patients and transgenic mouse model, as well as Aβ-treated cells. Indeed, Aβ bound to ATP synthase directly and reduced the O-GlcNAcylation of ATP5A by inhibition of direct interaction between ATP5A and mitochondrial O-GlcNAc transferase, resulting in decreased ATP production and ATPase activity. Furthermore, treatment of O-GlcNAcase inhibitor rescued the Aβ-induced impairment in ATP production and ATPase activity. These results indicate that Aβ-mediated reduction of ATP synthase activity in AD pathology results from direct binding between Aβ and ATP synthase and inhibition of O-GlcNAcylation of Thr432 residue on ATP5A. PMID:26358770

  17. The Effect of ATP on the Photoconversion of Protochlorophyllide in Isolated Etioplasts of Zea mays1

    PubMed Central

    Horton, Peter; Leech, Rachel M.

    1975-01-01

    The transformation of protochlorophyllide (PChle) into chlorophyllide (Chle) has been studied in isolated etioplasts from Zea mays. ATP (1.5mm) prevented the transformation of photoconvertible PChle 650 to PChle 630 in aged etioplasts. Curve analysis indicated that the ATP effect on photoconvertibility could be entirely accounted for by changes in the proportions of PChle 630 and PChle 650 and examination of the isolated pigments revealed that only unphytylated PChle could be activated for photoconversion by ATP. In etioplasts aged for 5 hours, ATP also stimulated photoconversion of PChle 630 into Chle 670. The process was temperature-sensitive and involved PChle 650 and Chle 680 as intermediates. AMP alone had no effect, but inhibited ATP retardation of PChle loss. ADP had a similar but lesser effect than ATP. The ADP response, but not the ATP response, was considerably enhanced in the presence of an ATP-generating system (phosphoenolpyruvate/pyruvate kinase). UTP, GTP, and CTP gave 40 to 50% of the ATP response with intact etioplasts. In envelope-free etioplasts, ATP gave the greatest response but the other nucleotides were now 80% as effective as ATP. After primary photoconversion, ATP stimulated resynthesis of PChle 650. It is proposed that ATP both gives the holochrome the ability to bind to the PChle molecule and enables additional association of the pigment-protein complex to form PChle 650. PMID:16659239

  18. Evidence for Extracellular ATP as a Stress Signal in a Single-Celled Organism

    PubMed Central

    Sivaramakrishnan, Venketesh

    2015-01-01

    ATP is omnipresent in biology and acts as an extracellular signaling molecule in mammals. Information regarding the signaling function of extracellular ATP in single-celled eukaryotes is lacking. Here, we explore the role of extracellular ATP in cell volume recovery during osmotic swelling in the amoeba Dictyostelium. Release of micromolar ATP could be detected during cell swelling and regulatory cell volume decrease (RVD) phases during hypotonic challenge. Scavenging ATP with apyrase caused profound cell swelling and loss of RVD. Apyrase-induced swelling could be rescued by 100 μM βγ-imidoATP. N-Ethylmalemide (NEM), an inhibitor of vesicular exocytosis, caused heightened cell swelling, loss of RVD, and inhibition of ATP release. Amoebas with impaired contractile vacuole (CV) fusion (drainin knockout [KO] cells) displayed increased swelling but intact ATP release. One hundred micromolar Gd3+ caused cell swelling while blocking any recovery by βγ-imidoATP. ATP release was 4-fold higher in the presence of Gd3+. Cell swelling was associated with an increase in intracellular nitric oxide (NO), with NO-scavenging agents causing cell swelling. Swelling-induced NO production was inhibited by both apyrase and Gd3+, while NO donors rescued apyrase- and Gd3+-induced swelling. These data suggest extracellular ATP released during cell swelling is an important signal that elicits RVD. Though the cell surface receptor for ATP in Dictyostelium remains elusive, we suggest ATP operates through a Gd3+-sensitive receptor that is coupled with intracellular NO production. PMID:26048010

  19. Model-aided atpE gene knockout strategy in Escherichia coli for enhanced succinic acid production from glycerol.

    PubMed

    Mienda, Bashir Sajo; Shamsir, Mohd Shahir; Md Illias, Rosli

    2016-08-01

    Succinic acid is an important platform chemical with a variety of applications. Model-guided metabolic engineering strategies in Escherichia coli for strain improvement to increase succinic acid production using glucose and glycerol remain largely unexplored. Herein, we report what are, to our knowledge, the first metabolic knockout of the atpE gene to have increased succinic acid production using both glucose and alternative glycerol carbon sources in E. coli. Guided by a genome-scale metabolic model, we engineered the E. coli host to enhance anaerobic production of succinic acid by deleting the atpE gene, thereby generating additional reducing equivalents by blocking H(+) conduction across the mutant cell membrane. This strategy produced 1.58 and .49 g l(-1) of succinic acid from glycerol and glucose substrate, respectively. This work further elucidates a model-guided and/or system-based metabolic engineering, involving only a single-gene deletion strategy for enhanced succinic acid production in E. coli. PMID:26513379

  20. Kinetic and hysteretic behavior of ATP hydrolysis of the highly stable dimeric ATP synthase of Polytomella sp.

    PubMed

    Villavicencio-Queijeiro, Alexa; Pardo, Juan Pablo; González-Halphen, Diego

    2015-06-01

    The F1FO-ATP synthase of the colorless alga Polytomella sp. exhibits a robust peripheral arm constituted by nine atypical subunits only present in chlorophycean algae. The isolated dimeric enzyme exhibits a latent ATP hydrolytic activity which can be activated by some detergents. To date, the kinetic behavior of the algal ATPase has not been studied. Here we show that while the soluble F1 sector exhibits Michaelis-Menten kinetics, the dimer exhibits a more complex behavior. The kinetic parameters (Vmax and Km) were obtained for both the F1 sector and the dimeric enzyme as isolated or activated by detergent, and this activation was also seen on the enzyme reconstituted in liposomes. Unlike other ATP synthases, the algal dimer hydrolyzes ATP on a wide range of pH and temperature. The enzyme was inhibited by oligomycin, DCCD and Mg-ADP, although oligomycin induced a peculiar inhibition pattern that can be attributed to structural differences in the algal subunit-c. The hydrolytic activity was temperature-dependent and exhibited activation energy of 4 kcal/mol. The enzyme also exhibited a hysteretic behavior with a lag phase strongly dependent on temperature but not on pH, that may be related to a possible regulatory role in vivo. PMID:25843420

  1. Loss of the gene for the alpha subunit of ATP synthase (ATP5A1) from the W chromosome in the African grey parrot (Psittacus erithacus).

    PubMed

    de Kloet, S R

    2001-08-01

    This study describes the results of an analysis using Southern blotting, the polymerase chain reaction, and sequencing which shows that the African grey parrot (Psittacus erithacus) lacks the W-chromosomal gene for the alpha subunit of mitochondrial ATP synthase (ATP5A1W). Additional evidence shows that in other psittacines a fragment of the ATP5A1W gene contains five times as many nonsynonymous nucleotide replacements as the homologous fragment of the Z gene. Therefore, whereas in these other psittacines the corresponding ATP5A1Z protein fragment is highly conserved and varies by only a few, moderately conservative amino acid substitutions, the homologous ATP5A1W fragments contain a considerable number of, sometimes highly nonconservative, amino acid replacements. In one of these species, the ringneck parakeet (Psittacula krameri), the ATP5A1W gene is present in an inactive form because of the presence of a nonsense codon. Other changes, possibly leading to an inactive ATP5A1W gene product, involve the substitution of arginine residues by cysteine in the ATP5A1W protein of the mitred conure (Aratinga mitrata) and the blue and gold macaw (Ara ararauna). The data suggest also that although the divergence of the psittacine ATP5A1W and ATP5A1Z genes preceded the origin of the psittacidae, this divergence occurred independently of a similar process in the myna (Gracula religiosa), the outgroup used in this study. PMID:11479684

  2. Asymmetric ring structure of Vps4 required for ESCRT-III disassembly

    NASA Astrophysics Data System (ADS)

    Caillat, Christophe; Macheboeuf, Pauline; Wu, Yuanfei; McCarthy, Andrew A.; Boeri-Erba, Elisabetta; Effantin, Gregory; Göttlinger, Heinrich G.; Weissenhorn, Winfried; Renesto, Patricia

    2015-12-01

    The vacuolar protein sorting 4 AAA-ATPase (Vps4) recycles endosomal sorting complexes required for transport (ESCRT-III) polymers from cellular membranes. Here we present a 3.6-Å X-ray structure of ring-shaped Vps4 from Metallosphera sedula (MsVps4), seen as an asymmetric pseudohexamer. Conserved key interface residues are shown to be important for MsVps4 assembly, ATPase activity in vitro, ESCRT-III disassembly in vitro and HIV-1 budding. ADP binding leads to conformational changes within the protomer, which might propagate within the ring structure. All ATP-binding sites are accessible and the pseudohexamer binds six ATP with micromolar affinity in vitro. In contrast, ADP occupies one high-affinity and five low-affinity binding sites in vitro, consistent with conformational asymmetry induced on ATP hydrolysis. The structure represents a snapshot of an assembled Vps4 conformation and provides insight into the molecular motions the ring structure undergoes in a concerted action to couple ATP hydrolysis to ESCRT-III substrate disassembly.

  3. Asymmetric ring structure of Vps4 required for ESCRT-III disassembly

    PubMed Central

    Caillat, Christophe; Macheboeuf, Pauline; Wu, Yuanfei; McCarthy, Andrew A.; Boeri-Erba, Elisabetta; Effantin, Gregory; Göttlinger, Heinrich G.; Weissenhorn, Winfried; Renesto, Patricia

    2015-01-01

    The vacuolar protein sorting 4 AAA–ATPase (Vps4) recycles endosomal sorting complexes required for transport (ESCRT-III) polymers from cellular membranes. Here we present a 3.6-Å X-ray structure of ring-shaped Vps4 from Metallosphera sedula (MsVps4), seen as an asymmetric pseudohexamer. Conserved key interface residues are shown to be important for MsVps4 assembly, ATPase activity in vitro, ESCRT-III disassembly in vitro and HIV-1 budding. ADP binding leads to conformational changes within the protomer, which might propagate within the ring structure. All ATP-binding sites are accessible and the pseudohexamer binds six ATP with micromolar affinity in vitro. In contrast, ADP occupies one high-affinity and five low-affinity binding sites in vitro, consistent with conformational asymmetry induced on ATP hydrolysis. The structure represents a snapshot of an assembled Vps4 conformation and provides insight into the molecular motions the ring structure undergoes in a concerted action to couple ATP hydrolysis to ESCRT-III substrate disassembly. PMID:26632262

  4. Activation of ATP binding for the autophosphorylation of DosS, a Mycobacterium tuberculosis histidine kinase lacking an ATP lid motif.

    PubMed

    Cho, Ha Yeon; Lee, Young-Hoon; Bae, Young-Seuk; Kim, Eungbin; Kang, Beom Sik

    2013-05-01

    The sensor histidine kinases of Mycobacterium tuberculosis, DosS and DosT, are responsible for sensing hypoxic conditions and consist of sensor and kinase cores responsible for accepting signals and phosphorylation activity, respectively. The kinase core contains a dimerization and histidine phosphate-accepting (DHp) domain and an ATP binding domain (ABD). The 13 histidine kinase genes of M. tuberculosis can be grouped based on the presence or absence of the ATP lid motif and F box (elements known to play roles in ATP binding) in their ABDs; DosS and DosT have ABDs lacking both these elements, and the crystal structures of their ABDs indicated that they were unsuitable for ATP binding, as a short loop covers the putative ATP binding site. Although the ABD alone cannot bind ATP, the kinase core is functional in autophosphorylation. Appropriate spatial arrangement of the ABD and DHp domain within the kinase core is required for both autophosphorylation and ATP binding. An ionic interaction between Arg(440) in the DHp domain and Glu(537) in the short loop of the ABD is available and may open the ATP binding site, by repositioning the short loop away from the site. Mutations at Arg(440) and Glu(537) reduce autophosphorylation activity. Unlike other histidine kinases containing an ATP lid, which protects bound ATP, DosS is unable to accept ATP until the ABD is properly positioned relative to the histidine; this may prevent unexpected ATP reactions. ATP binding can, therefore, function as a control mechanism for histidine kinase activity. PMID:23486471

  5. Identification of a mitochondrial ATP synthase-adenine nucleotide translocator complex in Leishmania.

    PubMed

    Detke, Siegfried; Elsabrouty, Rania

    2008-01-01

    The ATP synthasome is a macromolecular complex consisting of ATP synthase, adenine nucleotide translocator and phosphate carrier. To determine if this complex is evolutionary old or young, we searched for its presence in Leishmania, a mitochondria containing protozoan which evolved from the main eukaryote line soon after eukaryotes split from prokaryotes. Sucrose gradient centrifugation showed that the distribution of ANT among the fractions coincided with the distribution of ATP synthase. In addition, ATP synthase co-precipitated with FLAG tagged and wild type adenine nucleotide translocator isolated with anti FLAG and anti adenine nucleotide translocator antibodies, respectively. These data indicate that the adenine nucleotide translocator interacted with the ATP synthase to form a stable structure referred to as the ATP synthasome. The presence of the ATP synthasome in Leishmania, an organism branching off the main line of eukaryotes early in the development of eukaryotes, as well as in higher eukaryotes suggests that the ATP synthasome is a phylogenetically ancient structure. PMID:17920025

  6. Hydrostatic pressure activates ATP-sensitive K+ channels in lung epithelium by ATP release through pannexin and connexin hemichannels.

    PubMed

    Richter, Katrin; Kiefer, Kevin P; Grzesik, Benno A; Clauss, Wolfgang G; Fronius, Martin

    2014-01-01

    Lungs of air-breathing vertebrates are constantly exposed to mechanical forces and therefore are suitable for investigation of mechanotransduction processes in nonexcitable cells and tissues. Freshly dissected Xenopus laevis lungs were used for transepithelial short-circuit current (ISC) recordings and were exposed to increased hydrostatic pressure (HP; 5 cm fluid column, modified Ussing chamber). I(SC) values obtained under HP (I(5cm)) were normalized to values before HP (I(0cm)) application (I(5cm)/I(0cm)). Under control conditions, HP decreased I(SC) (I(5cm)/I(0cm)=0.84; n=68; P<0.0001). This effect was reversible and repeatable ≥30 times. Preincubation with ATP-sensitive K(+) channel (K(ATP)) inhibitors (HMR1098 and glibenclamide) prevented the decrease in I(SC) (I(5cm)/I(0cm): HMR1098=1.19, P<0.0001; glibenclamide=1.11, P<0.0001). Similar effects were observed with hemichannel inhibitors (I(5cm)/I(0cm): meclofenamic acid=1.09, P<0.0001; probenecid=1.0, P<0.0001). The HP effect was accompanied by release of ATP (P<0.05), determined by luciferin-luciferase luminescence in perfusion solution from the luminal side of an Ussing chamber. ATP release was abrogated by both meclofenamic acid and probenecid. RT-PCR experiments revealed the expression of pannexin and connexin hemichannels and KATP subunit transcripts in X. laevis lung. These data show an activation of KATP in pulmonary epithelial cells in response to HP that is induced by ATP release through mechanosensitive pannexin and connexin hemichannels. These findings represent a novel mechanism of mechanotransduction in nonexcitable cells. PMID:24048216

  7. Dynamic Regulation of Cell Volume and Extracellular ATP of Human Erythrocytes

    PubMed Central

    Leal Denis, M. Florencia; Alvarez, H. Ariel; Lauri, Natalia; Alvarez, Cora L.; Chara, Osvaldo; Schwarzbaum, Pablo J.

    2016-01-01

    Introduction The peptide mastoparan 7 (MST7) triggered in human erythrocytes (rbcs) the release of ATP and swelling. Since swelling is a well-known inducer of ATP release, and extracellular (ATPe), interacting with P (purinergic) receptors, can affect cell volume (Vr), we explored the dynamic regulation between Vr and ATPe. Methods and Treatments We made a quantitative assessment of MST7-dependent kinetics of Vr and of [ATPe], both in the absence and presence of blockers of ATP efflux, swelling and P receptors. Results In rbcs 10 μM MST7 promoted acute, strongly correlated changes in [ATPe] and Vr. Whereas MST7 induced increases of 10% in Vr and 190 nM in [ATPe], blocking swelling in a hyperosmotic medium + MST7 reduced [ATPe] by 40%. Pre-incubation of rbcs with 10 μM of either carbenoxolone or probenecid, two inhibitors of the ATP conduit pannexin 1, reduced [ATPe] by 40–50% and swelling by 40–60%, while in the presence of 80 U/mL apyrase, an ATPe scavenger, cell swelling was prevented. While exposure to 10 μM NF110, a blocker of ATP-P2X receptors mediating sodium influx, reduced [ATPe] by 48%, and swelling by 80%, incubation of cells in sodium free medium reduced swelling by 92%. Analysis and Discussion Results were analyzed by means of a mathematical model where ATPe kinetics and Vr kinetics were mutually regulated. Model dependent fit to experimental data showed that, upon MST7 exposure, ATP efflux required a fast 1960-fold increase of ATP permeability, mediated by two kinetically different conduits, both of which were activated by swelling and inactivated by time. Both experimental and theoretical results suggest that, following MST7 exposure, ATP is released via two conduits, one of which is mediated by pannexin 1. The accumulated ATPe activates P2X receptors, followed by sodium influx, resulting in cell swelling, which in turn further activates ATP release. Thus swelling and P2X receptors constitute essential components of a positive feedback loop

  8. Cytotoxicity of mitochondria-targeted resveratrol derivatives: interactions with respiratory chain complexes and ATP synthase.

    PubMed

    Sassi, Nicola; Mattarei, Andrea; Azzolini, Michele; Szabo', Ildiko'; Paradisi, Cristina; Zoratti, Mario; Biasutto, Lucia

    2014-10-01

    We recently reported that mitochondria-targeted derivatives of resveratrol are cytotoxic in vitro, selectively inducing mostly necrotic death of fast-growing and tumoral cells when supplied in the low μM range (N. Sassi et al., Curr. Pharm. Des. 2014). Cytotoxicity is due to H2O2 produced upon accumulation of the compounds into mitochondria. We investigate here the mechanisms underlying ROS generation and mitochondrial depolarization caused by these agents. We find that they interact with the respiratory chain, especially complexes I and III, causing superoxide production. "Capping" free hydroxyls with acetyl or methyl groups increases their effectiveness as respiratory chain inhibitors, promoters of ROS generation and cytotoxic agents. Exposure to the compounds also induces an increase in the occurrence of short transient [Ca(2+)] "spikes" in the cells. This increase is unrelated to ROS production, and it is not the cause of cell death. These molecules furthermore inhibit the F0F1 ATPase. When added to oligomycin-treated cells, the acetylated/methylated ones cause a recovery of the cellular oxygen consumption rates depressed by oligomycin. Since a protonophoric futile cycle which might account for the uncoupling effect is impossible, we speculate that the compounds may cause the transformation of the ATP synthase and/or respiratory chain complex(es) into a conduit for uncoupled proton translocation. Only in the presence of excess oligomycin the most effective derivatives appear to induce the mitochondrial permeability transition (MPT) within the cells. This may be considered to provide circumstantial support for the idea that the ATP synthase is the molecular substrate for the MPT pore. PMID:24997425

  9. Subtype-specific control of P2X receptor channel signaling by ATP and Mg2+

    PubMed Central

    Li, Mufeng; Silberberg, Shai D.; Swartz, Kenton J.

    2013-01-01

    The identity and forms of activating ligands for ion channels are fundamental to their physiological roles in rapid electrical signaling. P2X receptor channels are ATP-activated cation channels that serve important roles in sensory signaling and inflammation, yet the active forms of the nucleotide are unknown. In physiological solutions, ATP is ionized and primarily found in complex with Mg2+. Here we investigated the active forms of ATP and found that the action of MgATP2− and ATP4− differs between subtypes of P2X receptors. The slowly desensitizing P2X2 receptor can be activated by free ATP, but MgATP2− promotes opening with very low efficacy. In contrast, both free ATP and MgATP2− robustly open the rapidly desensitizing P2X3 subtype. A further distinction between these two subtypes is the ability of Mg2+ to regulate P2X3 through a distinct allosteric mechanism. Importantly, heteromeric P2X2/3 channels present in sensory neurons exhibit a hybrid phenotype, characterized by robust activation by MgATP2− and weak regulation by Mg2+. These results reveal the existence of two classes of homomeric P2X receptors with differential sensitivity to MgATP2− and regulation by Mg2+, and demonstrate that both restraining mechanisms can be disengaged in heteromeric channels to form fast and sensitive ATP signaling pathways in sensory neurons. PMID:23959888

  10. Fullerene derived molecularly imprinted polymer for chemosensing of adenosine-5'-triphosphate (ATP).

    PubMed

    Sharma, Piyush S; Dabrowski, Marcin; Noworyta, Krzysztof; Huynh, Tan-Phat; Kc, Chandra B; Sobczak, Janusz W; Pieta, Piotr; D'Souza, Francis; Kutner, Wlodzimierz

    2014-09-24

    For molecular imprinting of oxidatively electroactive analytes by electropolymerization, we used herein reductively electroactive functional monomers. As a proof of concept, we applied C60 fullerene adducts as such for the first time. For that, we derivatized C60 to bear either an uracil or an amide, or a carboxy addend for recognition of the adenosine-5'-triphosphate (ATP) oxidizable analyte with the ATP-templated molecularly imprinted polymer (MIP-ATP). Accordingly, the ATP complex with all of the functional monomers formed in solution was potentiodynamically electropolymerized to deposit an MIP-ATP film either on an Au electrode of the quartz crystal resonator or on a Pt disk electrode for the piezoelectric microgravimetry (PM) or capacitive impedimetry (CI) determination of ATP, respectively, under the flow-injection analysis (FIA) conditions. The apparent imprinting factor for ATP was ∼4.0. After extraction of the ATP template, analytical performance of the resulting chemosensors, including detectability, sensitivity, and selectivity, was characterized. The limit of detection was 0.3 and 0.03mM ATP for the PM and CI chemosensor, respectively. The MIP-ATP film discriminated structural analogues of ATP quite well. The Langmuir, Freundlich, and Langmuir-Freundlich isotherms were fitted to the experimental data of the ATP sorption and sorption stability constants appeared to be nearly independent of the adopted sorption model. PMID:25172817

  11. Measurement and interpretation of microbial adenosine tri-phosphate (ATP) in aquatic environments.

    PubMed

    Hammes, Frederik; Goldschmidt, Felix; Vital, Marius; Wang, Yingying; Egli, Thomas

    2010-07-01

    There is a widespread need for cultivation-free methods to quantify viability of natural microbial communities in aquatic environments. Adenosine tri-phosphate (ATP) is the energy currency of all living cells, and therefore a useful indicator of viability. A luminescence-based ATP kit/protocol was optimised in order to detect ATP concentrations as low as 0.0001 nM with a standard deviation of <5%. Using this method, more than 100 water samples from a variety of aquatic environments (drinking water, groundwater, bottled water, river water, lake water and wastewater effluent) were analysed for extracellular ATP and microbial ATP in comparison with flow-cytometric (FCM) parameters. Microbial ATP concentrations ranged between 3% and 97% of total ATP concentrations, and correlated well (R(2)=0.8) with the concentrations of intact microbial cells (after staining with propidium iodide). From this correlation, we calculated an average ATP-per-cell value of 1.75x10(-10)nmol/cell. An even better correlation (R(2)=0.88) was observed between intact biovolume (derived from FCM scatter data) and microbial ATP concentrations, and an average ATP-per-biovolume value of 2.95x10(-9)nmol/microm(3) was calculated. These results support the use of ATP analysis for both routine monitoring and research purposes, and contribute towards a better interpretation of ATP data. PMID:20605621

  12. 7 CFR 3300.88 - Fees for U.S. ATP certificates.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 15 2010-01-01 2010-01-01 false Fees for U.S. ATP certificates. 3300.88 Section 3300... EQUIPMENT TO BE USED FOR SUCH CARRIAGE (ATP); INSPECTION, TESTING, AND CERTIFICATION OF SPECIAL EQUIPMENT Other Provisions § 3300.88 Fees for U.S. ATP certificates. The fee schedule for issuance of U.S....

  13. Characterization of an ATP translocase identified in the plant pathogen, Candidatus Liberibacter asiaticus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ATP/ADP translocases allow for the transport of ATP across a lipid bilayer, which is normally impermeable to this molecule due to its size and charge. These transport proteins appear to be unique to mitochondria, plant plastids, and obligate-intracellular bacteria. Of the bacterial ATP/ADP translo...

  14. 42 CFR 88.1 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ...) through (1)(xi)of this definition. (2) Mental health conditions: (i) Posttraumatic stress disorder. (ii) Major depressive disorder. (iii) Panic disorder. (iv) Generalized anxiety disorder. (v) Anxiety disorder . (vi) Depression . (vii) Acute stress disorder. (viii) Dysthymic disorder. (ix) Adjustment disorder....

  15. 12 CFR 3.202 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... least two tranches that reflect different levels of seniority; (3) Performance of the securitization... in ); (iii) An employee benefit plan as defined in paragraphs (3) and (32) of section 3 of ERISA, a... 12 Banks and Banking 1 2014-01-01 2014-01-01 false Definitions. 3.202 Section 3.202 Banks...

  16. 24 CFR 290.3 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... Disposition of Multifamily Projects § 290.3 Definitions. The terms Department and URA are defined in 24 CFR... consumer cooperative as defined under 24 CFR part 213. It may include mutual housing associations. HUD... section 236(f)(2) of the National Housing Act (hereinafter, RAP); (iii) Housing assistance payments...

  17. 24 CFR 290.3 - Definitions.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Disposition of Multifamily Projects § 290.3 Definitions. The terms Department and URA are defined in 24 CFR... consumer cooperative as defined under 24 CFR part 213. It may include mutual housing associations. HUD... section 236(f)(2) of the National Housing Act (hereinafter, RAP); (iii) Housing assistance payments...

  18. 24 CFR 290.3 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... Disposition of Multifamily Projects § 290.3 Definitions. The terms Department and URA are defined in 24 CFR... consumer cooperative as defined under 24 CFR part 213. It may include mutual housing associations. HUD... section 236(f)(2) of the National Housing Act (hereinafter, RAP); (iii) Housing assistance payments...

  19. 24 CFR 290.3 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... Disposition of Multifamily Projects § 290.3 Definitions. The terms Department and URA are defined in 24 CFR... consumer cooperative as defined under 24 CFR part 213. It may include mutual housing associations. HUD... section 236(f)(2) of the National Housing Act (hereinafter, RAP); (iii) Housing assistance payments...

  20. 7 CFR 1944.506 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... income, computed in accordance with 7 CFR part 3550, subpart B, does not exceed the maximum low-income... distributing any gains or profits to its members. (f) Rural area. The definition in 7 CFR part 3550 applies. (g...; (ii) Monitoring payment of taxes and insurance; (iii) Home maintenance and managment; and (iv)...

  1. 21 CFR 17.3 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...) Defective, for the purposes of interpreting 21 U.S.C. 333(g)(1)(B)(iii), includes any defect in performance... or respondent includes an individual, partnership, corporation, association, scientific or academic... 21 Food and Drugs 1 2010-04-01 2010-04-01 false Definitions. 17.3 Section 17.3 Food and Drugs...

  2. 5 CFR 831.1901 - Definitions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...) RETIREMENT State Income Tax Withholding § 831.1901 Definitions. For the purpose of this subpart: Agreement means the Federal-State agreement contained in this subpart. Annuitant means an employee or Member... III, chapter 83 of title 5, United States Code. Effective date means, with respect to a request...

  3. 5 CFR 831.1901 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ...) RETIREMENT State Income Tax Withholding § 831.1901 Definitions. For the purpose of this subpart: Agreement means the Federal-State agreement contained in this subpart. Annuitant means an employee or Member... III, chapter 83 of title 5, United States Code. Effective date means, with respect to a request...

  4. 5 CFR 831.1901 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ...) RETIREMENT State Income Tax Withholding § 831.1901 Definitions. For the purpose of this subpart: Agreement means the Federal-State agreement contained in this subpart. Annuitant means an employee or Member... III, chapter 83 of title 5, United States Code. Effective date means, with respect to a request...

  5. 5 CFR 831.1901 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ...) RETIREMENT State Income Tax Withholding § 831.1901 Definitions. For the purpose of this subpart: Agreement means the Federal-State agreement contained in this subpart. Annuitant means an employee or Member... III, chapter 83 of title 5, United States Code. Effective date means, with respect to a request...

  6. 5 CFR 831.1901 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...) RETIREMENT State Income Tax Withholding § 831.1901 Definitions. For the purpose of this subpart: Agreement means the Federal-State agreement contained in this subpart. Annuitant means an employee or Member... III, chapter 83 of title 5, United States Code. Effective date means, with respect to a request...

  7. 14 CFR 414.3 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... activities for which a license is required under 14 CFR chapter III. A safety approval does not relieve its... 14 Aeronautics and Space 4 2013-01-01 2013-01-01 false Definitions. 414.3 Section 414.3 Aeronautics and Space COMMERCIAL SPACE TRANSPORTATION, FEDERAL AVIATION ADMINISTRATION, DEPARTMENT...

  8. 34 CFR 5.2 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Secretary, Department of Education AVAILABILITY OF INFORMATION TO THE PUBLIC General Provisions § 5.2 Definitions. As used in this part: (a) Act or FOIA means the Freedom of Information Act, as amended, 5 U.S.C... under a records management contract with the Federal Government; and (iii) Documentary...

  9. 34 CFR 5.2 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... Secretary, Department of Education AVAILABILITY OF INFORMATION TO THE PUBLIC General Provisions § 5.2 Definitions. As used in this part: (a) Act or FOIA means the Freedom of Information Act, as amended, 5 U.S.C... under a records management contract with the Federal Government; and (iii) Documentary...

  10. 17 CFR 41.43 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... considered a customer of the security futures intermediary absent the partnership relationship; and (iii) Any... T means Regulation T promulgated by the Board of Governors of the Federal Reserve System, 12 CFR... Customer Accounts and Margin Requirements § 41.43 Definitions. (a) For purposes of this Regulation...

  11. 10 CFR 580.02 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... paragraph (b) of this section. (b) The following definitions are applicable to this part: (1) Commercial... Secretary of Agriculture determines is necessary for full food and fiber production. (3) Essential... commercial establishment in amounts of less than 50 Mcf on a peak day; or (iii) In any school or hospital;...

  12. 10 CFR 580.02 - Definitions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... paragraph (b) of this section. (b) The following definitions are applicable to this part: (1) Commercial... Secretary of Agriculture determines is necessary for full food and fiber production. (3) Essential... commercial establishment in amounts of less than 50 Mcf on a peak day; or (iii) In any school or hospital;...

  13. 10 CFR 580.02 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... paragraph (b) of this section. (b) The following definitions are applicable to this part: (1) Commercial... Secretary of Agriculture determines is necessary for full food and fiber production. (3) Essential... commercial establishment in amounts of less than 50 Mcf on a peak day; or (iii) In any school or hospital;...

  14. 10 CFR 580.02 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... paragraph (b) of this section. (b) The following definitions are applicable to this part: (1) Commercial... Secretary of Agriculture determines is necessary for full food and fiber production. (3) Essential... commercial establishment in amounts of less than 50 Mcf on a peak day; or (iii) In any school or hospital;...

  15. 46 CFR 160.076-5 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... of the PFD (I, II, or III). PFD means personal flotation device as defined in 33 CFR 175.13. PFD... 46 Shipping 6 2012-10-01 2012-10-01 false Definitions. 160.076-5 Section 160.076-5 Shipping COAST...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Inflatable Recreational Personal Flotation Devices §...

  16. 46 CFR 160.076-5 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... classification of the PFD (I, II, or III). PFD means personal flotation device as defined in 33 CFR 175.13. PFD... 46 Shipping 6 2010-10-01 2010-10-01 false Definitions. 160.076-5 Section 160.076-5 Shipping COAST...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Inflatable Recreational Personal Flotation Devices §...

  17. 46 CFR 160.076-5 - Definitions.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... classification of the PFD (I, II, or III). PFD means personal flotation device as defined in 33 CFR 175.13. PFD... 46 Shipping 6 2011-10-01 2011-10-01 false Definitions. 160.076-5 Section 160.076-5 Shipping COAST...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Inflatable Recreational Personal Flotation Devices §...

  18. Calcium induced ATP synthesis: Isotope effect, magnetic parameters and mechanism

    NASA Astrophysics Data System (ADS)

    Buchachenko, A. L.; Kuznetsov, D. A.; Breslavskaya, N. N.; Shchegoleva, L. N.; Arkhangelsky, S. E.

    2011-03-01

    ATP synthesis by creatine kinase with calcium ions is accompanied by 43Ca/ 40Ca isotope effect: the enzyme with 43Ca 2+ was found to be 2.0 ± 0.3 times more active than enzymes, in which Ca 2+ ions have nonmagnetic nuclei 40Ca. The effect demonstrates that primary reaction in ATP synthesis is electron transfer between reaction partners, Сa( HO)n2+ ( n ⩽ 3) and Ca 2+(ADP) 3-. It generates ion-radical pair, in which spin conversion results in the isotope effect. Magnetic parameters (g-factors and HFC constants a( 43Ca) and a( 31P)) confirm that namely terminal oxygen atom of the ADP ligand in the complex Ca 2+(ADP) 3- donates electron to the Ca( HO)n2+ ion.

  19. Pathogenic VCP Mutations Induce Mitochondrial Uncoupling and Reduced ATP Levels

    PubMed Central

    Bartolome, Fernando; Wu, Hsiu-Chuan; Burchell, Victoria S.; Preza, Elisavet; Wray, Selina; Mahoney, Colin J.; Fox, Nick C.; Calvo, Andrea; Canosa, Antonio; Moglia, Cristina; Mandrioli, Jessica; Chiò, Adriano; Orrell, Richard W.; Houlden, Henry; Hardy, John; Abramov, Andrey Y.; Plun-Favreau, Helene

    2013-01-01

    Summary Valosin-containing protein (VCP) is a highly expressed member of the type II AAA+ ATPase family. VCP mutations are the cause of inclusion body myopathy, Paget’s disease of the bone, and frontotemporal dementia (IBMPFD) and they account for 1%–2% of familial amyotrophic lateral sclerosis (ALS). Using fibroblasts from patients carrying three independent pathogenic mutations in the VCP gene, we show that VCP deficiency causes profound mitochondrial uncoupling leading to decreased mitochondrial membrane potential and increased mitochondrial oxygen consumption. This mitochondrial uncoupling results in a significant reduction of cellular ATP production. Decreased ATP levels in VCP-deficient cells lower their energy capacity, making them more vulnerable to high energy-demanding processes such as ischemia. Our findings propose a mechanism by which pathogenic VCP mutations lead to cell death. PMID:23498975

  20. Bioluminescence microscopy: application to ATP measurements in single living cells

    NASA Astrophysics Data System (ADS)

    Brau, Frederic; Helle, Pierre; Bernengo, Jean C.

    1997-12-01

    Bioluminescence microscopy can be used to measure intracellular cofactors and ionic concentrations (Ca2+, K+, ATP, NADH), as an alternative to micro- spectrophotometry and micro-fluorimetry, due to the development of sensitive detectors (cooled photomultipliers tubes and CCD). The main limitation comes from the very small and brief intensity of the emitted light. Our instrumentation based on an inverted microscope, equipped with high aperture immersion lenses is presented. Light intensity measurements are carried out through a photomultiplier sorted for low dark current and cooled at -5 degree(s)C to reduce thermal noise. Our first aim is to quantify ATP on single living cells using the firefly luciferin-luciferase couple. Experimental and kinetic aspects are presented to emphasize the potentialities of the technique.

  1. Statistical Mechanics Analysis of ATP Binding to a Multisubunit Enzyme

    NASA Astrophysics Data System (ADS)

    Zhang, Yun-Xin

    2014-10-01

    Due to inter-subunit communication, multisubunit enzymes usually hydrolyze ATP in a concerted fashion. However, so far the principle of this process remains poorly understood. In this study, from the viewpoint of statistical mechanics, a simple model is presented. In this model, we assume that the binding of ATP will change the potential of the corresponding enzyme subunit, and the degree of this change depends on the state of its adjacent subunits. The probability of enzyme in a given state satisfies the Boltzmann's distribution. Although it looks much simple, this model can fit the recent experimental data of chaperonin TRiC/CCT well. From this model, the dominant state of TRiC/CCT can be obtained. This study provide a new way to understand biophysical processe by statistical mechanics analysis.

  2. Arsenic Binding and Transfer by the ArsD As(III) Metallochaperone†

    PubMed Central

    Yang, Jianbo; Rawat, Swati; Stemmler, Timothy L.; Rosen, Barry P.

    2010-01-01

    ArsD is a metallochaperone that delivers trivalent metalloids [As(III) or Sb(III)] to the ArsA ATPase, the catalytic subunit of the ArsAB pump encoded by the arsRDABC operon of Escherichia coli plasmid R773. Interaction with ArsD increases the affinity of ArsA for As(III), conferring resistance to environmental concentrations of arsenic. Previous genetic analysis suggested that ArsD residues Cys12, Cys13, and Cys18 are involved in the transfer of As(III) to ArsA. Here X-ray absorption spectroscopy was used to show that As(III) is coordinated with three sulfur atoms, consistent with the three cysteine residues forming the As(III) binding site. Two single-tryptophan derivatives of ArsD exhibited quenching of intrinsic protein fluorescence upon binding of As(III) or Sb(III), which allowed estimation of the rates of binding and affinities for metalloids. Substitution of Cys12, Cys13, or Cys18 decreased the affinity for As(III) more than 10-fold. Reduced glutathione greatly increased the rate of binding of As(III) to ArsD but did not affect binding of As(III) to ArsA. This suggests that in vivo cytosolic As(III) might be initially bound to GSH and transferred to ArsD and then to ArsAB, which pumps the metalloid out of the cell. The As(III) chelator dimercaptosuccinic acid did not block the transfer from ArsD to ArsA, consistent with channeling of the metalloid from one protein to the other, as opposed to release and rebinding of the metalloid. Finally, transfer of As(III) from ArsD to ArsA occurred in the presence of MgATP at 23 °C but not at 4 °C. Neither MgADP nor MgATP-γ-S could replace MgATP. These results suggest that transfer occurs with a conformation of ArsA that transiently forms during the catalytic cycle. PMID:20361763

  3. Arsenic binding and transfer by the ArsD As(III) metallochaperone.

    PubMed

    Yang, Jianbo; Rawat, Swati; Stemmler, Timothy L; Rosen, Barry P

    2010-05-01

    ArsD is a metallochaperone that delivers trivalent metalloids [As(III) or Sb(III)] to the ArsA ATPase, the catalytic subunit of the ArsAB pump encoded by the arsRDABC operon of Escherichia coli plasmid R773. Interaction with ArsD increases the affinity of ArsA for As(III), conferring resistance to environmental concentrations of arsenic. Previous genetic analysis suggested that ArsD residues Cys12, Cys13, and Cys18 are involved in the transfer of As(III) to ArsA. Here X-ray absorption spectroscopy was used to show that As(III) is coordinated with three sulfur atoms, consistent with the three cysteine residues forming the As(III) binding site. Two single-tryptophan derivatives of ArsD exhibited quenching of intrinsic protein fluorescence upon binding of As(III) or Sb(III), which allowed estimation of the rates of binding and affinities for metalloids. Substitution of Cys12, Cys13, or Cys18 decreased the affinity for As(III) more than 10-fold. Reduced glutathione greatly increased the rate of binding of As(III) to ArsD but did not affect binding of As(III) to ArsA. This suggests that in vivo cytosolic As(III) might be initially bound to GSH and transferred to ArsD and then to ArsAB, which pumps the metalloid out of the cell. The As(III) chelator dimercaptosuccinic acid did not block the transfer from ArsD to ArsA, consistent with channeling of the metalloid from one protein to the other, as opposed to release and rebinding of the metalloid. Finally, transfer of As(III) from ArsD to ArsA occurred in the presence of MgATP at 23 degrees C but not at 4 degrees C. Neither MgADP nor MgATP-gamma-S could replace MgATP. These results suggest that transfer occurs with a conformation of ArsA that transiently forms during the catalytic cycle. PMID:20361763

  4. Actions of pinacidil at a reduced potassium concentration: a direct cardiac effect possibly involving the ATP-dependent potassium channel.

    PubMed

    Chi, L; Black, S C; Kuo, P I; Fagbemi, S O; Lucchesi, B R

    1993-02-01

    We investigated the effects of the ATP-dependent K+ channel antagonist glyburide and the ATP-dependent K+ channel agonist pinacidil in a Langendorff-perfused rabbit isolated heart subjected to a period of global hypoxia. A class III antiarrhythmic drug, E-4031, also was studied in this model. These studies aimed to define the mechanism of action of putative profibrillatory actions of pinacidil and the mechanism for the antifibrillatory effect of the class III antiarrhythmic drug, E-4031, in the hypoxic heart. After stabilization and determination of baseline functional parameters under normoxic perfusion conditions (95% O2/5% CO2), hearts were subjected to global hypoxia by switching to a 95% N2/5% CO2 saturated perfusion medium for a period of 12 min. After the hypoxic period, normoxia was re-established by switching to the oxygen-carbon dioxide saturated buffer medium for a period of 40 min. The oxygen tension of the perfusion buffer was reduced from approximately 400 mm Hg to below 50 mm Hg during the hypoxic period. All hearts subjected to hypoxia had reduced function: the left ventricular developed pressure and +/- dP/dt were reduced significantly. Myocardial tissue ATP concentrations were reduced (> 50%) in hearts subjected to hypoxia. Under conditions of hypoxic/reoxygenation and in the presence of a low (2.5 mM) potassium concentration ([K+]0), pinacidil (1.25 microM) facilitated the induction of ventricular fibrillation (80% fibrillation in the presence of pinacidil vs. 20% in the absence of pinacidil). Glyburide (10 microM) and E-4031 (1 and 10 microM) significantly reduced the incidence of ventricular fibrillation associated with pinacidil (20% fibrillation in the presence of hypoxia, pinacidil, and glyburide or 10 microM E-4031). Opening of the ATP-dependent K+ channel by pinacidil under normoxia and low K+ also facilitated the induction of ventricular fibrillation (60% ventricular fibrillation). Pinacidil failed to induce ventricular fibrillation under

  5. Inhibition of Escherichia coli ATP synthase by amphibian antimicrobial peptides.

    PubMed

    Laughlin, Thomas F; Ahmad, Zulfiqar

    2010-04-01

    Previously melittin, the alpha-helical basic honey bee venom peptide, was shown to inhibit F(1)-ATPase by binding at the beta-subunit DELSEED motif of F(1)F(o)-ATP synthase. Herein, we present the inhibitory effects of the basic alpha-helical amphibian antimicrobial peptides, ascaphin-8, aurein 2.2, aurein 2.3, carein 1.8, carein 1.9, citropin 1.1, dermaseptin, maculatin 1.1, maganin II, MRP, or XT-7, on purified F(1) and membrane bound F(1)F(0)Escherichia coli ATP synthase. We found that the extent of inhibition by amphibian peptides is variable. Whereas MRP-amide inhibited ATPase essentially completely (approximately 96% inhibition), carein 1.8 did not inhibit at all (0% inhibition). Inhibition by other peptides was partial with a range of approximately 13-70%. MRP-amide was also the most potent inhibitor on molar scale (IC(50) approximately 3.25 microM). Presence of an amide group at the c-terminal of peptides was found to be critical in exerting potent inhibition of ATP synthase ( approximately 20-40% additional inhibition). Inhibition was fully reversible and found to be identical in both F(1)F(0) membrane preparations as well as in isolated purified F(1). Interestingly, growth of E. coli was abrogated in the presence of ascaphin-8, aurein 2.2, aurein 2.3, citropin 1.1, dermaseptin, magainin II-amide, MRP, MRP-amide, melittin, or melittin-amide but was unaffected in the presence of carein 1.8, carein 1.9, maculatin 1.1, magainin II, or XT-7. Hence inhibition of F(1)-ATPase and E. coli cell growth by amphibian antimicrobial peptides suggests that their antimicrobial/anticancer properties are in part linked to their actions on ATP synthase. PMID:20100509

  6. Inhibition of Escherichia coli ATP synthase by amphibian antimicrobial peptides

    PubMed Central

    Laughlin, Thomas F.; Ahmad, Zulfiqar

    2010-01-01

    Previously melittin, the α-helical basic honey bee venom peptide, was shown to inhibit F1-ATPase by binding at the β-subunit DELSEED motif of F1Fo ATP synthase. Herein, we present the inhibitory effects of the basic α-helical amphibian antimicrobial peptides, ascaphin-8, aurein 2.2, aurein 2.3, carein 1.8, carein 1.9, citropin 1.1, dermaseptin, maculatin 1.1, maganin II, MRP, or XT-7, on purified F1 and membrane bound F1Fo E. coli ATP synthase. We found that the extent of inhibition by amphibian peptides is variable. Whereas MRP-amide inhibited ATPase essentially completely (~96% inhibition), carein 1.8 did not inhibit at all (0% inhibition). Inhibition by other peptides was partial with a range of ~13% to 70%. MRP-amide was also the most potent inhibitor on molar scale (IC50 ~3.25 µM). Presence of an amide group at the c-terminal of peptides was found to be critical in exerting potent inhibition of ATP synthase (~20–40% additional inhibition). Inhibition was fully reversible and found to be identical in both F1Fo membrane preparations as well as in isolated purified F1. Interestingly, growth of Escherichia coli was abrogated in the presence of ascaphin-8, aurein 2.2, aurein 2.3, citropin 1.1, dermaseptin, magainin II-amide, MRP, MRP-amide, melittin, or melittin-amide but was unaffected in the presence of carein 1.8, carein 1.9, maculatin 1.1, magainin II, or XT-7. Hence inhibition of F1-ATPase and E. coli cell growth by amphibian antimicrobial peptides suggests that their antimicrobial/anticancer properties are in part linked to their actions on ATP synthase. PMID:20100509

  7. The second metal-binding site of 70 kDa heat-shock protein is essential for ADP binding, ATP hydrolysis and ATP synthesis.

    PubMed Central

    Wu, Xueji; Yano, Mihiro; Washida, Hiroyo; Kido, Hiroshi

    2004-01-01

    The chaperone activity of Hsp70 (70 kDa heat-shock protein) in protein folding and its conformational switch, including oligomeric and monomeric interconversion, are regulated by the hydrolysis of ATP and the ATP-ADP exchange cycle. The crystal structure of human ATPase domain shows two metal-binding sites, the first for ATP binding and a second, in close proximity to the first, whose function remains unknown [Sriram, Osipiuk, Freeman, Morimoto and Joachimiak (1997) Structure 5, 403-414]. In this study, we have characterized the second metal-binding motif by site-directed mutagenesis and the kinetics of ATP and ADP binding, and found that the second metal-binding site, comprising a loop co-ordinated by His-227, Glu-231 and Asp-232, participates both in ATP hydrolysis and ATP-synthetic activities, in co-operation with the first metal-binding site. The first metal-binding site, a catalytic centre, is essential for ATP binding and the second site for ADP binding in the reactions of ATP hydrolysis and ATP synthesis. PMID:14664695

  8. TCDD decreases ATP levels and increases reactive oxygen production through changes in mitochondrial F F{sub 1}-ATP synthase and ubiquinone

    SciTech Connect

    Shertzer, Howard G. . E-mail: shertzhg@ucmail.uc.edu; Genter, Mary Beth; Shen, Dongxiao; Nebert, Daniel W.; Chen, Ying; Dalton, Timothy P.

    2006-12-15

    Mitochondria generate ATP and participate in signal transduction and cellular pathology and/or cell death. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) decreases hepatic ATP levels and generates mitochondrial oxidative DNA damage, which is exacerbated by increasing mitochondrial glutathione redox state and by inner membrane hyperpolarization. This study identifies mitochondrial targets of TCDD that initiate and sustain reactive oxygen production and decreased ATP levels. One week after treating mice with TCDD, liver ubiquinone (Q) levels were significantly decreased, while rates of succinoxidase and Q-cytochrome c oxidoreductase activities were increased. However, the expected increase in Q reduction state following TCDD treatment did not occur; instead, Q was more oxidized. These results could be explained by an ATP synthase defect, a premise supported by the unusual finding that TCDD lowers ATP/O ratios without concomitant changes in respiratory control ratios. Such results suggest either a futile cycle in ATP synthesis, or hydrolysis of newly synthesized ATP prior to release. The TCDD-mediated decrease in Q, concomitant with an increase in respiration, increases complex 3 redox cycling. This acts in concert with glutathione to increase membrane potential and reactive oxygen production. The proposed defect in ATP synthase explains both the greater respiratory rates and the lower tissue ATP levels.

  9. TCDD decreases ATP levels and increases reactive oxygen production through changes in mitochondrial F0F1-ATP synthase and ubiquinone

    PubMed Central

    Shertzer, Howard G.; Genter, Mary Beth; Shen, Dongxiao; Nebert, Daniel W.; Chen, Ying; Dalton, Timothy P.

    2007-01-01

    Mitochondria generate ATP and participate in signal transduction and cellular pathology and/or cell death. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) decreases hepatic ATP levels and generates mitochondrial oxidative DNA damage, which is exacerbated by increasing mitochondrial glutathione redox state and by inner-membrane hyperpolarization. This study identifies mitochondrial targets of TCDD that initiate and sustain reactive oxygen production and decreased ATP levels. One week after treating mice with TCDD, liver ubiquinone (Q) levels were significantly decreased, while rates of succinoxidase and Q-cytochrome c oxidoreductase activities were increased. However, the expected increase in Q reduction state following TCDD treatment did not occur; instead, Q was more oxidized. These results could be explained by an ATP synthase defect, a premise supported by the unusual finding that TCDD lowers ATP/O ratios without concomitant changes in respiratory control ratios. Such results suggest either a futile cycle in ATP synthesis, or hydrolysis of newly-synthesized ATP prior to release. The TCDD-mediated decrease in Q, concomitant with an increase in respiration, increases complex 3 redox-cycling. This acts in concert with glutathione to increase membrane potential and reactive oxygen production. The proposed defect in ATP synthase explains both the greater respiratory rates and the lower tissue ATP levels. PMID:17109908

  10. Polarized ATP distribution in urothelial mucosal and serosal space is differentially regulated by stretch and ectonucleotidases.

    PubMed

    Yu, Weiqun

    2015-11-15

    Purinergic signaling is a major pathway in regulating bladder function, and mechanical force stimulates urothelial ATP release, which plays an important role in bladder mechanotransduction. Although urothelial ATP release was first reported almost 20 years ago, the way in which release is regulated by mechanical force, and the presence of ATP-converting enzymes in regulating the availability of released ATP is still not well understood. Using a set of custom-designed Ussing chambers with the ability to manipulate mechanical forces applied on the urothelial tissue, we have demonstrated that it is stretch and not hydrostatic pressure that induces urothelial ATP release. The experiments reveal that urothelial ATP release is tightly controlled by stretch speed, magnitude, and direction. We have further shown that stretch-induced urothelial ATP release is insensitive to temperature (4°C). Interestingly, stretch-induced ATP release shows polarized distribution, with the ATP concentration in mucosal chamber (nanomolar level) about 10 times higher than the ATP concentration in serosal chamber (subnanomolar level). Furthermore, we have consistently observed differential ATP lifetime kinetics in the mucosal and serosal chambers, which is consistent with our immunofluorescent localization data, showing that ATP-converting enzymes ENTPD3 and alkaline phosphatase are expressed on urothelial basal surface, but not on the apical membrane. In summary, our data indicate that urothelial ATP release is finely regulated by stretch speed, magnitude, and direction, and extracellular ATP signaling is likely to be differentially regulated by ectonucleotidase, which results in temporally and spatially distinct ATP kinetics in response to mechanical stretch. PMID:26336160

  11. Light Effect on Water Viscosity: Implication for ATP Biosynthesis

    NASA Astrophysics Data System (ADS)

    Sommer, Andrei P.; Haddad, Mike Kh.; Fecht, Hans-Jörg

    2015-07-01

    Previous work assumed that ATP synthase, the smallest known rotary motor in nature, operates at 100% efficiency. Calculations which arrive to this result assume that the water viscosity inside mitochondria is constant and corresponds to that of bulk water. In our opinion this assumption is not satisfactory for two reasons: (1) There is evidence that the water in mitochondria prevails to 100% as interfacial water. (2) Laboratory experiments which explore the properties of interfacial water suggest viscosities which exceed those of bulk water, specifically at hydrophilic interfaces. Here, we wish to suggest a physicochemical mechanism which assumes intramitochondrial water viscosity gradients and consistently explains two cellular responses: The decrease and increase in ATP synthesis in response to reactive oxygen species and non-destructive levels of near-infrared (NIR) laser light, respectively. The mechanism is derived from the results of a new experimental method, which combines the technique of nanoindentation with the modulation of interfacial water layers by laser irradiation. Results, including the elucidation of the principle of light-induced ATP production, are expected to have broad implications in all fields of medicine.

  12. Sperm motility and ATP content in seminal hyperviscosity.

    PubMed

    Mendeluk, G R; Munuce, M J; Carizza, C; Sardi, M; Bregni, C

    1997-01-01

    Objective spermatic motility (Hamilton Thorne Research), the rapid progressive spermatozoa (grade A) recovery after swim-up, and the spermatozoa ATP content (bioluminescence) were studied in normoviscous and hyperviscous asthenospermic samples. The amplitude of lateral head displacement (ALH) was significantly lower in hyperviscous semen (normal: 4.6 +/- 0.7 microns [n = 20], high: 3.5 +/- 1.2 microns [n = 16]; p < .05). The grade A recovery percentage after swim-up was significantly higher in semens with high consistency (normal: 71.0 +/- 38.0 [n = 14], high: 181.3 +/- 108.9 [n = 6]; p < .05). The ATP content per living spermatozoa was in the normal consistency group 449.4 +/- 65.1 pmol per million living spermatozoa (n = 29) and in the high consistency batch 605.1 +/- 242.8 (n = 9), p < .05. In asthenospermia, the spermatozoa from hyperviscous samples have minor ALH values, better response to swim-up, and high ATP content than those from normoviscous ejaculates. PMID:9352034

  13. Allosteric regulation of focal adhesion kinase by PIP₂ and ATP.

    PubMed

    Zhou, Jing; Bronowska, Agnieszka; Le Coq, Johanne; Lietha, Daniel; Gräter, Frauke

    2015-02-01

    Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that regulates cell signaling, proliferation, migration, and development. A major mechanism of regulation of FAK activity is an intramolecular autoinhibitory interaction between two of its domains--the catalytic and FERM domains. Upon cell adhesion to the extracellular matrix, FAK is being translocated toward focal adhesion sites and activated. Interactions of FAK with phosphoinositide phosphatidylinsositol-4,5-bis-phosphate (PIP₂) are required to activate FAK. However, the molecular mechanism of the activation remains poorly understood. Recent fluorescence resonance energy transfer experiments revealed a closure of the FERM-kinase interface upon ATP binding, which is reversed upon additional binding of PIP₂. Here, we addressed the allosteric regulation of FAK by performing all-atom molecular-dynamics simulations of a FAK fragment containing the catalytic and FERM domains, and comparing the dynamics in the absence or presence of ATP and PIP₂. As a major conformational change, we observe a closing and opening motion upon ATP and additional PIP₂ binding, respectively, in good agreement with the fluorescence resonance energy transfer experiments. To reveal how the binding of the regulatory PIP₂ to the FERM F2 lobe is transduced to the very distant F1/N-lobe interface, we employed force distribution analysis. We identified a network of mainly charged residue-residue interactions spanning from the PIP₂ binding site to the distant interface between the kinase and FERM domains, comprising candidate residues for mutagenesis to validate the predicted mechanism of FAK activation. PMID:25650936

  14. Human ATP-binding cassette (ABC) transporter family

    PubMed Central

    2009-01-01

    There exist four fundamentally different classes of membrane-bound transport proteins: ion channels; transporters; aquaporins; and ATP-powered pumps. ATP-binding cassette (ABC) transporters are an example of ATP-dependent pumps. ABC transporters are ubiquitous membrane-bound proteins, present in all prokaryotes, as well as plants, fungi, yeast and animals. These pumps can move substrates in (influx) or out (efflux) of cells. In mammals, ABC transporters are expressed predominantly in the liver, intestine, blood-brain barrier, blood-testis barrier, placenta and kidney. ABC proteins transport a number of endogenous substrates, including inorganic anions, metal ions, peptides, amino acids, sugars and a large number of hydrophobic compounds and metabolites across the plasma membrane, and also across intracellular membranes. The human genome contains 49 ABC genes, arranged in eight subfamilies and named via divergent evolution. That ABC genes are important is underscored by the fact that mutations in at least I I of these genes are already known to cause severe inherited diseases (eg cystic fibrosis and X-linked adrenoleukodystrophy [X-ALD]). ABC transporters also participate in the movement of most drugs and their metabolites across cell surface and cellular organelle membranes; thus, defects in these genes can be important in terms of cancer therapy, pharmacokinetics and innumerable pharmacogenetic disorders. PMID:19403462

  15. Light Effect on Water Viscosity: Implication for ATP Biosynthesis

    PubMed Central

    Sommer, Andrei P.; Haddad, Mike Kh.; Fecht, Hans-Jörg

    2015-01-01

    Previous work assumed that ATP synthase, the smallest known rotary motor in nature, operates at 100% efficiency. Calculations which arrive to this result assume that the water viscosity inside mitochondria is constant and corresponds to that of bulk water. In our opinion this assumption is not satisfactory for two reasons: (1) There is evidence that the water in mitochondria prevails to 100% as interfacial water. (2) Laboratory experiments which explore the properties of interfacial water suggest viscosities which exceed those of bulk water, specifically at hydrophilic interfaces. Here, we wish to suggest a physicochemical mechanism which assumes intramitochondrial water viscosity gradients and consistently explains two cellular responses: The decrease and increase in ATP synthesis in response to reactive oxygen species and non-destructive levels of near-infrared (NIR) laser light, respectively. The mechanism is derived from the results of a new experimental method, which combines the technique of nanoindentation with the modulation of interfacial water layers by laser irradiation. Results, including the elucidation of the principle of light-induced ATP production, are expected to have broad implications in all fields of medicine. PMID:26154113

  16. Multiple chromatographic forms of ATP citrate lyase from rat liver.

    PubMed Central

    Corrigan, A P; Rider, C C

    1983-01-01

    ATP citrate lyase is shown to exist as multiple forms in extracts of rat liver. DEAE-Sephadex ion-exchange chromatography of liver supernatants reveals two peaks of activity. A minor, basic, component, comprising 14% of the recovered activity, is eluted without retention, whereas the major, acidic, form is eluted by a KCl gradient. Gel filtration of similar extracts shows the presence of a high-Mr form of ATP citrate lyase (Mr around 10(7) in addition to the tetrameric enzyme (Mr 4.1 X 10(5). This associated state, which represents 10% of the total activity, is unstable, breaking down to the tetramer, and appears to be disrupted by Mg2+. The basic form changes in the partially purified state to give the acidic form. Most of the high-Mr enzyme is acidic in nature. No evidence could be found for an association of the enzyme with mitochondrial or microsomal membranes. ATP citrate lyase from rat brain also shows two peaks of activity on DEAE-Sephadex ion-exchange chromatography, but the activity is distributed between the peaks in almost equal proportions. However, only the tetrameric enzyme was observed on gel filtration. PMID:6615476

  17. Overview of photo-induced therapy for ATP production

    NASA Astrophysics Data System (ADS)

    Abdalla, Mohamed; Nagy, A.; Ye, W. N.; Mussivand, T.

    2012-10-01

    The purpose of this report is to provide a review of the effects of low-power photo-induced therapy using lasers of different device parameters such as intensity, wavelength, lasing mechanism (i.e., pulsed or continuous) on the production of Adenosine triphosphate (ATP) in mammalian cells. This is a very important research topic as it is suggested in literature that there might be a relationship between the ATP levels and specific diseases. It has been shown that the ATP production was enhanced at wavelengths ranging between 600 nm and 1000 nm (also known as the optical window), in particular at 600nm, 632.8nm, 635nm, 650nm, and 904nm. However, certain experiments showed that the effectiveness of the photo-induced therapy was also dependent on the dosage and the duration of the supplied light. We present the research conclusions drawn from the experiments reported within the last decade, and provide a list of potential medical treatment(s) for patients using visible and near infrared (NIR) light.

  18. Differential proteomic profiling unveils new molecular mechanisms associated with mitochondrial complex III deficiency

    PubMed Central

    Morán, María; López-Bernardo, Elia; Cadenas, Susana; Hidalgo, Beatriz; Sánchez, Ricardo; Seneca, Sara; Arenas, Joaquín; Martín, Miguel A.; Ugalde, Cristina

    2014-01-01

    We have analyzed the cellular pathways and metabolic adaptations that take place in primary skin fibroblasts from patients with mutations in BCS1L, a major genetic cause of mitochondrial complex III enzyme deficiency. Mutant fibroblasts exhibited low oxygen consumption rates and intracellular ATP levels, indicating that the main altered molecular event probably is a limited respiration-coupled ATP production through the OXPHOS system. Two-dimensional DIGE and MALDI-TOF/TOF mass spectrometry analyses unambiguously identified 39 proteins whose expression was significantly altered in complex III-deficient fibroblasts. Extensive statistical and cluster analyses revealed a protein profile characteristic for the BCS1L mutant fibroblasts that included alterations in energy metabolism, cell signaling and gene expression regulation, cytoskeleton formation and maintenance, and intracellular stress responses. The physiological validation of the predicted functional adaptations of human cultured fibroblasts to complex III deficiency confirmed the up-regulation of glycolytic enzyme activities and the accumulation of branched-chain among other amino acids, suggesting the activation of anaerobic glycolysis and cellular catabolic states, in particular protein catabolism, together with autophagy as adaptive responses to mitochondrial respiratory chain dysfunction and ATP deficiency. Our data point to an overall metabolic and genetic reprogramming that could contribute to explain the clinical manifestations of complex III deficiency in patients. PMID:25239759

  19. SUPERSTARS III: K-2.

    ERIC Educational Resources Information Center

    North Carolina State Dept. of Public Education, Raleigh.

    SUPERSTARS III is a K-8 program designed as an enrichment opportunity for self-directed learners in mathematics. The basic purpose of SUPERSTARS III is to provide the extra challenge that self-motivated students need in mathematics and to do so in a structured, long-term program that does not impinge on the normal classroom routine or the…

  20. CITY III Player's Manual.

    ERIC Educational Resources Information Center

    Envirometrics, Inc., Washington, DC.

    CITY III is a computer-assisted simulation game in which participants make decisions affecting the economic, governmental, and social conditions of a simulated urban area. In CITY III, the computer stores all the relevant statistics for the area, updates data when changes are made, and prints out yearly reports. The computer also simulates…

  1. CITY III Operator's Manual.

    ERIC Educational Resources Information Center

    Envirometrics, Inc., Washington, DC.

    CITY III is a computer-assisted simulation game of an urban system involving player operation of and interaction with economic, social, and government components. The role of operator in the game is to take the handwritten inputs (decisions) from the CITY III participants, process them, and return output which initiates the next round of…

  2. atpE gene as a new useful specific molecular target to quantify Mycobacterium in environmental samples

    PubMed Central

    2013-01-01

    Background The environment is the likely source of many pathogenic mycobacterial species but detection of mycobacteria by bacteriological tools is generally difficult and time-consuming. Consequently, several molecular targets based on the sequences of housekeeping genes, non-functional RNA and structural ribosomal RNAs have been proposed for the detection and identification of mycobacteria in clinical or environmental samples. While certain of these targets were proposed as specific for this genus, most are prone to false positive results in complex environmental samples that include related, but distinct, bacterial genera. Nowadays the increased number of sequenced genomes and the availability of software for genomic comparison provide tools to develop novel, mycobacteria-specific targets, and the associated molecular probes and primers. Consequently, we conducted an in silico search for proteins exclusive to Mycobacterium spp. genomes in order to design sensitive and specific molecular targets. Results Among the 3989 predicted proteins from M. tuberculosis H37Rv, only 11 proteins showed 80% to 100% of similarity with Mycobacterium spp. genomes, and less than 50% of similarity with genomes of closely related Corynebacterium, Nocardia and Rhodococcus genera. Based on DNA sequence alignments, we designed primer pairs and a probe that specifically detect the atpE gene of mycobacteria, as verified by quantitative real-time PCR on a collection of mycobacteria and non-mycobacterial species. The real-time PCR method we developed was successfully used to detect mycobacteria in tap water and lake samples. Conclusions The results indicate that this real-time PCR method targeting the atpE gene can serve for highly specific detection and precise quantification of Mycobacterium spp. in environmental samples. PMID:24299240

  3. ATP-Responsive DNA-Graphene Hybrid Nanoaggregates for Anticancer Drug Delivery

    PubMed Central

    Mo, Ran; Jiang, Tianyue; Sun, Wujin; Gu, Zhen

    2015-01-01

    Stimuli-triggered drug delivery systems are primarily focused on the applications of the tumor microenvironmental or cellular physiological cues to enhance the release of drugs at the target site. In this study, we applied adenosine-5′-triphosphate (ATP), the primary “energy molecule”, as a trigger for enhanced release of preloaded drugs responding to the intracellular ATP concentration that is significantly higher than the extracellular level. A new ATP-responsive anticancer drug delivery strategy utilizing DNA-graphene crosslinked hybrid nanoaggregates as carriers was developed for controlled release of doxorubicin (DOX), which consists of graphene oxide (GO), two single-stranded DNA (ssDNA, denoted as DNA1 and DNA2) and ATP aptamer. The single-stranded DNA1 and DNA2 together with the ATP aptamer serve as the linkers upon hybridization for controlled assembly of the DNA-GO nanoaggregates, which effectively inhibited the release of DOX from the GO nanosheets. In the presence of ATP, the responsive formation of the ATP/ATP aptamer complex causes the dissociation of the aggregates, which promoted the release of DOX in the environment with a high ATP concentration such as cytosol compared with that in the ATP-deficient extracellular fluid. This supports the development of a novel ATP-responsive platform for targeted on-demand delivery of anticancer drugs inside specific cells. PMID:25736497

  4. Blockade of adenosine receptors unmasks a stimulatory effect of ATP on cardiac contractility.

    PubMed Central

    Mantelli, L.; Amerini, S.; Filippi, S.; Ledda, F.

    1993-01-01

    1. The effects of ATP, alpha,beta-methylene ATP and beta,gamma-methylene ATP on the contractile tension of guinea-pig isolated left atria were evaluated. 2. ATP (1-100 microM) produced a concentration-dependent negative inotropic effect; this response was converted to a positive inotropic effect in the presence of the antagonist of adenosine A1 receptors, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 0.1 microM), and in the presence of 8-phenyltheophylline (10 microM), an antagonist of A1 and A2 receptors. 3. The positive inotropic effect of ATP was antagonized by the P2 receptor antagonist, suramin (500 microM). Reactive blue 2 (30-500 microM), a putative P2y receptor antagonist, concentration-dependently reduced and finally abolished the effect of ATP. 4. In the presence of 8-phenyltheophylline, the stable analogues of ATP, alpha,beta-methylene ATP and beta,gamma-methylene ATP (1-30 microM), produced a concentration-dependent increase in atrial contractility of a lesser degree than that induced by ATP. 5. The results suggest that when inhibitory adenosine receptors are blocked, ATP produces a positive inotropic effect, probably mediated by P2y receptor stimulation. PMID:8401938

  5. Arrangement of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1

    PubMed Central

    Althoff, Thorsten; Mills, Deryck J; Popot, Jean-Luc; Kühlbrandt, Werner

    2011-01-01

    The respiratory chain in the inner mitochondrial membrane contains three large multi-enzyme complexes that together establish the proton gradient for ATP synthesis, and assemble into a supercomplex. A 19-Å 3D map of the 1.7-MDa amphipol-solubilized supercomplex I1III2IV1 from bovine heart obtained by single-particle electron cryo-microscopy reveals an amphipol belt replacing the membrane lipid bilayer. A precise fit of the X-ray structures of complex I, the complex III dimer, and monomeric complex IV indicates distances of 13 nm between the ubiquinol-binding sites of complexes I and III, and of 10–11 nm between the cytochrome c binding sites of complexes III and IV. The arrangement of respiratory chain complexes suggests two possible pathways for efficient electron transfer through the supercomplex, of which the shorter branch through the complex III monomer proximal to complex I may be preferred. PMID:21909073

  6. Coassembly of Photosystem II and ATPase as Artificial Chloroplast for Light-Driven ATP Synthesis.

    PubMed

    Feng, Xiyun; Jia, Yi; Cai, Peng; Fei, Jinbo; Li, Junbai

    2016-01-26

    Adenosine triphosphate (ATP) is one of the most important energy sources in living cells, which can drive serial key biochemical processes. However, generation of a proton gradient for ATP production in an artificial way poses a great challenge. In nature, photophosphorylation occurring in chloroplasts is an ideal prototype of ATP production. In this paper we imitate the light-to-ATP conversion process occurring in the thylakoid membrane by construction of FoF1-ATPase proteoliposome-coated PSII-based microspheres with well-defined core@shell structures using molecular assembly. Under light illumination, PSII can split water into protons, oxygen, and electrons and can generate a proton gradient for ATPase to produce ATP. Thus, an artificially designed chloroplast for PSII-driven ATP synthesis is realized. This biomimetic system will help to understand the photophosphorylation process and may facilitate the development of ATP-driven devices by remote light control. PMID:26615669

  7. Lysosomal ATP imaging in living cells by a water-soluble cationic polythiophene derivative.

    PubMed

    Huang, Bing-Huan; Geng, Zhi-Rong; Ma, Xiao-Yan; Zhang, Cui; Zhang, Zhi-Yang; Wang, Zhi-Lin

    2016-09-15

    Lysosomes in astrocytes and microglia can release ATP as the signaling molecule for the cells through ca(2+)-dependent exocytosis in response to various stimuli. At present, fluorescent probes that can detect ATP in lysosomes have not been reported. In this work, we have developed a new water-soluble cationic polythiophene derivative that can be specifically localized in lysosomes and can be utilized as a fluorescent probe to sense ATP in cells. PEMTEI exhibits high selectivity and sensitivity to ATP at physiological pH values and the detection limit of ATP is as low as 10(-11)M. The probe has low cytotoxicity, good permeability and high photostability in living cells and has been applied successfully to real-time monitoring of the change in concentrations of ATP in lysosomes though fluorescence microscopy. We also demonstrated that lysosomes in Hela cells can release ATP through Ca(2+)-dependent exocytosis in response to drug stimuli. PMID:27131993

  8. [Stabilization of Cadmium Contaminated Soils by Ferric Ion Modified Attapulgite (Fe/ATP)--Characterizations and Stabilization Mechanism].

    PubMed

    Rong, Yang; Li, Rong-bo; Zhou, Yong-li; Chen, Jing; Wang, Lin-ling; Lu, Xiao-hua

    2015-08-01

    Ferric ion modified attapulgite (Fe/ATP) was prepared by impregnation and its structure and morphology were characterized. The toxicity characteristic leaching procedure (TCLP) was used to evaluate the effect of Cadmium( Cd) stabilization in soil with the addition of attapulgite (ATP) and Fe/ATP. The stabilization mechanism of Cd was further elucidated by comparing the morphologies and structure of ATP and Fe/ATP before and after Cd adsorption. Fe/ATP exhibited much better adsorption capacity than ATP, suggesting different adsorption mechanisms occurred between ATP and Fe/ATP. The leaching concentrations of Cd in soil decreased by 45% and 91% respectively, with the addition of wt. 20% ATP and Fe/ATP. The former was attributed to the interaction between Cd2 and --OH groups by chemical binding to form inner-sphere complexes in ATP and the attachment between Cd2+ and the defect sites in ATP framework. Whereas Cd stabilization with Fe/ATP was resulted from the fact that the active centers (--OH bonds or O- sites) on ATP could react with Fe3+ giving Fe--O--Cd-- bridges, which helped stabilize Cd in surface soil. What'more, the ferric oxides and metal hydroxides on the surface of ATP could interact with Cd, probably by the formation of cadmium ferrite. In conclusion, Fe/ATP, which can be easily prepared, holds promise as a potential low-cost and environmental friendly stabilizing agent for remediation of soil contaminated with heavy metals. PMID:26592037

  9. An ATP-activated channel is involved in mitogenic stimulation of human T lymphocytes.

    PubMed

    Baricordi, O R; Ferrari, D; Melchiorri, L; Chiozzi, P; Hanau, S; Chiari, E; Rubini, M; Di Virgilio, F

    1996-01-15

    We investigated the effect of pharmacologic modulation of the ATP receptor on intracellular ion changes and proliferative response of human peripheral blood lymphocytes (PBLs) and purified T lymphocytes. Extracellular ATP (ATPe) triggered in these cells an increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) and plasma membrane depolarization. Whereas both Ca2+ release from intracellular stores and influx across the plasma membrane were detected in the whole PBL population, only Ca2+ influx was observed in T cells. In the presence of near physiologic extracellular Na+ concentrations (125 mmol/L), Ca2+ permeability through the ATPe-gated channel was very low, suggesting a higher selectivity for monovalent over divalent cations. The selective P2Z agonist benzoylbenzoic ATP (BzATP) increased [Ca2+]i in the presence but not the absence of extracellular Ca2+ and also caused plasma membrane depolarization. The covalent blocker oxidized ATP (oATP), an inhibitor of P2X and P2Z receptors, prevented Ca2+ influx and plasma membrane depolarization, but had no effect on Ca2+ release from stores. Stimulation with ATPe alone had no significant effects on PBL 3H-thymidine incorporation. On the contrary, ATPe or BzATP had a synergistic effect on DNA synthesis stimulated by selective T-cell mitogens such as phytohemagglutinin, anti-CD3 monoclonal antibody, or allogenic PBLs (mixed lymphocyte cultures). Treatment with oATP inhibited mitogenic stimulation by these receptor-directed agents but not by the combined application of the Ca2+ ionophore ionomycin and phorbol myristate acetate. Interleukin-2 partially relieved inhibition by oATP. These results suggest that human T lymphocytes express a plasma membrane channel gated by ATPe that is involved in mitogenic stimulation. PMID:8555491

  10. Structural Characterization of Two Metastable ATP-Bound States of P-Glycoprotein

    PubMed Central

    O’Mara, Megan L.; Mark, Alan E.

    2014-01-01

    ATP Binding Cassette (ABC) transporters couple the binding and hydrolysis of ATP to the transport of substrate molecules across the membrane. The mechanism by which ATP binding and/or hydrolysis drives the conformational changes associated with substrate transport has not yet been characterized fully. Here, changes in the conformation of the ABC export protein P-glycoprotein on ATP binding are examined in a series of molecular dynamics simulations. When one molecule of ATP is placed at the ATP binding site associated with each of the two nucleotide binding domains (NBDs), the membrane-embedded P-glycoprotein crystal structure adopts two distinct metastable conformations. In one, each ATP molecule interacts primarily with the Walker A motif of the corresponding NBD. In the other, the ATP molecules interacts with both Walker A motif of one NBD and the Signature motif of the opposite NBD inducing the partial dimerization of the NBDs. This interaction is more extensive in one of the two ATP binding site, leading to an asymmetric structure. The overall conformation of the transmembrane domains is not altered in either of these metastable states, indicating that the conformational changes associated with ATP binding observed in the simulations in the absence of substrate do not lead to the outward-facing conformation and thus would be insufficient in themselves to drive transport. Nevertheless, the metastable intermediate ATP-bound conformations observed are compatible with a wide range of experimental cross-linking data demonstrating the simulations do capture physiologically important conformations. Analysis of the interaction between ATP and its cofactor Mg2+ with each NBD indicates that the coordination of ATP and Mg2+ differs between the two NBDs. The role structural asymmetry may play in ATP binding and hydrolysis is discussed. Furthermore, we demonstrate that our results are not heavily influenced by the crystal structure chosen for initiation of the simulations

  11. The a subunit of the A1AO ATP synthase of Methanosarcina mazei Gö1 contains two conserved arginine residues that are crucial for ATP synthesis.

    PubMed

    Gloger, Carolin; Born, Anna-Katharina; Antosch, Martin; Müller, Volker

    2015-01-01

    Like the evolutionary related F1FO ATP synthases and V1VO ATPases, the A1AO ATP synthases from archaea are multisubunit, membrane-bound transport machines that couple ion flow to the synthesis of ATP. Although the subunit composition is known for at least two species, nothing is known so far with respect to the function of individual subunits or amino acid residues. To pave the road for a functional analysis of A1AO ATP synthases, we have cloned the entire operon from Methanosarcina mazei into an expression vector and produced the enzyme in Escherichia coli. Inverted membrane vesicles of the recombinants catalyzed ATP synthesis driven by NADH oxidation as well as artificial driving forces. [Formula: see text] as well as ΔpH were used as driving forces which is consistent with the inhibition of NADH-driven ATP synthesis by protonophores. Exchange of the conserved glutamate in subunit c led to a complete loss of ATP synthesis, proving that this residue is essential for H+ translocation. Exchange of two conserved arginine residues in subunit a has different effects on ATP synthesis. The role of these residues in ion translocation is discussed. PMID:25724672

  12. Synthesis of bisphosphonate derivatives of ATP by T4 DNA ligase, ubiquitin activating enzyme (E1) and other ligases.

    PubMed

    Günther Sillero, María A; de Diego, Anabel; Pérez-Zúñiga, Francisco J; Sillero, Antonio

    2008-05-15

    T4 DNA ligase and the ubiquitin activating enzyme (E1), catalyze the synthesis of ATP beta,gamma-bisphosphonate derivatives. Concerning T4 DNA ligase: (i) etidronate (pC(OH)(CH(3))p) displaced the AMP moiety of the complex E-AMP in a concentration dependent manner; (ii) the K(m) values and the rate of synthesis k(cat) (s(-1)), determined for the following compounds were, respectively: etidronate, 0.73+/-0.09 mM and (70+/-10)x10(-3) s(-1); clodronate (pCCl(2)p), 0.08+/-0.01 mM and (4.1+/-0.3)x10(-3) s(-1); methylenebisphosphonate (pCH(2)p), 0.024+/-0.001 mM and (0.6+/-0.1)x10(-3) s(-1); tripolyphosphate (P(3)) (in the synthesis of adenosine 5'-tetraphosphate, p(4)A), 1.30+/-0.30 mM and (6.2+/-1.1)x10(-3) s(-1); (iii) in the presence of GTP and ATP, inhibition of the synthesis of Ap(4)G was observed with clodronate but not with pamidronate (pC(OH)(CH(2)-CH(2)-NH(3))p). Concerning the ubiquitin activating enzyme (E1): methylenebisphosphonate was the only bisphosphonate, out of the ones tested, that served as substrate for the synthesis of an ATP derivative (K(m)=0.36+/-0.09 mM and k(cat)=0.15+/-0.02 s(-1)). None of the above bisphosphonates were substrates of the reaction catalyzed by luciferase or by acyl-CoA synthetase. The ability of acetyl-CoA synthetase to use methylenebisphosphonate as substrate depended on the commercial source of the enzyme. In our view this report widens our knowledge of the enzymes able to metabolize bisphosphonates, a therapeutic tool widely used in the treatment of osteoporosis. PMID:18378215

  13. Fo-driven Rotation in the ATP Synthase Direction against the Force of F1 ATPase in the FoF1 ATP Synthase*

    PubMed Central

    Martin, James; Hudson, Jennifer; Hornung, Tassilo; Frasch, Wayne D.

    2015-01-01

    Living organisms rely on the FoF1 ATP synthase to maintain the non-equilibrium chemical gradient of ATP to ADP and phosphate that provides the primary energy source for cellular processes. How the Fo motor uses a transmembrane electrochemical ion gradient to create clockwise torque that overcomes F1 ATPase-driven counterclockwise torque at high ATP is a major unresolved question. Using single FoF1 molecules embedded in lipid bilayer nanodiscs, we now report the observation of Fo-dependent rotation of the c10 ring in the ATP synthase (clockwise) direction against the counterclockwise force of ATPase-driven rotation that occurs upon formation of a leash with Fo stator subunit a. Mutational studies indicate that the leash is important for ATP synthase activity and support a mechanism in which residues aGlu-196 and cArg-50 participate in the cytoplasmic proton half-channel to promote leash formation. PMID:25713065

  14. Potentiation of disease-associated cystic fibrosis transmembrane conductance regulator mutants by hydrolyzable ATP analogs.

    PubMed

    Miki, Haruna; Zhou, Zhen; Li, Min; Hwang, Tzyh-Chang; Bompadre, Silvia G

    2010-06-25

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the ATP-binding cassette transporter superfamily. CFTR is gated by ATP binding and hydrolysis at its two nucleotide-binding domains (NBDs), which dimerize in the presence of ATP to form two ATP-binding pockets (ABP1 and ABP2). Mutations reducing the activity of CFTR result in the genetic disease cystic fibrosis. Two of the most common mutations causing a severe phenotype are G551D and DeltaF508. Previously we found that the ATP analog N(6)-(2-phenylethyl)-ATP (P-ATP) potentiates the activity of G551D by approximately 7-fold. Here we show that 2'-deoxy-ATP (dATP), but not 3'-deoxy-ATP, increases the activity of G551D-CFTR by approximately 8-fold. We custom synthesized N(6)-(2-phenylethyl)-2'-deoxy-ATP (P-dATP), an analog combining the chemical modifications in dATP and P-ATP. This new analog enhances G551D current by 36.2 +/- 5.4-fold suggesting an independent but energetically additive action of these two different chemical modifications. We show that P-dATP binds to ABP1 to potentiate the activity of G551D, and mutations in both sides of ABP1 (W401G and S1347G) decrease its potentiation effect, suggesting that the action of P-dATP takes place at the interface of both NBDs. Interestingly, P-dATP completely rectified the gating abnormality of DeltaF508-CFTR by increasing its activity by 19.5 +/- 3.8-fold through binding to both ABPs. This result highlights the severity of the gating defect associated with DeltaF508, the most prevalent disease-associated mutation. The new analog P-dATP can be not only an invaluable tool to study CFTR gating, but it can also serve as a proof-of-principle that, by combining elements that potentiate the channel activity independently, the increase in chloride transport necessary to reach a therapeutic target is attainable. PMID:20406820

  15. Potentiation of Disease-associated Cystic Fibrosis Transmembrane Conductance Regulator Mutants by Hydrolyzable ATP Analogs*

    PubMed Central

    Miki, Haruna; Zhou, Zhen; Li, Min; Hwang, Tzyh-Chang; Bompadre, Silvia G.

    2010-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the ATP-binding cassette transporter superfamily. CFTR is gated by ATP binding and hydrolysis at its two nucleotide-binding domains (NBDs), which dimerize in the presence of ATP to form two ATP-binding pockets (ABP1 and ABP2). Mutations reducing the activity of CFTR result in the genetic disease cystic fibrosis. Two of the most common mutations causing a severe phenotype are G551D and ΔF508. Previously we found that the ATP analog N6-(2-phenylethyl)-ATP (P-ATP) potentiates the activity of G551D by ∼7-fold. Here we show that 2′-deoxy-ATP (dATP), but not 3′-deoxy-ATP, increases the activity of G551D-CFTR by ∼8-fold. We custom synthesized N6-(2-phenylethyl)-2′-deoxy-ATP (P-dATP), an analog combining the chemical modifications in dATP and P-ATP. This new analog enhances G551D current by 36.2 ± 5.4-fold suggesting an independent but energetically additive action of these two different chemical modifications. We show that P-dATP binds to ABP1 to potentiate the activity of G551D, and mutations in both sides of ABP1 (W401G and S1347G) decrease its potentiation effect, suggesting that the action of P-dATP takes place at the interface of both NBDs. Interestingly, P-dATP completely rectified the gating abnormality of ΔF508-CFTR by increasing its activity by 19.5 ± 3.8-fold through binding to both ABPs. This result highlights the severity of the gating defect associated with ΔF508, the most prevalent disease-associated mutation. The new analog P-dATP can be not only an invaluable tool to study CFTR gating, but it can also serve as a proof-of-principle that, by combining elements that potentiate the channel activity independently, the increase in chloride transport necessary to reach a therapeutic target is attainable. PMID:20406820

  16. Convergent Evolution of Fern-Specific Mitochondrial Group II Intron atp1i361g2 and Its Ancient Source Paralogue rps3i249g2 and Independent Losses of Intron and RNA Editing among Pteridaceae.

    PubMed

    Zumkeller, Simon Maria; Knoop, Volker; Knie, Nils

    2016-01-01

    Mitochondrial intron patterns are highly divergent between the major land plant clades. An intron in the atp1 gene, atp1i361g2, is an example for a group II intron specific to monilophytes (ferns). Here, we report that atp1i361g2 is lost independently at least 4 times in the fern family Pteridaceae. Such plant organelle intron losses have previously been found to be accompanied by loss of RNA editing sites in the flanking exon regions as a consequence of genomic recombination of mature cDNA. Instead, we now observe that RNA editing events in both directions of pyrimidine exchange (C-to-U and U-to-C) are retained in atp1 exons after loss of the intron in Pteris argyraea/biaurita and in Actiniopteris and Onychium We find that atp1i361g2 has significant similarity with intron rps3i249g2 present in lycophytes and gymnosperms, which we now also find highly conserved in ferns. We conclude that atp1i361g2 may have originated from the more ancestral rps3i249g2 paralogue by a reverse splicing copy event early in the evolution of monilophytes. Secondary structure elements of the two introns, most characteristically their domains III, show strikingly convergent evolution in the monilophytes. Moreover, the intron paralogue rps3i249g2 reveals relaxed evolution in taxa where the atp1i361g2 paralogue is lost. Our findings may reflect convergent evolution of the two related mitochondrial introns exerted by co-evolution with an intron-binding protein simultaneously acting on the two paralogues. PMID:27492234

  17. Convergent Evolution of Fern-Specific Mitochondrial Group II Intron atp1i361g2 and Its Ancient Source Paralogue rps3i249g2 and Independent Losses of Intron and RNA Editing among Pteridaceae

    PubMed Central

    Zumkeller, Simon Maria; Knoop, Volker; Knie, Nils

    2016-01-01

    Mitochondrial intron patterns are highly divergent between the major land plant clades. An intron in the atp1 gene, atp1i361g2, is an example for a group II intron specific to monilophytes (ferns). Here, we report that atp1i361g2 is lost independently at least 4 times in the fern family Pteridaceae. Such plant organelle intron losses have previously been found to be accompanied by loss of RNA editing sites in the flanking exon regions as a consequence of genomic recombination of mature cDNA. Instead, we now observe that RNA editing events in both directions of pyrimidine exchange (C-to-U and U-to-C) are retained in atp1 exons after loss of the intron in Pteris argyraea/biaurita and in Actiniopteris and Onychium. We find that atp1i361g2 has significant similarity with intron rps3i249g2 present in lycophytes and gymnosperms, which we now also find highly conserved in ferns. We conclude that atp1i361g2 may have originated from the more ancestral rps3i249g2 paralogue by a reverse splicing copy event early in the evolution of monilophytes. Secondary structure elements of the two introns, most characteristically their domains III, show strikingly convergent evolution in the monilophytes. Moreover, the intron paralogue rps3i249g2 reveals relaxed evolution in taxa where the atp1i361g2 paralogue is lost. Our findings may reflect convergent evolution of the two related mitochondrial introns exerted by co-evolution with an intron-binding protein simultaneously acting on the two paralogues. PMID:27492234

  18. Purification and cloning of a soluble ATP-diphosphohydrolase (apyrase) from potato tubers (Solanum tuberosum).

    PubMed

    Handa, M; Guidotti, G

    1996-01-26

    A soluble ATP-diphosphohydrolase (apyrase, EC 3.6.1.5) has been purified from potato tubers. Solanum tuberosum, to a specific activity of 10,000 mumol P(i)/mg/min. The cDNA corresponding to the potato apyrase has been isolated and termed RROP1. The deduced amino acid sequence contains a putative signal sequence, two hydrophobic regions at the carboxy terminus, two potential Asn-linked glycosylation sites, and four regions in the amino-terminal half that we term ACR (apyrase conserved regions) 1-4 that are highly conserved in known apyrases and related enzymes; garden pea nucleoside triphosphatase, Toxoplasma gondii nucleoside triphosphate hydrolases, and Saccharomyces cerevisiae golgi guanosine diphosphatase. A yeast 71.9-kDa hypothetical protein on chromosome V, a Caenorhabditis elegans hypothetical 61.3-kDa protein on chromosome III, and human CD39, a lymphoid cell activation antigen, also share the conserved ACR regions, but their ability to hydrolyze nucleotides has not been assessed. PMID:8579614

  19. Small molecules ATP-competitive inhibitors of FLT3: a chemical overview.

    PubMed

    Schenone, S; Brullo, C; Botta, M

    2008-01-01

    FLT3 is a tyrosine kinase (TK), member of the class III TK receptor family, normally expressed in hematopoietic, immune and neural systems, also playing an important role in the pathogenesis of acute leukemias, particularly acute myeloid leukemia (AML), where it is present in constitutively activated mutated forms, correlated with poor prognosis, in a notable percentage of patients. For these reasons FLT3 soon appeared as a promising target for the therapeutic intervention for this severe and aggressive malignancy; the recent determination of the crystal structure of the autoinhibited form of FLT3 gave new trend for the design and the synthesis of potent inhibitors. Small molecules tyrosine kinase inhibitors represent one of the largest drug family currently targeted by pharmaceutical companies for the treatment of cancer. Exciting examples of such molecules have reached advanced clinical trials and have been recently approved by FDA for the treatment of different solid or haematological tumors. Usually TK inhibitors share common features, namely two hydrophobic/aromatic regions bearing one or more hydrogen bonding substituents. These two regions can be connected by different spacers and almost all the molecules contain a component resembling the ATP purine structure. This review will deal with FLT3 synthetic inhibitors, reporting not only the most important molecules that are in clinical trials, but also the new compounds that have appeared in literature in the last few years. Our attention will be focused on chemical structures, mechanisms of action and structure-activity relationships. PMID:19075657

  20. The transport mechanism of the mitochondrial ADP/ATP carrier.

    PubMed

    Kunji, Edmund R S; Aleksandrova, Antoniya; King, Martin S; Majd, Homa; Ashton, Valerie L; Cerson, Elizabeth; Springett, Roger; Kibalchenko, Mikhail; Tavoulari, Sotiria; Crichton, Paul G; Ruprecht, Jonathan J

    2016-10-01

    The mitochondrial ADP/ATP carrier imports ADP from the cytosol and exports ATP from the mitochondrial matrix, which are key transport steps for oxidative phosphorylation in eukaryotic organisms. The transport protein belongs to the mitochondrial carrier family, a large transporter family in the inner membrane of mitochondria. It is one of the best studied members of the family and serves as a paradigm for the molecular mechanism of mitochondrial carriers. Structurally, the carrier consists of three homologous domains, each composed of two transmembrane α-helices linked with a loop and short α-helix on the matrix side. The transporter cycles between a cytoplasmic and matrix state in which a central substrate binding site is alternately accessible to these compartments for binding of ADP or ATP. On both the cytoplasmic and matrix side of the carrier are networks consisting of three salt bridges each. In the cytoplasmic state, the matrix salt bridge network is formed and the cytoplasmic network is disrupted, opening the central substrate binding site to the intermembrane space and cytosol, whereas the converse occurs in the matrix state. In the transport cycle, tighter substrate binding in the intermediate states allows the interconversion of conformations by lowering the energy barrier for disruption and formation of these networks, opening and closing the carrier to either side of the membrane in an alternating way. Conversion between cytoplasmic and matrix states might require the simultaneous rotation of three domains around a central translocation pathway, constituting a unique mechanism among transport proteins. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou. PMID:27001633

  1. Membrane-associated proteolytic activity in Escherichia coli that is stimulated by ATP

    SciTech Connect

    Klemes, Y.; Voellmy, R.W.; Goldberg, A.L.

    1986-05-01

    The degradation of proteins in bacteria requires metabolism energy. One important enzyme in this process is protease La, a soluble ATP-dependent protease encoded by the lon gene. However, lon mutants that lack a functional protease La still show some ATP-dependent protein breakdown. The authors have reported an ATP-stimulated endoproteolytic activity associated with the inner membrane of E. coli. This ATP-stimulated activity is found in normal levels in membranes derived from lon mutants, including strains carrying insertions in the lon gene. The membrane-bound activity hydrolyzes /sup 14/C-methylglobin at a linear rate for up to 3 hours. These fractions also contain appreciable proteolytic activity that is not affected by ATP. The stimulation by ATP requires the presence of Mg/sup 2 +/. Nonhydrolyzable ATP analogs (e.g. AMPPNP or ATP-..gamma..-S) and ADP do not enhance proteolysis. Unlike protease La, the membrane-associated enzyme does not degrade the fluorometric substrate, Glt-Ala-Ala-Phe-MNA, in an ATP-stimulated fashion, and its level is not influenced by high temperature of by the gene which regulates the heat-shock response. The enzyme is inhibited by dichloroisocoumarin and certain peptide chloromethyl ketones. They conclude that E. coli contain at least two ATP-dependent proteases with distinct specificities: one is soluble and the other is membrane-associated.

  2. Dorsal horn neurons release extracellular ATP in a VNUT-dependent manner that underlies neuropathic pain.

    PubMed

    Masuda, Takahiro; Ozono, Yui; Mikuriya, Satsuki; Kohro, Yuta; Tozaki-Saitoh, Hidetoshi; Iwatsuki, Ken; Uneyama, Hisayuki; Ichikawa, Reiko; Salter, Michael W; Tsuda, Makoto; Inoue, Kazuhide

    2016-01-01

    Activation of purinergic receptors in the spinal cord by extracellular ATP is essential for neuropathic hypersensitivity after peripheral nerve injury (PNI). However, the cell type responsible for releasing ATP within the spinal cord after PNI is unknown. Here we show that PNI increases expression of vesicular nucleotide transporter (VNUT) in the spinal cord. Extracellular ATP content ([ATP]e) within the spinal cord was increased after PNI, and this increase was suppressed by exocytotic inhibitors. Mice lacking VNUT did not show PNI-induced increase in [ATP]e and had attenuated hypersensitivity. These phenotypes were recapitulated in mice with specific deletion of VNUT in spinal dorsal horn (SDH) neurons, but not in mice lacking VNUT in primary sensory neurons, microglia or astrocytes. Conversely, ectopic VNUT expression in SDH neurons of VNUT-deficient mice restored PNI-induced increase in [ATP]e and pain. Thus, VNUT is necessary for exocytotic ATP release from SDH neurons which contributes to neuropathic pain. PMID:27515581

  3. Controlled rotation of the F1-ATPase reveals differential and continuous binding changes for ATP synthesis

    PubMed Central

    Adachi, Kengo; Oiwa, Kazuhiro; Yoshida, Masasuke; Nishizaka, Takayuki; Kinosita, Kazuhiko

    2012-01-01

    F1-ATPase is an ATP-driven rotary molecular motor that synthesizes ATP when rotated in reverse. To elucidate the mechanism of ATP synthesis, we imaged binding and release of fluorescently labelled ADP and ATP while rotating the motor in either direction by magnets. Here we report the binding and release rates for each of the three catalytic sites for 360° of the rotary angle. We show that the rates do not significantly depend on the rotary direction, indicating ATP synthesis by direct reversal of the hydrolysis-driven rotation. ADP and ATP are discriminated in angle-dependent binding, but not in release. Phosphate blocks ATP binding at angles where ADP binding is essential for ATP synthesis. In synthesis rotation, the affinity for ADP increases by >104, followed by a shift to high ATP affinity, and finally the affinity for ATP decreases by >104. All these angular changes are gradual, implicating tight coupling between the rotor angle and site affinities. PMID:22929779

  4. Modeling the effects of hypoxia on ATP turnover in exercising muscle

    NASA Technical Reports Server (NTRS)

    Arthur, P. G.; Hogan, M. C.; Bebout, D. E.; Wagner, P. D.; Hochachka, P. W.

    1992-01-01

    Most models of metabolic control concentrate on the regulation of ATP production and largely ignore the regulation of ATP demand. We describe a model, based on the results of Hogan et al. (J. Appl. Physiol. 73: 728-736, 1992), that incorporates the effects of ATP demand. The model is developed from the premise that a unique set of intracellular conditions can be measured at each level of ATP turnover and that this relationship is best described by energetic state. Current concepts suggest that cells are capable of maintaining oxygen consumption in the face of declines in the concentration of oxygen through compensatory changes in cellular metabolites. We show that these compensatory changes can cause significant declines in ATP demand and result in a decline in oxygen consumption and ATP turnover. Furthermore we find that hypoxia does not directly affect the rate of anaerobic ATP synthesis and associated lactate production. Rather, lactate production appears to be related to energetic state, whatever the PO2. The model is used to describe the interaction between ATP demand and ATP supply in determining final ATP turnover.

  5. Dorsal horn neurons release extracellular ATP in a VNUT-dependent manner that underlies neuropathic pain

    PubMed Central

    Masuda, Takahiro; Ozono, Yui; Mikuriya, Satsuki; Kohro, Yuta; Tozaki-Saitoh, Hidetoshi; Iwatsuki, Ken; Uneyama, Hisayuki; Ichikawa, Reiko; Salter, Michael W.; Tsuda, Makoto; Inoue, Kazuhide

    2016-01-01

    Activation of purinergic receptors in the spinal cord by extracellular ATP is essential for neuropathic hypersensitivity after peripheral nerve injury (PNI). However, the cell type responsible for releasing ATP within the spinal cord after PNI is unknown. Here we show that PNI increases expression of vesicular nucleotide transporter (VNUT) in the spinal cord. Extracellular ATP content ([ATP]e) within the spinal cord was increased after PNI, and this increase was suppressed by exocytotic inhibitors. Mice lacking VNUT did not show PNI-induced increase in [ATP]e and had attenuated hypersensitivity. These phenotypes were recapitulated in mice with specific deletion of VNUT in spinal dorsal horn (SDH) neurons, but not in mice lacking VNUT in primary sensory neurons, microglia or astrocytes. Conversely, ectopic VNUT expression in SDH neurons of VNUT-deficient mice restored PNI-induced increase in [ATP]e and pain. Thus, VNUT is necessary for exocytotic ATP release from SDH neurons which contributes to neuropathic pain. PMID:27515581

  6. Copper transport during lactation in transgenic mice expressing the human ATP7A protein

    PubMed Central

    Llanos, Roxana M.; Michalczyk, Agnes A.; Freestone, David J.; Currie, Scott; Linder, Maria C.; Ackland, M. Leigh; Mercer, Julian F.B.

    2008-01-01

    Both copper transporting ATPases, ATP7A and ATP7B, are expressed in mammary epithelial cells but their role in copper delivery to milk has not been clarified. We investigated the role of ATP7A in delivery of copper to milk using transgenic mice that over-express human ATP7A. In mammary gland of transgenic mice, human ATP7A protein was 10- to 20-fold higher than in control mice, and was localized to the basolateral membrane of mammary epithelial cells in lactating mice. The copper concentration in the mammary gland of transgenic dams and stomach contents of transgenic pups was significantly reduced compared to non-transgenic mice. The mRNA levels of endogenous Atp7a, Atp7b, and Ctr1 copper transporters in the mammary gland were not altered by the expression of the ATP7A transgene, and the protein levels of Atp7b and ceruloplasmin were similar in transgenic and non-transgenic mice. These data suggest that ATP7A plays a role in removing excess copper from the mammary epithelial cells rather than supplying copper to milk. PMID:18515074

  7. Oxidized ATP. An irreversible inhibitor of the macrophage purinergic P2Z receptor.

    PubMed

    Murgia, M; Hanau, S; Pizzo, P; Rippa, M; Di Virgilio, F

    1993-04-15

    The effects of oxidized ATP (oATP) on responses triggered by extracellular adenosine 5'-triphosphate (ATPe) were investigated in the mouse macrophage-like cell line J774. ATPe induced in this cell line two kinds of responses mediated by two different P2 purinergic receptors: 1) an early permeabilization of the plasma membrane to extracellular hydrophilic markers of M(r) up to 900 mediated by P2Z receptors; and 2) a fast mobilization of Ca2+ from intracellular stores mediated by P2Y receptors. Low oATP concentrations (100 microM) completely blocked the first response without affecting the second. ATPe-dependent cell swelling, vacuolization, and lysis were also inhibited. Antagonism developed slowly, as an incubation at 37 degrees C for at least 2 h in the presence of oATP was needed and was irreversible, thus suggesting that the inhibitory action was due to covalent modification of the receptor. The rate of hydrolysis of exogenous ATP was slightly decreased by oATP, indicating a minor blocking effect of this compound on plasma membrane ecto-ATPases in the concentration range tested. These observations suggest that oATP may be a potentially very useful tool for isolation and characterization of the P2Z purinergic receptor. PMID:8463330

  8. CFTR-independent ATP release from epithelial cells triggered by mechanical stimuli.

    PubMed

    Grygorczyk, R; Hanrahan, J W

    1997-03-01

    Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated ATP efflux has been proposed as an autocrine mechanism for regulating chloride secretion through other types of chloride channels. Although we found in previous studies that wild-type CFTR channels bathed with high-ATP solutions do not conduct ATP at rates that can be measured with the patch-clamp technique, those experiments would not have detected very small or electroneutral ATP fluxes through CFTR or ATP efflux through other pathways that might be regulated by CFTR. To examine these possibilities, we have now used a sensitive luciferase luminometric assay to measure ATP efflux from epithelial and nonepithelial cell lines. Adenosine 3',5'-cyclic monophosphate (cAMP) stimulation did not raise external ATP concentration above the background noise in any of the cell lines tested [T84, Calu-3, 9HTEo- and sigma CFTE29o- (colonic and airway human epithelial cells, respectively), NIH/3T3 fibroblasts, and Chinese hamster ovary cells], and variations in ATP release were not correlated with CFTR expression. The rate of ATP release was unaffected by cAMP but was exquisitely sensitive to mechanical perturbations in both CFTR expressing and nonexpressing cells. Mechanically induced, CFTR-independent ATP release may be a physiologically relevant mechanism of epithelial regulation, which has not previously been fully appreciated. PMID:9124508

  9. ATP synthesis and export in heart left ventricle mitochondria from spontaneously hypertensive rat.

    PubMed

    Atlante, A; Seccia, T M; Pierro, P; Vulpis, V; Marra, E; Pirrelli, A; Passarella, S

    1998-04-01

    Use was made of mitochondria isolated from heart left ventricles of either spontaneously hypertensive or age-matched Wistar-Kyoto rats used as a control to find out whether hypertrophy (5-week-old rats) or hypertrophy/hypertension (24-week-old rats) can cause change in the mechanisms by which ATP is synthesised via ATP synthase and subsequently exported via the ADP/ATP translocator outside mitochondria. To do this, photometric measurements were made of the rate of ATP appearance in the extramitochondrial phase, which occurs as a result of ADP addition to mitochondria. In mitochondria from spontaneously hypertensive rats deficit of ATP production was found dependent on changes in the KmADP and Vmax values of both the ADP/ATP translocator and the ATP synthase. The ADP/ATP translocator was found to determine the rate of ATP production outside mitochondria in all the tested samples. In an initial investigation carried out to ascertain how cell ATP deficit can be counterbalanced, an increase in both adenylate kinase and creatine kinase activities was found in both hypertrophy and hypertrophy/hypertension. A possible increase in anaerobic glycolysis was also suggested by the increased lactate dehydrogenase activity. PMID:9852286

  10. Induction of Posttranslational Modifications of Mitochondrial Proteins by ATP Contributes to Negative Regulation of Mitochondrial Function

    PubMed Central

    Zhang, Yong; Zhao, Zhiyun; Ke, Bilun; Wan, Lin; Wang, Hui; Ye, Jianping

    2016-01-01

    It is generally accepted that ATP regulates mitochondrial function through the AMPK signaling pathway. However, the AMPK-independent pathway remains largely unknown. In this study, we investigated ATP surplus in the negative regulation of mitochondrial function with a focus on pyruvate dehydrogenase (PDH) phosphorylation and protein acetylation. PDH phosphorylation was induced by a high fat diet in the liver of obese mice, which was associated with ATP elevation. In 1c1c7 hepatoma cells, the phosphorylation was induced by palmitate treatment through induction of ATP production. The phosphorylation was associated with a reduction in mitochondria oxygen consumption after 4 h treatment. The palmitate effect was blocked by etomoxir, which inhibited ATP production through suppression of fatty acid β-oxidation. The PDH phosphorylation was induced by incubation of mitochondrial lysate with ATP in vitro without altering the expression of PDH kinase 2 (PDK2) and 4 (PDK4). In addition, acetylation of multiple mitochondrial proteins was induced by ATP in the same conditions. Acetyl-CoA exhibited a similar activity to ATP in induction of the phosphorylation and acetylation. These data suggest that ATP elevation may inhibit mitochondrial function through induction of the phosphorylation and acetylation of mitochondrial proteins. The results suggest an AMPK-independent mechanism for ATP regulation of mitochondrial function. PMID:26930489

  11. Copper directs ATP7B to the apical domain of hepatic cells via basolateral endosomes.

    PubMed

    Nyasae, Lydia K; Schell, Michael J; Hubbard, Ann L

    2014-12-01

    Physiologic Cu levels regulate the intracellular location of the Cu ATPase ATP7B. Here, we determined the routes of Cu-directed trafficking of endogenous ATP7B in the polarized hepatic cell line WIF-B and in the liver in vivo. Copper (10 µm) caused ATP7B to exit the trans-Golgi network (TGN) in vesicles, which trafficked via large basolateral endosomes to the apical domain within 1 h. Although perturbants of luminal acidification had little effect on the TGN localization of ATP7B in low Cu, they blocked delivery to the apical membrane in elevated Cu. If the vesicular proton-pump inhibitor bafilomycin-A1 (Baf) was present with Cu, ATP7B still exited the TGN, but accumulated in large endosomes located near the coverslip, in the basolateral region. Baf washout restored ATP7B trafficking to the apical domain. If ATP7B was staged apically in high Cu, Baf addition promoted the accumulation of ATP7B in subapical endosomes, indicating a blockade of apical recycling, with concomitant loss of ATP7B at the apical membrane. The retrograde pathway to the TGN, induced by Cu removal, was far less affected by Baf than the anterograde (Cu-stimulated) case. Overall, loss of acidification-impaired Cu-regulated trafficking of ATP7B at two main sites: (i) sorting and exit from large basolateral endosomes and (ii) recycling via endosomes near the apical membrane. PMID:25243755

  12. Ordered ATP hydrolysis in the gamma complex clamp loader AAA+ machine.

    PubMed

    Johnson, Aaron; O'Donnell, Mike

    2003-04-18

    The gamma complex couples ATP hydrolysis to the loading of beta sliding clamps onto DNA for processive replication. The gamma complex structure shows that the clamp loader subunits are arranged as a circular heteropentamer. The three gamma motor subunits bind ATP, the delta wrench opens the beta ring, and the delta' stator modulates the delta-beta interaction. Neither delta nor delta' bind ATP. This report demonstrates that the delta' stator contributes a catalytic arginine for hydrolysis of ATP bound to the adjacent gamma(1) subunit. Thus, the delta' stator contributes to the motor function of the gamma trimer. Mutation of arginine 169 of gamma, which removes the catalytic arginines from only the gamma(2) and gamma(3) ATP sites, abolishes ATPase activity even though ATP site 1 is intact and all three sites are filled. This result implies that hydrolysis of the three ATP molecules occurs in a particular order, the reverse of ATP binding, where ATP in site 1 is not hydrolyzed until ATP in sites 2 and/or 3 is hydrolyzed. Implications of these results to clamp loaders of other systems are discussed. PMID:12582167

  13. Purinergic and muscarinic modulation of ATP release from the urothelium and its paracrine actions.

    PubMed

    Sui, Guiping; Fry, Chris H; Montgomery, Bruce; Roberts, Max; Wu, Rui; Wu, Changhao

    2014-02-01

    The urothelium is a newly recognized sensory structure that detects bladder fullness. Pivotal to this sensory role is the release of ATP from the urothelium. However, the routes for urothelial ATP release, its modulation by receptor-mediated pathways, and the autocrine/paracrine role of ATP are poorly understood, especially in native tissue. We examined the action of key neurotransmitters: purinergic and muscarinic agonists on ATP release and its paracrine effect. Guinea pig and human urothelial mucosa were mounted in a perfusion trough; superfusate ATP was measured using a luciferin-luciferase assay, and tissue contractions were recorded with a tension transducer. Intracellular Ca²⁺ was measured in isolated urothelial cells with fura-2. The P2Y agonist UTP but not the P2X agonist α,β-methylene-ATP generated ATP release. The muscarinic agonist carbachol and the M₂-preferential agonist oxotremorine also generated ATP release, which was antagonized by the M₂-specific agent methoctramine. Agonist-evoked ATP release was accompanied by mucosal contractions. Urothelial ATP release was differentially mediated by intracellular Ca²⁺ release, cAMP, exocytosis, or connexins. Urothelium-attached smooth muscle exhibited spontaneous contractions that were augmented by subthreshold concentrations of carbachol, which had little direct effect on smooth muscle. This activity was attenuated by desensitizing P2X receptors on smooth muscle. Urothelial ATP release was increased in aging bladders. Purinergic and muscarinic agents produced similar effects in human urothelial tissue. This is the first demonstration of specific modulation of urothelial ATP release in native tissue by purinergic and muscarinic neurotransmitters via distinct mechanisms. Released ATP produces paracrine effects on underlying tissues. This process is altered during aging and has relevance to human bladder pathologies. PMID:24285497

  14. Purinergic and muscarinic modulation of ATP release from the urothelium and its paracrine actions

    PubMed Central

    Sui, Guiping; Fry, Chris H.; Montgomery, Bruce; Roberts, Max; Wu, Rui

    2013-01-01

    The urothelium is a newly recognized sensory structure that detects bladder fullness. Pivotal to this sensory role is the release of ATP from the urothelium. However, the routes for urothelial ATP release, its modulation by receptor-mediated pathways, and the autocrine/paracrine role of ATP are poorly understood, especially in native tissue. We examined the action of key neurotransmitters: purinergic and muscarinic agonists on ATP release and its paracrine effect. Guinea pig and human urothelial mucosa were mounted in a perfusion trough; superfusate ATP was measured using a luciferin-luciferase assay, and tissue contractions were recorded with a tension transducer. Intracellular Ca2+ was measured in isolated urothelial cells with fura-2. The P2Y agonist UTP but not the P2X agonist α,β-methylene-ATP generated ATP release. The muscarinic agonist carbachol and the M2-preferential agonist oxotremorine also generated ATP release, which was antagonized by the M2-specific agent methoctramine. Agonist-evoked ATP release was accompanied by mucosal contractions. Urothelial ATP release was differentially mediated by intracellular Ca2+ release, cAMP, exocytosis, or connexins. Urothelium-attached smooth muscle exhibited spontaneous contractions that were augmented by subthreshold concentrations of carbachol, which had little direct effect on smooth muscle. This activity was attenuated by desensitizing P2X receptors on smooth muscle. Urothelial ATP release was increased in aging bladders. Purinergic and muscarinic agents produced similar effects in human urothelial tissue. This is the first demonstration of specific modulation of urothelial ATP release in native tissue by purinergic and muscarinic neurotransmitters via distinct mechanisms. Released ATP produces paracrine effects on underlying tissues. This process is altered during aging and has relevance to human bladder pathologies. PMID:24285497

  15. Stable ATP binding mediated by a partial NBD dimer of the CFTR chloride channel.

    PubMed

    Tsai, Ming-Feng; Li, Min; Hwang, Tzyh-Chang

    2010-05-01

    Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the adenosine triphosphate (ATP) binding cassette (ABC) superfamily, is an ATP-gated chloride channel. Like other ABC proteins, CFTR encompasses two nucleotide binding domains (NBDs), NBD1 and NBD2, each accommodating an ATP binding site. It is generally accepted that CFTR's opening-closing cycles, each completed within 1 s, are driven by rapid ATP binding and hydrolysis events in NBD2. Here, by recording CFTR currents in real time with a ligand exchange protocol, we demonstrated that during many of these gating cycles, NBD1 is constantly occupied by a stably bound ATP or 8-N(3)-ATP molecule for tens of seconds. We provided evidence that this tightly bound ATP or 8-N(3)-ATP also interacts with residues in the signature sequence of NBD2, a telltale sign for an event occurring at the NBD1-NBD2 interface. The open state of CFTR has been shown to represent a two-ATP-bound NBD dimer. Our results indicate that upon ATP hydrolysis in NBD2, the channel closes into a "partial NBD dimer" state where the NBD interface remains partially closed, preventing ATP dissociation from NBD1 but allowing the release of hydrolytic products and binding of the next ATP to occur in NBD2. Opening and closing of CFTR can then be coupled to the formation and "partial" separation of the NBD dimer. The tightly bound ATP molecule in NBD1 can occasionally dissociate from the partial dimer state, resulting in a nucleotide-free monomeric state of NBDs. Our data, together with other structural/functional studies of CFTR's NBDs, suggest that this process is poorly reversible, implying that the channel in the partial dimer state or monomeric state enters the open state through different pathways. We therefore proposed a gating model for CFTR with two distinct cycles. The structural and functional significance of our results to other ABC proteins is discussed. PMID:20421370

  16. Terrestrial evolution of polymerization of amino acids - Heat to ATP

    NASA Technical Reports Server (NTRS)

    Fox, S. W.; Nakashima, T.

    1981-01-01

    Sets of amino acids containing sufficient trifunctional monomer are thermally polymerized at temperatures such as 65 deg; the amino acids order themselves. Various polymers have diverse catalytic activities. The polymers aggregate, in aqueous solution, to cell-like structures having those activities plus emergent properties, e.g. proliferatability. Polyamino acids containing sufficient lysine catalyze conversion of free amino acids, by ATP, to small peptides and a high molecular weight fraction. The lysine-rich proteinoid is active in solution, within suspensions of cell-like particles, or in other particles composed of lysine-rich proteinoid and homopolyribonucleotide. Selectivities are observed. An archaic polyamino acid prelude to coded protein synthesis is indicated.

  17. Detection and quantification of ATP in human blood serum.

    PubMed

    Akdeniz, Ali; Caglayan, Mehmet Gokhan; Polivina, Irina; Anzenbacher, Pavel

    2016-08-21

    Two fluorometric sensors based on the tri-serine tri-lactone scaffold and thiourea or sulfonamide moieties serving as hydrogen bond donors allowing for anion binding are described. The sensor utilizing thiourea as a recognition moiety shows fluorescence enhancement while the sensor with sulfonamide shows quenching upon addition of phosphates. Sensor arrays composed of two sensors are able to discriminate structurally similar organic phosphates in the presence of interferents in human blood serum. The quantitative analysis of ATP in human blood serum shows high accuracy (the root mean square error of prediction, 1.65%) without requiring any sample pretreatment. PMID:27454442

  18. Detection of ATP hydrolysis through motion of nanoconfined DNA

    NASA Astrophysics Data System (ADS)

    Roushan, Maedeh; Livshits, Gideon; Azad, Zubair; Wang, Hong; Riehn, Robert

    Confinement of DNA to nanochannels with a cross-section of 100 ×100 nm2 and hundreds of micrometer long has previously been used to investigate the equilibrium binding properties of proteins to DNA. Here we report on the observation that a range of proteins which catalyze a modification of DNA, and that do so by hydrolyzing ATP, cause a net directed motion of nanochannel-confined DNA. We present a model for this observation that does not require any motor-like action of the protein and that is purely dependent on the catalytic properties.

  19. Antithrombin III blood test

    MedlinePlus

    ... AT III) is a protein that helps control blood clotting. A blood test can determine the amount of ... may mean you have an increased risk of blood clotting. This can occur when there is not enough ...

  20. Antithrombin III blood test

    MedlinePlus

    ... be due to: Bone marrow transplant Disseminated intravascular coagulation (DIC) AT III deficiency, an inherited condition Liver ... Schmaier AH, Miller JL. Coagulation and fibrinolysis. In: McPherson ... Management by Laboratory Methods . 22nd ed. Philadelphia, PA: ...

  1. Fragments of ATP synthase mediate plant perception of insect attack

    PubMed Central

    Schmelz, Eric A.; Carroll, Mark J.; LeClere, Sherry; Phipps, Stephen M.; Meredith, Julia; Chourey, Prem S.; Alborn, Hans T.; Teal, Peter E. A.

    2006-01-01

    Plants can perceive a wide range of biotic attackers and respond with targeted induced defenses. Specificity in plant non-self-recognition occurs either directly by perception of pest-derived elicitors or indirectly through resistance protein recognition of host targets that are inappropriately proteolyzed. Indirect plant perception can occur during interactions with pathogens, yet evidence for analogous events mediating the detection of insect herbivores remains elusive. Here we report indirect perception of herbivory in cowpea (Vigna unguiculata) plants attacked by fall armyworm (Spodoptera frugiperda) larvae. We isolated and identified a disulfide-bridged peptide (+ICDINGVCVDA−), termed inceptin, from S. frugiperda larval oral secretions that promotes cowpea ethylene production at 1 fmol leaf−1 and triggers increases in the defense-related phytohormones salicylic acid and jasmonic acid. Inceptins are proteolytic fragments of chloroplastic ATP synthase γ-subunit regulatory regions that mediate plant perception of herbivory through the induction of volatile, phenylpropanoid, and protease inhibitor defenses. Only S. frugiperda larvae that previously ingested chloroplastic ATP synthase γ-subunit proteins and produced inceptins significantly induced cowpea defenses after herbivory. Digestive fragments of an ancient and essential plant enzyme, inceptin functions as a potent indirect signal initiating specific plant responses to insect attack. PMID:16720701

  2. Minimum energy reaction profiles for ATP hydrolysis in myosin.

    PubMed

    Grigorenko, Bella L; Kaliman, Ilya A; Nemukhin, Alexander V

    2011-11-01

    The minimum energy reaction profiles corresponding to two possible reaction mechanisms of adenosine triphosphate (ATP) hydrolysis in myosin are computed in this work within the framework of the quantum mechanics-molecular mechanics (QM/MM) method by using the same partitioning of the model system to the QM and MM parts and the same computational protocol. On the first reaction route, one water molecule performs nucleophilic attack at the phosphorus center P(γ) from ATP while the second water molecule in the closed protein cleft serves as a catalytic base assisted by the Glu residue from the myosin salt bridge. According to the present QM/MM calculations consistent with the results of kinetic studies this reaction pathway is characterized by a low activation energy barrier about 10 kcal/mol. The computed activation energy barrier for the second mechanism, which assumes the penta-coordinated oxyphosphorane transition state upon involvement of single water molecule in the reaction, is considerably higher than that for the two-water mechanism. PMID:21839658

  3. Mechanosensitive ATP Release Maintains Proper Mucus Hydration of Airways

    PubMed Central

    Button, Brian; Okada, Seiko F.; Frederick, Charles Brandon; Thelin, William R.; Boucher, Richard C.

    2013-01-01

    The clearance of mucus from the airways protects the lungs from inhaled noxious and infectious materials. Proper hydration of the mucus layer enables efficient mucus clearance through beating of cilia on airway epithelial cells, and reduced clearance of excessively concentrated mucus occurs in patients with chronic obstructive pulmonary disease and cystic fibrosis. Key steps in the mucus transport process are airway epithelia sensing and responding to changes in mucus hydration. We reported that extracellular adenosine triphosphate (ATP) and adenosine were important luminal auto-crine and paracrine signals that regulated the hydration of the surface of human airway epithelial cultures through their action on apical membrane purinoceptors. Mucus hydration in human airway epithelial cultures was sensed by an interaction between cilia and the overlying mucus layer: Changes in mechanical strain, proportional to mucus hydration, regulated ATP release rates, adjusting fluid secretion to optimize mucus layer hydration. This system provided a feedback mechanism by which airways maintained mucus hydration in an optimum range for cilia propulsion. Understanding how airway epithelia can sense and respond to changes in mucus properties helps us to understand how the mucus clearance system protects the airways in health and how it fails in lung diseases such as cystic fibrosis. PMID:23757023

  4. Efficacy and limitations of an ATP-based monitoring system.

    PubMed

    Turner, Danielle E; Daugherity, Erin K; Altier, Craig; Maurer, Kirk J

    2010-03-01

    Monitoring of sanitation is an essential function of laboratory animal facilities. The purpose of the current study was to assess the ability of an ATP-based system to detect microbes and organic contaminants. Serial dilutions of Escherichia coli, Staphylococcus aureus, Toxocara canis eggs, Toxoplasma gondii tachyzoites, epithelial cells, and rodent blood, urine, and feces were analyzed according to the manufacturer's recommendations. The limit of E. coli detection was 10(4) organisms; sonication of E. coli significantly improved detection, indicating incomplete bacterial lysis in the detection system. Detection of S. aureus was significantly greater than that of E. coli with a limit of detection of 10(2); sonication did not alter results. In contrast, detection of T. canis, T. gondii, RBC, and epithelial cells was robust and ranged from 2 T. canis eggs to 10 epithelial cells. Urine was weakly detected, with a limit of detection at 1:10 dilution. Detection of all cell types except epithelia had a strong linear correlation to total cell number. In addition, our data demonstrate that the efficacy of the detection system can be affected adversely by residual disinfectants and that sample-bearing swabs are stable for more than 7 h after swabbing. These data demonstrate that this ATP based system sensitively detects pure cells and organic contaminants with a strong degree of linear predictability. A limitation of the system is its inability to detect gram-negative bacteria efficiently because of incomplete cell lysis. PMID:20353694

  5. How azide inhibits ATP hydrolysis by the F-ATPases

    PubMed Central

    Bowler, Matthew W.; Montgomery, Martin G.; Leslie, Andrew G. W.; Walker, John E.

    2006-01-01

    In the structure of bovine F1-ATPase determined at 1.95-Å resolution with crystals grown in the presence of ADP, 5′-adenylyl-imidodiphosphate, and azide, the azide anion interacts with the β-phosphate of ADP and with residues in the ADP-binding catalytic subunit, βDP. It occupies a position between the catalytically essential amino acids, β-Lys-162 in the P loop and the “arginine finger” residue, α-Arg-373, similar to the site occupied by the γ-phosphate in the ATP-binding subunit, βTP. Its presence in the βDP-subunit tightens the binding of the side chains to the nucleotide, enhancing its affinity and thereby stabilizing the state with bound ADP. This mechanism of inhibition appears to be common to many other ATPases, including ABC transporters, SecA, and DNA topoisomerase IIα. It also explains the stimulatory effect of azide on ATP-sensitive potassium channels by enhancing the binding of ADP. PMID:16728506

  6. Mechanosensitive ATP release maintains proper mucus hydration of airways.

    PubMed

    Button, Brian; Okada, Seiko F; Frederick, Charles Brandon; Thelin, William R; Boucher, Richard C

    2013-06-11

    The clearance of mucus from the airways protects the lungs from inhaled noxious and infectious materials. Proper hydration of the mucus layer enables efficient mucus clearance through beating of cilia on airway epithelial cells, and reduced clearance of excessively concentrated mucus occurs in patients with chronic obstructive pulmonary disease and cystic fibrosis. Key steps in the mucus transport process are airway epithelia sensing and responding to changes in mucus hydration. We reported that extracellular adenosine triphosphate (ATP) and adenosine were important luminal autocrine and paracrine signals that regulated the hydration of the surface of human airway epithelial cultures through their action on apical membrane purinoceptors. Mucus hydration in human airway epithelial cultures was sensed by an interaction between cilia and the overlying mucus layer: Changes in mechanical strain, proportional to mucus hydration, regulated ATP release rates, adjusting fluid secretion to optimize mucus layer hydration. This system provided a feedback mechanism by which airways maintained mucus hydration in an optimum range for cilia propulsion. Understanding how airway epithelia can sense and respond to changes in mucus properties helps us to understand how the mucus clearance system protects the airways in health and how it fails in lung diseases such as cystic fibrosis. PMID:23757023

  7. Activation of the heat-stable polypeptide of the ATP-dependent proteolytic system.

    PubMed Central

    Ciechanover, A; Heller, H; Katz-Etzion, R; Hershko, A

    1981-01-01

    It had been shown previously that the heat-stable polypeptide of the ATP-dependent proteolytic system of reticulocytes, designated APF-1, forms covalent conjugates with protein substrates in an ATP-requiring process. We now describe an enzyme that carries out the activation by ATP of the polypeptide with pyrophosphate displacement. The formation of AMP-polypeptide and transfer of the polypeptide to a secondary acceptor are suggested by an APF-1 requirement for ATP-PPi and ATP-AMP exchange reactions, respectively. With radiolabeled polypeptide, an ATP-dependent labeling of the enzyme was shown to be by a linkage that is acid stable but is labile to treatment with mild alkali, hydroxylamine, borohydride, or mercuric salts. It therefore appears that the AMP-polypeptide undergoes attack by an -SH group of the enzyme to form a thiolester. PMID:6262770

  8. Application of ATP measurements to the microbiological evaluation of a petroleum reservoir

    SciTech Connect

    Jones, P.M.

    1981-06-01

    The objective of the work reported in this document was to determine whether the bioluminescent luciferin/luciferase based adenosine triphosphate (ATP) assay could be used as a rapid field tests for determining the presence and numbers of microorganisms in oil field fluids. The ATP-photometric technique employed is based on the ATP-mediated bioluminescent oxidation of firefly luciferin. Light production is stoichiometrically related to ATP concentration; ATP concentration is related to numbers of living organisms present in a sample. Samples used in this study comprised reservoir fluids collected from several Southern California oilfields. Based on experimental evidence, it was concluded that the ATP assay could be profitably applied to Microbially Enhanced Oil Recovery (MEOR) process monitoring and control. The theoretical basis for the assay, field-usage methodologies, and fundamentals of data interpretation are presented to make the document usable as a field manual.

  9. Genetic effects of ATP1A2 in familial hemiplegic migraine type II and animal models

    PubMed Central

    2013-01-01

    Na+/K+-ATPase alpha 2 (Atp1a2) is an integral plasma membrane protein belonging to the P-type ATPase family that is responsible for maintaining the sodium (Na+) and potassium (K+) gradients across cellular membranes with hydrolysis of ATP. Atp1a2 contains two subunits, alpha and beta, with each having various isoforms and differential tissue distribution. In humans, mutations in ATP1A2 are associated with a rare form of hereditary migraines with aura known as familial hemiplegic migraine type II. Genetic studies in mice have revealed other neurological effects of Atp1a2 in mice including anxiety, fear, and learning and motor function disorders. This paper reviews the recent findings in the literature concerning Atp1a2. PMID:23561701

  10. Structure guided simulations illuminate the mechanism of ATP transport through VDAC1

    PubMed Central

    Choudhary, O.P.; Paz, A.; Adelman, J.L.; Colletier, J.P.; Abramson, J.; Grabe, M.

    2014-01-01

    The voltage-dependent anion channel (VDAC) mediates metabolite and ion flow across the outer mitochondrial membrane of all eukaryotic cells. The open channel passes millions of ATP molecules per second, while the closed state exhibits no detectable ATP flux. High-resolution structures of VDAC1 revealed a 19-stranded β-barrel with an α-helix partially occupying the central pore. To understand ATP permeation through VDAC, we solved the crystal structure of mouse VDAC1 (mVDAC1) in the presence of ATP, revealing a low-affinity binding site. Guided by these coordinates, we initiated hundreds of molecular dynamics (MD) simulations to construct a Markov State Model (MSM) of ATP permeation. These simulations indicate that ATP flows through VDAC using multiple pathways, consistent with our structural data and experimentally determined physiological rates. PMID:24908397

  11. ATP synthase subunit-β down-regulation aggravates diabetic nephropathy

    PubMed Central

    Guan, Siao-Syun; Sheu, Meei-Ling; Wu, Cheng-Tien; Chiang, Chih-Kang; Liu, Shing-Hwa

    2015-01-01

    In this study, we investigated the role of ATP synthase subunit-β (ATP5b) in diabetic nephropathy. Histopathological changes, fibrosis, and protein expressions of α-smooth muscle actin (α-SMA), advanced glycation end-products (AGEs), and ATP5b were obviously observed in the kidneys of db/db diabetic mice as compared with the control db/m+ mice. The increased ATP5b expression was majorly observed in diabetic renal tubules and was notably observed to locate in cytoplasm of tubule cells, but no significant increase of ATP5b in diabetic glomeruli. AGEs significantly increased protein expression of ATP5b and fibrotic factors and decreased ATP content in cultured renal tubular cells via an AGEs-receptor for AGEs (RAGE) axis pathway. Oxidative stress was also induced in diabetic kidneys and AGEs-treated renal tubular cells. The increase of ATP5b and CTGF protein expression in AGEs-treated renal tubular cells was reversed by antioxidant N-acetylcysteine. ATP5b-siRNA transfection augmented the increased protein expression of α-SMA and CTGF and CTGF promoter activity in AGEs-treated renal tubular cells. The in vivo ATP5b-siRNA delivery significantly enhanced renal fibrosis and serum creatinine in db/db mice with ATP5b down-regulation. These findings suggest that increased ATP5b plays an important adaptive or protective role in decreasing the rate of AGEs-induced renal fibrosis during diabetic condition. PMID:26449648

  12. 31P magnetization transfer measurements of Pi→ATP flux in exercising human muscle

    PubMed Central

    Savage, David B.; Williams, Guy B.; Porter, David; Carpenter, T. Adrian; Brindle, Kevin M.; Kemp, Graham J.

    2016-01-01

    Fundamental criticisms have been made over the use of 31P magnetic resonance spectroscopy (MRS) magnetization transfer estimates of inorganic phosphate (Pi)→ATP flux (VPi-ATP) in human resting skeletal muscle for assessing mitochondrial function. Although the discrepancy in the magnitude of VPi-ATP is now acknowledged, little is known about its metabolic determinants. Here we use a novel protocol to measure VPi-ATP in human exercising muscle for the first time. Steady-state VPi-ATP was measured at rest and over a range of exercise intensities and compared with suprabasal oxidative ATP synthesis rates estimated from the initial rates of postexercise phosphocreatine resynthesis (VATP). We define a surplus Pi→ATP flux as the difference between VPi-ATP and VATP. The coupled reactions catalyzed by the glycolytic enzymes GAPDH and phosphoglycerate kinase (PGK) have been shown to catalyze measurable exchange between ATP and Pi in some systems and have been suggested to be responsible for this surplus flux. Surplus VPi-ATP did not change between rest and exercise, even though the concentrations of Pi and ADP, which are substrates for GAPDH and PGK, respectively, increased as expected. However, involvement of these enzymes is suggested by correlations between absolute and surplus Pi→ATP flux, both at rest and during exercise, and the intensity of the phosphomonoester peak in the 31P NMR spectrum. This peak includes contributions from sugar phosphates in the glycolytic pathway, and changes in its intensity may indicate changes in downstream glycolytic intermediates, including 3-phosphoglycerate, which has been shown to influence the exchange between ATP and Pi catalyzed by GAPDH and PGK. PMID:26744504

  13. Studies on the activation by ATP of the 26 S proteasome complex from rat skeletal muscle.

    PubMed Central

    Dahlmann, B; Kuehn, L; Reinauer, H

    1995-01-01

    The 26 S proteasome complex is thought to catalyse the breakdown of ubiquitinated proteins within eukaryotic cells. In addition it has been found that the complex also degrades short-lived proteins such as ornithine decarboxylase in a ubiquitin-independent manner. Both proteolytic processes are paralleled by the hydrolysis of ATP. Here we show that ATP also affects the hydrolytic activity towards fluorigenic peptide substrates by the 26 S proteasome complex from rat skeletal muscle tissue. Low concentrations of ATP (about 25 microM) optimally activate the so-called chymotryptic and tryptic activity by increasing the rate of peptide hydrolysis but not peptidylglutamylpeptide hydrolysis. Activation of the enzyme by ATP is transient but this effect can be enhanced and prolonged by including in the assay an ATP-regenerating system, indicating that ATP is hydrolysed by the 26 S proteasome complex. Although ATP cannot be substituted for by adenosine 5'-[beta,gamma-methylene]triphosphate or AMP, hydrolysis of the phosphoanhydride bond of ATP seems not to be necessary for the activation process of the proteasome complex, a conclusion drawn from the findings that ATP analogues such as adenosine 5'-[beta,gamma-imido]triphosphate, adenosine 5'-O-[gamma-thio]triphosphate, adenosine 5'-O-[beta-thio]-diphosphate and adenosine 5'-[alpha,beta-methylene]triphosphate give the same effect as ATP, and vanadate does not prevent ATP activation. These effects are independent of the presence of Mg2+. Thus, ATP and other nucleotides may act as allosteric activators of peptide-hydrolysing activities of the 26 S proteasome complex as has also been found with the lon protease from Escherichia coli. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:7619056

  14. Functional production of the Na+ F1F(O) ATP synthase from Acetobacterium woodii in Escherichia coli requires the native AtpI.

    PubMed

    Brandt, Karsten; Müller, Daniel B; Hoffmann, Jan; Hübert, Christine; Brutschy, Bernd; Deckers-Hebestreit, Gabriele; Müller, Volker

    2013-02-01

    The Na(+) F(1)F(O) ATP synthase of the anaerobic, acetogenic bacterium Acetobacterium woodii has a unique F(O)V(O) hybrid rotor that contains nine copies of a F(O)-like c subunit and one copy of a V(O)-like c(1) subunit with one ion binding site in four transmembrane helices whose cellular function is obscure. Since a genetic system to address the role of different c subunits is not available for this bacterium, we aimed at a heterologous expression system. Therefore, we cloned and expressed its Na(+) F(1)F(O) ATP synthase operon in Escherichia coli. A Δatp mutant of E. coli produced a functional, membrane-bound Na(+) F(1)F(O) ATP synthase that was purified in a single step after inserting a His(6)-tag to its β subunit. The purified enzyme was competent in Na(+) transport and contained the F(O)V(O) hybrid rotor in the same stoichiometry as in A. woodii. Deletion of the atpI gene from the A. woodii operon resulted in a loss of the c ring and a mis-assembled Na(+) F(1)F(O) ATP synthase. AtpI from E. coli could not substitute AtpI from A. woodii. These data demonstrate for the first time a functional production of a F(O)V(O) hybrid rotor in E. coli and revealed that the native AtpI is required for assembly of the hybrid rotor. PMID:23054076

  15. New soluble ATP-dependent protease, Ti, in Escherichia coli that is distinct from protease La

    SciTech Connect

    Chung, C.H.; Hwang, B.J.; Park, W.J.; Goldberg, A.L.

    1987-05-01

    E. coli must contain other ATP-requiring proteolytic systems in addition to protease La (the lon gene product). A new ATP-dependent protease was purified from lon cells which lack protease La, as shown by immuno-blotting. This enzyme hydrolyzes (TH)casein to acid-soluble products in the presence of ATP (or dATP) and MgS . Nonhydrolyzable ATP analogs, other nucleoside triphosphates and AMP can not replace ATP. Therefore, ATP hydrolysis appears necessary for proteolysis. The enzyme appears to be a serine protease, but also contains essential thiol residues. Unlike protease La, it is not inhibited by vanadate, heparin, or the defective R9 subunit of protease La. On gel filtration, this enzyme has an apparent Mr of 340,000 and is comprised of two components of 190,000D and 130,000D, which can be separated by phosphocellulose chromatography. By themselves, these components do not show ATP-dependent proteolysis, but when mixed, full activity is restored. These finding and similar ones of Maurizi and Gottesman indicate that E. coli contain two soluble ATP-dependent proteases, which function by different mechanisms. This new enzyme may contribute to the rapid breakdown of abnormal polypeptides or of normal proteins during starvation. The authors propose to name it protease Ti.

  16. ATP-containing vesicles in stria vascular marginal cell cytoplasms in neonatal rat cochlea are lysosomes

    PubMed Central

    Liu, Jun; Liu, Wenjing; Yang, Jun

    2016-01-01

    We confirmed that ATP is released from cochlear marginal cells in the stria vascular but the cell organelle in which ATP stores was not identified until now. Thus, we studied the ATP-containing cell organelles and suggest that these are lysosomes. Primary cultures of marginal cells of Sprague-Dawley rats aged 1–3 days was established. Vesicles within marginal cells stained with markers were identified under confocal laser scanning microscope and transmission electron microscope (TEM). Then ATP release from marginal cells was measured after glycyl-L-phenylalanine-ß- naphthylamide (GPN) treatment using a bioluminescent assay. Quinacrine-stained granules within marginal cells were labeled with LysoTracker, a lysosome tracer, and lysosomal-associated membrane protein 1(LAMP1), but not labeled with the mitochondrial tracer MitoTracker. Furthermore, LysoTracker-labelled puncta showed accumulation of Mant-ATP, an ATP analog. Treatment with 200 μM GPN quenched fluorescently labeled puncta after incubation with LysoTracker or quinacrine, but not MitoTracker. Quinacrine-labeled organelles observed by TEM were lysosomes, and an average 27.7 percent increase in ATP luminescence was observed in marginal cells extracellular fluid after GPN treatment. ATP-containing vesicles in cochlear marginal cells of the stria vascular from neonatal rats are likely lysosomes. ATP release from marginal cells may be via Ca2+-dependent lysosomal exocytosis. PMID:26864824

  17. Lipid-mediated Protein-protein Interactions Modulate Respiration-driven ATP Synthesis

    PubMed Central

    Nilsson, Tobias; Lundin, Camilla Rydström; Nordlund, Gustav; Ädelroth, Pia; von Ballmoos, Christoph; Brzezinski, Peter

    2016-01-01

    Energy conversion in biological systems is underpinned by membrane-bound proton transporters that generate and maintain a proton electrochemical gradient across the membrane which used, e.g. for generation of ATP by the ATP synthase. Here, we have co-reconstituted the proton pump cytochrome bo3 (ubiquinol oxidase) together with ATP synthase in liposomes and studied the effect of changing the lipid composition on the ATP synthesis activity driven by proton pumping. We found that for 100 nm liposomes, containing 5 of each proteins, the ATP synthesis rates decreased significantly with increasing fractions of DOPA, DOPE, DOPG or cardiolipin added to liposomes made of DOPC; with e.g. 5% DOPG, we observed an almost 50% decrease in the ATP synthesis rate. However, upon increasing the average distance between the proton pumps and ATP synthases, the ATP synthesis rate dropped and the lipid dependence of this activity vanished. The data indicate that protons are transferred along the membrane, between cytochrome bo3 and the ATP synthase, but only at sufficiently high protein densities. We also argue that the local protein density may be modulated by lipid-dependent changes in interactions between the two proteins complexes, which points to a mechanism by which the cell may regulate the overall activity of the respiratory chain. PMID:27063297

  18. Structural Basis for a Unique ATP Synthase Core Complex from Nanoarcheaum equitans.

    PubMed

    Mohanty, Soumya; Jobichen, Chacko; Chichili, Vishnu Priyanka Reddy; Velázquez-Campoy, Adrián; Low, Boon Chuan; Hogue, Christopher W V; Sivaraman, J

    2015-11-01

    ATP synthesis is a critical and universal life process carried out by ATP synthases. Whereas eukaryotic and prokaryotic ATP synthases are well characterized, archaeal ATP synthases are relatively poorly understood. The hyperthermophilic archaeal parasite, Nanoarcheaum equitans, lacks several subunits of the ATP synthase and is suspected to be energetically dependent on its host, Ignicoccus hospitalis. This suggests that this ATP synthase might be a rudimentary machine. Here, we report the crystal structures and biophysical studies of the regulatory subunit, NeqB, the apo-NeqAB, and NeqAB in complex with nucleotides, ADP, and adenylyl-imidodiphosphate (non-hydrolysable analog of ATP). NeqB is ∼20 amino acids shorter at its C terminus than its homologs, but this does not impede its binding with NeqA to form the complex. The heterodimeric NeqAB complex assumes a closed, rigid conformation irrespective of nucleotide binding; this differs from its homologs, which require conformational changes for catalytic activity. Thus, although N. equitans possesses an ATP synthase core A3B3 hexameric complex, it might not function as a bona fide ATP synthase. PMID:26370083

  19. Activity-driven local ATP synthesis is required for synaptic function

    PubMed Central

    Rangaraju, Vidhya; Calloway, Nathaniel; Ryan, Timothy A.

    2014-01-01

    Summary Cognitive function is tightly related to metabolic state but the locus of this control is not well understood. Synapses are thought to present large ATP demands however it is unclear how fuel availability and electrical activity impact synaptic ATP levels, and how ATP availability controls synaptic function. We developed a quantitative genetically-encoded optical reporter of presynaptic ATP, Syn-ATP, and find that electrical activity imposes large metabolic demands that are met via activity-driven control of both glycolysis and mitochondrial function. We discovered that the primary source of activity-driven metabolic demand is the synaptic vesicle cycle. In metabolically intact synapses, activity-driven ATP synthesis is well matched to the energetic needs of synaptic function which at steady state results in ~ 106 free ATPs per nerve terminal. Despite this large reservoir of ATP we find that several key aspects of presynaptic function are severely impaired following even brief interruptions in activity-stimulated ATP synthesis. PMID:24529383

  20. Metallothionein prevents cardiac pathological changes in diabetes by modulating nitration and inactivation of cardiac ATP synthase.

    PubMed

    Cong, Weitao; Zhao, Ting; Zhu, Zhongxin; Huang, Binbin; Ma, Weide; Wang, Yuehui; Tan, Yi; Chakrabarti, Subrata; Li, Xiaokun; Jin, Litai; Cai, Lu

    2014-04-01

    Mitochondrial ATP production is the main energy source for the cell. Diabetes reduces the efficient generation of ATP, possibly due to the inactivation of ATP synthase. However, the exact mechanism by which diabetes induces inactivation of ATP synthase remains unknown, as well as whether such inactivation has a role in the development of pathological abnormalities of the diabetic heart. To address these issues, we used cardiac metallothionein-transgenic (MT-TG) and wild-type (WT) mice with streptozotocin-induced diabetes, since we have demonstrated previously that diabetes-induced cardiac damage and remodeling were found in WT diabetic mice, but not in MT-TG diabetic mice. Immunohistochemical and biochemical assays were used to compare pathological and biochemical changes of the heart between MT-TG and WT diabetic mice, and a proteomic assay to evaluate ATP synthase expression and tyrosine nitration, with its activity. LC/MS analysis revealed that diabetes increased tyrosine nitration of the ATP synthase α subunit at Tyr(271), Tyr(311), and Tyr(476), and the β subunit at Tyr(269) and Tyr(508), and also significantly reduced ATP synthase activity by ~32%. These changes were not observed in MT-TG diabetic mice. Furthermore, parallel experiments with induced expression of cardiac MT by zinc supplementation in diabetic mice produced similar effects. These results suggest that MT can preserve ATP synthase activity in streptozotocin-induced diabetes, probably through the inhibition of ATP synthase nitration. PMID:24629910

  1. Multiscale approach to link red blood cell dynamics, shear viscosity, and ATP release

    PubMed Central

    Forsyth, Alison M.; Wan, Jiandi; Owrutsky, Philip D.; Abkarian, Manouk; Stone, Howard A.

    2011-01-01

    RBCs are known to release ATP, which acts as a signaling molecule to cause dilation of blood vessels. A reduction in the release of ATP from RBCs has been linked to diseases such as type II diabetes and cystic fibrosis. Furthermore, reduced deformation of RBCs has been correlated with myocardial infarction and coronary heart disease. Because ATP release has been linked to cell deformation, we undertook a multiscale approach to understand the links between single RBC dynamics, ATP release, and macroscopic viscosity all at physiological shear rates. Our experimental approach included microfluidics, ATP measurements using a bioluminescent reaction, and rheology. Using microfluidics technology with high-speed imaging, we visualize the deformation and dynamics of single cells, which are known to undergo motions such as tumbling, swinging, tanktreading, and deformation. We report that shear thinning is not due to cellular deformation as previously believed, but rather it is due to the tumbling-to-tanktreading transition. In addition, our results indicate that ATP release is constant at shear stresses below a threshold (3 Pa), whereas above the threshold ATP release is increased and accompanied by large cellular deformations. Finally, performing experiments with well-known inhibitors, we show that the Pannexin 1 hemichannel is the main avenue for ATP release both above and below the threshold, whereas, the cystic fibrosis transmembrane conductance regulator only contributes to deformation-dependent ATP release above the stress threshold. PMID:21690355

  2. Inhibition of ATP release from Erythrocytes: A role for EPACs and PKC

    PubMed Central

    Adderley, Shaquria P.; Sridharan, Meera; Bowles, Elizabeth A.; Stephenson, Alan H.; Sprague, Randy S.; Ellsworth, Mary L.

    2010-01-01

    Objective Here we demonstrate that, in human erythrocytes, increases in cAMP that are not localized to a specific receptor-mediated signaling pathway for ATP release can activate effector proteins resulting in inhibition of ATP release. Specifically we sought to establish that exchange proteins activated by cAMP (EPACs) inhibit ATP release via activation of protein kinase C (PKC). Methods ATP release stimulated by iloprost (ILO), or isoproterenol (ISO), was determined in the absence and presence of selective phosphodiesterase inhibitors and/or the EPAC activator, 8CPT2OMecAMP (8CPT). To determine whether EPACs inhibit ATP release via activation of PKC, erythrocytes were incubated with phorbol 12-myristate 13-acetate (PMA) prior to either forskolin or ILO in the absence and presence of a PKC inhibitor, calphostin C (CALC). Results Selective inhibition of PDEs in one pathway inhibited ATP release in response to activation of the other cAMP-dependent pathway. 8CPT and PMA inhibited both ILO- and ISO-induced ATP release. Inhibition of ATP release with 8CPT was rescued by CALC. Conclusion These results support the hypothesis that cAMP not localized to a specific signaling pathway can activate EPACs which inhibit ATP release via activation of PKC and suggest a novel role for EPACs in erythrocytes. PMID:21166931

  3. Helical arrays of U-shaped ATP synthase dimers form tubular cristae in ciliate mitochondria

    PubMed Central

    Mühleip, Alexander W.; Joos, Friederike; Wigge, Christoph; Frangakis, Achilleas S.; Kühlbrandt, Werner; Davies, Karen M.

    2016-01-01

    F1Fo-ATP synthases are universal energy-converting membrane protein complexes that synthesize ATP from ADP and inorganic phosphate. In mitochondria of yeast and mammals, the ATP synthase forms V-shaped dimers, which assemble into rows along the highly curved ridges of lamellar cristae. Using electron cryotomography and subtomogram averaging, we have determined the in situ structure and organization of the mitochondrial ATP synthase dimer of the ciliate Paramecium tetraurelia. The ATP synthase forms U-shaped dimers with parallel monomers. Each complex has a prominent intracrista domain, which links the c-ring of one monomer to the peripheral stalk of the other. Close interaction of intracrista domains in adjacent dimers results in the formation of helical ATP synthase dimer arrays, which differ from the loose dimer rows in all other organisms observed so far. The parameters of the helical arrays match those of the cristae tubes, suggesting the unique features of the P. tetraurelia ATP synthase are directly responsible for generating the helical tubular cristae. We conclude that despite major structural differences between ATP synthase dimers of ciliates and other eukaryotes, the formation of ATP synthase dimer rows is a universal feature of mitochondria and a fundamental determinant of cristae morphology. PMID:27402755

  4. Helical arrays of U-shaped ATP synthase dimers form tubular cristae in ciliate mitochondria.

    PubMed

    Mühleip, Alexander W; Joos, Friederike; Wigge, Christoph; Frangakis, Achilleas S; Kühlbrandt, Werner; Davies, Karen M

    2016-07-26

    F1Fo-ATP synthases are universal energy-converting membrane protein complexes that synthesize ATP from ADP and inorganic phosphate. In mitochondria of yeast and mammals, the ATP synthase forms V-shaped dimers, which assemble into rows along the highly curved ridges of lamellar cristae. Using electron cryotomography and subtomogram averaging, we have determined the in situ structure and organization of the mitochondrial ATP synthase dimer of the ciliate Paramecium tetraurelia. The ATP synthase forms U-shaped dimers with parallel monomers. Each complex has a prominent intracrista domain, which links the c-ring of one monomer to the peripheral stalk of the other. Close interaction of intracrista domains in adjacent dimers results in the formation of helical ATP synthase dimer arrays, which differ from the loose dimer rows in all other organisms observed so far. The parameters of the helical arrays match those of the cristae tubes, suggesting the unique features of the P. tetraurelia ATP synthase are directly responsible for generating the helical tubular cristae. We conclude that despite major structural differences between ATP synthase dimers of ciliates and other eukaryotes, the formation of ATP synthase dimer rows is a universal feature of mitochondria and a fundamental determinant of cristae morphology. PMID:27402755

  5. Mechanisms of ATP release and signalling in the blood vessel wall

    PubMed Central

    Lohman, Alexander W.; Billaud, Marie; Isakson, Brant E.

    2012-01-01

    The nucleotide adenosine 5′-triphosphate (ATP) has classically been considered the cell's primary energy currency. Importantly, a novel role for ATP as an extracellular autocrine and/or paracrine signalling molecule has evolved over the past century and extensive work has been conducted to characterize the ATP-sensitive purinergic receptors expressed on almost all cell types in the body. Extracellular ATP elicits potent effects on vascular cells to regulate blood vessel tone but can also be involved in vascular pathologies such as atherosclerosis. While the effects of purinergic signalling in the vasculature have been well documented, the mechanism(s) mediating the regulated release of ATP from cells in the blood vessel wall and circulation are now a key target of investigation. The aim of this review is to examine the current proposed mechanisms of ATP release from vascular cells, with a special emphasis on the transporters and channels involved in ATP release from vascular smooth muscle cells, endothelial cells, circulating red blood cells, and perivascular sympathetic nerves, including vesicular exocytosis, plasma membrane F1/F0-ATP synthase, ATP-binding cassette (ABC) transporters, connexin hemichannels, and pannexin channels. PMID:22678409

  6. Intracellular ATP levels are a pivotal determinant of chemoresistance in colon cancer cells.

    PubMed

    Zhou, Yunfei; Tozzi, Federico; Chen, Jinyu; Fan, Fan; Xia, Ling; Wang, Jinrong; Gao, Guang; Zhang, Aijun; Xia, Xuefeng; Brasher, Heather; Widger, William; Ellis, Lee M; Weihua, Zhang

    2012-01-01

    Altered metabolism in cancer cells is suspected to contribute to chemoresistance, but the precise mechanisms are unclear. Here, we show that intracellular ATP levels are a core determinant in the development of acquired cross-drug resistance of human colon cancer cells that harbor different genetic backgrounds. Drug-resistant cells were characterized by defective mitochondrial ATP production, elevated aerobic glycolysis, higher absolute levels of intracellular ATP, and enhanced HIF-1α-mediated signaling. Interestingly, direct delivery of ATP into cross-chemoresistant cells destabilized HIF-1α and inhibited glycolysis. Thus, drug-resistant cells exhibit a greater "ATP debt" defined as the extra amount of ATP needed to maintain homeostasis of survival pathways under genotoxic stress. Direct delivery of ATP was sufficient to render drug-sensitive cells drug resistant. Conversely, depleting ATP by cell treatment with an inhibitor of glycolysis, 3-bromopyruvate, was sufficient to sensitize cells cross-resistant to multiple chemotherapeutic drugs. In revealing that intracellular ATP levels are a core determinant of chemoresistance in colon cancer cells, our findings may offer a foundation for new improvements to colon cancer treatment. PMID:22084398

  7. Intracellular ATP Levels are a Pivotal Determinant of Chemoresistance in Colon Cancer Cells

    PubMed Central

    Zhou, Yunfei; Tozzi, Federico; Chen, Jinyu; Fan, Fan; Xia, Ling; Wang, Jinrong; Gao, Guang; Zhang, Aijun; Xia, Xuefeng; Brasher, Heather; Widger, William; Ellis, Lee M; Weihua, Zhang

    2013-01-01

    Altered metabolism in cancer cells is suspected to contribute to chemoresistance but the precise mechanisms are unclear. Here we show that intracellular ATP levels are a core determinant in the development of acquired cross-drug resistance of human colon cancer cells that harbor different genetic backgrounds. Drug-resistant cells were characterized by defective mitochondrial ATP production, elevated aerobic glycolysis, higher absolute levels of intracellular ATP and enhanced HIF-1α-mediated signaling. Interestingly, direct delivery of ATP into cross-chemoresistant cells destabilized HIF-1α and inhibited glycolysis. Thus, drug-resistant cells exhibit a greater “ATP debt” defined as the extra amount of ATP needed to maintain homeostasis of survival pathways under genotoxic stress. Direct delivery of ATP was sufficient to render drug-sensitive cells drug resistant. Conversely, depleting ATP by cell treatment with an inhibitor of glycolysis, 3-bromopyruvate, was sufficient to sensitize cells cross-resistant to multiple chemotherapeutic drugs. In revealing intracellular ATP levels are a core determinant of chemoresistance in colon cancer cells, our findings may offer a foundation for new improvements to colon cancer treatment. PMID:22084398

  8. A sodium-stimulated ATP synthase in the acetogenic bacterium Acetobacterium woodii.

    PubMed

    Heise, R; Reidlinger, J; Müller, V; Gottschalk, G

    1991-12-16

    Experiments with resting cells of Acetobacterium woodii were performed to elucidate the coupling ion used by the ATP synthase. A. woodii synthesized ATP in response to an artificial delta pH, indicating the presence of a proton-translocating ATPase. On the other hand, a delta pNa, as well as a proton diffusion potential, could serve as a driving force for ATP synthesis with the latter strictly dependent on Na+. These results are indicative for the presence of a Na(+)-translocating ATP synthase in A. woodii. PMID:1837273

  9. ATP Dependence of the ICl, swell Channel Varies with Rate of Cell Swelling

    PubMed Central

    Bond, Tamara; Basavappa, Srisaila; Christensen, Michael; Strange, Kevin

    1999-01-01

    Swelling-induced activation of the outwardly rectifying anion current, ICl, swell, is modulated by intracellular ATP. The mechanisms by which ATP controls channel activation, however, are unknown. Whole cell patch clamp was employed to begin addressing this issue. Endogenous ATP production was inhibited by dialyzing N1E115 neuroblastoma cells for 4–5 min with solutions containing (μM): 40 oligomycin, 5 iodoacetate, and 20 rotenone. The effect of ATP on current activation was observed in the absence of intracellular Mg2+, in cells exposed to extracellular metabolic inhibitors for 25–35 min followed by intracellular dialysis with oligomycin, iodoacetate, and rotenone, after substitution of ATP with the nonhydrolyzable analogue AMP-PNP, and in the presence of AMP-PNP and alkaline phosphatase to dephosphorylate intracellular proteins. These results demonstrate that the ATP dependence of the channel requires ATP binding rather than hydrolysis and/or phosphorylation reactions. When cells were swollen at 15–55%/min in the absence of intracellular ATP, current activation was slow (0.3–0.8 pA/pF per min). ATP concentration increased the rate of current activation up to maximal values of 4–6 pA/pF per min, but had no effect on the sensitivity of the channel to cell swelling. Rate of current activation was a saturable, hyperbolic function of ATP concentration. The EC50 for ATP varied inversely with the rate of cell swelling. Activation of current was rapid (4–6 pA/pF per min) in the absence of ATP when cells were swollen at rates ≥65%/min. Intracellular ATP concentration had no effect on current activation induced by high rates of swelling. Current activation was transient when endogenous ATP was dialyzed out of the cytoplasm of cells swollen at 15%/min. Rundown of the current was reversed by increasing the rate of swelling to 65%/min. These results indicate that the channel and/or associated regulatory proteins are capable of sensing the rate of cell volume

  10. Purification and characterization of pyrophosphate- and ATP-dependent phosphofructokinases from banana fruit.

    PubMed

    Turner, William L; Plaxton, William C

    2003-05-01

    Pyrophosphate-dependent phosphofructokinase (PFP; EC 2.7.1.90) and two isoforms of ATP-dependent phosphofructokinase (PFK I and PFK II; EC 2.7.1.11) from ripened banana ( Musa cavendishii L. cv. Cavendish) fruits were resolved via hydrophobic interaction fast protein liquid chromatography (FPLC), and further purified using anion-exchange and gel filtration FPLC. PFP was purified 1,158-fold to a final specific activity of 13.9 micromol fructose 1,6-bisphosphate produced (mg protein)(-1) x min(-1). Gel filtration FPLC and immunoblot analyses indicated that this PFP exists as a 490-kDa heterooctomer composed of equal amounts of 66- (alpha) and 60-kDa (beta) subunits. PFP displayed hyperbolic saturation kinetics for fructose 6-phosphate (Fru 6-P), PPi, fructose 1,6-bisphosphate, and Pi ( K(m) values = 32, 9.7, 25, and 410 microM, respectively) in the presence of saturating (5 microM) fructose 2,6-bisphosphate, which elicited a 24-fold enhancement of glycolytic PFP activity ( K(a)=8 nM). PFK I and PFK II were each purified about 350-fold to final specific activities of 5.5-6.0 micromol fructose 1,6-bisphosphate produced (mg protein)(-1) x min(-1). Analytical gel filtration yielded respective native molecular masses of 210 and 160 kDa for PFK I and PFK II. Several properties of PFK I and PFK II were consistent with their respective designation as plastid and cytosolic PFK isozymes. PFK I and PFK II exhibited: (i) pH optima of 8.0 and 7.3, respectively; (ii) hyperbolic saturation kinetics for ATP ( K(m)=34 and 21 microM, respectively); and (iii) sigmoidal saturation kinetics for Fru 6-P ( S0.5=540 and 90 microM, respectively). Allosteric effects of phospho enolpyruvate (PEP) and Pi on the activities of PFP, PFK I, and PFK II were characterized. Increasing concentrations of PEP or Pi progressively disrupted fructose 2,6-bisphosphate binding by PFP. PEP potently inhibited PFK I and to a lesser extent PFK II ( I50=2.3 and 900 microM, respectively), while Pi activated PFK I

  11. Two ATP Binding Cassette G Transporters, Rice ATP Binding Cassette G26 and ATP Binding Cassette G15, Collaboratively Regulate Rice Male Reproduction1[OPEN

    PubMed Central

    Zhao, Guochao; Shi, Jianxin; Liang, Wanqi; Xue, Feiyang; Luo, Qian; Zhu, Lu; Qu, Guorun; Chen, Mingjiao; Schreiber, Lukas; Zhang, Dabing

    2015-01-01

    Male reproduction in higher plants requires the support of various metabolites, including lipid molecules produced in the innermost anther wall layer (the tapetum), but how the molecules are allocated among different anther tissues remains largely unknown. Previously, rice (Oryza sativa) ATP binding cassette G15 (ABCG15) and its Arabidopsis (Arabidopsis thaliana) ortholog were shown to be required for pollen exine formation. Here, we report the significant role of OsABCG26 in regulating the development of anther cuticle and pollen exine together with OsABCG15 in rice. Cytological and chemical analyses indicate that osabcg26 shows reduced transport of lipidic molecules from tapetal cells for anther cuticle development. Supportively, the localization of OsABCG26 is on the plasma membrane of the anther wall layers. By contrast, OsABCG15 is polarly localized in tapetal plasma membrane facing anther locules. osabcg26 osabcg15 double mutant displays an almost complete absence of anther cuticle and pollen exine, similar to that of osabcg15 single mutant. Taken together, we propose that OsABCG26 and OsABCG15 collaboratively regulate rice male reproduction: OsABCG26 is mainly responsible for the transport of lipidic molecules from tapetal cells to anther wall layers, whereas OsABCG15 mainly is responsible for the export of lipidic molecules from the tapetal cells to anther locules for pollen exine development. PMID:26392263

  12. Functional redundancy between DNA ligases I and III in DNA replication in vertebrate cells

    PubMed Central

    Arakawa, Hiroshi; Bednar, Theresa; Wang, Minli; Paul, Katja; Mladenov, Emil; Bencsik-Theilen, Alena A.; Iliakis, George

    2012-01-01

    In eukaryotes, the three families of ATP-dependent DNA ligases are associated with specific functions in DNA metabolism. DNA ligase I (LigI) catalyzes Okazaki-fragment ligation at the replication fork and nucleotide excision repair (NER). DNA ligase IV (LigIV) mediates repair of DNA double strand breaks (DSB) via the canonical non-homologous end-joining (NHEJ) pathway. The evolutionary younger DNA ligase III (LigIII) is restricted to higher eukaryotes and has been associated with base excision (BER) and single strand break repair (SSBR). Here, using conditional knockout strategies for LIG3 and concomitant inactivation of the LIG1 and LIG4 genes, we show that in DT40 cells LigIII efficiently supports semi-conservative DNA replication. Our observations demonstrate a high functional versatility for the evolutionary new LigIII in DNA replication and mitochondrial metabolism, and suggest the presence of an alternative pathway for Okazaki fragment ligation. PMID:22127868

  13. Fusion Power Demonstration III

    SciTech Connect

    Lee, J.D.

    1985-07-01

    This is the third in the series of reports covering the Fusion Power Demonstration (FPD) design study. This volume considers the FPD-III configuration that incorporates an octopole end plug. As compared with the quadrupole end-plugged designs of FPD-I and FPD-II, this octopole configuration reduces the number of end cell magnets and shortens the minimum ignition length of the central cell. The end-cell plasma length is also reduced, which in turn reduces the size and cost of the end cell magnets and shielding. As a contiuation in the series of documents covering the FPD, this report does not stand alone as a design description of FPD-III. Design details of FPD-III subsystems that do not differ significantly from those of the FPD-II configuration are not duplicated in this report.

  14. Vacuolar ATPases, like F1,F0-ATPases, show a strong dependence of the reaction velocity on the binding of more than one ATP per enzyme.

    PubMed Central

    Kasho, V N; Boyer, P D

    1989-01-01

    Recent studies with vacuolar ATPases have shown that multiple copies catalytic subunits are present and that these have definite sequence homology with catalytic subunits of the F1,F0-ATPases. Experiments are reported that assess whether the vacuolar ATPases may have the unusual catalytic cooperativity with sequential catalytic site participation as in the binding change mechanism for the F1,F0-ATPases. The extent of reversal of bound ATP hydrolysis to bound ADP and Pi as medium ATP concentration was lowered was determined by 18O-exchange measurements for yeast and neurospora vacuolar ATPases. The results show a pronounced increase in the extent of water oxygen incorporation into the Pi formed as ATP concentration is decreased to the micromolar range. The F1,F0-ATPase from neurospora mitochondria showed an even more pronounced modulation, similar to that of other F1-type ATPases. The vacuolar ATPases thus appear to have a catalytic mechanism quite analogous to that of the F1,F0-ATPases. PMID:2530585

  15. Pioneer III Probe

    NASA Technical Reports Server (NTRS)

    1961-01-01

    Looking more like surgeons, these technicians wearing 'cleanroom' attire inspect the Pioneer III probe before shipping it to Cape Canaveral, Florida. Pioneer III was launched on December 6, 1958 aboard a Juno II rocket at the Atlantic Missile Range, Cape Canaveral, Florida. The mission objectives were to measure the radiation intensity of the Van Allen radiation belt, test long range communication systems, the launch vehicle and other subsystems. The Juno II failed to reach proper orbital escape velocity. The probe re-entered the Earth's atmosphere on December 7th ending its brief mission.

  16. Type III restriction-modification enzymes: a historical perspective

    PubMed Central

    Rao, Desirazu N.; Dryden, David T. F.; Bheemanaik, Shivakumara

    2014-01-01

    Restriction endonucleases interact with DNA at specific sites leading to cleavage of DNA. Bacterial DNA is protected from restriction endonuclease cleavage by modifying the DNA using a DNA methyltransferase. Based on their molecular structure, sequence recognition, cleavage position and cofactor requirements, restriction–modification (R–M) systems are classified into four groups. Type III R–M enzymes need to interact with two separate unmethylated DNA sequences in inversely repeated head-to-head orientations for efficient cleavage to occur at a defined location (25–27 bp downstream of one of the recognition sites). Like the Type I R–M enzymes, Type III R–M enzymes possess a sequence-specific ATPase activity for DNA cleavage. ATP hydrolysis is required for the long-distance communication between the sites before cleavage. Different models, based on 1D diffusion and/or 3D-DNA looping, exist to explain how the long-distance interaction between the two recognition sites takes place. Type III R–M systems are found in most sequenced bacteria. Genome sequencing of many pathogenic bacteria also shows the presence of a number of phase-variable Type III R–M systems, which play a role in virulence. A growing number of these enzymes are being subjected to biochemical and genetic studies, which, when combined with ongoing structural analyses, promise to provide details for mechanisms of DNA recognition and catalysis. PMID:23863841

  17. ATP-dependent chromatin remodeling in T cells

    PubMed Central

    Wurster, Andrea L.; Pazin, Michael J.

    2012-01-01

    One of the best studied systems for mammalian chromatin remodeling is transcriptional regulation during T cell development. The variety of these studies have led to important findings in T cell gene regulation and cell fate determination. Importantly, these findings have also advanced our knowledge of the function of remodeling enzymes in mammalian gene regulation. In this review, first we briefly present biochemical/cell-free analysis of 3 types of ATP dependent remodeling enzymes (SWI/SNF, Mi2, and ISWI), to construct an intellectual framework to understand how these enzymes might be working. Second, we compare and contrast the function of these enzymes, during early (thymic) and late (peripheral) T cell development. Finally, we examine some of the gaps in our present understanding. PMID:21999456

  18. Resonance phenomenon of the ATP motor as an ultrasensitive biosensor.

    PubMed

    Wang, Peirong; Zhang, Xiaoguang; Zhang, Xu; Wang, Xia; Li, Xueren; Yue, Jiachang

    2012-09-28

    We designed a rotary biosensor as a damping effector, with the rotation of the F(0)F(1)-ATPase driven by Adenosine Triphosphate (ATP) synthesis being indicated by the fluorescence intensity and a damping effect force being induced by the binding of an RNA molecule to its probe on the rotary biosensor. We found that the damping effect could contribute to the resonance phenomenon and energy transfer process of our rotary biosensor in the liquid phase. This result indicates that the ability of the rotary motor to operate in the vibration harmonic mode depends on the environmental conditions and mechanism in that a few molecules of the rotary biosensor could induce all of the sensor molecules to fluoresce together. These findings contribute to the theory study of the ATPase motor and future development of biosensors for ultrasensitive detection. PMID:22960174

  19. ATP binding cassette G transporters and plant male reproduction.

    PubMed

    Zhao, Guochao; Shi, Jianxin; Liang, Wanqi; Zhang, Dabing

    2016-01-01

    The function of ATP Binding Cassette G (ABCG) transporters in the regulation of plant vegetative organs development has been well characterized in various plant species. In contrast, their function in reproductive development particularly male reproductive development received considerably less attention till some ABCG transporters was reported to be associated with anther and pollen wall development in Arabidopsis thaliana and rice (Oryza sativa) during the past decade. This mini-review summarizes current knowledge of ABCG transporters regarding to their roles in male reproduction and underlying genetic and biochemical mechanisms, which makes it evident that ABCG transporters represent one of those conserved and divergent components closely related to male reproduction in plants. This mini-review also discusses the current challenges and future perspectives in this particular field. PMID:26906115

  20. NASA ATP Force Measurement Technology Capability Strategic Plan

    NASA Technical Reports Server (NTRS)

    Rhew, Ray D.

    2008-01-01

    The Aeronautics Test Program (ATP) within the National Aeronautics and Space Administration (NASA) Aeronautics Research Mission Directorate (ARMD) initiated a strategic planning effort to re-vitalize the force measurement capability within NASA. The team responsible for developing the plan included members from three NASA Centers (Langley, Ames and Glenn) as well as members from the Air Force s Arnold Engineering and Development Center (AEDC). After visiting and discussing force measurement needs and current capabilities at each participating facility as well as selected force measurement companies, a strategic plan was developed to guide future NASA investments. This paper will provide the details of the strategic plan and include asset management, organization and technology research and development investment priorities as well as efforts to date.

  1. Functional domains of an ATP-dependent DNA ligase.

    PubMed

    Doherty, A J; Wigley, D B

    1999-01-01

    The crystal structure of an ATP-dependent DNA ligase from bacteriophage T7 revealed that the protein comprised two structural domains. In order to investigate the biochemical activities of these domains, we have overexpressed them separately and purified them to homogeneity. The larger N-terminal domain retains adenylation and ligase activities, though both at a reduced level. The adenylation activity of the large domain is stimulated by the presence of the smaller domain, suggesting that a conformational change is required for adenylation in the full length protein. The DNA binding properties of the two fragments have also been studied. The larger domain is able to band shift both single and double-stranded DNA, while the smaller fragment is only able to bind to double-stranded DNA. These data suggest that the specificity of DNA ligases for nick sites in DNA is produced by a combination of these different DNA binding activities in the intact enzyme. PMID:9878388

  2. Invited review: Architectures and mechanisms of ATP binding cassette proteins.

    PubMed

    Hopfner, Karl-Peter

    2016-08-01

    ATP binding cassette (ABC) ATPases form chemo-mechanical engines and switches that function in a broad range of biological processes. Most prominently, a very large family of integral membrane NTPases-ABC transporters-catalyzes the import or export of a diverse molecules across membranes. ABC proteins are also important components of the chromosome segregation, recombination, and DNA repair machineries and regulate or catalyze critical steps of ribosomal protein synthesis. Recent structural and mechanistic studies draw interesting architectural and mechanistic parallels between diverse ABC proteins. Here, I review this state of our understanding how NTP-dependent conformational changes of ABC proteins drive diverse biological processes. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 492-504, 2016. PMID:27037766

  3. Pyridoxal 5'-phosphate is an ATP-receptor antagonist in freshly isolated rat cardiomyocytes.

    PubMed

    Wang, X; Dakshinamurti, K; Musat, S; Dhalla, N S

    1999-05-01

    Although extracellular ATP is considered to exert a positive inotropic action on the myocardium through purinoceptors, very little information is available regarding interventions which may modify the actions of ATP on the heart. We report here that pyridoxal 5'-phosphate (PLP), an active form of vitamin B6, shows antagonism towards ATP-induced positive inotropic effect in isolated perfused rat hearts, ATP-induced increase in [Ca2+] in freshly isolated adult cardiomyocytes and ATP-binding in cardiac sarcolemma; ED50 for PLP in each of these cases varied from 10-15 microM. PLP (5-50 microM) was observed to antagonize the positive inotropic effect of ATP but did not modify the action of isoproterenol in the isolated perfused heart. Preincubation of cardiomyocytes with 1-50 microM PLP prevented the ATP-induced increase in [Ca2+]i in a concentration-dependent manner but showed no effect on the KCl-induced increase in [Ca2+]i. Creatine phosphate and Na2HPO4 as well as vitamin B6-related compounds, such as pyridoxine, pyridoxal, 4-deoxypyridoxine and isonicotinic acid hydrazide showed no effect on the ATP-induced increase in [Ca2+]i in cardiomyocytes. Furthermore, different concentrations of PLP (1-50 microM) were shown to inhibit the specific ATP gamma S binding at both the high and low affinity sites in the cardiac sarcolemmal membrane; adrenoceptor and Ca2+-channel inhibitors did not affect the ATP-binding. It is concluded that PLP may antagonize the actions of ATP on the heart in a selective manner and both pyridoxal and phosphate moieties are essential for its action. Furthermore, it is suggested that PLP may serve as a valuable tool for monitoring the role of purinoceptors in cellular function. PMID:10336844

  4. Ca2+ responses to ATP via purinoceptors in the early embryonic chick retina.

    PubMed Central

    Sugioka, M; Fukuda, Y; Yamashita, M

    1996-01-01

    1. The action of adenosine triphosphate on cytoplasmic Ca2+ concentration ([Ca2+]i) was studied in the retinal cell of early embryonic chicks with fura-2 fluorescence measurements. The fluorescence was measured from the whole neural retina dissected from chick embryos at embryonic day three (E3). 2. Bath application of ATP (> or = 30 microM; EC50, 128 microM) raised [Ca2+]i by the release of Ca2+ from intracellular Ca2+ stores, since the Ca2+ response to ATP occurred even in a Ca(2+)-free medium. 3. The Ca2+ response to ATP was mediated by P2U purinoceptors. An agonist for P2U purinoceptors, uridine triphosphate (UTP), evoked Ca2+ rises more potently (> or = 3 microM; EC50, 24 microM) than ATP. Agonists for P2X purinoceptors, alpha, beta-methylene ATP and beta, gamma-methylene ATP, or an agonist for P2Y purinoceptors, 2-methylthio ATP (500 microM each), caused no Ca2+ response. Suramin (100 microM) and Reactive Blue 2 (50 microM) almost completely blocked the Ca2+, responses to 500 microM ATP and 200 microM UTP. 4. The developmental profile of the Ca2+ response to ATP was studied from E3 to E13. The Ca2+ response to ATP was largest at E3, drastically declined towards E8 and decreased further until E11-13. 5. These results suggest that the Ca2+ mobilization by ATP via P2U purinoceptors is characteristic of early embryonic retinal cells. PMID:8799905

  5. ATP secretion in the male reproductive tract: essential role of CFTR.

    PubMed

    Ruan, Ye Chun; Shum, Winnie W C; Belleannée, Clémence; Da Silva, Nicolas; Breton, Sylvie

    2012-09-01

    Extracellular ATP is essential for the function of the epididymis and spermatozoa, but ATP release in the epididymis remains uncharacterized. We investigated here whether epithelial cells release ATP into the lumen of the epididymis, and we examined the role of the cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-) and HCO(3)(-) conducting ion channel known to be associated with male fertility, in this process. Immunofluorescence labelling of mouse cauda epididymidis showed expression of CFTR in principal cells but not in other epithelial cells. CFTR mRNA was not detectable in clear cells isolated by fluorescence-activated cell sorting (FACS) from B1-EGFP mice, which express enhanced green fluorescent protein (EGFP) exclusively in these cells in the epididymis. ATP release was detected from the mouse epididymal principal cell line (DC2) and increased by adrenaline and forskolin. Inhibition of CFTR with CFTR(inh172) and transfection with CFTR-specific siRNAs in DC2 cells reduced basal and forskolin-activated ATP release. CFTR-dependent ATP release was also observed in primary cultures of mouse epididymal epithelial cells. In addition, steady-state ATP release was detected in vivo in mice, by measuring ATP concentration in a solution perfused through the lumen of the cauda epididymidis tubule and collected by cannulation of the vas deferens. Luminal CFTR(inh172) reduced the ATP concentration detected in the perfusate. This study shows that CFTR is involved in the regulation of ATP release from principal cells in the cauda epididymidis. Given that mutations in CFTR are a leading cause of male infertility, we propose that defective ATP signalling in the epididymis might contribute to dysfunction of the male reproductive tract associated with these mutations. PMID:22711960

  6. Activation by ATP of a P2U 'nucleotide' receptor in an exocrine cell.

    PubMed Central

    Martin, S. C.; Shuttleworth, T. J.

    1995-01-01

    1. We employed the perforated patch whole-cell technique to investigate the effects of ATP and other related nucleotides on membrane conductances in avian exocrine salt gland cells. 2. ATP (10 microM-1 mM) evoked an increase in maxi-K+ and Cl- conductances with a reversal potential of -35 mV. At lower concentrations of ATP (< or = 100 microM) responses were generally oscillatory with a sustained response observed at higher concentrations (> or = 200 microM). 3. Both oscillatory and sustained responses were abolished by the removal of bath Ca2+. In cells preincubated in extracellular saline containing reduced Ca2+, the application of ATP resulted in a transient increase in current. 4. As increasing concentrations of ATP (and related nucleotides) evoked a graded sequence of events with little run-down we were able to establish a rank order of potency in single cells. The order of potency of ATP analogues and agonists of the various P2-receptor subtypes was UTP > ATP = 2-methylthio-ATP > ADP. Adenosine (1 microM-1 mM), AMP (1 microM-1 mM), alpha,beta-methylene-ATP (1 microM-1 mM) and beta,gamma-methylene-ATP (1 microM-1 mM) were without effect. 5. In conclusion, although unable to preclude a role for a P2Y-receptor, our results suggest that ATP binds to a P2U-receptor increasing [Ca2+]i and subsequently activating Ca(2+)-sensitive K+ and Cl- currents. PMID:7670734

  7. Effect of ATP concentration on CFTR Cl- channels: a kinetic analysis of channel regulation.

    PubMed Central

    Winter, M C; Sheppard, D N; Carson, M R; Welsh, M J

    1994-01-01

    Phosphorylated cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels require nucleoside triphosphates, such as ATP, to open. As the concentration of intracellular ATP increases, the probability of the channel being open (Po) increases. To better understand how ATP regulates the channel, we studied excised inside-out membrane patches that contained single, phosphorylated CFTR Cl- channels and examined the kinetics of gating at different concentrations of ATP. As the ATP concentration increased from 0.1 to 3 mM the mean closed time decreased, but mean open time did not change. Analysis of the data using histograms of open- and closed-state durations, the maximum likelihood method, and the log-likelihood ratio test suggested that channel behavior could be described by a model containing one open and two closed states (C1<==>C2<==>O). ATP regulated phosphorylated channels at the transition between the closed states C1 and C2: as the concentration of ATP increased, the rate of transition from C1 to C2 (C1-->C2) increased. In contrast, transitions from C2 to C1 and between C2 and the open state (O) were not significantly altered by ATP. Addition of ADP in the presence of ATP decreased the transition rate from C1 to C2 without affecting other transition rates. These data suggest that ATP regulates CFTR Cl- channels through an interaction that increases the rate of transition from the closed state to a bursting state in which the channel flickers back and forth between an open and a closed state (C2). This transition may reflect ATP binding or perhaps a step subsequent to binding. Images FIGURE 3 FIGURE 5 PMID:7520292

  8. The Phylogenetic Signature Underlying ATP Synthase c-Ring Compliance.

    PubMed

    Pandini, Alessandro; Kleinjung, Jens; Taylor, Willie R; Junge, Wolfgang; Khan, Shahid

    2015-09-01

    The proton-driven ATP synthase (FOF1) is comprised of two rotary, stepping motors (FO and F1) coupled by an elastic power transmission. The elastic compliance resides in the rotor module that includes the membrane-embedded FO c-ring. Proton transport by FO is firmly coupled to the rotation of the c-ring relative to other FO subunits (ab2). It drives ATP synthesis. We used a computational method to investigate the contribution of the c-ring to the total elastic compliance. We performed principal component analysis of conformational ensembles built using distance constraints from the bovine mitochondrial c-ring x-ray structure. Angular rotary twist, the dominant ring motion, was estimated to show that the c-ring accounted in part for the measured compliance. Ring rotation was entrained to rotation of the external helix within each hairpin-shaped c-subunit in the ring. Ensembles of monomer and dimers extracted from complete c-rings showed that the coupling between collective ring and the individual subunit motions was independent of the size of the c-ring, which varies between organisms. Molecular determinants were identified by covariance analysis of residue coevolution and structural-alphabet-based local dynamics correlations. The residue coevolution gave a readout of subunit architecture. The dynamic couplings revealed that the hinge for both ring and subunit helix rotations was constructed from the proton-binding site and the adjacent glycine motif (IB-GGGG) in the midmembrane plane. IB-GGGG motifs were linked by long-range couplings across the ring, while intrasubunit couplings connected the motif to the conserved cytoplasmic loop and adjacent segments. The correlation with principal collective motions shows that the couplings underlie both ring rotary and bending motions. Noncontact couplings between IB-GGGG motifs matched the coevolution signal as well as contact couplings. The residue coevolution reflects the physiological importance of the dynamics that may

  9. Dual contractile effects of ATP released by field stimulation revealed by effects of alpha,beta-methylene ATP and suramin in rat tail artery.

    PubMed Central

    Bao, J. X.; Stjärne, L.

    1993-01-01

    1. The field stimulation-induced release of endogenous ATP and noradrenaline (NA) and contractile response in rat isolated tail artery were examined. The release of ATP was studied by extracellular electrophysiological recording and that of NA by a novel voltammetrical technique. The effects of the P2-purinceptor antagonist, suramin, on these parameters were compared with those of alpha,beta-methylene ATP, a P2X-purinoceptor desensitizing agent. 2. Neither alpha,beta-methylene ATP (10 microM) nor suramin (100-500 microM) had significant effects on the extracellularly recorded nerve terminal action potential but both abolished the ATP-induced excitatory junction current caused by stimulation at 0.1 Hz. Neither agent affected significantly the voltammetrically measured release of NA induced by 10 or 100 pulses at 20 Hz. 3. Combined blockade of both postjunctional alpha 1- and alpha 2-adrenoceptors by prazosin and yohimbine (both 0.1 microM) profoundly depressed the contractile response to 10 pulses at 20 Hz. The small and fast residual contraction in the presence of these agents was abolished by alpha,beta-methylene ATP (10 microM) and inhibited by suramin in a concentration-dependent manner (10-500 microM; IC50 75 microM) and was hence probably caused by ATP or a related nucleotide. 4. When added first, alpha,beta-methylene ATP (10 microM) or suramin (100-500 microM) delayed the onset and enhanced the amplitude of the neurogenic contraction. This enhanced response was abolished by further addition of prazosin and yohimbine (both 0.1 microM). 5. The K+ channel blocker, tetraethylammonium (10 mM), dramatically enhanced the contractile response to 100 pulses at 1 Hz and caused it to become diphasic.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8306081

  10. Diverse roles of K(ATP) channels learned from Kir6.2 genetically engineered mice.

    PubMed

    Seino, S; Iwanaga, T; Nagashima, K; Miki, T

    2000-03-01

    The regulation of insulin secretion from pancreatic beta-cells depends critically on the activities of their plasma membrane ion channels. ATP-sensitive K+ channels (K(ATP) channels) are present in many cells and regulate a variety of cellular functions by coupling cell metabolism with membrane potential. The activity of the K(ATP) channels in pancreatic beta-cells is regulated by changes in the ATP and ADP concentrations (ATP/ADP ratio) caused by glucose metabolism. Thus, the K(ATP) channels are the ATP and ADP sensors in the regulation of glucose-induced insulin secretion. K(ATP) channels are also the target of sulfonylureas, which are widely used in the treatment of type 2 diabetes. Molecular cloning of the two subunits of the pancreatic beta-cell K(ATP) channel, Kir6.2 (an inward rectifier K+ channel member) and SUR1 (a receptor for sulfonylureas), has provided great insight into its structure and function. Kir6.2 subunits form the K+ ion-permeable pore and primarily confer inhibition of the channels by ATP, while SUR1 subunits confer activation of the channels by MgADP and K+ channel openers, such as diazoxide, as well as inhibition by sulfonylureas. The SUR1 subunits also enhance the sensitivity of the channels to ATP. To determine the physiological roles of K(ATP) channels directly, we have generated two kinds of genetically engineered mice: mice expressing a dominant-negative form of Kir6.2 specifically in the pancreatic beta-cells (Kir6.2G132S Tg mice) and mice lacking Kir6.2 (Kir6.2 knockout mice). Studies of these mice elucidated various roles of the K(ATP) channels in endocrine pancreatic function: 1) the K(ATP) channels are the major determinant of the resting membrane potential of pancreatic beta-cells, 2) both glucose- and sulfonylurea-induced membrane depolarization of beta-cells require closure of the K(ATP) channels, 3) both glucose- and sulfonylurea-induced rises in intracellular calcium concentration in beta-cells require closure of the K(ATP

  11. Summary of Session III

    SciTech Connect

    Furman, M.A.

    2002-06-19

    This is a summary of the talks presented in Session III ''Simulations of Electron-Cloud Build Up'' of the Mini-Workshop on Electron-Cloud Simulations for Proton and Positron Beams ECLOUD-02, held at CERN, 15-18 April 2002.

  12. The Apple III.

    ERIC Educational Resources Information Center

    Ditlea, Steve

    1982-01-01

    Describes and evaluates the features, performance, peripheral devices, available software, and capabilities of the Apple III microcomputer. The computer's operating system, its hardware, and the commercially produced software it accepts are discussed. Specific applications programs for financial planning, accounting, and word processing are…

  13. CITY III Director's Guide.

    ERIC Educational Resources Information Center

    Envirometrics, Inc., Washington, DC.

    CITY III is a computer-assisted simulation game which allows the participants to make decisions affecting various aspects of the economic, governmental, and social sectors of a simulated urban area. The game director selects one of five possible starting city configurations, may set a number of conditions in the city before the start of play, and…

  14. Proposed role of ATP in protein breakdown: conjugation of protein with multiple chains of the polypeptide of ATP-dependent proteolysis.

    PubMed Central

    Hershko, A; Ciechanover, A; Heller, H; Haas, A L; Rose, I A

    1980-01-01

    The heat-stable polypeptide ATP-dependent proteolysis factor 1 (APF-1) of the reticulocyte proteolytic system forms covalent compounds with proteins in an ATP-requiring reaction. APF-1 and lysozyme, a good substrate for ATP-dependent proteolysis, form multiple conjugates, as was shown by comigration of label from each upon gel electrophoresis. Multiple bands were also seen with other substrates of the ATP-dependent proteolytic system, such as globin or alpha-lactalbumin. Analysis of the ratio of APF-1 to lysozyme radioactivities and of the molecular weights of the bands indicated that they consist of increasing numbers of the APF-1 polypeptide bound to one molecule of lysozyme. The covalent linkage is probably of an isopeptide nature, because it is stable to hydroxylamine and alkali, and polylysine is able to give conjugates of APF-1. Removal of ATP after formation of the 125I-labeled APF-1 conjugates with endogenous proteins caused the regeneration of APF-1, indicating presence of an amidase. This reaction is thought to compete with proteases that may act on APF-1-protein conjugates, especially those containing several APF-1 ligands. A sequence of reactions in which the linkage of APF-1 to the substrate is followed by the proteolytic breakdown of the substrate is proposed to explain the role of ATP. Images PMID:6990414

  15. Structural basis of asymmetric DNA methylation and ATP-triggered long-range diffusion by EcoP15I

    PubMed Central

    Gupta, Yogesh K.; Chan, Siu-Hong; Xu, Shuang-yong; Aggarwal, Aneel K.

    2015-01-01

    Type III R–M enzymes were identified >40 years ago and yet there is no structural information on these multisubunit enzymes. Here we report the structure of a Type III R–M system, consisting of the entire EcoP15I complex (Mod2Res1) bound to DNA. The structure suggests how ATP hydrolysis is coupled to long-range diffusion of a helicase on DNA, and how a dimeric methyltransferase functions to methylate only one of the two DNA strands. We show that the EcoP15I motor domains are specifically adapted to bind double-stranded DNA and to facilitate DNA sliding via a novel ‘Pin' domain. We also uncover unexpected ‘division of labour', where one Mod subunit recognizes DNA, while the other Mod subunit methylates the target adenine—a mechanism that may extend to adenine N6 RNA methylation in mammalian cells. Together the structure sheds new light on the mechanisms of both helicases and methyltransferases in DNA and RNA metabolism. PMID:26067164

  16. Structural basis of asymmetric DNA methylation and ATP-triggered long-range diffusion by EcoP15I

    NASA Astrophysics Data System (ADS)

    Gupta, Yogesh K.; Chan, Siu-Hong; Xu, Shuang-Yong; Aggarwal, Aneel K.

    2015-06-01

    Type III R-M enzymes were identified >40 years ago and yet there is no structural information on these multisubunit enzymes. Here we report the structure of a Type III R-M system, consisting of the entire EcoP15I complex (Mod2Res1) bound to DNA. The structure suggests how ATP hydrolysis is coupled to long-range diffusion of a helicase on DNA, and how a dimeric methyltransferase functions to methylate only one of the two DNA strands. We show that the EcoP15I motor domains are specifically adapted to bind double-stranded DNA and to facilitate DNA sliding via a novel `Pin' domain. We also uncover unexpected `division of labour', where one Mod subunit recognizes DNA, while the other Mod subunit methylates the target adenine--a mechanism that may extend to adenine N6 RNA methylation in mammalian cells. Together the structure sheds new light on the mechanisms of both helicases and methyltransferases in DNA and RNA metabolism.

  17. Intracellular ATP Decrease Mediates NLRP3 Inflammasome Activation upon Nigericin and Crystal Stimulation.

    PubMed

    Nomura, Johji; So, Alexander; Tamura, Mizuho; Busso, Nathalie

    2015-12-15

    Activation of the nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome initiates an inflammatory response, which is associated with host defense against pathogens and the progression of chronic inflammatory diseases such as gout and atherosclerosis. The NLRP3 inflammasome mediates caspase-1 activation and subsequent IL-1β processing in response to various stimuli, including extracellular ATP, although the roles of intracellular ATP (iATP) in NLRP3 activation remain unclear. In this study, we found that in activated macrophages artificial reduction of iATP by 2-deoxyglucose, a glycolysis inhibitor, caused mitochondrial membrane depolarization, leading to IL-1β secretion via NLRP3 and caspase-1 activation. Additionally, the NLRP3 activators nigericin and monosodium urate crystals lowered iATP through K(+)- and Ca(2+)-mediated mitochondrial dysfunction, suggesting a feedback loop between iATP loss and lowering of mitochondrial membrane potential. These results demonstrate the fundamental roles of iATP in the maintenance of mitochondrial function and regulation of IL-1β secretion, and they suggest that maintenance of the intracellular ATP pools could be a strategy for countering NLRP3-mediated inflammation. PMID:26546608

  18. Structure and Mechanism of Soybean ATP Sulfurylase and the Committed Step in Plant Sulfur Assimilation*

    PubMed Central

    Herrmann, Jonathan; Ravilious, Geoffrey E.; McKinney, Samuel E.; Westfall, Corey S.; Lee, Soon Goo; Baraniecka, Patrycja; Giovannetti, Marco; Kopriva, Stanislav; Krishnan, Hari B.; Jez, Joseph M.

    2014-01-01

    Enzymes of the sulfur assimilation pathway are potential targets for improving nutrient content and environmental stress responses in plants. The committed step in this pathway is catalyzed by ATP sulfurylase, which synthesizes adenosine 5′-phosphosulfate (APS) from sulfate and ATP. To better understand the molecular basis of this energetically unfavorable reaction, the x-ray crystal structure of ATP sulfurylase isoform 1 from soybean (Glycine max ATP sulfurylase) in complex with APS was determined. This structure revealed several highly conserved substrate-binding motifs in the active site and a distinct dimerization interface compared with other ATP sulfurylases but was similar to mammalian 3′-phosphoadenosine 5′-phosphosulfate synthetase. Steady-state kinetic analysis of 20 G. max ATP sulfurylase point mutants suggests a reaction mechanism in which nucleophilic attack by sulfate on the α-phosphate of ATP involves transition state stabilization by Arg-248, Asn-249, His-255, and Arg-349. The structure and kinetic analysis suggest that ATP sulfurylase overcomes the energetic barrier of APS synthesis by distorting nucleotide structure and identifies critical residues for catalysis. Mutations that alter sulfate assimilation in Arabidopsis were mapped to the structure, which provides a molecular basis for understanding their effects on the sulfur assimilation pathway. PMID:24584934

  19. Structural, Biochemical and Genetic Characterization of Dissimilatory ATP Sulfurylase from Allochromatium vinosum

    PubMed Central

    Parey, Kristian; Demmer, Ulrike; Warkentin, Eberhard; Wynen, Astrid; Ermler, Ulrich; Dahl, Christiane

    2013-01-01

    ATP sulfurylase (ATPS) catalyzes a key reaction in the global sulfur cycle by reversibly converting inorganic sulfate (SO42−) with ATP to adenosine 5′-phosphosulfate (APS) and pyrophosphate (PPi). In this work we report on the sat encoded dissimilatory ATP sulfurylase from the sulfur-oxidizing purple sulfur bacterium Allochromatium vinosum. In this organism, the sat gene is located in one operon and co-transcribed with the aprMBA genes for membrane-bound APS reductase. Like APS reductase, Sat is dispensible for growth on reduced sulfur compounds due to the presence of an alternate, so far unidentified sulfite-oxidizing pathway in A. vinosum. Sulfate assimilation also proceeds independently of Sat by a separate pathway involving a cysDN-encoded assimilatory ATP sulfurylase. We produced the purple bacterial sat-encoded ATP sulfurylase as a recombinant protein in E. coli, determined crucial kinetic parameters and obtained a crystal structure in an open state with a ligand-free active site. By comparison with several known structures of the ATPS-APS complex in the closed state a scenario about substrate-induced conformational changes was worked out. Despite different kinetic properties ATPS involved in sulfur-oxidizing and sulfate-reducing processes are not distinguishable on a structural level presumably due to the interference between functional and evolutionary processes. PMID:24073218

  20. Regulation of calreticulin–major histocompatibility complex (MHC) class I interactions by ATP

    PubMed Central

    Wijeyesakere, Sanjeeva Joseph; Gagnon, Jessica K.; Arora, Karunesh; Brooks, Charles L.; Raghavan, Malini

    2015-01-01

    The MHC class I peptide loading complex (PLC) facilitates the assembly of MHC class I molecules with peptides, but factors that regulate the stability and dynamics of the assembly complex are largely uncharacterized. Based on initial findings that ATP, in addition to MHC class I-specific peptide, is able to induce MHC class I dissociation from the PLC, we investigated the interaction of ATP with the chaperone calreticulin, an endoplasmic reticulum (ER) luminal, calcium-binding component of the PLC that is known to bind ATP. We combined computational and experimental measurements to identify residues within the globular domain of calreticulin, in proximity to the high-affinity calcium-binding site, that are important for high-affinity ATP binding and for ATPase activity. High-affinity calcium binding by calreticulin is required for optimal nucleotide binding, but both ATP and ADP destabilize enthalpy-driven high-affinity calcium binding to calreticulin. ATP also selectively destabilizes the interaction of calreticulin with cellular substrates, including MHC class I molecules. Calreticulin mutants that affect ATP or high-affinity calcium binding display prolonged associations with monoglucosylated forms of cellular MHC class I, delaying MHC class I dissociation from the PLC and their transit through the secretory pathway. These studies reveal central roles for ATP and calcium binding as regulators of calreticulin–substrate interactions and as key determinants of PLC dynamics. PMID:26420867

  1. Effects of Extracellular ATP on Bovine Lung Endothelial and Epithelial Cell Monolayer Morphologies, Apoptoses, and Permeabilities▿

    PubMed Central

    McClenahan, David; Hillenbrand, Kati; Kapur, Arvinder; Carlton, David; Czuprynski, Charles

    2009-01-01

    Pneumonia in cattle is an important disease both economically and in terms of animal welfare. Recent evidence in other species has shown ATP to be an important modulator of inflammation in the lung, where it is released by activated alveolar macrophages and damaged lung cells. Whether ATP serves a similar process during infection in the bovine lung is unknown. In the present study, we examined the effects of ATP treatment on the morphology, apoptosis, and permeability of bovine pulmonary epithelial (BPE) cells and bovine pulmonary microvascular endothelial cells (BPMEC). Monolayers of BPE cells underwent striking morphological changes when exposed to ATP that included separation of the cells. Neither BPE cells nor BPMEC exhibited increased apoptosis in response to ATP. BPE cell and BPMEC monolayers displayed virtually identical increases in permeability when exposed to ATP, with a 50% change occurring within the first hour of exposure. Both cell types contained mRNA for the P2X7 receptor, a known receptor for ATP. In BPE cells, but not BPMEC, the change in permeability in response to ATP was reversed by the addition of a P2X7 receptor antagonist. If similar permeability changes occur in vivo, they could be a factor in vascular leakage into lung airspaces during pneumonia. PMID:18987163

  2. ATP as a biomarker of viable microorganisms in clean-room facilities.

    PubMed

    Venkateswaran, Kasthuri; Hattori, Noriaki; La Duc, Myron T; Kern, Roger

    2003-03-01

    A new firefly luciferase bioluminescence assay method that differentiates free extracellular ATP (dead cells, etc.) from intracellular ATP (viable microbes) was used to determine the viable microbial cleanliness of various clean-room facilities. For comparison, samples were taken from both clean-rooms, where the air was filtered to remove particles >0.5 microm, and ordinary rooms with unfiltered air. The intracellular ATP was determined after enzymatically degrading the sample's free ATP. Also for comparison, cultivable microbial populations were counted on nutrient-rich trypticase soy agar (TSA) plates. Both the cultivable and ATP-based determinations indicate that the microbial burden was lower in clean-room facilities than in ordinary rooms. However, there was no direct correlation between the two sets of measurements because the two assays measured very different populations. A large fraction of the samples yielded no colony formers on TSA, but were positive for intracellular ATP. Subsequently, genomic DNA was isolated directly from selected samples and 16S rDNA fragments were cloned and sequenced, identifying nearest neighbors, many of which are known to be noncultivable in the media employed. It was concluded that viable microbial contamination can be reliably monitored by measurement of intracellular ATP, and that this method may be considered superior to cultivable colony counts due to its speed and its ability to report the presence of viable but noncultivable organisms. When the detection of nonviable microbes is of interest, the ATP assay can be supplemented with DNA analysis. PMID:12531506

  3. A lipid switch unlocks Parkinson’s disease-associated ATP13A2

    PubMed Central

    Holemans, Tine; Sørensen, Danny Mollerup; van Veen, Sarah; Martin, Shaun; Hermans, Diane; Kemmer, Gerdi Christine; Van den Haute, Chris; Baekelandt, Veerle; Günther Pomorski, Thomas; Agostinis, Patrizia; Wuytack, Frank; Palmgren, Michael; Eggermont, Jan; Vangheluwe, Peter

    2015-01-01

    ATP13A2 is a lysosomal P-type transport ATPase that has been implicated in Kufor–Rakeb syndrome and Parkinson’s disease (PD), providing protection against α-synuclein, Mn2+, and Zn2+ toxicity in various model systems. So far, the molecular function and regulation of ATP13A2 remains undetermined. Here, we demonstrate that ATP13A2 contains a unique N-terminal hydrophobic extension that lies on the cytosolic membrane surface of the lysosome, where it interacts with the lysosomal signaling lipids phosphatidic acid (PA) and phosphatidylinositol(3,5)bisphosphate [PI(3,5)P2]. We further demonstrate that ATP13A2 accumulates in an inactive autophosphorylated state and that PA and PI(3,5)P2 stimulate the autophosphorylation of ATP13A2. In a cellular model of PD, only catalytically active ATP13A2 offers cellular protection against rotenone-induced mitochondrial stress, which relies on the availability of PA and PI(3,5)P2. Thus, the N-terminal binding of PA and PI(3,5)P2 emerges as a key to unlock the activity of ATP13A2, which may offer a therapeutic strategy to activate ATP13A2 and thereby reduce α-synuclein toxicity or mitochondrial stress in PD or related disorders. PMID:26134396

  4. Specific requirement for ATP at an early step of in vitro transcription of human mitochondrial DNA

    SciTech Connect

    Narasimhan, N.; Attardi, G.

    1987-06-01

    The ATP concentrations allowing transcription of heavy- and light-strand of human mtDNA in a HeLa cell mitochrondrial lysate were found to cover a broad range, with a maximum around 2.5 mM, and with reproducible differences in the ATP response curves for the two transcription events. Direct measurements showed that nonspecific ATP degradation during the assay did not account for the high ATP requirement. 5'-Adenylyl imidodiphosphate (p(NH)ppA), an ATP analog with a nonhydrolyzable ..beta..-..gamma.. bond, was unable to substitute for ATP in supporting mtDNA transcription but greatly stimulated this transcription in the presence of a low concentration of exogenous APT, measured with (/sup 32/P)-labeled nucleotides. Evidence was obtained indicating that p(NH)ppA did not support an early event in mtDNA transcription (formation of preinitiation complex or initiation), whereas this analog could substitute effectively for ATP in the subsequent elongation steps. These results pointed to a specific requirement for ATP at an early step of the transcription process.

  5. Three-Dimensional Structures Reveal Multiple ADP/ATP Binding Modes

    SciTech Connect

    C Simmons; C Magee; D Smith; L Lauman; J Chaput; J Allen

    2011-12-31

    The creation of synthetic enzymes with predefined functions represents a major challenge in future synthetic biology applications. Here, we describe six structures of de novo proteins that have been determined using protein crystallography to address how simple enzymes perform catalysis. Three structures are of a protein, DX, selected for its stability and ability to tightly bind ATP. Despite the addition of ATP to the crystallization conditions, the presence of a bound but distorted ATP was found only under excess ATP conditions, with ADP being present under equimolar conditions or when crystallized for a prolonged period of time. A bound ADP cofactor was evident when Asp was substituted for Val at residue 65, but ATP in a linear configuration is present when Phe was substituted for Tyr at residue 43. These new structures complement previously determined structures of DX and the protein with the Phe 43 to Tyr substitution [Simmons, C. R., et al. (2009) ACS Chem. Biol. 4, 649-658] and together demonstrate the multiple ADP/ATP binding modes from which a model emerges in which the DX protein binds ATP in a configuration that represents a transitional state for the catalysis of ATP to ADP through a slow, metal-free reaction capable of multiple turnovers. This unusual observation suggests that design-free methods can be used to generate novel protein scaffolds that are tailor-made for catalysis.

  6. Characterization of the Saccharomyces cerevisiae ATP-Interactome using the iTRAQ-SPROX Technique.

    PubMed

    Geer, M Ariel; Fitzgerald, Michael C

    2016-02-01

    The stability of proteins from rates of oxidation (SPROX) technique was used in combination with an isobaric mass tagging strategy to identify adenosine triphosphate (ATP) interacting proteins in the Saccharomyces cerevisiae proteome. The SPROX methodology utilized in this work enabled 373 proteins in a yeast cell lysate to be assayed for ATP interactions (both direct and indirect) using the non-hydrolyzable ATP analog, adenylyl imidodiphosphate (AMP-PNP). A total of 28 proteins were identified with AMP-PNP-induced thermodynamic stability changes. These protein hits included 14 proteins that were previously annotated as ATP-binding proteins in the Saccharomyces Genome Database (SGD). The 14 non-annotated ATP-binding proteins included nine proteins that were previously found to be ATP-sensitive in an earlier SPROX study using a stable isotope labeling with amino acids in cell culture (SILAC)-based approach. A bioinformatics analysis of the protein hits identified here and in the earlier SILAC-SPROX experiments revealed that many of the previously annotated ATP-binding protein hits were kinases, ligases, and chaperones. In contrast, many of the newly discovered ATP-sensitive proteins were not from these protein classes, but rather were hydrolases, oxidoreductases, and nucleic acid-binding proteins. Graphical Abstract ᅟ. PMID:26530046

  7. Autism Post-Mortem Neuroinformatic Resource: The Autism Tissue Program (ATP) Informatics Portal

    ERIC Educational Resources Information Center

    Brimacombe, Michael B.; Pickett, Richard; Pickett, Jane

    2007-01-01

    The Autism Tissue Program (ATP) was established to oversee and manage brain donations related to neurological research in autism. The ATP Informatics Portal (www.atpportal.org) is an integrated data access system based on Oracle technology, developed to provide access for researchers to information on this rare tissue resource. It also permits…

  8. A SPECTROPHOTOMETRIC ASSAY TO MEASURE RUBISCO ACTIVASE ACTIVATION ACTIVITY UNDER VARYING ATP:ADP RATIOS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ratio of ATP to ADP in the stroma is an important regulatory mechanism for controlling the activation state of Rubisco via Rubisco activase (activase). Understanding the response of activase to a varying ATP:ADP ratio should reveal insights into the regulation of photosynthesis. However, the cur...

  9. Structure and mechanism of soybean ATP sulfurylase and the committed step in plant sulfur assimilation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enzymes of the sulfur assimilation pathway are potential targets for improving nutrient content and environmental stress responses in plants. The committed step in this pathway is catalyzed by ATP sulfurylase, which synthesizes adenosine-5'-phosphosulfate (APS) from sulfate and ATP. To better unde...

  10. The Contribution of Red Blood Cell Dynamics to Intrinsic Viscosity and Functional ATP Release

    NASA Astrophysics Data System (ADS)

    Forsyth, Alison; Abkarian, Manouk; Wan, Jiandi; Stone, Howard

    2010-11-01

    In shear flow, red blood cells (RBCs) exhibit a variety of behaviors such as rouleaux formation, tumbling, swinging, and tank-treading. The physiological consequences of these dynamic behaviors are not understood. In vivo, ATP is known to signal vasodilation; however, to our knowledge, no one has deciphered the relevance of RBC microrheology to the functional release of ATP. Previously, we correlated RBC deformation and ATP release in microfluidic constrictions (Wan et al., 2008). In this work, a cone-plate rheometer is used to shear a low hematocrit solution of RBCs at varying viscosity ratios (λ) between the inner cytoplasmic hemoglobin and the outer medium, to determine the intrinsic viscosity of the suspension. Further, using a luciferin-luciferase enzymatic reaction, we report the relative ATP release at varying shear rates. Results indicate that for λ = 1.6, 3.8 and 11.1, ATP release is constant up to 500 s-1, which suggests that the tumbling-tanktreading transition does not alter ATP release in pure shear. For lower viscosity ratios, λ = 1.6 and 3.8, at 500 s-1 a change in slope occurs in the intrinsic viscosity data and is marked by an increase in ATP release. Based on microfluidic observations, this simultaneous change in viscosity and ATP release occurs within the tank-treading regime.

  11. Synergistic binding of glucose and aluminium ATP to hexokinase from Saccharomyces cerevisiae.

    PubMed

    Woolfitt, A R; Kellett, G L; Hoggett, J G

    1988-08-10

    The binding of glucose, AlATP and AlADP to the monomeric and dimeric forms of the native yeast hexokinase PII isoenzyme and to the proteolytically modified SII monomeric form was monitored at pH 6.7 by the concomitant quenching of intrinsic protein fluorescence. No fluorescence changes were observed when free enzyme was mixed with AlATP at concentrations up to 7500 microM. In the presence of saturating concentrations of glucose, the maximal quenching of fluorescence induced by AlATP was between 1.5 and 3.5% depending on species, and the average value of [L]0.5, the concentration of ligand at half-saturation, over all monomeric species was 0.9 +/- 0.4 microM. The presence of saturating concentrations of AlATP diminished [L]0.5 for glucose binding by between 260- and 670-fold for hexokinase PII and SII monomers, respectively (dependent on the ionic strength), and by almost 4000-fold for PII dimer. The data demonstrate extremely strong synergistic interactions in the binding of glucose and AlATP to yeast hexokinase, arising as a consequence of conformational changes in the free enzyme induced by glucose and in enzyme-glucose complex induced by AlATP. The synergistic interactions of glucose and AlATP are related to their kinetic synergism and to the ability of AlATP to act as a powerful inhibitor of the hexokinase reaction. PMID:3042027

  12. Development of an ATP measurement method suitable for xenobiotic treatment activated sludge biomass.

    PubMed

    Nguyen, Lan Huong; Chong, Nyuk-Min

    2015-09-01

    Activated sludge consumes a large amount of energy to degrade a xenobiotic organic compound. By tracking the energy inventory of activated sludge biomass during the sludge's degradation of a xenobiotic, any disadvantageous effect on the sludge's performance caused by energy deficiency can be observed. The purpose of this study was to develop a reliable and accurate method for measuring the ATP contents of activated sludge cells that were to degrade a xenobiotic organic. Cell disruption and cellular ATP extraction were performed by a protocol with which xenobiotic degrading activated sludge biomass was washed with SDS, treated by Tris and TCA, and followed by bead blasting. The suspension of disrupted cells was filtered before the filtrate was injected into HPLC that was set at optimal conditions to measure the ATP concentration therein. This extraction protocol and HPLC measurement of ATP was evaluated for its linearity, limits of detection, and reproducibility. Evaluation test results reported a R(2) of 0.999 of linear fit of ATP concentration versus activated sludge concentration, a LOD=0.00045mg/L, a LOQ=0.0015mg/L for HPLC measurement of ATP, a MDL=0.46mg/g SS for ATP extraction protocol, and a recovery efficiency of 96.4±2%. This method of ATP measurement was simple, rapid, reliable, and was unburdened of some limitations other methods may have. PMID:26210584

  13. Phylogeny and Identification of Enterococci by atpA Gene Sequence Analysis

    PubMed Central

    Naser, S.; Thompson, F. L.; Hoste, B.; Gevers, D.; Vandemeulebroecke, K.; Cleenwerck, I.; Thompson, C. C.; Vancanneyt, M.; Swings, J.

    2005-01-01

    The relatedness among 91 Enterococcus strains representing all validly described species was investigated by comparing a 1,102-bp fragment of atpA, the gene encoding the alpha subunit of ATP synthase. The relationships observed were in agreement with the phylogeny inferred from 16S rRNA gene sequence analysis. However, atpA gene sequences were much more discriminatory than 16S rRNA for species differentiation. All species were differentiated on the basis of atpA sequences with, at a maximum, 92% similarity. Six members of the Enterococcus faecium species group (E. faecium, E. hirae, E. durans, E. villorum, E. mundtii, and E. ratti) showed >99% 16S rRNA gene sequence similarity, but the highest value of atpA gene sequence similarity was only 89.9%. The intraspecies atpA sequence similarities for all species except E. faecium strains varied from 98.6 to 100%; the E. faecium strains had a lower atpA sequence similarity of 96.3%. Our data clearly show that atpA provides an alternative tool for the phylogenetic study and identification of enterococci. PMID:15872246

  14. Electrochemical monitoring systems of demembranated flagellate algal motility for ATP sensing.

    PubMed

    Shitanda, Isao; Tanaka, Koji; Hoshi, Yoshinao; Itagaki, Masayuki

    2014-02-21

    The ATP-induced behavior of the unicellular flagellate alga Chlamydomonas reinhardtii was recorded as changes in the redox currents for a coexisting redox marker. The ATP concentration was estimated using the presented compact electrochemical system, which is based on monitoring of the motility of the flagellates. PMID:24336166

  15. Effect of visible laser light on ATP level of anaemic red blood cell.

    PubMed

    Suardi, Nursakinah; Sodipo, Bashiru Kayode; Mustafa, Mohd Zulkifli; Ali, Zalila

    2016-09-01

    In this work we present influence of visible laser light on ATP level and viability of anaemic red blood cell (RBC). The visible laser lights used in this work are 460nm and 532nm. The responses of ATP level in anaemic and normal RBC before and after laser irradiation at different exposure time (30, 40, 50 and 60s) were observed. Three aliquots were prepared from the ethylenediaminetetraacetic acid (EDTA) blood sample. One served as a control (untreated) and another two were irradiated with 460nm and 560nm lasers. Packed RBC was prepared to study ATP level in the RBC using CellTiter-GloLuminescent cell Viability Assay kit. The assay generates a glow type signal produced by luciferase reaction, which is proportional to the amount of ATP present in RBCs. Paired t-test were done to analyse ATP level before and after laser irradiation. The results revealed laser irradiation improve level of ATP in anaemic RBC. Effect of laser light on anaemic RBCs were significant over different exposure time for both 460nm (p=0.000) and 532nm (p=0.003). The result of ATP level is further used as marker for RBC viability. The influence of ATP level and viability were studied. Optical densities obtained from the data were used to determine cell viability of the samples. Results showed that laser irradiation increased viability of anaemic RBC compared to normal RBC. PMID:27508880

  16. ATP as a biomarker of viable microorganisms in clean-room facilities

    NASA Technical Reports Server (NTRS)

    Venkateswaran, Kasthuri; Hattori, Noriaki; La Duc, Myron T.; Kern, Roger

    2003-01-01

    A new firefly luciferase bioluminescence assay method that differentiates free extracellular ATP (dead cells, etc.) from intracellular ATP (viable microbes) was used to determine the viable microbial cleanliness of various clean-room facilities. For comparison, samples were taken from both clean-rooms, where the air was filtered to remove particles >0.5 microm, and ordinary rooms with unfiltered air. The intracellular ATP was determined after enzymatically degrading the sample's free ATP. Also for comparison, cultivable microbial populations were counted on nutrient-rich trypticase soy agar (TSA) plates. Both the cultivable and ATP-based determinations indicate that the microbial burden was lower in clean-room facilities than in ordinary rooms. However, there was no direct correlation between the two sets of measurements because the two assays measured very different populations. A large fraction of the samples yielded no colony formers on TSA, but were positive for intracellular ATP. Subsequently, genomic DNA was isolated directly from selected samples and 16S rDNA fragments were cloned and sequenced, identifying nearest neighbors, many of which are known to be noncultivable in the media employed. It was concluded that viable microbial contamination can be reliably monitored by measurement of intracellular ATP, and that this method may be considered superior to cultivable colony counts due to its speed and its ability to report the presence of viable but noncultivable organisms. When the detection of nonviable microbes is of interest, the ATP assay can be supplemented with DNA analysis.

  17. Characterization of the Saccharomyces cerevisiae ATP-Interactome using the iTRAQ-SPROX Technique

    NASA Astrophysics Data System (ADS)

    Geer, M. Ariel; Fitzgerald, Michael C.

    2016-02-01

    The stability of proteins from rates of oxidation (SPROX) technique was used in combination with an isobaric mass tagging strategy to identify adenosine triphosphate (ATP) interacting proteins in the Saccharomyces cerevisiae proteome. The SPROX methodology utilized in this work enabled 373 proteins in a yeast cell lysate to be assayed for ATP interactions (both direct and indirect) using the non-hydrolyzable ATP analog, adenylyl imidodiphosphate (AMP-PNP). A total of 28 proteins were identified with AMP-PNP-induced thermodynamic stability changes. These protein hits included 14 proteins that were previously annotated as ATP-binding proteins in the Saccharomyces Genome Database (SGD). The 14 non-annotated ATP-binding proteins included nine proteins that were previously found to be ATP-sensitive in an earlier SPROX study using a stable isotope labeling with amino acids in cell culture (SILAC)-based approach. A bioinformatics analysis of the protein hits identified here and in the earlier SILAC-SPROX experiments revealed that many of the previously annotated ATP-binding protein hits were kinases, ligases, and chaperones. In contrast, many of the newly discovered ATP-sensitive proteins were not from these protein classes, but rather were hydrolases, oxidoreductases, and nucleic acid-binding proteins.

  18. ATP: A Coherent View for School Advanced Level Studies in Biology.

    ERIC Educational Resources Information Center

    Gayford, Chris

    1986-01-01

    Discusses how instruction of biological concepts as ATP cellular energetics is related to fundamental physical science understandings. Reviews areas of common misconceptions and confusions. Summarizes results of a study which investigated students' knowledge and perception of difficulty associated with the topic of energy and ATP. (ML)

  19. Cation Transport Coupled to ATP Hydrolysis by the (Na, K)-ATPase: An Integrated, Animated Model

    ERIC Educational Resources Information Center

    Leone, Francisco A.; Furriel, Rosa P. M.; McNamara, John C.; Horisberger, Jean D.; Borin, Ivana A.

    2010-01-01

    An Adobe[R] animation is presented for use in undergraduate Biochemistry courses, illustrating the mechanism of Na[superscript +] and K[superscript +] translocation coupled to ATP hydrolysis by the (Na, K)-ATPase, a P[subscript 2c]-type ATPase, or ATP-powered ion pump that actively translocates cations across plasma membranes. The enzyme is also…

  20. 75 FR 25294 - Notice Pursuant to the National Cooperative Research and Production Act of 1993-High Definition...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-07

    ... published a notice in the Federal Register pursuant to Section 6(b) of the Act on March 10, 2008 (73 FR... published in the Federal Register pursuant to Section 6(b) of the Act on October 21, 2008 (73 FR 62542... Definition Metrology and Process-2 Micron Manufacturing Under ATP Award No. 70NANB77041 Notice is...

  1. ATP-dependent release of glucocorticoid receptors from the nuclear matrix.

    PubMed Central

    Tang, Y; DeFranco, D B

    1996-01-01

    Glucocorticoid receptors (GRs) have the capacity to shuttle between the nuclear and cytoplasmic compartments, sharing that trait with other steroid receptors and unrelated nuclear proteins of diverse function. Although nuclear import of steroid receptors, like that of nearly all other karyophilic proteins examined to date, requires ATP, there appear to be different energetic requirements for export of proteins, including steroid receptors, from nuclei. In an attempt to reveal which steps, if any, in the nuclear export pathway utilized by steroid receptors require ATP, we have used indirect immunofluorescence to visualize GRs within cells subjected to a reversible ATP depletion. Under conditions which lead to >95% depletion of cellular ATP levels within 90 min, GRs remain localized within nuclei and do not efflux into the cytoplasm. Under analogous conditions of ATP depletion, transfected progesterone receptors are also retained within nuclei. Importantly, GRs which accumulate within nuclei of ATP-depleted cells are distinguished from nuclear receptors in metabolically active cells by their resistance to in situ extraction with a hypotonic, detergent-containing buffer. GRs in ATP-depleted cells are not permanently trapped in this nuclear compartment, as nuclear receptors rapidly regain their capacity to be extracted upon restoration of cellular ATP, even in the absence of de novo protein synthesis. More extensive extraction of cells with high salt and detergent, coupled with DNase I digestion, established that a significant fraction of GRs in ATP-depleted cells are associated with an RNA-containing nuclear matrix. Quantitative Western blot (immunoblot) analysis confirmed the dramatic increase in GR binding to the nuclear matrix of ATP-depleted cells, while confocal microscopy revealed that GRs are bound to the matrix throughout all planes of the nucleus. ATP depletion does not lead to wholesale collapse of nuclear proteins onto the matrix, as the interaction of a

  2. Mechanism of action of ATP on canine pulmonary vagal C fibre nerve terminals.

    PubMed Central

    Pelleg, A; Hurt, C M

    1996-01-01

    1. The effects of extracellular adenosine 5'-triphosphate (ATP) on pulmonary vagal afferent fibres (n = 46) was studied in a canine model in vivo (n = 38). 2. ATP (3-6 mumol kg-1), administered as a rapid bolus into the right atrium, elicited a transient burst of action potentials in cervical vagal fibres, which was not affected by either blockade of ganglionic transmission (hexamethonium) or a drop in arterial blood pressure (nitroglycerine). 3. The fibres with ATP-sensitive terminals were otherwise quiescent with no activity related to either cardiac or respiratory cycles and their conduction velocity was 0.85 +/- 0.13 m s-1 (n = 7). 4. Inflation of the lungs to 2-3 times the tidal volume triggered brief bursts of action potentials in these fibres. 5. Capsaicin (10 micrograms kg-1), given as a rapid bolus into the right atrium, elicited a burst of action potentials in these ATP-sensitive fibres. 6. Smaller amounts of ATP and capsaicin (0.5-3 mumol kg-1 and 1-5 micrograms kg-1, respectively) had similar effects when the two compounds were given into the right pulmonary artery. 7. Adenosine, adenosine 5'-monophosphate, or adenosine 5'-diphosphate did not excite these fibres (n = 30). 8. The non-degradable analogue of ATP alpha,beta-methylene ATP (alpha,beta-mATP) was tenfold more potent than ATP while beta,gamma-methylene ATP (beta,gamma-mATP) was in active. 9. The selective P2x-purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid markedly attenuated the effect of ATP but not of capsaicin. The P2Y-purinoceptor antagonist Reactive Blue 2 was without effect. 10. Pretreatment with pertussis toxin (PTX) did not affect this action of ATP. 11. In the canine lungs ATP activates vagal C fibre nerve terminals. This action is mediated by P2X-purinoceptors and is independent of a PTX-sensitive guanine nucleotide binding protein (G protein). PMID:8745294

  3. Light-harvesting chlorophyll pigments enable mammalian mitochondria to capture photonic energy and produce ATP.

    PubMed

    Xu, Chen; Zhang, Junhua; Mihai, Doina M; Washington, Ilyas

    2014-01-15

    Sunlight is the most abundant energy source on this planet. However, the ability to convert sunlight into biological energy in the form of adenosine-5'-triphosphate (ATP) is thought to be limited to chlorophyll-containing chloroplasts in photosynthetic organisms. Here we show that mammalian mitochondria can also capture light and synthesize ATP when mixed with a light-capturing metabolite of chlorophyll. The same metabolite fed to the worm Caenorhabditis elegans leads to increase in ATP synthesis upon light exposure, along with an increase in life span. We further demonstrate the same potential to convert light into energy exists in mammals, as chlorophyll metabolites accumulate in mice, rats and swine when fed a chlorophyll-rich diet. Results suggest chlorophyll type molecules modulate mitochondrial ATP by catalyzing the reduction of coenzyme Q, a slow step in mitochondrial ATP synthesis. We propose that through consumption of plant chlorophyll pigments, animals, too, are able to derive energy directly from sunlight. PMID:24198392

  4. ROLE OF ATP IN REGULATING RENAL MICROVASCULAR FUNCTION AND IN HYPERTENSION

    PubMed Central

    Guan, Zhengrong; Inscho, Edward W.

    2011-01-01

    Adenosine triphosphate (ATP) is an essential energy substrate for cellular metabolism but it can also influence many biological processes when released into the extracellular milieu. Research has established that extracellular ATP acts as an autocrine/paracrine factor that regulates many physiological functions. Alternatively, excessive extracellular ATP levels contribute to pathophysiological processes such as inflammation, cell proliferation and apoptosis, and atherosclerosis. Renal P2 receptors are widely distributed throughout glomeruli, vasculature and tubular segments, and participate in controlling renal vascular resistance, mediating renal autoregulation, and regulating tubular transport function. This review will focus on the role of ATP-P2 receptor signaling in regulating renal microvascular function and autoregulation, recent advances on the role of ATP-P2 signaling in hypertension-associated renal vascular injury, and emerging new directions. PMID:21768526

  5. Measuring ATP Concentration in a Small Number of Murine Hematopoietic Stem Cells.

    PubMed

    Szade, Krzysztof; Zukowska, Monika; Jozkowicz, Alicja; Dulak, Jozef

    2016-01-01

    The metabolism of quiescent adult stem cells differs from the metabolism of differentiated cells. The metabolic processes are tightly regulated and their alterations disturb function of stem cells. One of the indicators of metabolic status of cells is the ATP level. While the method of measuring the ATP levels has been known for many years, estimating ATP levels in small population of defined stem cells isolated directly from the tissue has remained challenging. Here, we show our method of measuring the ATP levels in hematopoietic stem cells sorted from murine bone marrow. We used magnetic sorting as well as cell sorter and adopted the commonly used bioluminescence-based detection kits in described protocol. Our strategy allows to measure ATP levels in 1000 highly purified HSC. PMID:27138010

  6. Failure of the Cystic Fibrosis Transmembrane Conductance Regulator to Conduct ATP

    NASA Astrophysics Data System (ADS)

    Reddy, M. M.; Quinton, P. M.; Haws, C.; Wine, J. J.; Grygorczyk, R.; Tabcharani, J. A.; Hanrahan, J. W.; Gunderson, K. L.; Kopito, R. R.

    1996-03-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is chloride ion channel regulated by protein kinase A and adenosine triphosphate (ATP). Loss of CFTR-mediated chloride ion conductance from the apical plasma membrane of epithelial cells is a primary physiological lesion in cystic fibrosis. CFTR has also been suggested to function as an ATP channel, although the size of the ATP anion is much larger than the estimated size of the CFTR pore. ATP was not conducted through CFTR in intact organs, polarized human lung cell lines, stably transfected mammalian cell lines, or planar lipid bilayers reconstituted with CFTR protein. These findings suggest that ATP permeation through the CFTR is unlikely to contribute to the normal function of CFTR or to the pathogenesis of cystic fibrosis.

  7. Interdependence of ATP signalling and pannexin channels; the servant was really the master all along?

    PubMed

    Jackson, Michael F

    2015-12-15

    Pannexin channels are recognized as important conduits for the release of ATP, which contributes to purinergic signalling. Pathologically, ATP release via these channels acts as a find-me signal for apoptotic cell clearance. Accordingly, there is considerable and growing interest in understanding the function and regulation of pannexin channels. In a recent issue of the Biochemical Journal, Boyce et al. provide evidence that the surface expression of pannexin channels is regulated by extracellular ATP. They propose a model in which ATP triggers pannexin channel internalization through a pathway involving clathrin- and caveolin-independent entry into early endosomes. Intriguingly, their evidence suggests that internalization is initiated through the association of ATP with pannexin channels themselves as well as ionotropic purinergic receptor 7 (P2X7) receptors. PMID:26613946

  8. Coupling of CFTR Cl- channel gating to an ATP hydrolysis cycle.

    PubMed

    Baukrowitz, T; Hwang, T C; Nairn, A C; Gadsby, D C

    1994-03-01

    For cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels to open, they must be phosphorylated by protein kinase A and then exposed to a hydrolyzable nucleoside triphosphate, such as ATP. To test whether channel opening is linked to ATP hydrolysis, we applied VO4 and BeF3 to CFTR channels in inside-out patches excised from cardiac myocytes. These inorganic phosphate analogs interrupt ATP hydrolysis cycles by binding tightly in place of the released hydrolysis product, inorganic phosphate. The analogs acted only on CFTR channels opened by ATP and locked them open, increasing their mean open time by 2-3 orders of magnitude. These findings establish that opening and closing of CFTR channels are coupled to an ATP hydrolysis cycle. PMID:7512348

  9. ATP release mechanisms of endothelial cell-mediated stimulus-dependent hyperalgesia

    PubMed Central

    Joseph, Elizabeth K.; Green, Paul G.; Levine, Jon D.

    2014-01-01

    Endothelin-1 acts on endothelial cells to enhance mechanical stimulation-induced release of ATP, which in turn can act on sensory neurons innervating blood vessels to contribute to vascular pain, a phenomenon we have referred to as stimulus-dependent hyperalgesia (SDH). In the present study we evaluated the role of the major classes of ATP release mechanisms to SDH: vesicular exocytosis, plasma membrane associated ATP synthase, ATP-Binding Cassette (ABC) transporters, and ion channels. Inhibitors of vesicular exocytosis (i.e., monensin, brefeldin A and bafilomycin), plasma membrane associated ATPase (i.e., oligomycin and pigment epithelium-derived factor-derived peptide 34-mer) and connexin ion channels (carbenoxolone and flufenamic acid), but not ABC transporters (i.e., dipyridamole, nicardipine or CFTRinh-172) attenuated stimulus-dependent hyperalgesia. These studies support a role of ATP in SDH, and suggest novel targets for the treatment of vascular pain syndromes. PMID:24793242

  10. Hexokinase inhibits flux of fluorescently labeled ATP through mitochondrial outer membrane porin.

    PubMed

    Perevoshchikova, Irina V; Zorov, Savva D; Kotova, Elena A; Zorov, Dmitry B; Antonenko, Yuri N

    2010-06-01

    Mitochondrial function requires maintaining metabolite fluxes across the mitochondrial outer membrane, which is mediated primarily by the voltage dependent anion channel (VDAC). We applied fluorescence correlation spectroscopy (FCS) to study regulation of the VDAC functional state by monitoring distribution of fluorescently labeled ATP (BODIPY-FL-ATP) in isolated intact rat liver and heart mitochondria. Addition of mitochondria to BODIPY-FL-ATP solution resulted in accumulation of the fluorescent probe in these organelles. The addition of hexokinase II (HKII) isolated from rat heart led to a decrease in the BODIPY-FL-ATP accumulation, while a 15-residue peptide corresponding to the N-terminal domain of hexokinase did not produce this effect. Therefore, the hexokinase-induced inhibition of the ATP flow mediated by VDAC was revealed in isolated mitochondria. PMID:20412805

  11. ATP hydrolysis assists phosphate release and promotes reaction ordering in F1-ATPase

    PubMed Central

    Li, Chun-Biu; Ueno, Hiroshi; Watanabe, Rikiya; Noji, Hiroyuki; Komatsuzaki, Tamiki

    2015-01-01

    F1-ATPase (F1) is a rotary motor protein that can efficiently convert chemical energy to mechanical work of rotation via fine coordination of its conformational motions and reaction sequences. Compared with reactant binding and product release, the ATP hydrolysis has relatively little contributions to the torque and chemical energy generation. To scrutinize possible roles of ATP hydrolysis, we investigate the detailed statistics of the catalytic dwells from high-speed single wild-type F1 observations. Here we report a small rotation during the catalytic dwell triggered by the ATP hydrolysis that is indiscernible in previous studies. Moreover, we find in freely rotating F1 that ATP hydrolysis is followed by the release of inorganic phosphate with low synthesis rates. Finally, we propose functional roles of the ATP hydrolysis as a key to kinetically unlock the subsequent phosphate release and promote the correct reaction ordering. PMID:26678797

  12. Physiological heart activation by adrenaline involves parallel activation of ATP usage and supply.

    PubMed

    Korzeniewski, Bernard; Deschodt-Arsac, Véronique; Calmettes, Guillaume; Franconi, Jean-Michel; Diolez, Philippe

    2008-07-15

    During low-to-high work transition in adult mammalian heart in vivo the concentrations of free ADP, ATP, PCr (phosphocreatine), P(i) and NADH are essentially constant, in striking contrast with skeletal muscle. The direct activation by calcium ions of ATP usage and feedback activation of ATP production by ADP (and P(i)) alone cannot explain this perfect homoeostasis. A comparison of the response to adrenaline (increase in rate-pressure product and [PCr]) of the intact beating perfused rat heart with the elasticities of the PCr producer and consumer to PCr concentration demonstrated that both the ATP/PCr-producing block and ATP/PCr-consuming block are directly activated to a similar extent during physiological heart activation. Our finding constitutes a direct evidence for the parallel-activation mechanism of the regulation of oxidative phosphorylation in heart postulated previously in a theoretical way. PMID:18377364

  13. ATP hydrolysis assists phosphate release and promotes reaction ordering in F1-ATPase

    NASA Astrophysics Data System (ADS)

    Li, Chun-Biu; Ueno, Hiroshi; Watanabe, Rikiya; Noji, Hiroyuki; Komatsuzaki, Tamiki

    2015-12-01

    F1-ATPase (F1) is a rotary motor protein that can efficiently convert chemical energy to mechanical work of rotation via fine coordination of its conformational motions and reaction sequences. Compared with reactant binding and product release, the ATP hydrolysis has relatively little contributions to the torque and chemical energy generation. To scrutinize possible roles of ATP hydrolysis, we investigate the detailed statistics of the catalytic dwells from high-speed single wild-type F1 observations. Here we report a small rotation during the catalytic dwell triggered by the ATP hydrolysis that is indiscernible in previous studies. Moreover, we find in freely rotating F1 that ATP hydrolysis is followed by the release of inorganic phosphate with low synthesis rates. Finally, we propose functional roles of the ATP hydrolysis as a key to kinetically unlock the subsequent phosphate release and promote the correct reaction ordering.

  14. Modeling regulation of cardiac KATP and L-type Ca2+ currents by ATP, ADP, and Mg2+

    NASA Technical Reports Server (NTRS)

    Michailova, Anushka; Saucerman, Jeffrey; Belik, Mary Ellen; McCulloch, Andrew D.

    2005-01-01

    Changes in cytosolic free Mg(2+) and adenosine nucleotide phosphates affect cardiac excitability and contractility. To investigate how modulation by Mg(2+), ATP, and ADP of K(ATP) and L-type Ca(2+) channels influences excitation-contraction coupling, we incorporated equations for intracellular ATP and MgADP regulation of the K(ATP) current and MgATP regulation of the L-type Ca(2+) current in an ionic-metabolic model of the canine ventricular myocyte. The new model: 1), quantitatively reproduces a dose-response relationship for the effects of changes in ATP on K(ATP) current, 2), simulates effects of ADP in modulating ATP sensitivity of K(ATP) channel, 3), predicts activation of Ca(2+) current during rapid increase in MgATP, and 4), demonstrates that decreased ATP/ADP ratio with normal total Mg(2+) or increased free Mg(2+) with normal ATP and ADP activate K(ATP) current, shorten action potential, and alter ionic currents and intracellular Ca(2+) signals. The model predictions are in agreement with experimental data measured under normal and a variety of pathological conditions.

  15. Extracellular ATP promotes stomatal opening of Arabidopsis thaliana through heterotrimeric G protein α subunit and reactive oxygen species.

    PubMed

    Hao, Li-Hua; Wang, Wei-Xia; Chen, Chen; Wang, Yu-Fang; Liu, Ting; Li, Xia; Shang, Zhong-Lin

    2012-07-01

    In recent years, adenosine tri-phosphate (ATP) has been reported to exist in apoplasts of plant cells as a signal molecule. Extracellular ATP (eATP) plays important roles in plant growth, development, and stress tolerance. Here, extracellular ATP was found to promote stomatal opening of Arabidopsis thaliana in light and darkness. ADP, GTP, and weakly hydrolyzable ATP analogs (ATPγS, Bz-ATP, and 2meATP) showed similar effects, whereas AMP and adenosine did not affect stomatal movement. Apyrase inhibited stomatal opening. ATP-promoted stomatal opening was blocked by an NADPH oxidase inhibitor (diphenylene iodonium) or deoxidizer (dithiothreitol), and was impaired in null mutant of NADPH oxidase (atrbohD/F). Added ATP triggered ROS generation in guard cells via NADPH oxidase. ATP also induced Ca(2+) influx and H(+) efflux in guard cells. In atrbohD/F, ATP-induced ion flux was strongly suppressed. In null mutants of the heterotrimeric G protein α subunit, ATP-promoted stomatal opening, cytoplasmic ROS generation, Ca(2+) influx, and H(+) efflux were all suppressed. These results indicated that eATP-promoted stomatal opening possibly involves the heterotrimeric G protein, ROS, cytosolic Ca(2+), and plasma membrane H(+)-ATPase. PMID:22138967

  16. Modeling regulation of cardiac KATP and L-type Ca2+ currents by ATP, ADP, and Mg2+.

    PubMed

    Michailova, Anushka; Saucerman, Jeffrey; Belik, Mary Ellen; McCulloch, Andrew D

    2005-03-01

    Changes in cytosolic free Mg(2+) and adenosine nucleotide phosphates affect cardiac excitability and contractility. To investigate how modulation by Mg(2+), ATP, and ADP of K(ATP) and L-type Ca(2+) channels influences excitation-contraction coupling, we incorporated equations for intracellular ATP and MgADP regulation of the K(ATP) current and MgATP regulation of the L-type Ca(2+) current in an ionic-metabolic model of the canine ventricular myocyte. The new model: 1), quantitatively reproduces a dose-response relationship for the effects of changes in ATP on K(ATP) current, 2), simulates effects of ADP in modulating ATP sensitivity of K(ATP) channel, 3), predicts activation of Ca(2+) current during rapid increase in MgATP, and 4), demonstrates that decreased ATP/ADP ratio with normal total Mg(2+) or increased free Mg(2+) with normal ATP and ADP activate K(ATP) current, shorten action potential, and alter ionic currents and intracellular Ca(2+) signals. The model predictions are in agreement with experimental data measured under normal and a variety of pathological conditions. PMID:15738467

  17. Hyper III on ramp

    NASA Technical Reports Server (NTRS)

    1969-01-01

    The Hyper III was a full-scale lifting-body remotely piloted research vehicle (RPRV) built at what was then the NASA Flight Research Center located at Edwards Air Force Base in Southern California. The Flight Research Center (FRC--as Dryden was named from 1959 until 1976) already had experience with testing small-scale aircraft using model-airplane techniques, but the first true remotely piloted research vehicle was the Hyper III, which flew only once in December 1969. At that time, the Center was engaged in flight research with a variety of reentry shapes called lifting bodies, and there was a desire both to expand the flight research experience with maneuverable reentry vehicles, including a high-performance, variable-geometry craft, and to investigate a remotely piloted flight research technique that made maximum use of a research pilot's skill and experience by placing him 'in the loop' as if he were in the cockpit. (There have been, as yet, no female research pilots assigned to Dryden.) The Hyper III as originally conceived was a stiletto-shaped lifting body that had resulted from a study at NASA's Langley Research Center in Hampton, Virginia. It was one of a number of hypersonic, cross-range reentry vehicles studied at Langley. (Hypersonic means Mach 5--five times the speed of sound--or faster; cross-range means able to fly a considerable distance to the left or right of the initial reentry path.) The FRC added a small, deployable, skewed wing to compensate for the shape's extremely low glide ratio. Shop personnel built the 32-foot-long Hyper III and covered its tubular frame with dacron, aluminum, and fiberglass, for about $6,500. Hyper III employed the same '8-ball' attitude indicator developed for control-room use when flying the X-15, two model-airplane receivers to command the vehicle's hydraulic controls, and a telemetry system (surplus from the X-15 program) to transmit 12 channels of data to the ground not only for display and control but for data

  18. Use of ATP to characterize biomass viability in freely suspended and immobilized cell bioreactors

    SciTech Connect

    Gikas, P.; Livingston, A.G. . Dept. of Chemical Engineering)

    1993-12-01

    This work describes investigations into the viability of cells growing on 3,4-dichloroaniline (34DCA). Two bio-reactors are employed for microbial growth, a continuous stirred tank (CST) bioreactor with a 2-L working volume, and a three-phase air lift (TPAL) bioreactor with a 3-L working volume. Experiments have been performed at several dilution rates between 0.027 and 0.115 h[sup [minus]1] in the CST bioreactor and between 0.111 and 0.500h[sup [minus]1] in the TPAL bioreactor. The specific ATP concentration was calculated at each dilution rate in the suspended biomass in both bioreactors as well as in the immobilized biomass in the TPAL bioreactor. The cultures were inspected under an electron microscope to monitor compositional changes. Results from the CST bioreactor showed that the biomass-specific ATP concentration increases from 0.44 to 1.86 mg ATP g[sup [minus]1] dry weight (dw) as dilution rate increases from 0.027 to 0.115 h[sup [minus]1]. At this upper dilution rate the cells were washed out. The specific ATP concentration reached a limiting average value of 1.73 mg ATP g[sup [minus]1] dw, which is assumed to be the quantity of ATP in 100% viable biomass, In the TPAL bioreactor, the ATP level increased with dilution rat in both the immobilized and suspended biomass. The specific ATP concentration in the immobilized biomass increased from approximately 0.051 mg ATP g[sup [minus]1] dw at dilution rates between 0.111 and 0.200 h[sup [minus]1] to approximately 0.119 mg ATP g[sup [minus]1] dw at dilution rates between 0.300 and 0.500 h[sup [minus]1].

  19. The molecular motor F-ATP synthase is targeted by the tumoricidal protein HAMLET.

    PubMed

    Ho, James; Sielaff, Hendrik; Nadeem, Aftab; Svanborg, Catharina; Grüber, Gerhard

    2015-05-22

    HAMLET (human alpha-lactalbumin made lethal to tumor cells) interacts with multiple tumor cell compartments, affecting cell morphology, metabolism, proteasome function, chromatin structure and viability. This study investigated if these diverse effects of HAMLET might be caused, in part, by a direct effect on the ATP synthase and a resulting reduction in cellular ATP levels. A dose-dependent reduction in cellular ATP levels was detected in A549 lung carcinoma cells, and by confocal microscopy, co-localization of HAMLET with the nucleotide-binding subunits α (non-catalytic) and β (catalytic) of the energy converting F1F0 ATP synthase was detected. As shown by fluorescence correlation spectroscopy, HAMLET binds to the F1 domain of the F1F0 ATP synthase with a dissociation constant (KD) of 20.5μM. Increasing concentrations of the tumoricidal protein HAMLET added to the enzymatically active α3β3γ complex of the F-ATP synthase lowered its ATPase activity, demonstrating that HAMLET binding to the F-ATP synthase effects the catalysis of this molecular motor. Single-molecule analysis was applied to study HAMLET-α3β3γ complex interaction. Whereas the α3β3γ complex of the F-ATP synthase rotated in a counterclockwise direction with a mean rotational rate of 3.8±0.7s(-1), no rotation could be observed in the presence of bound HAMLET. Our findings suggest that direct effects of HAMLET on the F-ATP synthase may inhibit ATP-dependent cellular processes. PMID:25681694

  20. Extracellular ATP has a potent effect to enhance cytosolic calcium and contractility in single ventricular myocytes.

    PubMed

    Danziger, R S; Raffaeli, S; Moreno-Sanchez, R; Sakai, M; Capogrossi, M C; Spurgeon, H A; Hansford, R G; Lakatta, E G

    1988-08-01

    The effect of extracellular ATP on the contraction of single rat cardiac myocytes was investigated, together with the effect on the transient change in cytosolic Ca2+ (Cai) elicited by excitation and on the relationship between these two parameters. In unstimulated single myocytes, ATP caused a small increase in Cai (measured as the ratio of fluorescence of Indo-1 at 410 to that at 490 nm. In myocytes bathed in a medium containing 1.0 mM [Ca2+] at 23 degrees C and stimulated at 1 Hz, ATP (1 microM) resulted in a two-threefold increase in amplitude of contraction, as measured by video cinemicrographic techniques. The duration of the Cai-transient was not altered but its amplitude was markedly enhanced, as was the amplitude of contraction. The relation between Cai and contraction-amplitude was not altered by ATP, when measured over a range of extracellular [Ca2+], suggesting that ATP does not affect the myofilament-Ca2+ interaction. The primary site of action of ATP in increasing Cai is at the sarcolemma since the addition to suspensions of myocytes of caffeine (10 mM), which depletes the sarcoplasmic reticulum Ca2+ load, does not prevent the subsequent increase of Cai due to ATP. Further, lowering of the extracellular [Ca2+] to less than 1 microM with EGTA abolishes the response of Cai to ATP, though not the response to caffeine. Thus in rat cardiac myocytes ATP stimulates trans-sarcolemmal influx of Ca2+: ADP, AMP and adenosine are ineffective. ATP markedly augments the amplitude of the Cai transient elicited by electrical stimulation thus rendering it a potent inotropic agent. PMID:3191528

  1. ATP and related purines stimulate motility, spatial congregation, and coalescence in red algal spores.

    PubMed

    Huidobro-Toro, Juan P; Donoso, Verónica; Flores, Verónica; Santelices, Bernabé

    2015-04-01

    Adenosine 5'-triphosphate (ATP) is a versatile extracellular signal along the tree of life, whereas cAMP plays a major role in vertebrates as an intracellular messenger for hormones, transmitters, tastants, and odorants. Since red algal spore coalescence may be considered analogous to the congregation process of social amoeba, which is stimulated by cAMP, we ascertained whether exogenous applications of ATP, cAMP, adenine, or adenosine modified spore survival and motility, spore settlement and coalescence. Concentration-response studies were performed with carpospores of Mazzaella laminarioides (Gigartinales), incubated with and without added purines. Stirring of algal blades released ADP/ATP to the cell media in a time-dependent manner. 10-300 μM ATP significantly increased spore survival; however, 1,500 μM ATP, cAMP or adenine induced 100% mortality within less than 24 h; the exception was adenosine, which up to 3,000 μM, did not alter spore survival. ATP exposure elicited spore movement with speeds of 2.2-2.5 μm · s(-1) . 14 d after 1,000 μM ATP addition, spore abundance in the central zone of the plaques was increased 2.7-fold as compared with parallel controls. Likewise, 1-10 μM cAMP or 30-100 μM adenine also increased central zone spore abundance, albeit these purines were less efficacious than ATP; adenosine up to 3,000 μM did not influence settlement. Moreover, 1,000 μM ATP markedly accelerated coalescence, the other purines caused a variable effect. We conclude that exogenous cAMP, adenine, but particularly ATP, markedly influence red algal spore physiology; effects are compatible with the expression of one or more membrane purinoceptor(s), discarding adenosine receptor participation. PMID:26986520

  2. Computational Analysis of the Ligand Binding Site of the Extracellular ATP Receptor, DORN1.

    PubMed

    Nguyen, Cuong The; Tanaka, Kiwamu; Cao, Yangrong; Cho, Sung-Hwan; Xu, Dong; Stacey, Gary

    2016-01-01

    DORN1 (also known as P2K1) is a plant receptor for extracellular ATP, which belongs to a large gene family of legume-type (L-type) lectin receptor kinases. Extracellular ATP binds to DORN1 with strong affinity through its lectin domain, and the binding triggers a variety of intracellular activities in response to biotic and abiotic stresses. However, information on the tertiary structure of the ligand binding site of DORN1is lacking, which hampers efforts to fully elucidate the mechanism of receptor action. Available data of the crystal structures from more than 50 L-type lectins enable us to perform an in silico study of molecular interaction between DORN1 and ATP. In this study, we employed a computational approach to develop a tertiary structure model of the DORN1 lectin domain. A blind docking analysis demonstrated that ATP binds to a cavity made by four loops (defined as loops A B, C and D) of the DORN1 lectin domain with high affinity. In silico target docking of ATP to the DORN1 binding site predicted interaction with 12 residues, located on the four loops, via hydrogen bonds and hydrophobic interactions. The ATP binding pocket is structurally similar in location to the carbohydrate binding pocket of the canonical L-type lectins. However, four of the residues predicted to interact with ATP are not conserved between DORN1 and the other carbohydrate-binding lectins, suggesting that diversifying selection acting on these key residues may have led to the ATP binding activity of DORN1. The in silico model was validated by in vitro ATP binding assays using the purified extracellular lectin domain of wild-type DORN1, as well as mutated DORN1 lacking key ATP binding residues. PMID:27583834

  3. Fueling type III secretion

    PubMed Central

    Lee, Pei-Chung

    2015-01-01

    Type III secretion systems are complex nanomachines that export proteins from the bacterial cytoplasm across the cell envelope in a single step. They are at the core of the machinery used to assemble the bacterial flagellum, and the needle complex many Gram-negative pathogens use to inject effector proteins into host cells and cause disease. Several models have been put forward to explain how this export is energized, and the mechanism has been the subject of considerable debate. Here we present an overview of these models and discuss their relative merits. Recent evidence suggests that the proton motive force is the primary energy source for type III secretion, although contribution from refolding of secreted proteins has not been ruled out. The mechanism, by which the proton motive force is converted to protein export, remains enigmatic. PMID:25701111

  4. Cranial mononeuropathy III - diabetic type

    MedlinePlus

    ... gov/ency/article/000692.htm Cranial mononeuropathy III - diabetic type To use the sharing features on this page, please enable JavaScript. Cranial mononeuropathy III -- diabetic type -- is usually a complication of diabetes that causes ...

  5. Medical tele-education system with superhigh-definition (SHD) image viewer

    NASA Astrophysics Data System (ADS)

    Tsumura, Hiroshi; Ashihara, Tsukasa; Urata, Yoji; Hata, Jun-ichi; Fukuhara, Yoshimi; Ono, Sadayasu

    1996-02-01

    We have been studying a medical tele-education support system by an individual tutoring system, called CALAT, and a super high definition (SHD) image processing system, called SuperFM-III. Now, we are in a trial operation to use the SuperFM-III for a super high definition image control viewer on the CALAT client side, and have created the courseware of the pathological images. In this paper, we show the concept and the implementation of this system.

  6. The Phylogenetic Signature Underlying ATP Synthase c-Ring Compliance

    SciTech Connect

    Pandini, Alessandro; Kleinjung, Jens; Taylor, Willie R.; Junge, Wolfgang; Khan, Shahid

    2015-09-01

    The proton-driven ATP synthase (FOF1) is comprised of two rotary, stepping motors (FO and F1) coupled by an elastic power transmission. The elastic compliance resides in the rotor module that includes the membrane-embedded FO c-ring. Proton transport by FO is firmly coupled to the rotation of the c-ring relative to other FO subunits (ab2). It drives ATP synthesis. We used a computational method to investigate the contribution of the c-ring to the total elastic compliance. We performed principal component analysis of conformational ensembles built using distance constraints from the bovine mitochondrial c-ring x-ray structure. Angular rotary twist, the dominant ring motion, was estimated to show that the c-ring accounted in part for the measured compliance. Ring rotation was entrained to rotation of the external helix within each hairpin-shaped c-subunit in the ring. Ensembles of monomer and dimers extracted from complete c-rings showed that the coupling between collective ring and the individual subunit motions was independent of the size of the c-ring, which varies between organisms. Molecular determinants were identified by covariance analysis of residue coevolution and structural-alphabet-based local dynamics correlations. The residue coevolution gave a readout of subunit architecture. The dynamic couplings revealed that the hinge for both ring and subunit helix rotations was constructed from the proton-binding site and the adjacent glycine motif (IB-GGGG) in the midmembrane plane. IB-GGGG motifs were linked by long-range couplings across the ring, while intrasubunit couplings connected the motif to the conserved cytoplasmic loop and adjacent segments. The correlation with principal collective motions shows that the couplings underlie both ring rotary and bending motions. Noncontact couplings between IB-GGGG motifs matched the coevolution signal as well as contact couplings

  7. The Phylogenetic Signature Underlying ATP Synthase c-Ring Compliance

    DOE PAGESBeta

    Pandini, Alessandro; Kleinjung, Jens; Taylor, Willie R.; Junge, Wolfgang; Khan, Shahid

    2015-09-01

    The proton-driven ATP synthase (FOF1) is comprised of two rotary, stepping motors (FO and F1) coupled by an elastic power transmission. The elastic compliance resides in the rotor module that includes the membrane-embedded FO c-ring. Proton transport by FO is firmly coupled to the rotation of the c-ring relative to other FO subunits (ab2). It drives ATP synthesis. We used a computational method to investigate the contribution of the c-ring to the total elastic compliance. We performed principal component analysis of conformational ensembles built using distance constraints from the bovine mitochondrial c-ring x-ray structure. Angular rotary twist, the dominant ringmore » motion, was estimated to show that the c-ring accounted in part for the measured compliance. Ring rotation was entrained to rotation of the external helix within each hairpin-shaped c-subunit in the ring. Ensembles of monomer and dimers extracted from complete c-rings showed that the coupling between collective ring and the individual subunit motions was independent of the size of the c-ring, which varies between organisms. Molecular determinants were identified by covariance analysis of residue coevolution and structural-alphabet-based local dynamics correlations. The residue coevolution gave a readout of subunit architecture. The dynamic couplings revealed that the hinge for both ring and subunit helix rotations was constructed from the proton-binding site and the adjacent glycine motif (IB-GGGG) in the midmembrane plane. IB-GGGG motifs were linked by long-range couplings across the ring, while intrasubunit couplings connected the motif to the conserved cytoplasmic loop and adjacent segments. The correlation with principal collective motions shows that the couplings underlie both ring rotary and bending motions. Noncontact couplings between IB-GGGG motifs matched the coevolution signal as well as contact couplings. The residue coevolution reflects the physiological importance of the dynamics

  8. A stable ATP binding to the nucleotide binding domain is important for reliable gating cycle in an ABC transporter CFTR

    PubMed Central

    Shimizu, Hiroyasu; Yu, Ying-Chun; Kono, Koichi; Kubota, Takahiro; Yasui, Masato; Li, Min

    2016-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, a member of ABC transporter superfamily, gates following ATP-dependent conformational changes of the nucleotide binding domains (NBD). Reflecting the hundreds of milliseconds duration of the channel open state corresponding to the dimerization of two NBDs, macroscopic WT-CFTR currents usually showed a fast, single exponential relaxation upon removal of cytoplasmic ATP. Mutations of tyrosine1219, a residue critical for ATP binding in second NBD (NBD2), induced a significant slow phase in the current relaxation, suggesting that weakening ATP binding affinity at NBD2 increases the probability of the stable open state. The slow phase was effectively diminished by a higher affinity ATP analogue. These data suggest that a stable binding of ATP to NBD2 is required for normal CFTR gating cycle, andthat the instability of ATP binding frequently halts the gating cycle in the open state presumably through a failure of ATP hydrolysis at NBD2. PMID:20628841

  9. A stable ATP binding to the nucleotide binding domain is important for reliable gating cycle in an ABC transporter CFTR.

    PubMed

    Shimizu, Hiroyasu; Yu, Ying-Chun; Kono, Koichi; Kubota, Takahiro; Yasui, Masato; Li, Min; Hwang, Tzyh-Chang; Sohma, Yoshiro

    2010-09-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, a member of ABC transporter superfamily, gates following ATP-dependent conformational changes of the nucleotide binding domains (NBD). Reflecting the hundreds of milliseconds duration of the channel open state corresponding to the dimerization of two NBDs, macroscopic WT-CFTR currents usually showed a fast, single exponential relaxation upon removal of cytoplasmic ATP. Mutations of tyrosine1219, a residue critical for ATP binding in second NBD (NBD2), induced a significant slow phase in the current relaxation, suggesting that weakening ATP binding affinity at NBD2 increases the probability of the stable open state. The slow phase was effectively diminished by a higher affinity ATP analogue. These data suggest that a stable binding of ATP to NBD2 is required for normal CFTR gating cycle, andthat the instability of ATP binding frequently halts the gating cycle in the open state presumably through a failure of ATP hydrolysis at NBD2. PMID:20628841

  10. Probing the ATP Site of GRP78 with Nucleotide Triphosphate Analogs.

    PubMed

    Hughes, Scott J; Antoshchenko, Tetyana; Chen, Yun; Lu, Hua; Pizarro, Juan C; Park, Hee-Won

    2016-01-01

    GRP78, a member of the ER stress protein family, can relocate to the surface of cancer cells, playing key roles in promoting cell proliferation and metastasis. GRP78 consists of two major functional domains: the ATPase and protein/peptide-binding domains. The protein/peptide-binding domain of cell-surface GRP78 has served as a novel functional receptor for delivering cytotoxic agents (e.g., a apoptosis-inducing peptide or taxol) across the cell membrane. Here, we report our study on the ATPase domain of GRP78 (GRP78ATPase), whose potential as a transmembrane delivery system of cytotoxic agents (e.g., ATP-based nucleotide triphosphate analogs) remains unexploited. As the binding of ligands (ATP analogs) to a receptor (GRP78ATPase) is a pre-requisite for internalization, we determined the binding affinities and modes of GRP78ATPase for ADP, ATP and several ATP analogs using surface plasmon resonance and x-ray crystallography. The tested ATP analogs contain one of the following modifications: the nitrogen at the adenine ring 7-position to a carbon atom (7-deazaATP), the oxygen at the β-γ bridge position to a carbon atom (AMPPCP), or the removal of the 2'-OH group (2'-deoxyATP). We found that 7-deazaATP displays an affinity and a binding mode that resemble those of ATP regardless of magnesium ion (Mg++) concentration, suggesting that GRP78 is tolerant to modifications at the 7-position. By comparison, AMPPCP's binding affinity was lower than ATP and Mg++-dependent, as the removal of Mg++ nearly abolished binding to GRP78ATPase. The AMPPCP-Mg++ structure showed evidence for the critical role of Mg++ in AMPPCP binding affinity, suggesting that while GRP78 is sensitive to modifications at the β-γ bridge position, these can be tolerated in the presence of Mg++. Furthermore, 2'-deoxyATP's binding affinity was significantly lower than those for all other nucleotides tested, even in the presence of Mg++. The 2'-deoxyATP structure showed the conformation of the bound

  11. Migration Type III

    NASA Astrophysics Data System (ADS)

    Artymowicz, Pawel

    2004-03-01

    Migration type IIIMigration of objects embedded in disks (and the accompanying eccentricity evolution) is becoming a major theme in planetary system formation.The underlying physics can be distilled into the notion of disk-planet coupling via Lindblad resonances, which launch waves, sometimes spectacular spiral shock waves in gas disks. The wave pattern exchanges angular momentum with the planet. That causes (i) migration, (ii) eccentricity evolution, and (iii) gap opening by sufficiently massive planets.A competing source of disk-planet interaction, the corotationaltorques, are much less conspicuous (corotation does not produce easilydetectable waves, as galaxy observers can attest) and have often been missed in the analysis of planet migration. If spiral waves are like waves at Goleta beach, then the corotation acts more like a stealthy riptide. Corotationalflows lie at the basis of a new, surprisingly rapid, mode of migration (type III),superseding the standard type II migration (with a gap), and revising the speed of type I migration (without a gap). The talk will contain results obtained at KITP, e.g., an analytical derivation of da/dt in type III motion. It will be illustrated by videos of high-resolution numerical simulations obtained with different implementations of the Piecewise Parabolic Method hydrodynamics.

  12. Mechanism of G551D-CFTR (cystic fibrosis transmembrane conductance regulator) potentiation by a high affinity ATP analog.

    PubMed

    Bompadre, Silvia G; Li, Min; Hwang, Tzyh-Chang

    2008-02-29

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel gated by ATP binding and hydrolysis at its nucleotide binding domains (NBD). The NBDs dimerize in a head-to-tail configuration, forming two ATP binding pockets (ABP) with the ATP molecules buried at the dimer interface. Previous studies have indicated that ABP2, formed by the Walker A and B motifs of NBD2 and the signature sequence of NBD1, is the site critical for the ATP-dependent opening of CFTR. The G551D mutation in ABP2, the third most common cystic fibrosis-associated mutation, abolishes ATP-dependent gating, resulting in an open probability that is approximately 100-fold lower than that of wild-type channels. Interestingly, we found that the ATP analog N6-(2-phenylethyl)-ATP (P-ATP) increases G551D currents mainly by increasing the open time of the channel. This effect is reduced when P-ATP is applied together with ATP, suggesting a competition between ATP and P-ATP for a common binding site. Introducing mutations that lower the nucleotide binding affinity at ABP2 did not alter significantly the effects of P-ATP on G551D-CFTR, whereas an equivalent mutation at ABP1 (consisting of the Walker A and B motifs of NBD1 and the signature sequence of NBD2) dramatically decreased the potency of P-ATP, indicating that ABP1 is the site where P-ATP binds to increase the activity of G551D-CFTR. These results substantiate the idea that nucleotide binding at ABP1 stabilizes the open channel conformation. Our observation that P-ATP enhances the G551D activity by binding at ABP1 implicates that ABP1 can potentially be a target for drugs to bind and increase the channel activity. PMID:18167357

  13. ATP-binding sites in brain p97/VCP (valosin-containing protein), a multifunctional AAA ATPase.

    PubMed Central

    Zalk, Ran; Shoshan-Barmatz, Varda

    2003-01-01

    VCP (valosin-containing protein) or p97 is a member of the AAA family (ATPases associated with a variety of cellular activities family), a diverse group of proteins sharing a key conserved AAA module containing duplicate putative ATP-binding sites. Although the functions of the AAA family are related to their putative ATP-binding sites, the binding of ATP to these sites has not yet been demonstrated. In the present study, the ATP-binding site(s) of brain VCP was characterized using the photoreactive ATP analogue, BzATP [3'- O -(4-benzoylbenzoyl)ATP]. Photo-activation of Bz-[alpha-(32)P]ATP resulted in its covalent binding to a 97-kDa purified soluble or membrane-associated protein, identified by amino acid sequencing as VCP. Bz-[alpha-(32)P]ATP covalently bound to the purified homo-hexameric VCP with an apparent high affinity (74-111 nM). A molar stoichiometry of 2.23+/-0.14 BzATP bound per homo-hexameric VCP (n =6) was determined using different methods for analysis of radiolabelling and protein determination. Nucleotides inhibited the binding of Bz-[alpha-(32)P]ATP to VCP with the following efficiency: BzATP>ATP>ADP>>adenosine 5'-[beta,gamma-imido]triphosphate>or=adenosine 5'-[beta,gamma-methylene]triphosphate, whereas AMP, GTP and CTP were ineffective. VCP was observed to possess very low ATPase activity, with nucleotide specificity similar to that for BzATP binding. Conformational changes induced by an alternating site mechanism for ATP binding are suggested as a molecular mechanism for coupling ATP binding to the diverse activities of the AAA family. PMID:12747802

  14. Use of ATP analogs to inhibit HIV-1 transcription.

    PubMed

    Narayanan, Aarthi; Sampey, Gavin; Van Duyne, Rachel; Guendel, Irene; Kehn-Hall, Kylene; Roman, Jessica; Currer, Robert; Galons, Hervé; Oumata, Nassima; Joseph, Benoît; Meijer, Laurent; Caputi, Massimo; Nekhai, Sergei; Kashanchi, Fatah

    2012-10-10

    Human immunodeficiency virus type 1 (HIV-1) is the etiological agent of AIDS. Chronic persistent infection is an important reason for the presence of "latent cell populations" even after Anti-Retroviral Therapy (ART). We have analyzed the effect of ATP analogs in inhibiting cdk9/T1 complex in infected cells. A third generation drug named CR8#13 is an effective inhibitor of Tat activated transcription. Following drug treatment, we observed a decreased loading of cdk9 onto the HIV-1 DNA. We found multiple novel cdk9/T1 complexes present in infected and uninfected cells with one complex being unique to infected cells. This complex is sensitive to CR8#13 in kinase assays. Treatment of PBMC with CR8#13 does not kill infected cells as compared to Flavopiridol. Interestingly, there is a difference in sensitivity of various clades to these analogs. Collectively, these results point to targeting novel complexes for inhibition of cellular proteins that are unique to infected cells. PMID:22771113

  15. An ATP System for Deep-Space Optical Communication

    NASA Technical Reports Server (NTRS)

    Lee, Shinhak; Irtuzm Gerardi; Alexander, James

    2008-01-01

    An acquisition, tracking, and pointing (ATP) system is proposed for aiming an optical-communications downlink laser beam from deep space. In providing for a direction reference, the concept exploits the mature technology of star trackers to eliminate the need for a costly and potentially hazardous laser beacon. The system would include one optical and two inertial sensors, each contributing primarily to a different portion of the frequency spectrum of the pointing signal: a star tracker (<10 Hz), a gyroscope (<50 Hz), and a precise fluid-rotor inertial angular-displacement sensor (sometimes called, simply, "angle sensor") for the frequency range >50 Hz. The outputs of these sensors would be combined in an iterative averaging process to obtain high-bandwidth, high-accuracy pointing knowledge. The accuracy of pointing knowledge obtainable by use of the system was estimated on the basis of an 8-cm-diameter telescope and known parameters of commercially available star trackers and inertial sensors: The single-axis pointing-knowledge error was found to be characterized by a standard deviation of 150 nanoradians - below the maximum value (between 200 and 300 nanoradians) likely to be tolerable in deep-space optical communications.

  16. Do ATP4- and Mg2+ bind stepwise to the F1-ATPase of Halobacterium saccharovorum?

    PubMed

    Schobert, B

    1998-06-01

    It is commonly believed that MgATP2- is the substrate of F1-ATPases and ATP4- acts as a competitive inhibitor. However, the velocity equation for such competitive inhibition is equivalent to that for a rapid equilibrium ordered binding mechanism in which ATP4- adds first and the binding of Mg2+ is dependent on the formation of the E x ATP4- complex. According to this ordered-binding model, solution formed MgATP2- is not recognized by the ATPase as a direct substrate, and the high-affinity binding of Mg2+ to the E x ATP4- complex is the key reaction towards the formation of the ternary complex. These models (and others) were tested with an F1- ATPase, isolated from Halobacterium saccharovorum, by evaluating the rate of ATP hydrolysis as a function of free [ATP4-] or free [Mg2+]. The rates were asymmetrical with respect to increasing [ATP4-] versus increasing [Mg2+]. For the ordered-binding alternative, a series of apparent dissociation constants were obtained for ATP4-(K(A)aPP), which decreased as [Mg2+] increased. From this family of K(A)aPP the true K(A) was retrieved by extrapolation to [Mg2+] = 0 and was found to be 0.2 mM. The dissociation constants for Mg2+, established from these experiments, were also apparent (K(B)aPP) and dependent on [ATP4-] as well as on the pH. The actual K(B) was established from a series of K(B)aPP by extrapolating to [ATP4-] = infinity and to the absence of competing protons, and was found to be 0.0041 mM. The pKa of the protonable group for Mg2+ binding is 8.2. For the competitive inhibition alternative, rearrangement of the constants and fitting to the velocity equation gave an actual binding constant for MgATP2- (K(EAB)) of 0.0016 mM and for ATP4- (K(EA)) of 0.2 mM. Decision between the two models has far-reaching mechanistic implications. In the competitive inhibition model MgATP2- binds with high affinity, but Mg2+ cannot bind once the E x ATP4- complex is formed, while in the ordered-binding model binding of Mg2+ requires that

  17. Response of ATP sulfurylase and serine acetyltransferase towards cadmium in hyperaccumulator Sedum alfredii Hance*

    PubMed Central

    Guo, Wei-dong; Liang, Jun; Yang, Xiao-e; Chao, Yue-en; Feng, Ying

    2009-01-01

    We studied the responses of the activities of adenosine-triphosphate (ATP) sulfurylase (ATPS) and serine acetyltransferase (SAT) to cadmium (Cd) levels and treatment time in hyperaccumulating ecotype (HE) Sedum alfredii Hance, as compared with its non-hyperaccumulating ecotype (NHE). The results show that plant growth was inhibited in NHE but promoted in HE when exposed to high Cd level. Cd concentrations in leaves and shoots rapidly increased in HE rather than in NHE, and they became much higher in HE than in NHE along with increasing treatment time and Cd supply levels. ATPS activity was higher in HE than in NHE in all Cd treatments, and increased with increasing Cd supply levels in both HE and NHE when exposed to Cd treatment within 8 h. However, a marked difference of ATPS activity between HE and NHE was found with Cd treatment for 168 h, where ATPS activity increased in HE but decreased in NHE. Similarly, SAT activity was higher in HE than in NHE at all Cd treatments, but was more sensitive in NHE than in HE. Both ATPS and SAT activities in NHE leaves tended to decrease with increasing treatment time after 8 h at all Cd levels. The results reveal the different responses in sulfur assimilation enzymes and Cd accumulation between HE and NHE. With increasing Cd stress, the activities of sulfur assimilation enzymes (ATPS and SAT) were induced in HE, which may contribute to Cd accumulation in the hyperaccumulator Sedum alfredii Hance. PMID:19353742

  18. Response of ATP sulfurylase and serine acetyltransferase towards cadmium in hyperaccumulator Sedum alfredii Hance.

    PubMed

    Guo, Wei-dong; Liang, Jun; Yang, Xiao-e; Chao, Yue-en; Feng, Ying

    2009-04-01

    We studied the responses of the activities of adenosine-triphosphate (ATP) sulfurylase (ATPS) and serine acetyltransferase (SAT) to cadmium (Cd) levels and treatment time in hyperaccumulating ecotype (HE) Sedum alfredii Hance, as compared with its non-hyperaccumulating ecotype (NHE). The results show that plant growth was inhibited in NHE but promoted in HE when exposed to high Cd level. Cd concentrations in leaves and shoots rapidly increased in HE rather than in NHE, and they became much higher in HE than in NHE along with increasing treatment time and Cd supply levels. ATPS activity was higher in HE than in NHE in all Cd treatments, and increased with increasing Cd supply levels in both HE and NHE when exposed to Cd treatment within 8 h. However, a marked difference of ATPS activity between HE and NHE was found with Cd treatment for 168 h, where ATPS activity increased in HE but decreased in NHE. Similarly, SAT activity was higher in HE than in NHE at all Cd treatments, but was more sensitive in NHE than in HE. Both ATPS and SAT activities in NHE leaves tended to decrease with increasing treatment time after 8 h at all Cd levels. The results reveal the different responses in sulfur assimilation enzymes and Cd accumulation between HE and NHE. With increasing Cd stress, the activities of sulfur assimilation enzymes (ATPS and SAT) were induced in HE, which may contribute to Cd accumulation in the hyperaccumulator Sedum alfredii Hance. PMID:19353742

  19. Comparison of ATP and Ergosterol as Indicators of Fungal Biomass Associated with Decomposing Leaves in Streams

    PubMed Central

    Suberkropp, K.; Gessner, M. O.; Chauvet, E.

    1993-01-01

    ATP and ergosterol were compared as indicators of fungal biomass associated with leaves decomposing in laboratory microcosms and streams. In all studies, the sporulation rates of the fungi colonizing leaves were also determined to compare patterns of fungal reproductive activity with patterns of mycelial growth. During leaf degradation, ATP concentrations exhibited significant, positive correlations with ergosterol concentrations in the laboratory and when leaves had been air dried prior to being submerged in a stream. However, when freshly shed leaves were submerged in a stream, concentrations of ATP and ergosterol were negatively correlated during degradation. This appeared to be due to the persistence of leaf-derived ATP in freshly shed leaves during the first 1 to 2 weeks in the stream. Estimates of fungal biomass from ergosterol concentrations of leaf litter were one to three times those calculated from ATP concentrations. ATP, ergosterol, and sporulation data generally provided similar information about the fungi associated with decomposing leaves in streams during periods when fungi were growing. Ergosterol concentrations provide a more accurate indication of fungal biomass in situations in which other organisms make significant contributions to ATP pools. PMID:16349069

  20. Structure and conformational states of the bovine mitochondrial ATP synthase by cryo-EM

    PubMed Central

    Zhou, Anna; Rohou, Alexis; Schep, Daniel G; Bason, John V; Montgomery, Martin G; Walker, John E; Grigorieff, Nikolaus; Rubinstein, John L

    2015-01-01

    Adenosine triphosphate (ATP), the chemical energy currency of biology, is synthesized in eukaryotic cells primarily by the mitochondrial ATP synthase. ATP synthases operate by a rotary catalytic mechanism where proton translocation through the membrane-inserted FO region is coupled to ATP synthesis in the catalytic F1 region via rotation of a central rotor subcomplex. We report here single particle electron cryomicroscopy (cryo-EM) analysis of the bovine mitochondrial ATP synthase. Combining cryo-EM data with bioinformatic analysis allowed us to determine the fold of the a subunit, suggesting a proton translocation path through the FO region that involves both the a and b subunits. 3D classification of images revealed seven distinct states of the enzyme that show different modes of bending and twisting in the intact ATP synthase. Rotational fluctuations of the c8-ring within the FO region support a Brownian ratchet mechanism for proton-translocation-driven rotation in ATP synthases. DOI: http://dx.doi.org/10.7554/eLife.10180.001 PMID:26439008

  1. ATP/ADP Ratio, the Missed Connection between Mitochondria and the Warburg Effect

    PubMed Central

    Maldonado, Eduardo N.; Lemasters, John J.

    2014-01-01

    Non-proliferating cells generate the bulk of cellular ATP by fully oxidizing respiratory substrates in mitochondria. Respiratory substrates cross the mitochondrial outer membrane through only one channel, the voltage dependent anion channel (VDAC). Once in the matrix, respiratory substrates are oxidized in the tricarboxylic acid cycle to generate mostly NADH that is further oxidized in the respiratory chain to generate a proton motive force comprised mainly of membrane potential (ΔΨ) to synthesize ATP. Mitochondrial ΔΨ then drives release of ATP−4 from the matrix in exchange for ADP−3 in the cytosol via the adenine nucleotide translocator (ANT) located in the mitochondrial inner membrane. Thus, mitochondrial function in non-proliferating cells drives a high cytosolic ATP/ADP ratio, essential to inhibit glycolysis. By contrast, the bioenergetics of the Warburg phenotype of proliferating cells is characterized by enhanced aerobic glycolysis and suppression of mitochondrial metabolism. Suppressed mitochondrial function leads to lower production of mitochondrial ATP and hence lower cytosolic ATP/ADP ratios that favor enhanced glycolysis. Thus, cytosolic ATP/ADP ratio is a key feature that determines if cell metabolism is predominantly oxidative or glycolytic. Here, we describe two novel mechanisms to explain the suppression of mitochondrial metabolism in cancer cells: the relative closure of VDAC by free tubulin and inactivation of ANT. Both mechanisms contribute to low ATP/ADP ratios that activate glycolysis. PMID:25229666

  2. Wilson Disease Protein ATP7B Utilizes Lysosomal Exocytosis to Maintain Copper Homeostasis

    PubMed Central

    Polishchuk, Elena V.; Concilli, Mafalda; Iacobacci, Simona; Chesi, Giancarlo; Pastore, Nunzia; Piccolo, Pasquale; Paladino, Simona; Baldantoni, Daniela; van IJzendoorn, Sven C.D.; Chan, Jefferson; Chang, Christopher J.; Amoresano, Angela; Pane, Francesca; Pucci, Piero; Tarallo, Antonietta; Parenti, Giancarlo; Brunetti-Pierri, Nicola; Settembre, Carmine; Ballabio, Andrea; Polishchuk, Roman S.

    2014-01-01

    Summary Copper is an essential yet toxic metal and its overload causes Wilson disease, a disorder due to mutations in copper transporter ATP7B. To remove excess copper into the bile, ATP7B traffics toward canalicular area of hepatocytes. However, the trafficking mechanisms of ATP7B remain elusive. Here, we show that, in response to elevated copper, ATP7B moves from the Golgi to lysosomes and imports metal into their lumen. ATP7B enables lysosomes to undergo exocytosis through the interaction with p62 subunit of dynactin that allows lysosome translocation toward the canalicular pole of hepatocytes. Activation of lysosomal exocytosis stimulates copper clearance from the hepatocytes and rescues the most frequent Wilson-disease-causing ATP7B mutant to the appropriate functional site. Our findings indicate that lysosomes serve as an important intermediate in ATP7B trafficking, whereas lysosomal exocytosis operates as an integral process in copper excretion and hence can be targeted for therapeutic approaches to combat Wilson disease. PMID:24909901

  3. Selective Migration of Subpopulations of Bone Marrow Cells along an SDF-1α and ATP Gradient.

    PubMed

    Laupheimer, Michael; Skorska, Anna; Große, Jana; Tiedemann, Gudrun; Steinhoff, Gustav; David, Robert; Lux, Cornelia A

    2014-01-01

    Both stem cell chemokine stromal cell-derived factor-1α (SDF-1α) and extracellular nucleotides such as adenosine triphosphate (ATP) are increased in ischemic myocardium. Since ATP has been reported to influence cell migration, we analysed the migratory response of bone marrow cells towards a combination of SDF-1 and ATP. Total nucleated cells (BM-TNCs) were isolated from bone marrow of cardiac surgery patients. Migration assays were performed in vitro. Subsequently, migrated cells were subjected to multicolor flow cytometric analysis of CD133, CD34, CD117, CD184, CD309, and CD14 expression. BM-TNCs migrated significantly towards a combination of SDF-1 and ATP. The proportions of CD34+ cells as well as subpopulations coexpressing multiple stem cell markers were selectively enhanced after migration towards SDF-1 or SDF-1 + ATP. After spontaneous migration, significantly fewer stem cells and CD184+ cells were detected. Direct incubation with SDF-1 led to a reduction of CD184+ but not stem cell marker-positive cells, while incubation with ATP significantly increased CD14+ percentage. In summary, we found that while a combination of SDF-1 and ATP elicited strong migration of BM-TNCs in vitro, only SDF-1 was responsible for selective attraction of hematopoietic stem cells. Meanwhile, spontaneous migration of stem cells was lower compared to BM-TNCs or monocytes. PMID:25610653

  4. Wilson disease protein ATP7B utilizes lysosomal exocytosis to maintain copper homeostasis.

    PubMed

    Polishchuk, Elena V; Concilli, Mafalda; Iacobacci, Simona; Chesi, Giancarlo; Pastore, Nunzia; Piccolo, Pasquale; Paladino, Simona; Baldantoni, Daniela; van IJzendoorn, Sven C D; Chan, Jefferson; Chang, Christopher J; Amoresano, Angela; Pane, Francesca; Pucci, Piero; Tarallo, Antonietta; Parenti, Giancarlo; Brunetti-Pierri, Nicola; Settembre, Carmine; Ballabio, Andrea; Polishchuk, Roman S

    2014-06-23

    Copper is an essential yet toxic metal and its overload causes Wilson disease, a disorder due to mutations in copper transporter ATP7B. To remove excess copper into the bile, ATP7B traffics toward canalicular area of hepatocytes. However, the trafficking mechanisms of ATP7B remain elusive. Here, we show that, in response to elevated copper, ATP7B moves from the Golgi to lysosomes and imports metal into their lumen. ATP7B enables lysosomes to undergo exocytosis through the interaction with p62 subunit of dynactin that allows lysosome translocation toward the canalicular pole of hepatocytes. Activation of lysosomal exocytosis stimulates copper clearance from the hepatocytes and rescues the most frequent Wilson-disease-causing ATP7B mutant to the appropriate functional site. Our findings indicate that lysosomes serve as an important intermediate in ATP7B trafficking, whereas lysosomal exocytosis operates as an integral process in copper excretion and hence can be targeted for therapeutic approaches to combat Wilson disease. PMID:24909901

  5. Extracellular ATP inhibits Schwann cell dedifferentiation and proliferation in an ex vivo model of Wallerian degeneration

    SciTech Connect

    Shin, Youn Ho; Lee, Seo Jin; Jung, Junyang

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer ATP-treated sciatic explants shows the decreased expression of p75NGFR. Black-Right-Pointing-Pointer Extracellular ATP inhibits the expression of phospho-ERK1/2. Black-Right-Pointing-Pointer Lysosomal exocytosis is involved in Schwann cell dedifferentiation. Black-Right-Pointing-Pointer Extracellular ATP blocks Schwann cell proliferation in sciatic explants. -- Abstract: After nerve injury, Schwann cells proliferate and revert to a phenotype that supports nerve regeneration. This phenotype-changing process can be viewed as Schwann cell dedifferentiation. Here, we investigated the role of extracellular ATP in Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Using several markers of Schwann cell dedifferentiation and proliferation in sciatic explants, we found that extracellular ATP inhibits Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Furthermore, the blockage of lysosomal exocytosis in ATP-treated sciatic explants is sufficient to induce Schwann cell dedifferentiation. Together, these findings suggest that ATP-induced lysosomal exocytosis may be involved in Schwann cell dedifferentiation.

  6. ATP-sensitive potassium channels: uncovering novel targets for treating depression.

    PubMed

    Fan, Yi; Kong, Hui; Ye, Xinhai; Ding, Jianhua; Hu, Gang

    2016-07-01

    ATP-sensitive potassium (K-ATP) channels have been shown to couple membrane electrical activity to energy metabolism in a variety of cells and are important in several physiological systems. In the brain, K-ATP channels are strongly expressed in the neuronal circuitry. The distributional profile and functional significance of K-ATP channels suggest that they may be involved in stress-induced depression. First, we showed that chronic mild stress (CMS) significantly increased the expression of hippocampal Kir6.2 and Kir6.1 subunits of K-ATP channels. Next, using Kir6.2 knockout (Kir6.2(-/-)) mice, we presented that Kir6.2 deficiency resulted in antidepressant-like behaviors under non-stress conditions, but aggravated depressive behaviors accompanied by the loss of CA3 neuron and the reduction of brain-derived neurotrophic factor in hippocampus under chronic stress. Finally, we demonstrated that the K-ATP channel opener iptakalim, as well as a classical antidepressant fluoxetine, can reverse CMS-induced depression-related behaviors and counteract the deleterious effects of stress on hippocampus in wild-type mice, but only partially alleviate these symptoms in Kir6.2(-/-) mice. Collectively, our findings demonstrate that K-ATP channels are involved in the pathogenesis of depression and may be a promising target for the therapy of depression. PMID:26289962

  7. Impairment of vesicular ATP release affects glucose metabolism and increases insulin sensitivity

    PubMed Central

    Sakamoto, Shohei; Miyaji, Takaaki; Hiasa, Miki; Ichikawa, Reiko; Uematsu, Akira; Iwatsuki, Ken; Shibata, Atsushi; Uneyama, Hisayuki; Takayanagi, Ryoichi; Yamamoto, Akitsugu; Omote, Hiroshi; Nomura, Masatoshi; Moriyama, Yoshinori

    2014-01-01

    Neuroendocrine cells store ATP in secretory granules and release it along with hormones that may trigger a variety of cellular responses in a process called purinergic chemical transmission. Although the vesicular nucleotide transporter (VNUT) has been shown to be involved in vesicular storage and release of ATP, its physiological relevance in vivo is far less well understood. In Vnut knockout (Vnut−/−) mice, we found that the loss of functional VNUT in adrenal chromaffin granules and insulin granules in the islets of Langerhans led to several significant effects. Vesicular ATP accumulation and depolarization-dependent ATP release were absent in the chromaffin granules of Vnut−/− mice. Glucose-responsive ATP release was also absent in pancreatic β-cells in Vnut−/− mice, while glucose-responsive insulin secretion was enhanced to a greater extent than that in wild-type tissue. Vnut−/− mice exhibited improved glucose tolerance and low blood glucose upon fasting due to increased insulin sensitivity. These results demonstrated an essential role of VNUT in vesicular storage and release of ATP in neuroendocrine cells in vivo and suggest that vesicular ATP and/or its degradation products act as feedback regulators in catecholamine and insulin secretion, thereby regulating blood glucose homeostasis. PMID:25331291

  8. Selective Migration of Subpopulations of Bone Marrow Cells along an SDF-1α and ATP Gradient

    PubMed Central

    Laupheimer, Michael; Skorska, Anna; Große, Jana; Tiedemann, Gudrun; Steinhoff, Gustav; David, Robert; Lux, Cornelia A.

    2014-01-01

    Both stem cell chemokine stromal cell-derived factor-1α (SDF-1α) and extracellular nucleotides such as adenosine triphosphate (ATP) are increased in ischemic myocardium. Since ATP has been reported to influence cell migration, we analysed the migratory response of bone marrow cells towards a combination of SDF-1 and ATP. Total nucleated cells (BM-TNCs) were isolated from bone marrow of cardiac surgery patients. Migration assays were performed in vitro. Subsequently, migrated cells were subjected to multicolor flow cytometric analysis of CD133, CD34, CD117, CD184, CD309, and CD14 expression. BM-TNCs migrated significantly towards a combination of SDF-1 and ATP. The proportions of CD34+ cells as well as subpopulations coexpressing multiple stem cell markers were selectively enhanced after migration towards SDF-1 or SDF-1 + ATP. After spontaneous migration, significantly fewer stem cells and CD184+ cells were detected. Direct incubation with SDF-1 led to a reduction of CD184+ but not stem cell marker-positive cells, while incubation with ATP significantly increased CD14+ percentage. In summary, we found that while a combination of SDF-1 and ATP elicited strong migration of BM-TNCs in vitro, only SDF-1 was responsible for selective attraction of hematopoietic stem cells. Meanwhile, spontaneous migration of stem cells was lower compared to BM-TNCs or monocytes. PMID:25610653

  9. L-ATP is recognized by some cellular and viral enzymes: does chance drive enzymic enantioselectivity?

    PubMed Central

    Verri, A; Montecucco, A; Gosselin, G; Boudou, V; Imbach, J L; Spadari, S; Focher, F

    1999-01-01

    We demonstrate that l-ATP is recognized by some enzymes that are involved in the synthesis of nucleotides and nucleic acids. l-ATP, as well as its natural d-enantiomer, acts as a phosphate donor in the reaction catalysed by human deoxycytidine kinase, whereas it is not recognized by either enantioselective human thymidine kinase or non-enantioselective herpes virus thymidine kinase. l-ATP strongly inhibits (Ki 80 microM) the synthesis of RNA primers catalysed by DNA primase associated with human DNA polymerase alpha, whereas RNA synthesis catalysed by Escherichia coli RNA polymerase is completely unaffected. Moreover, l-ATP competitively inhibits ATP-dependent T4 DNA ligase (Ki 25 microM), suggesting that it interacts with the ATP-binding site of the enzyme. Kinetic studies demonstrated that l-ATP cannot be used as a cofactor in the ligase-catalysed joining reaction. On the other hand, l-AMP is used by T4 DNA ligase to catalyse the reverse reaction, even though a high level of intermediate circular nicked DNA molecules accumulates. Our results suggest that a lack of enantioselectivity of enzymes is more common than was believed a few years ago, and, given the absence of selective constraints against l-nucleosides in Nature, this may depend on chance more than on evolutionary strategy. PMID:9895305

  10. Fine-tuned ATP signals are acute mediators in osteocyte mechanotransduction.

    PubMed

    Kringelbach, Tina M; Aslan, Derya; Novak, Ivana; Ellegaard, Maria; Syberg, Susanne; Andersen, Christina K B; Kristiansen, Kim A; Vang, Ole; Schwarz, Peter; Jørgensen, Niklas R

    2015-12-01

    Osteocytes are considered the primary mechanosensors of bone, but the signaling pathways they apply in mechanotransduction are still incompletely investigated and characterized. A growing body of data strongly indicates that P2 receptor signaling among osteoblasts and osteoclasts has regulatory effects on bone remodeling. Therefore, we hypothesized that ATP signaling is also applied by osteocytes in mechanotransduction. We applied a short fluid pulse on MLO-Y4 osteocyte-like cells during real-time detection of ATP and demonstrated that mechanical stimulation activates the acute release of ATP and that these acute ATP signals are fine-tuned according to the magnitude of loading. ATP release was then challenged by pharmacological inhibitors, which indicated a vesicular release pathway for acute ATP signals. Finally, we showed that osteocytes express functional P2X2 and P2X7 receptors and respond to even low concentrations of nucleotides by increasing intracellular calcium concentration. These results indicate that in osteocytes, vesicular ATP release is an acute mediator of mechanical signals and the magnitude of loading. These and previous results, therefore, implicate purinergic signaling as an early signaling pathway in osteocyte mechanotransduction. PMID:26327582

  11. Imaging of ATP membrane transport with dual micro-disk electrodes and scanning electrochemical microscopy.

    PubMed

    Kueng, Angelika; Kranz, Christine; Mizaikoff, Boris

    2005-08-15

    Extracellular adenosine-5'-triphosphate (ATP) is involved in a variety of relevant regulatory mechanisms at a cellular level and has therefore been focus of extensive research. One of the major challenges associated with measuring this key regulatory analyte is the ability to detect and localize extracellular ATP with sufficient spatial and temporal resolution in physiological environments. In this study, scanning electrochemical microscopy (SECM) utilizing an amperometric micro-biosensor based on co-immobilization of the enzymes glucose oxidase and hexokinase is applied for imaging ATP transport through a porous polycarbonate membrane under physiologically relevant conditions. The enzymatic biosensor operates on competitive consumption of the substrate glucose between the immobilized enzymes glucose oxidase and hexokinase involving ATP as a co-substrate. Quantitative determination of the ATP concentration is based on a linear correlation between the glucose consumption and the ATP level. Integration of the amperometric ATP micro-biosensor into a dual micro-disk electrode configuration is achieved by immobilizing the enzymes at one of the micro-disk electrodes while the second disk serves as an unmodified amperometric probe for controlled positioning of the micro-biosensor in close proximity to the sample surface enabling quantification of the obtained current signal. PMID:16023962

  12. Catalytic signal amplification for the discrimination of ATP and ADP using functionalised gold nanoparticles.

    PubMed

    Pezzato, Cristian; Chen, Jack L-Y; Galzerano, Patrizia; Salvi, Michela; Prins, Leonard J

    2016-07-12

    Diagnostic assays that incorporate a signal amplification mechanism permit the detection of analytes with enhanced selectivity. Herein, we report a gold nanoparticle-based chemical system able to differentiate ATP from ADP by means of catalytic signal amplification. The discrimination between ATP and ADP is of relevance for the development of universal assays for the detection of enzymes which consume ATP. For example, protein kinases are a class of enzymes critical for the regulation of cellular functions, and act to modulate the activity of other proteins by transphosphorylation, transferring a phosphate group from ATP to give ADP as a byproduct. The system described here exploits the ability of cooperative catalytic head groups on gold nanoparticles to very efficiently catalyze chromogenic reactions such as the transphosphorylation of 2-hydroxypropyl-4-nitrophenyl phosphate (HPNPP). A series of chromogenic substrates have been synthesized and evaluated by means of Michaelis-Menten kinetics (compounds 2, 4-6). 2-Hydroxypropyl-(3-trifluoromethyl-4-nitro)phenyl phosphate (5) was found to display higher reactivity (kcat) and higher binding affinity (KM) when compared to HPNPP. This higher binding affinity allows phosphate 5 to compete with ATP and ADP to different extents for binding on the monolayer surface, thus enabling a catalytically amplified signal only when ATP is absent. Overall, this represents a viable new approach for monitoring the conversion of ATP into ADP with high sensitivity. PMID:27336846

  13. Crystallization of the c14-rotor of the chloroplast ATP synthase reveals that it contains pigments

    PubMed Central

    Varco-Merth, Benjamin; Fromme, Raimund; Wang, Meitian; Fromme, Petra

    2012-01-01

    The ATP synthase is one of the most important enzymes on earth as it couples the transmembrane electrochemical potential of protons to the synthesis of ATP from ADP and inorganic phosphate, providing the main ATP source of almost all higher life on earth. During ATP synthesis, stepwise protonation of a conserved carboxylate on each protein subunit of an oligomeric ring of 10–15 c-subunits is commonly thought to drive rotation of the rotor moiety (c10–14γε) relative to stator moiety (α3β3δab2). Here we report the isolation and crystallization of the c14-ring of subunit c from the spinach chloroplast enzyme diffracting as far as 2.8 Å. Though ATP synthase was not previously known to contain any pigments, the crystals of the c-subunit possessed a strong yellow color. The pigment analysis revealed that they contain 1 chlorophyll and 2 carotenoids, thereby showing for the first time that the chloroplast ATP synthase contains cofactors, leading to the question of the possible roles of the functions of the pigments in the chloroplast ATP synthase. PMID:18515064

  14. Homocysteine induces cardiac hypertrophy by up-regulating ATP7a expression

    PubMed Central

    Cao, Zhanwei; Zhang, Yanzhou; Sun, Tongwen; Zhang, Shuguang; Yu, Weiya; Zhu, Jie

    2015-01-01

    Aims: The aim of the study is to investigate the molecular mechanism by which homocysteine (Hcy) induces cardiac hypertrophy. Methods: Primary cardiomyocytes were obtained from baby Sprague-Dawley rats within 3 days after birth. Flow cytometry was used to measure cell sizes. Quantitative real-time polymerase chain reaction was performed to measure the expression of β-myosin heavy chain and atrial natriuretic peptide genes. Western blotting assay was employed to determine ATP7a protein expression. Cytochrome C oxidase (COX) activity test was used to evaluate the activity of COX. Atomic absorption spectroscopy was performed to determine copper content. siRNAs were used to target-silence the expression of ATP7a. Results: Hcy induced cardiac hypertrophy and increased the expression of cardiac hypertrophy-related genes. ATP7a was a key factor in cardiac hypertrophy induced by Hcy. Reduced ATP7a expression inhibited cardiac hypertrophy induced by Hcy. Elevated ATP7a expression induced by Hcy inhibited COX activity. Enhanced ATP7a expression inhibited COX activity by lowering intracellular copper content. Conclusions: Hcy elevates ATP7a protein expression, reduces copper content, and lowers COX activity, finally leading to cardiac hypertrophy. PMID:26722473

  15. The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria*

    PubMed Central

    Bhattacharjee, Ashima; Yang, Haojun; Duffy, Megan; Robinson, Emily; Conrad-Antoville, Arianrhod; Lu, Ya-Wen; Capps, Tony; Braiterman, Lelita; Wolfgang, Michael; Murphy, Michael P.; Yi, Ling; Kaler, Stephen G.; Lutsenko, Svetlana; Ralle, Martina

    2016-01-01

    Copper-transporting ATPase ATP7A is essential for mammalian copper homeostasis. Loss of ATP7A activity is associated with fatal Menkes disease and various other pathologies. In cells, ATP7A inactivation disrupts copper transport from the cytosol into the secretory pathway. Using fibroblasts from Menkes disease patients and mouse 3T3-L1 cells with a CRISPR/Cas9-inactivated ATP7A, we demonstrate that ATP7A dysfunction is also damaging to mitochondrial redox balance. In these cells, copper accumulates in nuclei, cytosol, and mitochondria, causing distinct changes in their redox environment. Quantitative imaging of live cells using GRX1-roGFP2 and HyPer sensors reveals highest glutathione oxidation and elevation of H2O2 in mitochondria, whereas the redox environment of nuclei and the cytosol is much less affected. Decreasing the H2O2 levels in mitochondria with MitoQ does not prevent glutathione oxidation; i.e. elevated copper and not H2O2 is a primary cause of glutathione oxidation. Redox misbalance does not significantly affect mitochondrion morphology or the activity of respiratory complex IV but markedly increases cell sensitivity to even mild glutathione depletion, resulting in loss of cell viability. Thus, ATP7A activity protects mitochondria from excessive copper entry, which is deleterious to redox buffers. Mitochondrial redox misbalance could significantly contribute to pathologies associated with ATP7A inactivation in tissues with paradoxical accumulation of copper (i.e. renal epithelia). PMID:27226607

  16. Gating of the CFTR Cl- channel by ATP-driven nucleotide-binding domain dimerisation.

    PubMed

    Hwang, Tzyh-Chang; Sheppard, David N

    2009-05-15

    The cystic fibrosis transmembrane conductance regulator (CFTR) plays a fundamental role in fluid and electrolyte transport across epithelial tissues. Based on its structure, function and regulation, CFTR is an ATP-binding cassette (ABC) transporter. These transporters are assembled from two membrane-spanning domains (MSDs) and two nucleotide-binding domains (NBDs). In the vast majority of ABC transporters, the NBDs form a common engine that utilises the energy of ATP hydrolysis to pump a wide spectrum of substrates through diverse transmembrane pathways formed by the MSDs. By contrast, in CFTR the MSDs form a pathway for passive anion flow that is gated by cycles of ATP binding and hydrolysis by the NBDs. Here, we consider how the interaction of ATP with two ATP-binding sites, formed by the NBDs, powers conformational changes in CFTR structure to gate the channel pore. We explore how conserved sequences from both NBDs form ATP-binding sites at the interface of an NBD dimer and highlight the distinct roles that each binding site plays during the gating cycle. Knowledge of how ATP gates the CFTR Cl- channel is critical for understanding CFTR's physiological role, its malfunction in disease and the mechanism of action of small molecules that modulate CFTR channel gating. PMID:19332488

  17. Gating of the CFTR Cl− channel by ATP-driven nucleotide-binding domain dimerisation

    PubMed Central

    Hwang, Tzyh-Chang; Sheppard, David N

    2009-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) plays a fundamental role in fluid and electrolyte transport across epithelial tissues. Based on its structure, function and regulation, CFTR is an ATP-binding cassette (ABC) transporter. These transporters are assembled from two membrane-spanning domains (MSDs) and two nucleotide-binding domains (NBDs). In the vast majority of ABC transporters, the NBDs form a common engine that utilises the energy of ATP hydrolysis to pump a wide spectrum of substrates through diverse transmembrane pathways formed by the MSDs. By contrast, in CFTR the MSDs form a pathway for passive anion flow that is gated by cycles of ATP binding and hydrolysis by the NBDs. Here, we consider how the interaction of ATP with two ATP-binding sites, formed by the NBDs, powers conformational changes in CFTR structure to gate the channel pore. We explore how conserved sequences from both NBDs form ATP-binding sites at the interface of an NBD dimer and highlight the distinct roles that each binding site plays during the gating cycle. Knowledge of how ATP gates the CFTR Cl− channel is critical for understanding CFTR's physiological role, its malfunction in disease and the mechanism of action of small molecules that modulate CFTR channel gating. PMID:19332488

  18. Impairment of vesicular ATP release affects glucose metabolism and increases insulin sensitivity.

    PubMed

    Sakamoto, Shohei; Miyaji, Takaaki; Hiasa, Miki; Ichikawa, Reiko; Uematsu, Akira; Iwatsuki, Ken; Shibata, Atsushi; Uneyama, Hisayuki; Takayanagi, Ryoichi; Yamamoto, Akitsugu; Omote, Hiroshi; Nomura, Masatoshi; Moriyama, Yoshinori

    2014-01-01

    Neuroendocrine cells store ATP in secretory granules and release it along with hormones that may trigger a variety of cellular responses in a process called purinergic chemical transmission. Although the vesicular nucleotide transporter (VNUT) has been shown to be involved in vesicular storage and release of ATP, its physiological relevance in vivo is far less well understood. In Vnut knockout (Vnut(-/-)) mice, we found that the loss of functional VNUT in adrenal chromaffin granules and insulin granules in the islets of Langerhans led to several significant effects. Vesicular ATP accumulation and depolarization-dependent ATP release were absent in the chromaffin granules of Vnut(-/-) mice. Glucose-responsive ATP release was also absent in pancreatic β-cells in Vnut(-/-) mice, while glucose-responsive insulin secretion was enhanced to a greater extent than that in wild-type tissue. Vnut(-/-) mice exhibited improved glucose tolerance and low blood glucose upon fasting due to increased insulin sensitivity. These results demonstrated an essential role of VNUT in vesicular storage and release of ATP in neuroendocrine cells in vivo and suggest that vesicular ATP and/or its degradation products act as feedback regulators in catecholamine and insulin secretion, thereby regulating blood glucose homeostasis. PMID:25331291

  19. Biosynthesis reaction mechanism and kinetics of deoxynucleoside triphosphates, dATP and dGTP.

    PubMed

    Bao, Jie; Ryu, Dewey D Y

    2005-02-20

    The enzyme reaction mechanism and kinetics for biosyntheses of deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) from the corresponding deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP) catalyzed by pyruvate kinase were studied. A kinetic model for this synthetic reaction was developed based on a Bi-Bi random rapid equilibrium mechanism. Kinetic constants involved in this pyruvate kinase catalyzed phosphorylation reactions of deoxynucleoside diphosphates including the maximum reaction velocity, Michaelis-Menten constants, and inhibition constants for dATP and dGTP biosyntheses were experimentally determined. These kinetic constants for dATP and dGTP biosyntheses are of the same order of magnitude but significantly different between the two reactions. Kinetic constants involved in ATP and GTP biosyntheses as reported in literature are about one order of magnitude different from those involved in dATP and dGTP biosyntheses. This enzyme reaction requires Mg2+ ion and the optimal Mg2+ concentration was also determined. The experimental results showed a very good agreement with the simulation results obtained from the kinetic model developed. This kinetic model can be applied to the practical application of a pyruvate kinase reaction system for production of dATP and dGTP. There is a significant advantage of using enzymatic biosyntheses of dATP and dGTP as compared to the chemical method that has been in commercial use. PMID:15643625

  20. ATP monitoring technology for microbial growth control in potable water systems

    NASA Astrophysics Data System (ADS)

    Whalen, Patrick A.; Whalen, Philip J.; Cairns, James E.

    2006-05-01

    ATP (Adenosine Triphosphate) is the primary energy transfer molecule present in all living biological cells on Earth. ATP cannot be produced or maintained by anything but a living organism, and as such, its measurement is a direct indication of biological activity. The main advantage of ATP as a biological indicator is the speed of the analysis - from collecting the sample to obtaining the result, only minutes are required. The technology to measure ATP is already widely utilized to verify disinfection efficacy in the food industry and is also commonly applied in industrial water processes such as cooling water systems to monitor microbial growth and biocide applications. Research has indicated that ATP measurement technology can also play a key role in such important industries as potable water distribution and biological wastewater treatment. As will be detailed in this paper, LuminUltra Technologies has developed and applied ATP measurement technologies designed for any water type, and as such can provide a method to rapidly and accurately determine the level of biological activity in drinking water supplies. Because of its speed and specificity to biological activity, ATP measurement can play a key role in defending against failing drinking water quality, including those encountered during routine operation and also bioterrorism.

  1. The reaction process of firefly bioluminescence triggered by photolysis of caged-ATP.

    PubMed

    Kageyama, Takeshi; Tanaka, Masatoshi; Sekiya, Takao; Ohno, Shin-Ya; Wada, Naohisa

    2011-01-01

    The reaction process of firefly bioluminescence was studied by photolyzing caged-ATP to adenosine triphosphate (ATP) within 100 ms. The intensity of luminescence increases markedly to reach a maximum within 1 s, maintains almost the same intensity up to 5 s and then decays monotonically. The rise γ(1) and decay γ(2) rate constants were determined to be about 5 s(-1) and 1 × 10(-2) s(-1), respectively, so as to phenomenologically fit the time course. A second luminescence peak appears after around 350 s. The dependence of the rate constants on the concentrations of reactants and a viscous reagent revealed that two kinds of reaction contribute the observed time course: (1) an intrinsic reaction by ATP photolyzed from caged-ATP that is already trapped in luciferase; and (2) a diffusion-controlled reaction by free ATP in the buffer solution outside luciferase. Numerical analysis based on reaction kinetics related γ(1) and γ(2) to the rate constants of a three-step reaction model, and accurately described the effects of concentration of reactants and a viscous reagent on the time courses of bioluminescence. Thus, it has been clearly concluded that the binding mode of caged-ATP at the catalytic center of luciferase is very different from that of ATP. PMID:21208215

  2. Thiol modulation of the chloroplast ATP synthase is dependent on the energization of thylakoid membranes.

    PubMed

    Konno, Hiroki; Nakane, Takeshi; Yoshida, Masasuke; Ueoka-Nakanishi, Hanayo; Hara, Satoshi; Hisabori, Toru

    2012-04-01

    Thiol modulation of the chloroplast ATP synthase γ subunit has been recognized as an important regulatory system for the activation of ATP hydrolysis activity, although the physiological significance of this regulation system remains poorly characterized. Since the membrane potential required by this enzyme to initiate ATP synthesis for the reduced enzyme is lower than that needed for the oxidized form, reduction of this enzyme was interpreted as effective regulation for efficient photophosphorylation. However, no concrete evidence has been obtained to date relating to the timing and mode of chloroplast ATP synthase reduction and oxidation in green plants. In this study, thorough analysis of the redox state of regulatory cysteines of the chloroplast ATP synthase γ subunit in intact chloroplasts and leaves shows that thiol modulation of this enzyme is pivotal in prohibiting futile ATP hydrolysis activity in the dark. However, the physiological importance of efficient ATP synthesis driven by the reduced enzyme in the light could not be demonstrated. In addition, we investigated the significance of the electrochemical proton gradient in reducing the γ subunit by the reduced form of thioredoxin in chloroplasts, providing strong insights into the molecular mechanisms underlying the formation and reduction of the disulfide bond on the γ subunit in vivo. PMID:22362842

  3. Hybrid rotors in F1F(o) ATP synthases: subunit composition, distribution, and physiological significance.

    PubMed

    Brandt, Karsten; Müller, Volker

    2015-09-01

    The c ring of the Na+ F1F(o) ATP synthase from the anaerobic acetogenic bacterium Acetobacterium woodii is encoded by three different genes: atpE1, atpE2 and atpE3. Subunit c1 is similar to typical V-type c subunits and has four transmembrane helices with one ion binding site. Subunit c2 and c3 are identical at the amino acid level and are typical F-type c subunits with one ion binding site in two transmembrane helices. All three constitute a hybrid F(o)V(o) c ring, the first found in nature. To analyze whether other species may have similar hybrid rotors, we searched every genome sequence publicly available as of 23 February 2015 for F1F(o) ATPase operons that have more than one gene encoding the c subunit. This revealed no other species that has three different c subunit encoding genes but twelve species that encode one F(o)- and one V(o)-type c subunit in one operon. Their c subunits have the conserved binding motif for Na+. The organisms are all anaerobic. The advantage of hybrid c rings for the organisms in their environments is discussed. PMID:25838297

  4. Control of a Salmonella virulence locus by an ATP-sensing leader messenger RNA.

    PubMed

    Lee, Eun-Jin; Groisman, Eduardo A

    2012-06-14

    The facultative intracellular pathogen Salmonella enterica resides within a membrane-bound compartment inside macrophages. This compartment must be acidified for Salmonella to survive within macrophages, possibly because acidic pH promotes expression of Salmonella virulence proteins. We reasoned that Salmonella might sense its surroundings have turned acidic not only upon protonation of the extracytoplasmic domain of a protein sensor but also by an increase in cytosolic ATP levels, because conditions that enhance the proton gradient across the bacterial inner membrane stimulate ATP synthesis. Here we report that an increase in cytosolic ATP promotes transcription of the coding region for the virulence gene mgtC, which is the most highly induced horizontally acquired gene when Salmonella is inside macrophages. This transcript is induced both upon media acidification and by physiological conditions that increase ATP levels independently of acidification. ATP is sensed by the coupling/uncoupling of transcription of the unusually long mgtC leader messenger RNA and translation of a short open reading frame located in this region. A mutation in the mgtC leader messenger RNA that eliminates the response to ATP hinders mgtC expression inside macrophages and attenuates Salmonella virulence in mice. Our results define a singular example of an ATP-sensing leader messenger RNA. Moreover, they indicate that pathogens can interpret extracellular cues by the impact they have on cellular metabolites. PMID:22699622

  5. Molecular mechanism of ATP binding and ion channel activation in P2X receptors

    SciTech Connect

    Hattori, Motoyuki; Gouaux, Eric

    2012-10-24

    P2X receptors are trimeric ATP-activated ion channels permeable to Na{sup +}, K{sup +} and Ca{sup 2+}. The seven P2X receptor subtypes are implicated in physiological processes that include modulation of synaptic transmission, contraction of smooth muscle, secretion of chemical transmitters and regulation of immune responses. Despite the importance of P2X receptors in cellular physiology, the three-dimensional composition of the ATP-binding site, the structural mechanism of ATP-dependent ion channel gating and the architecture of the open ion channel pore are unknown. Here we report the crystal structure of the zebrafish P2X4 receptor in complex with ATP and a new structure of the apo receptor. The agonist-bound structure reveals a previously unseen ATP-binding motif and an open ion channel pore. ATP binding induces cleft closure of the nucleotide-binding pocket, flexing of the lower body {beta}-sheet and a radial expansion of the extracellular vestibule. The structural widening of the extracellular vestibule is directly coupled to the opening of the ion channel pore by way of an iris-like expansion of the transmembrane helices. The structural delineation of the ATP-binding site and the ion channel pore, together with the conformational changes associated with ion channel gating, will stimulate development of new pharmacological agents.

  6. The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria.

    PubMed

    Bhattacharjee, Ashima; Yang, Haojun; Duffy, Megan; Robinson, Emily; Conrad-Antoville, Arianrhod; Lu, Ya-Wen; Capps, Tony; Braiterman, Lelita; Wolfgang, Michael; Murphy, Michael P; Yi, Ling; Kaler, Stephen G; Lutsenko, Svetlana; Ralle, Martina

    2016-08-01

    Copper-transporting ATPase ATP7A is essential for mammalian copper homeostasis. Loss of ATP7A activity is associated with fatal Menkes disease and various other pathologies. In cells, ATP7A inactivation disrupts copper transport from the cytosol into the secretory pathway. Using fibroblasts from Menkes disease patients and mouse 3T3-L1 cells with a CRISPR/Cas9-inactivated ATP7A, we demonstrate that ATP7A dysfunction is also damaging to mitochondrial redox balance. In these cells, copper accumulates in nuclei, cytosol, and mitochondria, causing distinct changes in their redox environment. Quantitative imaging of live cells using GRX1-roGFP2 and HyPer sensors reveals highest glutathione oxidation and elevation of H2O2 in mitochondria, whereas the redox environment of nuclei and the cytosol is much less affected. Decreasing the H2O2 levels in mitochondria with MitoQ does not prevent glutathione oxidation; i.e. elevated copper and not H2O2 is a primary cause of glutathione oxidation. Redox misbalance does not significantly affect mitochondrion morphology or the activity of respiratory complex IV but markedly increases cell sensitivity to even mild glutathione depletion, resulting in loss of cell viability. Thus, ATP7A activity protects mitochondria from excessive copper entry, which is deleterious to redox buffers. Mitochondrial redox misbalance could significantly contribute to pathologies associated with ATP7A inactivation in tissues with paradoxical accumulation of copper (i.e. renal epithelia). PMID:27226607

  7. Chemical Properties And Toxicity of Chromium(III) Nutritional Supplements

    SciTech Connect

    Levina, A.; Lay, P.A.

    2009-05-19

    The status of Cr(III) as an essential micronutrient for humans is currently under question. No functional Cr(III)-containing biomolecules have been definitively described as yet, and accumulated experience in the use of Cr(III) nutritional supplements (such as [Cr(pic){sub 3}], where pic = 2-pyridinecarboxylato) has shown no measurable benefits for nondiabetic people. Although the use of large doses of Cr(III) supplements may lead to improvements in glucose metabolism for type 2 diabetics, there is a growing concern over the possible genotoxicity of these compounds, particularly of [Cr(pic){sub 3}]. The current perspective discusses chemical transformations of Cr(III) nutritional supplements in biological media, with implications for both beneficial and toxic actions of Cr(III) complexes, which are likely to arise from the same biochemical mechanisms, dependent on concentrations of the reactive species. These species include: (1) partial hydrolysis products of Cr(III) nutritional supplements, which are capable of binding to biological macromolecules and altering their functions; and (2) highly reactive Cr(VI/V/IV) species and organic radicals, formed in reactions of Cr(III) with biological oxidants. Low concentrations of these species are likely to cause alterations in cell signaling (including enhancement of insulin signaling) through interactions with the active centers of regulatory enzymes in the cell membrane or in the cytoplasm, while higher concentrations are likely to produce genotoxic DNA lesions in the cell nucleus. These data suggest that the potential for genotoxic side-effects of Cr(III) complexes may outweigh their possible benefits as insulin enhancers, and that recommendations for their use as either nutritional supplements or antidiabetic drugs need to be reconsidered in light of these recent findings.

  8. The Metalloprotease Encoded by ATP23 Has a Dual Function in Processing and Assembly of Subunit 6 of Mitochondrial ATPase

    PubMed Central

    Zeng, Xiaomei; Neupert, Walter

    2007-01-01

    In the present study we have identified a new metalloprotease encoded by the nuclear ATP23 gene of Saccharomyces cerevisiae that is essential for expression of mitochondrial ATPase (F1-FO complex). Mutations in ATP23 cause the accumulation of the precursor form of subunit 6 and prevent assembly of FO. Atp23p is associated with the mitochondrial inner membrane and is conserved from yeast to humans. A mutant harboring proteolytically inactive Atp23p accumulates the subunit 6 precursor but is nonetheless able to assemble a functional ATPase complex. These results indicate that removal of the subunit 6 presequence is not an essential event for ATPase biogenesis and that Atp23p, in addition to its processing activity, must provide another important function in FO assembly. The product of the yeast ATP10 gene was previously shown to interact with subunit 6 and to be required for its association with the subunit 9 ring. In this study one extra copy of ATP23 was found to be an effective suppressor of an atp10 null mutant, suggesting an overlap in the functions of Atp23p and Atp10p. Atp23p may, therefore, also be a chaperone, which in conjunction with Atp10p mediates the association of subunit 6 with the subunit 9 ring. PMID:17135290

  9. Mutation in cysteine bridge domain of the gamma-subunit affects light regulation of the ATP synthase in Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The chloroplast ATP synthase functions to synthesize ATP from ADP and free phosphate coupled by the electrochemical potential across the thylakoid membrane in the light. The light-dependent regulation of ATP synthase activity is carried out in part through redox modulation of a cysteine bridge in CF...

  10. Time-resolved Measurements of Intracellular ATP in the Yeast Saccharomyces cerevisiae using a New Type of Nanobiosensor*

    PubMed Central

    Özalp, Veli C.; Pedersen, Tina R.; Nielsen, Lise J.; Olsen, Lars F.

    2010-01-01

    Adenosine 5′-triphosphate is a universal molecule in all living cells, where it functions in bioenergetics and cell signaling. To understand how the concentration of ATP is regulated by cell metabolism and in turn how it regulates the activities of enzymes in the cell it would be beneficial if we could measure ATP concentration in the intact cell in real time. Using a novel aptamer-based ATP nanosensor, which can readily monitor intracellular ATP in eukaryotic cells with a time resolution of seconds, we have performed the first on-line measurements of the intracellular concentration of ATP in the yeast Saccharomyces cerevisiae. These ATP measurements show that the ATP concentration in the yeast cell is not stationary. In addition to an oscillating ATP concentration, we also observe that the concentration is high in the starved cells and starts to decrease when glycolysis is induced. The decrease in ATP concentration is shown to be caused by the activity of membrane-bound ATPases such as the mitochondrial F0F1 ATPase-hydrolyzing ATP and the plasma membrane ATPase (PMA1). The activity of these two ATPases are under strict control by the glucose concentration in the cell. Finally, the measurements of intracellular ATP suggest that 2-deoxyglucose (2-DG) may have more complex function than just a catabolic block. Surprisingly, addition of 2-DG induces only a moderate decline in ATP. Furthermore, our results suggest that 2-DG may inhibit the activation of PMA1 after addition of glucose. PMID:20880841

  11. Definition of "experimental procedures".

    PubMed

    2009-11-01

    This Practice Committee Opinion provides a revised definition of "experimental procedures." This version replaces the document "Definition of Experimental" that was published most recently in November 2008. PMID:19836733

  12. ATP appears to act via different receptors in terminals vs. somata of the Hypothalamic Neurohypophysial System

    PubMed Central

    Knott, Thomas K.; Hussy, Nicolas; Cuadra, Adolfo E.; Lee, Ryan H.; Ortiz-Miranda, Sonia; Custer, Edward E.; Lemos, José R.

    2012-01-01

    ATP-induced ionic currents were investigated in isolated terminals and somata of the Hypothalamic Neurohypophysial System (HNS). Both terminals and somata showed inward rectification of the ATP-induced currents and reversal near 0 mV. In terminals, ATP dose-dependently evoked an inactivating, inward current. However, in hypothalamic somata ATP evoked a very slowly inactivating, inward current with a higher density, and different dose dependence; EC50 of 50 μM in somata vs. 9.6 μM in terminals. The ATP induced currents, in both the HNS terminals and somata, were highly and reversibly inhibited by suramin, suggesting the involvement of a P2X receptor. However, the suramin inhibition was significantly different in the two HNS compartments: IC50 of 3.6 μM in somata vs 11.6 μM in terminals. Also, both HNS compartments show significantly different responses to the purinergic receptor agonists ATP-γ-S and Benzoyl-benzoyl-ATP. Finally, there was an initial desensitization to ATP upon successive stimulations in the terminals which was not observed in the somata. These differences in EC50, inactivation, desensitization, and agonist sensitivity in terminals vs. somata indicate that different P2X receptors mediate the responses in these two compartments of HNS neurons. Previous work has revealed mRNA transcripts for multiple purinergic receptors in micropunches of the hypothalamus. In the HNS terminals, the P2X purinergic receptor types P2X2, 3, 4, and 7 but not 6 have been shown to exist in AVP terminals. Immonohistochemistry now indicates that P2X4R is only present in AVP terminals and that the P2X7R is found in both AVP and OT terminals and somata. We speculate that these differences in receptor types reflects the specific function of endogenous ATP in the terminals vs. somata of these CNS neurons. PMID:22340013

  13. Critical roles of interdomain interactions for modulatory ATP binding to sarcoplasmic reticulum Ca2+-ATPase.

    PubMed

    Clausen, Johannes D; Holdensen, Anne Nyholm; Andersen, Jens Peter

    2014-10-17

    ATP has dual roles in the reaction cycle of sarcoplasmic reticulum Ca(2+)-ATPase. Upon binding to the Ca2E1 state, ATP phosphorylates the enzyme, and by binding to other conformational states in a non-phosphorylating modulatory mode ATP stimulates the dephosphorylation and other partial reaction steps of the cycle, thereby ensuring a high rate of Ca(2+) transport under physiological conditions. The present study elucidates the mechanism underlying the modulatory effect on dephosphorylation. In the intermediate states of dephosphorylation the A-domain residues Ser(186) and Asp(203) interact with Glu(439) (N-domain) and Arg(678) (P-domain), respectively. Single mutations to these residues abolish the stimulation of dephosphorylation by ATP. The double mutation swapping Asp(203) and Arg(678) rescues ATP stimulation, whereas this is not the case for the double mutation swapping Ser(186) and Glu(439). By taking advantage of the ability of wild type and mutant Ca(2+)-ATPases to form stable complexes with aluminum fluoride (E2·AlF) and beryllium fluoride (E2·BeF) as analogs of the E2·P phosphoryl transition state and E2P ground state, respectively, of the dephosphorylation reaction, the mutational effects on ATP binding to these intermediates are demonstrated. In the wild type Ca(2+)-ATPase, the ATP affinity of the E2·P phosphoryl transition state is higher than that of the E2P ground state, thus explaining the stimulation of dephosphorylation by nucleotide-induced transition state stabilization. We find that the Asp(203)-Arg(678) and Ser(186)-Glu(439) interdomain bonds are critical, because they tighten the interaction with ATP in the E2·P phosphoryl transition state. Moreover, ATP binding and the Ser(186)-Glu(439) bond are mutually exclusive in the E2P ground state. PMID:25193668

  14. Unexpected role of the copper transporter ATP7A in PDGF-induced vascular smooth

    SciTech Connect

    Ashino, T.; Varadarajan, S.; Urao, N.; Oshikawa, J.; Chen, G. -F.; Wang, H.; Huo, Y.; Finney, L.; Vogt, S.; McKinney, R. D.; Maryon, E. B.; Kaplan, J. H.; Ushio-Fukai, M.; Fukai, T.

    2010-09-09

    Copper, an essential nutrient, has been implicated in vascular remodeling and atherosclerosis with unknown mechanism. Bioavailability of intracellular copper is regulated not only by the copper importer CTR1 (copper transporter 1) but also by the copper exporter ATP7A (Menkes ATPase), whose function is achieved through copper-dependent translocation from trans-Golgi network (TGN). Platelet-derived growth factor (PDGF) promotes vascular smooth muscle cell (VSMC) migration, a key component of neointimal formation. To determine the role of copper transporter ATP7A in PDGF-induced VSMC migration. Depletion of ATP7A inhibited VSMC migration in response to PDGF or wound scratch in a CTR1/copper-dependent manner. PDGF stimulation promoted ATP7A translocation from the TGN to lipid rafts, which localized at the leading edge, where it colocalized with PDGF receptor and Rac1, in migrating VSMCs. Mechanistically, ATP7A small interfering RNA or CTR small interfering RNA prevented PDGF-induced Rac1 translocation to the leading edge, thereby inhibiting lamellipodia formation. In addition, ATP7A depletion prevented a PDGF-induced decrease in copper level and secretory copper enzyme precursor prolysyl oxidase (Pro-LOX) in lipid raft fraction, as well as PDGF-induced increase in LOX activity. In vivo, ATP7A expression was markedly increased and copper accumulation was observed by synchrotron-based x-ray fluorescence microscopy at neointimal VSMCs in wire injury model. These findings suggest that ATP7A plays an important role in copper-dependent PDGF-stimulated VSMC migration via recruiting Rac1 to lipid rafts at the leading edge, as well as regulating LOX activity. This may contribute to neointimal formation after vascular injury. Our findings provide insight into ATP7A as a novel therapeutic target for vascular remodeling and atherosclerosis.

  15. Imaging and characterization of stretch-induced ATP release from alveolar A549 cells

    PubMed Central

    Grygorczyk, Ryszard; Furuya, Kishio; Sokabe, Masahiro

    2013-01-01

    Mechano-transduction at cellular and tissue levels often involves ATP release and activation of the purinergic signalling cascade. In the lungs, stretch is an important physical stimulus but its impact on ATP release, the underlying release mechanisms and transduction pathways are poorly understood. Here, we investigated the effect of unidirectional stretch on ATP release from human alveolar A549 cells by real-time luciferin–luciferase bioluminescence imaging coupled with simultaneous infrared imaging, to monitor the extent of cell stretch and to identify ATP releasing cells. In subconfluent (<90%) cell cultures, single 1 s stretch (10–40%)-induced transient ATP release from a small fraction (≤1.5%) of cells that grew in number dose-dependently with increasing extent of