Sample records for atp iii definition

  1. Prevalence of the Metabolic Syndrome using the Modified ATP III Definitions for Workers in Japan, Korea and Mongolia

    Microsoft Academic Search

    Kuninori Shiwaku; Akiko Nogi; Keiko Kitajima; Erdembileg Anuurad; Byambaa Enkhmaa; Masayuki Yamasaki; Jung-Man Kim; In-Shik Kim; Sung-Kook Lee; Tsendsuren Oyunsuren; Yosuke Yamane

    2005-01-01

    Prevalence of the Metabolic Syndrome using the Modified ATP III Definitions for Workers in Japan, Korea and Mongolia: Kuninori SHIWAKU, et al. Department of Environmental and Preventive Medicine, Shimane University School of Medicine— A clustering of insulin resistance, hypertension and dyslipidemia has been labeled as the metabolic syndrome. Asians have a lower frequency of obesity than do Caucasians, but have

  2. Cytoplasmic Transfer of the mtDNA nt 8993 T --> G (ATP6) Point Mutation Associated with Leigh Syndrome into mtDNA-Less Cells Demonstrates Cosegregation with a Decrease in State III Respiration and ADP\\/O Ratio

    Microsoft Academic Search

    Ian Trounce; Stephanie Neill; Douglas C. Wallace

    1994-01-01

    A point mutation in the mtDNA-encoded ATP6 gene (T --> G at nt 8993) associated with Leigh syndrome in two pedigrees was found to decrease ADP-stimulated (state III) respiration and the ratio of ADP molecules phosphorylated to oxygen atoms reduced (ADP\\/O ratio) but did not affect 2,4-dinitrophenol (DNP)-uncoupled respiration, suggesting a defective mitochondrial H^+-translocating ATP synthase. Intact mitochondria isolated from

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

    Microsoft Academic Search

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

    2009-01-01

    Reconstruction of the highly unstable, anteriorly decompressed cervical spine poses biomechanical challenges to current stabilization\\u000a strategies, including circumferential instrumented fusion, to prevent failure. To avoid secondary posterior surgery, particularly\\u000a in the elderly population, while increasing primary construct rigidity of anterior-only reconstructions, the authors introduced\\u000a the concept of anterior transpedicular screw (ATPS) fixation and plating. We demonstrated its morphological feasibility, its

  4. ATP synthase.

    PubMed

    Junge, Wolfgang; Nelson, Nathan

    2015-06-01

    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

  5. Optical ATP Biosensor for Extracellular ATP Measurement

    PubMed Central

    Wang, C.; Huang, C.-Y.C; Lin, W-C

    2013-01-01

    Extracellular Adenosine-5?-triphosphate (ATP) is an important multi-functional molecule which can mediate numerous physiological activities by activating purinergic P2 receptors. The objective of this study was to develop a novel optical ATP sensor for in-situ extracellular ATP measurement in biological tissues. The optical ATP sensor was made by applying two layers of sol-gel coating to the end of an optical fiber probe end. The first layer contained ruthenium complex for sensing changes in oxygen concentration which resulted from oxidation of ATP by glycerol kinase and glycerol 3-phosphate oxidase entrapped in the second layer. It was demonstrated that the optical ATP sensor was capable of detecting ATP concentration at a broad range of 10?3 mM to 1.5 mM. A compensation method was established to enable the optical sensor to determine ATP concentration at different oxygen levels. This study also demonstrated the capability of ATP sensor to measure extracellular ATP content in biological tissues (i.e., porcine intervertebral disc). In addition, it was shown that the optical ATP sensor was not affected by pH and derivatives of extracellular ATP. Therefore, the newly developed optical ATP sensor is a good option for in-situ extracellular ATP measurement. PMID:23357001

  6. Cx43 hemichannels are permeable to ATP

    PubMed Central

    Kang, Jian; Kang, Ning; Lovatt, Ditte; Torres, Arnulfo; Zhao, Zhuo; Lin, Jane; Nedergaard, Maiken

    2013-01-01

    Astrocytes are electrically non-excitable cells that communicate by means of Ca2+ signaling. Long-distance intercellular Ca2+ waves are initiated by release of ATP and activation of purinergic receptors on nearby cells. Previous studies have implicated connexin 43 (Cx43) in ATP release, but definitive proof that ATP exits through Cx43 hemichannels does not exist. Here we show that ATP anions can permeate through Cx43 hemichannels using several alternative approaches. First, openings of Cx43 hemichannels were detected in both cell-attached and inside-out patch recordings in C6 cells expressing Cx43, but not in C6 cells expressing Cx43-eGFP (enhanced green fluorescent protein) or a C-terminus truncation mutant of Cx43. Second, Cx43 hemichannel openings were inhibited by three structurally different gap-junction channel blockers, but not by the P2X7 blocker Brilliant blue G. Third, bioluminescence imaging of ATP combined with single channel recording in the inside-out patch configuration showed that ATP efflux coincided with channel openings and was absent when the Cx43 hemichannel was closed. Fourth, ion replacement experiments confirmed that Cx43 hemichannels are permeable to ATP. In sum, these observations provide the first direct evidence for efflux of ATP through Cx43 hemichannels. Furthermore, a putative Cx43 hemichannel with characteristics identical to the Cx43 hemichannel in C6 cells was identified in the membrane of hippocampal astrocytes in acutely prepared slices. PMID:18448647

  7. Impact of metabolic syndrome definitions on prevalence estimates: a study in a Portuguese community.

    PubMed

    Santos, Ana-Cristina; Barros, Henrique

    2007-12-01

    This study compared the prevalence of metabolic syndrome (MS) according to World Health Organization (WHO), National Cholesterol Education Program Third Adult Treatment Panel (NCEP-ATP III), International Diabetes Federation (IDF) and American Heart Association/ National Heart, Lung and Blood Institute (AHA/NHLBI) definitions, to evaluate how well the different classifications agreed. The study also compared their 10-year predicted risk of coronary heart disease (CHD) with the Framingham risk score (FRS). Some 886 women and 547 men aged 18-92 years were included in the study. Demographic and personal medical history data were obtained at interview. Four operational definitions of MS were used (those of the WHO, NCEP-ATP III, AHA/NHLBI and IDF). The prevalence of metabolic syndrome was found to be 26.4% (WHO criteria), 24.0% (NCEP-ATP III criteria), 41.9% (IDF criteria) and 37.2% (AHA/NHLBI criteria). According to the definition used, central obesity ranged from 41.9% to 75.1% and high blood pressure from 52.9% to 65.8%. Agreement between classifications ranged from 75.2% (kappa=0.47) to 90.4% (kappa=0.80) and was lower in males. The 10-year predicted risk of CHD by FRS was similar between the different definitions. IDF and AHA/NHLBI definitions resulted in a higher prevalence of MS than the NCEP-ATP III or WHO definition. Overall, however, good agreement was found between definitions, and the predicted 10-year of CHD risk was similar. PMID:18158702

  8. Molecular Structure of ATP

    NSDL National Science Digital Library

    2003-05-09

    In plant cells, ATP is produced in the cristae of mitochondria and chloroplasts. Christae are the multiply-folded inner membranes of a cell's mitochondrion, which are finger-like projections. The walls of the cristae are the site of the cell's energy production (it is where ATP is generated). Chloroplasts are made up of stacks of thylakoid disks that contain chlorophyll. Production of ATP molecules from sunlight takes place on thylakoid disks. The mechanism of ATP synthesis is the same in both mitochondria and chloroplasts. An important role of ATP as a plant molecule is to provide energy for biosynthesis. Interestingly enough, this chemical energy can also be converted into light energy in the reaction catalyzed by luciferase. Each molecule of ATP consumed in the reaction produces one photon of light.

  9. agentTool III: From Process Definition to Code Generation Juan C. Garcia-Ojeda Scott A. DeLoach Robby

    E-print Network

    Deloach, Scott A.

    in 2000 ­ 2001 at the Air Force Institute of Technology and is currently a project of the Multiagent13931393 agentTool III: From Process Definition to Code Generation Juan C. Garcia-Ojeda Scott A. De of multiagent systems following the Organization-based Multiagent Systems Engineering (O-MaSE) methodology

  10. 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

  11. Prognostic factors for survival in stage III non-small-cell lung cancer treated with definitive radiation therapy: Impact of tumor volume

    SciTech Connect

    Basaki, Kiyoshi [Department of Radiology, Hirosaki University School of Medicine, Aomori (Japan)]. E-mail: basaki-rad@umin.ac.jp; Abe, Yoshinao [Department of Radiology, Hirosaki University School of Medicine, Aomori (Japan); Aoki, Masahiko [Department of Radiology, Hirosaki University School of Medicine, Aomori (Japan); Kondo, Hidehiro [Department of Radiology, Hirosaki University School of Medicine, Aomori (Japan); Hatayama, Yoshiomi [Department of Radiology, Hirosaki University School of Medicine, Aomori (Japan); Nakaji, Shigeyuki [Department of Social Medicine, Hirosaki University School of Medicine, Aomori (Japan)

    2006-02-01

    Purpose: To investigate the impact of tumor volume on overall survival in patients with Stage III non-small-cell lung cancer (NSCLC) treated with definitive radiation therapy (RT). Methods and Materials: Between May 1997 and February 2003, 71 patients with Stage III NSCLC were treated with radiation therapy of 60 Gy or more. The total target dose was between 60 and 77 Gy (average, 66.3 Gy). Chemotherapy was used in 45 cases. The primary tumor and nodal volume were identified in pretreatment computed tomography scans. Univariate and multivariate analyses were used to evaluate the impact of tumor volume on survival after RT. Results: The overall 2-year survival rate was 23%, with a median survival time of 14 months. The median survival times were 10 months and 19 months with large primary tumor volume more than median volume and smaller primary tumor volume, respectively. At a univariate analysis, the total tumor volume (TTV) (p < 0.0003) and the primary tumor volume (p < 0.00008) were significant and the nodal volume was not. At multivariate analyses, both the TTV and the primary tumor volume were significant prognostic factors. Conclusion: The primary tumor volume as well as TTV is a significant prognostic factor on survival in patients with Stage III NSCLC treated with RT and should be recorded in clinical results when the survivals are compared among clinical studies.

  12. ATP release via anion channels.

    PubMed

    Sabirov, Ravshan Z; Okada, Yasunobu

    2005-12-01

    ATP serves not only as an energy source for all cell types but as an 'extracellular messenger' for autocrine and paracrine signalling. It is released from the cell via several different purinergic signal efflux pathways. ATP and its Mg(2+) and/or H(+) salts exist in anionic forms at physiological pH and may exit cells via some anion channel if the pore physically permits this. In this review we survey experimental data providing evidence for and against the release of ATP through anion channels. CFTR has long been considered a probable pathway for ATP release in airway epithelium and other types of cells expressing this protein, although non-CFTR ATP currents have also been observed. Volume-sensitive outwardly rectifying (VSOR) chloride channels are found in virtually all cell types and can physically accommodate or even permeate ATP(4-) in certain experimental conditions. However, pharmacological studies are controversial and argue against the actual involvement of the VSOR channel in significant release of ATP. A large-conductance anion channel whose open probability exhibits a bell-shaped voltage dependence is also ubiquitously expressed and represents a putative pathway for ATP release. This channel, called a maxi-anion channel, has a wide nanoscopic pore suitable for nucleotide transport and possesses an ATP-binding site in the middle of the pore lumen to facilitate the passage of the nucleotide. The maxi-anion channel conducts ATP and displays a pharmacological profile similar to that of ATP release in response to osmotic, ischemic, hypoxic and salt stresses. The relation of some other channels and transporters to the regulated release of ATP is also discussed. PMID:18404516

  13. 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

  14. Comparison and correlation of binding mode of ATP in the kinase domains of Hexokinase family

    PubMed Central

    Kumar, Yellapu Nanda; Kumar, Pasupuleti Santhosh; Sowjenya, Gopal; Rao, Valasani Koteswara; Yeswanth, Sthanikam; Prasad, Uppu Venkateswara; Pradeepkiran, Jangampalli Adi; Sarma, PVGK; Bhaskar, Matcha

    2012-01-01

    Hexokinases (HKs) are the enzymes that catalyses the ATP dependent phosphorylation of Hexose sugars to Hexose-6-Phosphate (Hex-6-P). There exist four different forms of HKs namely HK-I, HK-II, HK-III and HK-IV and all of them share a common ATP binding site core surrounded by more variable sequence that determine substrate affinities. Although they share a common binding site but they differ in their kinetic functions, hence the present study is aimed to analyze the binding mode of ATP. The analysis revealed that the four ATP binding domains are showing 13 identical, 7 similar and 6 dissimilar residues with similar structural conformation. Molecular docking of ATP into the kinase domains using Molecular Operating Environment (MOE) soft ware tool clearly showed the variation in the binding mode of ATP with variable docking scores. This probably explains the variable phosphorylation rates among hexokinases family. PMID:22829728

  15. 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.

  16. Metabolic syndrome in a sample of the 6- to 16-year-old overweight or obese pediatric population: a comparison of two definitions

    PubMed Central

    Saffari, Fatemeh; Jalilolghadr, Shabnam; Esmailzadehha, Neda; Azinfar, Peyman

    2012-01-01

    Purpose The purpose of this study was to estimate the presence of metabolic syndrome (MS) in a group of children and adolescents with a body mass index (BMI) above the 85th percentile for their age and sex in Qazvin Province, Iran; to evaluate the relationship between obesity and metabolic abnormalities; and to compare two proposed definitions of MS. Patients and methods The study was conducted on 100 healthy subjects aged between 6 and 16 years (average age, 10.52 ± 2.51 years) with a high BMI for their age and sex. Fifty- eight percent of subjects were female. Physical examination including evaluation of weight, height, BMI, and blood pressure measurement was performed (“overweight” was defined as a BMI between the 85th and 95th percentiles for children of the same age and sex; “obese” was defined as a BMI over the 95th percentile for children of the same age and sex). Blood levels of glucose, insulin, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, and uric acid were measured after a 12-hour overnight fast. The authors used and compared two definitions of MS: the National Cholesterol Education Program’s Adult Treatment Panel III (NCEP ATP III) criteria and a modified definition by Weiss et al. Variables were compared using the Student’s t-test and chi-square and Mann-Whitney U tests, and agreement between the two definitions was analyzed using kappa values. Results The subjects had a mean BMI of 26.02 ± 4.38 and 80% had obesity. Insulin resistance was found in 81% of the study population. MS was present in ten (50%) of the overweight and 53 (66.2%) of the obese subjects using the NCEP ATP III criteria. MS was present in five (25%) of the overweight and 34 (42.5%) of the obese subjects using the definition by Weiss et al. The overall kappa value for the two definitions of MS was 0.533. There were no statistically significant differences between the two definitions of MS in participants. Conclusion The prevalence of MS in children and adolescents depends on the criteria chosen and their respective cutoff points. The NCEP ATP III criteria, the parameters of which include higher cutoff values for high-density lipoprotein cholesterol and triglycerides, detected the higher prevalence and therefore the NCEP ATP III criteria are able to diagnose a larger number of children and adolescents at metabolic risk. PMID:22346358

  17. Energy transduction in ATP synthase.

    PubMed

    Elston, T; Wang, H; Oster, G

    1998-01-29

    Mitochondria, bacteria and chloroplasts use the free energy stored in transmembrane ion gradients to manufacture ATP by the action of ATP synthase. This enzyme consists of two principal domains. The asymmetric membrane-spanning F0 portion contains the proton channel, and the soluble F1 portion contains three catalytic sites which cooperate in the synthetic reactions. The flow of protons through F0 is thought to generate a torque which is transmitted to F1 by an asymmetric shaft, the coiled-coil gamma-subunit. This acts as a rotating 'cam' within F1, sequentially releasing ATPs from the three active sites. The free-energy difference across the inner membrane of mitochondria and bacteria is sufficient to produce three ATPs per twelve protons passing through the motor. It has been suggested that this proton motive force biases the rotor's diffusion so that F0 constitutes a rotary motor turning the gamma shaft. Here we show that biased diffusion, augmented by electrostatic forces, does indeed generate sufficient torque to account for ATP production. Moreover, the motor's reversibility-supplying torque from ATP hydrolysis in F1 converts the motor into an efficient proton pump-can also be explained by our model. PMID:9461222

  18. ATP synthesis by decarboxylation phosphorylation.

    PubMed

    Dimroth, Peter; von Ballmoos, Christoph

    2008-01-01

    Adenosine triphosphate (ATP) is used as a general energy source by all living cells. The free energy released by hydrolyzing its terminal phosphoric acid anhydride bond to yield ADP and phosphate is utilized to drive various energy-consuming reactions. The ubiquitous F(1)F(0) ATP synthase produces the majority of ATP by converting the energy stored in a transmembrane electrochemical gradient of H(+) or Na(+) into mechanical rotation. While the mechanism of ATP synthesis by the ATP synthase itself is universal, diverse biological reactions are used by different cells to energize the membrane. Oxidative phosphorylation in mitochondria or aerobic bacteria and photophosphorylation in plants are well-known processes. Less familiar are fermentation reactions performed by anaerobic bacteria, wherein the free energy of the decarboxylation of certain metabolites is converted into an electrochemical gradient of Na(+) ions across the membrane (decarboxylation phosphorylation). This chapter will focus on the latter mechanism, presenting an updated survey on the Na(+)-translocating decarboxylases from various organisms. In the second part, we provide a detailed description of the F(1)F(0) ATP synthases with special emphasis on the Na(+)-translocating variant of these enzymes. PMID:18049805

  19. Implications of Recent Clinical Trials for the National Cholesterol Education Program Adult Treatment Panel III Guidelines

    Microsoft Academic Search

    Scott M. Grundy; James I. Cleeman; C. Noel Bairey Merz; H. Bryan Brewer Jr; Luther T. Clark; Donald B. Hunninghake; Richard C. Pasternak; Sidney C. Smith Jr; Neil J. Stone

    2004-01-01

    The Adult Treatment Panel III (ATP III) of the National Cholesterol Education Program issued an evidence-based set of guidelines on cholesterol management in 2001. Since the publication of ATP III, 5 major clinical trials of statin therapy with clinical end points have been published. These trials addressed issues that were not examined in previous clinical trials of cholesterol-lowering therapy. The

  20. 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 [Department of Radiology, Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto 860 (Japan); Uji, Yasuyoshi; Taen, Akira [Department of Oral and Maxillofacial Surgery, Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto 860 (Japan)

    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.

  1. Crystal structure of ATP sulfurylase from Saccharomyces cerevisiae, a key enzyme in sulfate activation

    PubMed Central

    Ullrich, Tobias C.; Blaesse, Michael; Huber, Robert

    2001-01-01

    ATP sulfurylases (ATPSs) are ubiquitous enzymes that catalyse the primary step of intracellular sulfate activation: the reaction of inorganic sulfate with ATP to form adenosine-5?-phosphosulfate (APS) and pyrophosphate (PPi). With the crystal structure of ATPS from the yeast Saccharomyces cerevisiae, we have solved the first structure of a member of the ATP sulfurylase family. We have analysed the crystal structure of the native enzyme at 1.95 ? resolution using multiple isomorphous replacement (MIR) and, subsequently, the ternary enzyme product complex with APS and PPi bound to the active site. The enzyme consists of six identical subunits arranged in two stacked rings in a D3 symmetric assembly. Nucleotide binding causes significant conformational changes, which lead to a rigid body structural displacement of domains III and IV of the ATPS monomer. Despite having similar folds and active site design, examination of the active site of ATPS and comparison with known structures of related nucleotidylyl transferases reveal a novel ATP binding mode that is peculiar to ATP sulfuryl-ases. PMID:11157739

  2. Optimization of ATP synthase function in mitochondria and chloroplasts via the adenylate kinase equilibrium.

    PubMed

    Igamberdiev, Abir U; Kleczkowski, Leszek A

    2015-01-01

    The bulk of ATP synthesis in plants is performed by ATP synthase, the main bioenergetics engine of cells, operating both in mitochondria and in chloroplasts. The reaction mechanism of ATP synthase has been studied in detail for over half a century; however, its optimal performance depends also on the steady delivery of ATP synthase substrates and the removal of its products. For mitochondrial ATP synthase, we analyze here the provision of stable conditions for (i) the supply of ADP and Mg(2+), supported by adenylate kinase (AK) equilibrium in the intermembrane space, (ii) the supply of phosphate via membrane transporter in symport with H(+), and (iii) the conditions of outflow of ATP by adenylate transporter carrying out the exchange of free adenylates. We also show that, in chloroplasts, AK equilibrates adenylates and governs Mg(2+) contents in the stroma, optimizing ATP synthase and Calvin cycle operation, and affecting the import of inorganic phosphate in exchange with triose phosphates. It is argued that chemiosmosis is not the sole component of ATP synthase performance, which also depends on AK-mediated equilibrium of adenylates and Mg(2+), adenylate transport, and phosphate release and supply. PMID:25674099

  3. 'Domino' systems biology and the 'A' of ATP.

    PubMed

    Verma, Malkhey; Zakhartsev, Maksim; Reuss, Matthias; Westerhoff, Hans V

    2013-01-01

    We develop a strategic 'domino' approach that starts with one key feature of cell function and the main process providing for it, and then adds additional processes and components only as necessary to explain provoked experimental observations. The approach is here applied to the energy metabolism of yeast in a glucose limited chemostat, subjected to a sudden increase in glucose. The puzzles addressed include (i) the lack of increase in adenosine triphosphate (ATP) upon glucose addition, (ii) the lack of increase in adenosine diphosphate (ADP) when ATP is hydrolyzed, and (iii) the rapid disappearance of the 'A' (adenine) moiety of ATP. Neither the incorporation of nucleotides into new biomass, nor steady de novo synthesis of adenosine monophosphate (AMP) explains. Cycling of the 'A' moiety accelerates when the cell's energy state is endangered, another essential domino among the seven required for understanding of the experimental observations. This new domino analysis shows how strategic experimental design and observations in tandem with theory and modeling may identify and resolve important paradoxes. It also highlights the hitherto unexpected role of the 'A' component of ATP. PMID:23031542

  4. The Chloroplast atpA Gene Cluster in Chlamydomonas reinhardtii1

    PubMed Central

    Drapier, Dominique; Suzuki, Hideki; Levy, Haim; Rimbault, Blandine; Kindle, Karen L.; Stern, David B.; Wollman, Francis-André

    1998-01-01

    Most chloroplast genes in vascular plants are organized into polycistronic transcription units, which generate a complex pattern of mono-, di-, and polycistronic transcripts. In contrast, most Chlamydomonas reinhardtii chloroplast transcripts characterized to date have been monocistronic. This paper describes the atpA gene cluster in the C. reinhardtii chloroplast genome, which includes the atpA, psbI, cemA, and atpH genes, encoding the ?-subunit of the coupling-factor-1 (CF1) ATP synthase, a small photosystem II polypeptide, a chloroplast envelope membrane protein, and subunit III of the CF0 ATP synthase, respectively. We show that promoters precede the atpA, psbI, and atpH genes, but not the cemA gene, and that cemA mRNA is present only as part of di-, tri-, or tetracistronic transcripts. Deletions introduced into the gene cluster reveal, first, that CF1-? can be translated from di- or polycistronic transcripts, and, second, that substantial reductions in mRNA quantity have minimal effects on protein synthesis rates. We suggest that posttranscriptional mRNA processing is common in C. reinhardtii chloroplasts, permitting the expression of multiple genes from a single promoter. PMID:9625716

  5. Electrical power fuels rotary ATP synthase.

    PubMed

    Dimroth, Peter; von Ballmoos, Christoph; Meier, Thomas; Kaim, Georg

    2003-12-01

    ATP synthesis by F-type ATP synthases consumes energy stored in a transmembrane electrochemical gradient of protons or sodium ions. The electric component of the ion motive force is crucial for ATP synthesis. Here, we incorporate recent results on structure and function of the F(0) domain and present a mechanism for torque generation with the fundamental nature of the membrane potential as driving force in the core. PMID:14656431

  6. Assembly of F1F0-ATP synthases.

    PubMed

    Rühle, Thilo; Leister, Dario

    2015-09-01

    F1F0-ATP synthases are multimeric protein complexes and common prerequisites for their correct assembly are (i) provision of subunits in appropriate relative amounts, (ii) coordination of membrane insertion and (iii) avoidance of assembly intermediates that uncouple the proton gradient or wastefully hydrolyse ATP. Accessory factors facilitate these goals and assembly occurs in a modular fashion. Subcomplexes common to bacteria and mitochondria, but in part still elusive in chloroplasts, include a soluble F1 intermediate, a membrane-intrinsic, oligomeric c-ring, and a membrane-embedded subcomplex composed of stator subunits and subunit a. The final assembly step is thought to involve association of the preformed F1-c10-14 with the ab2 module (or the ab8-stator module in mitochondria) - mediated by binding of subunit ? in bacteria or OSCP in mitochondria, respectively. Despite the common evolutionary origin of F1F0-ATP synthases, the set of auxiliary factors required for their assembly in bacteria, mitochondria and chloroplasts shows clear signs of evolutionary divergence. This article is part of a Special Issue entitled: Chloroplast Biogenesis. PMID:25667968

  7. Catalytic strategy used by the myosin motor to hydrolyze ATP

    PubMed Central

    Kiani, Farooq Ahmad; Fischer, Stefan

    2014-01-01

    Myosin is a molecular motor responsible for biological motions such as muscle contraction and intracellular cargo transport, for which it hydrolyzes adenosine 5'-triphosphate (ATP). Early steps of the mechanism by which myosin catalyzes ATP hydrolysis have been investigated, but still missing are the structure of the final ADP·inorganic phosphate (Pi) product and the complete pathway leading to it. Here, a comprehensive description of the catalytic strategy of myosin is formulated, based on combined quantum–classical molecular mechanics calculations. A full exploration of catalytic pathways was performed and a final product structure was found that is consistent with all experiments. Molecular movies of the relevant pathways show the different reorganizations of the H-bond network that lead to the final product, whose ?-phosphate is not in the previously reported HP?O42? state, but in the H2P?O4? state. The simulations reveal that the catalytic strategy of myosin employs a three-pronged tactic: (i) Stabilization of the ?-phosphate of ATP in a dissociated metaphosphate (P?O3?) state. (ii) Polarization of the attacking water molecule, to abstract a proton from that water. (iii) Formation of multiple proton wires in the active site, for efficient transfer of the abstracted proton to various product precursors. The specific role played in this strategy by each of the three loops enclosing ATP is identified unambiguously. It explains how the precise timing of the ATPase activation during the force generating cycle is achieved in myosin. The catalytic strategy described here for myosin is likely to be very similar in most nucleotide hydrolyzing enzymes. PMID:25006262

  8. Sodium-sodium exchange through the sodium pump: the roles of ATP and ADP.

    PubMed Central

    Cavieres, J D; Glynn, I M

    1979-01-01

    1. We have developed a procedure for preparing resealed red cell ghosts that contain ADP but very little ATP. 2. The procedure involves (i) lysis of the cells in a very large volume of lysing solution, (ii) resuspension of the ghosts in a small volume, (iii) the incorporation into the ghosts, before they are resealed, of the adenylate kinase inhibitor P1,P5-di(adenosine-5'-)pentaphosphate (AP5A) and of hexokinase, and (iv) the removal of traces of ATP, formed by residual adenylate kinase activity, by the addition of glucose. 3. Measurements of sodium efflux from ghosts prepared in this way show that sodium-sodium exchange through the sodium pump does not occur in the absence of ATP even if ADP is present. 4. The beta:gamma imido analogue of ATP (AMP.PNP), which is incapable of phosphorylating sodium, potassium-ATPase, cannot replace ATP in supporting sodium-sodium exchange. 5. These findings support the hypothesis that the outward movement of sodium ions through the sodium pump is associated with the transfer of a phosphoryl group from ATP to the enzyme, and that the inward movement of sodium ions through the pump is associated with the return of a phosphoryl group from the phosphoenzyme to ADP. PMID:536926

  9. Historical review: ATP as a neurotransmitter

    E-print Network

    Burnstock, Geoffrey

    signalling is now recognized to be involved in a wide range of activities of the nervous system, including of a transmitter role for ATP in the nervous system by demonstrating the release of ATP during antidromic and angiogenesis, pain and mechanosensory transduction and the physiology of the special senses. In this article, I

  10. Communication Definitions... general definition

    E-print Network

    Jones, Ian L.

    Communication Definitions... general definition "the process of conveying information from a sender to a receiver with the use of a medium in which the communicated information is understood the same way by both sender and receiver" (Wikipedia)! Biological communication Action by one organism (individual

  11. Diacylglycerol Signaling Underlies Astrocytic ATP Release

    PubMed Central

    Mungenast, Alison E.

    2011-01-01

    Astrocytes have the ability to modulate neuronal excitability and synaptic transmission by the release of gliotransmitters. The importance of ATP released downstream of the activation of Gq-coupled receptors has been well established, but the mechanisms by which this release is regulated are unclear. The current work reveals that the elevation of diacylglycerol (DAG) in astrocytes induces vesicular ATP release. Unexpectedly, DAG-induced ATP release was found to be independent of PKC activation, but dependent upon activation of a C1 domain-containing protein. Astrocytes express the C1 domain-containing protein Munc13-1, which has been implicated in neuronal transmitter release, and RNAi-targeted downregulation of Munc13-1 inhibits astrocytic ATP release. These studies demonstrate that elevations of DAG induce the exocytotic release of ATP in astrocytes, likely via a Munc13-1-dependent mechanism. PMID:21826278

  12. ATP hydrolysis and synthesis by the membrane-bound ATP synthetase complex of Methanobacterium thermoautotrophicum.

    PubMed Central

    Doddema, H J; Hutten, T J; van der Drift, C; Vogels, G D

    1978-01-01

    The membrane-bound ATP synthetase complex of Methanobacterium thermoautotrophicum showed maximum activity for ATP hydrolysis at pH 8, at temperatures between 65 and 70 degrees C, and at an ATP-Mg2+ ratio of 0.5. Anaerobic conditions were not prerequisite for enzyme activity. The enzyme showed a Km value for ATP of 2 mM, and activity was Mg2+ dependent; Mn2+, Co2+, Ca2+, and Zn2+ could replace Mg2+ to some extent. Other nucleoside triphosphates could be hydrolyzed. N,N'-dicyclohexylcarbodiimide inhibited ATP hydrolysis. A proton-motive force, artificially imposed by a pH shift or valinomycin, resulted in ATP synthesis in whole cells. The ATP synthetase complex of the thermophilic methanogenic bacterium is similar to those described in aerobic and anaerobic microorganisms. PMID:30747

  13. Comparisons of Different Metabolic Syndrome Definitions and Associations with Coronary Heart Disease, Stroke, and Peripheral Arterial Disease in a Rural Chinese Population

    PubMed Central

    Wen, Jiangping; Yang, Jingang; Shi, Yujie; Liang, Yuanbo; Wang, Fenghua; Duan, Xinrong; Lu, Xilin; Tao, Qiushan; Lu, Xinxin; Tian, Yaping; Wang, Ningli

    2015-01-01

    Objectives We estimated the prevalence of metabolic syndrome (MetS) and compared associations of different MetS definitions with coronary heart disease (CHD), stroke, and peripheral arterial disease (PAD) in a rural Chinese population. Methods Among 4,748 residents (2,145 men and 2,603 women) aged 30+ years in rural China from 2006 to 2007, the prevalence of MetS was estimated by using five different definitions: modified World Health Organization (WHO), Chinese Diabetes Society (CDS), the updated National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATP III) for Asian-Americans, International Diabetes Federation (IDF), and Joint Interim Statement (JIS). Multivariable logistic regression analyses were implemented to estimate the association between MetS and the prevalence of CHD, stroke and PAD, respectively. Results Prevalence of MetS in men was 11.5% (WHO), 14.8% (CDS), 32.4% (NCEP-ATP III), 27.5% (IDF) and 39.7% (JIS) and in women was 15.7% (WHO), 20.7% (CDS), 54.2% (NCEP-ATP III), 51.5% (IDF) and 54.2% (JIS), respectively. Respective ORs (95% CI) for associating MetS with CHD in men were 1.79 (1.02-3.17), 1.25 (0.69-2.26), 1.61 (1.01-2.58), 1.84 (1.14-2.96), and 1.53 (0.96-2.43). Corresponding ORs (95% CI) for stroke in men were 2.18 (95% CI 1.20 to 3.97), 2.20 (95% CI 1.25 to 3.89), 1.71 (95% CI 1.02 to 2.84), 1.30 (95% CI 0.77 to 2.23), and 1.61 (95% CI 0.97 to 2.68), respectively. In women, CHD and stroke were significantly associated with MetS using all five definitions of MetS. In addition, PAD was associated with all five MetS definitions in men, but not in women. Only hyperglycemia and BMI were significantly associated with PAD in women. Conclusions In this rural Chinese population, the JIS, IDF and CDS criteria may not be more suitable than WHO and updated NCEP-ATPIII definitions for screening high-risk individuals and estimating the risk of CHD and stroke from MetS, especially in men. PMID:25961739

  14. Negative-feedback regulation of ATP release: ATP release from cardiomyocytes is strictly regulated during ischemia.

    PubMed

    Kunugi, Satohiko; Iwabuchi, Sadahiro; Matsuyama, Daisuke; Okajima, Takaharu; Kawahara, Koichi

    2011-12-16

    Extracellular ATP acts as a potent agonist on cardiomyocytes, inducing a broad range of physiological responses via P2 purinoceptors. Its concentration in the interstitial space within the heart is elevated during ischemia or hypoxia due to its release from a number of cell types, including cardiomyocytes. However, the exact mechanism responsible for the release of ATP from cardiomyocytes during ischemia is not known. In this study, we investigated whether and how the release of ATP was strictly regulated during ischemia in cultured neonatal rat cardiomyocytes. Ischemia was mimicked by oxygen-glucose deprivation (OGD). Exposure of cardiomyocytes to OGD resulted in an increase in the concentration of extracellular ATP shortly after the onset of OGD (15 min), and the increase was reversed by treatment with blockers of maxi-anion channels. Unexpectedly, at 1 and 2h after the onset of OGD, the blocking of maxi-anion channels increased the concentration of extracellular ATP, and the increase was significantly suppressed by co-treatment with blockers of hemichannels, suggesting that ATP release via maxi-anion channels was involved in the suppression of ATP release via hemichannels during persistent OGD. Here we show the possibility that the release of ATP from cardiomyocytes was strictly regulated during ischemia by negative-feedback mechanisms; that is, maxi-anion channel-derived ATP-induced suppression of ATP release via hemichannels in cardiomyocytes. PMID:22133679

  15. 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

  16. Interdependence of mitochondrial ATP production and extramitochondrial ATP utilization in intact spermatozoa.

    PubMed

    Halangk, W; Bohnensack, R; Kunz, W

    1985-07-17

    The dependence of both respiration and total activity of ATP-consuming reactions on the cellular adenine nucleotide pattern was investigated in intact bovine spermatozoa. ATP consumption was manipulated by inhibition with vanadate and activation with caffeine, leading to a decrease or increase in the rate of respiration up to 70% or 20%, respectively. Oligomycin blocked the respiration to the same extent as did vanadate, suggesting that the total extramitochondrial ATP-consuming activity is vanadate-sensitive. The major part of ATP utilization must be linked to dynein ATPase, since inhibition of (Na+, K+) ATPase by ouabain showed only a small effect on respiration (-17%). Being a potent inhibitor of dynein ATPase, vanadate drastically reduced the amount of motile cells, whereas caffeine tended to increase the intensity of motion. The effects of vanadate or caffeine on respiration were paralleled by changes in cellular ATP, reflecting the response of mitochondrial respiration on the cellular ATP/ADP ratio. Respiration was found to depend on changes in the ATP/ADP ratio in the range from about 3 (+caffeine) to 9 (+vanadate). The range of response of ATP consumption to the ATP/ADP ratio was determined by varying the mitochondrial ATP production via the concentration of lactate which was used as substrate. The measured effects on both respiratory rate and ATP/ADP ratio suggested that ATP consumption was markedly dependent on ATP/ADP ratios below 5. It is concluded that lactate concentrations above 1 mM sufficiently supply bovine spermatozoa with substrate and the energy turnover is mainly limited by the activity of dynein ATPase rather than by the capacity of mitochondrial oxidative phosphorylation. PMID:3848331

  17. 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.

  18. The Rotary Mechanism of the ATP Synthase

    PubMed Central

    Nakamoto, Robert K.; Scanlon, Joanne A. Baylis; Al-Shawi, Marwan K.

    2008-01-01

    The FOF1 ATP synthase is a large complex of at least 22 subunits, more than half of which are in the membranous FO sector. This nearly ubiquitous transporter is responsible for the majority of ATP synthesis in oxidative and photo-phosphorylation, and its overall structure and mechanism have remained conserved throughout evolution. Most examples utilize the proton motive force to drive ATP synthesis except for a few bacteria, which use a sodium motive force. A remarkable feature of the complex is the rotary movement of an assembly of subunits that plays essential roles in both transport and catalytic mechanisms. This review addresses the role of rotation in catalysis of ATP synthesis/hydrolysis and the transport of protons or sodium. PMID:18515057

  19. Mitochondrial ATP synthase: architecture, function and pathology

    Microsoft Academic Search

    An I. Jonckheere; Jan A. M. Smeitink; Richard J. T. Rodenburg

    Human mitochondrial (mt) ATP synthase, or complex V consists of two functional domains: F1, situated in the mitochondrial matrix, and Fo, located in the inner mitochondrial membrane. Complex V uses the energy created by the proton electrochemical gradient to\\u000a phosphorylate ADP to ATP. This review covers the architecture, function and assembly of complex V. The role of complex V di-and

  20. Plasma membrane signal-transducing ATP

    Microsoft Academic Search

    A. A. Karelin; A. G. Globa; V. S. Demidova

    1999-01-01

    Plasma membranes of target cells generate considerable amounts of ATP in response to binding of growth factors, cytokines,\\u000a and oncoproteins. Plasma membrane ATP is formed at the stage of ligand-receptor signal transduction by the anaerobic pathway\\u000a with the involvement of plasma membrane redox systems and Na+ (but not adenylate cyclase). The assumption on the involvement of transitory reversed Na+, K+-ATPase

  1. Sequence and structural analysis of kinase ATP pocket residues

    Microsoft Academic Search

    Anna Vulpetti; Roberta Bosotti

    2004-01-01

    Protein kinases represent one of the largest known families of enzymes. Most kinases bind ATP and most synthetic kinase inhibitors are ATP-competitive, which makes selectivity a potential problem. However, despite the high sequence similarity in the ATP binding pocket, several groups including ours have been able to develop highly potent and selective ATP-competitive inhibitors. To systematically aid the design of

  2. Introduction Definitions

    E-print Network

    Herwig, Wendt

    Introduction Definitions Bootstrap Results Conclusions and Perspectives Bootstrap for Log Wavelet;Introduction Definitions Bootstrap Results Conclusions and Perspectives Motivation Motivation Multifractal, Patrice Abry Bootstrap for Log Wavelet Leaders Cumulant based MFA #12;Introduction Definitions Bootstrap

  3. Assembly of the rotor component of yeast mitochondrial ATP synthase is enhanced when Atp9p is supplied by Atp9p-Cox6p complexes.

    PubMed

    Su, Chen-Hsien; McStay, Gavin P; Tzagoloff, Alexander

    2014-11-01

    The Atp9p ring is one of several assembly modules of yeast mitochondrial ATP synthase. The ring, composed of 10 copies of Atp9p, is part of the rotor that couples proton translocation to synthesis or hydrolysis of ATP. We present evidence that before its assembly with other ATP synthase modules, most of Atp9p is present in at least three complexes with masses of 200-400 kDa that co-immunopurify with Cox6p. Pulse-labeling analysis disclosed a time-dependent reduction of radiolabeled Atp9p in the complexes and an increase of Atp9p in the ring form of wild type yeast and of mss51, pet111, and pet494 mutants lacking Cox1p, Cox2p, and Cox3p, respectively. Ring formation was not significantly different from wild type in an mss51 or atp10 mutant. The atp10 mutation blocks the interaction of the Atp9p ring with other modules of the ATP synthase. In contrast, ring formation was reduced in a cox6 mutant, consistent with a role of Cox6p in oligomerization of Atp9p. Cox6p involvement in ATP synthase assembly is also supported by studies showing that ring formation in cells adapting from fermentative to aerobic growth was less efficient in mitochondria of the cox6 mutant than the parental respiratory-competent strain or a cox4 mutant. We speculate that the constitutive and Cox6p-independent rate of Atp9p oligomerization may be sufficient to produce the level of ATP synthase needed for maintaining a membrane potential but limiting for optimal oxidative phosphorylation. PMID:25253699

  4. Mechanisms for ATP-dependent chromatin remodelling.

    PubMed

    Whitehouse, I; Flaus, A; Havas, K; Owen-Hughes, T

    2000-01-01

    Gene regulation involves the generation of a local chromatin topology that is conducive to transcription. Several classes of chromatin remodelling activity have been shown to play a role in this process. ATP-dependent chromatin-remodelling activities use energy derived from the hydrolysis of ATP to alter the structure of chromatin, making it more accessible for transcription factor binding. The yeast SWI-SWF complex is the founding member of this family of ATP-dependent chromatin-remodelling activities. We have developed a model system to study the ability of the SWI-SWF complex to alter chromatin structure. Using this system, we find that SWI-SWF is able to alter the position of nucleosomes along the DNA. This is consistent with recent reports that other ATP-dependent chromatin-remodelling activities can alter the positions of nucleosomes along DNA. This suggests that nucleosome mobilization may be a general feature of the activity of ATP-dependent chromatin-remodelling activities. Some of the mechanisms by which nucleosomes may be moved along DNA are discussed. PMID:10961923

  5. Regulation of mitochondrial ATP synthase in cardiac pathophysiology

    PubMed Central

    Long, Qinqiang; Yang, Kevin; Yang, Qinglin

    2015-01-01

    Mitochondrial function is paramount to energy homeostasis, metabolism, signaling, and apoptosis in cells. Mitochondrial complex V (ATP synthase), a molecular motor, is the ultimate ATP generator and a key determinant of mitochondrial function. ATP synthase catalyzes the final coupling step of oxidative phosphorylation to supply energy in the form of ATP. Alterations at this step will crucially impact mitochondrial respiration and hence cardiac performance. It is well established that cardiac contractility is strongly dependent on the mitochondria, and that myocardial ATP depletion is a key feature of heart failure. ATP synthase dysfunction can cause and exacerbate human diseases, such as cardiomyopathy and heart failure. While ATP synthase has been extensively studied, essential questions related to how the regulation of ATP synthase determines energy metabolism in the heart linger and therapies targeting this important mechanism remain scarce. This review will visit the main findings, identify unsolved issues and provide insights into potential future perspectives related to the regulation of ATP synthase and cardiac pathophysiology.

  6. 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

  7. SAGE III

    Atmospheric Science Data Center

    2014-12-04

    SAGE III Data and Information The Stratospheric Aerosol and Gas ... Guide Documents:  Project Guide Data Products User's Guide  (PDF) Relevant Documents:  ... Join SAGE III News List SAGE I Data Table SAGE II Data Table SAGE III Home Page ...

  8. An ATP synthase harboring an atypical ?-subunit is involved in ATP synthesis in tomato fruit chromoplasts.

    PubMed

    Pateraki, Irini; Renato, Marta; Azcón-Bieto, Joaquín; Boronat, Albert

    2013-04-01

    Chromoplasts are non-photosynthetic plastids specialized in the synthesis and accumulation of carotenoids. During fruit ripening, chloroplasts differentiate into photosynthetically inactive chromoplasts in a process characterized by the degradation of the thylakoid membranes, and by the active synthesis and accumulation of carotenoids. This transition renders chromoplasts unable to photochemically synthesize ATP, and therefore these organelles need to obtain the ATP required for anabolic processes through alternative sources. It is widely accepted that the ATP used for biosynthetic processes in non-photosynthetic plastids is imported from the cytosol or is obtained through glycolysis. In this work, however, we show that isolated tomato (Solanum lycopersicum) fruit chromoplasts are able to synthesize ATP de novo through a respiratory pathway using NADPH as an electron donor. We also report the involvement of a plastidial ATP synthase harboring an atypical ?-subunit induced during ripening, which lacks the regulatory dithiol domain present in plant and algae chloroplast ?-subunits. Silencing of this atypical ?-subunit during fruit ripening impairs the capacity of isolated chromoplast to synthesize ATP de novo. We propose that the replacement of the ?-subunit present in tomato leaf and green fruit chloroplasts by the atypical ?-subunit lacking the dithiol domain during fruit ripening reflects evolutionary changes, which allow the operation of chromoplast ATP synthase under the particular physiological conditions found in this organelle. PMID:23302027

  9. 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 ?-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

  10. Introduction -Definition

    E-print Network

    Aslaksen, Helmer

    #12;Introduction - Definition - Characteristics - Good kaleidoscope angle The two-mirror system - process of reflection The three-mirror system - summation of good kaleidoscope angles - process kaleidoscopes Steps in making a kaleidoscope Bibliography #12;Introduction Definition Kaleidoscopes are devices

  11. 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

  12. Changes in dermal interstitial ATP levels during local heating of human skin.

    PubMed

    Gifford, Jayson R; Heal, Cory; Bridges, Jarom; Goldthorpe, Scott; Mack, Gary W

    2012-12-15

    Heating skin is believed to activate vanilloid type III and IV transient receptor potential ion channels (TRPV3, TRPV4, respectively), resulting in the release of ATP into the interstitial fluid. We examined the hypothesis that local skin heating would result in an accumulation of ATP in the interstitial fluid that would be related with a rise in skin blood flow (SkBF) and temperature sensation. Two microdialysis probes were inserted into the dermis on the dorsal aspect of the forearm in 15 young, healthy subjects. The probed skin was maintained at 31°C, 35°C, 39°C and 43°C for 8 min periods, during which SkBF was monitored as cutaneous vascular conductance (CVC). Dialysate was collected and analysed for ATP ([ATP](d)) using a luciferase-based assay, and ratings of perceived warmth were taken at each temperature. At a skin temperature of 31°C, [ATP](d) averaged 18.93 ± 4.06 nm and CVC averaged 12.57 ± 1.59% peak. Heating skin to 35°C resulted in an increase in CVC (17.63 ± 1.27% peak; P < 0.05), but no change in [ATP](d). Heating skin to 39°C and 43°C resulted in a decreased [ATP](d) (5.88 ± 1.68 nm and 8.75 ± 3.44 nm, respectively; P < 0.05), which was accompanied by significant elevations in CVC (38.90 ± 1.37% peak and 60.32 ± 1.95% peak, respectively; P < 0.05). Ratings of perceived warmth increased in proportion to the increase in skin temperature (r(2) = 0.75, P < 0.05). In conclusion, our data indicate that an accumulation of interstitial ATP does not occur during local heating, and therefore does not have a role in temperature sensation or the dilator response in human skin. Nevertheless, the low threshold of dilatation (35°C) indicates a possible role for the TRPV3, TRPV4 channels or the sensitization of other ion channels in mediating the dilator response. PMID:23045344

  13. ATP synthesis without R210 of subunit a in the Escherichia coli ATP synthase

    PubMed Central

    Ishmukhametov, Robert R.; Pond, J. Blake; Al-Huqail, Asma; A. Galkin, Mikhail; Vik, Steven B.

    2008-01-01

    Interactions between subunit a and oligomeric subunit c are essential for the coupling of proton translocation to rotary motion in the ATP synthase. A pair of previously described mutants, R210Q/Q252R and P204T/R210Q/Q252R (Hatch et al., J. Biol. Chem. (1995) 270:29417-29412) have been constructed and further analyzed. These mutants, in which the essential arginine of subunit a, R210, was switched with a conserved glutamine residue, Q252, are shown here to be capable of both ATP synthesis by oxidative phosphorylation, and ATP-driven proton translocation. In addition, lysine can replace the arginine at position 252 with partial retention of both activities. The pH dependence of ATP-driven proton translocation was determined after purification of mutant enzymes, and reconstitution into liposomes. Proton translocation by the lysine mutant, and to a lesser extent the arginine mutant, dropped off sharply above pH 7.5, consistent with the requirement for a positive charge during function. Finally, the rates of ATP synthesis and of ATP-driven proton translocation were completely inhibited by treatment with DCCD (N, N’-dicyclohexyl carbodiimide), while rates of ATP hydrolysis by the mutants were not significantly affected, indicating that DCCD modification disrupts the F1-Fo interface. The results suggest that minimal requirements for proton translocation by the ATP synthase include a positive charge in subunit a and a weak interface between subunit a and oligomeric subunit c. PMID:18068111

  14. ATP synthase: an electrochemical ransducer with rotatory mechanics

    Microsoft Academic Search

    Wolfgang Junge; Holger Lill; Siegfried Engelbrecht

    1997-01-01

    ATP synthase (F0F1-ATPase) uses proton- or sodium-motive force to produce ATP from ADP and P1. Three lines of experiment have recently demonstrated large-scale intersubunit rotation during ATP hydrolysis by F1. We discuss how ion flow through the membrane-intrinsic portion, F0, may generate torque and how this might be transmitted between stator and rotor to finally expel spontaneously formed ATP from

  15. Renal cell-to-cell communication via extracellular ATP.

    PubMed

    Komlosi, Peter; Fintha, Attila; Bell, P Darwin

    2005-04-01

    In the kidney, macula densa cells communicate with the mesangial cell-afferent arteriolar smooth muscle cell complex through ATP signaling. This signaling process involves release of ATP across the macula densa basolateral membrane through a maxi anion channel and the interaction of ATP with purinergic P2 receptors. PMID:15772296

  16. Physiological levels of ATP Negatively Regulate Proteasome Function

    PubMed Central

    Huang, Hongbiao; Zhang, Xiaoyan; Li, Shujue; Liu, Ningning; Lian, Wen; McDowell, Emily; Zhou, Ping; Zhao, Canguo; Guo, Haiping; Zhang, Change; Yang, Changshan; Wen, Guangmei; Dong, Xiaoxian; Lu, Li; Ma, Ningfang; Dong, Weihua; Dou, Q. Ping; Wang, Xuejun; Liu, Jinbao

    2010-01-01

    Intracellular protein degradation by the ubiquitin-proteasome system is ATP-dependent and the optimal ATP concentration to activate proteasome function in vitro is ~100 ?M. Intracellular ATP levels are generally in the low millimolar range but ATP at a level within this range was shown to inhibit proteasome peptidase activities in vitro. Here we report new evidence that supports a hypothesis that intracellular ATP at the physiological levels bidirectionally regulates 26S proteasome proteolytic function in the cell. First, we confirmed that ATP exerted bidirectional regulation on the 26S proteasome in vitro, with the optimal ATP concentration (between 50–100 ?M) stimulating proteasome chymotrypsin-like activities. Second, we found that manipulating intracellular ATP levels also led to bidirectional changes in the levels of proteasome-specific protein substrates in cultured cells. Finally, measures to increase intracellular ATP enhanced, while decreasing intracellular ATP attenuated, the ability of proteasome inhibition to induce cell death. These data strongly suggest that endogenous ATP within the physiological concentration range can exert a negative impact on proteasome activities, allowing the cell to rapidly up-regulate proteasome activity upon ATP reduction under stress conditions. PMID:20805844

  17. 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…

  18. 50 CFR 230.2 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ...person described in paragraph (1) of this definition. Calf means any whale less than 1 year old or having milk in its stomach. Commission means the International Whaling Commission established by article III of the Convention. Convention...

  19. 50 CFR 230.2 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ...person described in paragraph (1) of this definition. Calf means any whale less than 1 year old or having milk in its stomach. Commission means the International Whaling Commission established by article III of the Convention. Convention...

  20. 50 CFR 230.2 - Definitions.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ...person described in paragraph (1) of this definition. Calf means any whale less than 1 year old or having milk in its stomach. Commission means the International Whaling Commission established by article III of the Convention. Convention...

  1. 48 CFR 34.101 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ...Federal Acquisition Regulations System FEDERAL ACQUISITION REGULATION...CATEGORIES OF CONTRACTING MAJOR SYSTEM ACQUISITION Testing, Qualification...Developed Under Title III, Defense Production Act 34.101 Definitions...subsystem integral to a major system, and other property...

  2. Clostridium pasteurianum F1Fo ATP Synthase: Operon, Composition, and Some Properties

    Microsoft Academic Search

    Amaresh Das; Lars G. Ljungdahl

    2003-01-01

    The atp operon encoding F1Fo ATP synthase in the fermentative obligate anaerobic bacterium Clostridium pasteurianum was sequenced. It consisted of nine genes arranged in the order atpI(i), atpB(a), atpE(c), atpF(b), atpH(), atpA(), atpG(), atpD(), and atpC(), which was identical to that found in many bacteria. Reverse transcription-PCR confirmed the presence of the transcripts of all nine genes. The amount of

  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. ATP-dependent proteinases in bacteria.

    PubMed

    Hlavácek, O; Váchová, L

    2002-01-01

    Cytoplasmic proteolysis is an indispensable process for proper function of a cell. Degradation of many intracellular proteins is initiated by ATP-dependent proteinases, which are involved in the regulation of the level of proteins with short half-lives. In addition, they remove many damaged and abnormal proteins and thus play also an important role during stress. ATP-dependent proteinases are large multi-subunit assemblies composed of proteolytic core domains and ATPase-containing regulatory domains on a single polypeptide chain or on distinct subunits, which can act as molecular chaperones. This review briefly summarizes the data about four main groups of these proteinases in bacteria (i.e. Lon, Clp family, HslUV and FtsH) and characterizes their structure, mechanism of action and properties. PMID:12094726

  5. ATP modulates the growth of specific microbial strains.

    PubMed

    Li, Ming; Lee, Sung-Kwon; Yang, Seung Hwan; Ko, Jung Hwan; Han, Jeong Sun; Kim, Tae-Jong; Suh, Joo-Won

    2011-01-01

    The regulatory function of extracellular ATP (exATP) in bacteria is unknown, but recent studies have demonstrated exATP induced enhanced secondary metabolite production and morphological differentiation in Streptomyces coelicolor. The growth of Streptomyces coelicolor, however, was unaffected by exATP, although changes in growth are common phenotypes. To identify bacteria whose growth is altered by exATP, we measured exATP-induced population changes in fast-growing microbes and actinomycetes in compost. Compared with the water-treated control, the addition of 10 ml 100 ?M ATP to 10 g of compost enhanced the actinomycetes population by 30% and decreased fast-growing microbial numbers by 20%. Eight microbes from each group were selected from the most populated colony, based on appearance. Of the eight isolated fast-growing microbes, the 16S rRNA sequences of three isolates were similar to the plant pathogens Serratia proteamaculans and Sphingomonas melonis, and one was close to a human pathogen, Elizabethkingia meningoseptica. The growth of all fast-growing microbes was inhibited by ATP, which was confirmed in Pseudomonas syringae DC3000, a pathogenic plant bacterium. The growth of six of eight isolated actinomycetes strains, all of which were identified as close to Streptomyces neyagawaensis, was enhanced by ATP treatment. This study suggests that exATP regulates bacterial physiology and that the exATP response system is a target for the control of bacterial ecology. PMID:20512647

  6. ATP synthases from archaea: the beauty of a molecular motor.

    PubMed

    Grüber, Gerhard; Manimekalai, Malathy Sony Subramanian; Mayer, Florian; Müller, Volker

    2014-06-01

    Archaea live under different environmental conditions, such as high salinity, extreme pHs and cold or hot temperatures. How energy is conserved under such harsh environmental conditions is a major question in cellular bioenergetics of archaea. The key enzymes in energy conservation are the archaeal A1AO ATP synthases, a class of ATP synthases distinct from the F1FO ATP synthase ATP synthase found in bacteria, mitochondria and chloroplasts and the V1VO ATPases of eukaryotes. A1AO ATP synthases have distinct structural features such as a collar-like structure, an extended central stalk, and two peripheral stalks possibly stabilizing the A1AO ATP synthase during rotation in ATP synthesis/hydrolysis at high temperatures as well as to provide the storage of transient elastic energy during ion-pumping and ATP synthesis/-hydrolysis. High resolution structures of individual subunits and subcomplexes have been obtained in recent years that shed new light on the function and mechanism of this unique class of ATP synthases. An outstanding feature of archaeal A1AO ATP synthases is their diversity in size of rotor subunits and the coupling ion used for ATP synthesis with H(+), Na(+) or even H(+) and Na(+) using enzymes. The evolution of the H(+) binding site to a Na(+) binding site and its implications for the energy metabolism and physiology of the cell are discussed. PMID:24650628

  7. Yeast ADP/ATP Carrier Isoform 2

    PubMed Central

    Clémençon, Benjamin; Rey, Martial; Trézéguet, Véronique; Forest, Eric; Pelosi, Ludovic

    2011-01-01

    The mitochondrial ADP/ATP carrier, or Ancp, is a member of the mitochondrial carrier family responsible for exchanging ADP and ATP across the mitochondrial inner membrane. ADP/ATP transport involves Ancp switching between two conformational states. These can be analyzed using specific inhibitors, carboxyatractyloside (CATR) and bongkrekic acid (BA). The high resolution three-dimensional structure of bovine Anc1p (bAnc1p), as a CATR-carrier complex, has been solved. However, because the structure of the BA-carrier complex has not yet been determined, the detailed mechanism of transport remains unknown. Recently, sample processing for hydrogen/deuterium exchange experiments coupled to mass spectrometry was improved, providing novel insights into bAnc1p conformational transitions due to inhibitor binding. In this work we performed both hydrogen/deuterium exchange-mass spectrometry experiments and genetic manipulations. Because these are very difficult to apply with bovine Anc1p, we used Saccharomyces cerevisiae Anc isoform 2 (ScAnc2p). Significant differences in solvent accessibility were observed throughout the amino acid sequence for ScAnc2p complexed to either CATR or BA. Interestingly, in detergent solution, the conformational dynamics of ScAnc2p were dissimilar to those of bAnc1p, in particular for the upper half of the cavity, toward the intermembrane space, and the m2 loop, which is thought to be easily accessible to the solvent from the matrix in bAnc1p. Our study then focused on the methionyl residues of the Ancp signature sequence, RRRMMM. All our results indicate that the methionine cluster is involved in the ADP/ATP transport mechanism and confirm that the Ancp cavity is a highly dynamic structure. PMID:21868387

  8. Proteomic Analysis of Extracellular ATP-Regulated Proteins Identifies ATP Synthase ?-Subunit as a Novel Plant Cell Death Regulator*

    PubMed Central

    Chivasa, Stephen; Tomé, Daniel F. A.; Hamilton, John M.; Slabas, Antoni R.

    2011-01-01

    Extracellular ATP is an important signal molecule required to cue plant growth and developmental programs, interactions with other organisms, and responses to environmental stimuli. The molecular targets mediating the physiological effects of extracellular ATP in plants have not yet been identified. We developed a well characterized experimental system that depletes Arabidopsis cell suspension culture extracellular ATP via treatment with the cell death-inducing mycotoxin fumonisin B1. This provided a platform for protein profile comparison between extracellular ATP-depleted cells and fumonisin B1-treated cells replenished with exogenous ATP, thus enabling the identification of proteins regulated by extracellular ATP signaling. Using two-dimensional difference in-gel electrophoresis and matrix-assisted laser desorption-time of flight MS analysis of microsomal membrane and total soluble protein fractions, we identified 26 distinct proteins whose gene expression is controlled by the level of extracellular ATP. An additional 48 proteins that responded to fumonisin B1 were unaffected by extracellular ATP levels, confirming that this mycotoxin has physiological effects on Arabidopsis that are independent of its ability to trigger extracellular ATP depletion. Molecular chaperones, cellular redox control enzymes, glycolytic enzymes, and components of the cellular protein degradation machinery were among the extracellular ATP-responsive proteins. A major category of proteins highly regulated by extracellular ATP were components of ATP metabolism enzymes. We selected one of these, the mitochondrial ATP synthase ?-subunit, for further analysis using reverse genetics. Plants in which the gene for this protein was knocked out by insertion of a transfer-DNA sequence became resistant to fumonisin B1-induced cell death. Therefore, in addition to its function in mitochondrial oxidative phosphorylation, our study defines a new role for ATP synthase ?-subunit as a pro-cell death protein. More significantly, this protein is a novel target for extracellular ATP in its function as a key negative regulator of plant cell death. PMID:21156838

  9. Unique rotary ATP synthase and its biological diversity.

    PubMed

    von Ballmoos, Christoph; Cook, Gregory M; Dimroth, Peter

    2008-01-01

    F1F0 ATP synthases convert energy stored in an electrochemical gradient of H+ or Na+ across the membrane into mechanical rotation, which is subsequently converted into the chemical bond energy of ATP. The majority of cellular ATP is produced by the ATP synthase in organisms throughout the biological kingdom and therefore under diverse environmental conditions. The ATP synthase of each particular cell is confronted with specific challenges, imposed by the specific environment, and thus by necessity must adapt to these conditions for optimal operation. Examples of these adaptations include diverse mechanisms for regulating the ATP hydrolysis activity of the enzyme, the utilization of different coupling ions with distinct ion binding characteristics, different ion-to-ATP ratios reflected by variations in the size of the rotor c ring, the mode of ion delivery to the binding sites, and the different contributions of the electrical and chemical gradients to the driving force. PMID:18573072

  10. [Structure and function of ATP7A and ATP7B proteins--Cu-transporting ATPases].

    PubMed

    Lenartowicz, Ma?gorzata; Krzeptowski, Wojciech

    2010-01-01

    Living organisms have developed refined and geneticaly controlled mechanisms of the copper metabolism and transport. ATP7A and ATP7B proteins play the key role in copper homeostasis in the organism. Both proteins are P-type Cu-transporting ATPases and use the energy of ATP hydrolysis to transfer the copper ions across the cellular membranes. Both proteins are localised in Golgi aparatus and involved in regulation of overall copper status in the body and their function is the export of excess copper from the cells and delivery of copper ions to Cu-dependent enzymes. Moreover in organism Cu-transporting ATPases are involved in absorption of dietary copper, Cu removal with the bile, placental copper transport and its secretion to the milk during lactation. Moreover it is known that Cu-transporting ATPases play a role in generation of anti-cancer drug resistance. Disturbances of ATP7A and ATP7B function caused by mutations lead to severe metabolic diseases Menkes and Wilson diseases, respectively. PMID:21117320

  11. Early opening of sarcolemmal ATP-sensitive potassium channels is not a key step in PKC-mediated cardioprotection.

    PubMed

    Brennan, Sean; Jackson, Robert; Patel, Manish; Sims, Mark W; Hudman, Diane; Norman, Robert I; Lodwick, David; Rainbow, Richard D

    2015-02-01

    ATP-sensitive potassium (KATP) channels are abundantly expressed in the myocardium. Although a definitive role for the channel remains elusive they have been implicated in the phenomenon of cardioprotection, but the precise mechanism is unclear. We set out to test the hypothesis that the channel protects by opening early during ischemia to shorten action potential duration and reduce electrical excitability thus sparing intracellular ATP. This could reduce reperfusion injury by improving calcium homeostasis. Using a combination of contractile function analysis, calcium fluorescence imaging and patch clamp electrophysiology in cardiomyocytes isolated from adult male Wistar rats, we demonstrated that the opening of sarcolemmal KATP channels was markedly delayed after cardioprotective treatments: ischemic preconditioning, adenosine and PMA. This was due to the preservation of intracellular ATP for longer during simulated ischemia therefore maintaining sarcolemmal KATP channels in the closed state for longer. As the simulated ischemia progressed, KATP channels opened to cause contractile, calcium transient and action potential failure; however there was no indication of any channel activity early during simulated ischemia to impart an energy sparing hyperpolarization or action potential shortening. We present compelling evidence to demonstrate that an early opening of sarcolemmal KATP channels during simulated ischemia is not part of the protective mechanism imparted by ischemic preconditioning or other PKC-dependent cardioprotective stimuli. On the contrary, channel opening was actually delayed. We conclude that sarcolemmal KATP channel opening is a consequence of ATP depletion, not a primary mechanism of ATP preservation in these cells. PMID:25450614

  12. 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

  13. Membrane embedded location of Na+ or H+ binding sites on the rotor ring of F1F0 ATP synthases.

    PubMed

    von Ballmoos, Christoph; Meier, Thomas; Dimroth, Peter

    2002-11-01

    Recent crosslinking studies indicated the localization of the coupling ion binding site in the Na+-translocating F1F0 ATP synthase of Ilyobacter tartaricus within the hydrophobic part of the bilayer. Similarly, a membrane embedded H+-binding site is accepted for the H+-translocating F1F0 ATP synthase of Escherichia coli. For a more definite analysis, we performed parallax analysis of fluorescence quenching with ATP synthases from both I. tartaricus and E. coli. Both ATP synthases were specifically labelled at their c subunit sites with N-cyclohexyl-N'-(1-pyrenyl)carbodiimide, a fluorescent analogue of dicyclohexylcarbodiimide and the enzymes were reconstituted into proteoliposomes. Using either soluble quenchers or spinlabelled phospholipids, we observed a deeply membrane embedded binding site, which was quantitatively determined for I. tartaricus and E. coli to be 1.3 +/- 2.4 A and 1.8 +/- 2.8 A from the bilayer center apart, respectively. These data show a conserved topology among enzymes of different species. We further demonstrated the direct accessibility for Na+ ions to the binding sites in the reconstituted I. tartaricus c11 oligomer in the absence of any other subunits, pointing to intrinsic rotor channels. The common membrane embedded location of the binding site of ATP synthases suggest a common mechanism for ion transfer across the membrane. PMID:12423357

  14. BPA Definitions

    SciTech Connect

    United States. Bonneville Power Administration.

    1993-12-01

    The Bonneville Power Administration`s definition of terms are documented here. The terms primarily focus on transmission, generation and distribution, but also cover BPA services and responsibilities.

  15. Clusterin and COMMD1 Independently Regulate Degradation of the Mammalian Copper ATPases ATP7A and ATP7B*

    PubMed Central

    Materia, Stephanie; Cater, Michael A.; Klomp, Leo W. J.; Mercer, Julian F. B.; La Fontaine, Sharon

    2012-01-01

    ATP7A and ATP7B are copper-transporting P1B-type ATPases (Cu-ATPases) that are critical for regulating intracellular copper homeostasis. Mutations in the genes encoding ATP7A and ATP7B lead to copper deficiency and copper toxicity disorders, Menkes and Wilson diseases, respectively. Clusterin and COMMD1 were previously identified as interacting partners of these Cu-ATPases. In this study, we confirmed that clusterin and COMMD1 interact to down-regulate both ATP7A and ATP7B. Overexpression and knockdown of clusterin/COMMD1 decreased and increased, respectively, endogenous levels of ATP7A and ATP7B, consistent with a role in facilitating Cu-ATPase degradation. We demonstrate that whereas the clusterin/ATP7B interaction was enhanced by oxidative stress or mutation of ATP7B, the COMMD1/ATP7B interaction did not change under oxidative stress conditions, and only increased with ATP7B mutations that led to its misfolding. Clusterin and COMMD1 facilitated the degradation of ATP7B containing the same Wilson disease-causing C-terminal mutations via different degradation pathways, clusterin via the lysosomal pathway and COMMD1 via the proteasomal pathway. Furthermore, endogenous ATP7B existed in a complex with clusterin and COMMD1, but these interactions were neither competitive nor cooperative and occurred independently of each other. Together these data indicate that clusterin and COMMD1 represent alternative and independent systems regulating Cu-ATPase quality control, and consequently contributing to the maintenance of copper homeostasis. PMID:22130675

  16. Kinetic mechanism of the dimeric ATP sulfurylase from plants.

    PubMed

    Ravilious, Geoffrey E; Herrmann, Jonathan; Goo Lee, Soon; Westfall, Corey S; Jez, Joseph M

    2013-01-01

    In plants, sulfur must be obtained from the environment and assimilated into usable forms for metabolism. ATP sulfurylase catalyses the thermodynamically unfavourable formation of a mixed phosphosulfate anhydride in APS (adenosine 5'-phosphosulfate) from ATP and sulfate as the first committed step of sulfur assimilation in plants. In contrast to the multi-functional, allosterically regulated ATP sulfurylases from bacteria, fungi and mammals, the plant enzyme functions as a mono-functional, non-allosteric homodimer. Owing to these differences, here we examine the kinetic mechanism of soybean ATP sulfurylase [GmATPS1 (Glycine max (soybean) ATP sulfurylase isoform 1)]. For the forward reaction (APS synthesis), initial velocity methods indicate a single-displacement mechanism. Dead-end inhibition studies with chlorate showed competitive inhibition versus sulfate and non-competitive inhibition versus APS. Initial velocity studies of the reverse reaction (ATP synthesis) demonstrate a sequential mechanism with global fitting analysis suggesting an ordered binding of substrates. ITC (isothermal titration calorimetry) showed tight binding of APS to GmATPS1. In contrast, binding of PPi (pyrophosphate) to GmATPS1 was not detected, although titration of the E•APS complex with PPi in the absence of magnesium displayed ternary complex formation. These results suggest a kinetic mechanism in which ATP and APS are the first substrates bound in the forward and reverse reactions, respectively. PMID:23789618

  17. Coupled ATP and potassium efflux from intercalated cells

    PubMed Central

    Holtzclaw, J. David; Cornelius, Ryan J.; Hatcher, Lori I.

    2011-01-01

    Increased flow in the distal nephron induces K secretion through the large-conductance, calcium-activated K channel (BK), which is primarily expressed in intercalated cells (IC). Since flow also increases ATP release from IC, we hypothesized that purinergic signaling has a role in shear stress (?; 10 dynes/cm2) -induced, BK-dependent, K efflux. We found that 10 ?M ATP led to increased IC Ca concentration, which was significantly reduced in the presence of the P2 receptor blocker suramin or calcium-free buffer. ATP also produced BK-dependent K efflux, and IC volume decrease. Suramin inhibited ?-induced K efflux, suggesting that K efflux is at least partially dependent on purinergic signaling. BK-?4 small interfering (si) RNA, but not nontarget siRNA, decreased ATP secretion and both ATP-dependent and ?-induced K efflux. Similarly, carbenoxolone (25 ?M), which blocks connexins, putative ATP pathways, blocked ?-induced K efflux and ATP secretion. Compared with BK-?4?/? mice, wild-type mice with high distal flows exhibited significantly more urinary ATP excretion. These data demonstrate coupled electrochemical efflux between K and ATP as part of the mechanism for ?-induced ATP release in IC. PMID:21454249

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

    PubMed Central

    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 (SO42-), 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 SO42--activation and yields activated high-energy compound adenosine-5?-phosphosulfate that is reduced to sulfide (S2-) 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

  19. Sequence and structural analysis of kinase ATP pocket residues.

    PubMed

    Vulpetti, Anna; Bosotti, Roberta

    2004-10-01

    Protein kinases represent one of the largest known families of enzymes. Most kinases bind ATP and most synthetic kinase inhibitors are ATP-competitive, which makes selectivity a potential problem. However, despite the high sequence similarity in the ATP binding pocket, several groups including ours have been able to develop highly potent and selective ATP-competitive inhibitors. To systematically aid the design of specific inhibitors in our protein kinase projects, we aligned all known three-dimensional structures and all known sequences of human protein kinases. We identified a set of 38 residues that make up the ATP pocket and analyzed the variability among these residues. The most variable residues in the ATP pocket are targeted to design specificity into inhibitors in our various kinase projects. PMID:15474052

  20. Evaluation of ATP content in hair bulbs in human scalp.

    PubMed

    Mikhal'chik, E V; Suprun, M V; Fedorkova, M V; Ibragimova, G A; Dmitrieva, E I; Lipatova, V A; Kutsev, S I

    2014-05-01

    The content of ATP in scalp hair bulbs in humans was measured in the hair roots from 15 healthy volunteers. Light and electron microscopy confirmed the presence of outer and an inner root sheaths in the root of pulled out anagen hair. Incubation of samples in buffer solution led to extraction of ATP, which was measured by the chemiluminescent method. Mechanic disintegration of hair bulbs and their freezing-defrosting did not increase ATP output. The results of microscopy indicated that ATP extraction procedure was associated with separation of the outer radical sheath from the inner one without impairing the structure of the inner sheath. The mean content of ATP was 12 ± 2 pmol per bulb. The use of pulled out hair bulbs for ATP measurements simplified the procedure as involved no surgical removal of follicles. PMID:24909726

  1. PROTECTED VETERAN DEFINITIONS TITLE DEFINITION

    E-print Network

    Tipple, Brett

    .S. military, ground, naval, or air service who served in the Republic of Vietnam between February 28, 1961PROTECTED VETERAN DEFINITIONS TITLE DEFINITION Veteran of the Vietnam Era Veteran of the U through May7, 1975, who: 1. served on active duty for a period of more than 180 days and was discharged

  2. Efficient Purification and Reconstitution of ATP Binding Cassette Transporter B6 (ABCB6) for Functional and Structural Studies*

    PubMed Central

    Chavan, Hemantkumar; Taimur Khan, Mohiuddin Md.; Tegos, George; Krishnamurthy, Partha

    2013-01-01

    The mitochondrial ATP binding cassette transporter ABCB6 has been associated with a broad range of physiological functions, including growth and development, therapy-related drug resistance, and the new blood group system Langereis. ABCB6 has been proposed to regulate heme synthesis by shuttling coproporphyrinogen III from the cytoplasm into the mitochondria. However, direct functional information of the transport complex is not known. To understand the role of ABCB6 in mitochondrial transport, we developed an in vitro system with pure and active protein. ABCB6 overexpressed in HEK293 cells was solubilized from mitochondrial membranes and purified to homogeneity. Purified ABCB6 showed a high binding affinity for MgATP (Kd = 0.18 ?m) and an ATPase activity with a Km of 0.99 mm. Reconstitution of ABCB6 into liposomes allowed biochemical characterization of the ATPase including (i) substrate-stimulated ATPase activity, (ii) transport kinetics of its proposed endogenous substrate coproporphyrinogen III, and (iii) transport kinetics of substrates identified using a high throughput screening assay. Mutagenesis of the conserved lysine to alanine (K629A) in the Walker A motif abolished ATP hydrolysis and substrate transport. These results suggest a direct interaction between mitochondrial ABCB6 and its transport substrates that is critical for the activity of the transporter. Furthermore, the simple immunoaffinity purification of ABCB6 to near homogeneity and efficient reconstitution of ABCB6 into liposomes might provide the basis for future studies on the structure/function of ABCB6. PMID:23792964

  3. ATP7B detoxifies silver in ciliated airway epithelial cells

    SciTech Connect

    Ibricevic, Aida, E-mail: aidaibricevic@hotmail.co [Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110 (United States); Brody, Steven L., E-mail: sbrody@dom.wustl.ed [Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110 (United States); Youngs, Wiley J., E-mail: youngs@uakron.ed [Department of Chemistry, University of Akron, Akron, OH 44325 (United States); Cannon, Carolyn L., E-mail: carolyn.cannon@utsouthwestern.ed [Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110 (United States)

    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.

  4. 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

  5. ATP Alone Triggers the Outward Facing Conformation of the Maltose ATP-binding Cassette Transporter*

    PubMed Central

    Bao, Huan; Duong, Franck

    2013-01-01

    The maltose transporter MalFGK2 is a study prototype for ABC importers. During catalysis, the MalFG membrane domain alternates between inward and outward facing conformations when the MalK dimer closes and hydrolyzes ATP. Because a rapid ATP hydrolysis depends on MalE and maltose, it has been proposed that closed liganded MalE facilitates the transition to the outward facing conformation. Here we find that, in contrast to the expected, ATP is sufficient for the closure of MalK and for the conversion of MalFG to the outward facing state. The outward facing transporter binds MalE with nanomolar affinity, yet neither MalE nor maltose is necessary or facilitates the transition. Thus, the rapid hydrolysis of ATP observed in the presence of MalE and maltose is not because closed liganded MalE accelerates the formation of the outward facing conformation. These findings have fundamental implications for the description of the transport reaction. PMID:23243313

  6. Essentials for ATP synthesis by F1F0 ATP synthases.

    PubMed

    von Ballmoos, Christoph; Wiedenmann, Alexander; Dimroth, Peter

    2009-01-01

    The majority of cellular energy in the form of adenosine triphosphate (ATP) is synthesized by the ubiquitous F(1)F(0) ATP synthase. Power for ATP synthesis derives from an electrochemical proton (or Na(+)) gradient, which drives rotation of membranous F(0) motor components. Efficient rotation not only requires a significant driving force (DeltamuH(+)), consisting of membrane potential (Deltapsi) and proton concentration gradient (DeltapH), but also a high proton concentration at the source P side. In vivo this is maintained by dynamic proton movements across and along the surface of the membrane. The torque-generating unit consists of the interface of the rotating c ring and the stator a subunit. Ion translocation through this unit involves a sophisticated interplay between the c-ring binding sites, the stator arginine, and the coupling ions on both sides of the membrane. c-ring rotation is transmitted to the eccentric shaft gamma-subunit to elicit conformational changes in the catalytic sites of F(1), leading to ATP synthesis. PMID:19489730

  7. ATP5H/KCTD2 locus is associated with Alzheimer's disease risk

    PubMed Central

    Boada, M; Antúnez, C; Ramírez-Lorca, R; DeStefano, A L; González-Pérez, A; Gayán, J; López-Arrieta, J; Ikram, M A; Hernández, I; Marín, J; Galán, J J; Bis, J C; Mauleón, A; Rosende-Roca, M; Moreno-Rey, C; Gudnasson, V; Morón, F J; Velasco, J; Carrasco, J M; Alegret, M; Espinosa, A; Vinyes, G; Lafuente, A; Vargas, L; Fitzpatrick, A L; Launer, L J; Sáez, M E; Vázquez, E; Becker, J T; López, O L; Serrano-Ríos, M; Tárraga, L; van Duijn, C M; Real, L M; Seshadri, S; Ruiz, A

    2014-01-01

    To identify loci associated with Alzheimer disease, we conducted a three-stage analysis using existing genome-wide association studies (GWAS) and genotyping in a new sample. In Stage I, all suggestive single-nucleotide polymorphisms (at P<0.001) in a previously reported GWAS of seven independent studies (8082 Alzheimer's disease (AD) cases; 12?040 controls) were selected, and in Stage II these were examined in an in silico analysis within the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium GWAS (1367 cases and 12904 controls). Six novel signals reaching P<5 × 10?6 were genotyped in an independent Stage III sample (the Fundació ACE data set) of 2200 sporadic AD patients and 2301 controls. We identified a novel association with AD in the adenosine triphosphate (ATP) synthase, H+ transporting, mitochondrial F0 (ATP5H)/Potassium channel tetramerization domain-containing protein 2 (KCTD2) locus, which reached genome-wide significance in the combined discovery and genotyping sample (rs11870474, odds ratio (OR)=1.58, P=2.6 × 10?7 in discovery and OR=1.43, P=0.004 in Fundació ACE data set; combined OR=1.53, P=4.7 × 10?9). This ATP5H/KCTD2 locus has an important function in mitochondrial energy production and neuronal hyperpolarization during cellular stress conditions, such as hypoxia or glucose deprivation. PMID:23857120

  8. ATP5H/KCTD2 locus is associated with Alzheimer's disease risk.

    PubMed

    Boada, M; Antúnez, C; Ramírez-Lorca, R; DeStefano, A L; González-Pérez, A; Gayán, J; López-Arrieta, J; Ikram, M A; Hernández, I; Marín, J; Galán, J J; Bis, J C; Mauleón, A; Rosende-Roca, M; Moreno-Rey, C; Gudnasson, V; Morón, F J; Velasco, J; Carrasco, J M; Alegret, M; Espinosa, A; Vinyes, G; Lafuente, A; Vargas, L; Fitzpatrick, A L; Launer, L J; Sáez, M E; Vázquez, E; Becker, J T; López, O L; Serrano-Ríos, M; Tárraga, L; van Duijn, C M; Real, L M; Seshadri, S; Ruiz, A

    2014-06-01

    To identify loci associated with Alzheimer disease, we conducted a three-stage analysis using existing genome-wide association studies (GWAS) and genotyping in a new sample. In Stage I, all suggestive single-nucleotide polymorphisms (at P<0.001) in a previously reported GWAS of seven independent studies (8082 Alzheimer's disease (AD) cases; 12?040 controls) were selected, and in Stage II these were examined in an in silico analysis within the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium GWAS (1367 cases and 12904 controls). Six novel signals reaching P<5 × 10(-6) were genotyped in an independent Stage III sample (the Fundació ACE data set) of 2200 sporadic AD patients and 2301 controls. We identified a novel association with AD in the adenosine triphosphate (ATP) synthase, H+ transporting, mitochondrial F0 (ATP5H)/Potassium channel tetramerization domain-containing protein 2 (KCTD2) locus, which reached genome-wide significance in the combined discovery and genotyping sample (rs11870474, odds ratio (OR)=1.58, P=2.6 × 10(-7) in discovery and OR=1.43, P=0.004 in Fundació ACE data set; combined OR=1.53, P=4.7 × 10(-9)). This ATP5H/KCTD2 locus has an important function in mitochondrial energy production and neuronal hyperpolarization during cellular stress conditions, such as hypoxia or glucose deprivation. PMID:23857120

  9. 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

  10. 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. (inventors)

    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.

  11. Imaging changes in the cytosolic ATP-to-ADP ratio

    PubMed Central

    Tantama, Mathew; Yellen, Gary

    2015-01-01

    Adenosine triphosphate (ATP) is a central metabolite that plays fundamental roles as an energy transfer molecule, a phosphate donor, and a signaling molecule inside cells. The phosphoryl group transfer potential of ATP provides a thermodynamic driving force for many metabolic reactions, and phosphorylation of both small metabolites and large proteins can serve as a regulatory modification. In the process of phosphoryl transfer from ATP, the diphosphate ADP is produced, and as a result, the ATP-to-ADP ratio is an important physiological control parameter. The ATP-to-ADP ratio is directly proportional to cellular energy charge and phosphorylation potential. Furthermore, several ATP-dependent enzymes and signaling proteins are regulated by ADP, and their activation profiles are a function of the ATP-to-ADP ratio. Finally, regeneration of ATP from ADP can serve as an important readout of energy metabolism and mitochondrial function. We therefore developed a genetically-encoded fluorescent biosensor tuned to sense ATP-to-ADP ratios in the physiological range of healthy mammalian cells. Here we present a protocol for using this biosensor to visualize energy status using live-cell fluorescence microscopy. PMID:25416365

  12. 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.

  13. 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-?1 (TGF-?1) 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-?1 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

  14. Glycolytic atp production estimated from 31 p magnetic resonance spectroscopy measurements during ischemic exercise in vivo

    Microsoft Academic Search

    H. Wackerhage; K. Mueller; U. Hoffmann; D. Leyk; D. Essfeld; J. Zange

    1996-01-01

    In an oxygen-depleted muscle, glycolytically produced ATP is inversely related to the ([ATP] + creatine phosphate [PCr]) decrease because ATP, PCr, and glycolysis are virtually the only energy sources under these conditions. In particular, the onset of glycolysis or any appreciable increase in the rate of glycolytic ATP production will lead to a slower rate of ([ATP] + [PCr]) breakdown

  15. ATP7A gene addition to the choroid plexus results in long-term rescue of the lethal copper transport defect in a Menkes disease mouse model.

    PubMed

    Donsante, Anthony; Yi, Ling; Zerfas, Patricia M; Brinster, Lauren R; Sullivan, Patricia; Goldstein, David S; Prohaska, Joseph; Centeno, Jose A; Rushing, Elisabeth; Kaler, Stephen G

    2011-12-01

    Menkes disease is a lethal infantile neurodegenerative disorder of copper metabolism caused by mutations in a P-type ATPase, ATP7A. Currently available treatment (daily subcutaneous copper injections) is not entirely effective in the majority of affected individuals. The mottled-brindled (mo-br) mouse recapitulates the Menkes phenotype, including abnormal copper transport to the brain owing to mutation in the murine homolog, Atp7a, and dies by 14 days of age. We documented that mo-br mice on C57BL/6 background were not rescued by peripheral copper administration, and used this model to evaluate brain-directed therapies. Neonatal mo-br mice received lateral ventricle injections of either adeno-associated virus serotype 5 (AAV5) harboring a reduced-size human ATP7A (rsATP7A) complementary DNA (cDNA), copper chloride, or both. AAV5-rsATP7A showed selective transduction of choroid plexus epithelia and AAV5-rsATP7A plus copper combination treatment rescued mo-br mice; 86% survived to weaning (21 days), median survival increased to 43 days, 37% lived beyond 100 days, and 22% survived to the study end point (300 days). This synergistic treatment effect correlated with increased brain copper levels, enhanced activity of dopamine-?-hydroxylase, a copper-dependent enzyme, and correction of brain pathology. Our findings provide the first definitive evidence that gene therapy may have clinical utility in the treatment of Menkes disease. PMID:21878905

  16. ATP7A Gene Addition to the Choroid Plexus Results in Long-term Rescue of the Lethal Copper Transport Defect in a Menkes Disease Mouse Model

    PubMed Central

    Donsante, Anthony; Yi, Ling; Zerfas, Patricia M; Brinster, Lauren R; Sullivan, Patricia; Goldstein, David S; Prohaska, Joseph; Centeno, Jose A; Rushing, Elisabeth; Kaler, Stephen G

    2011-01-01

    Menkes disease is a lethal infantile neurodegenerative disorder of copper metabolism caused by mutations in a P-type ATPase, ATP7A. Currently available treatment (daily subcutaneous copper injections) is not entirely effective in the majority of affected individuals. The mottled-brindled (mo-br) mouse recapitulates the Menkes phenotype, including abnormal copper transport to the brain owing to mutation in the murine homolog, Atp7a, and dies by 14 days of age. We documented that mo-br mice on C57BL/6 background were not rescued by peripheral copper administration, and used this model to evaluate brain-directed therapies. Neonatal mo-br mice received lateral ventricle injections of either adeno-associated virus serotype 5 (AAV5) harboring a reduced-size human ATP7A (rsATP7A) complementary DNA (cDNA), copper chloride, or both. AAV5-rsATP7A showed selective transduction of choroid plexus epithelia and AAV5-rsATP7A plus copper combination treatment rescued mo-br mice; 86% survived to weaning (21 days), median survival increased to 43 days, 37% lived beyond 100 days, and 22% survived to the study end point (300 days). This synergistic treatment effect correlated with increased brain copper levels, enhanced activity of dopamine-?-hydroxylase, a copper-dependent enzyme, and correction of brain pathology. Our findings provide the first definitive evidence that gene therapy may have clinical utility in the treatment of Menkes disease. PMID:21878905

  17. The dark side of extracellular ATP in kidney diseases.

    PubMed

    Solini, Anna; Usuelli, Vera; Fiorina, Paolo

    2015-05-01

    Intracellular ATP is the most vital source of cellular energy for biologic systems, whereas extracellular ATP is a multifaceted mediator of several cell functions via its interaction, in an autocrine or paracrine manner, with P2 purinergic receptors expressed on the cell surface. These ionotropic and metabotropic P2 purinergic receptors modulate a variety of physiologic events upon the maintenance of a highly sensitive "set point," the derangement of which may lead to the development of key pathogenic mechanisms during acute and chronic diseases. Growing evidence suggests that extracellular ATP signaling via P2 purinergic receptors may be involved in different renal pathologic conditions. For these reasons, investigators and pharmaceutical companies are actively exploring novel strategies to antagonize or block these receptors with the goal of reducing extracellular ATP production or accelerating extracellular ATP clearance. Targeting extracellular ATP signaling, particularly through the P2X7 receptor, has considerable translational potential, given that novel P2X7-receptor inhibitors are already available for clinical use (e.g., CE224,535, AZD9056, and GSK1482160). This review summarizes the current evidence regarding the involvement of extracellular ATP and its P2 purinergic receptor-mediated signaling in physiologic and pathologic processes in the kidney; potential therapeutic options targeting extracellular ATP purinergic receptors are analyzed as well. PMID:25452669

  18. Synphilin-1 Binds ATP and Regulates Intracellular Energy Status

    PubMed Central

    Li, Tianxia; Liu, Jingnan; Smith, Wanli W.

    2014-01-01

    Recent studies have suggested that synphilin-1, a cytoplasmic protein, is involved in energy homeostasis. Overexpression of synphilin-1 in neurons results in hyperphagia and obesity in animal models. However, the mechanism by which synphilin-1 alters energy homeostasis is unknown. Here, we used cell models and biochemical approaches to investigate the cellular functions of synphilin-1 that may affect energy balance. Synphilin-1 was pulled down by ATP-agarose beads, and the addition of ATP and ADP reduced this binding, indicating that synphilin-1 bound ADP and ATP. Synphilin-1 also bound GMP, GDP, and GTP but with a lower affinity than it bound ATP. In contrast, synphilin-1 did not bind with CTP. Overexpression of synphilin-1 in HEK293T cells significantly increased cellular ATP levels. Genetic alteration to abolish predicted ATP binding motifs of synphilin-1 or knockdown of synphilin-1 by siRNA reduced cellular ATP levels. Together, these data demonstrate that synphilin-1 binds and regulates the cellular energy molecule, ATP. These findings provide a molecular basis for understanding the actions of synphilin-1 in energy homeostasis. PMID:25545246

  19. Constitutive and agonist stimulated ATP secretion in leukocytes

    PubMed Central

    Campwala, Hinnah; Fountain, Samuel J.

    2013-01-01

    Release and reception of extracellular ATP by leukocytes plays a critical role in immune responses to infection, injury and cardiovascular disease. Leukocytes of both the innate, adaptive immune and central nervous system express a repertoire of cell surface receptors for ATP (P2X and P2Y receptors) and its metabolites. ATP acts as a damage-associated molecule pattern (DAMP) released by injured or dying cells. Detection of released ATP by neighboring leukocytes initiates inflammation and wound healing. However, recent evidence from our group and others suggests ATP release by leukocytes themselves serves to regulate homeostatic mechanisms and coordinate responses to external pro-inflammatory cues. Examples include the homeostatic control of intracellular calcium and regulation of migratory guidance during chemotactic response to external cues. Though there has been some progress in elucidating ATP release mechanisms of some mammalian cells types, release conduits and coupling signal transduction machinery remain larger elusive for leukocytes. Our recent studies suggest a role for secretory lysosomes in releasing ATP in monocytes. Though poorly defined, targeting ATP release mechanisms in leukocytes have great anti-inflammatory potential. PMID:23713132

  20. ATP Synthase: Two rotary molecular motors working together

    E-print Network

    Wang, Hongyun

    by which the protein operates (Figure 1b). Indeed, it turns out that ATP synthase is two rotary engines1 ATP Synthase: Two rotary molecular motors working together George Oster Hongyun Wang University this in a surprising way: by converting the electromotive force into a rotary torque that is used to promote phosphate

  1. ATP synthase--a marvellous rotary engine of the cell.

    PubMed

    Yoshida, M; Muneyuki, E; Hisabori, T

    2001-09-01

    ATP synthase can be thought of as a complex of two motors--the ATP-driven F1 motor and the proton-driven Fo motor--that rotate in opposite directions. The mechanisms by which rotation and catalysis are coupled in the working enzyme are now being unravelled on a molecular scale. PMID:11533724

  2. ATP synthase — a marvellous rotary engine of the cell

    Microsoft Academic Search

    Masasuke Yoshida; Eiro Muneyuki; Toru Hisabori

    2001-01-01

    ATP synthase can be thought of as a complex of two motors — the ATP-driven F1 motor and the proton-driven Fo motor — that rotate in opposite directions. The mechanisms by which rotation and catalysis are coupled in the working enzyme are now being unravelled on a molecular scale.

  3. Extracellular ATP: a potential regulator of plant cell death.

    PubMed

    Feng, Hanqing; Guan, Dongdong; Bai, Jingyue; Sun, Kun; Jia, Lingyun

    2015-08-01

    Adenosine 5'-triphosphate (ATP) has been regarded as an intracellular energy currency molecule for many years. In recent decades, it has been determined that ATP is released into the extracellular milieu by animal, plant and microbial cells. In animal cells, this extracellular ATP (eATP) functions as a signalling compound to mediate many cellular processes through its interaction with membrane-associated receptor proteins. It has also been reported that eATP is a signalling molecule required for the regulation of plant growth, development and responses to environmental stimuli. Recently, the first plant receptor for eATP was identified in Arabidopsis thaliana. Interestingly, some studies have shown that eATP is of particular importance in the control of plant cell death. In this review article, we summarize and discuss the theoretical and experimental advances that have been made with regard to the roles and mechanisms of eATP in plant cell death. We also make an attempt to address some speculative aspects to help develop and expand future research in this area. PMID:25395168

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

    PubMed

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

    2004-09-15

    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 (approximately 390 pS). The channel was selective to anions and showed significant permeability to ATP4- (PATP/PCl approximately 0.1) and MgATP2- (PATP/PCl approximately 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

  5. ZEPLIN-III

    SciTech Connect

    St Murphy, A. J. [School of Physics and Astronomy, University of Edinburgh, Edinburgh, EH9 3JZ (United Kingdom)

    2009-09-08

    The direct detection of weakly interacting massive particles by a terrestrial device is widely recognised as a definitive proof of the cold dark matter hypothesis and a robust test of physics beyond the Standard Model. ZEPLIN-III is one of the latest generation of instruments specifically designed for this objective. This instrument has developed the two-phase liquid-gas xenon technology, and features high-field extraction, open plan geometry and low background components. Here we present the status of the project as of February 2008.

  6. Modelling the ATP production in mitochondria

    E-print Network

    Saa, Alberto

    2012-01-01

    We revisit here the mathematical model for ATP production in mitochondria introduced recently by Bertram, Pedersen, Luciani, and Sherman (BPLS) as a simplification of the more complete but intricate Magnus and Keizer's model. We correct some inaccuracies in the BPLS original approximations and then analyze some of the dynamical properties of the model. We infer from exhaustive numerical explorations that the enhanced BPLS equations have a unique attractor fixed point for physiologically acceptable ranges of mitochondrial variables and respiration inputs. We determine, in the stationary regime, the dependence of the mitochondrial variables on the respiration inputs, namely the cytosolic concentration of calcium ${\\rm Ca}_{\\rm c}$ and the substrate fructose 1,6-bisphosphate FBP. The same effect of calcium saturation reported for the original BPLS model is observed here. We find out, however, an interesting non-stationary effect: the inertia of the model tends to increase considerably for high concentrations of ...

  7. Changing paradigms from a historical DSM-III and DSM-IV view toward an evidence-based definition of premature ejaculation. Part II--proposals for DSM-V and ICD11

    Microsoft Academic Search

    Marcel D. Waldinger; Dave H. Schweitzer

    2006-01-01

    In the Diagnostic and Statistical Manual of Mental Disorders (DSM), a descriptive definition for\\u000apremature ejaculation (PE) that was based on historical assumptions has been accepted.\\u000aAim. To formulate a new functional definition of PE in the DSM.\\u000aMethods. A “syndrome” approach instead of a “complaint” approach is applied and evidence-based data from\\u000aepidemiological and clinical studies are used.\\u000aResults.

  8. Performance and specificity of the covalently linked immunomagnetic separation-ATP method for rapid detection and enumeration of enterococci in coastal environments.

    PubMed

    Zimmer-Faust, Amity G; Thulsiraj, Vanessa; Ferguson, Donna; Jay, Jennifer A

    2014-05-01

    The performance and specificity of the covalently linked immunomagnetic separation-ATP (Cov-IMS/ATP) method for the detection and enumeration of enterococci was evaluated in recreational waters. Cov-IMS/ATP performance was compared with standard methods: defined substrate technology (Enterolert; IDEXX Laboratories), membrane filtration (EPA Method 1600), and an Enterococcus-specific quantitative PCR (qPCR) assay (EPA Method A). We extend previous studies by (i) analyzing the stability of the relationship between the Cov-IMS/ATP method and culture-based methods at different field sites, (ii) evaluating specificity of the assay for seven ATCC Enterococcus species, (iii) identifying cross-reacting organisms binding the antibody-bead complexes with 16S rRNA gene sequencing and evaluating specificity of the assay to five nonenterococcus species, and (iv) conducting preliminary tests of preabsorption as a means of improving the assay. Cov-IMS/ATP was found to perform consistently and with strong agreement rates (based on exceedance/compliance with regulatory limits) of between 83% and 100% compared to the culture-based Enterolert method at a variety of sites with complex inputs. The Cov-IMS/ATP method is specific to five of seven different Enterococcus spp. tested. However, there is potential for nontarget bacteria to bind the antibody, which may be reduced by purification of the IgG serum with preabsorption at problematic sites. The findings of this study help to validate the Cov-IMS/ATP method, suggesting a predictable relationship between the Cov-IMS/ATP method and traditional culture-based methods, which will allow for more widespread application of this rapid and field-portable method for coastal water quality assessment. PMID:24561583

  9. Mechanism of ATP loss in nonoxidative contracting muscle

    NSDL National Science Digital Library

    2011-03-01

    The transition from rest to intense exercise is a challenge to cellular energetics (11, 13, 15). The metabolic fuels, i.e., the sources of ATP to sustain muscular contraction, are creatine phosphate and glycogen. Two anaerobic metabolic paths, leading to ATP generation, are catalyzed by creatine kinase and by the 12 enzymes of nonoxidative glycolysis, starting from glycogen. There is now general agreement that, unless replenished, creatine phosphate can sustain heavy muscle contraction for only 3Â?4 s. Thereafter, nonoxidative glycolysis becomes the main ATP source, until the onset of fatigue. This article aimed to relate the path of ATP generation during glycogen utilization as a metabolic fuel with that of ATP breakdown in nonoxidative contracting muscle.

  10. Novel mitochondrial mutations in the ATP6 and ATP8 genes in patients with breast cancer

    PubMed Central

    GRZYBOWSKA-SZATKOWSKA, LUDMI?A; ?LASKA, BRYGIDA; RZYMOWSKA, JOLANTA; BRZOZOWSKA, ANNA; FLORIA?CZYK, BOLES?AW

    2014-01-01

    The role of the mitochondria in the process of carcinogenesis, mainly oxidative phosphorylation, mostly concerns their participation in the production of free radicals and ATP and in the process of apoptosis. The purpose of this study was to detect potential changes in the genes encoding the subunits 6 and 8 of the ATP synthase and their impact on the enzyme’s biochemical properties, structure and function in patients with breast tumors. The tested material was mitochondrial DNA (mtDNA) isolated from specimens of ductal carcinoma (carcinoma ductale) Tp1-2Np0-1Mp0, blood and non-cancerous tissue of mammary gland (control), sampled from 50 patients who had been operated for breast cancer. In the case of missense-type changes in the mtDNA, protein prediction software was used to assess their effect on the biochemical properties of the protein, its structure and function. We identified 8 changes in the ATP6 gene in 36/50 examined breast cancer cell samples and 5 changes in the ATP8 gene (10/50). Most of them were homoplasmic changes of missense type. Four of the changes (A8439C, G8858C, C9130G and T9119G) had not been described in the literature before. The identified mutations and polymorphisms, especially those of missense type, can affect mitochondrial functions, especially if the conservative domain of the protein is concerned. Replacement of ‘wild-type’ mtDNA by mutated mtDNA can be an important event in carcinogenesis. PMID:25110199

  11. Novel mitochondrial mutations in the ATP6 and ATP8 genes in patients with breast cancer.

    PubMed

    Grzybowska-Szatkowska, Ludmi?a; Slaska, Brygida; Rzymowska, Jolanta; Brzozowska, Anna; Floria?czyk, Boles?aw

    2014-10-01

    The role of the mitochondria in the process of carcinogenesis, mainly oxidative phosphorylation, mostly concerns their participation in the production of free radicals and ATP and in the process of apoptosis. The purpose of this study was to detect potential changes in the genes encoding the subunits 6 and 8 of the ATP synthase and their impact on the enzyme's biochemical properties, structure and function in patients with breast tumors. The tested material was mitochondrial DNA (mtDNA) isolated from specimens of ductal carcinoma (carcinoma ductale) Tp1-2Np0-1Mp0, blood and non-cancerous tissue of mammary gland (control), sampled from 50 patients who had been operated for breast cancer. In the case of missense-type changes in the mtDNA, protein prediction software was used to assess their effect on the biochemical properties of the protein, its structure and function. We identified 8 changes in the ATP6 gene in 36/50 examined breast cancer cell samples and 5 changes in the ATP8 gene (10/50). Most of them were homoplasmic changes of missense type. Four of the changes (A8439C, G8858C, C9130G and T9119G) had not been described in the literature before. The identified mutations and polymorphisms, especially those of missense type, can affect mitochondrial functions, especially if the conservative domain of the protein is concerned. Replacement of 'wild-type' mtDNA by mutated mtDNA can be an important event in carcinogenesis. PMID:25110199

  12. Structure and expression of the atp operon coding for F1F0-ATP synthase from the antibiotic-producing actinomycete Nonomuraea sp. ATCC 39727.

    PubMed

    Gaballo, Antonio; Abbrescia, Anna; Palese, Luigi L; Micelli, Loris; di Summa, Roberta; Alifano, Pietro; Papa, Sergio

    2006-09-01

    Nonomuraea sp. ATCC 39727 is a poorly characterized actinomycete, producer of the glycopeptide antibiotic A40926. In this study, the nucleotide sequence of the atp operon coding for F1F0-ATP synthase of Nonomuraea sp. ATCC 39727 was determined. It consisted of ten open reading frames arranged in the order atpI (encoding the i protein), orfX, atpB (a subunit), atpE (c subunit), atpF (b subunit), atpH (delta subunit), atpA (alpha subunit), atpG (gamma subunit), atpD (beta subunit) and atpC (epsilon subunit). The orfX coded for a putative small hydrophobic 71 amino acid peptide of unknown function related to several bacterial permeases. Its presence appeared to be a distinctive feature of the atp operon of phylogenetically distant actinobacteria. Transcription of the atp operon was evaluated. The results of northern blot and RT-PCR experiments demonstrated that the atp genes were co-transcribed into a single polycistronic mRNA. Real-time RT-PCR data provided evidence showing that transcription of the atp operon was biphasic during Nonomuraea growth. The amount of the atpD transcript decreased at the end of the exponential growth phase, and then moderately increased during the early stationary phase when, in contrast, the levels of ctaC, encoding the cytochrome c oxidase subunit II, progressively decreased. Western blot analysis confirmed that ATP synthase was also present in the membrane during the stationary phase. These results together with previous data demonstrate that oligomycin-sensitive ATP-driven proton pumping activity remained constant in the stationary phase; in contrast, the activity and cytochrome content of the respiratory enzymes became negligible. PMID:16545948

  13. Allosteric modulation of GABAA receptors by extracellular ATP

    PubMed Central

    2014-01-01

    Background The ?-aminobutyric acid type A receptor (GABAAR) is the primary receptor mediating fast synaptic inhibition in the brain and plays a critical role in modulation of neuronal excitability and neural networks. Previous studies have demonstrated that ATP and its nucleotide analogs may regulate the function of GABAARs via Ca2+-dependent intracellular mechanisms, which require activation of purinergic 2 (P2) receptors or cross-talk between two receptors. Results Here, we report a potentiation of GABAARs by extracellular ATP via a previously un-recognized allosteric mechanism. Using cultured hippocampal neurons as well as HEK293 cells transiently expressing GABAARs, we demonstrate that extracellular ATP potentiates GABAAR mediated currents in a dose-dependent manner with an EC50 of 2.1?±?0.2 mM. The potentiation was mediated by a postsynaptic mechanism that was not dependent on activation of either ecto-protein kinase or P2 receptors. Single channel recordings from cell-free excised membrane patches under outside-out mode or isolated membrane patches under cell-attached mode suggest that the ATP modulation of GABA currents is achieved through a direct action of ATP on the channels themselves and manifested by increasing the single channel open probability without alteration of its conductance. Moreover, this ATP potentiation of GABAAR could be reconstituted in HEK293 cells that transiently expressed recombinant rat GABAARs. Conclusions Our data strongly suggest that extracellular ATP allosterically potentiates GABAAR-gated chloride channels. This novel mode of ATP-mediated modulation of GABAARs may play an important role in regulating neuronal excitability and thereby in fine-tuning the excitation-inhibition balance under conditions where a high level of extracellular ATP is ensured. PMID:24456563

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

    SciTech Connect

    Chalut, J. [Grace Dearborn Inc., Mississauga, Ontario (Canada); Small, G. [Grace Dearborn, Lake Zurich, IL (United States); Payton, J. [Grace Dearborn, Arlington Heights, IL (United States)

    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.

  15. Dual recognition unit strategy improves the specificity of the adenosine triphosphate (ATP) aptamer biosensor for cerebral ATP assay.

    PubMed

    Yu, Ping; He, Xiulan; Zhang, Li; Mao, Lanqun

    2015-01-20

    Adenosine triphosphate (ATP) aptamer has been widely used as a recognition unit for biosensor development; however, its relatively poor specificity toward ATP against adenosine-5'-diphosphate (ADP) and adenosine-5'-monophosphate (AMP) essentially limits the application of the biosensors in real systems, especially in the complex cerebral system. In this study, for the first time, we demonstrate a dual recognition unit strategy (DRUS) to construct a highly selective and sensitive ATP biosensor by combining the recognition ability of aptamer toward A nucleobase and of polyimidazolium toward phosphate. The biosensors are constructed by first confining the polyimidazolium onto a gold surface by surface-initiated atom transfer radical polymerization (SI-ATRP), and then the aptamer onto electrode surface by electrostatic self-assembly to form dual-recognition-unit-functionalized electrodes. The constructed biosensor based on DRUS not only shows an ultrahigh sensitivity toward ATP with a detection limit down to the subattomole level but also an ultrahigh selectivity toward ATP without interference from ADP and AMP. The constructed biosensor is used for selective and sensitive sensing of the extracellular ATP in the cerebral system by combining in vivo microdialysis and can be used as a promising neurotechnology to probing cerebral ATP concentration. PMID:25495279

  16. Kinetics of the ATP and dATP-mediated formation of a functionally-active RecA-ssDNA complex.

    PubMed

    Nayak, Sunil; Bryant, Floyd R

    2015-08-01

    The kinetics of the ATP and dATP-mediated formation of a functionally-active RecA-ssDNA complex were examined by stopped-flow fluorescence spectroscopy, using a modified version of the RecA protein that contains a fluorescent reporter group in the ssDNA binding site. The results indicated that: i) an active RecA-ssDNA complex was formed more rapidly on dT200 than on dT50 when either ATP or dATP was provided as the nucleotide cofactor, and ii) active complex formation occurred more rapidly with dATP than with ATP on either dT50 or dT200. The dependence on both the identity of the nucleotide cofactor and the length of the ssDNA effector indicated that active complex formation occurs by a cooperative mechanism and that dATP is more effective than ATP in mediating the interactions between RecA monomers that drive this process. Interestingly, the time courses of dATP-mediated active complex formation were closely similar to those that were obtained with ATP?S, an effectively non-hydrolyzable ATP analog that strongly stabilizes the active conformation of the RecA-ssDNA complex. These results provide mechanistic insight into the enhanced ssDNA binding and DNA strand exchange activities that are observed when dATP is provided in place of ATP in RecA biochemical assays. PMID:26100204

  17. Astrocytes Protect Neurons against Methylmercury via ATP/P2Y1 Receptor-Mediated Pathways in Astrocytes

    PubMed Central

    Shibata, Keisuke; Imura, Yoshio; Morizawa, Yosuke; Gachet, Christian; Koizumi, Schuichi

    2013-01-01

    Methylmercury (MeHg) is a well known environmental pollutant that induces serious neuronal damage. Although MeHg readily crosses the blood-brain barrier, and should affect both neurons and glial cells, how it affects glia or neuron-to-glia interactions has received only limited attention. Here, we report that MeHg triggers ATP/P2Y1 receptor signals in astrocytes, thereby protecting neurons against MeHg via interleukin-6 (IL-6)-mediated pathways. MeHg increased several mRNAs in astrocytes, among which IL-6 was the highest. For this, ATP/P2Y1 receptor-mediated mechanisms were required because the IL-6 production was (i) inhibited by a P2Y1 receptor antagonist, MRS2179, (ii) abolished in astrocytes obtained from P2Y1 receptor-knockout mice, and (iii) mimicked by exogenously applied ATP. In addition, (iv) MeHg released ATP by exocytosis from astrocytes. As for the intracellular mechanisms responsible for IL-6 production, p38 MAP kinase was involved. MeHg-treated astrocyte-conditioned medium (ACM) showed neuro-protective effects against MeHg, which was blocked by anti-IL-6 antibody and was mimicked by the application of recombinant IL-6. As for the mechanism of neuro-protection by IL-6, an adenosine A1 receptor-mediated pathway in neurons seems to be involved. Taken together, when astrocytes sense MeHg, they release ATP that autostimulates P2Y1 receptors to upregulate IL-6, thereby leading to A1 receptor-mediated neuro-protection against MeHg. PMID:23469098

  18. 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

  19. Reverse engineering a protein: the mechanochemistry of ATP synthase.

    PubMed

    Oster, G; Wang, H

    2000-05-31

    ATP synthase comprises two rotary motors in one. The F(1) motor can generate a mechanical torque using the hydrolysis energy of ATP. The F(o) motor generates a rotary torque in the opposite direction, but it employs a transmembrane proton motive force. Each motor can be reversed: The F(o) motor can drive the F(1) motor in reverse to synthesize ATP, and the F(1) motor can drive the F(o) motor in reverse to pump protons. Thus ATP synthase exhibits two of the major energy transduction pathways employed by the cell to convert chemical energy into mechanical force. Here we show how a physical analysis of the F(1) and F(o) motors can provide a unified view of the mechanochemical principles underlying these energy transducers. PMID:10838060

  20. Bacterial Na(+)-ATP synthase has an undecameric rotor.

    PubMed

    Stahlberg, H; Müller, D J; Suda, K; Fotiadis, D; Engel, A; Meier, T; Matthey, U; Dimroth, P

    2001-03-01

    Synthesis of adenosine triphosphate (ATP) by the F(1)F(0) ATP synthase involves a membrane-embedded rotary engine, the F(0) domain, which drives the extra-membranous catalytic F(1) domain. The F(0) domain consists of subunits a(1)b(2) and a cylindrical rotor assembled from 9-14 alpha-helical hairpin-shaped c-subunits. According to structural analyses, rotors contain 10 c-subunits in yeast and 14 in chloroplast ATP synthases. We determined the rotor stoichiometry of Ilyobacter tartaricus ATP synthase by atomic force microscopy and cryo-electron microscopy, and show the cylindrical sodium-driven rotor to comprise 11 c-subunits. PMID:11266365

  1. Aeronautics Test Program (ATP) Corporate Management of Aeronautical

    E-print Network

    Aeronautics Test Program (ATP) Corporate Management of Aeronautical Facilities Blair Gloss Program #12;Goals Corporate Management of Aeronautical Facilities · Increase the probability of having Chief, Research Testing Div. Jeffrey E. Haas Glenn Research Center Aeronautics Test Program Organization

  2. Aeronautics Test Program (ATP) Corporate Management of Aeronautical Facilities

    E-print Network

    Aeronautics Test Program (ATP) Corporate Management of Aeronautical Facilities 44th AIAA Aerospace Activity (NATA) · Summary #12;Goals Corporate Management of Aeronautical Facilities · Increase vision and plan · NASA Aeronautics Research Mission Directorate (ARMD) commitment to sustain facilities

  3. 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

  4. 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 ?3 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

  5. 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 of the Fo sector. The absence of genes encoding orthologs of the novel subunits even in apicomplexans suggests that the Tetrahymena ATP synthase, despite core similarities, is a unique enzyme exhibiting dramatic differences compared to the conventional complexes found in metazoan, fungal, and plant mitochondria, as well as in prokaryotes. These findings have significant implications for the origins and evolution of a central player in bioenergetics. PMID:20644710

  6. Highly divergent mitochondrial ATP synthase complexes in Tetrahymena thermophila.

    PubMed

    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 F(1) sector catalyzes ATP synthesis, whereas the F(o) sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F(1) and F(o) 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 F(o) 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 of the F(o) sector. The absence of genes encoding orthologs of the novel subunits even in apicomplexans suggests that the Tetrahymena ATP synthase, despite core similarities, is a unique enzyme exhibiting dramatic differences compared to the conventional complexes found in metazoan, fungal, and plant mitochondria, as well as in prokaryotes. These findings have significant implications for the origins and evolution of a central player in bioenergetics. PMID:20644710

  7. Clinical application of adenosine and ATP for pain control

    Microsoft Academic Search

    Masakazu Hayashida; Ken-ichi Fukuda; Atsuo Fukunaga

    2005-01-01

    This review summarizes clinical application of adenosine and adenosine 5?-triphosphate (ATP) in pain conditions. Investigations have been performed in patients with acute perioperative pain or chronic neuropathic pain treated with intravenous adenosine or ATP, or intrathecal adenosine. Characteristic central adenosine A1 receptor-mediated pain-relieving effects have been observed after intravenous adenosine infusion in human inflammation\\/sensitization pain models and in patients with

  8. ATP-regulated K+ channels in cardiac muscle

    Microsoft Academic Search

    A. Noma

    1983-01-01

    An outward current of unknown nature increases significantly when cardiac cells are treated with cyanide or subjected to hypoxia1-4, and decreases on intracellular injection of ATP5. We report here that application of the patch-clamp technique to CN-treated mammalian heart cells reveals specific K+ channels which are depressed by intracellular ATP (ATPi) at levels greater than 1 mM. For these channels,

  9. Contribution of intracellular ATP to cisplatin resistance of tumor cells.

    PubMed

    Schneider, Verena; Krieger, Michaela L; Bendas, Gerd; Jaehde, Ulrich; Kalayda, Ganna V

    2013-02-01

    Decreased cellular accumulation of cisplatin is a frequently observed mechanism of resistance to the drug. Beside passive diffusion, several cellular proteins using ATP hydrolysis as an energy source are assumed to be involved in cisplatin transport in and out of the cell. This investigation aimed at clarifying the contribution of intracellular ATP as an indicator of energy-dependent transport to cisplatin resistance using the A2780 human ovarian adenocarcinoma cell line and its cisplatin-resistant variant A2780cis. Depletion of intracellular ATP with oligomycin significantly decreased cellular platinum accumulation (measured by flameless atomic absorption spectrometry) in sensitive but not in resistant cells, and did not affect cisplatin efflux in both cell lines. Inhibition of Na(+),K(+)-ATPase with ouabain reduced platinum accumulation in A2780 cells but to a lesser extent compared with oligomycin. Western blot analysis revealed lower expression of Na(+),K(+)-ATPase ?(1) subunit in resistant cells compared with sensitive counterparts. The basal intracellular ATP level (determined using a bioluminescence-based assay) was significantly higher in A2780cis cells than in A2780 cells. Our results highlight the importance of ATP-dependent transport, among other processes mediated by Na(+),K(+)-ATPase, for cisplatin influx in sensitive cells. Cellular platinum accumulation in resistant cells is reduced and less dependent on energy sources, which may partly result from Na(+),K(+)-ATPase downregulation. Our data suggest the involvement of other ATP-dependent processes beside those regulated by Na(+),K(+)-ATPase. Higher basal ATP level in cisplatin-resistant cells, which appears to be a consequence of enhanced mitochondrial ATP production, may represent a survival mechanism established during development of resistance. PMID:23183891

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

    Microsoft Academic Search

    Judith Rishpon; Patrick J. O'Hara; Noam Lahav; James G. Lawless

    1982-01-01

    Summary The adsorption of ATP and ADP on montmorillonite, kaolinite, and A1(OH)3 was studied as a funtion of pH and, for montmorillonite and kaolinite, as a funtion of the ionic composition of the system. The three minerals exhibit different adsorption charcteristics. Mg2+- and Zn2+-montmorillonite adsorb ATP and ADP more than Na+-montmorillonite, presumably because of complex formation. In kaolinite, the effect

  11. Application of Hybrid Functional Groups to Predict ATP Binding Proteins

    PubMed Central

    Mbah, Andreas N.

    2014-01-01

    The ATP binding proteins exist as a hybrid of proteins with Walker A motif and universal stress proteins (USPs) having an alternative motif for binding ATP. There is an urgent need to find a reliable and comprehensive hybrid predictor for ATP binding proteins using whole sequence information. In this paper the open source LIBSVM toolbox was used to build a classifier at 10-fold cross-validation. The best hybrid model was the combination of amino acid and dipeptide composition with an accuracy of 84.57% and Mathews correlation coefficient (MCC) value of 0.693. This classifier proves to be better than many classical ATP binding protein predictors. The general trend observed is that combinations of descriptors performed better and improved the overall performances of individual descriptors, particularly when combined with amino acid composition. The work developed a comprehensive model for predicting ATP binding proteins irrespective of their functional motifs. This model provides a high probability of success for molecular biologists in predicting and selecting diverse groups of ATP binding proteins irrespective of their functional motifs. PMID:24729962

  12. Energy transduction in the F1 motor of ATP synthase.

    PubMed

    Wang, H; Oster, G

    1998-11-19

    ATP synthase is the universal enzyme that manufactures ATP from ADP and phosphate by using the energy derived from a transmembrane protonmotive gradient. It can also reverse itself and hydrolyse ATP to pump protons against an electrochemical gradient. ATP synthase carries out both its synthetic and hydrolytic cycles by a rotary mechanism. This has been confirmed in the direction of hydrolysis after isolation of the soluble F1 portion of the protein and visualization of the actual rotation of the central 'shaft' of the enzyme with respect to the rest of the molecule, making ATP synthase the world's smallest rotary engine. Here we present a model for this engine that accounts for its mechanochemical behaviour in both the hydrolysing and synthesizing directions. We conclude that the F1 motor achieves its high mechanical torque and almost 100% efficiency because it converts the free energy of ATP binding into elastic strain, which is then released by a coordinated kinetic and tightly coupled conformational mechanism to create a rotary torque. PMID:9834036

  13. Energy transduction in the F1 motor of ATP synthase

    NASA Astrophysics Data System (ADS)

    Wang, Hongyun; Oster, George

    1998-11-01

    ATP synthase is the universal enzyme that manufactures ATP from ADP and phosphate by using the energy derived from a transmembrane protonmotive gradient. It can also reverse itself and hydrolyse ATP to pump protons against an electrochemical gradient. ATP synthase carries out both its synthetic and hydrolytic cycles by a rotary mechanism. This has been confirmed in the direction of hydrolysis, after isolation of the soluble F1 portion of the protein and visualization of the actual rotation of the central `shaft' of the enzyme with respect to the rest of the molecule, making ATP synthase the world's smallest rotary engine. Here we present a model for this engine that accounts for its mechanochemical behaviour in both the hydrolysing and synthesizing directions. We conclude that the F1 motor achieves its high mechanical torque and almost 100% efficiency because it converts the free energy of ATP binding into elastic strain, which is then released by a coordinated kinetic and tightly coupled conformational mechanism to create a rotary torque.

  14. ATP and heat production in human skeletal muscle during dynamic exercise: higher efficiency of anaerobic than aerobic ATP resynthesis

    PubMed Central

    Krustrup, Peter; Ferguson, Richard A; Kjær, Michael; Bangsbo, Jens

    2003-01-01

    The aim of the present study was to simultaneously examine skeletal muscle heat production and ATP turnover in humans during dynamic exercise with marked differences in aerobic metabolism. This was done to test the hypothesis that efficiency is higher in anaerobic than aerobic ATP resynthesis. Six healthy male subjects performed 90 s of low intensity knee-extensor exercise with (OCC) and without thigh occlusion (CON-LI) as well as 90 s of high intensity exercise (CON-HI) that continued from the CON-LI bout. Muscle heat production was determined by continuous measurements of muscle heat accumulation and heat release to the blood. Muscle ATP production was quantified by repeated measurements of thigh oxygen uptake as well as blood and muscle metabolite changes. All temperatures of the thigh were equalized to ?37 °C prior to exercise by a water-perfused heating cuff. Oxygen uptake accounted for 80 ± 2 and 59 ± 4 %, respectively, of the total ATP resynthesis in CON-LI and CON-HI, whereas it was negligible in OCC. The rise in muscle temperature was lower (P < 0.05) in OCC than CON-LI (0.32 ± 0.04 vs. 0.37 ± 0.03 °C). The mean rate of heat production was also lower (P < 0.05) in OCC than CON-LI (36 ± 4 vs. 57 ± 4 J s?1). Mechanical efficiency was 52 ± 4 % after 15 s of OCC and remained constant, whereas it decreased (P < 0.05) from 56 ± 5 to 32 ± 3 % during CON-LI. During CON-HI, mechanical efficiency transiently increased (P < 0.05) to 47 ± 4 %, after which it decreased (P < 0.05) to 36 ± 3 % at the end of CON-HI. Assuming a fully coupled mitochondrial respiration, the ATP turnover per unit of work was calculated to be unaltered during OCC (?20 mmol ATP kJ?1), whereas it increased (P < 0.05) from 21 ± 4 to 29 ± 3 mmol ATP kJ?1 during CON-LI and further (P < 0.05) to 37 ± 3 mmol ATP kJ?1 during CON-HI. The present data confirm the hypothesis that heat loss is lower in anaerobic ATP resynthesis than in oxidative phosphorylation and can in part explain the finding that efficiency declines markedly during dynamic exercise. In addition, the rate of ATP turnover apparently increases during constant load low intensity exercise. Alternatively, mitochondrial efficiency is lowered as exercise progresses, since ATP turnover was unaltered during the ischaemic exercise bout. PMID:12651917

  15. The ATP:Co(I)rrinoid Adenosyltransferase (CobA) Enzyme of Salmonella enterica Requires the 2 -OH Group of ATP for Function

    E-print Network

    Rayment, Ivan

    The ATP:Co(I)rrinoid Adenosyltransferase (CobA) Enzyme of Salmonella enterica Requires the 2 -OH of Salmonella enterica serovar Typhimurium LT2 for its nucleotide substrate was tested using ATP analogs

  16. What limits the allotopic expression of nucleus-encoded mitochondrial genes? The case of the chimeric Cox3 and Atp6 genes

    Microsoft Academic Search

    Francisco Figueroa-Martínez; Miriam Vázquez-Acevedo; Paulina Cortés-Hernández; José J. García-Trejo; Edgar Davidson; Michael P. King; Diego González-Halphen

    2011-01-01

    Allotopic expression is potentially a gene therapy for mtDNA-related diseases. Some OXPHOS proteins like ATP6 (subunit a of complex V) and COX3 (subunit III of complex IV) that are typically mtDNA-encoded, are naturally nucleus-encoded in the alga Chlamydomonas reinhardtii. The mitochondrial proteins whose genes have been relocated to the nucleus exhibit long mitochondrial targeting sequences ranging from 100 to 140

  17. Molecular mechanisms of ATP secretion during immunogenic cell death

    PubMed Central

    Martins, I; Wang, Y; Michaud, M; Ma, Y; Sukkurwala, A Q; Shen, S; Kepp, O; Métivier, D; Galluzzi, L; Perfettini, J-L; Zitvogel, L; Kroemer, G

    2014-01-01

    The immunogenic demise of cancer cells can be induced by various chemotherapeutics, such as anthracyclines and oxaliplatin, and provokes an immune response against tumor-associated antigens. Thus, immunogenic cell death (ICD)-inducing antineoplastic agents stimulate a tumor-specific immune response that determines the long-term success of therapy. The release of ATP from dying cells constitutes one of the three major hallmarks of ICD and occurs independently of the two others, namely, the pre-apoptotic exposure of calreticulin on the cell surface and the postmortem release of high-mobility group box 1 (HMBG1) into the extracellular space. Pre-mortem autophagy is known to be required for the ICD-associated secretion of ATP, implying that autophagy-deficient cancer cells fail to elicit therapy-relevant immune responses in vivo. However, the precise molecular mechanisms whereby ATP is actively secreted in the course of ICD remain elusive. Using a combination of pharmacological screens, silencing experiments and techniques to monitor the subcellular localization of ATP, we show here that, in response to ICD inducers, ATP redistributes from lysosomes to autolysosomes and is secreted by a mechanism that requires the lysosomal protein LAMP1, which translocates to the plasma membrane in a strictly caspase-dependent manner. The secretion of ATP additionally involves the caspase-dependent activation of Rho-associated, coiled-coil containing protein kinase 1 (ROCK1)-mediated, myosin II-dependent cellular blebbing, as well as the opening of pannexin 1 (PANX1) channels, which is also triggered by caspases. Of note, although autophagy and LAMP1 fail to influence PANX1 channel opening, PANX1 is required for the ICD-associated translocation of LAMP1 to the plasma membrane. Altogether, these findings suggest that caspase- and PANX1-dependent lysosomal exocytosis has an essential role in ATP release as triggered by immunogenic chemotherapy. PMID:23852373

  18. Preservation of myocardial ATP. Comparison of blood vs crystalloid cardioplegia.

    PubMed

    Catinella, F P; Cunningham, J N; Knopp, E A; Laschinger, J C; Spencer, F C

    1983-04-01

    Preservation of myocardial high-energy phosphates correlates with the heart's ability to resume normal function following aortic crossclamping (AXC). The ability of the canine myocardium to synthesize and maintain ATP during 180 minutes of AXC was evaluated in 12 hearts subjected to either blood or crystalloid cardioplegic arrest. Group 1 hearts were arrested by infusion of 750 ml of blood potassium cardioplegia (BKC) solution into the aortic root initially and every 30 minutes, as were group 2 (six) hearts but with a crystalloid cardioplegia (CC) solution. Transmural left ventricular biopsy specimens were obtained for ATP analysis prior to AXC (control), before and after cardioplegia injections 2, 4, and 6, prior to unclamping (180 minutes of AXC), and 30 minutes following reperfusion. ATP levels increased significantly above control (p less than 0.005) during the 180 minutes of AXC immediately following infusion of BKC. At the end of 180 minutes of AXC and following 30 minutes of reperfusion, ATP was noted to be normal in this group (p = NS). In contrast, ATP levels fell significantly (p less than 0.005) during the period of aortic cross-clamping in the crystalloid cardioplegia group and did not return to normal even after 30 minutes of reperfusion (p less than 0.005). We concluded that BKC, by presenting the arrested myocyte with adequate oxygen and substrate, allows for synthesis and preservation of myocardial ATP during periods of AXC as long as three hours. In this respect, it should be regarded as superior to CC, which permits a statistically significant depletion of ATP (p less than 0.005) uncorrected, even after 30 minutes of reperfusion in the beating, nonworking state. PMID:6831955

  19. Two distinct proton binding sites in the ATP synthase family.

    PubMed

    von Ballmoos, Christoph; Dimroth, Peter

    2007-10-23

    The F1F0 ATP synthase utilizes energy stored in an electrochemical gradient of protons (or Na+ ions) across the membrane to synthesize ATP from ADP and phosphate. Current models predict that the protonation/deprotonation of specific acidic c ring residues is at the core of the proton translocation mechanism by this enzyme. To probe the mode of proton binding, we measured the covalent modification of the acidic c ring residues with the inhibitor dicyclohexylcarbodiimide (DCCD) over the pH range from 5 to 11. With the H+-translocating ATP synthase from the archaeum Halobacterium salinarium or the Na+-translocating ATP synthase from Ilyobacter tartaricus, the pH profile of DCCD labeling followed a titration curve with a pKa around neutral, reflecting protonation of the acidic c ring residues. However, with the ATP synthases from Escherichia coli, mitochondria, or chloroplasts, a clearly different, bell-shaped pH profile for DCCD labeling was observed which is not compatible with carboxylate protonation but might be explained by the coordination of a hydronium ion as proposed earlier [Boyer, P. D. (1988) Trends Biochem. Sci. 13, 5-7]. Upon site-directed mutagenesis of single binding site residues of the structurally resolved c ring, the sigmoidal pH profile for DCCD labeling could be converted to a more bell-shaped one, demonstrating that the different ion binding modes are based on subtle changes in the amino acid sequence of the protein. The concept of two different binding sites in the ATP synthase family is supported by the ATP hydrolysis pH profiles of the investigated enzymes. PMID:17910472

  20. Regulation of ATP/ADP in pancreatic islets.

    PubMed

    Sweet, Ian R; Cook, Daniel L; DeJulio, Eric; Wallen, Angela R; Khalil, Gamal; Callis, James; Reems, JoAnna

    2004-02-01

    ATP and ADP levels are critical regulators of glucose-stimulated insulin secretion. In many aerobic cell types, the phosphorylation potential (ATP/ADP/P(i)) is controlled by sensing mechanisms inherent in mitochondrial metabolism that feed back and induce compensatory changes in electron transport. To determine whether such regulation may contribute to stimulus-secretion coupling in islet cells, we used a recently developed flow culture system to continuously and noninvasively measure cytochrome c redox state and oxygen consumption as indexes of electron transport in perifused isolated rat islets. Increasing substrate availability by increasing glucose increased cytochrome c reduction and oxygen consumption, whereas increasing metabolic demand with glibenclamide increased oxygen consumption but not cytochrome c reduction. The data were analyzed using a kinetic model of the dual control of electron transport and oxygen consumption by substrate availability and energy demand, and ATP/ADP/P(i) was estimated as a function of time. ATP/ADP/P(i) increased in response to glucose and decreased in response to glibenclamide, consistent with what is known about the effects of these agents on energy state. Therefore, a simple model representing the hypothesized role of mitochondrial coupling in governing phosphorylation potential correctly predicted the directional changes in ATP/ADP/P(i). Thus, the data support the notion that mitochondrial-coupling mechanisms, by virtue of their role in establishing ATP and ADP levels, may play a role in mediating nutrient-stimulated insulin secretion. Our results also offer a new method for continuous noninvasive measures of islet cell phosphorylation potential, a critical metabolic variable that controls insulin secretion by ATP-sensitive K(+)-dependent and -independent mechanisms. PMID:14747291

  1. Coupling of proton ow to ATP synthesis in Rhodobacter capsulatus: F0F1-ATP synthase is absent from about half of chromatophores

    E-print Network

    Steinhoff, Heinz-Jürgen

    Coupling of proton £ow to ATP synthesis in Rhodobacter capsulatus: F0F1-ATP synthase is absent from the enzyme consists of a membrane-embedded proton- translocating F0 portion and a protruding hydrophilic F1 the kinetics of the total proton flow through F0F1 into (i) those coupled to the ATP synthesis and (ii) the de

  2. 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

  3. 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

  4. ATP synthases: cellular nanomotors characterized by LILBID mass spectrometry

    PubMed Central

    Hoffmann, Jan; Sokolova, Lucie; Preiss, Laura; Hicks, David B.; Krulwich, Terry A.; Morgner, Nina; Wittig, Ilka; Schägger, Hermann; Meier, Thomas; Brutschy, Bernd

    2010-01-01

    Mass spectrometry of membrane protein complexes is still a methodological challenge due to hydrophobic and hydrophilic parts of the species and the fact that all subunits are bound non-covalently together. The present study with the novel laser induced liquid bead ion desorption mass spectrometry (LILBID-MS) reports on the determination of the subunit composition of the F1Fo-ATP synthase from Bacillus pseudofirmus OF4, that of both bovine heart and, for the first time, of human heart mitochondrial F1Fo-ATP synthases. Under selected buffer conditions the mass of the intact F1Fo-ATP synthase of B. pseudofirmus OF4 could be measured, allowing the analysis of complex subunit stoichiometry. The agreement with theoretical masses derived from sequence databases is very good. A comparison of the ATP synthase subunit composition of 5 different ATPases reveals differences in the complexity of eukaryotic and bacterial ATP synthases. However, whereas the overall construction of eukaryotic enzymes is more complex than the bacterial ones, functionally important subunits are conserved among all ATPases. PMID:20820587

  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. Capture and quality control mechanisms for ATP binding

    PubMed Central

    Li, Li; Martinis, Susan A.

    2013-01-01

    The catalytic events in members of the nucleotidylyl transferase superfamily are initiated by a millisecond binding of ATP in the active site. Through metadynamics simulations on a class I aminoacyl-tRNA synthetase (aaRSs), the largest group in the superfamily, we calculate the free energy landscape of ATP selection and binding. Mutagenesis studies and fluorescence spectroscopy validated the identification of the most populated intermediate states. The rapid first binding step involves formation of encounter complexes captured through a fly-casting mechanism that acts up on the triphosphate moiety of ATP. In the slower nucleoside binding step, a conserved histidine in the HxxH motif orients the incoming ATP through base-stacking interactions resulting in a deep minimum in the free energy surface. Mutation of this histidine significantly decreases the binding affinity measured experimentally and computationally. The metadynamics simulations further reveal an intermediate quality control state that the synthetases and most likely other members of the superfamily use to select ATP over other nucleoside triphosphates. PMID:23276298

  7. Expanding the clinical phenotypes of MT-ATP6 mutations.

    PubMed

    López-Gallardo, Ester; Emperador, Sonia; Solano, Abelardo; Llobet, Laura; Martín-Navarro, Antonio; López-Pérez, Manuel José; Briones, Paz; Pineda, Mercedes; Artuch, Rafael; Barraquer, Elena; Jericó, Ivonne; Ruiz-Pesini, Eduardo; Montoya, Julio

    2014-12-01

    Mitochondrial DNA mutations at MT-ATP6 gene are relatively common in individuals suffering from striatal necrosis syndromes. These patients usually do not show apparent histochemical and/or biochemical signs of oxidative phosphorylation dysfunction. Because of this, MT-ATP6 is not typically analyzed in many other mitochondrial disorders that have not been previously associated to mutations in this gene. To correct this bias, we have performed a screening of the MT-ATP6 gene in a large collection of patients suspected of suffering different mitochondrial DNA (mtDNA) disorders. In three cases, biochemical, molecular-genetics and other analyses in patient tissues and cybrids were also carried out. We found three new pathologic mutations. Two of them in patients showing phenotypes that have not been commonly associated to mutations in the MT-ATP6 gene. These results remark the importance of sequencing the MT-ATP6 gene in patients with striatal necrosis syndromes, but also within other mitochondrial pathologies. This gene should be sequenced at least in all those patients suspected of suffering an mtDNA disorder disclosing normal results for histochemical and biochemical analyses of respiratory chain. PMID:24986921

  8. ATP-dependent bioluminescence in the firefly squid, Watasenia scintillans.

    PubMed

    Tsuji, F I

    1985-07-01

    The Japanese firefly squid, Watasenia scintillans, emits intense flashes of light from three tiny luminous organs that are located at the tip of each of a pair of ventral arms. Light is also produced from hundreds of other minute organs that are scattered over the body. The luminescence is due to an ATP-dependent reaction, with an optimal pH of 8.80. The decay of light intensity follows first-order kinetics and the decay constant is independent of initial ATP concentration. The light emission also requires MgCl(2), a soluble component, and an insoluble component that is membrane bound. Squids represent a major group of organisms unrelated to fireflies in which ATP is required for bioluminescence. PMID:16593580

  9. ATP-dependent bioluminescence in the firefly squid, Watasenia scintillans

    PubMed Central

    Tsuji, Frederick I.

    1985-01-01

    The Japanese firefly squid, Watasenia scintillans, emits intense flashes of light from three tiny luminous organs that are located at the tip of each of a pair of ventral arms. Light is also produced from hundreds of other minute organs that are scattered over the body. The luminescence is due to an ATP-dependent reaction, with an optimal pH of 8.80. The decay of light intensity follows first-order kinetics and the decay constant is independent of initial ATP concentration. The light emission also requires MgCl2, a soluble component, and an insoluble component that is membrane bound. Squids represent a major group of organisms unrelated to fireflies in which ATP is required for bioluminescence. Images PMID:16593580

  10. Dodecamer rotor ring defines H+/ATP ratio for ATP synthesis of prokaryotic V-ATPase from Thermus thermophilus

    PubMed Central

    Toei, Masashi; Gerle, Christoph; Nakano, Masahiro; Tani, Kazutoshi; Gyobu, Nobuhiko; Tamakoshi, Masatada; Sone, Nobuhito; Yoshida, Masasuke; Fujiyoshi, Yoshinori; Mitsuoka, Kaoru; Yokoyama, Ken

    2007-01-01

    ATP synthesis by V-ATPase from the thermophilic bacterium Thermus thermophilus driven by the acid-base transition was investigated. The rate of ATP synthesis increased in parallel with the increase in proton motive force (PMF) >110 mV, which is composed of a difference in proton concentration (?pH) and the electrical potential differences (??) across membranes. The optimum rate of synthesis reached 85 s?1, and the H+/ATP ratio of 4.0 ± 0.1 was obtained. ATP was synthesized at a considerable rate solely by ?pH, indicating ?? was not absolutely required for synthesis. Consistent with the H+/ATP ratio, cryoelectron micrograph images of 2D crystals of the membrane-bound rotor ring of the V-ATPase at 7.0-? resolution showed the presence of 12 Vo-c subunits, each composed of two transmembrane helices. These results indicate that symmetry mismatch between the rotor and catalytic domains is not obligatory for rotary ATPases/synthases. PMID:18077374

  11. Dactinomycin impairs cellular respiration and reduces accompanying ATP formation.

    PubMed

    Tao, Zhimin; Ahmad, Syed S; Penefsky, Harvey S; Goodisman, Jerry; Souid, Abdul-Kader

    2006-01-01

    The effect of dactinomycin on cellular respiration and accompanying ATP formation was investigated in Jurkat and HL-60 cells. Cellular mitochondrial oxygen consumption (measured by a homemade phosphorescence analyzer) and ATP content (measured by the luciferin-luciferase bioluminescence system) were determined as functions of time t during continuous exposure to the drug. The rate of respiration, k, was the negative of the slope of [O2] versus t. Oxygen consumption and ATP content were diminished by cyanide, confirming that both processes involved oxidations in the mitochondrial respiratory chain. In the presence of dactinomycin, k decreased gradually with t, the decrease being more pronounced at higher drug concentrations. Cellular ATP remained constant for 5 h in untreated cells, but in the presence of 20 microM dactinomycin it decreased gradually (to one-tenth the value at 5 h for untreated cells). The drug-induced inhibition of respiration and decrease in ATP were blocked by the pancaspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethyl ketone (zVAD-fmk). A rapid but temporary decrease in cellular ATP observed on the addition of zVAD-fmk was shown to be due to DMSO (added with zVAD-fmk). The effect of dactinomycin on respiration differed from that of doxorubicin. Plots of [O2] versus t were curved for dactinomycin so that k decreased gradually with t. The corresponding plots for doxorubicin were well fit by two straight lines; so k was constant for approximately 150 min, at which time k decreased, remaining constant at a lower level thereafter. The results for cells treated with mixtures of the two drugs indicated that the drugs acted synergistically. These results show the onset and severity of mitochondrial dysfunction in cells undergoing apoptosis induced by dactinomycin. PMID:17140264

  12. 4. System Definition 16 4. System Definition

    E-print Network

    Berlin,Technische Universität

    4. System Definition 16 _____________________________________________________________________________ 4. System Definition Based on our knowledge that we can "join" the dynamic characteristics-up, complete system. A general and simple example of substructuring is the source-transmission element

  13. Design and development of robust ATP subsystem for the Altair UAV-to-ground lasercomm 2.5-Gbps demonstration

    NASA Astrophysics Data System (ADS)

    Ortiz, Gerardo G.; Lee, Shinhak; Monacos, Steve P.; Wright, Malcolm W.; Biswas, Abhijit

    2003-07-01

    A robust acquisition, tracking and pointing (ATP) subsystem is being developed for the 2.5 Gigabit per second (Gbps) Unmanned-Aerial-Vehicle (UAV) to ground free-space optical communications link project. The demonstration will gather HDTV images of regions of geological interest (e.g. volcanic) and then downlink those images to ground receivers at a range of 50 km while the UAV is at an altitude of 18 km. With a 200 mW downlink laser at 1550 nm for a BER of 1E-9, the pointing requirements on the flight terminal are a jitter error of 19.5 urad and a bias error of 14.5 urad with a probability of pointing induced fades of 0.1 %. In order to mitigate the effect of atmospheric fades and deal with UAV flight and vibration uncertainties (relatively new craft) the ATP subsystem requirements have been set to a stringent level in order to assure success of the communication link. The design, analysis and development of this robust ATP subsystem will be described in this paper. The key innovations that have been developed to make the ATP subsystem robust are i) the application of inertial sensors to make the acquisition and tracking functions tolerant to atmospheric fades, ii) the usage of active exposure control to provide a 16 dB dynamic range on the Focal Plane Array (FPA) tracking window, and iii) the introduction of a second ultra wide field of view camera to assure acquisition of the ground beacon.

  14. [ATP and polyphosphate-dependent bacterial NAD+-kinases].

    PubMed

    Filippovich, S Iu; Afanas'eva, T P; Bachurina, G P; Kritski?, M S

    2000-01-01

    Measurable levels of activity of NAD+ kinases of actinomycetes Micrococcus luteus and Corynebacterium ammoniagenes were observed after substituting inorganic tripolyphosphate for ATP, whereas the enzyme from the eubacterium Escherichia coli was not active with this substrate. Gradient PAGE found two molecular isoforms of NAD+ kinase in C. ammoniagenes and E. coli; four forms were found in M. luteus. All isoforms of this enzyme found in C. ammoniagenes and M. luteus displayed a NADP-synthesizing activity in the presence of either ATP or tripolyphosphate. Because of its capability of utilizing inorganic tripolyphosphate, M. luteus is the most promising NADP producer organism. PMID:10779996

  15. Human K ATP channelopathies: diseases of metabolic homeostasis

    Microsoft Academic Search

    Timothy M. Olson; Andre Terzic

    2010-01-01

    Assembly of an inward rectifier K+ channel pore (Kir6.1\\/Kir6.2) and an adenosine triphosphate (ATP)-binding regulatory subunit (SUR1\\/SUR2A\\/SUR2B) forms ATP-sensitive\\u000a K+ (KATP) channel heteromultimers, widely distributed in metabolically active tissues throughout the body. KATP channels are metabolism-gated biosensors functioning as molecular rheostats that adjust membrane potential-dependent functions\\u000a to match cellular energetic demands. Vital in the adaptive response to (patho)physiological stress, KATP

  16. Resveratrol plus ethanol counteract the ethanol-induced impairment of energy metabolism: ³¹P NMR study of ATP and sn-glycerol-3-phosphate on isolated and perfused rat liver.

    PubMed

    Gallis, Jean-Louis; Serhan, Nizar; Gin, Henri; Couzigou, Patrice; Beauvieux, Marie-Christine

    2012-03-01

    The effects of trans-resveratrol (RSV) combined with ethanol (EtOH) were evaluated by (31)P NMR on total ATP and sn-glycerol-3-phosphate (sn-G3P) contents measured in real time in isolated and perfused whole liver of the rat. Mitochondrial ATP turnover was assessed by using specific inhibitors of glycolytic and mitochondrial ATP supply (iodacetate and KCN, respectively). In RSV alone, the slight decrease in ATP content (-14±5% of the initial content), sn-G3P content and ATP turnover were similar to those in the Krebs-Henseleit buffer control. Compared to control, EtOH alone (14 or 70 mmol/L) induced a decrease in ATP content (-24.95±2.95% of initial content, p<0.05) and an increase in sn-G3P (+158±22%), whereas ATP turnover tended to be increased. RSV (20 ?mol/L) combined with EtOH, (i) maintained ATP content near 100%, (ii) induced a 1.6-fold increase in mitochondrial ATP turnover (p=0.049 and p=0.004 vs EtOH 14 and 70 mmol/L alone, respectively) and (iii) led to an increase in sn-G3P (+49±9% and +81±6% for 14 and 70 mmol/L EtOH, respectively). These improvements were obtained only when glycolysis was efficient at the time of addition of EtOH+RSV. Glycolysis inhibition by iodacetate (IAA) evidenced an almost 21% contribution of this pathway to ATP content. RSV alone or RSV+EtOH prevented the ATP decrease induced by IAA addition (p<0.05 vs control). This is the first demonstration of the combined effects of RSV and EtOH on liver energy metabolism. RSV increased (i) the flux of substrates through ATP producing pathways (glycolysis and phosphorylative oxidation) probably via the activation of AMPkinase, and (ii) maintained the glycolysis deviation to sn-G3P linked to NADH+H? re-oxidation occurring during EtOH detoxication, thus reducing the energy cost due to the latter. PMID:22227530

  17. Extracellular ATP in the Immune System: More Than Just a "Danger Signal"

    NSDL National Science Digital Library

    Alain Trautmann (France; Universit預aris Descartes REV)

    2009-02-03

    Extracellular adenosine 5?-triphosphate (eATP) is ubiquitously used for cell-to-cell communication. The low concentration of eATP ([eATP]) that exists in a “halo” surrounding resting cells signals the presence of neighboring living cells. Transient increases in [eATP] are used for basic physiological signaling, namely, in the nervous and vascular systems. Larger increases in [eATP] that are associated with cell death serve as a key “danger” signal in inflammatory processes. Two studies now point to roles for ATP in the immune system: providing a costimulatory signal to T cells and driving the differentiation of intestinal T helper 17 (TH17) cells.

  18. 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

  19. Phospholipid Flippase ATP10A Translocates Phosphatidylcholine and Is Involved in Plasma Membrane Dynamics.

    PubMed

    Naito, Tomoki; Takatsu, Hiroyuki; Miyano, Rie; Takada, Naoto; Nakayama, Kazuhisa; Shin, Hye-Won

    2015-06-12

    We showed previously that ATP11A and ATP11C have flippase activity toward aminophospholipids (phosphatidylserine (PS) and phosphatidylethanolamine (PE)) and ATP8B1 and that ATP8B2 have flippase activity toward phosphatidylcholine (PC) (Takatsu, H., Tanaka, G., Segawa, K., Suzuki, J., Nagata, S., Nakayama, K., and Shin, H. W. (2014) J. Biol. Chem. 289, 33543-33556). Here, we show that the localization of class 5 P4-ATPases to the plasma membrane (ATP10A and ATP10D) and late endosomes (ATP10B) requires an interaction with CDC50A. Moreover, exogenous expression of ATP10A, but not its ATPase-deficient mutant ATP10A(E203Q), dramatically increased PC flipping but not flipping of PS or PE. Depletion of CDC50A caused ATP10A to be retained at the endoplasmic reticulum instead of being delivered to the plasma membrane and abrogated the increased PC flipping activity observed by expression of ATP10A. These results demonstrate that ATP10A is delivered to the plasma membrane via its interaction with CDC50A and, specifically, flips PC at the plasma membrane. Importantly, expression of ATP10A, but not ATP10A(E203Q), dramatically altered the cell shape and decreased cell size. In addition, expression of ATP10A, but not ATP10A(E203Q), delayed cell adhesion and cell spreading onto the extracellular matrix. These results suggest that enhanced PC flipping activity due to exogenous ATP10A expression alters the lipid composition at the plasma membrane, which may in turn cause a delay in cell spreading and a change in cell morphology. PMID:25947375

  20. Cardiac ATP-sensitive K+ channels. Evidence for preferential regulation by glycolysis

    PubMed Central

    1989-01-01

    The ability of glycolysis, oxidative phosphorylation, the creatine kinase system, and exogenous ATP to suppress ATP-sensitive K+ channels and prevent cell shortening were compared in patch-clamped single guinea pig ventricular myocytes. In cell-attached patches on myocytes permeabilized at one end with saponin, ATP-sensitive K+ channels were activated by removing ATP from the bath, and could be closed equally well by exogenous ATP or substrates for endogenous ATP production by glycolysis (with the mitochondrial inhibitor FCCP present), mitochondrial oxidative phosphorylation, or the creatine kinase system. In the presence of an exogenous ATP-consuming system, however, glycolytic substrates (with FCCP present) were superior to substrates for either oxidative phosphorylation or the creatine kinase system at suppressing ATP-sensitive K+ channels. All three groups of substrates were equally effective at preventing cell shortening. In 6 of 38 excised inside-out membrane patches, ATP-sensitive K+ channels activated by removing ATP from the bath were suppressed by a complete set of substrates for the ATP-producing steps of glycolysis but not by individual glycolytic substrates, which is consistent with the presence of key glycolytic enzymes located near the channels in these patches. Under whole-cell voltage-clamp conditions, inclusion of 15 mM ATP in the patch electrode solution dialyzing the interior of the cell did not prevent activation of the ATP-sensitive K+ current under control conditions or during exposure to complete metabolic inhibition. In isolated arterially perfused rabbit interventricular septa, selective inhibition of glycolysis caused an immediate increase in 42K+ efflux rate, which was prevented by 100 microM glyburide, a known blocker of ATP-sensitive K+ channels. These observations suggest that key glycolytic enzymes are associated with cardiac. ATP-sensitive K+ channels and under conditions in which intracellular competition for ATP is high (e.g., in beating heart) that act as a preferential source of ATP for these channels. PMID:2512370

  1. 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.

  2. P2Y Receptor Modulation of ATP Release in the Urothelium

    PubMed Central

    Mansfield, Kylie J.; Hughes, Jessica R.

    2014-01-01

    The release of ATP from the urothelium in response to stretch during filling demonstrates the importance of the purinergic system for the physiological functioning of the bladder. This study examined the effect of P2 receptor agonists on ATP release from two urothelial cell lines (RT4 and UROtsa cells). Hypotonic Krebs was used as a stretch stimulus. Incubation of urothelial cells with high concentrations of the P2Y agonist ADP induced ATP release to a level that was 40-fold greater than hypotonic-stimulated ATP release (P < 0.0011, ADP EC50 1.8?µM). Similarly, an increase in ATP release was also observed with the P2Y agonist, UTP, up to a maximum of 70% of the hypotonic response (EC50 0.62?µM). Selective P2 receptor agonists, ??-methylene-ATP, ATP-?-S, and 2-methylthio-ADP had minimal effects on ATP release. ADP-stimulated ATP release was significantly inhibited by suramin (100?µM, P = 0.002). RT4 urothelial cells break down nucleotides (100?µM) including ATP, ADP, and UTP to liberate phosphate. Phosphate liberation was also demonstrated from endogenous nucleotides with approximately 10% of the released ATP broken down during the incubation. These studies demonstrate a role for P2Y receptor activation in stimulation of ATP release and emphasize the complexity of urothelial P2 receptor signalling. PMID:24829920

  3. 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 ? and ? 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 ? (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.

  4. Perspective How does ATP hydrolysis control actin's associations?

    E-print Network

    Spudich, James A.

    Perspective How does ATP hydrolysis control actin's associations? Elena P. Sablin*, John F. Dawson in the DNaseI-binding loop from a random coil to a helix in the ADP-bound actin (3) was suggested (4 is similar to that used by other families of nucleotide- hydrolyzing proteins (NTPases). Various families

  5. ATP 3-20.16 Mobile Gun System Platoon

    E-print Network

    US Army Corps of Engineers

    ATP 3-20.16 Mobile Gun System Platoon February 2013 Headquarters, Department of the Army of the Army Washington, DC, 15 February 2013 Mobile Gun System Platoon Contents Page PREFACE OF THE MOBILE GUN SYSTEM PLATOON ...2-1 Section I ­ Text References.......................................2

  6. Transient accumulation of elastic energy in proton translocating ATP synthase

    E-print Network

    Steinhoff, Heinz-Jürgen

    Hypothesis Transient accumulation of elastic energy in proton translocating ATP synthase Dmitry A,18^20]. We analyzed the transient elastic storage of energy derived from four proton-translocation steps between FH and FI (see Fig. 1) gives a clue to how the elastic energy storage might function. The proton

  7. ATP-induced noncooperative thermal unfolding of hen lysozyme

    SciTech Connect

    Liu, Honglin; Yin, Peidong; He, Shengnan; Sun, Zhihu [National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, Anhui 230029 (China)] [National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, Anhui 230029 (China); Tao, Ye; Huang, Yan; Zhuang, Hao [Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049 (China)] [Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049 (China); Zhang, Guobin [National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, Anhui 230029 (China)] [National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, Anhui 230029 (China); Wei, Shiqiang, E-mail: sqwei@ustc.edu.cn [National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, Anhui 230029 (China)] [National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, Anhui 230029 (China)

    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.

  8. 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

  9. Minireview R67 ATP synthase: two motors, two fuels

    E-print Network

    Oster, George

    microscopy. What emerged was a picture of F1 in hydrolysis mode as a rotary engine that advanced in three is the universal protein responsible for ATP synthesis. The enzyme comprises two reversible rotary motors: Fo's catalytic rates and coordinated their cycles of hydrolysis by a rotary mechanism [6,7]. The subunit

  10. Extracellular ATP signaling in plants Kiwamu Tanaka1

    E-print Network

    Jones, Alan M.

    Extracellular ATP signaling in plants Kiwamu Tanaka1 , Simon Gilroy2 , Alan M. Jones3 and Gary Stacey1 1 Division of Plant Sciences, University of Missouri, Columbia, MO 65211, USA 2 Botany Department a number of cellular responses in plants and animals. Some of the molecular components for purinergic sig

  11. Distinct Wilson-disease mutations in ATP7B are associated with enhanced binding to COMMD1 and reduced stability of ATP7B

    PubMed Central

    de Bie, Prim; van de Sluis, Bart; Burstein, Ezra; van de Berghe, Peter V.E.; Muller, Patricia; Berger, Ruud; Gitlin, Jonathan D.; Wijmenga, Cisca; Klomp, Leo W.J.

    2008-01-01

    Background/Aims Wilson disease is characterized by hepatic copper overload and caused by mutations in the gene encoding the copper transporting P-type ATPase ATP7B. ATP7B interacts with COMMD1, a protein that is deleted in Bedlington terriers with hereditary copper toxicosis. Here we characterized the implications of the interaction between COMMD1 and ATP7B in relation to the pathogenesis of Wilson disease. Methods GST pull-down experiments, co-immunoprecipitations, immunofluoresensce microscopy, site-directed mutagenesis and biosynthetic labeling experiments were performed to characterize the interaction between COMMD1 and ATP7B and the effects of Wilson disease causing mutations. Results COMMD1 specifically interacted with the amino-terminal region of ATP7B. This interaction was independent of intracellular copper levels and of the expression of the copper chaperone ATOX1. Four Wilson disease patient derived mutations in this region of ATP7B significantly increased its binding to COMMD1. Two of these mutations also resulted in mislocalization and increased degradation rate of ATP7B. Although COMMD1 did not affect copper-induced trafficking of ATP7B, it markedly decreased the stability of newly synthesized ATP7B. Conclusions Our data implicate COMMD1 in the pathogenesis of Wilson disease and indicate that COMMD1 exerts its regulatory role in copper homeostasis through the regulation of ATP7B stability. PMID:17919502

  12. 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

  13. Crystal structures of actin-related protein 2 3 complex with bound ATP or ADP

    E-print Network

    the electron density maps. This flexibility may explain why Arp2 does not hydrolyze ATP until the complex a conformational change that may occur in actin and Arps when ATP is hydrolyzed and phosphate dissociates

  14. Recombinant Expression, Purification, and Reconstitution of the Chloroplast ATP Synthase c-subunit Ring

    E-print Network

    Thorpe, Michael

    Recombinant Expression, Purification, and Reconstitution of the Chloroplast ATP Synthase c chloroplast ATP synthase has successfully been expressed in Escherichia coli and purified in mg quantities alpha- helical secondary structure. Recent experiments appear to indicate that this monomeric

  15. Regulation of extracellular ATP in human erythrocytes infected with Plasmodium falciparum.

    PubMed

    Alvarez, Cora Lilia; Schachter, Julieta; de Sá Pinheiro, Ana Acacia; Silva, Leandro de Souza; Verstraeten, Sandra Viviana; Persechini, Pedro Muanis; Schwarzbaum, Pablo Julio

    2014-01-01

    In human erythrocytes (h-RBCs) various stimuli induce increases in [cAMP] that trigger ATP release. The resulting pattern of extracellular ATP accumulation (ATPe kinetics) depends on both ATP release and ATPe degradation by ectoATPase activity. In this study we evaluated ATPe kinetics from primary cultures of h-RBCs infected with P. falciparum at various stages of infection (ring, trophozoite and schizont stages). A "3V" mixture containing isoproterenol (?-adrenergic agonist), forskolin (adenylate kinase activator) and papaverine (phosphodiesterase inhibitor) was used to induce cAMP-dependent ATP release. ATPe kinetics of r-RBCs (ring-infected RBCs), t-RBCs (trophozoite-infected RBCs) and s-RBCs (schizont-infected RBCs) showed [ATPe] to peak acutely to a maximum value followed by a slower time dependent decrease. In all intraerythrocytic stages, values of ?ATP1 (difference between [ATPe] measured 1 min post-stimulus and basal [ATPe]) increased nonlinearly with parasitemia (from 2 to 12.5%). Under 3V exposure, t-RBCs at parasitemia 94% (t94-RBCs) showed 3.8-fold higher ?ATP1 values than in h-RBCs, indicative of upregulated ATP release. Pre-exposure to either 100 µM carbenoxolone, 100 nM mefloquine or 100 µM NPPB reduced ?ATP1 to 83-87% for h-RBCs and 63-74% for t94-RBCs. EctoATPase activity, assayed at both low nM concentrations (300-900 nM) and 500 µM exogenous ATPe concentrations increased approx. 400-fold in t94-RBCs, as compared to h-RBCs, while intracellular ATP concentrations of t94-RBCs were 65% that of h-RBCs. In t94-RBCs, production of nitric oxide (NO) was approx. 7-fold higher than in h-RBCs, and was partially inhibited by L-NAME pre-treatment. In media with L-NAME, ?ATP1 values were 2.7-times higher in h-RBCs and 4.2-times higher in t94-RBCs, than without L-NAME. Results suggest that P. falciparum infection of h-RBCs strongly activates ATP release via Pannexin 1 in these cells. Several processes partially counteracted ATPe accumulation: an upregulated ATPe degradation, an enhanced NO production, and a decreased intracellular ATP concentration. PMID:24858837

  16. Anomalous influence of reduced internal ATP levels on sodium efflux in Myxicola giant axons

    Microsoft Academic Search

    R. A. Sjodin; O. E. Ortiz; J. G. Montes

    1989-01-01

    Summary Giant axons from the marine annelid,Myxicola infundibulum, were internally dialyzed with ATP-free media and with media with lower than normal ATP levels in an attempt to determine quantitatively the ATP requirement of the Na pump in these cells. This was accomplished by using22Na ions to measure Na efflux. When [ATP]i in dialysis fluid fell to values within the range

  17. ATP Requirements and Small Interfering RNA Structure in the RNA Interference Pathway

    Microsoft Academic Search

    Antti Nykänen; Benjamin Haley; Phillip D. Zamore

    2001-01-01

    We examined the role of ATP in the RNA interference (RNAi) pathway. Our data reveal two ATP-dependent steps and suggest that the RNAi reaction comprises at least four sequential steps: ATP-dependent processing of double-stranded RNA into small interfering RNAs (siRNAs), incorporation of siRNAs into an inactive ?360 kDa protein\\/RNA complex, ATP-dependent unwinding of the siRNA duplex to generate an active

  18. ATP pools and transients in the blue-green alga, Anabaena cylindrica

    Microsoft Academic Search

    P. J. Bottomley; W. D. P. Stewart

    1976-01-01

    Anabaena cylindrica grown in steady state continuous culture has an extractable ATP pool, measured on the basis of the luciferin-luciferase assay of 165±35 nmoles ATP mg chla-1. This pool is maintained by a dynamic balance between the rate of ATP synthesis and the rate of ATP utilization. Phosphorylating mechanisms which can maintain the pool in the short term are total

  19. Human Keratinocyte ATP2C1 Localizes to the Golgi and Controls Golgi Ca2+ Stores

    Microsoft Academic Search

    Martin J. Behne; Chia-Ling Tu; Ida Aronchik; Ervin Epstein; Graham Bench; Daniel D. Bikle; Tullio Pozzan; Theodora M. Mauro

    2003-01-01

    Hailey–Hailey disease (MIM16960) is a blistering skin disease caused by mutations in the Ca2+ ATPase ATP2C1. We found that the abnormal Ca2+ signaling seen in Hailey–Hailey disease keratinocytes correlates with decreased protein levels of ATP2C1. Human ATP2C1 protein approximated 115 kDa in size. The ATP2C1 is localized to the Golgi apparatus in human keratinocytes, similar to its localization in yeast

  20. ATP-induced currents in submucous plexus neurons of the guinea pig small intestine

    Microsoft Academic Search

    A. V. Glushakov; A. I. Melishchuk; V. I. Skok

    1996-01-01

    ATP-induced membrane durrents in the submucous neurons of the guinea pig small intestine were studied using the whole-cell patch-clamp recording technique. Being applied at ?50 mV. ATP activated an inward non-selective cationic current in 68.3% of the investigated neurons. An increase in ATP concentration within the 1–1,000 µM range resulted in the s-like increase in the amplitude of ATP-induced current.

  1. Inhibition of chemokine expression in rat inflamed paws by systemic use of the antihyperalgesic oxidized ATP

    Microsoft Academic Search

    Alessandro Fulgenzi; Giacomo Dell'Antonio; Chiara Foglieni; Elena Dal Cin; Paolo Ticozzi; Josè S Franzone; Maria Elena Ferrero

    2005-01-01

    BACKGROUND: We previously showed that local use of periodate oxidized ATP (oATP, a selective inhibitor of P2X7 receptors for ATP) in rat paw treated with Freund's adjuvant induced a significant reduction of hyperalgesia Herein we investigate the role of oATP, in the rat paws inflamed by carrageenan, which mimics acute inflammation in humans. RESULTS: Local, oral or intravenous administration of

  2. Mitochondrial electroporation and in organello RNA editing of chimeric atp6 transcripts

    Microsoft Academic Search

    Matthias Staudinger; Nina Bolle; Frank Kempken

    2005-01-01

    The Sorghum bicolor atp6-1 gene and chimeric atp6 genes with additional maize sequences were introduced into isolated maize mitochondria via electroporation. Transcripts isolated after in vitro incubation of the transformed organelles were then analysed for RNA editing. Transcripts of the S. bicolor atp6-1 gene, and the RNAs obtained from most of chimeric sorghum-maize atp6 gene constructs tested, were not edited.

  3. Extracellular ATP signaling during differentiation of C2C12 skeletal muscle cells: role in proliferation

    Microsoft Academic Search

    Tiziana Martinello; Maria Cristina Baldoin; Laura Morbiato; Maddalena Paganin; Elena Tarricone; Giorgio Schiavo; Elisa Bianchini; Dorianna Sandonà; Romeo Betto

    2011-01-01

    Evidence shows that extracellular ATP signals influence myogenesis, regeneration and physiology of skeletal muscle. Present\\u000a work was aimed at characterizing the extracellular ATP signaling system of skeletal muscle C2C12 cells during differentiation.\\u000a We show that mechanical and electrical stimulation produces substantial release of ATP from differentiated myotubes, but not\\u000a from proliferating myoblasts. Extracellular ATP-hydrolyzing activity is low in myoblasts and

  4. Identification of two nuclear genes (ATP11, ATP12) required for assembly of the yeast F1-ATPase.

    PubMed Central

    Ackerman, S H; Tzagoloff, A

    1990-01-01

    Nuclear respiratory-deficient mutants of Saccharomyces cerevisiae (pet mutants) have been screened for defects in the mitochondrial ATPase. Mutants in two complementation groups were found to have 10% or less of wild-type ATPase activity. The two wild-type nuclear genes defined by the mutants have been designated ATP11 and ATP12. The proteins encoded by the two genes are not subunits of the ATPase but rather appear to exercise an important function at a late stage in the synthesis of F1 after transport of the subunits into the internal compartment of mitochondria. Mitochondria of atp11 and atp12 mutants have only marginally reduced levels of the alpha and beta subunits of F1. Both proteins are processed to their mature size but are not part of a native F1 structure or associated with the mitochondrial membrane. The most reasonable explanation for the mutant phenotype is a block in the assembly of the F1 oligomer. Images PMID:2142305

  5. 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, ?H*, ?S* and ?G* 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.

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

    PubMed

    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, ?H(*), ?S(*) and ?G(*) 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. PMID:24632173

  7. Apyrase Suppression Raises Extracellular ATP Levels and Induces Gene Expression and Cell Wall Changes

    E-print Network

    Webb, Lauren J.

    ATP into their extracellular matrix (ECM) or the growth medium when they are wounded (Song et al, Texas 78712 Plant cells release ATP into their extracellular matrix as they grow, and extracellular ATPApyrase Suppression Raises Extracellular ATP Levels and Induces Gene Expression and Cell Wall

  8. ATP increases within the lumen of the endoplasmic reticulum upon intracellular Ca2+ release

    PubMed Central

    Vishnu, Neelanjan; Jadoon Khan, Muhammad; Karsten, Felix; Groschner, Lukas N.; Waldeck-Weiermair, Markus; Rost, Rene; Hallström, Seth; Imamura, Hiromi; Graier, Wolfgang F.; Malli, Roland

    2014-01-01

    Multiple functions of the endoplasmic reticulum (ER) essentially depend on ATP within this organelle. However, little is known about ER ATP dynamics and the regulation of ER ATP import. Here we describe real-time recordings of ER ATP fluxes in single cells using an ER-targeted, genetically encoded ATP sensor. In vitro experiments prove that the ATP sensor is both Ca2+ and redox insensitive, which makes it possible to monitor Ca2+-coupled ER ATP dynamics specifically. The approach uncovers a cell type–specific regulation of ER ATP homeostasis in different cell types. Moreover, we show that intracellular Ca2+ release is coupled to an increase of ATP within the ER. The Ca2+-coupled ER ATP increase is independent of the mode of Ca2+ mobilization and controlled by the rate of ATP biosynthesis. Furthermore, the energy stress sensor, AMP-activated protein kinase, is essential for the ATP increase that occurs in response to Ca2+ depletion of the organelle. Our data highlight a novel Ca2+-controlled process that supplies the ER with additional energy upon cell stimulation. PMID:24307679

  9. 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

  10. 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

  11. Failure of the Cystic Fibrosis Transmembrane Conductance Regulator to Conduct ATP

    Microsoft Academic Search

    M. M. Reddy; P. M. Quinton; C. Haws; J. J. Wine; R. Grygorczyk; J. A. Tabcharani; J. W. Hanrahan; K. L. Gunderson; R. R. Kopito

    1996-01-01

    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

  12. ORIGINAL INVESTIGATION Further characterization of ATP6V0A2-related autosomal

    E-print Network

    ORIGINAL INVESTIGATION Further characterization of ATP6V0A2-related autosomal recessive cutis laxa heterogeneous disorders. Mutations in the ATP6V0A2 gene were found to underlie both, autosomal recessive cutis laxa type 2 (ARCL2), Debre´ type, and wrinkly skin syndrome (WSS). The ATP6V0A2 gene encodes the a2

  13. Use of atp6 in Fungal Phylogenetics: An Example from the Boletales

    E-print Network

    Bruns, Tom

    Use of atp6 in Fungal Phylogenetics: An Example from the Boletales Annette M. Kretzer1 and Thomas D sequences have been deter- mined for atp6 from Suillus luteus and cox3 from Suillus sinuspaulianus, respectively. These se- quences were used to design PCR primers for the amplification of partial atp6 and cox3

  14. Release of ATP in rat vas deferens: origin and role of calcium

    Microsoft Academic Search

    Anna Kordelia Kurz; Ralph Biiltmann; Bernd Driessen; Ivar von Kiigelgen; Klaus Starke

    1994-01-01

    Release of endogenous ATP elicited by electrical (neural) stimulation and exogenous agonists was studied in the rat isolated vas deferens. The aims were to dissect neural and postjunctional contributions to the nerve activity-evoked overflow of ATP and to clarify the role of transmitter receptors and calcium in postjunctional ATP release.

  15. Loss of the Gene for the ? Subunit of ATP Synthase (ATP5A1) from the W Chromosome in the African Grey Parrot ( Psittacus erithacus )

    Microsoft Academic Search

    Siwo R. de Kloet

    2001-01-01

    .   This study describes the results of an analysis using Southern blotting, the polymerase chain reaction, and sequencing which\\u000a 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\\u000a in other psittacines a fragment of the ATP5A1W gene contains five times as many nonsynonymous nucleotide

  16. ATP Regulation of Type-1 Inositol 1,4,5-Trisphosphate Receptor Activity Does Not Require Walker A-type ATP-binding Motifs*

    PubMed Central

    Betzenhauser, Matthew J.; Wagner, Larry E.; Park, Hyung Seo; Yule, David I.

    2009-01-01

    ATP is known to increase the activity of the type-1 inositol 1,4,5-trisphosphate receptor (InsP3R1). This effect is attributed to the binding of ATP to glycine rich Walker A-type motifs present in the regulatory domain of the receptor. Only two such motifs are present in neuronal S2+ splice variant of InsP3R1 and are designated the ATPA and ATPB sites. The ATPA site is unique to InsP3R1, and the ATPB site is conserved among all three InsP3R isoforms. Despite the fact that both the ATPA and ATPB sites are known to bind ATP, the relative contribution of these two sites to the enhancing effects of ATP on InsP3R1 function is not known. We report here a mutational analysis of the ATPA and ATPB sites and conclude neither of these sites is required for ATP modulation of InsP3R1. ATP augmented InsP3-induced Ca2+ release from permeabilized cells expressing wild type and ATP-binding site-deficient InsP3R1. Similarly, ATP increased the single channel open probability of the mutated InsP3R1 to the same extent as wild type. ATP likely exerts its effects on InsP3R1 channel function via a novel and as yet unidentified mechanism. PMID:19386591

  17. ATP binding to a multisubunit enzyme: statistical thermodynamics analysis

    E-print Network

    Yunxin Zhang

    2012-03-22

    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 thermodynamics, 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 provided a new way to understand biophysical processes by statistical thermodynamics analysis.

  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, ?a( 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. 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.

  20. ATP-Dependent Bioluminescence in the Firefly Squid, Watasenia Scintillans

    Microsoft Academic Search

    Frederick I. Tsuji

    1985-01-01

    The Japanese firefly squid, Watasenia scintillans, emits intense flashes of light from three tiny luminous organs that are located at the tip of each of a pair of ventral arms. Light is also produced from hundreds of other minute organs that are scattered over the body. The luminescence is due to an ATP-dependent reaction, with an optimal pH of 8.80.

  1. ATP as a mediator of macula densa cell signalling.

    PubMed

    Bell, P Darwin; Komlosi, Peter; Zhang, Zhi-Ren

    2009-12-01

    Within each nephro-vascular unit, the tubule returns to the vicinity of its own glomerulus. At this site, there are specialised tubular cells, the macula densa cells, which sense changes in tubular fluid composition and transmit information to the glomerular arterioles resulting in alterations in glomerular filtration rate and blood flow. Work over the last few years has characterised the mechanisms that lead to the detection of changes in luminal sodium chloride and osmolality by the macula densa cells. These cells are true "sensor cells" since intracellular ion concentrations and membrane potential reflect the level of luminal sodium chloride concentration. An unresolved question has been the nature of the signalling molecule(s) released by the macula densa cells. Currently, there is evidence that macula densa cells produce nitric oxide via neuronal nitric oxide synthase (nNOS) and prostaglandin E(2) (PGE(2)) through cyclooxygenase 2 (COX 2)-microsomal prostaglandin E synthase (mPGES). However, both of these signalling molecules play a role in modulating or regulating the macula-tubuloglomerular feedback system. Direct macula densa signalling appears to involve the release of ATP across the basolateral membrane through a maxi-anion channel in response to an increase in luminal sodium chloride concentration. ATP that is released by macula densa cells may directly activate P2 receptors on adjacent mesangial cells and afferent arteriolar smooth muscle cells, or the ATP may be converted to adenosine. However, the critical step in signalling would appear to be the regulated release of ATP across the basolateral membrane of macula densa cells. PMID:19330465

  2. ATP-dependent proteases that also chaperone protein biogenesis

    Microsoft Academic Search

    Carolyn K. Suzuki; Martijn Rep; Jan Maarten van Dijl; Kitaru Suda; Leslie A. Grivell; Gottfried Schatz

    1997-01-01

    The ATP-dependent proteases Clp and FtsH from bacteria, as well as mitochondrial homologs of FtsH and Lon from yeast, may act as chaperones; they mediate not only proteolysis, but also the insertion of proteins into membranes and the disassembly or oligomerization of protein complexes. The coordination of such processes with selective proteolysis may function in the quality control of protein

  3. The chloroplast ATP synthase: Structural changes during catalysis

    Microsoft Academic Search

    Mark L. Richter; Fei Gao

    1996-01-01

    This article summarizes some of the evidence for the existence of light-driven structural changes in the? and? subunits of the chlorplast ATP synthase. Formation of a transmembrane proton gradient results in: (1) a change in the position of the? subunit such that it becomes exposed to polyclonal antibodies and to reagents which selectively modify?Lys109; (2) enhanced solvent accessibility of several

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

    PubMed

    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

  5. 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

  6. Toward a Multiscale Description of Microvascular Flow Regulation: O2-Dependent Release of ATP from Human Erythrocytes and the Distribution of ATP in Capillary Networks

    PubMed Central

    Goldman, Daniel; Fraser, Graham M.; Ellis, Christopher G.; Sprague, Randy S.; Ellsworth, Mary L.; Stephenson, Alan H.

    2012-01-01

    Integration of the numerous mechanisms that have been suggested to contribute to optimization of O2 supply to meet O2 need in skeletal muscle requires a systems biology approach which permits quantification of these physiological processes over a wide range of length scales. Here we describe two individual computational models based on in vivo and in vitro studies which, when incorporated into a single robust multiscale model, will provide information on the role of erythrocyte-released ATP in perfusion distribution in skeletal muscle under both physiological and pathophysiological conditions. Healthy human erythrocytes exposed to low O2 tension release ATP via a well characterized signaling pathway requiring activation of the G-protein, Gi, and adenylyl cyclase leading to increases in cAMP. This cAMP then activates PKA and subsequently CFTR culminating in ATP release via pannexin 1. A critical control point in this pathway is the level of cAMP which is regulated by pathway-specific phosphodiesterases. Using time constants (~100?ms) that are consistent with measured erythrocyte ATP release, we have constructed a dynamic model of this pathway. The model predicts levels of ATP release consistent with measurements obtained over a wide range of hemoglobin O2 saturations (sO2). The model further predicts how insulin, at concentrations found in pre-diabetes, enhances the activity of PDE3 and reduces intracellular cAMP levels leading to decreased low O2-induced ATP release from erythrocytes. The second model, which couples O2 and ATP transport in capillary networks, shows how intravascular ATP and the resulting conducted vasodilation are affected by local sO2, convection and ATP degradation. This model also predicts network-level effects of decreased ATP release resulting from elevated insulin levels. Taken together, these models lay the groundwork for investigating the systems biology of the regulation of microvascular perfusion distribution by erythrocyte-derived ATP. PMID:22934004

  7. Definitions of dwelling

    E-print Network

    Olgyay, Victor W. (Victor Wayne)

    1986-01-01

    Home is an elusive concept. In one manner it is highly specific and individual in its definition, and in other aspects it is ubiquitous, present in our every act. In this thesis I explore several possible definitions of ...

  8. 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

  9. 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

  10. Touch induces ATP release in Arabidopsis roots that is modulated by the heterotrimeric G-protein complex

    E-print Network

    Jones, Alan M.

    an ATP-dependent touch response system in which ATP release is fine tuned by the G-pro- tein complex. 2Touch induces ATP release in Arabidopsis roots that is modulated by the heterotrimeric G a heterotrimeric G-protein. Obstacle avoidance may utilize a touch-induced release of ATP to the extracellular

  11. ATP-synthase of Rhodobacter capsulatus: coupling of proton ow through FH to reactions in FI under the ATP synthesis and slip conditions

    E-print Network

    Steinhoff, Heinz-Jürgen

    ATP-synthase of Rhodobacter capsulatus: coupling of proton £ow through FH to reactions in FI under proton-translocating FH and the peripheral catalytic FI. The bacterial FH is formed by three di- and O-subunits of FI interact with the cIP-ring of FH to form the rotor of the ATP-synthase, whereas

  12. Definitions Main Result

    E-print Network

    Heubach, Silvia

    Background Definitions Main Result Special Types of Patterns Summary Avoidance of partially ordered Avoidance of partially ordered patterns in compositions #12;Background Definitions Main Result Special Types of Patterns Summary Outline 1 Background 2 Definitions 3 Main Result Preliminaries Main Result 4 Special Types

  13. Definitions Appendix B

    E-print Network

    APPENDIX B Definitions #12;Appendix B Definitions Adsorption - partitioning of a dissolved species.e., smaller than clay size), that can be easily suspended. original sense, the definition of a colloid different from that of the uncontaminated portion of the aquifer. Fulvic Acids - breakdown products

  14. Relation Definition Theory.

    ERIC Educational Resources Information Center

    Herrmann, Douglas J.; Chaffin, Roger

    The relation definition theory proposed in this paper is explicitly different from previous semantic memory theories since it is the first to make a relation's definition the basis of semantic processing. The paper suggests that this relation definition theory successfully predicts relation similarity on the basis of one key primary assumption:…

  15. Hypersensitivity to hypercapnia: definition/(s).

    PubMed

    Vickers, Kristin

    2012-05-15

    Empirical evidence indicates that panic disorder (PD) patients experience hypersensitivity to hypercapnia, a condition in which the blood level of carbon dioxide exceeds the normal value. The importance of this research line is substantial and indeed, hypercapnic hypersensitivity has been advanced as a possible endophenotype of panic. Definitions of "hypersensitivity," however, have varied. The purpose of this brief review is to delineate and critique different definitions of hypercapnic hypersensitivity. Several definitions - panic attack rate, panic symptoms including dyspnea, subjective anxiety, and respiratory disturbance - are explored. The review concludes that although no ideal definition has emerged, marked anxiety post-hypercapnia has substantial support as a putative trait marker of PD. The term "subjective hypersensitivity" (Coryell et al., 2001) is re-introduced to denote pronounced anxiety post-hypercapnia and recommended for use along with its previous definition: increased self-reported anxiety measured on a continuous visual analog scale, already widely in use. Due to the well-established link between panic and respiration, definitional candidates focusing on aberrant respiratory response - less investigated as trait markers of PD in high risk studies - warrant scrutiny as well. Several reasons why definitional clarity might be beneficial are presented, along with ideas for future research. PMID:22401967

  16. Interactions of a Photo-Affinity ATP Analog with Cation-Stimulated Adenosine Triphosphatases of Human Red Cell Membranes

    PubMed Central

    Haley, Boyd E.; Hoffman, Joseph F.

    1974-01-01

    To identify and isolate ATP binding and hydrolyzing sites of human red cell membranes we have synthesized a photo-activated ATP analog, 8-azido adenosine triphosphate (N3ATP). In the absence of ultraviolet light it is a substrate for both the Mg-ATPase and the ouabain-sensitive, Na,K-ATPase. Hydrolysis of N3ATP is prevented by increasing concentrations of ATP. Photolysis of N3ATP with red cell membranes results in covalent incorporation and irreversible inhibition of both ATPase activities. Also, only three protein components of the red cell membranes are labeled. This labeling is completely abolished by appropriate concentrations of ATP. PMID:4279407

  17. [Comparison on mitochondrial ATP6, ATP8 and Cyt b genes between Chinese Tibetans in three different zones: detecting the signature of natural selection on mitochondrial genome].

    PubMed

    Gu, Ming-Liang; Wang, Ye-Jun; Shi, Lei; Zhang, Yong-Biao; Chu, Jia-You

    2009-02-01

    Mitochondrial DNA (mtDNA) differs from nuclear genome in many aspects such as lack of recombination, thus the investigation of mtDNA plays an essential role in human evolutionary history. We compared different sequences (approximately 2 kb) of ATP6, ATP8 and Cyt b genes in mtDNA among Tibetans in three different zones and found that the whole mtDNA sequences of the three genes, ATP6 and ATP 8 genes deviate gradually from neutral model with the increase of altitudes, yet no differences were observed. Also we found that the effect of purifying selection on Cyt b gene was elevated with the decrease of altitudes. Meanwhile, there was a possibility for the adaptive selection in ATP6 gene, which had an enhanced trend with the increase of altitudes. Thus, the geographic environment is the main determinant for selection, namely, different geographic environment has direct effect on selection. PMID:19273422

  18. The efflux of a fluorescent probe is catalyzed by an ATP-driven extrusion system in Lactococcus lactis.

    PubMed Central

    Molenaar, D; Bolhuis, H; Abee, T; Poolman, B; Konings, W N

    1992-01-01

    Many bacteria, both gram positive and gram negative, extrude in an energy-dependent manner the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5[and -6]-carboxyfluorescein (BCECF) (D. Molenaar, T. Abee, and W. N. Konings, Biochim. Biophys. Acta 1115:75-83, 1991). This efflux was studied in detail in Lactococcus lactis, and several indications that a transport system is involved were found. This transport system is most likely driven by ATP or a related compound. The evidence is that BCECF extrusion (i) occurs against a BCECF gradient, (ii) is strictly correlated with ATP concentration and not with the proton motive force, and (iii) is inhibited by vanadate and to a lesser extent by N,N'-dicyclohexylcarbodiimide. Most convincingly, a UV mutant with a strongly reduced efflux rate was isolated. Such a mutant was isolated from a BCECF-loaded and lactose-energized population by selection of highly fluorescent cells in a flow cytometer-cell sorter. The physiological function of this extrusion system is unknown, but its characteristics classify it among the traffic ATPases. PMID:1577684

  19. 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

  20. 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.

  1. Stimulation of ATP secretion in the liver by therapeutic bile acids.

    PubMed Central

    Nathanson, M H; Burgstahler, A D; Masyuk, A; Larusso, N F

    2001-01-01

    ATP receptors are ubiquitously expressed and are potential targets for the therapy of a number of disorders. However, delivery of ATP or other nucleotides to specific tissues is problematic, and no pharmacological means to stimulate the release of endogenous ATP has been described. We examined the effects of the bile acid ursodeoxycholic acid (UDCA) on ATP release into bile, since this bile acid is the only agent known to be of therapeutic benefit in secretory disorders of the liver, and since its mechanism of action is not established. Both UDCA and its taurine conjugate stimulated secretion of ATP by isolated rat hepatocytes, and produced measurable increases in ATP in bile of isolated rat liver. Perfusion of ATP into microdissected bile-duct segments induced Ca(2+) signalling in bile-duct epithelia, while perfusion of bile acid did not. Thus UDCA may promote bile flow by inducing hepatocytes to release ATP into bile, which then stimulates fluid and electrolyte secretion by bile-duct epithelia downstream via changes in cytosolic Ca(2+). Moreover, these findings demonstrate the feasibility of using pharmacological means to induce secretion of endogenous ATP. Since the liver and other epithelial organs express luminal ATP receptors, these findings more generally suggest that a mechanism exists for pharmacological activation of this paracrine signalling pathway. This strategy may be useful for treatment of cystic fibrosis and other secretory disorders of the liver and other epithelial tissues. PMID:11485545

  2. ATP binds to proteasomal ATPases in pairs with distinct functional effects implying an ordered reaction cycle

    PubMed Central

    Smith, David M.; Fraga, Hugo; Reis, Christian; Kafri, Galit; Goldberg, Alfred L.

    2011-01-01

    In the eukaryotic 26S proteasome, the 20S particle is regulated by six AAA ATPase subunits, and in archaea by a homologous ring complex, PAN. To clarify the role of ATP in proteolysis, we studied how nucleotides bind to PAN. Although PAN has six identical subunits it binds ATPs in pairs, and its subunits exhibit three conformational states with high, low, or no affinity for ATP. When PAN binds two ATP?S molecules, or two ATP?S plus two ADP molecules it is maximally active in binding protein substrates, associating with the 20S particle, and promoting 20S gate-opening. However, binding of four ATP?S molecules reduces these functions. The 26S proteasome shows similar nucleotide dependence. These findings imply an ordered cyclical mechanism in which two ATPase subunits bind ATP simultaneously and dock into the 20S. These results can explain how these hexameric ATPases interact with and “wobble” on top of the heptameric 20S proteasome. PMID:21335235

  3. Rho Signaling Regulates Pannexin 1-mediated ATP Release from Airway Epithelia*

    PubMed Central

    Seminario-Vidal, Lucia; Okada, Seiko F.; Sesma, Juliana I.; Kreda, Silvia M.; van Heusden, Catharina A.; Zhu, Yunxiang; Jones, Lisa C.; O'Neal, Wanda K.; Penuela, Silvia; Laird, Dale W.; Boucher, Richard C.; Lazarowski, Eduardo R.

    2011-01-01

    ATP released from airway epithelial cells promotes purinergic receptor-regulated mucociliary clearance activities necessary for innate lung defense. Cell swelling-induced membrane stretch/strain is a common stimulus that promotes airway epithelial ATP release, but the mechanisms transducing cell swelling into ATP release are incompletely understood. Using knockdown and knockout approaches, we tested the hypothesis that pannexin 1 mediates ATP release from hypotonically swollen airway epithelia and investigated mechanisms regulating this activity. Well differentiated primary cultures of human bronchial epithelial cells subjected to hypotonic challenge exhibited enhanced ATP release, which was paralleled by the uptake of the pannexin probe propidium iodide. Both responses were reduced by pannexin 1 inhibitors and by knocking down pannexin 1. Importantly, hypotonicity-evoked ATP release from freshly excised tracheas and dye uptake in primary tracheal epithelial cells were impaired in pannexin 1 knockout mice. Hypotonicity-promoted ATP release and dye uptake in primary well differentiated human bronchial epithelial cells was accompanied by RhoA activation and myosin light chain phosphorylation and was reduced by the RhoA dominant negative mutant RhoA(T19N) and Rho and myosin light chain kinase inhibitors. ATP release and Rho activation were reduced by highly selective inhibitors of transient receptor potential vanilloid 4 (TRPV4). Lastly, knocking down TRPV4 impaired hypotonicity-evoked airway epithelial ATP release. Our data suggest that TRPV4 and Rho transduce cell membrane stretch/strain into pannexin 1-mediated ATP release in airway epithelia. PMID:21606493

  4. Air-Stimulated ATP Release from Keratinocytes Occurs through Connexin Hemichannels

    PubMed Central

    Barr, Travis P.; Albrecht, Phillip J.; Hou, Quanzhi; Mongin, Alexander A.; Strichartz, Gary R.; Rice, Frank L.

    2013-01-01

    Cutaneous ATP release plays an important role in both epidermal stratification and chronic pain, but little is known about ATP release mechanisms in keratinocytes that comprise the epidermis. In this study, we analyzed ATP release from cultured human neonatal keratinocytes briefly exposed to air, a process previously demonstrated to trigger ATP release from these cells. We show that exposing keratinocytes to air by removing media for 15 seconds causes a robust, long-lasting ATP release. This air-stimulated ATP release was increased in calcium differentiated cultures which showed a corresponding increase in connexin 43 mRNA, a major component of keratinocyte hemichannels. The known connexin hemichannel inhibitors 1-octanol and carbenoxolone both significantly reduced air-stimulated ATP release, as did two drugs traditionally used as ABC transporter inhibitors (glibenclamide and verapamil). These same 4 inhibitors also prevented an increase in the uptake of a connexin permeable dye induced by air exposure, confirming that connexin hemichannels are open during air-stimulated ATP release. In contrast, activity of the MDR1 ABC transporter was reduced by air exposure and the drugs that inhibited air-stimulated ATP release had differential effects on this transporter. These results indicate that air exposure elicits non-vesicular release of ATP from keratinocytes through connexin hemichannels and that drugs used to target connexin hemichannels and ABC transporters may cross-inhibit. Connexins represent a novel, peripheral target for the treatment of chronic pain and dermatological disease. PMID:23457608

  5. ATP Antagonizes Thrombin-Induced Signal Transduction through 12(S)-HETE and cAMP

    PubMed Central

    Burzaco, Jaione; Conde, Manuel; Parada, Luis A.; Zugaza, José L.; Dehaye, Jean-Paul; Marino, Aida

    2013-01-01

    In this study we have investigated the role of extracellular ATP on thrombin induced-platelet aggregation (TIPA) in washed human platelets. ATP inhibited TIPA in a dose-dependent manner and this inhibition was abolished by apyrase but not by adenosine deaminase (ADA) and it was reversed by extracellular magnesium. Antagonists of P2Y1 and P2Y12 receptors had no effect on this inhibition suggesting that a P2X receptor controlled ATP-mediated TIPA inhibition. ATP also blocked inositol phosphates (IP1, IP2, IP3) generation and [Ca2+]i mobilization induced by thrombin. Thrombin reduced cAMP levels which were restored in the presence of ATP. SQ-22536, an adenylate cyclase (AC) inhibitor, partially reduced the inhibition exerted by ATP on TIPA. 12-lipoxygenase (12-LO) inhibitors, nordihidroguaretic acid (NDGA) and 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15(S)-HETE), strongly prevented ATP-mediated TIPA inhibition. Additionally, ATP inhibited the increase of 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE) induced by thrombin. Pretreatment with both SQ-22536 and NDGA almost completely abolished ATP-mediated TIPA inhibition. Our results describe for the first time that ATP implicates both AC and 12-LO pathways in the inhibition of human platelets aggregation in response to agonists. PMID:23826207

  6. Controlled rotation of the F?-ATPase reveals differential and continuous binding changes for ATP synthesis.

    PubMed

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

    2012-01-01

    F(1)-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 >10(4), followed by a shift to high ATP affinity, and finally the affinity for ATP decreases by >10(4). All these angular changes are gradual, implicating tight coupling between the rotor angle and site affinities. PMID:22929779

  7. ATP7A-related copper transport diseases—emerging concepts and future trends

    PubMed Central

    Kaler, Stephen G.

    2014-01-01

    This Review summarizes recent advances in understanding copper-transporting ATPase 1 (ATP7A), and examines the neurological phenotypes associated with dysfunction of this protein. Involvement of ATP7A in axonal outgrowth, synapse integrity and neuronal activation underscores the fundamental importance of copper metabolism to neurological function. Defects in ATP7A cause Menkes disease, an infantile-onset, lethal condition. Neonatal diagnosis and early treatment with copper injections enhance survival in patients with this disease, and can normalize clinical outcomes if mutant ATP7A molecules retain small amounts of residual activity. Gene replacement rescues a mouse model of Menkes disease, suggesting a potential therapeutic approach for patients with complete loss-of-function ATP7A mutations. Remarkably, a newly discovered ATP7A disorder—isolated distal motor neuropathy—has none of the characteristic clinical or biochemical abnormalities of Menkes disease or its milder allelic variant occipital horn syndrome (OHS), instead resembling Charcot–Marie–Tooth disease type 2. These findings indicate that ATP7A has a crucial but previously unappreciated role in motor neuron maintenance, and that the mechanism underlying ATP7A-related distal motor neuropathy is distinct from Menkes disease and OHS pathophysiology. Collectively, these insights refine our knowledge of the neurology of ATP7A-related copper transport diseases and pave the way for further progress in understanding ATP7A function. PMID:21221114

  8. How Reliable Are ATP Bioluminescence Meters in Assessing Decontamination of Environmental Surfaces in Healthcare Settings?

    PubMed Central

    Omidbakhsh, Navid; Ahmadpour, Faraz; Kenny, Nicole

    2014-01-01

    Background Meters based on adenosine triphosphate (ATP) bioluminescence measurements in relative light units (RLU) are often used to rapidly assess the level of cleanliness of environmental surfaces in healthcare and other settings. Can such ATP measurements be adversely affected by factors such as soil and cleaner-disinfectant chemistry? Objective This study tested a number of leading ATP meters for their sensitivity, linearity of the measurements, correlation of the readings to the actual microbial contamination, and the potential disinfectant chemicals’ interference in their readings. Methods First, solutions of pure ATP in various concentrations were used to construct a standard curve and determine linearity and sensitivity. Serial dilutions of a broth culture of Staphylococcus aureus, as a representative nosocomial pathogen, were then used to determine if a given meter’s ATP readings correlated with the actual CFUs. Next, various types of disinfectant chemistries were tested for their potential to interfere with the standard ATP readings. Results All four ATP meters tested herein demonstrated acceptable linearity and repeatability in their readings. However, there were significant differences in their sensitivity to detect the levels of viable microorganisms on experimentally contaminated surfaces. Further, most disinfectant chemistries tested here quenched the ATP readings variably in different ATP meters evaluated. Conclusions Apart from their limited sensitivity in detecting low levels of microbial contamination, the ATP meters tested were also prone to interference by different disinfectant chemistries. PMID:24940751

  9. Comparative analysis of cytosolic and mitochondrial ATP synthesis in embryonic and postnatal hippocampal neuronal cultures

    PubMed Central

    Surin, Alexander M.; Khiroug, Serguei; Gorbacheva, Lubov R.; Khodorov, Boris I.; Pinelis, Vsevolod G.; Khiroug, Leonard

    2013-01-01

    ATP in neurons is commonly believed to be synthesized mostly by mitochondria via oxidative phosphorylation. Neuronal mitochondria have been studied primarily in culture, i.e., in neurons isolated either from embryos or from neonatal pups. Although it is generally assumed that both embryonic and postnatal cultured neurons derive their ATP from mitochondrial oxidative phosphorylation, this has never been tested experimentally. We expressed the FRET-based ATP sensor AT1.03 in cultured hippocampal neurons isolated either from E17 to E18 rat embryos or from P1 to P2 rat pups and monitored [ATP]c simultaneously with mitochondrial membrane potential (??m; TMRM) and NAD(P)H autofluorescence. In embryonic neurons, transient glucose deprivation induced a near-complete decrease in [ATP]c, which was partially reversible and was accelerated by inhibition of glycolysis with 2-deoxyglucose. In the absence of glucose, pyruvate did not cause any significant increase in [ATP]c in 84% of embryonic neurons, and inhibition of mitochondrial ATP synthase with oligomycin failed to decrease [ATP]c. Moreover, ??m was significantly reduced by oligomycin, indicating that mitochondria acted as consumers rather than producers of ATP in embryonic neurons. In sharp contrast, in postnatal neurons pyruvate added during glucose deprivation significantly increased [ATP]c (by 54 ± 8%), whereas oligomycin induced a sharp decline in [ATP]c and increased ??m. These signs of oxidative phosphorylation were observed in all tested P1–P2 neurons. Measurement of ??m with the potential-sensitive probe JC-1 revealed that neuronal mitochondrial membrane potential was significantly reduced in embryonic cultures compared to the postnatal ones, possibly due to increased proton permeability of inner mitochondrial membrane. We conclude that, in embryonic, but not postnatal neuronal cultures, ATP synthesis is predominantly glycolytic and the oxidative phosphorylation-mediated synthesis of ATP by mitochondrial F1Fo-ATPase is insignificant. PMID:23335879

  10. Urinary ATP Synthase Subunit ? Is a Novel Biomarker of Renal Mitochondrial Dysfunction in Acute Kidney Injury.

    PubMed

    Whitaker, Ryan M; Korrapati, Midhun C; Stallons, Lindsey J; Jesinkey, Sean R; Arthur, John M; Beeson, Craig C; Zhong, Zhi; Schnellmann, Rick G

    2015-05-01

    Although the importance of mitochondrial dysfunction in acute kidney injury (AKI) has been documented, noninvasive early biomarkers of mitochondrial damage are needed. We examined urinary ATP synthase subunit ? (ATPS?) as a biomarker of renal mitochondrial dysfunction during AKI. Mice underwent sham surgery or varying degrees (5, 10, or 15?min ischemia) of ischemia/reperfusion (I/R)-induced AKI. Serum creatinine, BUN, and neutrophil gelatinase-associated lipocalin were elevated only in the 15?min I/R group at 24?h. Immunoblot analysis of urinary ATPS? revealed two bands (full length ?52?kDa and cleaved ?25?kDa), both confirmed as ATPS? by LC-MS/MS, that increased at 24?h in 10- and 15-min I/R groups. These changes were associated with mitochondrial dysfunction evidenced by reduced renal cortical expression of mitochondrial proteins, ATPS? and COX1, proximal tubular oxygen consumption, and ATP. Furthermore, in the 15-min I/R group, urinary ATPS? was elevated until 72?h before returning to baseline 144?h after reperfusion with recovery of renal function. Evaluation of urinary ATPS? in a nonalcoholic steatohepatitis model of liver injury only revealed cleaved ATPS?, suggesting specificity of full-length ATPS? for renal injury. Immunoblot analyses of patient urine samples collected 36?h after cardiac surgery revealed increased urinary ATPS? levels in patients with postcardiac surgery-induced AKI. LC-MS/MS urinalysis in human subjects with AKI confirmed increased ATPS?. These translational studies provide evidence that ATPS? may be a novel and sensitive urinary biomarker of renal mitochondrial dysfunction and could serve as valuable tool for the testing of potential therapies for AKI and chemical-induced nephrotoxicity. PMID:25666834

  11. 77 FR 39388 - Removal of Category IIIa, IIIb, and IIIc Definitions; Confirmation of Effective Date and Response...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-03

    ...have no impact on the recognition of any CAT III a, b, or c operational approval for...definition relaxation will result in blending the Cat III operational and system performance distinctions...have no impact on the recognition of any CAT III a, b, or c operational approval...

  12. Angiostatin-Like Activity of a Monoclonal Antibody to the Catalytic Subunit of F1F0 ATP Synthase

    Microsoft Academic Search

    Miriam L. Wahl; Yvonne M. Mowery; Siqing Shan; Somnath Mukhopadhyay; Susana C. Hilderbrand; Daniel J. Kenan; Barbara D. Lipes; Carrie E. Johnson; Michael F. Marusich; Roderick A. Capaldi; Mark W. Dewhirst; Salvatore V. Pizzo; Mitosciences LLC

    2007-01-01

    The antiangiogenic protein angiostatin inhibits ATP synthase on the endothelial cell surface, blocking cellular proliferation. To examine the specificity of this interaction, we generated monoclonal antibodies (mAb) directed against ATP synthase. mAb directed against the B-catalytic subunit of ATP synthase (MAb3D5AB1) inhibits the activity of the F1 domain of ATP synthase and recognizes the catalytic B-subunit of ATP synthase. We

  13. Extracellular ATP promotes stomatal opening of Arabidopsis thaliana through heterotrimeric G protein ? 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 (ATP?S, 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 ? 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

  14. Assessing ATP binding and hydrolysis by NLR proteins

    PubMed Central

    Mo, Jinyao; Duncan, Joseph A.

    2014-01-01

    Summary Nucleotide-binding and leucine rich repeat domain-containing proteins (NLR) are central to the formation of many inflammasome complexes. Several inflammasome forming NLR proteins are known to be ATPases, but the nucleotide binding specificity of many remains to be characterized. The oligomerization of NLR proteins and assembly of inflammasomes require the ATP (or other nucleotide) binding activity of the NLR proteins. Quantitative and qualitative studies of the nucleotide binding properties of these proteins are useful tools in studying the regulation of inflammasome activity, and will be outlined in this Chapter. PMID:23852603

  15. 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.

  16. Argininosuccinate synthetase: a stereochemical study using chiral ATP analogs 

    E-print Network

    Hess, Tamara Louise Chapman

    1984-01-01

    AND METHODS V1 V1. 1 1. X 14 HPLC Assays Citrulline Assays Pi Assays Synthesis of 0-ci(gulline Synthesis of ATPap 02 Synthesis of [u- 0, 0]ATP Assay for Myokinase Activity Assay for Synthetase Activity Using ATPaS (Sp) and IBO citrulline..., and Boehringer. HPLC Assays Assays were conducted on a Gilson HPLC system employing a IYhatman Partisil 10 SAX anion exchange column. The concentrations of various nucleotides were measured by monitoring the absorbance at 260nm. The characteristic retention...

  17. Nonsynaptic and nonvesicular ATP release from neurons and relevance to neuron–glia signaling

    PubMed Central

    Fields, R. Douglas

    2011-01-01

    Studies on the release of ATP from neurons began with the earliest investigations of quantal neurotransmitter release in the 1950s, but in contrast to ATP release from other cells, studies of ATP release from neurons have been narrowly constrained to one mechanism, vesicular release. This is a consequence of the prominence of synaptic transmission in neuronal communication, but nonvesicular mechanisms for ATP release from neurons are likely to have a broader range of functions than synaptic release. Investigations of activity-dependent communication between axons and myelinating glia have stimulated a search for mechanisms that could release ATP from axons and other nonsynaptic regions in response to action potential firing. This has identified volume-activated anion channels as an important mechanism in activity-dependent ATP release from axons, and renewed interest in micromechanical changes in axons that accompany action potential firing. PMID:21320624

  18. The interaction of potassium ions and ATP on the sodium pump of resealed red cell ghosts.

    PubMed Central

    Eisner, D A; Richards, D E

    1981-01-01

    1. Ouabain-sensitive K or Rb influx was measure into ghosts resealed to contain ATP concentrations of 1 micrometers-3 mM and no K. 2. Increasing ATP from 1 to 100 micro M, at saturation external K, increased K influx about twentyfold while have no effect on the ratio of ouabain-sensitive K influx to ouabain-sensitive ATPase activity. 3. Increasing external K decreased the apparent affinity for ATP. Similarly increasing ATP decreased the apparent affinity for external K. 4. The K influx can be empirically described as: influx = VmaxK2/(K + Kapp)2. Increasing ATP increased Vmax and (Kapp)2 by the same amount. 5. These results are consistent with a consecutive model for the Na pump in which an ATP-dependent reaction follows a K-activated dephosphorylation. PMID:7320919

  19. Effect of external torque on the ATP-driven rotation of F1-ATPase.

    PubMed

    Watanabe-Nakayama, Takahiro; Toyabe, Shoichi; Kudo, Seishi; Sugiyama, Shigeru; Yoshida, Masasuke; Muneyuki, Eiro

    2008-02-22

    F(1)-ATPase is a rotary molecular motor powered by the torque generated by another rotary motor F(0) to synthesize ATP in vivo. Therefore elucidation of the behavior of F(1) under external torque is very important. Here, we applied controlled external torque by electrorotation and investigated the ATP-driven rotation for the first time. The rotation was accelerated by assisting torque and decelerated by hindering torque, but F(1) rarely showed rotations in the ATP synthesis direction. This is consistent with the prediction by models based on the assumption that the rotation is tightly coupled to ATP hydrolysis and synthesis. At low ATP concentrations (2 and 5 microM), 120 degrees stepwise rotation was observed. Due to the temperature rise during experiment, quantitative interpretation of the data is difficult, but we found that the apparent rate constant of ATP binding clearly decreased by hindering torque and increased by assisting torque. PMID:18083117

  20. ATP Dependence of Na+-Driven Cl–HCO3 Exchange in Squid Axons

    PubMed Central

    Davis, Bruce A.; Hogan, Emilia M.; Russell, John M.; Boron, Walter F.

    2010-01-01

    Squid giant axons recover from acid loads by activating a Na+-driven Cl–HCO3 exchanger. We internally dialyzed axons to an intracellular pH (pHi) of 6.7, halted dialysis and monitored the pHi recovery (increase) in the presence of ATP or other nucleotides, using cyanide to block oxidative phosphorylation. We computed the equivalent acid-extrusion rate (JH) from the rate of pHi increase and intracellular buffering power. In experimental series 1, we used dialysis to vary [ATP]i, finding that Michaelis-Menten kinetics describes JH vs. [ATP]i, with an apparent Vmax of 15.6 pmole cm?2 s?1 and Km of 124 µM. In series 2, we examined ATP?S, AMP-PNP, AMP-PCP, AMP-CPP, GMP-PNP, ADP, ADP?S and GDP?S to determine if any, by themselves, could support transport. Only ATP?S (8 mM) supported acid extrusion; ATP?S also supported the HCO3?-dependent 36Cl efflux expected of a Na+-driven Cl–HCO3 exchanger. Finally, in series 3, we asked whether any nucleotide could alter JH in the presence of a background [ATP]i of ~230 µM (control JH = 11.7 pmol cm?2 s?1). We found JH was decreased modestly by 8 mM AMP-PNP (JH = 8.0 pmol cm?2 s?1) but increased modestly by 1 mM ADP?S (JH = 16.0 pmol cm?2 s?1). We suggest that ATP?S leads to stable phosphorylation of the transporter or an essential activator. PMID:18478173

  1. Modulation of K channels in dialyzed squid axons. ATP-mediated phosphorylation

    PubMed Central

    1989-01-01

    In squid axons, internally applied ATP potentiates the magnitude of the potassium conductance and slows down its activation kinetics. This effect was characterized using internally dialyzed axons under voltage- clamp conditions. Both amplitude potentiation and kinetic slow-down effects are very selective towards ATP, other nucleotides like GTP and ITP are ineffective in millimolar concentrations. The current potentiation Km for ATP is near 10 microM with no further effects for concentrations greater than 100 microM. ATP effect is most likely produced via a phosphorylative reaction because Mg ion is an obligatory requirement and nonhydrolyzable ATP analogues are without effect. In the presence of ATP, the K current presents more delay, resembling a Cole-Moore effect due to local hyperpolarization of the channel. ATP effect induces a 10-20 mV shift in both activation and inactivation parameters towards more depolarized potentials. As a consequence of this shift, conductance-voltage curves with and without ATP cross at approximately -40 mV. This result is consistent with the hyperpolarization observed with ATP depletion, which is reversed by ATP addition. At potentials around the resting value, addition of ATP removes almost completely K current slow inactivation. It is suggested that a change in the amount of the slow inactivation is responsible for the differences in current amplitude with and without ATP, possibly as a consequence of the additional negative charge carried by the phosphate group. However, a modification of the local potential is not enough to explain completely the differences under the two conditions. PMID:2769224

  2. Vitro packaging of bacteriophage T7 DNA requires ATP. [Escherichia Coli

    SciTech Connect

    Masker, W.E.

    1982-07-01

    Removal of nucleoside triphosphates from extracts prepared from bacteriophage T7-infected Escherichia coli results in a stringent requirement for added ATP to form infective phage particles by in vitro packaging of bacteriophage T7 DNA. Optimal packaging efficiency was achieved at a concentration of about 1.25 mM. Other nucleoside triphosphates could be substituted for ATP, but none of the common nucleoside triphosphates was as effective as ATP in promoting in vitro encapsulation.

  3. ATP Transport Through a Single Mitochondrial Channel, VDAC, Studied by Current Fluctuation Analysis

    Microsoft Academic Search

    Tatiana K. Rostovtseva; Sergey M. Bezrukov

    1998-01-01

    The “molecular Coulter counter” concept has been used to study transport of ATP molecules through the nanometer-scale aqueous pore of the voltage-dependent mitochondrial ion channel, VDAC. We examine the ATP-induced current fluctuations and the change in average current through a single fully open channel reconstituted into a planar lipid bilayer. At high salt concentration (1M NaCl), the addition of ATP

  4. ATP as a biomarker of viable microorganisms in clean-room facilities

    Microsoft Academic Search

    Kasthuri Venkateswaran; Noriaki Hattori; Myron T. La Duc; Roger Kern

    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 ?m, and ordinary rooms with unfiltered air. The intracellular ATP was

  5. Role of ATP in UV-induced DNA excision repair in human cells

    SciTech Connect

    Dresler, S.L.

    1986-05-01

    In permeable human fibroblasts, UV-induced DNA excision repair is dependent on ATP, with a K/sub m/ of approximately 1 mM. Omission of ATP from the reaction mix completely inhibits damage-specific incision of DNA, but has little effect on repair patch synthesis proceeding from previously incised sites. UV-induced excision repair in permeable xeroderma pigmentosum (XP) cells complemented with T4 UV endonuclease is also totally dependent on ATP. Because the T4 enzyme is not ATP-dependent, ATP must be required for an endogenous activity other than the incision of damaged DNA. Alkaline elution reveals that, in the absence of ATP, T4 UV endonuclease does incise the DNA of permeable UV-irradiated XP cells, but that the incision rate is stimulated approximately 2-fold by the addition of ATP. This 2-fold stimulation of incision can not, however, be responsible for the absolute ATP dependence of excision repair in UV endonuclease-complemented XP cells. Apparently, although T4 UV endonuclease can incise damaged nuclear DNA in the absence of ATP, the incised sites must also be altered in an ATP-dependent reaction before subsequent steps of the repair process can proceed. This conclusion, coupled with the fact that ATP stimulates incision of damaged nuclear DNA by T4 UV endonuclease and is absolutely required for incision of damaged nuclear DNA by the endogenous human UV endonuclease, suggests that an important function of the early ATP-dependent step in UV-induced excision repair is to make damaged sites in DNA accessible to repair enzymes.

  6. A broad bean mitochondrial atp6 gene with an unusually simple, non-conserved 5? region

    Microsoft Academic Search

    Jane L. Macfarlane; Jill A. Wahleithner; David R. Wolstenholme

    1990-01-01

    A nucleotide sequence of broad bean mitochondrial DNA (mtDNA) that contains an atp6 gene of 876 ntp is presented. Relative to other plant atp6 genes, this broad bean gene comprises a 90 ntp non-conserved 5' region, a 759 ntp highly conserved central region and a 27 ntp non-conserved 3' region. The non-conserved, 5' region of the broad bean atp6 gene

  7. The pea mitochondrial atp6 : RNA editing and similarity of presequences in the Vicieae tribe

    Microsoft Academic Search

    Marta Gibala; Bartosz Szczesny; Jan Kieleczawa; Hanna Janska

    2004-01-01

    The atp6 gene has been identified as a single-copy sequence in the mitochondrial genome of the pea. An unexpected finding concerns the atp6 5? extension which is known to be poorly conserved at the sequence level, even between closely related plant species. We have shown that the presequences of ATP6 from the pea and other species belonging to the Vicieae

  8. Polymorphisms in canine ATP7B: Candidate modifier of copper toxicosis in the Bedlington terrier

    Microsoft Academic Search

    Veronica A. Coronado; Brian O’Neill; Manoj Nanji; Diane W. Cox

    2008-01-01

    A COMMD1(MURR1) deletion has been reported as the cause of copper toxicosis (CT) in Bedlington terriers. Recent studies identified Bedlington terriers with copper accumulation without homozygous COMMD1 deletions. Wilson disease in humans is a copper storage disorder similar to CT caused by mutations in ATP7B, and COMMD1 has been shown to interact with the ATP7B protein. ATP7B may act as

  9. ATP-Loaded Liposomes Effectively Protect the Myocardium in Rabbits with an Acute Experimental Myocardial Infarction

    Microsoft Academic Search

    Daya D. Verma; William C. Hartner; Tatayana S. Levchenko; Eugene A. Bernstein; Vladimir P. Torchilin

    2005-01-01

    Purpose. We assessed whether the infusion of ATP-loaded liposomes (ATP-L) can limit the fraction of the irreversibly damaged myocardium in rabbits with an experimental myocardial infarction. Methods. ATP-L, empty liposomes (EL), or KrebsYHenseleit (KH) buffer were administered by intracoronary infusion, followed by 30 min of occlusion and 3 h of reperfusion. Unisperse Blue dye was used to demarcate the net

  10. Potentiation by cadmium ion of ATP-evoked dopamine release in rat phaeochromocytoma cells.

    PubMed Central

    Ikeda, M.; Koizumi, S.; Nakazawa, K.; Inoue, K.; Ito, K.; Inoue, K.

    1996-01-01

    1. The effects of cadmium ion (Cd2+) on release of dopamine and on an inward current evoked by extracellular ATP were investigated in rat phaeochromocytoma PC12 cells. 2. Cd2+ (100 microM-3 mM) potentiated the dopamine release evoked by 30 microM ATP from the cells. Cd2+ (100 microM) shifted the concentration-response curve of ATP-evoked dopamine release to the left without affecting the maximal response. 3. Suramin (30 microM) completely abolished the dopamine release evoked by 30 microM ATP but only partially inhibited the release evoked by 100 microM ATP consistent with its role as a competitive antagonist. The response evoked by 30 microM ATP in the presence of Cd2+ (300 microM) was comparable to that observed with 100 microM ATP alone; however, only the former was almost completely inhibited by suramin. 4. Cd2+ (100 microM) potentiated an inward current activated by 30 microM ATP alone. A higher concentration of Cd2+ (300 microM) had a smaller effect on amplitude potentiation but significantly prolonged the duration of the current. 5. The time-course of the ATP-evoked dopamine release was investigated using a real-time monitoring system for dopamine release. Although Cd2+ (300 microM) had little effect on the time-course of activation the ATP-evoked dopamine release, it produced a long-lasting dopamine release which slowly returned to the baseline. 6. Taken together, these observations suggest that Cd2+ enhances ATP-evoked dopamine release by affecting P2-purinoceptor/channels. The enhancement may be attributed to a Cd(2+)-dependent increase in sensitivity to ATP. PMID:8851516

  11. ORIGINAL RESEARCH The E646D-ATP13A4 Mutation Associated with Autism Reveals

    E-print Network

    Crawford, Dorota A.

    . Keywords P5-type ATPase Á ATP13A4 ATPase Á fura-2 AM Á Intracellular free calcium Á Ratiometric calcium in mammals, includes Cd2? -, Cu2? -, and K? -ATP- ases, P2, also found in mammals, consists of Ca2? -, H? / K was measured in COS-7 cells over-expressing mouse ATP13A4 using ratiometric cal- cium imaging with fura-2 AM

  12. The genes for the eight subunits of the membrane bound ATP synthase of Escherichia coli

    Microsoft Academic Search

    Flemming G. Hansen; Jørgen Nielsen; Erik Riise; Kaspar Meyenburg

    1981-01-01

    The genes for the eight subunits of the membrane bound ATP synthase of Escherichia coli (Ca++, Mg++ dependent ATPase, EC 3.6.1.3) were mapped through genetic, physical and functional analysis of specialized transducing phages ?asn (von Meyenburg et al. 1978). The ATP synthase genes, designated atp1, are located at 83.2 min in a segment of the chromosome between 3.5 and 11.3

  13. 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

  14. A tridecameric c ring of the adenosine triphosphate (ATP) synthase from the thermoalkaliphilic Bacillus sp. strain TA2.A1 facilitates ATP synthesis at low electrochemical proton potential.

    PubMed

    Meier, Thomas; Morgner, Nina; Matthies, Doreen; Pogoryelov, Denys; Keis, Stefanie; Cook, Gregory M; Dimroth, Peter; Brutschy, Bernhard

    2007-09-01

    Despite the thermodynamic problem imposed on alkaliphilic bacteria of synthesizing adenosine triphosphate (ATP) against a large inverted pH gradient and consequently a low electrochemical proton potential, these bacteria still utilize a proton-coupled F(1)F(o)-ATP synthase to synthesize ATP. One potential solution to this apparent thermodynamic problem would be the operation of a larger oligomeric c ring, which would raise the ion to ATP ratio, thus facilitating the conversion of a low electrochemical potential into a significant phosphorylation potential. To address this hypothesis, we have purified the oligomeric c ring from the thermoalkaliphilic bacterium Bacillus sp. strain TA2.A1 and determined the number of c-subunits using a novel mass spectrometry method, termed 'laser-induced liquid bead ion desorption' (LILBID). This technique allows the mass determination of non-covalently assembled, detergent-solubilized membrane protein complexes, and hence enables an accurate determination of c ring stoichiometries. We show that the Bacillus sp. strain TA2.A1 ATP synthase harbours a tridecameric c ring. The operation of a c ring with 13 subunits renders the thermodynamic problem of ATP synthesis at alkaline pH less severe and may represent a strategy for ATP synthesis at low electrochemical potential. PMID:17645441

  15. What limits the allotopic expression of nucleus-encoded mitochondrial genes? The case of the chimeric Cox3 and Atp6 genes.

    PubMed

    Figueroa-Martínez, Francisco; Vázquez-Acevedo, Miriam; Cortés-Hernández, Paulina; García-Trejo, José J; Davidson, Edgar; King, Michael P; González-Halphen, Diego

    2011-01-01

    Allotopic expression is potentially a gene therapy for mtDNA-related diseases. Some OXPHOS proteins like ATP6 (subunit a of complex V) and COX3 (subunit III of complex IV) that are typically mtDNA-encoded, are naturally nucleus-encoded in the alga Chlamydomonas reinhardtii. The mitochondrial proteins whose genes have been relocated to the nucleus exhibit long mitochondrial targeting sequences ranging from 100 to 140 residues and a diminished overall mean hydrophobicity when compared with their mtDNA-encoded counterparts. We explored the allotopic expression of the human gene products COX3 and ATP6 that were re-designed for mitochondrial import by emulating the structural properties of the corresponding algal proteins. In vivo and in vitro data in homoplasmic human mutant cells carrying either a T8993G mutation in the mitochondrial atp6 gene or a 15bp deletion in the mtDNA-encoded cox3 gene suggest that these human mitochondrial proteins re-designed for nuclear expression are targeted to the mitochondria, but fail to functionally integrate into their corresponding OXPHOS complexes. PMID:20854934

  16. 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 ?atp 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 ? 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

  17. Clusterin (Apolipoprotein J), a Molecular Chaperone That Facilitates Degradation of the Copper-ATPases ATP7A and ATP7B*

    PubMed Central

    Materia, Stephanie; Cater, Michael A.; Klomp, Leo W. J.; Mercer, Julian F. B.; La Fontaine, Sharon

    2011-01-01

    The copper-transporting P1B-type ATPases (Cu-ATPases) ATP7A and ATP7B are key regulators of physiological copper levels. They function to maintain intracellular copper homeostasis by delivering copper to secretory compartments and by trafficking toward the cell periphery to export excess copper. Mutations in the genes encoding ATP7A and ATP7B lead to copper deficiency and toxicity disorders, Menkes and Wilson diseases, respectively. This report describes the interaction between the Cu-ATPases and clusterin and demonstrates a chaperone-like role for clusterin in facilitating their degradation. Clusterin interacted with both ATP7A and ATP7B in mammalian cells. This interaction increased under conditions of oxidative stress and with mutations in ATP7B that led to its misfolding and mislocalization. A Wilson disease patient mutation (G85V) led to enhanced ATP7B turnover, which was further exacerbated when cells overexpressed clusterin. We demonstrated that clusterin-facilitated degradation of mutant ATP7B is likely to involve the lysosomal pathway. The knockdown and overexpression of clusterin increased and decreased, respectively, the Cu-ATPase-mediated copper export capacity of cells. These results highlight a new role for intracellular clusterin in mediating Cu-ATPase quality control and hence in the normal maintenance of copper homeostasis, and in promoting cell survival in the context of disease. Based on our findings, it is possible that variations in clusterin expression and function could contribute to the variable clinical expression of Menkes and Wilson diseases. PMID:21242307

  18. Adenosine uptake is the major effector of extracellular ATP toxicity in human cervical cancer cells

    PubMed Central

    Mello, Paola de Andrade; Filippi-Chiela, Eduardo Cremonese; Nascimento, Jéssica; Beckenkamp, Aline; Santana, Danielle Bertodo; Kipper, Franciele; Casali, Emerson André; Nejar Bruno, Alessandra; Paccez, Juliano Domiraci; Zerbini, Luiz Fernando; Wink, Marcia Rosângela; Lenz, Guido; Buffon, Andréia

    2014-01-01

    In cervical cancer, HPV infection and disruption of mechanisms involving cell growth, differentiation, and apoptosis are strictly linked with tumor progression and invasion. Tumor microenvironment is ATP and adenosine rich, suggesting a role for purinergic signaling in cancer cell growth and death. Here we investigate the effect of extracellular ATP on human cervical cancer cells. We find that extracellular ATP itself has a small cytotoxic effect, whereas adenosine formed from ATP degradation by ectonucleotidases is the main factor responsible for apoptosis induction. The level of P2×7 receptor seemed to define the main cytotoxic mechanism triggered by ATP, since ATP itself eliminated a small subpopulation of cells that express high P2×7 levels, probably through its activation. Corroborating these data, blockage or knockdown of P2×7 only slightly reduced ATP cytotoxicity. On the other hand, cell viability was almost totally recovered with dipyridamole, an adenosine transporter inhibitor. Moreover, ATP-induced apoptosis and signaling—p53 increase, AMPK activation, and PARP cleavage—as well as autophagy induction were also inhibited by dipyridamole. In addition, inhibition of adenosine conversion into AMP also blocked cell death, indicating that metabolization of intracellular adenosine originating from extracellular ATP is responsible for the main effects of the latter in human cervical cancer cells. PMID:25103241

  19. Oxygen-glucose deprivation induces ATP release via maxi-anion channels in astrocytes.

    PubMed

    Liu, Hong-Tao; Sabirov, Ravshan Z; Okada, Yasunobu

    2008-06-01

    ATP represents a major gliotransmitter that serves as a signaling molecule for the cross talk between glial and neuronal cells. ATP has been shown to be released by astrocytes in response to a number of stimuli under nonischemic conditions. In this study, using a luciferin-luciferase assay, we found that mouse astrocytes in primary culture also exhibit massive release of ATP in response to ischemic stress mimicked by oxygen-glucose deprivation (OGD). Using a biosensor technique, the local ATP concentration at the surface of single astrocytes was found to increase to around 4 muM. The OGD-induced ATP release was inhibited by Gd(3+) and arachidonic acid but not by blockers of volume-sensitive outwardly rectifying Cl(-) channels, cystic fibrosis transmembrane conductance regulator (CFTR), multidrug resistance-related protein (MRP), connexin or pannexin hemichannels, P2X(7) receptors, and exocytotic vesicular transport. In cell-attached patches on single astrocytes, OGD caused activation of maxi-anion channels that were sensitive to Gd(3+) and arachidonic acid. The channel was found to be permeable to ATP(4-) with a permeability ratio of P(ATP)/P(Cl) = 0.11. Thus, it is concluded that ischemic stress induces ATP release from astrocytes and that the maxi-anion channel may serve as a major ATP-releasing pathway under ischemic conditions. PMID:18368522

  20. 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

  1. Enhancement of ATP-activated current by protons in dorsal root ganglion neurons

    Microsoft Academic Search

    C. Li; Robert W. Peoples; Forrest F. Weight

    1997-01-01

    The effect of pH on ATP-activated current in bullfrog dorsal root ganglion neurons was studied using the whole-cell patch-clamp\\u000a technique. ATP-activated current amplitude was highly dependent upon extracellular pH. An acid pH increased, whereas alkaline\\u000a pH decreased, ATP-activated current amplitude. The half-maximal pH (EC50) for potentiation of 2.5 ?M ATP-activated current was 7.2. Acidification alone did not activate detectable current

  2. Neurokinin B potentiates ATP-activated currents in rat DRG neurons.

    PubMed

    Wang, M J; Xiong, S H; Li, Z W

    2001-12-27

    This study aimed to explore whether NKB could modulate the responses mediated by ATP receptor (P2X purinoceptor). Whole-cell patch clamp and repatch experiments were performed on cultured rat DRG neurons. The majority of neurons examined were sensitive both to ATP and to NKB (77.1%, 54/70). NKB preapplied could potentiate ATP-activated currents (I(ATP)) markedly; this effect was concentration-dependent and could be blocked by SR 142801, an NK3 receptor antagonist. Preapplication of 0.001, 0.01, 0.1 and 1.0 microM NKB increased ATP-activated currents by 55.1+/-18.8, 75.2+/-17.4, 84.1+/-18.8 and 81.0+/-21.7%, respectively. The concentration-response curves for ATP with and without preapplication of NKB show that: (1) preapplication of NKB shifted the curve upwards; (2) the maximal amplitude of I(ATP) with NKB preapplication increased by 78.5%, while the threshold value remained unchanged; (3) the EC(50) values of the two curves were very close (44 vs. 42 microM). Intracellular dialysis of H-7 by using repatch clamp technique could block the potentiation of I(ATP) by NKB. It suggests that this potentiating effect was caused by phosphorylation of ATP receptor, which resulted from the activation of G protein coupled NK3 receptor and consequential intracellular signal transduction cascade. PMID:11743983

  3. Use of firefly luciferase for ATP measurement: other nucleotides enhance turnover.

    PubMed

    Ford, S R; Chenault, K H; Bunton, L S; Hampton, G J; McCarthy, J; Hall, M S; Pangburn, S J; Buck, L M; Leach, F R

    1996-01-01

    Firefly luciferase utilizes only ATP and a few closely related nucleotides as substrates for the formation of luciferyl adenylate which is an intermediate in the bioluminescent reaction sequence that oxidizes firefly luciferin. The enzyme shows two different time courses of light production depending on ATP concentration used: a flash with high concentrations of ATP (> 8 microM) or a fairly constant production of light with lower concentrations of ATP (< 1 microM). Many nucleotides, nucleotide-containing substances and other compounds, when added either prior to or 1 min after the addition of ATP, change the time course of light production. When added before ATP, these compounds yield a reaction mixture in which light production is fairly constant (at the level characteristic of the flash observed with that ATP concentration). When the compounds are added after ATP addition, light production is markedly stimulated and the higher rate of light production is maintained for several minutes. There is an increase in quanta of light produced per luciferase dimer from 1 to 5/min with the addition of any of several nucleotide analogues. These results are consistent with a stimulated release of the inhibitory product oxyluciferin, allowing turnover of the enzyme. This enzyme turnover permits more light output at high ATP concentrations, thus enhancing the sensitivity of enzyme determination. PMID:8844345

  4. Autocrine/paracrine stimulation of purinergic receptors in osteoblasts: contribution of vesicular ATP release.

    PubMed

    Romanello, Milena; Codognotto, Andrea; Bicego, Massimiliano; Pines, Alex; Tell, Gianluca; D'Andrea, Paola

    2005-06-17

    Extracellular nucleotides such as ATP and UTP are released in response to mechanical stimulation in different cell systems. It is becoming increasingly evident that ATP release plays a role in autocrine and paracrine stimulation of osteoblasts. Mechanical stimulation, as shear stress, membrane stretch or hypo-osmotic swelling, as well as oscillatory fluid flow, stimulates ATP release from different osteoblastic cell lines. Human osteoblast-like initial transfectant (HOBIT) cells release ATP in response to mechanical stimulation. In the present study, we show that HOBIT cells are activated by nanomolar levels of extracellular ATP, concentrations that can be detected under resting conditions and increase following hypotonic shock. Cell activation by hypotonic medium induced intracellular Ca2+ oscillations, and Egr-1 synthesis and DNA-binding activity. Quinacrine staining of living, resting cells revealed a granular fluorescence, typical of ATP-storing vesicles. Monensin prevented quinacrine staining and considerably inhibited hypotonic-induced ATP release. Finally, elevated levels of cytosolic Ca2+ activated massive ATP release and a dose-dependent loss of quinacrine granules. The contribution of a vesicular mechanism for ATP release is proposed to sustain paracrine osteoblast activation. PMID:15883034

  5. Chromatophore Vesicles of Rhodobacter capsulatus Contain on Average One FOF1-ATP Synthase Each

    E-print Network

    Steinhoff, Heinz-Jürgen

    to ATP synthesis, can be spectrophotometrically monitored by electrochromic absorption transients molecules per chromatophore vesicle. Kinetic analysis of the electrochromic transients plus/minus specific

  6. Extracellular ATP acts as a damage-associated molecular pattern (DAMP) signal in plants

    PubMed Central

    Tanaka, Kiwamu; Choi, Jeongmin; Cao, Yangrong; Stacey, Gary

    2014-01-01

    As sessile organisms, plants have evolved effective mechanisms to protect themselves from environmental stresses. Damaged (i.e., wounded) plants recognize a variety of endogenous molecules as danger signals, referred to as damage-associated molecular patterns (DAMPs). ATP is among the molecules that are released by cell damage, and recent evidence suggests that ATP can serve as a DAMP. Although little studied in plants, extracellular ATP is well known for its signaling roles in animals, including acting as a DAMP during the inflammatory response and wound healing. If ATP acts outside the cell, then it is reasonable to expect that it is recognized by a plasma membrane-localized receptor. Recently, DORN1, a lectin receptor kinase, was shown to recognize extracellular ATP in Arabidopsis. DORN1 is the founding member of a new purinoceptor subfamily, P2K (P2 receptor kinase), which is plant-specific. P2K1 (DORN1) is required for ATP-induced cellular responses (e.g., cytosolic Ca2+ elevation, MAPK phosphorylation, and gene expression). Genetic analysis of loss-of-function mutants and overexpression lines showed that P2K1 participates in the plant wound response, consistent with the role of ATP as a DAMP. In this review, we summarize past research on the roles and mechanisms of extracellular ATP signaling in plants, and discuss the direction of future research on extracellular ATP as a DAMP signal. PMID:25232361

  7. Inhibition by ATP of calcium oscillations in rat cultured hippocampal neurones

    PubMed Central

    Koizumi, Schuichi; Inoue, Kazuhide

    1997-01-01

    The effect of adenosine 5?-triphosphate (ATP) on glutamatergic synaptic transmission in hippocampus was examined by an indicator of intracellular Ca2+ oscillations. These oscillations were postsynaptic responses by glutamate released from presynaptic sites. ATP completely inhibited the oscillations in a concentration-dependent manner. The ATP-induced inhibition was mediated via P2-purinoceptors since ATP exhibited the inhibitory action even in the presence of P1-purinoceptor antagonists. Also non-hydrolysable ATP analogues and uridine 5?-triphosphate (UTP) inhibited the oscillation. The rank order of agonist potency of ATP analogues for inhibition of the Ca2+ oscillation was as follows: 2-methyl-thio-adenosine 5?-triphosphate?ATP>adenosine 5?-O-(3-thiotriphosphate)>UTP>?,?-methylene-adenosine 5?-triphosphate. These inhibitory effects were insensitive to suramin. Judging from this rank order of potency, the inhibitory P2-purinoceptor could be assigned to a subclass of GTP-binding protein coupled-type receptors. The site of action of ATP was thought to be presynaptic since ATP did not affect the postsynaptic Ca2+ responses by glutamate. These results suggest the existence of a presynaptic inhibitory P2-receptor that inhibits glutamate release in the hippocampus. PMID:9298528

  8. Binding of 3',4'-dideoxykanamycin B to ATP and its related compounds.

    PubMed

    Inaba, A; Aramaki, Y; Takahashi, M; Tsuchiya, S

    1986-10-13

    The interactions of aminoglycoside, 3',4'-dideoxykanamycin B(DKB) with ATP and its related compounds were investigated. ATP, ADP, cyclic AMP and FAD bound to the DKB-conjugated Sepharose 4B column. The binding of DKB to ATP was also confirmed by equilibrium gel filtration. In the acidic pH region, the fluorescence of nucleotides was quenched by DKB. The Stern-Volmer plots showed that the molar ratios of the complexes were 1:1. The apparent stability constant was dependent on the number of the phosphate groups of nucleotides and was in the order of ATP greater than ADP greater than AMP. PMID:3020330

  9. 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

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

    PubMed

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

    2011-11-16

    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 I(1)III(2)IV(1) 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

  11. Nuclear and mitochondrial subunits from the white shrimp Litopenaeus vannamei F 0 F 1 ATP-synthase complex: cDNA sequence, molecular modeling, and mRNA quantification of atp9 and atp6

    Microsoft Academic Search

    Adriana Muhlia-Almazan; Oliviert Martinez-Cruz; Fernando Garcia-Carreño; Rodrigo Arreola; Rogerio Sotelo-Mundo; Gloria Yepiz-Plascencia

    2008-01-01

    We studied for the first time the ATP-synthase complex from shrimp as a model to understand the basis of crustacean bioenergetics\\u000a since they are exposed to endogenous processes as molting that demand high amount of energy. We analyzed the cDNA sequence\\u000a of two subunits of the Fo sector from mitochondrial ATP-synthase in the white shrimp Litopenaeus vannamei. The nucleus encoded

  12. Calcium handling and purinoceptor subtypes involved in ATP-induced contraction in rat small mesenteric arteries.

    PubMed Central

    Lagaud, G J; Stoclet, J C; Andriantsitohaina, R

    1996-01-01

    1. The relationship between the stimulation of ATP receptors, the increase in intracellular free calcium concentration ([Ca2+]i; measured using the fluorescent indicator fura-2), contraction and the subtypes of purinoceptors involved were investigated in the small mesenteric artery of the rat. 2. In normal physiological solution, ATP (0.001-3 mM) caused concentration-dependent increases in both [Ca2+]i and contraction. Both responses produced by ATP (1 mM) were inhibited by 50% in the presence of nitrendipine (1 microM) and were abolished in the presence of nitrendipine plus SK&F 96365 (30 microM). 3. In Ca(2+)-free medium, ATP (3 mM) elicited a transient increase in both [Ca2+]i and tension which were abolished by caffeine and decreased by 65% by thapsigargin (1 microM). Moreover, ATP (1 and 3 mM) produced increases in the [3H]D-myo-inositol 1,4,5-trisphosphate ([3H]IP3) content of vessels in a concentration-dependent manner. 4. Treatment of the vessels with Bordetella pertussis toxin (PTX) inhibited contractions to ATP linked to the influx of calcium through nitrendipine-sensitive mechanisms, but not those linked to the release of Ca2+ from intracellular stores nor the capacity of ATP in increasing IP3 content of the vessels. 5. The order of potency of ATP and its analogues in eliciting contraction was alpha, beta-methylene-ATP (alpha, beta-MeATP) > 2-methylthio-ATP (2-MeSATP) > ATP = ADP. The response to ATP was inhibited by suramin. Reactive Blue 2 (up to 100 microM) did not affect the contractile response to ATP. Pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid 4-sodium (PPADS) and alpha, beta-MeATP abolished the response to low concentrations of ATP and reduced contractions elicited by high concentrations of ATP. 6. After blockade of P2X-purinoceptors with PPADS, the order of potency of ATP and its analogues was 2-MeSATP > ATP = ADP. UTP produced concentration-dependent contractions which were not affected by suramin, Reactive Blue 2, PPADS or alpha, beta-MeATP, suggesting the presence of P2U-purinoceptors. 7. The results suggest that low concentrations of ATP activate P2X-purinoceptors and produce an influx of calcium through both voltage-dependent calcium channels sensitive to nitrendipine and through receptor-operated calcium channels sensitive to SK&F 96365. High concentrations of ATP activate P2Y-purinoceptors which promote firstly a nitrendipine-sensitive calcium influx via a PTX-sensitive G protein and secondly a release of Ca2+ from an internal source via the production of IP3. PMID:8734982

  13. Characterization of [35S]-ATP?S and [3H]-?,?-MeATP binding sites in rat brain cortical synaptosomes: regulation of ligand binding by divalent cations

    PubMed Central

    Schäfer, Rainer; Reiser, Georg

    1997-01-01

    We made a comparative analysis of the binding characteristics of the radioligands [35S]-ATP?S and [3H]-?,?-MeATP in order to test whether these ligands can be used to analyse P2-purinoceptors in synaptosomal membranes from rat brain cortex. Synaptosomes possess sites with high affinity for [35S]-ATP?S (Kd=22.2±9.1?nM, Bmax=14.8 pmol?mg?1 protein). The rank order of the competition potency of the different compounds (ATP?S, ATP, ATP?S>ADP?S, 2-MeSATP>deoxyATP, ADP>>UTP, ?,?-MeATP, AMP, Reactive Blue-2, suramin, isoPPADS) is consistent with pharmacological properties of P2Y-purinoceptors. Under identical conditions [35S]-ATP?S and [3H]-?,?-MeATP bind to different binding sites at synaptosomal membranes from rat brain cortex. The affinity of the [3H]-?,?-MeATP binding sites (Kd=13.7±1.8?nM, Bmax=6.34±0.28?pmol?mg?1 protein) was 38 fold higher than the potency of ?,?-MeATP to displace [35S]-ATP?S binding (Ki=0.52??M). ATP and ADP?S competed at both binding sites with different affinities, 60 fold and 175 fold, respectively. The other agonists tested (2-MeSATP, UTP, GTP) did not affect specific [35H]-?,?-MeATP binding at concentrations up to 100??M. The antagonists (suramin, isoPPADS, Evan's Blue) showed completely different affinities for both binding sites. Binding of [35S]-ATP?S on synaptosomes was regulated by GTP, which is indicative for G-protein coupled receptors. The Kd value for the high affinity binding site was reduced in the presence of GTP about 5 fold (from 1.8?nM to 8.6?nM). In the presence of Mg2+ the affinity was increased (Kd 1.8?nM versus 22?nM in the absence of Mg2+). The binding of both radioligands was regulated in an opposite manner by physiological concentrations of Ca2+ and Mg2+. Binding of [3H]-?,?-MeATP to synaptosomal membranes was increased 3 fold by raising the Ca2+ concentration from 10??M to 1?mM, whereas the addition of Mg2+ in the same concentration range resulted in an 80% reduction of the binding. In contrast, [35S]-ATP?S binding was not influenced at the same range of Ca2+ or Mg2+ concentrations (10??M to 1?mM). The addition of Mg2+ (5?mM) increased the affinity of [35S]-ATP?S for the high affinity site 10 fold. Diadenosine polyphosphates had a bimodal effect on [35S]-ATP?S binding to synaptosomal membranes. AP5A and Ap6A enhanced binding of [35S]-ATP?S 1.6 fold in a concentration range between 0.1 and 50??M. Ap3A was a weak inhibitor with a Ki value of 7.2??M. Ap4A, AP5A and Ap6A inhibited with Ki values>100??M. These data support the concept that diadenosine polyphosphates do not directly interact with ATP?S binding sites. In conclusion, on the basis of present knowledge of the interaction of P2-purinoceptor active compounds with P2X- and/or P2Y-purinoceptors, our data strongly suggest that [35S]-ATP?S is a useful tool to study P2Y-purinoceptors. Thus, the [35S]-ATP?S binding site might to a large extent represent P2Y-purinoceptors in synaptosomes from rat brain cortex. The nucleotide binding is regulated by G proteins, indicated by the effects of GTP/Mg2+ on binding. PMID:9222547

  14. Identification of ATP-Binding Regions in the RyR1 Ca2+ Release Channel

    PubMed Central

    Popova, Olga B.; Baker, Mariah R.; Tran, Tina P.; Le, Tri; Serysheva, Irina I.

    2012-01-01

    ATP is an important modulator of gating in type 1 ryanodine receptor (RyR1), also known as a Ca2+ release channel in skeletal muscle cells. The activating effect of ATP on this channel is achieved by directly binding to one or more sites on the RyR1 protein. However, the number and location of these sites have yet to be determined. To identify the ATP-binding regions within RyR1 we used 2N3ATP-2?,3?-Biotin-LC-Hydrazone (BioATP-HDZ), a photo-reactive ATP analog to covalently label the channel. We found that BioATP-HDZ binds RyR1 specifically with an IC50?=?0.6±0.2 mM, comparable with the reported EC50 for activation of RyR1 with ATP. Controlled proteolysis of labeled RyR1 followed by sequence analysis revealed three fragments with apparent molecular masses of 95, 45 and 70 kDa that were crosslinked by BioATP-HDZ and identified as RyR1 sequences. Our analysis identified four glycine-rich consensus motifs that can potentially constitute ATP-binding sites and are located within the N-terminal 95-kDa fragment. These putative nucleotide-binding sequences include amino acids 699–704, 701–706, 1081–1084 and 1195–1200, which are conserved among the three RyR isoforms. Located next to the N-terminal disease hotspot region in RyR1, these sequences may communicate the effects of ATP-binding to channel function by tuning conformational motions within the neighboring cytoplasmic regulatory domains. Two other labeled fragments lack ATP-binding consensus motifs and may form non-canonical ATP-binding sites. Based on domain topology in the 3D structure of RyR1 it is also conceivable that the identified ATP-binding regions, despite their wide separation in the primary sequence, may actually constitute the same non-contiguous ATP-binding pocket within the channel tetramer. PMID:23144945

  15. Inhibition of Tonoplast ATPase by 2?,3?-Dialdehyde Derivative of ATP 1

    PubMed Central

    Chow, Wing Ming; Tzeng, Chi Meng; Chen, Chung Shung; Kuo, Soong Yu; Wang, May Yun; Pan, Rong Long

    1992-01-01

    The 2?,3?-dialdehyde derivative of ATP (dial-ATP) has been shown to be an affinity label for the ATP binding site of the H+-ATPase from tonoplast of etiolated mung bean seedlings (Vigna radiata L.). The dial-ATP caused marked inactivation of enzymatic activities of both membrane-bound and soluble ATPase and its associated proton translocation. The inactivation was reversible, but could be stabilized by NaBH4. The sodium dodecyl sulfatepolyacrylamide gel electrophoresis pattern revealed that the dial-ATP binding site was in the large (A) subunit of ATPase. The inhibition could be substantially protected by its physiological substrate ATP, pyrophosphate, and nucleotides in the decreasing order: ATP > pyrophosphate > ADP = AMP > GTP > CTP = UTP. A Lineweaver-Burk plot showed that the mode of inhibition was competitive with respect to ATP. Loss of ATPase activity followed pseudo-first order kinetics with a Ki of 4.1 millimolar, a minimum inactivation half-time of 20 seconds, and a pseudo-first order rate constant of 0.035 s?1. The double logarithmic plot of apparent rate constant versus dial-ATP concentration gave a slope of 0.927, indicating that inactivation results from reaction of at least one lysine residue at the catalytic site of the large subunit. Labeling studies with [3H]dial-ATP indicate that the incorporation of approximately 1 mole of dial-ATP per mole ATPase is sufficient to completely inhibit the ATPase. A working model of nonequivalent subunits for enzymatic mechanism of vacuolar ATPase is suggested. PMID:16668647

  16. ATP Binding and Hydrolysis Properties of ABCB10 and Their Regulation by Glutathione

    PubMed Central

    Qiu, Wei; Liesa, Marc; Carpenter, Elizabeth P.; Shirihai, Orian S.

    2015-01-01

    ABCB10 (ATP binding cassette sub-family B10) is a mitochondrial inner-membrane ABC transporter. ABCB10 has been shown to protect the heart from the impact of ROS during ischemia-reperfusion and to allow for proper hemoglobin synthesis during erythroid development. ABC transporters are proteins that increase ATP binding and hydrolysis activity in the presence of the transported substrate. However, molecular entities transported by ABCB10 and its regulatory mechanisms are currently unknown. Here we characterized ATP binding and hydrolysis properties of ABCB10 by using the 8-azido-ATP photolabeling technique. This technique can identify potential ABCB10 regulators, transported substrates and amino-acidic residues required for ATP binding and hydrolysis. We confirmed that Gly497 and Lys498 in the Walker A motif, Glu624 in the Walker B motif and Gly602 in the C-Loop motif of ABCB10 are required for proper ATP binding and hydrolysis activity, as their mutation changed ABCB10 8-Azido-ATP photo-labeling. In addition, we show that the potential ABCB10 transported entity and heme precursor delta-aminolevulinic acid (dALA) does not alter 8-azido-ATP photo-labeling. In contrast, oxidized glutathione (GSSG) stimulates ATP hydrolysis without affecting ATP binding, whereas reduced glutathione (GSH) inhibits ATP binding and hydrolysis. Indeed, we detectABCB10 glutathionylation in Cys547 and show that it is one of the exposed cysteine residues within ABCB10 structure. In all, we characterize essential residues for ABCB10 ATPase activity and we provide evidence that supports the exclusion of dALA as a potential substrate directly transported by ABCB10. Last, we show the first molecular mechanism by which mitochondrial oxidative status, through GSH/GSSG, can regulate ABCB10. PMID:26053025

  17. Contribution of ATP and nitric oxide to NANC inhibitory transmission in rat pyloric sphincter.

    PubMed Central

    Soediono, P; Burnstock, G

    1994-01-01

    1. Changes in isometric tension were recorded from circular muscle strips of rat pyloric sphincter in vitro, in response to electrical field stimulation and exogenously applied muscle relaxants. 2. Concentration-response relationships were studied for relaxation to exogenously applied adenosine 5'-triphosphate (ATP) and two analogues, 2-methylthioATP (2-MeSATP) and alpha,beta-methylene ATP (alpha,beta-MeATP). These drugs evoked concentration-dependent relaxation of rat pyloric sphincter with an order of potency 2-MeSATP > ATP >> alpha,beta-MeATP, indicating the presence of P2y-purinoceptors. The IC50 value of each nucleotide was: 2-MeSATP, 5.0 x 10(-8); ATP, 7.9 x 10(-6) M; alpha,beta-MeATP showed only slight activity at a concentration of 0.1 mM. 3. Frequency-response relationships for relaxations evoked by electrical field stimulation (EFS) were studied in the absence and presence of 10 microM NG-nitro-L-arginine methyl ester (L-NAME, an inhibitor of nitric oxide (NO) synthesis) and 20 microM reactive blue 2 (a P2y-purinoceptor antagonist). It was found that these substances significantly reduced the relaxant response of rat pyloric sphincter to EFS by 40% and 50% respectively. In the presence of both L-NAME and reactive blue 2 the responses were reduced by 75%. 4. Concentration-response relationships were studied for ATP and 2-MeSATP in the presence of L-NAME. It was found that L-NAME did not significantly inhibit the relaxant responses to these drugs. 5. Concentration-response relationships for ATP and noradrenaline were studied in the presence of reactive blue 2 (20 microM); the P2y-antagonist significantly inhibited the relaxant response to ATP, but not that to noradrenaline.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 6 PMID:7532079

  18. Macula densa basolateral ATP release is regulated by luminal [NaCl] and dietary salt intake.

    PubMed

    Komlosi, Peter; Peti-Peterdi, Janos; Fuson, Amanda L; Fintha, Attila; Rosivall, Laszlo; Bell, Phillip Darwin

    2004-06-01

    One component of the macula densa (MD) tubuloglomerular feedback (TGF) signaling pathway may involve basolateral release of ATP through a maxi-anion channel. Release of ATP has previously been studied during a maximal luminal NaCl concentration ([NaCl](L)) stimulus (20-150 mmol/l). Whether MD ATP release occurs during changes in [NaCl](L) within the physiological range (20-60 mmol/l) has not been examined. Also, because TGF is known to be enhanced by low dietary salt intake, we examined the pattern of MD ATP release from salt-restricted rabbits. Fluorescence microscopy, with fura 2-loaded cultured mouse mesangial cells as biosensors, was used to assess ATP release from the isolated, perfused thick ascending limb containing the MD segment. The mesangial biosensor cells, which contain purinergic receptors and elevate intracellular Ca(2+) concentration ([Ca(2+)](i)) on ATP binding, were placed adjacent to the MD basolateral membrane. Elevations in [NaCl](L) between 0 and 80 mmol/l, in 20-mmol/l increments, caused stepwise increases in [Ca(2+)](i), with the highest increase at [NaCl](L) of approximately 60 mmol/l. Luminal furosemide at 10(-4) mol/l blocked ATP release, which suggests that the efflux of ATP required MD Na-2Cl-K cotransport. A low-salt diet for 1 wk increased the magnitude of [NaCl](L)-dependent elevations in biosensor [Ca(2+)](i) by twofold, whereas high-salt intake had no effect. In summary, ATP release occurs over the same range of [NaCl](L) (20-60 mmol/l) previously reported for TGF responses, and, similar to TGF, ATP release was enhanced by dietary salt restriction. Thus these two findings are consistent with the role of MD ATP release as a signaling component of the TGF pathway. PMID:14749255

  19. Three-Dimensional Structure of ATP:Corrinoid Adenosyltransferase from Salmonella typhimurium in Its Free State, Complexed with MgATP, or Complexed

    E-print Network

    Rayment, Ivan

    Three-Dimensional Structure of ATP:Corrinoid Adenosyltransferase from Salmonella typhimurium in ItsVised Manuscript ReceiVed NoVember 6, 2000 ABSTRACT: In Salmonella typhimurium, formation of the cobalt-carbon bond

  20. Identification of the initiation codon for the atpB gene in Chlamydomonas chloroplasts excludes translation of a precursor form of the beta subunit of the ATP synthase.

    PubMed

    Rimbault, B; Esposito, D; Drapier, D; Choquet, Y; Stern, D; Wollman, F A

    2000-11-01

    The chloroplast atpB gene of Chlamydomonas reinhardtii, which encodes the beta subunit of the ATP synthase, contains three in-frame ATGs that are candidate translation initiation codons. An earlier study revealed that the N terminus of the assembled beta subunit maps at the +2 position with respect to the second in-frame methionine codon (Fiedler et al. 1995). Using chloroplast transformation, we have examined the possibility that either of the two additional in-frame ATG codons is competent for translation initiation. We provide evidence that translation of atpB is initiated exclusively at the second ATG codon. We conclude that the beta subunit is not synthesized with an N-terminal leader before its assembly into a functional ATP synthase complex. PMID:11129053

  1. Human oocytes express ATP-sensitive K+ channels

    PubMed Central

    Du, Qingyou; Jovanovi?, Sofija; Sukhodub, Andriy; Barratt, Evelyn; Drew, Ellen; Whalley, Katherine M.; Kay, Vanessa; McLaughlin, Marie; Telfer, Evelyn E.; Barratt, Christopher L.R.; Jovanovi?, Aleksandar

    2010-01-01

    BACKGROUND ATP-sensitive K+ (KATP) channels link intracellular metabolism with membrane excitability and play crucial roles in cellular physiology and protection. The KATP channel protein complex is composed of pore forming, Kir6.x (Kir6.1 or Kir6.2) and regulatory, SURx (SUR2A, SUR2B or SUR1), subunits that associate in different combinations. The objective of this study was to determine whether mammalian oocytes (human, bovine, porcine) express KATP channels. METHODS Supernumerary human oocytes at different stages of maturation were obtained from patients undergoing assisted conception treatments. Bovine and porcine oocytes in the germinal vesicle (GV) stage were obtained by aspirating antral follicles from abattoir-derived ovaries. The presence of mRNA for KATP channel subunits was determined using real-time RT–PCR with primers specific for Kir6.2, Kir6.1, SUR1, SUR2A and SUR2B. To assess whether functional KATP channels are present in human oocytes, traditional and perforated patch whole cell electrophysiology and immunoprecipitation/western blotting were used. RESULTS Real-time PCR revealed that mRNA for Kir6.1, Kir6.2, SUR2A and SUR2B, but not SUR1, were present in human oocytes of different stages. Only SUR2B and Kir6.2 mRNAs were detected in GV stage bovine and porcine oocytes. Immunoprecipitation with SUR2 antibody and western blotting with Kir6.1 antibody identified bands corresponding to these subunits in human oocytes. In human oocytes, 2,4-dinitrophenol (400 µM), a metabolic inhibitor known to decrease intracellular ATP and activate KATP channels, increased whole cell K+ current. On the other hand, K+ current induced by low intracellular ATP was inhibited by extracellular glibenclamide (30 µM), an oral antidiabetic known to block the opening of KATP channels. CONCLUSIONS In conclusion, mammalian oocytes express KATP channels. This opens a new avenue of research into the complex relationship between metabolism and membrane excitability in oocytes under different conditions, including conception. PMID:20847183

  2. A practical implementation and exploitation of ATPS for intensive processing of biological feedstock: A novel approach for heavily biological feedstock loaded ATPS

    Microsoft Academic Search

    Pitchaivelu Selvakumar; Tau Chuan Ling; Simon Walker; Andrew Lyddiatt

    2010-01-01

    Aqueous two-phase systems (ATPS) are sensitive to loading with more than modest quantities (>5%, w\\/w, of system) of biological feedstock due to the contribution of contained macromolecular components (additional to phase-forming chemicals) to the phase diagram in respect of the binodal, tie-line length (TLL) and volume ratio. The present work demonstrates the method in the development and monitoring of ATPS

  3. Definitions of Entomological Terms

    NSDL National Science Digital Library

    0000-00-00

    A list of of morphological definitions and word roots useful to Entomology students and teachers. The list contains concise and easily understandable definitions for a number of morphological and physiological terms and specifies where on the insect these terms apply. A good reference for students in introductory entomology or insect morphology classes. Requires Adobe Acrobat Reader or equivalent software to read .pdf documents.

  4. Cachexia: A new definition

    Microsoft Academic Search

    William J. Evans; John E. Morley; Josep Argiles; Connie Bales; Vickie Baracos; Denis Guttridge; Aminah Jatoi; Kamyar Kalantar-Zadeh; Herbert Lochs; Giovanni Mantovani; Daniel Marks; William E. Mitch; Maurizio Muscaritoli; Armine Najand; Piotr Ponikowski; Filippo Rossi Fanelli; Morrie Schambelan; Annemie Schols; Michael Schuster; David Thomas; Robert Wolfe; Stefan D. Anker

    2008-01-01

    Summary On December 13th and 14th a group of scientists and clinicians met in Washington, DC, for the cachexia consensus conference. At the present time, there is no widely agreed upon operational definition of cachexia. The lack of a definition accepted by clinician and researchers has limited identification and treatment of cachectic patient as well as the development and approval

  5. Dynamics of Situation Definition

    ERIC Educational Resources Information Center

    Park, Dongseop; Moro, Yuji

    2006-01-01

    Situation definition is the process and product of actors' interpretive activities toward a given situation. By reviewing a number of psychological studies conducted in experimental settings, we found that the studies have only explicated a part of the situation definition process and have neglected its dynamic aspects. We need to focus on the…

  6. Pre-steady-state analysis of ATP hydrolysis by Saccharomyces cerevisiae DNA topoisomerase II. 2. Kinetic mechanism for the sequential hydrolysis of two ATP.

    PubMed

    Harkins, T T; Lewis, T J; Lindsley, J E

    1998-05-19

    In the preceding paper, we showed that DNA topoisomerase II from Saccharomyces cerevisiae binds two ATP and rapidly hydrolyzes at least one of them before encountering a slow step in the reaction mechanism. These data are potentially consistent with two different types of reaction pathways: (1) sequential ATP hydrolysis or (2) simultaneous hydrolysis of both ATP. Here, we present results that are consistent only with topoisomerase II hydrolyzing its two bound ATP sequentially. Additionally, these results indicate that the products of the first hydrolysis are released from the enzyme before the second ATP is hydrolyzed. Release of products from both the first and second hydrolyses contributes to the rate-determining process. The proposed mechanism for ATP hydrolysis by topoisomerase II is complex, having nine rate constants. To calculate values for each of these rate constants, a technique of kinetic parameter estimation was developed. This technique involved using singular perturbation theory in order to estimate rate constants, and consequently identify kinetic steps following the rate-determining step. PMID:9585544

  7. Human nongastric H -K -ATPase: transport properties of ATP1al1 assembled with different -subunits

    E-print Network

    Brand, Paul H.

    Human nongastric H -K -ATPase: transport properties of ATP1al1 assembled with different -subunits March 2002 Crambert, Gilles, Jean-Daniel Horisberger, Nikolai N. Modyanov, and Ka¨thi Geering. Human of human nongastric H -K -ATPase, ATP1al1 (AL1), and of the Na -K -ATPase 1-subunit ( 1) expressed

  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. A lipid switch unlocks Parkinson's disease-associated ATP13A2.

    PubMed

    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

    2015-07-21

    ATP13A2 is a lysosomal P-type transport ATPase that has been implicated in Kufor-Rakeb syndrome and Parkinson's disease (PD), providing protection against ?-synuclein, Mn(2+), and Zn(2+) 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 ?-synuclein toxicity or mitochondrial stress in PD or related disorders. PMID:26134396

  10. An increase in the myocardial PCr/ATP ratio in GLUT4 null mice.

    PubMed

    Weiss, Robert G; Chatham, John C; Georgakopolous, Dimitrios; Charron, Maureen J; Wallimann, Theo; Kay, Laurence; Walzel, Bernd; Wang, Yibin; Kass, David A; Gerstenblith, Gary; Chacko, V P

    2002-04-01

    ATP and creatine phosphate (PCr) are prime myocardial high-energy phosphates. Their relative concentrations are conserved among mammalian species and across a range of physiologic cardiac workloads. The cardiac PCr/ATP ratio is decreased with several pathologic conditions, such as ischemia and heart failure, but there are no reports of an increase in the cardiac PCr/ATP ratio in any species or with interventions. We studied the in vivo energetics in transgenic mice lacking expression of the glucose transport protein GLUT4 (G4N) and observed a significant 60% increase in the myocardial PCr/ATP ratio in G4N that was confirmed in three different experimental settings including intact animals. The higher PCr/ATP in G4N is cardiac-specific and is due to higher total cardiac creatine (CR) concentrations in G4N than in wild-type (WT). However, [ATP], [ADP], and -DG(-ATP) did not differ between the strains. Expression of the creatine transport protein (CreaT) that is responsible for creatine uptake in myocytes was preserved in G4N cardiac tissue. These observations demonstrate, for the first time to our knowledge, that G4N manifest a unique increase in the cardiac PCr/ATP ratio, which suggests a novel genetic strategy for increasing myocardial creatine levels. PMID:11919171

  11. ATP, an extracellular signaling molecule in red blood cells: a messenger for malaria?

    PubMed

    Ramdani, Ghania; Langsley, Gordon

    2014-01-01

    Adenosine 5' triphosphate (ATP), discovered in 1929 by Karl Lohmannest, is described as an essential energy source for cells. In the biochemistry of all living organisms, ATP hydrolysis provides the energy required for the chemical reactions of metabolism. It is the precursor of a number of essential enzyme cofactors, such as nicotinamide adenine dinucleotide (NAD + ) and coenzyme A [NAD + , flavin adenine dinucleotide (FAD), and is ATP coenzyme A are all formed from ATP] and is the source of the phosphoryl group in most kinase-mediated phosphorylation reactions. Another essential, but less known function is that ATP plays a very important role as an extracellular signaling molecule, allowing cells and tissues to communicate. ATP is converted into cAMP, a major second messenger involved in many cellular processes, by adenylyl cyclase, a membrane-associated enzyme. In this review, we describe the role of ATP as a beneficial extracellular molecule released by healthy red blood cells (RBCs) in response to hypoxia to mediate a vasodilator signal, by oxidatively stressed RBCs, and by Plasmodium falciparum-infected RBCs (iRBCs), and its similarity with released ATP that by the combined action of the ectonucleotidases CD39 and CD73 is converted to adenosine that mediates sickling in sickle cell disease (SCD). PMID:25179714

  12. 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…

  13. 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.

  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. ATP-evoked Increases in Intracellular Calcium in Neurons and Glia from the Dorsal Spinal Cord

    Microsoft Academic Search

    Mw Salter; Janice L. Hicks

    1994-01-01

    ATP has been proposed as a possible chemical mediator of synaptic transmission in the spinal dorsal horn on the basis that it is released in dorsal horn synaptosomes in a Ca2+- dependent manner and that its effects mimic those of syn- aptic inputs to dorsal horn neurons. In the present study we examined the actions of ATP on neurons and

  16. Effects of pyridoxine-pyrrolidon-carboxylate on hepatic and cerebral ATP levels in ethanol treated rats.

    PubMed

    Felicioli, R; Saracchi, I; Flagiello, A M; Bartoli, C

    1980-06-01

    ATP levels were measured in the liver and brain of rats acutely intoxicated with ethanol. The pretreatment of the animals with pyridoxine-pyrrolidon carboxylate (Metadoxine) prevented the marked fall in ATP concentration caused by ethanol in both organs. PMID:7192694

  17. Molecular, Functional, and Pathological Aspects of the Mitochondrial ADP/ATP Carrier

    NSDL National Science Digital Library

    C. Dahout-Gonzalez (UMR 5092 CEA-CNRS-Université Joseph Fourier Département de Réponse et Dynamique Cellulaires)

    2006-08-01

    In providing the cell with ATP generated by oxidative phosphorylation, the mitochondrial ADP/ATP carrier plays a central role in aerobic eukaryotic cells. Combining biochemical, genetic, and structural approaches contributes to understanding the molecular mechanism of this essential transport system, the dysfunction of which is implicated in neuromuscular diseases.

  18. Visualizing Arp2/3 complex activation mediated by binding of ATP and WASp using

    E-print Network

    Visualizing Arp2/3 complex activation mediated by binding of ATP and WASp using structural mass: ATP and WASp-family proteins. However, the mechanism of activation remains largely hypothetical. We of nucleotide- and WASp-binding on Arp2/3. These results represent two significant advances in such footprinting

  19. Significance of ATP, carbon, and caloric content of meiobenthic nematodes in partitioning benthic biomass

    Microsoft Academic Search

    J. P. Sikora; W. B. Sikora; C. W. Erkenbrecher; B. C. Coull

    1977-01-01

    Benthic, free-living marine nematodes from two stations, one subtidal, one intertidal, in the North Inlet Estuary, South Carolina, USA, have been characterized by measurement of adenosine triphosphate (ATP), carbon, caloric content, and ash-free dry weight. Two methods of extracting the organisms from the sediment were used. Resulting carbon to ATP ratios and population density data from ongoing North Inlet meiofauna

  20. Pannexin 1 is the conduit for low oxygen tension-induced ATP release from human erythrocytes

    PubMed Central

    Adderley, Shaquria P.; Bowles, Elizabeth A.; Egan, Terrance M.; Stephenson, Alan H.; Ellsworth, Mary L.; Sprague, Randy S.

    2010-01-01

    Erythrocytes release ATP in response to exposure to the physiological stimulus of lowered oxygen (O2) tension as well as pharmacological activation of the prostacyclin receptor (IPR). ATP release in response to these stimuli requires activation of adenylyl cyclase, accumulation of cAMP, and activation of protein kinase A. The mechanism by which ATP, a highly charged anion, exits the erythrocyte in response to lowered O2 tension or receptor-mediated IPR activation by iloprost is unknown. It was demonstrated previously that inhibiting pannexin 1 with carbenoxolone inhibits hypotonically induced ATP release from human erythrocytes. Here we demonstrate that three structurally dissimilar compounds known to inhibit pannexin 1 prevent ATP release in response to lowered O2 tension but not to iloprost-induced ATP release. These results suggest that pannexin 1 is the conduit for ATP release from erythrocytes in response to lowered O2 tension. However, the identity of the conduit for iloprost-induced ATP release remains unknown. PMID:20622111

  1. Neuromodulator role of zinc and copper during prolonged ATP applications to P2X 4 purinoceptors

    Microsoft Academic Search

    Claudio Coddou; Bernardo Morales; J. Pablo Huidobro-Toro

    2003-01-01

    To further elucidate the modulator role of trace metals such as zinc and copper on the activity of nucleotide purinoceptors, the action of these metals was assessed during prolonged ATP applications to rat P2X4 purinoceptors expressed in Xenopus laevis oocytes. Application of ATP for 3 min resulted in a biphasic effect; a fast transient peak was followed by a slower

  2. Algorithmic TamperProof (ATP) Security: Theoretical Foundations for Security Against

    E-print Network

    Lysyanskaya, Anna

    ­Verlag Berlin Heidelberg 2004 #12; Algorithmic Tamper­Proof (ATP) Security 259 secret information. now#. a closer analysis, the secure hardware assumption encompasses two dif­ ferent components, informally: Read­proofAlgorithmic Tamper­Proof (ATP) Security: Theoretical Foundations for Security Against Hardware

  3. Control of dynamic CFTR selectivity by glutamate and ATP in epithelial cells

    Microsoft Academic Search

    M. M. Reddy; P. M. Quinton

    2003-01-01

    Cystic fibrosis is caused by mutations in cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel. Phosphorylation and ATP hydrolysis are generally believed to be indispensable for activating CFTR. Here we report phosphorylation- and ATP-independent activation of CFTR by cytoplasmic glutamate that exclusively elicits Cl-, but not HCO3-, conductance in the human sweat duct. We also report that the anion

  4. RNA editing of mitochondrial functional genes atp6 and cox2 in maize ( Zea mays L.)

    Microsoft Academic Search

    Jing Wang; Mo Ju Cao; Guang Tang Pan; Yan Li Lu; Ting Zhao Rong

    2009-01-01

    RNA editing of two mitochondrial or organs genes, atp6 and cox2, in different tissues were analyzed using homonucleic but alloplasmic, and homoplasmic but heteronucleic maize (zea mays L.) as experimental materials. A total of 18 and 26 editing sites for atp6 conservative region transcript were identified by direct and clone sequencing, respectively. By direct sequencing 23 and 22 editing sites

  5. 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…

  6. Rows of ATP Synthase Dimers in Native Mitochondrial Inner Membranes Nikolay Buzhynskyy,* Pierre Sens,y

    E-print Network

    Sens, Pierre

    organization hasbeen elucidated.However, the supramolecular assembly of ATP synthases in biological membranes) and complexed with F1 (11). A large body of indirect evidence has accumulated showing that the ATP synthase forms dimers and oligomers crucial for mitochondria morphology, structure, and function (12­15).In

  7. A nuclear encoded chloroplast ATP synthase mutant of Zea mays L

    Microsoft Academic Search

    Craig S. Echt; Mary L. Polacco; Myron G. Neuffer

    1987-01-01

    Numerous Escherichia coli mutants have been used to determine the genetics and sequence of assembly of the prokaryotic proton-translocating ATP synthase complex. Similar studies with the analogous chloroplast ATP synthase in higher plants have not been possible due to lack of suitable mutants. We describe here a preliminary characterization of cfr, a nuclear mutation in Zea mays L. that appears

  8. The ion channel of F-ATP synthase is the target of toxic organotin compounds

    PubMed Central

    von Ballmoos, Christoph; Brunner, Josef; Dimroth, Peter

    2004-01-01

    ATP is the universal energy currency of living cells, and the majority of it is synthesized by the F1F0 ATP synthase. Inhibitors of this enzyme are therefore potentially detrimental for all life forms. Tributyltin chloride (TBT-Cl) inhibits ATP hydrolysis by the Na+-translocating ATP synthase of Ilyobacter tartaricus or the H+-translocating counterpart of Escherichia coli with apparent Ki of 200 nM. To target the site of this inhibition, we synthesized a tritium-labeled derivative of TBT-Cl in which one of the butyl groups was replaced by a photoactivatable aryldiazirine residue. Upon illumination, subunit a of the ATP synthase becomes specifically modified, and this labeling is suppressed in the presence of the original inhibitor. In case of the Na+ ATP synthase, labeling is also suppressed in the presence of Na+ ions, suggesting an interference in Na+ or TBT-Cl binding to subunit a. This interference is corroborated by the protection of ATP hydrolysis from TBT-Cl inhibition by 105 mM Na+. TBT-Cl strongly inhibits Na+ exchange by the reconstituted I. tartaricus ATP synthase. Taken together these results indicate that the subunit a ion channel is the target site for ATPase inhibition by toxic organotin compounds. An inhibitor interacting specifically with this site has not been reported previously. PMID:15277681

  9. The ion channel of F-ATP synthase is the target of toxic organotin compounds.

    PubMed

    von Ballmoos, Christoph; Brunner, Josef; Dimroth, Peter

    2004-08-01

    ATP is the universal energy currency of living cells, and the majority of it is synthesized by the F1F0 ATP synthase. Inhibitors of this enzyme are therefore potentially detrimental for all life forms. Tributyltin chloride (TBT-Cl) inhibits ATP hydrolysis by the Na(+)-translocating ATP synthase of Ilyobacter tartaricus or the H(+)-translocating counterpart of Escherichia coli with apparent Ki of 200 nM. To target the site of this inhibition, we synthesized a tritium-labeled derivative of TBT-Cl in which one of the butyl groups was replaced by a photoactivatable aryldiazirine residue. Upon illumination, subunit a of the ATP synthase becomes specifically modified, and this labeling is suppressed in the presence of the original inhibitor. In case of the Na+ ATP synthase, labeling is also suppressed in the presence of Na+ ions, suggesting an interference in Na+ or TBT-Cl binding to subunit a. This interference is corroborated by the protection of ATP hydrolysis from TBT-Cl inhibition by 105 mM Na+. TBT-Cl strongly inhibits Na+ exchange by the reconstituted I. tartaricus ATP synthase. Taken together these results indicate that the subunit a ion channel is the target site for ATPase inhibition by toxic organotin compounds. An inhibitor interacting specifically with this site has not been reported previously. PMID:15277681

  10. Functional asymmetry of the F(0) motor in bacterial ATP synthases.

    PubMed

    Wiedenmann, Alexander; Dimroth, Peter; von Ballmoos, Christoph

    2009-04-01

    F(1)F(0) ATP synthases use the electrochemical potential of H(+) or Na(+) across biological membranes to synthesize ATP by a rotary mechanism. In bacteria, the enzymes can act in reverse as ATP-driven ion pumps creating the indispensable membrane potential. Here, we demonstrate that the F(0) parts of a Na(+)- and H(+)-dependent enzyme display major asymmetries with respect to their mode of operation, reflected by the requirement of approximately 100 times higher Na(+) or H(+) concentrations for the synthesis compared with the hydrolysis of ATP. A similar asymmetry is observed during ion transport through isolated F(0) parts, indicating different affinities for the binding sites in the a/c interface. Together with further data, we propose a model that provides a rationale for a differential usage of membrane potential and ion gradient during ATP synthesis as observed experimentally. The functional asymmetry might also reflect an important property of the ATP synthesis mechanism in vivo. In Escherichia coli, we observed respiratory chain-driven ATP production at pH 7-8, while P-site pH values < 6.5 were required for ATP synthesis in vitro. This discrepancy is discussed with respect to the hypothesis that during respiration lateral proton diffusion could lead to significant acidification at the membrane surface. PMID:19317834

  11. Reverse engineering a protein: the mechanochemistry of ATP synthase George Oster *, Hongyun Wang

    E-print Network

    Oster, George

    Review Reverse engineering a protein: the mechanochemistry of ATP synthase George Oster *, Hongyun direction, but it employs a transmembrane proton motive force. Each motor can be reversed: The Fo motor can drive the F1 motor in reverse to synthesize ATP, and the F1 motor can drive the Fo motor in reverse

  12. ATP-dependent Translocation of Proteins along Single-stranded DNA: Models and Methods of Analysis

    E-print Network

    Lohman, Timothy M.

    size for translocation through global non-linear least-squares fitting of the full time-courses. q 2004 time-course for ATP hydrolysis by PcrA exhibited a burst phase, indicating several turnovers of ATP per enzyme, followed by a slower linear phase. The amplitude of the burst phase increased with increasing ss

  13. ATP-Dependent Silver Transport across the Basolateral Membrane of Rainbow Trout Gills

    E-print Network

    Grosell, Martin

    ATP-Dependent Silver Transport across the Basolateral Membrane of Rainbow Trout Gills N. R. Bury Received February 2, 1999; accepted May 19, 1999 ATP-Dependent Silver Transport across the Basolateral Mem. Appl. Pharmacol. 159, 1­8. Silver has been shown to be extremely toxic to freshwater teleosts, acting

  14. Alternative translational initiation of ATP sulfurylase underlying dual localization of sulfate assimilation pathways in plastids and cytosol in Arabidopsis thaliana.

    PubMed

    Bohrer, Anne-Sophie; Yoshimoto, Naoko; Sekiguchi, Ai; Rykulski, Nicholas; Saito, Kazuki; Takahashi, Hideki

    2014-01-01

    Plants assimilate inorganic sulfate into sulfur-containing vital metabolites. ATP sulfurylase (ATPS) is the enzyme catalyzing the key entry step of the sulfate assimilation pathway in both plastids and cytosol in plants. Arabidopsis thaliana has four ATPS genes (ATPS1, -2, -3, and -4) encoding ATPS pre-proteins containing N-terminal transit peptide sequences for plastid targeting, however, the genetic identity of the cytosolic ATPS has remained unverified. Here we show that Arabidopsis ATPS2 dually encodes plastidic and cytosolic ATPS isoforms, differentiating their subcellular localizations by initiating translation at AUG(Met1) to produce plastid-targeted ATPS2 pre-proteins or at AUG(Met52) or AUG(Met58) within the transit peptide to have ATPS2 stay in cytosol. Translational initiation of ATPS2 at AUG(Met52) or AUG(Met58) was verified by expressing a tandem-fused synthetic gene, ATPS2 (5'UTR-His12) :Renilla luciferase:ATPS2 (Ile13-Val77) :firefly luciferase, under a single constitutively active CaMV 35S promoter in Arabidopsis protoplasts and examining the activities of two different luciferases translated in-frame with split N-terminal portions of ATPS2. Introducing missense mutations at AUG(Met52) and AUG(Met58) significantly reduced the firefly luciferase activity, while AUG(Met52) was a relatively preferred site for the alternative translational initiation. The activity of luciferase fusion protein starting at AUG(Met52) or AUG(Met58) was not modulated by changes in sulfate conditions. The dual localizations of ATPS2 in plastids and cytosol were further evidenced by expression of ATPS2-GFP fusion proteins in Arabidopsis protoplasts and transgenic lines, while they were also under control of tissue-specific ATPS2 promoter activity found predominantly in leaf epidermal cells, guard cells, vascular tissues and roots. PMID:25601874

  15. Evaluation of intramitochondrial ATP levels identifies G0/G1 switch gene 2 as a positive regulator of oxidative phosphorylation.

    PubMed

    Kioka, Hidetaka; Kato, Hisakazu; Fujikawa, Makoto; Tsukamoto, Osamu; Suzuki, Toshiharu; Imamura, Hiromi; Nakano, Atsushi; Higo, Shuichiro; Yamazaki, Satoru; Matsuzaki, Takashi; Takafuji, Kazuaki; Asanuma, Hiroshi; Asakura, Masanori; Minamino, Tetsuo; Shintani, Yasunori; Yoshida, Masasuke; Noji, Hiroyuki; Kitakaze, Masafumi; Komuro, Issei; Asano, Yoshihiro; Takashima, Seiji

    2014-01-01

    The oxidative phosphorylation (OXPHOS) system generates most of the ATP in respiring cells. ATP-depleting conditions, such as hypoxia, trigger responses that promote ATP production. However, how OXPHOS is regulated during hypoxia has yet to be elucidated. In this study, selective measurement of intramitochondrial ATP levels identified the hypoxia-inducible protein G0/G1 switch gene 2 (G0s2) as a positive regulator of OXPHOS. A mitochondria-targeted, FRET-based ATP biosensor enabled us to assess OXPHOS activity in living cells. Mitochondria-targeted, FRET-based ATP biosensor and ATP production assay in a semiintact cell system revealed that G0s2 increases mitochondrial ATP production. The expression of G0s2 was rapidly and transiently induced by hypoxic stimuli, and G0s2 interacts with OXPHOS complex V (FoF1-ATP synthase). Furthermore, physiological enhancement of G0s2 expression prevented cells from ATP depletion and induced a cellular tolerance for hypoxic stress. These results show that G0s2 positively regulates OXPHOS activity by interacting with FoF1-ATP synthase, which causes an increase in ATP production in response to hypoxic stress and protects cells from a critical energy crisis. These findings contribute to the understanding of a unique stress response to energy depletion. Additionally, this study shows the importance of assessing intramitochondrial ATP levels to evaluate OXPHOS activity in living cells. PMID:24344269

  16. Evaluation of intramitochondrial ATP levels identifies G0/G1 switch gene 2 as a positive regulator of oxidative phosphorylation

    PubMed Central

    Kioka, Hidetaka; Kato, Hisakazu; Fujikawa, Makoto; Tsukamoto, Osamu; Suzuki, Toshiharu; Imamura, Hiromi; Nakano, Atsushi; Higo, Shuichiro; Yamazaki, Satoru; Matsuzaki, Takashi; Takafuji, Kazuaki; Asanuma, Hiroshi; Asakura, Masanori; Minamino, Tetsuo; Shintani, Yasunori; Yoshida, Masasuke; Noji, Hiroyuki; Kitakaze, Masafumi; Komuro, Issei; Asano, Yoshihiro; Takashima, Seiji

    2014-01-01

    The oxidative phosphorylation (OXPHOS) system generates most of the ATP in respiring cells. ATP-depleting conditions, such as hypoxia, trigger responses that promote ATP production. However, how OXPHOS is regulated during hypoxia has yet to be elucidated. In this study, selective measurement of intramitochondrial ATP levels identified the hypoxia-inducible protein G0/G1 switch gene 2 (G0s2) as a positive regulator of OXPHOS. A mitochondria-targeted, FRET-based ATP biosensor enabled us to assess OXPHOS activity in living cells. Mitochondria-targeted, FRET-based ATP biosensor and ATP production assay in a semiintact cell system revealed that G0s2 increases mitochondrial ATP production. The expression of G0s2 was rapidly and transiently induced by hypoxic stimuli, and G0s2 interacts with OXPHOS complex V (FoF1-ATP synthase). Furthermore, physiological enhancement of G0s2 expression prevented cells from ATP depletion and induced a cellular tolerance for hypoxic stress. These results show that G0s2 positively regulates OXPHOS activity by interacting with FoF1-ATP synthase, which causes an increase in ATP production in response to hypoxic stress and protects cells from a critical energy crisis. These findings contribute to the understanding of a unique stress response to energy depletion. Additionally, this study shows the importance of assessing intramitochondrial ATP levels to evaluate OXPHOS activity in living cells. PMID:24344269

  17. 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

  18. Conformational Motion of the ABC Transporter MsbA Induced by ATP Hydrolysis

    PubMed Central

    Borbat, Peter P; Surendhran, Kavitha; Bortolus, Marco; Zou, Ping; Freed, Jack H; Mchaourab, Hassane S

    2007-01-01

    We measured the amplitude of conformational motion in the ATP-binding cassette (ABC) transporter MsbA upon lipopolysaccharide (LPS) binding and following ATP turnover by pulse double electron-electron resonance and fluorescence homotransfer. The distance constraints from both methods reveal large-scale movement of opposite signs in the periplasmic and cytoplasmic part of the transporter upon ATP hydrolysis. LPS induces distinct structural changes that are inhibited by trapping of the transporter in an ATP post-hydrolysis intermediate. The formation of this intermediate involves a 33-Å distance change between the two ABCs, which is consistent with a dimerization-dissociation cycle during transport that leads to their substantial separation in the absence of nucleotides. Our results suggest that ATP-powered transport entails LPS sequestering into the open cytoplasmic chamber prior to its translocation by alternating access of the chamber, made possible by 10–20-Å conformational changes. PMID:17927448

  19. Involvement of ATP synthase ? subunit in chikungunya virus entry into insect cells.

    PubMed

    Fongsaran, Chanida; Jirakanwisal, Krit; Kuadkitkan, Atichat; Wikan, Nitwara; Wintachai, Phitchayapak; Thepparit, Chutima; Ubol, Sukathida; Phaonakrop, Narumon; Roytrakul, Sittiruk; Smith, Duncan R

    2014-12-01

    Chikungunya virus (CHIKV), the virus responsible for the disease chikungunya fever in humans, is transmitted by Aedes mosquitoes. While significant progress has been made in understanding the process by which CHIKV enters into mammalian cells, far less progress has been made in understanding the CHIKV entry process in insect cells. This study sought to identify mosquito-cell-expressed CHIKV-binding proteins through a combination of virus overlay protein binding assays (VOPBA) and mass spectroscopy. A 50-kDa CHIKV-binding protein was identified as the ATP synthase ? subunit (ATPS?). Co-immunoprecipitation studies confirmed the interaction, and colocalization analysis showed cell-surface and intracellular co-localization between CHIKV and ATPS?. Both antibody inhibition and siRNA-mediated downregulation experiments targeted to ATPS? showed a significant reduction in viral entry and virus production. These results suggest that ATPS? is a CHIKV-binding protein capable of mediating the entry of CHIKV into insect cells. PMID:25168043

  20. 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

  1. 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

  2. Prostacyclin receptor-mediated ATP release from erythrocytes requires the voltage-dependent anion channel

    PubMed Central

    Bowles, Elizabeth A.; Richards, Jennifer P.; Krantic, Medina; Davis, Katie L.; Dietrich, Kristine A.; Stephenson, Alan H.; Ellsworth, Mary L.; Sprague, Randy S.

    2012-01-01

    Erythrocytes have been implicated as controllers of vascular caliber by virtue of their ability to release the vasodilator ATP in response to local physiological and pharmacological stimuli. The regulated release of ATP from erythrocytes requires activation of a signaling pathway involving G proteins (Gi or Gs), adenylyl cyclase, protein kinase A, and the cystic fibrosis transmembrane conductance regulator as well as a final conduit through which this highly charged anion exits the cell. Although pannexin 1 has been shown to be the final conduit for ATP release from human erythrocytes in response to reduced oxygen tension, it does not participate in transport of ATP following stimulation of the prostacyclin (IP) receptor in these cells, which suggests that an additional protein must be involved. Using antibodies directed against voltage-dependent anion channel (VDAC)1, we confirm that this protein is present in human erythrocyte membranes. To address the role of VDAC in ATP release, two structurally dissimilar VDAC inhibitors, Bcl-xL BH44–23 and TRO19622, were used. In response to the IP receptor agonists, iloprost and UT-15C, ATP release was inhibited by both VDAC inhibitors although neither iloprost-induced cAMP accumulation nor total intracellular ATP concentration were altered. Together, these findings support the hypothesis that VDAC is the ATP conduit in the IP receptor-mediated signaling pathway in human erythrocytes. In addition, neither the pannexin inhibitor carbenoxolone nor Bcl-xL BH44–23 attenuated ATP release in response to incubation of erythrocytes with the ?-adrenergic receptor agonist isoproterenol, suggesting the presence of yet another channel for ATP release from human erythrocytes. PMID:22159995

  3. Intracellular ATP does not inhibit Slo2.1 K+ channels

    PubMed Central

    Garg, Priyanka; Sanguinetti, Michael C.

    2014-01-01

    Abstract Under normal physiological conditions, the open probability of Slo2.1 K+ channels is low. Elevation of cytosolic [Na+] and [Cl?] caused by ischemia or rapid electrical pacing of cells increases the open probability of Slo2.1 channels and the resulting outward current can stabilize the resting state of cells. Initial characterization of heterologously expressed human Slo2.1 indicated that these channels were inhibited by physiological levels of intracellular ATP. However, a subsequent study found that intracellular ATP had no effect on Slo2.1 channels. Here, we re?examine the effects of intracellular ATP on cloned human Slo2.1 channels heterologously expressed in Xenopus oocytes. Our studies provide both direct and indirect evidence that changes in intracellular [ATP] have no effect on Slo2.1 channels. First, we directly examined the effects of intracellular ATP on Slo2.1 channel activity in excised inside?out macropatches from Xenopus oocytes. Application of 5 mmol/L ATP to the intracellular solution did not inhibit Slo2.1 currents activated by niflumic acid. Second, we lowered the [ATP]i in whole oocytes using the metabolic inhibitor NaN3. Depletion of [ATP]i in oocytes by 3 mmol/L NaN3 rapidly activated heterologously expressed KATP channels, but did not increase wild?type Slo2.1 channel currents activated by niflumic acid or currents conducted by constitutively active mutant (E275D) Slo2.1 channels. Third, mutation of a conserved residue in the ATP binding consensus site in the C?terminal domain of the channel did not enhance the magnitude of Slo2.1 current as expected if binding to this site inhibited channel function. We conclude that Slo2.1 channels are not inhibited by intracellular ATP. PMID:25214519

  4. Role of luminal ATP in regulating electrogenic Na(+) absorption in guinea pig distal colon.

    PubMed

    Yamamoto, Takeshi; Suzuki, Yuichi

    2002-08-01

    Extracellular ATP regulates a variety of functions in epithelial tissues by activating the membrane P2-receptor. The purpose of this study was to investigate the autocrine/paracrine regulation by luminal ATP of electrogenic amiloride-sensitive Na(+) absorption in the distal colon from guinea pigs treated with aldosterone by measuring the amiloride-sensitive short-circuit current (I(sc)) and (22)Na(+) flux in vitro with the Ussing chamber technique. ATP added to the luminal side inhibited the amiloride-sensitive I(sc) and (22)Na(+) absorption to a similar degree. The concentration dependence of the inhibitory effect of ATP on amiloride-sensitive I(sc) had an IC(50) value of 20-30 microM, with the maximum inhibition being approximately 50%. The effects of different nucleotides and of a nucleoside were also studied, the order of potency being ATP = UTP > ADP > adenosine. The effects of ATP were slightly, but significantly, reduced in the presence of suramin in the luminal solution. The inhibitory effect of luminal ATP was more potent in the absence of both Mg2+ and Ca2+ from the luminal solution. Pretreatment of the tissue with ionomycin or thapsigargin in the absence of serosal Ca2+ did not affect the percent inhibition of amiloride-sensitive I(sc) induced by ATP. Mechanical perturbation with a hypotonic luminal solution caused a reduction in amiloride-sensitive I(sc), this effect being prevented by the presence of hexokinase, an ATP-scavenging enzyme. These results suggest that ATP released into the luminal side by hypotonic stimulation could exert an inhibitory effect on the electrogenic Na(+) absorption. This effect was probably mediated by a P2Y(2) receptor on the apical membrane of colonic epithelial cells, and a change in the intracellular Ca2+ concentration may not be necessary for this process. PMID:12121876

  5. The F0F1-ATP Synthase Complex Contains Novel Subunits and Is Essential for Procyclic Trypanosoma

    E-print Network

    Schnaufer, Achim

    sensitive to the ATP synthase inhibitor oligomycin even in the presence of glucose contrary to earlier-ATP synthase (respiratory complex V). It has a complete respiratory chain that oxidizes the reduced

  6. Inhibition of ATP hydrolysis by thermoalkaliphilic F1Fo-ATP synthase is controlled by the C terminus of the epsilon subunit.

    PubMed

    Keis, Stefanie; Stocker, Achim; Dimroth, Peter; Cook, Gregory M

    2006-06-01

    The F(1)F(o)-ATP synthases of alkaliphilic bacteria exhibit latent ATPase activity, and for the thermoalkaliphile Bacillus sp. strain TA2.A1, this activity is intrinsic to the F(1) moiety. To study the mechanism of ATPase inhibition, we developed a heterologous expression system in Escherichia coli to produce TA2F(1) complexes from this thermoalkaliphile. Like the native F(1)F(o)-ATP synthase, the recombinant TA2F(1) was blocked in ATP hydrolysis activity, and this activity was stimulated by the detergent lauryldimethylamine oxide. To determine if the C-terminal domain of the epsilon subunit acts as an inhibitor of ATPase activity and if an electrostatic interaction plays a role, a TA2F(1) mutant with either a truncated epsilon subunit [i.e., TA2F(1)(epsilon(DeltaC))] or substitution of basic residues in the second alpha-helix of epsilon with nonpolar alanines [i.e., TA2F(1)(epsilon(6A))] was constructed. Both mutants showed ATP hydrolysis activity at low and high concentrations of ATP. Treatment of the purified F(1)F(o)-ATP synthase and TA2F(1)(epsilon(WT)) complex with proteases revealed that the epsilon subunit was resistant to proteolytic digestion. In contrast, the epsilon subunit of TA2F(1)(epsilon(6A)) was completely degraded by trypsin, indicating that the C-terminal arm was in a conformation where it was no longer protected from proteolytic digestion. In addition, ATPase activity was not further activated by protease treatment when compared to the untreated control, supporting the observation that epsilon was responsible for inhibition of ATPase activity. To study the effect of the alanine substitutions in the epsilon subunit in the entire holoenzyme, we reconstituted recombinant TA2F(1) complexes with F(1)-stripped native membranes of strain TA2.A1. The reconstituted TA2F(o)F(1)(epsilon(WT)) was blocked in ATP hydrolysis and exhibited low levels of ATP-driven proton pumping consistent with the F(1)F(o)-ATP synthase in native membranes. Reconstituted TA2F(o)F(1)(epsilon(6A)) exhibited ATPase activity that correlated with increased ATP-driven proton pumping, confirming that the epsilon subunit also inhibits ATPase activity of TA2F(o)F(1). PMID:16707672

  7. Use of ATP, dATP and their alpha-thio derivatives to study DNA ligase adenylation.

    PubMed Central

    Montecucco, A; Lestingi, M; Pedrali-Noy, G; Spadari, S; Ciarrocchi, G

    1990-01-01

    Bacteriophage-T4 and human type I DNA ligases were found capable of self-adenylating upon exposure to both ribo- and deoxyribo-[alpha-35S]thio-ATP. However, the joining reaction does not take place in the presence of the deoxyribotriphosphates. Enzyme adenylation is reversed in all cases by an excess of PPi, but the rate of reversion is lower with thio derivatives. Therefore thio derivatives can be used to study the adenylation of DNA ligases and to search for specific inhibitors of the first step of the ligation reaction. In addition we show that thio derivatives can be used to detect DNA ligase adenylation activity covalently bound to a solid matrix. Images Fig. 2. Fig. 5. PMID:2222417

  8. ATP stimulates glucose transport through activation of P2 purinergic receptors in C 2C 12 skeletal muscle cells

    Microsoft Academic Search

    Min Suk Kim; Jinhwa Lee; Joohun Ha; Sung Soo Kim; Yoon Kong; Yong Ho Cho; Hyung Hwan Baik; Insug Kang

    2002-01-01

    Extracellular ATP acts as a signal that regulates a variety of cellular processes via binding to P2 purinergic receptors (P2 receptors). We herein investigated the effects and signaling pathways of ATP on glucose uptake in C2C12 skeletal muscle cells. ATP as well as P2 receptor agonists (ATP-? S) stimulated the rate of glucose uptake, while P2 receptor antagonists (suramin) inhibited

  9. ATP6 homoplasmic mutations inhibit and destabilize the human F1F0-ATP synthase without preventing enzyme assembly and oligomerization.

    PubMed

    Cortés-Hernández, Paulina; Vázquez-Memije, Martha E; García, José J

    2007-01-12

    The molecular pathogenic mechanism of the human mitochondrial diseases neurogenic ataxia and retinitis pigmentosa and maternally inherited Leigh syndrome was determined in cultured human cells harboring homoplasmic T8993G/T8993C point mutations in the mitochondrial ATP6 gene, which encodes subunit 6 of the F1F0-ATP synthase. Immunoprecipitation and blue native electrophoresis showed that F1F0-ATP synthase assembles correctly in homoplasmic mutant mitochondria. The mutants exhibited a tendency to have an increased sensitivity to subsaturating amounts of oligomycin; this provided further evidence for complete assembly and tight coupling between the F1 and F0 sectors. Furthermore, human ATP synthase dimers and higher homo-oligomers were observed for the first time, and it was demonstrated that the mutant enzymes retain enough structural integrity to oligomerize. A reproducible increase in the proportion of oligomeric-to-monomeric enzyme was found for the T8993G mutant suggesting that F1F0 oligomerization is regulated in vivo and that it can be modified in pathological conditions. Despite correct assembly, the T8993G mutation produced a 60% inhibition in ATP synthesis turnover. In vitro denaturing conditions showed F1F0 instability conferred by the mutations, although this instability did not produce enzyme disassembly in the conditions used for determination of ATP synthesis. Taken together, the data show that the primary molecular pathogenic mechanism of these deleterious human mitochondrial mutations is functional inhibition in a correctly assembled ATP synthase. Structural instability may play a role in the progression of the disease under potentially denaturing conditions, as discussed. PMID:17121862

  10. ATP Binding to the Motor Domain from an ABC Transporter Drives Formation of a Nucleotide Sandwich Dimer

    Microsoft Academic Search

    Paul C Smith; Nathan Karpowich; Linda Millen; Jonathan E Moody; Jane Rosen; Philip J Thomas; John F Hunt

    2002-01-01

    It has been proposed that the reaction cycle of ATP binding cassette (ABC) transporters is driven by dimerization of their ABC motor domains upon binding ATP at their mutual interface. However, no such ATP sandwich complex has been observed for an ABC from an ABC transporter. In this paper, we report the crystal structure of a stable dimer formed by

  11. RecA as a Motor Protein TESTING MODELS FOR THE ROLE OF ATP HYDROLYSIS IN DNA STRAND EXCHANGE*

    E-print Network

    Cox, Michael M.

    demonstrated by the use of the ATP analog ATP S (which is bound but not appreciably hydrolyzed by RecA) or the RecA mutant protein K72R (which binds but does not hydrolyze ATP) (4­11). In contrast, extension

  12. 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...

  13. Definitions Derived from Neutrosophics

    E-print Network

    Florentin Smarandache

    2003-01-28

    Thirty-three new definitions are presented, derived from neutrosophic set, neutrosophic probability, neutrosophic statistics, and neutrosophic logic. Each one is independent, short, with references and cross references like in a dictionary style.

  14. Definitions Derived from Neutrosophics

    Microsoft Academic Search

    Florentin Smarandache

    2003-01-01

    Thirty-three new definitions are presented, derived from neutrosophic set, neutrosophic probability, neutrosophic statistics, and neutrosophic logic. Each one is independent, short, with references and cross references like in a dictionary style.

  15. Genomic definition of species

    SciTech Connect

    Crkvenjakov, R.; Drmanac, R.

    1991-07-01

    The subject of this paper is the definition of species based on the assumption that genome is the fundamental level for the origin and maintenance of biological diversity. For this view to be logically consistent it is necessary to assume the existence and operation of the new law which we call genome law. For this reason the genome law is included in the explanation of species phenomenon presented here even if its precise formulation and elaboration are left for the future. The intellectual underpinnings of this definition can be traced to Goldschmidt. We wish to explore some philosophical aspects of the definition of species in terms of the genome. The point of proposing the definition on these grounds is that any real advance in evolutionary theory has to be correct in both its philosophy and its science.

  16. Mutations in the ATP13A2 Gene and Parkinsonism: A Preliminary Review

    PubMed Central

    Yang, Xinglong; Xu, Yanming

    2014-01-01

    Parkinson's disease (PD) is a major neurodegenerative disorder for which the etiology and pathogenesis remain as elusive as for Alzheimer's disease. PD appears to be caused by genetic and environmental factors, and pedigree and cohort studies have identified numerous susceptibility genes and loci related to PD. Autosomal recessive mutations in the genes Parkin, Pink1, DJ-1, ATP13A2, PLA2G6, and FBXO7 have been linked to PD susceptibility. Such mutations in ATP13A2, also named PARK9, were first identified in 2006 in a Chilean family and are associated with a juvenile-onset, levodopa-responsive type of Parkinsonism called Kufor-Rakeb syndrome (KRS). KRS involves pyramidal degeneration, supranuclear palsy, and cognitive impairment. Here we review current knowledge about the ATP13A2 gene, clinical characteristics of patients with PD-associated ATP13A2 mutations, and models of how the ATP13A2 protein may help prevent neurodegeneration by inhibiting ?-synuclein aggregation and supporting normal lysosomal and mitochondrial function. We also discuss another ATP13A2 mutation that is associated with the family of neurodegenerative disorders called neuronal ceroid lipofuscinoses (NCLs), and we propose a single pathway whereby ATP13A2 mutations may contribute to NCLs and Parkinsonism. Finally, we highlight how studies of mutations in this gene may provide new insights into PD pathogenesis and identify potential therapeutic targets. PMID:25197640

  17. Characterization of a Thermophilic ATP-Dependent DNA Ligase from the Euryarchaeon Pyrococcus horikoshii

    PubMed Central

    Keppetipola, Niroshika; Shuman, Stewart

    2005-01-01

    Archaea encode a DNA ligase composed of a C-terminal catalytic domain typical of ATP-dependent ligases plus an N-terminal domain similar to that found in eukaryotic cellular and poxvirus DNA ligases. All archaeal DNA ligases characterized to date have ATP-dependent adenylyltransferase and nick-joining activities. However, recent reports of dual-specificity ATP/NAD+ ligases in two Thermococcus species and Pyrococcus abyssi and an ATP/ADP ligase in Aeropyrum pernix raise the prospect that certain archaeal enzymes might exemplify an undifferentiated ancestral stage in the evolution of ligase substrate specificity. Here we analyze the biochemical properties of Pyrococcus horikoshii DNA ligase. P. horikoshii ligase catalyzes autoadenylylation and nick sealing in the presence of a divalent cation and ATP; it is unable to utilize NAD+ or ADP to promote ligation in lieu of ATP. P. horikoshii ligase is thermophilic in vitro, with optimal adenylyltransferase activity at 90°C and nick-joining activity at 70 to 90°C. P. horikoshii ligase resembles the ligases of Methanobacterium thermautotrophicum and Sulfolobus shibatae in its strict specificity for ATP. PMID:16199559

  18. Dynamic electron microscopy of ATP-induced myosin head movement in living muscle thick?filaments

    PubMed Central

    Sugi, Haruo; Akimoto, Tsuyoshi; Sutoh, Kazuo; Chaen, Shigeru; Oishi, Noboru; Suzuki, Suechika

    1997-01-01

    Although muscle contraction is known to result from movement of the myosin heads on the thick filaments while attached to the thin filaments, the myosin head movement coupled with ATP hydrolysis still remains to be investigated. Using a gas environmental (hydration) chamber, in which biological specimens can be kept in wet state, we succeeded in recording images of living muscle thick filaments with gold position markers attached to the myosin heads. The position of individual myosin heads did not change appreciably with time in the absence of ATP, indicating stability of the myosin head mean position. On application of ATP, the position of individual myosin heads was found to move by ?20 nm along the filament axis, whereas no appreciable movement of the filaments was detected. The ATP-induced myosin head movement was not observed in filaments in which ATPase activity of the myosin heads was eliminated. Application of ADP produced no appreciable myosin head movement. These results show that the ATP-induced myosin head movement takes place in the absence of the thin filaments. Because ATP reacts rapidly with the myosin head (M) to form the complex (M?ADP?Pi) with an average lifetime of >10 s, the observed myosin head movement may be mostly associated with reaction, M + ATP ? M?ADP?Pi. This work will open a new research field to study dynamic structural changes of individual biomolecules, which are kept in a living state in an electron microscope. PMID:9113997

  19. Calcium-Binding Properties of Sarcoplasmic Reticulum As Influenced by ATP, Caffeine, Quinine, and Local Anesthetics

    PubMed Central

    Carvalho, Arselio P.

    1968-01-01

    Calcium retained at binding sites of the sarcoplasmic reticulum membranes isolated from rabbit skeletal muscle requires 10-5 - 10-4 M ATP to exchange with 45Ca added to the medium. The ATP requirement for Ca exchangeability was observed with respect to the "intrinsic" Ca of the reticulum membranes and the fraction of Ca that is "actively" bound in the presence of ATP. Furthermore, a concentration of free Ca in the medium higher than 10-8 M is required for ATP to promote Ca exchangeability. This exchangeability is not influenced by caffeine, quinine, procaine, and tetracaine, and Ca that is either nonexchangeable (in the absence of ATP) or exchangeable (in the presence of ATP) is released by 1–5 mM quinine or tetracaine, but neither caffeine (6 mM) nor procaine (2–5 mM) has this effect. Quinine or tetracaine also releases Ca and Mg bound passively to the reticulum membranes. A possible role of ATP in maintaining the integrity of cellular membranes is discussed, and the effects of caffeine, quinine, and of local anesthetics on the binding of Ca by the isolated reticulum are related to the effects of these agents on 45Ca fluxes and on the twitch output observed in whole muscles. PMID:5682486

  20. ATP release and autocrine signaling through P2X4 receptors regulate ?? T cell activation

    PubMed Central

    Manohar, Monali; Hirsh, Mark I.; Chen, Yu; Woehrle, Tobias; Karande, Anjali A.; Junger, Wolfgang G.

    2012-01-01

    Purinergic signaling plays a key role in a variety of physiological functions, including regulation of immune responses. Conventional ?? T cells release ATP upon TCR cross-linking; ATP binds to purinergic receptors expressed by these cells and triggers T cell activation in an autocrine and paracrine manner. Here, we studied whether similar purinergic signaling pathways also operate in the “unconventional” ?? T lymphocytes. We observed that ?? T cells purified from peripheral human blood rapidly release ATP upon in vitro stimulation with anti-CD3/CD28-coated beads or IPP. Pretreatment of ?? T cells with 10panx-1, CBX, or Bf A reversed the stimulation-induced increase in extracellular ATP concentration, indicating that panx-1, connexin hemichannels, and vesicular exocytosis contribute to the controlled release of cellular ATP. Blockade of ATP release with 10panx-1 inhibited Ca2+ signaling in response to TCR stimulation. qPCR revealed that ?? T cells predominantly express purinergic receptor subtypes A2a, P2X1, P2X4, P2X7, and P2Y11. We found that pharmacological inhibition of P2X4 receptors with TNP-ATP inhibited transcriptional up-regulation of TNF-? and IFN-? in ?? T cells stimulated with anti-CD3/CD28-coated beads or IPP. Our data thus indicate that purinergic signaling via P2X4 receptors plays an important role in orchestrating the functional response of circulating human ?? T cells. PMID:22753954

  1. ATP decreases mechanical sensitivity of muscle thin-fiber afferents in rats.

    PubMed

    Matsuda, Teru; Kubo, Asako; Taguchi, Toru; Mizumura, Kazue

    2015-08-01

    ATP is an energy rich substance contained in cells in the order of mM. It is released when cells are damaged and when muscle is compressed or contracted. Subcutaneous injection of ATP induces pain-related behavior and hyperalgesia to mechanical and heat stimulation in rats. However, the effects of ATP in muscle have not been fully studied. In the present study we examined the effects of ATP on muscle C-fiber afferent activities using single fiber recordings, and on nociceptive behavior. Muscle C-fiber activities were recorded in vitro using extensor digitorum longus muscle-common peroneal nerve preparations excised from rats deeply anesthetized with pentobarbital. ATP (100?M and 1mM, but not 1?M) superfused for 5min before the mechanical stimulation suppressed the mechanical responses of muscle thin fibers irrespective of whether they excited the fiber. This suppressive effect was reversed by P2X receptor antagonists PPADS (100?M) and suramin (300?M). We also found that subcutaneous injection of ATP (10mM) induced nociceptive behavior, whereas intramuscular injection had no effect. These findings showed that effects of ATP on muscle afferents differ from those on cutaneous afferents. PMID:25862944

  2. Molecular mechanism of ATP binding and ion channel activation in P2X receptors

    SciTech Connect

    Hattori, Motoyuki; Gouaux, Eric (Oregon HSU)

    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.

  3. Involvement of anion channels in mediating elicitor-induced ATP efflux in Salvia miltiorrhiza hairy roots.

    PubMed

    Wu, Shu-Jing; Siu, Ka-Chai; Wu, Jian-Yong

    2011-01-15

    This study examines the roles of anion channels and ATP binding cassette (ABC) protein transporters in mediating elicitor-induced ATP release in Salvia miltiorrhiza hairy root cultures. The elicitor-induced ATP release was effectively blocked by two putative membrane anion channel blockers, niflumic acid and Zn(2+), but not by a specific Cl(-) channel blocker, phenylanthranilic acid. The elicitor-induced ATP release was also significantly suppressed by two ABC inhibitors, glibenclamide and ethacrynic acid. Notable ATP release from the hairy roots was also induced by verapamil (2mM), an ABC activator in animal cells. The verapamil-induced ATP release was effectively blocked by niflumic acid, but only slightly inhibited by the ABC inhibitors. Another notable effect of verapamil was the induction of exocytosis, the secretion of vesicle-like particles to the root surface. The verapamil-induced exocytosis was not inhibited by nifulumic acid and YE did not induce the exocytosis. Overall, the results suggest a significant role of anion channels, a possible involvement of ABC proteins and no significant involvement of exocytosis in mediating the ATP efflux in hairy root cells. PMID:20813428

  4. 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

  5. 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

  6. Tension Force-Induced ATP Promotes Osteogenesis Through P2X7 Receptor in Osteoblasts

    PubMed Central

    Kariya, Taro; Tanabe, Natsuko; Shionome, Chieko; Manaka, Soichiro; Kawato, Takayuki; Zhao, Ning; Maeno, Masao; Suzuki, Naoto; Shimizu, Noriyoshi

    2015-01-01

    Orthodontic tooth movement induces alveolar bone resorption and formation by mechanical stimuli. Force exerted on the traction side promotes bone formation. Adenosine triphosphate (ATP) is one of the key mediators that respond to bone cells by mechanical stimuli. However, the effect of tension force (TF)-induced ATP on osteogenesis is inadequately understood. Accordingly, we investigated the effect of TF on ATP production and osteogenesis in MC3T3-E1 cells. Cells were incubated in the presence or absence of P2X7 receptor antagonist A438079, and then stimulated with or without cyclic TF (6% or 18%) for a maximum of 24?h using Flexercell Strain Unit 3000. TF significantly increased extracellular ATP release compared to control. Six percent TF had maximum effect on ATP release compared to 18% TF and control. Six percent TF induced the expression of Runx2 and Osterix. Six percent TF also increased the expression of extracellular matrix proteins (ECMPs), ALP activity, and the calcium content in ECM. A438079 blocked the stimulatory effect of 6% TF on the expression of Runx2, Osterix and ECMPs, ALP activity, and calcium content in ECM. This study indicated that TF-induced extracellular ATP is released in osteoblasts, suggesting that TF-induced ATP promotes osteogenesis by autocrine action through P2X7 receptor in osteoblasts. J. Cell. Biochem. 116: 12–21, 2015. © 2014 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc. PMID:24905552

  7. 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.

  8. Tension force-induced ATP promotes osteogenesis through P2X7 receptor in osteoblasts.

    PubMed

    Kariya, Taro; Tanabe, Natsuko; Shionome, Chieko; Manaka, Soichiro; Kawato, Takayuki; Zhao, Ning; Maeno, Masao; Suzuki, Naoto; Shimizu, Noriyoshi

    2015-01-01

    Orthodontic tooth movement induces alveolar bone resorption and formation by mechanical stimuli. Force exerted on the traction side promotes bone formation. Adenosine triphosphate (ATP) is one of the key mediators that respond to bone cells by mechanical stimuli. However, the effect of tension force (TF)-induced ATP on osteogenesis is inadequately understood. Accordingly, we investigated the effect of TF on ATP production and osteogenesis in MC3T3-E1 cells. Cells were incubated in the presence or absence of P2X7 receptor antagonist A438079, and then stimulated with or without cyclic TF (6% or 18%) for a maximum of 24?h using Flexercell Strain Unit 3000. TF significantly increased extracellular ATP release compared to control. Six percent TF had maximum effect on ATP release compared to 18% TF and control. Six percent TF induced the expression of Runx2 and Osterix. Six percent TF also increased the expression of extracellular matrix proteins (ECMPs), ALP activity, and the calcium content in ECM. A438079 blocked the stimulatory effect of 6% TF on the expression of Runx2, Osterix and ECMPs, ALP activity, and calcium content in ECM. This study indicated that TF-induced extracellular ATP is released in osteoblasts, suggesting that TF-induced ATP promotes osteogenesis by autocrine action through P2X7 receptor in osteoblasts. PMID:24905552

  9. 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

  10. Genetic dysfunction of MT-ATP6 causes axonal Charcot-Marie-Tooth disease

    PubMed Central

    Pitceathly, Robert D.S.; Murphy, Sinéad M.; Cottenie, Ellen; Chalasani, Annapurna; Sweeney, Mary G.; Woodward, Cathy; Mudanohwo, Ese E.; Hargreaves, Iain; Heales, Simon; Land, John; Holton, Janice L.; Houlden, Henry; Blake, Julian; Champion, Michael; Flinter, Frances; Robb, Stephanie A.; Page, Rupert; Rose, Michael; Palace, Jacqueline; Crowe, Carol; Longman, Cheryl; Lunn, Michael P.; Rahman, Shamima; Reilly, Mary M.

    2012-01-01

    Objective: Charcot-Marie-Tooth (CMT) disease is the most common inherited neuromuscular disorder, affecting 1 in 2,500 individuals. Mitochondrial DNA (mtDNA) mutations are not generally considered within the differential diagnosis of patients with uncomplicated inherited neuropathy, despite the essential requirement of ATP for axonal function. We identified the mtDNA mutation m.9185T>C in MT-ATP6, encoding the ATP6 subunit of the mitochondrial ATP synthase (OXPHOS complex V), at homoplasmic levels in a family with mitochondrial disease in whom a severe motor axonal neuropathy was a striking feature. This led us to hypothesize that mutations in the 2 mtDNA complex V subunit encoding genes, MT-ATP6 and MT-ATP8, might be an unrecognized cause of isolated axonal CMT and distal hereditary motor neuropathy (dHMN). Methods: A total of 442 probands with CMT type 2 (CMT2) (270) and dHMN (172) were screened for MT-ATP6/8 mutations after exclusion of mutations in known CMT2/dHMN genes. Mutation load was quantified using restriction endonuclease analysis. Blue-native gel electrophoresis (BN-PAGE) was performed to analyze the effects of m.9185T>C on complex V structure and function. Results: Three further probands with CMT2 harbored the m.9185T>C mutation. Some relatives had been classified as having dHMN. Patients could be separated into 4 groups according to their mutant m.9185T>C levels. BN-PAGE demonstrated both impaired assembly and reduced activity of the complex V holoenzyme. Conclusions: We have shown that m.9185T>C in MT-ATP6 causes CMT2 in 1.1% of genetically undefined cases. This has important implications for diagnosis and genetic counseling. Recognition that mutations in MT-ATP6 cause CMT2 enhances current understanding of the pathogenic basis of axonal neuropathy. PMID:22933740

  11. Mechanical stress induces release of ATP from Ehrlich ascites tumor cells.

    PubMed

    Pedersen, S; Pedersen, S F; Nilius, B; Lambert, I H; Hoffmann, E K

    1999-01-12

    The supernatant from a suspension of Ehrlich cells exposed to centrifugation at 700xg for 45 s induced a transient increase in the intracellular concentration of free, cytosolic Ca2+, [Ca2+]i, as well as activation of an outwardly rectifying whole-cell current when added to a suspension of non-stimulated cells. These effects were inhibited by suramin, a non-specific P2 receptor antagonist, and mimicked by ATP. Reversed phase HPLC analysis revealed that the supernatant from Ehrlich cells exposed to centrifugation contained 2. 6+/-0.2 microM ATP, and that the mechanical stress-induced release of ATP was inhibited by glibenclamide and verapamil, non-specific inhibitors of the cystic fibrosis transmembrane conductance regulator and P-glycoprotein, respectively. After trypan blue staining, less than 0.5% of the cells were unable to extrude the dye. Addition of extracellular ATP induced a suramin-sensitive, transient, concentration-dependent increase in [Ca2+]i, activation of an outwardly rectifying whole-cell current and a hyperpolarization of the plasma membrane. The ATP-induced hyperpolarization of the plasma membrane was strongly inhibited in the presence of charybdotoxin (ChTX), an inhibitor of several Ca2+-activated K+ channels, suggesting that stimulation of P2 receptors in Ehrlich cells evokes a Ca2+-activated K+ current. The relative potencies of several nucleotides (ATP, UTP, ADP, 2-MeSATP, alpha,beta-MeATP, bzATP) in eliciting an increase in [Ca2+]i, as well as the effect of repetitive addition of nucleotides were investigated. The results lead us to conclude that mechanical stimulation of Ehrlich cells leads to release of ATP, which in turn stimulates both P2Y1 and P2Y2 receptors, resulting in Ca2+ influx as well as release and activation of an outwardly rectifying whole-cell current. PMID:9889382

  12. Crystal structure of the bifunctional ATP sulfurylase-APS kinase from the chemolithotrophic thermophile Aquifex aeolicus.

    PubMed

    Yu, Zhihao; Lansdon, Eric B; Segel, Irwin H; Fisher, Andrew J

    2007-01-19

    The thermophilic chemolithotroph, Aquifex aeolicus, expresses a gene product that exhibits both ATP sulfurylase and adenosine-5'-phosphosulfate (APS) kinase activities. These enzymes are usually segregated on two separate proteins in most bacteria, fungi, and plants. The domain arrangement in the Aquifex enzyme is reminiscent of the fungal ATP sulfurylase, which contains a C-terminal domain that is homologous to APS kinase yet displays no kinase activity. Rather, in the fungal enzyme, the motif serves as a sulfurylase regulatory domain that binds the allosteric effector 3'-phosphoadenosine-5'-phosphosulfate (PAPS), the product of true APS kinase. Therefore, the Aquifex enzyme may represent an ancestral homolog of a primitive bifunctional enzyme, from which the fungal ATP sulfurylase may have evolved. In heterotrophic sulfur-assimilating organisms such as fungi, ATP sulfurylase catalyzes the first committed step in sulfate assimilation to produce APS, which is subsequently metabolized to generate all sulfur-containing biomolecules. In contrast, ATP sulfurylase in sulfur chemolithotrophs catalyzes the reverse reaction to produce ATP and sulfate from APS and pyrophosphate. Here, the 2.3 A resolution X-ray crystal structure of Aquifex ATP sulfurylase-APS kinase bifunctional enzyme is presented. The protein dimerizes through its APS kinase domain and contains ADP bound in all four active sites. Comparison of the Aquifex ATP sulfurylase active site with those from sulfate assimilators reveals similar dispositions of the bound nucleotide and nearby residues. This suggests that minor perturbations are responsible for optimizing the kinetic properties for the physiologically relevant direction. The APS kinase active-site lid adopts two distinct conformations, where one conformation is distorted by crystal contacts. Additionally, a disulfide bond is observed in one ATP-binding P-loop of the APS kinase active site. This linkage accounts for the low kinase activity of the enzyme under oxidizing conditions. The thermal stability of the Aquifex enzyme can be explained by the 43% decreased cavity volume found within the protein core. PMID:17095009

  13. Defining the Role of ATP Hydrolysis in Mitotic Segregation of Bacterial Plasmids

    PubMed Central

    Ah-Seng, Yoan; Rech, Jérôme; Lane, David; Bouet, Jean-Yves

    2013-01-01

    Hydrolysis of ATP by partition ATPases, although considered a key step in the segregation mechanism that assures stable inheritance of plasmids, is intrinsically very weak. The cognate centromere-binding protein (CBP), together with DNA, stimulates the ATPase to hydrolyse ATP and to undertake the relocation that incites plasmid movement, apparently confirming the need for hydrolysis in partition. However, ATP-binding alone changes ATPase conformation and properties, making it difficult to rigorously distinguish the substrate and cofactor roles of ATP in vivo. We had shown that mutation of arginines R36 and R42 in the F plasmid CBP, SopB, reduces stimulation of SopA-catalyzed ATP hydrolysis without changing SopA-SopB affinity, suggesting the role of hydrolysis could be analyzed using SopA with normal conformational responses to ATP. Here, we report that strongly reducing SopB-mediated stimulation of ATP hydrolysis results in only slight destabilization of mini-F, although the instability, as well as an increase in mini-F clustering, is proportional to the ATPase deficit. Unexpectedly, the reduced stimulation also increased the frequency of SopA relocation over the nucleoid. The increase was due to drastic shortening of the period spent by SopA at nucleoid ends; average speed of migration per se was unchanged. Reduced ATP hydrolysis was also associated with pronounced deviations in positioning of mini-F, though time-averaged positions changed only modestly. Thus, by specifically targeting SopB-stimulated ATP hydrolysis our study reveals that even at levels of ATPase which reduce the efficiency of splitting clusters and the constancy of plasmid positioning, SopB still activates SopA mobility and plasmid positioning, and sustains near wild type levels of plasmid stability. PMID:24367270

  14. Assessment of the role in protection and pathogenesis of the Chlamydia muridarum V-type ATP synthase subunit A (AtpA) (TC0582)

    PubMed Central

    Pal, Sukumar; Tifrea, Delia; Sun, Guifeng; Teng, Andy A.; Liang, Xiaowu; Felgner, Philip L.; de la Maza, Luis M.

    2014-01-01

    A novel Chlamydia muridarum antigen (TC0582) was used to vaccinate BALB/c mice. Mice were also immunized with other components of the ATP synthase complex (TC0580, TC0581, and TC0584), or with the major outer membrane protein (MOMP). TC0582 was also formulated in combination with TC0580, TC0581 or MOMP. TC0582 alone, or in combination with the other antigens, elicited strong Chlamydia-specific humoral and cellular immune responses. Vaccinated animals were challenged intranasally and the course of the infection was followed for 10 days. Based on percentage change in body weight, lung weight, and number of Chlamydia inclusion forming units recovered from the lungs, mice immunized with TC0582, TC0581 or MOMP, as single antigens, showed significant protection. Mice immunized with combinations of two antigens were also protected but the level of protection was not additive. TC0582 has sequence homology with the eukaryotic ATP synthase subunit A (AtpA). Therefore, to determine if immunization with TC0582, or with Chlamydia, elicited antibodies that cross-reacted with the mouse AtpA, the two proteins were printed on a microarray. Sera from mice immunized with TC0582 and/or live Chlamydia, strongly reacted with TC0582 but did not recognize the mouse AtpA. In conclusion, TC0582 may be considered as a Chlamydia vaccine candidate. PMID:24161793

  15. The ATP synthase (F1F0) of Streptomyces lividans: sequencing of the atp operon and phylogenetic considerations with subunit beta.

    PubMed

    Hensel, M; Lill, H; Schmid, R; Deckers-Hebestreit, G; Altendorf, K

    1995-01-11

    The DNA encoding the subunits of the ATP synthase (F1F0) of Streptomyces lividans 66 strain 1326 was identified using oligodeoxyribonucleotide probes derived from the N-terminal sequence of subunit gamma of the F1 complex. The complete nucleotide sequence of the operon was determined. The atp operon contains nine genes, atpIBEFHAGDC, encoding the eight structural components of the ATP synthase complex and the i protein, a polypeptide of unknown function. The gene order found is identical to that in other non-photosynthetic eubacteria. The determination of the N-terminal amino acid (aa) sequences of the F1 subunits alpha, beta, gamma, delta and epsilon allowed us to identify the translational start points and to define the primary structures of the proteins. The aa sequence deduced for subunit delta revealed an N-terminal extension of about 90 aa, which is not present in any delta subunit or OSCP (oligomycin sensitivity-conferral protein) of other species studied so far. The phylogenetic relationship of eu- and archaebacteria was investigated using sequencing data of the highly conserved beta subunit of different ATP synthases including that of S. lividans. The calculations revealed that S. lividans beta does not form a phylogenetic group together with the Gram+ taxa of low G+C contents, but is more closely related to the beta subunit of Rhodobacteria. PMID:7828915

  16. Mitochondrial ATP-Mg/Pi carrier SCaMC-3/Slc25a23 counteracts PARP-1-dependent fall in mitochondrial ATP caused by excitotoxic insults in neurons.

    PubMed

    Rueda, Carlos B; Traba, Javier; Amigo, Ignacio; Llorente-Folch, Irene; González-Sánchez, Paloma; Pardo, Beatriz; Esteban, José A; del Arco, Araceli; Satrústegui, Jorgina

    2015-02-25

    Glutamate excitotoxicity is caused by sustained activation of neuronal NMDA receptors causing a large Ca(2+) and Na(+) influx, activation of poly(ADP ribose) polymerase-1 (PARP-1), and delayed Ca(2+) deregulation. Mitochondria undergo early changes in membrane potential during excitotoxicity, but their precise role in these events is still controversial. Using primary cortical neurons derived from mice, we show that NMDA exposure results in a rapid fall in mitochondrial ATP in neurons deficient in SCaMC-3/Slc25a23, a Ca(2+)-regulated mitochondrial ATP-Mg/Pi carrier. This fall is associated with blunted increases in respiration and a delayed decrease in cytosolic ATP levels, which are prevented by PARP-1 inhibitors or by SCaMC-3 activity promoting adenine nucleotide uptake into mitochondria. SCaMC-3 KO neurons show an earlier delayed Ca(2+) deregulation, and SCaMC-3-deficient mitochondria incubated with ADP or ATP-Mg had reduced Ca(2+) retention capacity, suggesting a failure to maintain matrix adenine nucleotides as a cause for premature delayed Ca(2+) deregulation. SCaMC-3 KO neurons have higher vulnerability to in vitro excitotoxicity, and SCaMC-3 KO mice are more susceptible to kainate-induced seizures, showing that early PARP-1-dependent fall in mitochondrial ATP levels, counteracted by SCaMC-3, is an early step in the excitotoxic cascade. PMID:25716855

  17. A quantitative study of bioenergetics in skeletal muscle lacking carbonic anhydrase III using 31P magnetic resonance spectroscopy

    PubMed Central

    Liu, M.; Walter, G. A.; Pathare, N. C.; Forster, R. E.; Vandenborne, K.

    2007-01-01

    Oxidative slow skeletal muscle contains carbonic anhydrase III in high concentration, but its primary function remains unknown. To determine whether its lack handicaps energy metabolism and/or acid elimination, we measured the intracellular pH and energy phosphates by 31P magnetic resonance spectroscopy in hind limb muscles of wild-type and CA III knockout mice during and after ischemia and intense exercise (electrical stimulation). Thirty minutes of ischemia caused phosphocreatine (PCr) to fall and Pi to rise while pH and ATP remained constant in both strains of mice. PCr and Pi kinetics during ischemia and recovery were not significantly different between the two genotypes. From this we conclude that under neutral pH conditions resting muscle anaerobic metabolism, the rate of the creatine kinase reaction, intracellular buffering of protons, and phosphorylation of creatine by mitochondrial oxygen metabolism are not influenced by the lack of CA III. Two minutes of intense stimulation of the mouse gastrocnemius caused PCr, ATP, and pH to fall and ADP and Pi to rise, and these changes, with the exception of ATP, were all significantly larger in the CA III knockouts. The rate of return of pH and ADP to control values was the same in wild-type and mutant mice, but in the mutants PCr and Pi recovery were delayed in the first minute after stimulation. Because the tension decrease during fatigue is known to be the same in the two genotypes, we conclude that a lack of CA III impairs mitochondrial ATP synthesis. PMID:17182736

  18. Higher Dietary Fructose Is Associated with Impaired Hepatic ATP Homeostasis in Obese Individuals with Type 2 Diabetes

    PubMed Central

    Abdelmalek, Manal F.; Lazo, Mariana; Horska, Alena; Bonekamp, Susanne; Lipkin, Edward W.; Balasubramanyam, Ashok; Bantle, John P.; Johnson, Richard J.; Diehl, Anna Mae; Clark, Jeanne M.

    2012-01-01

    Fructose consumption predicts increased hepatic fibrosis in those with nonalcoholic fatty liver disease (NAFLD). Due to its ability to lower hepatic adenosine triphosphate (ATP) levels, habitual fructose consumption could result in more hepatic ATP depletion and impaired ATP recovery. The degree of ATP depletion following an intravenous fructose challenge test in low versus high fructose consumers was assessed. We evaluated diabetic adults enrolled in the Look AHEAD Fatty Liver Ancillary Study (n=244) for whom dietary fructose consumption estimated by a 130-item Food Frequency questionnaire, hepatic ATP measured by phosphorus MRS (31P MRS) and uric acid (UA) levels were performed (n=105). In a subset of participants (n=25), an intravenous fructose challenge was utilized to assess change in hepatic ATP content. The relationships between dietary fructose, UA and hepatic ATP depletion at baseline and following intravenous fructose challenge was evaluated in low (<15 g/d) vs. high (?15 g/d) fructose consumers. High dietary fructose consumers had slightly lower baseline hepatic ATP levels and a greater absolute change in hepatic ?-ATP/Pi ratio (0.08 vs. 0.03, p=0.05) and ?-ATP /Pi ratio following an intravenous fructose challenge (0.03 vs. 0.06, p=0.06). Patients with high UA (?5.5 mg/dl) showed a lower minimum liver ATP/Pi ratio post-fructose challenge (4.5 vs. 7.0, p = 0.04). Conclusions High fructose consumption depletes hepatic ATP and impairs recovery from ATP depletion following an intravenous fructose challenge. Subjects with high UA show a greater nadir in hepatic ATP in response to fructose. Both high dietary fructose intake and elevated UA level may predict more severe hepatic ATP depletion in response to fructose and hence may be risk factors for the development and progression of NAFLD. PMID:22467259

  19. Basal Release of ATP: An Autocrine-Paracrine Mechanism for Cell Regulation

    PubMed Central

    Corriden, Ross; Insel, Paul A.

    2011-01-01

    Cells release adenosine triphosphate (ATP), which activates plasma membrane–localized P2X and P2Y receptors and thereby modulates cellular function in an autocrine or paracrine manner. Release of ATP and the subsequent activation of P2 receptors help establish the basal level of activation (sometimes termed “the set point”) for signal transduction pathways and regulate a wide array of responses that include tissue blood flow, ion transport, cell volume regulation, neuronal signaling, and host-pathogen interactions. Basal release and autocrine or paracrine responses to ATP are multifunctional and evolutionarily conserved, and they provide an economical means for the modulation of cell, tissue, and organismal biology. PMID:20068232

  20. ATP-conducting maxi-anion channel: a new player in stress-sensory transduction.

    PubMed

    Sabirov, Ravshan Z; Okada, Yasunobu

    2004-02-01

    The regulated release of ATP is a fundamental process in cell-to-cell signaling. The electrogenic translocation of ATP via an anion channel has been suggested as one possible mechanism of the release. In this review, we survey possible candidate channels for this pathway. The maxi-anion channel characterized by an exceedingly large unitary conductance has been a stray channel with regard to its function. A newly discovered property, its ATP conductivity and its activation in response to stress signals, indicates that this channel has a central role in stress-sensory transduction for cell volume regulation and tubuloglomerular feedback. PMID:15040843

  1. Concept of Sustained Ordering and an ATP-related Mechanism of Life’s Origin

    PubMed Central

    Galimov, Erik M.

    2009-01-01

    This paper shows that the steady state of a system of conjugated reactions, which are characterized by disproportionation of entropy and proceed in the domain of linear interactions, is an attractor of ordering. Such systems are primed to produce ordering, and life is a specific manifestation of the sustained ordering inherent to the chemistry of carbon. The adenosine triphospate (ATP) molecule has properties which makes ATP hydrolysis to be most appropriate to form such a system in primitive world. Hence, ATP is suggested to play a key role in prebiological evolution. Principles of the origin and evolution of life following from the concept of ordering are stated. PMID:19564936

  2. 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…

  3. ATP-Binding Cassette Efflux Transporters in Human Placenta

    PubMed Central

    Ni, Zhanglin; Mao, Qingcheng

    2010-01-01

    Pregnant women are often complicated with diseases including viral or bacterial infections, epilepsy, hypertension, or pregnancy-induced conditions such as depression and gestational diabetes that require treatment with medication. In addition, substance abuse during pregnancy remains a major public health problem. Many drugs used by pregnant women are off label without the necessary dose, efficacy, and safety data required for rational dosing regimens of these drugs. Thus, a major concern arising from the widespread use of drugs by pregnant women is the transfer of drugs across the placental barrier, leading to potential toxicity to the developing fetus. Knowledge regarding the ATP-binding cassette (ABC) efflux transporters, which play an important role in drug transfer across the placental barrier, is absolutely critical for optimizing the therapeutic strategy to treat the mother while protecting the fetus during pregnancy. Such transporters include P-glycoprotein (P-gp, gene symbol ABCB1), the breast cancer resistance protein (BCRP, gene symbol ABCG2), and the multidrug resistance proteins (MRPs, gene symbol ABCCs). In this review, we summarize the current knowledge with respect to developmental expression and regulation, membrane localization, functional significance, and genetic polymorphisms of these ABC transporters in the placenta and their relevance to fetal drug exposure and toxicity. PMID:21118087

  4. ATP-dependent chromatin remodeling: genetics, genomics and mechanisms

    PubMed Central

    Hargreaves, Diana C; Crabtree, Gerald R

    2011-01-01

    Macromolecular assemblies that regulate chromatin structure using the energy of ATP hydrolysis have critical roles in development, cancer, and stem cell biology. The ATPases of this family are encoded by 27 human genes and are usually associated with several other proteins that are stable, non-exchangeable subunits. One fundamental mechanism used by these complexes is thought to be the movement or exchange of nucleosomes to regulate transcription. However, recent genetic studies indicate that chromatin remodelers may also be involved in regulating other aspects of chromatin structure during many cellular processes. The SWI/SNF family in particular appears to have undergone a substantial change in subunit composition and mechanism coincident with the evolutionary advent of multicellularity and the appearance of linking histones. The differential usage of this greater diversity of mammalian BAF subunits is essential for the development of specific cell fates, including the progression from pluripotency to multipotency to committed neurons. Recent human genetic screens have revealed that BRG1, ARID1A, BAF155, and hSNF5 are frequently mutated in tumors, indicating that BAF complexes also play a critical role in the initiation or progression of cancer. The mechanistic bases underlying the genetic requirements for BAF and other chromatin remodelers in development and cancer are relatively unexplored and will be a focus of this review. PMID:21358755

  5. Structured Analysis for Requirements Definition

    Microsoft Academic Search

    Douglas T. Ross; Kenneth E. Schoman Jr.

    1977-01-01

    Requirements definition encompasses all aspects of system development prior to actual system design. We see the lack of an adequate approach to requirements definition as the source of major difficulties in current systems worlk This paper examines the needs for requirements definition, and proposes meeting those objectives with three interrelated subjects: context analysis, functional specification, and design constraints. Requirements definition

  6. Aqueous Two-Phase System (ATPS) Containing Gemini (12-3-12,2Br-)and SDS 1: Phase Diagram and Properties of ATPS

    SciTech Connect

    Shang, Yazhuo; Liu, Honglai; Hu, Ying; Prausnitz, John M.

    2005-07-21

    Two phases coexist in an aqueous system that contains the two surfactants cationic gemini 12-3-12,2Br- and anionic SDS. An aqueous two-phase system (ATPS) is formed in a narrow region of the ternary phase diagram different from that of traditional aqueous cationic-anionic surfactant systems. In that region, the molar ratio of gemini to SDS varies with the total concentration of surfactants. ATPS not only has higher stability but also has longer phase separation time for the new systems than that of the traditional system. Furthermore, the optical properties of ATPS are different at different total concentrations. All of these experimental observations can be attributed to the unique properties of gemini surfactant and the synergy between the cationic gemini surfactant and the anionic surfactant SDS.

  7. Regulation of Dissimilatory Fe(III) Reduction Activity in Shewanella putrefaciens

    PubMed Central

    Arnold, Robert G.; Hoffmann, Michael R.; DiChristina, Thomas J.; Picardal, Flynn W.

    1990-01-01

    Under anaerobic conditions, Shewanella putrefaciens is capable of respiratory-chain-linked, high-rate dissimilatory iron reduction via both a constitutive and inducible Fe(III)-reducing system. In the presence of low levels of dissolved oxygen, however, iron reduction by this microorganism is extremely slow. Fe(II)-trapping experiments in which Fe(III) and O2 were presented simultaneously to batch cultures of S. putrefaciens indicated that autoxidation of Fe(II) was not responsible for the absence of Fe(III) reduction. Inhibition of cytochrome oxidase with CN? resulted in a high rate of Fe(III) reduction in the presence of dissolved O2, which suggested that respiratory control mechanisms did not involve inhibition of Fe(III) reductase activities or Fe(III) transport by molecular oxygen. Decreasing the intracellular ATP concentrations by using an uncoupler, 2,4-dinitrophenol, did not increase Fe(III) reduction, indicating that the reduction rate was not controlled by the energy status of the cell. Control of electron transport at branch points could account for the observed pattern of respiration in the presence of the competing electron acceptors Fe(III) and O2. PMID:16348289

  8. Subterfuge and Manipulation: Type III

    E-print Network

    Dangl, Jeff

    , Center for Gene Research & Biocomputing, Oregon State University, Corvallis, Oregon 97331; email: jeff for the virulence of Pseudomonas syringae, Xan- thomonas spp., Ralstonia solanacearum and Erwinia species. Type III FUNCTIONS OF TYPE III EFFECTORS IN VIRULENCE . . . . . . . . . . . . . . . . . . . 429 Subterfuge: Type III

  9. Spectrum of mutations in the ATP binding domain of ATP7B gene of Wilson Disease in a regional Indian cohort.

    PubMed

    Guggilla, Sreenivasa Rao; Senagari, Jalandhar Reddy; Rao, P N; Madireddi, Sujatha

    2015-09-10

    Wilson disease is an autosomal recessive disorder of abnormal copper accumulation in the liver, brain, kidney and cornea, resulting in hepatic and neurological abnormalities, which results from impaired ATP7B protein function due to mutations in candidate ATP7B gene, till date more than 500 disease causing mutations were found. In India most disease causing mutations were identified in ATP-BD. DNA samples of the 101 WD cases and 100 control population were analyzed for mutations. 11 mutations were identified in 57 chromosomes. Three novel mutations, c.3310T>A (p.Cys1104Ser), c.3337C>A (p.Leu1113Met) on exon 15 and c.3877G>A (p.Glu1293Lys) on exon 18 were identified for the first time in the ATP7B gene. Two mutations, c.3121C>T (p.Arg1041Trp) and c.3128T>C (p.Leu1043Pro) on exon 14 were discovered for the first time in Indian Wilson disease patients. Four previously reported mutations c.3008C>T, c.3029A>G on exon 13, c.3182G>A on exon 14 and c.3809A>G on exon 18 from South India were also found in this study. Our research has enriched the spectrum of mutations of the ATP7B gene in the south Indian population. The detection of new mutations in the ATP7B gene can aid in genetic counseling and clinical or/prenatal diagnosis. PMID:25982861

  10. 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

  11. WAIS-III and WMS-III performance in chronic Lyme disease.

    PubMed

    Keilp, John G; Corbera, Kathy; Slavov, Iordan; Taylor, Michael J; Sackeim, Harold A; Fallon, Brian A

    2006-01-01

    There is controversy regarding the nature and degree of intellectual and memory deficits in chronic Lyme disease. In this study, 81 participants with rigorously diagnosed chronic Lyme disease were administered the newest revisions of the Wechsler Adult Intelligence Scale (WAIS-III) and Wechsler Memory Scale (WMS-III), and compared to 39 nonpatients. On the WAIS-III, Lyme disease participants had poorer Full Scale and Performance IQ's. At the subtest level, differences were restricted to Information and the Processing Speed subtests. On the WMS-III, Lyme disease participants performed more poorly on Auditory Immediate, Immediate, Auditory Delayed, Auditory Recognition Delayed, and General Memory indices. Among WMS-III subtests, however, differences were restricted to Logical Memory (immediate and delayed) and Family Pictures (delayed only), a Visual Memory subtest. Discriminant analyses suggest deficits in chronic Lyme are best characterized as a combination of memory difficulty and diminished processing speed. Deficits were modest, between one-third and two-thirds of a standard deviation, consistent with earlier studies. Depression severity had a weak relationship to processing speed, but little other association to test performance. Deficits in chronic Lyme disease are consistent with a subtle neuropathological process affecting multiple performance tasks, although further work is needed to definitively rule out nonspecific illness effects. PMID:16433951

  12. Regulation of Cl- channels in normal and cystic fibrosis airway epithelial cells by extracellular ATP.

    PubMed Central

    Stutts, M J; Chinet, T C; Mason, S J; Fullton, J M; Clarke, L L; Boucher, R C

    1992-01-01

    The rate of Cl- secretion by human airway epithelium is determined, in part, by apical cell membrane Cl- conductance. In cystic fibrosis airway epithelia, defective regulation of Cl- conductance decreases the capability to secrete Cl-. Here we report that extracytosolic ATP in the luminal bath of cultured human airway epithelia increased transepithelial Cl- secretion and apical membrane Cl- permeability. Single-channel studies in excised membrane patches revealed that ATP increased the open probability of outward rectifying Cl- channels. The latter effect occurs through a receptor mechanism that requires no identified soluble second messengers and is insensitive to probes of G protein function. These results demonstrate a mode of regulation of anion channels by binding ATP at the extracellular surface. Regulation of Cl- conductance by external ATP is preserved in cystic fibrosis airway epithelia. Images PMID:1371880

  13. Bovine F1Fo ATP synthase monomers bend the lipid bilayer in 2D membrane crystals.

    PubMed

    Jiko, Chimari; Davies, Karen M; Shinzawa-Itoh, Kyoko; Tani, Kazutoshi; Maeda, Shintaro; Mills, Deryck J; Tsukihara, Tomitake; Fujiyoshi, Yoshinori; Kühlbrandt, Werner; Gerle, Christoph

    2015-01-01

    We have used a combination of electron cryo-tomography, subtomogram averaging, and electron crystallographic image processing to analyse the structure of intact bovine F1Fo ATP synthase in 2D membrane crystals. ATPase assays and mass spectrometry analysis of the 2D crystals confirmed that the enzyme complex was complete and active. The structure of the matrix-exposed region was determined at 24 Å resolution by subtomogram averaging and repositioned into the tomographic volume to reveal the crystal packing. F1Fo ATP synthase complexes are inclined by 16° relative to the crystal plane, resulting in a zigzag topology of the membrane and indicating that monomeric bovine heart F1Fo ATP synthase by itself is sufficient to deform lipid bilayers. This local membrane curvature is likely to be instrumental in the formation of ATP synthase dimers and dimer rows, and thus for the shaping of mitochondrial cristae. PMID:25815585

  14. Micromolar HgCl2 concentrations transitorily duplicate the ATP level in Saccharomyces cerevisiae cells.

    PubMed

    Silles, Eduardo; Osorio, Hugo; Maia, Rita; Günther Sillero, María A; Sillero, Antonio

    2005-08-01

    Low concentrations of HgCl2 elicited, in Saccharomyces cerevisiae, a transitory increase in the ATP level followed by a decrease of its concentration, until almost disappearance. At 1 microM HgCl2, the increase in ATP lasted for about 30 min, while at 10 microM the increase was only observed in the first 5 min of treatment. The initial burst of ATP was accompanied by a decrease in the level of hexose phosphates, whereas during the decrease of ATP an increase in the inosine and hexose phosphates levels took place. The treatment with HgCl2 inhibited the plasma membrane proton ATPase but not the activities of hexokinase or 6-phosphofructokinase. PMID:16023109

  15. Kinetic and Stability Properties of Penicillium chrysogenum ATP Sulfurylase Missing the C-terminal Regulatory Domain*

    E-print Network

    Fisher, Andrew J.

    -limiting in the wild type enzyme. Without the C-terminal domain, the composite k5 step (isomerization of the central kinase) REACTIONS 1 AND 2 ATP sulfurylase from the filamentous fungus, Penicillium chrysogenum

  16. ATP hydrolysis by the proteasome regulatory complex PAN serves multiple functions in protein degradation.

    PubMed

    Benaroudj, Nadia; Zwickl, Peter; Seemüller, Erika; Baumeister, Wolfgang; Goldberg, Alfred L

    2003-01-01

    To clarify the role of ATP in proteolysis, we studied archaeal 20S proteasomes and the PAN (proteasome-activating nucleotidase) regulatory complex, a homolog of the eukaryotic 19S ATPases. PAN's ATPase activity was stimulated similarly by globular (GFPssrA) and unfolded (casein) substrates, and by the ssrA recognition peptide. Denaturation of GFPssrA did not accelerate its degradation or eliminate the requirement for PAN and ATP. During degradation of one molecule of globular or unfolded substrates, 300-400 ATP molecules were hydrolyzed. An N-terminal deletion in the 20S alpha subunits caused opening of the substrate-entry channel and rapid degradation of unfolded proteins without PAN; however, degradation of globular GFPssrA still required PAN's ATPase activity, even after PAN-catalyzed unfolding. Thus, substrate binding activates ATP hydrolysis, which promotes three processes: substrate unfolding, gate opening in the 20S, and protein translocation. PMID:12535522

  17. The mitochondrial ADP/ATP carrier (SLC25 family): pathological implications of its dysfunction.

    PubMed

    Clémençon, Benjamin; Babot, Marion; Trézéguet, Véronique

    2013-01-01

    In aerobic eukaryotic cells, the high energy metabolite ATP is generated mainly within the mitochondria following the process of oxidative phosphorylation. The mitochondrial ATP is exported to the cytoplasm using a specialized transport protein, the ADP/ATP carrier, to provide energy to the cell. Any deficiency or dysfunction of this membrane protein leads to serious consequences on cell metabolism and can cause various diseases such as muscular dystrophy. Described as a decisive player in the programmed cell death, it was recently shown to play a role in cancer. The objective of this review is to summarize the current knowledge of the involvement of the ADP/ATP carrier, encoded by the SLC25A4, SLC25A5, SLC25A6 and SLC25A31 genes, in human diseases and of the efforts made at designing different model systems to study this carrier and the associated pathologies through biochemical, genetic, and structural approaches. PMID:23506884

  18. Proton slip in the ATP synthase of Rhodobacter capsulatus: induction, proton conduction, and nucleotide dependence

    E-print Network

    Steinhoff, Heinz-Jürgen

    Proton slip in the ATP synthase of Rhodobacter capsulatus: induction, proton conduction. Uncoupled proton leakage (slip) has only been observed in chloroplast enzyme at unphysiologically low nucleotide concentration. We investigated the properties of proton slip in chromatophores (sub

  19. FIREFLY LUCIFERASE ATP ASSAY DEVELOPMENT FOR MONITORING BACTERIAL CONCENTRATIONS IN WATER SUPPLIES

    EPA Science Inventory

    This research program was initiated to develop a rapid, automatable system for measuring total viable microorganisms in potable drinking water supplies using the firefly luciferase ATP assay. The assay was adapted to an automatable flow system that provided comparable sensitivity...

  20. Synthesis of peptides from amino acids and ATP with lysine-rich proteinoid

    NASA Technical Reports Server (NTRS)

    Nakashima, T.; Fox, S. W.

    1980-01-01

    The paper examines the synthesis of peptides from aminoacids and ATP with a lysine-rich protenoid. The latter in aqueous solution catalyzes the formation of peptides from free amino acids and ATP; this catalytic activity is not found in acidic protenoids, even though the latter contain a basic aminoacid. The pH optimum for the synthesis is about 11, but it is appreciable below 8 and above 13. Temperature data indicate an optimum at 20 C or above, with little increase in rate up to 60 C. Pyrophosphate can be used instead of ATP, but the yields are lower. The ATP-aided syntheses of peptides in aqueous solution occur with several types of proteinous aminoacids.

  1. Turnover of ATP synthase subunits in F1-depleted HeLa and yeast cells.

    PubMed

    Rak, Malgorzata; McStay, Gavin P; Fujikawa, Makoto; Yoshida, Masasuke; Manfredi, Giovanni; Tzagoloff, Alexander

    2011-08-19

    Mitochondrial translation of the Saccharomyces cerevisiae Atp6p subunit of F(1)-F(0) ATP synthase is regulated by the F(1) ATPase. Here we show normal expression of Atp6p in HeLa cells depleted of the F(1) ? subunit. Instead of being translationally down-regulated, HeLa cells lacking F(1) degrade Atp6p, thereby preventing proton leakage across the inner membrane. Mammalian mitochondria also differ in the way they minimize the harmful effect of unassembled F(1) ? subunit. While yeast mutants lacking ? subunit have stable aggregated F(1) ? subunit in the mitochondrial matrix, the human ? subunit is completely degraded in cells deficient in F(1) ? subunit. These results are discussed in light of the different properties of the proteins and environments in which yeast and human mitochondria exist. PMID:21784071

  2. ATP-Dependent Formation of Phosphatidylserine-Rich Vesicles from the Endoplasmic Reticulum of Leek Cells

    PubMed Central

    Sturbois-Balcerzak, Bénédicte; Vincent, Patrick; Maneta-Peyret, Lilly; Duvert, Michel; Satiat-Jeunemaitre, Béatrice; Cassagne, Claude; Moreau, Patrick

    1999-01-01

    Leek (Allium porrum) plasma membrane is enriched in phosphatidylserine (PS) by the vesicular pathway, in a way similar to that already observed in animal cells (B. Sturbois-Balcerzak, D.J. Morré, O. Loreau, J.P. Noel, P. Moreau, C. Cassagne [1995] Plant Physiol Biochem 33: 625–637). In this paper we document the formation of PS-rich small vesicles from leek endoplasmic reticulum (ER) membranes upon addition of ATP and other factors. The omission of ATP or its replacement by ATP?-S prevents vesicle formation. These vesicles correspond to small structures (70–80 nm) and their phospholipid composition, characterized by a PS enrichment, is compatible with a role in PS transport. Moreover, the PS enrichment over phosphatidylinositol in the ER-derived vesicles is the first example, to our knowledge, of phospholipid sorting from the ER to ER-derived vesicles in plant cells. PMID:10318702

  3. ORIGINAL ARTICLE ATP release from the human ureter on distension and P2X3

    E-print Network

    Burnstock, Geoffrey

    . Sections of ureter were stained using antibodies against P2X3 and capsaicin receptors (TRPV1). [ATP] rose TRPV1 transient receptor potential vanilloid 1 Introduction Pain due to a calculus causing an acute

  4. Crystal Structure of the Bifunctional ATP Sulfurylase APS kinase from the Chemolithotrophic

    E-print Network

    Fisher, Andrew J.

    by crystal contacts. Additionally, a disulfide bond is observed in one ATP-binding P-loop of the APS kinase, and most bacterial species, PAPS is the substrate for a thioredoxin-dependent reductase that yields sul

  5. Mitochondrial protein sorting as a therapeutic target for ATP synthase disorders

    PubMed Central

    Aiyar, Raeka S.; Bohnert, Maria; Duvezin-Caubet, Stéphane; Voisset, Cécile; Gagneur, Julien; Fritsch, Emilie S.; Couplan, Elodie; von der Malsburg, Karina; Funaya, Charlotta; Soubigou, Flavie; Courtin, Florence; Suresh, Sundari; Kucharczyk, Roza; Evrard, Justine; Antony, Claude; St.Onge, Robert P.; Blondel, Marc; di Rago, Jean-Paul; van der Laan, Martin; Steinmetz, Lars M.

    2014-01-01

    Mitochondrial diseases are systemic, prevalent and often fatal; yet treatments remain scarce. Identifying molecular intervention points that can be therapeutically targeted remains a major challenge, which we confronted via a screening assay we developed. Using yeast models of mitochondrial ATP synthase disorders, we screened a drug repurposing library, and applied genomic and biochemical techniques to identify pathways of interest. Here we demonstrate that modulating the sorting of nuclear-encoded proteins into mitochondria, mediated by the TIM23 complex, proves therapeutic in both yeast and patient-derived cells exhibiting ATP synthase deficiency. Targeting TIM23-dependent protein sorting improves an array of phenotypes associated with ATP synthase disorders, including biogenesis and activity of the oxidative phosphorylation machinery. Our study establishes mitochondrial protein sorting as an intervention point for ATP synthase disorders, and because of the central role of this pathway in mitochondrial biogenesis, it holds broad value for the treatment of mitochondrial diseases. PMID:25519239

  6. Overexpression of ATP Sulfurylase in Indian Mustard Leads to Increased Selenate Uptake, Reduction, and Tolerance1

    E-print Network

    in Indian mustard (Brassica juncea). Compared with that in untransformed plants, the ATP sulfurylase (Brassica juncea) has proved to be a particularly suitable species for Se remediation, with high rates of Se

  7. PARK9-associated ATP13A2 localizes to intracellular acidic vesicles and regulates cation homeostasis and neuronal integrity.

    PubMed

    Ramonet, David; Podhajska, Agata; Stafa, Klodjan; Sonnay, Sarah; Trancikova, Alzbeta; Tsika, Elpida; Pletnikova, Olga; Troncoso, Juan C; Glauser, Liliane; Moore, Darren J

    2012-04-15

    Mutations in the ATP13A2 gene (PARK9, OMIM 610513) cause autosomal recessive, juvenile-onset Kufor-Rakeb syndrome and early-onset parkinsonism. ATP13A2 is an uncharacterized protein belonging to the P(5)-type ATPase subfamily that is predicted to regulate the membrane transport of cations. The physiological function of ATP13A2 in the mammalian brain is poorly understood. Here, we demonstrate that ATP13A2 is localized to intracellular acidic vesicular compartments in cultured neurons. In the human brain, ATP13A2 is localized to pyramidal neurons within the cerebral cortex and dopaminergic neurons of the substantia nigra. ATP13A2 protein levels are increased in nigral dopaminergic and cortical pyramidal neurons of Parkinson's disease brains compared with normal control brains. ATP13A2 levels are increased in cortical neurons bearing Lewy bodies (LBs) compared with neurons without LBs. Using short hairpin RNA-mediated silencing or overexpression to explore the function of ATP13A2, we find that modulating the expression of ATP13A2 reduces the neurite outgrowth of cultured midbrain dopaminergic neurons. We also find that silencing of ATP13A2 expression in cortical neurons alters the kinetics of intracellular pH in response to cadmium exposure. Furthermore, modulation of ATP13A2 expression leads to reduced intracellular calcium levels in cortical neurons. Finally, we demonstrate that silencing of ATP13A2 expression induces mitochondrial fragmentation in neurons. Oppositely, overexpression of ATP13A2 delays cadmium-induced mitochondrial fragmentation in neurons consistent with a neuroprotective effect. Collectively, this study reveals a number of intriguing neuronal phenotypes due to the loss- or gain-of-function of ATP13A2 that support a role for this protein in regulating intracellular cation homeostasis and neuronal integrity. PMID:22186024

  8. Consequences of the pathogenic T9176C mutation of human mitochondrial DNA on yeast mitochondrial ATP synthase

    PubMed Central

    Kucharczyk, Roza; Ezkurdia, Nahia; Couplan, Elodie; Procaccio, Vincent; Ackerman, Sharon H.; Blondel, Marc; di Rago, Jean-Paul

    2010-01-01

    Summary Several human neurological disorders have been associated with various mutations affecting mitochondrial enzymes involved in cellular ATP production. One of these mutations, T9176C in the mitochondrial DNA (mtDNA), changes a highly conserved leucine residue into proline at position 217 of the mitochondrially encoded Atp6p (or a) subunit of the F1FO-ATP synthase. The consequences of this mutation on the mitochondrial ATP synthase are still poorly defined. To gain insight into the primary pathogenic mechanisms induced by T9176C, we have investigated the consequences of this mutation on the ATP synthase of yeast where Atp6p is also encoded by the mtDNA. In vitro, yeast atp6-T9176C mitochondria showed a 30% decrease in the rate of ATP synthesis. When forcing the F1FO complex to work in the reverse mode, i.e. F1-catalyzed hydrolysis of ATP coupled to proton transport out of the mitochondrial matrix, the mutant showed a normal proton-pumping activity and this activity was fully sensitive to oligomycin, an inhibitor of the ATP synthase proton channel. However, under conditions of maximal ATP hydrolytic activity, using non-osmotically protected mitochondria, the mutant ATPase activity was less efficiently inhibited by oligomycin (60% inhibition versus 85% for the wild type control). BN-PAGE analyses revealed that atp6-T9176C yeast accumulated rather good levels of fully assembled ATP synthase complexes. However, a number of subcomplexes (F1, Atp9p-ring, unassembled ?-F1 subunits) could be detected as well, presumably because of a decreased stability of Atp6p within the ATP synthase. Although the oxidative phosphorylation capacity was reduced in atp6-T9176C yeast, the number of ATP molecules synthesized per electron transferred to oxygen was similar compared with wild type yeast. It can therefore be inferred that the coupling efficiency within the ATP synthase was mostly unaffected and that the T9176C mutation did not increase the proton permeability of the mitochondrial inner membrane. PMID:20056103

  9. Consequences of the pathogenic T9176C mutation of human mitochondrial DNA on yeast mitochondrial ATP synthase.

    PubMed

    Kucharczyk, Roza; Ezkurdia, Nahia; Couplan, Elodie; Procaccio, Vincent; Ackerman, Sharon H; Blondel, Marc; di Rago, Jean-Paul

    2010-01-01

    Several human neurological disorders have been associated with various mutations affecting mitochondrial enzymes involved in cellular ATP production. One of these mutations, T9176C in the mitochondrial DNA (mtDNA), changes a highly conserved leucine residue into proline at position 217 of the mitochondrially encoded Atp6p (or a) subunit of the F1FO-ATP synthase. The consequences of this mutation on the mitochondrial ATP synthase are still poorly defined. To gain insight into the primary pathogenic mechanisms induced by T9176C, we have investigated the consequences of this mutation on the ATP synthase of yeast where Atp6p is also encoded by the mtDNA. In vitro, yeast atp6-T9176C mitochondria showed a 30% decrease in the rate of ATP synthesis. When forcing the F1FO complex to work in the reverse mode, i.e. F1-catalyzed hydrolysis of ATP coupled to proton transport out of the mitochondrial matrix, the mutant showed a normal proton-pumping activity and this activity was fully sensitive to oligomycin, an inhibitor of the ATP synthase proton channel. However, under conditions of maximal ATP hydrolytic activity, using non-osmotically protected mitochondria, the mutant ATPase activity was less efficiently inhibited by oligomycin (60% inhibition versus 85% for the wild type control). Blue Native Polyacrylamide Gel Electrophoresis analyses revealed that atp6-T9176C yeast accumulated rather good levels of fully assembled ATP synthase complexes. However, a number of sub-complexes (F1, Atp9p-ring, unassembled alpha-F1 subunits) could be detected as well, presumably because of a decreased stability of Atp6p within the ATP synthase. Although the oxidative phosphorylation capacity was reduced in atp6-T9176C yeast, the number of ATP molecules synthesized per electron transferred to oxygen was similar compared with wild type yeast. It can therefore be inferred that the coupling efficiency within the ATP synthase was mostly unaffected and that the T9176C mutation did not increase the proton permeability of the mitochondrial inner membrane. PMID:20056103

  10. DNA aptasensor for the detection of ATP based on quantum dots electrochemiluminescence

    NASA Astrophysics Data System (ADS)

    Huang, Haiping; Tan, Yanglan; Shi, Jianjun; Liang, Guoxi; Zhu, Jun-Jie

    2010-04-01

    A novel and facile strategy for the fabrication of aptamer-based adenosine 5'-triphosphate (ATP) biosensor was developed by a quantum dot (QD) electrochemiluminescence (ECL) technique. Different from the existing strategies for the development of aptasensors based on electrochemical, fluorescent or other methods, the strategy proposed here is essentially based on the aptamer-ATP specific affinity and the rules of Watson-Crick base pairing. After the thiol modified anti-ATP probes were immobilized onto the pretreated Au electrode, the electrode was incubated in ATP solution to form aptamer-ATP bioaffinity complexes. The complementary DNA (cDNA) oligonucleotides were hybridized with the free probes. As a result, the avidin-modified QDs were bound to the aptasensor through the biotin-avidin system in the existence of biotin-modified cDNA. The ECL signal of the aptasensor was responsive to the amount of QDs bound to the cDNA oligonucleotides, which was inversely proportional to the combined target analyte ATP. The QDs were characterized by high resolution transmission electron microscopy (HRTEM), ultraviolet (UV) and photoluminescence (PL) spectra. The preparation process for the aptasensor was monitored by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Possible interference, such as from the pH value of the electrolyte, the incubation time and the concentration of coreactant K2S2O8, on the aptasensor ECL response were investigated. The ATP concentration was measured through the decrease of ECL intensity. The ECL intensity of the aptasensor decreased with the increase of the logarithm of the ATP concentration over the 0.018-90.72 ?M range. In addition, the aptasensor exhibited excellent selectivity responses toward the target analyte. This study may offer a new and relatively general approach to expand the application of QD ECL in the aptasensor field.

  11. The Signaling Mechanism of Contraction Induced by ATP and UTP in Feline Esophageal Smooth Muscle Cells

    PubMed Central

    Kwon, Tae Hoon; Jung, Hyunwoo; Cho, Eun Jeong; Jeong, Ji Hoon; Sohn, Uy Dong

    2015-01-01

    P2 receptors are membrane-bound receptors for extracellular nucleotides such as ATP and UTP. P2 receptors have been classified as ligand-gated ion channels or P2X receptors and G protein-coupled P2Y receptors. Recently, purinergic signaling has begun to attract attention as a potential therapeutic target for a variety of diseases especially associated with gastroenterology. This study determined the ATP and UTP-induced receptor signaling mechanism in feline esophageal contraction. Contraction of dispersed feline esophageal smooth muscle cells was measured by scanning micrometry. Phosphorylation of MLC20 was determined by western blot analysis. ATP and UTP elicited maximum esophageal contraction at 30 s and 10 ?M concentration. Contraction of dispersed cells treated with 10 ?M ATP was inhibited by nifedipine. However, contraction induced by 0.1 ?M ATP, 0.1 ?M UTP and 10 ?M UTP was decreased by U73122, chelerythrine, ML-9, PTX and GDP?S. Contraction induced by 0.1 ?M ATP and UTP was inhibited by G?i3 or G?q antibodies and by PLC?1 or PLC?3 antibodies. Phosphorylated MLC20 was increased by ATP and UTP treatment. In conclusion, esophageal contraction induced by ATP and UTP was preferentially mediated by P2Y receptors coupled to G?i3 and G q proteins, which activate PLC?1 and PLC?3. Subsequently, increased intracellular Ca2+ and activated PKC triggered stimulation of MLC kinase and inhibition of MLC phosphatase. Finally, increased pMLC20 generated esophageal contraction. PMID:26013385

  12. Evidence for Ectopic Aerobic ATP Production on C6 Glioma Cell Plasma Membrane

    Microsoft Academic Search

    Silvia Ravera; Maria Grazia Aluigi; Daniela Calzia; Paola Ramoino; Alessandro Morelli; Isabella Panfoli

    2011-01-01

    Extracellular ATP plays a pivotal role as a signaling molecule in physiological and pathological conditions in the CNS. In\\u000a several glioma cell lines, ATP is a positive factor for one or more characteristics important for the abnormal growth and\\u000a survival of these cells. This work presents immunofluorescence and biochemical analyses suggesting that an aerobic metabolism,\\u000a besides mitochondria, is located also

  13. Comparative single-molecule and ensemble myosin enzymology: sulfoindocyanine ATP and ADP derivatives.

    PubMed Central

    Oiwa, K; Eccleston, J F; Anson, M; Kikumoto, M; Davis, C T; Reid, G P; Ferenczi, M A; Corrie, J E; Yamada, A; Nakayama, H; Trentham, D R

    2000-01-01

    Single-molecule and macroscopic reactions of fluorescent nucleotides with myosin have been compared. The single-molecule studies serve as paradigms for enzyme-catalyzed reactions and ligand-receptor interactions analyzed as individual stochastic processes. Fluorescent nucleotides, called Cy3-EDA-ATP and Cy5-EDA-ATP, were derived by coupling the dyes Cy3.29.OH and Cy5.29.OH (compounds XI and XIV, respectively, in, Bioconjug. Chem. 4:105-111)) with 2'(3')-O-[N-(2-aminoethyl)carbamoyl]ATP (EDA-ATP). The ATP(ADP) analogs were separated into their respective 2'- and 3'-O-isomers, the interconversion rate of which was 30[OH(-)] s(-1) (0.016 h(-1) at pH 7.1) at 22 degrees C. Macroscopic studies showed that 2'(3')-O-substituted nucleotides had properties similar to those of ATP and ADP in their interactions with myosin, actomyosin, and muscle fibers, although the ATP analogs did not relax muscle as well as ATP did. Significant differences in the fluorescence intensity of Cy3-nucleotide 2'- and 3'-O-isomers in free solution and when they interacted with myosin were evident. Single-molecule studies using total internal reflection fluorescence microscopy showed that reciprocal mean lifetimes of the nucleotide analogs interacting with myosin filaments were one- to severalfold greater than predicted from macroscopic data. Kinetic and equilibrium data of nucleotide-(acto)myosin interactions derived from single-molecule microscopy now have a biochemical and physiological framework. This is important for single-molecule mechanical studies of motor proteins. PMID:10827983

  14. ATP dependence of Na+-driven Cl-HCO3 exchange in squid axons.

    PubMed

    Davis, Bruce A; Hogan, Emilia M; Russell, John M; Boron, Walter F

    2008-04-01

    Squid giant axons recover from acid loads by activating a Na(+)-driven Cl-HCO(3) exchanger. We internally dialyzed axons to an intracellular pH (pH( i )) of 6.7, halted dialysis and monitored the pH(i) recovery (increase) in the presence of ATP or other nucleotides, using cyanide to block oxidative phosphorylation. We computed the equivalent acid-extrusion rate (J(H)) from the rate of pH(i) increase and intracellular buffering power. In experimental series 1, we used dialysis to vary [ATP](i), finding that Michaelis-Menten kinetics describes J (H) vs. [ATP](i), with an apparent V(max) of 15.6 pmole cm(-2 )s(-1) and K (m) of 124 microM. In series 2, we examined ATP gamma S, AMP-PNP, AMP-PCP, AMP-CPP, GMP-PNP, ADP, ADP beta S and GDP beta S to determine if any, by themselves, could support transport. Only ATP gamma S (8 mM) supported acid extrusion; ATP gamma S also supported the HCO (3)(-) -dependent (36)Cl efflux expected of a Na(+)-driven Cl-HCO(3) exchanger. Finally, in series 3, we asked whether any nucleotide could alter J (H) in the presence of a background [ATP](i) of approximately 230 microM (control J (H) = 11.7 pmol cm(-2 )s(-1)). We found J (H) was decreased modestly by 8 mM AMP-PNP (J (H) = 8.0 pmol cm(-2 )s(-1)) but increased modestly by 1 mM ADP beta S (J (H) = 16.0 pmol cm(-2 )s(-1)). We suggest that ATP gamma S leads to stable phosphorylation of the transporter or an essential activator. PMID:18478173

  15. Control of membrane potential and excitability of Chara cells with ATP and Mg 2+

    Microsoft Academic Search

    Teruo Shimmen; Masashi Tazawa

    1977-01-01

    Summmary Electric characteristics of internodalChara australis cells, from which the tonoplast had been removed by vacuolar perfusion with media containing EGTA, were studied in relation to intracellular concentrations of ATP and Mg2+ using the ordinary microelectrode method and the open-vacuole method developed by Tazawa, Kikuyama and Nakagawa (1975.Plant Cell Physiol.16:611). The concentration of ATP was decreased by introducing hexokinase and

  16. DEAD-box proteins can completely separate an RNA duplex using a single ATP

    PubMed Central

    Chen, Yingfeng; Potratz, Jeffrey P.; Tijerina, Pilar; Del Campo, Mark; Lambowitz, Alan M.; Russell, Rick

    2008-01-01

    DEAD-box proteins are ubiquitous in RNA metabolism and use ATP to mediate RNA conformational changes. These proteins have been suggested to use a fundamentally different mechanism from the related DNA and RNA helicases, generating local strand separation while remaining tethered through additional interactions with structured RNAs and RNA-protein (RNP) complexes. Here, we provide a critical test of this model by measuring the number of ATP molecules hydrolyzed by DEAD-box proteins as they separate short RNA helices characteristic of structured RNAs (6–11 bp). We show that the DEAD-box protein CYT-19 can achieve complete strand separation using a single ATP, and that 2 related proteins, Mss116p and Ded1p, display similar behavior. Under some conditions, considerably <1 ATP is hydrolyzed per separation event, even though strand separation is strongly dependent on ATP and is not supported by the nucleotide analog AMP-PNP. Thus, ATP strongly enhances strand separation activity even without being hydrolyzed, most likely by eliciting or stabilizing a protein conformation that promotes strand separation, and AMP-PNP does not mimic ATP in this regard. Together, our results show that DEAD-box proteins can disrupt short duplexes by using a single cycle of ATP-dependent conformational changes, strongly supporting and extending models in which DEAD-box proteins perform local rearrangements while remaining tethered to their target RNAs or RNP complexes. This mechanism may underlie the functions of DEAD-box proteins by allowing them to generate local rearrangements without disrupting the global structures of their targets. PMID:19088196

  17. The Therapeutic Potential of Exogenous Adenosine Triphosphate (ATP) for Cartilage Tissue Engineering

    PubMed Central

    Usprech, Jenna; Chu, Gavin; Giardini-Rosa, Renata; Martin, Kathleen

    2012-01-01

    Objective: While mechanical stimuli can be used to enhance the properties of engineered cartilage, a promising alternative may be to directly harness the underlying mechanotransduction pathways responsible. Our initial studies on the adenosine triphosphate (ATP)–purinergic receptor pathway demonstrated that stimulation by exogenous ATP improved tissue growth and properties but elicited matrix turnover under high doses (250 µM) potentially due to the accumulation of extracellular inorganic pyrophosphate (ePPi). Therefore, the purpose of this study was to identify the mechanism of ATP-mediated catabolism and determine a therapeutic dose to maximize the anabolic effect. Design: Isolated bovine articular chondrocytes were seeded in high-density, 3-dimensional culture supplemented with varying doses of ATP for 4 weeks. The effects on biosynthesis, matrix metalloproteinase 13 (MMP-13) protein activity, and PPi accumulation were determined. Separate monolayer experiments were conducted to determine the effect of ePPi on MMP-13 activity. Results: High doses of ATP resulted in an increase in ePPi accumulation (by 54%) and MMP-13 activity (by 39%). Monolayer experiments confirmed a link between increased ePPi accumulation and MMP-13 activity, which appeared to require calcium and was inhibited by the MEK1/2 inhibitor U0126. Cultures supplemented with 62.5 to 125 µM ATP favored an anabolic response, which represented the therapeutic dose range. Conclusions: A therapeutic dose range of exogenous ATP to improve the properties of engineered cartilage has been identified, and a possible catabolic mechanism involving excess PPi was determined. Future research into PPi signal transduction and pathological crystal formation is necessary to maximize the beneficial effect of exogenous ATP on chondrocyte cultures.

  18. Coordinating role of His216 in MgATP binding and cleavage in pyruvate carboxylase.

    PubMed

    Adina-Zada, Abdussalam; Jitrapakdee, Sarawut; Wallace, John C; Attwood, Paul V

    2014-02-18

    His216 is a well-conserved residue in pyruvate carboxylases and, on the basis of structures of the enzyme, appears to have a role in the binding of MgATP, forming an interaction with the 3'-hydroxyl group of the ribose ring. Mutation of this residue to asparagine results in a 9-fold increase in the Km for MgATP in its steady-state cleavage in the absence of pyruvate and a 3-fold increase in the Km for MgADP in its steady-state phosphorylation by carbamoyl phosphate. However, from single-turnover experiments of MgATP cleavage, the Kd of the enzyme·MgATP complex is essentially the same in the wild-type enzyme and H216N. Direct stopped-flow measurements of nucleotide binding and release using the fluorescent analogue FTP support these observations. However, the first-order rate constant for MgATP cleavage in the single-turnover experiments in H216N is only 0.75% of that for the wild-type enzyme, and thus, the MgATP cleavage step is rate-limiting in the steady state for H216N but not for the wild-type enzyme. Close examination of the structure of the enzyme suggested that His216 may also interact with Glu218, which in turn interacts with Glu305 to form a proton relay system involved in the deprotonation of bicarbonate. Single-turnover MgATP cleavage experiments with mutations of these two residues resulted in kinetic parameters similar to those observed in H216N. We suggest that the primary role of His216 is to coordinate the binding of MgATP and the deprotonation of bicarbonate in the reaction to form the putative carboxyphosphate intermediate by participation in a proton relay system involving Glu218 and Glu305. PMID:24460480

  19. The Saccharomyces cerevisiae ATP22 Gene Codes for the Mitochondrial ATPase Subunit 6Specific Translation Factor

    Microsoft Academic Search

    Xiaomei Zeng; Audrey Hourset; Alexander Tzagoloff

    2007-01-01

    Mutations in the Saccharomyces cerevisiae ATP22 gene were previously shown to block assembly of the F0 component of the mitochondrial proton-translocating ATPase. Further inquiries into the function of Atp22p have revealed that it is essential for translation of subunit 6 of the mitochondrial ATPase. The mutant phenotype can be partially rescued by the presence in the same cell of wild-type

  20. Hereditary spastic paraplegia-like disorder due to a mitochondrial ATP6 gene point mutation

    Microsoft Academic Search

    Christophe Verny; Naig Guegen; Valerie Desquiret; Arnaud Chevrollier; Adriana Prundean; Frederic Dubas; Julien Cassereau; Marc Ferre; Patrizia Amati-Bonneau; Dominique Bonneau; Pascal Reynier; Vincent Procaccio

    2011-01-01

    Hereditary spastic paraplegia refers to a genetically heterogeneous syndrome. We identified five members of a family suffering from a late-onset spastic paraplegia-like disorder, carrying the homoplasmic m.9176T>C mutation in the mitochondrial ATP6 gene. The clinical severity of the disease observed in the family was correlated with the biochemical and assembly defects of the ATP synthase. The m.9176T>C mutation has been

  1. Analysis of silent RNA editing sites in atp6 transcripts of Sorghum bicolor

    Microsoft Academic Search

    F. Kempken; G. Höfken; D. R. Pring

    1995-01-01

    We have observed numerous examples of silent or rare non-silent editing sites in the amino-extension and part of the conserved core of mitochondrial atp6 transcripts of Sorghum. In this region of the 1.4-kb atp6-2 mRNA (position 300 to 550) two editing sites, which alter the aminoacid sequence and occur in all cDNAs analysed, were already known, while nine others were

  2. Mitochondrial atp6 transcript editing during microgametogenesis in male-sterile sorghum

    Microsoft Academic Search

    Daryl Pring; Hoang Tang

    2001-01-01

    A marked reduction of mitochondrial atp6 transcript-editing capability in sorghum anthers and pollen has been invoked as a factor in the loss of viability of male gametophytes in lines carrying the IS1112C male-sterile cytoplasm. We initiated a systematic examination of transcript editing of sorghum atp6 during microgametogenesis, from microspores through pollen, in two sets of male-fertile and near-isogenic, male-sterile lines.

  3. Kinetic mechanism of myosinV-S1 using a new fluorescent ATP analogue.

    PubMed

    Forgacs, Eva; Cartwright, Suzanne; Kovács, Mihály; Sakamoto, Takeshi; Sellers, James R; Corrie, John E T; Webb, Martin R; White, Howard D

    2006-10-31

    We have used a new fluorescent ATP analogue, 3'-(7-diethylaminocoumarin-3-carbonylamino)-3'-deoxyadenosine-5'-triphosphate (deac-aminoATP), to study the ATP hydrolysis mechanism of the single headed myosinV-S1. Our study demonstrates that deac-aminoATP is an excellent substrate for these studies. Although the deac-amino nucleotides have a low quantum yield in free solution, there is a very large increase in fluorescence emission ( approximately 20-fold) upon binding to the myosinV active site. The fluorescence emission intensity is independent of the hydrolysis state of the nucleotide bound to myosinV-S1. The very good signal-to-noise ratio that is obtained with deac-amino nucleotides makes them excellent substrates for studying expressed proteins that can only be isolated in small quantities. The combination of the fast rate of binding and the favorable signal-to-noise ratio also allows deac-nucleotides to be used in chase experiments to determine the kinetics of ADP and Pi dissociation from actomyosin-ADP-Pi. Although phosphate dissociation from actomyosinV-ADP-Pi does not itself produce a fluorescence signal, it produces a lag in the signal for deac-aminoADP dissociation. The lag provides direct evidence that the principal pathway of product dissociation from actomyosinV-ADP-Pi is an ordered mechanism in which phosphate precedes ADP. Although the mechanism of hydrolysis of deac-aminoATP by (acto)myosinV-S1 is qualitatively similar to the ATP hydrolysis mechanism, there are significant differences in some of the rate constants. Deac-aminoATP binds 3-fold faster to myosinV-S1, and the rate of deac-aminoADP dissociation from actomyosinV-S1 is 20-fold slower. Deac-aminoATP supports motility by myosinV-HMM on actin at a rate consistent with the slower rate of deac-aminoADP dissociation. PMID:17059220

  4. Further characterization of ATP6V0A2-related autosomal recessive cutis laxa.

    PubMed

    Fischer, Björn; Dimopoulou, Aikaterini; Egerer, Johannes; Gardeitchik, Thatjana; Kidd, Alexa; Jost, Dominik; Kayserili, Hülya; Alanay, Yasemin; Tantcheva-Poor, Iliana; Mangold, Elisabeth; Daumer-Haas, Cornelia; Phadke, Shubha; Peirano, Reto I; Heusel, Julia; Desphande, Charu; Gupta, Neerja; Nanda, Arti; Felix, Emma; Berry-Kravis, Elisabeth; Kabra, Madhulika; Wevers, Ron A; van Maldergem, Lionel; Mundlos, Stefan; Morava, Eva; Kornak, Uwe

    2012-11-01

    Autosomal recessive cutis laxa (ARCL) syndromes are phenotypically overlapping, but genetically heterogeneous disorders. Mutations in the ATP6V0A2 gene were found to underlie both, autosomal recessive cutis laxa type 2 (ARCL2), Debré type, and wrinkly skin syndrome (WSS). The ATP6V0A2 gene encodes the a2 subunit of the V-type H(+)-ATPase, playing a role in proton translocation, and possibly also in membrane fusion. Here, we describe a highly variable phenotype in 13 patients with ARCL2, including the oldest affected individual described so far, who showed strikingly progressive dysmorphic features and heterotopic calcifications. In these individuals we identified 17 ATP6V0A2 mutations, 14 of which are novel. Furthermore, we demonstrate a localization of ATP6V0A2 at the Golgi-apparatus and a loss of the mutated ATP6V0A2 protein in patients' dermal fibroblasts. Investigation of brefeldin A-induced Golgi collapse in dermal fibroblasts as well as in HeLa cells deficient for ATP6V0A2 revealed a delay, which was absent in cells deficient for the ARCL-associated proteins GORAB or PYCR1. Furthermore, fibroblasts from patients with ATP6V0A2 mutations displayed elevated TGF-? signalling and increased TGF-?1 levels in the supernatant. Our current findings expand the genetic and phenotypic spectrum and suggest that, besides the known glycosylation defect, alterations in trafficking and signalling processes are potential key events in the pathogenesis of ATP6V0A2-related ARCL. PMID:22773132

  5. ATP-Sensitive Potassium Channels: A Review of their Cardioprotective Pharmacology

    Microsoft Academic Search

    Gary J Grover; Keith D Garlid

    2000-01-01

    G. J. Grover and K. A. Garlid. ATP-Sensitive Potassium Channels: A Review of their Cardioprotective Pharmacology. Journal of Molecular and Cellular Cardiology (2000) 32, 677–696. ATP-sensitive potassium channels (KATP) have been thought to be a mediator of cardioprotection for the last ten years. Significant progress has been made in learning the pharmacology of this channel as well as its molecular

  6. Intravascular ATP and the regulation of blood flow and oxygen delivery in humans.

    PubMed

    Crecelius, Anne R; Kirby, Brett S; Dinenno, Frank A

    2015-01-01

    Regulation of vascular tone is a complex response that integrates multiple signals that allow for blood flow and oxygen supply to match oxygen demand appropriately. Here, we discuss the potential role of intravascular adenosine triphosphate (ATP) as a primary factor in these responses and put forth the hypothesis that deficient ATP release contributes to impairments in vascular control exhibited in aged and diseased populations. PMID:25390296

  7. Cell Type-Specific Loss of atp6 RNA Editing in Cytoplasmic Male Sterile Sorghum bicolor

    Microsoft Academic Search

    Werner Howad; Frank Kempken

    1997-01-01

    RNA editing and cytoplasmic male sterility are two important phenomena in higher plant mitochondria. To determine whether correlations might exist between the two, RNA editing in different tissues of Sorghum bicolor was compared employing reverse transcription-PCR and subsequent sequence analysis. In etiolated shoots, RNA editing of transcripts of plant mitochondrial atp6, atp9, nad3, nad4, and rps12 genes was identical among

  8. Novel mutations of ATP2A2 gene in Japanese patients of Darier's disease

    Microsoft Academic Search

    Hidetoshi Takahashi; Yoshiaki Atsuta; Katsuhiko Sato; Akemi Ishida-Yamamoto; Hiroshi Suzuki; Hajime Iizuka

    2001-01-01

    Darier's disease (DD) is a rare, dominantly inherited skin disorder with abnormal keratinization and acantholysis. Recently, mutations of ATP2A2 encoding the sarco\\/endoplasmic reticulum Ca2+-ATPase type 2 isoform (SERCA2) have been reported in Caucasian DD families. In the present study, we examined the ATP2A2 gene mutations of three sporadic (AS1,AS3,AS4) and one familial (AS2) Japanese DD patients. Sequence analysis revealed that

  9. Distinct activities of CHD1 and ACF in ATP-dependent chromatin assembly

    Microsoft Academic Search

    Alexandra Lusser; Debra L Urwin; James T Kadonaga

    2005-01-01

    CHD1 is a chromodomain-containing protein in the SNF2-like family of ATPases. Here we show that CHD1 exists predominantly as a monomer and functions as an ATP-utilizing chromatin assembly factor. This reaction involves purified CHD1, NAP1 chaperone, core histones and relaxed DNA. CHD1 catalyzes the ATP-dependent transfer of histones from the NAP1 chaperone to the DNA by a processive mechanism that

  10. A theoretical study on the amount of ATP required for synthesis of microbial cell material

    Microsoft Academic Search

    A. H. Stouthamer

    1973-01-01

    The amount of ATP required for the formation of microbial cells growing under various conditions was calculated. It was assumed\\u000a that the chemical composition of the cell was the same under all these conditions. The analysis of the chemical composition\\u000a of microbial cells of Morowitz (1968) was taken as a base. It was assumed that 4 moles of ATP are

  11. ATP-Sensitive Potassium Channel Blockage Attenuates Cisplatin-Induced Renal Damage

    Microsoft Academic Search

    Andréa G. C. Flávio; Fernando Q. Cunha; Heloísa D. Colletta Francescato; Telma J. Soares; Roberto S. Costa; Fernando Barbosa Júnior; Terezila M. Coimbra

    2007-01-01

    Background: Cisplatin-induced renal damage was associated with an inflammatory process. ATP-sensitive potassium channels can be involved in neutrophil migration. This study evaluated the effects of glibenclamide, an ATP-sensitive potassium channel blocker, on cisplatin-induced renal damage. Methods: A total of 48 Wistar rats received glibenclamide (20 mg\\/kg\\/day, s.c.) and 24 h later, these animals, and an additional group of 45 rats,

  12. ATP-driven Rad50 conformations regulate DNA tethering, end resection, and ATM checkpoint signaling

    PubMed Central

    Deshpande, Rajashree A; Williams, Gareth J; Limbo, Oliver; Williams, R Scott; Kuhnlein, Jeff; Lee, Ji-Hoon; Classen, Scott; Guenther, Grant; Russell, Paul; Tainer, John A; Paull, Tanya T

    2014-01-01

    The Mre11-Rad50 complex is highly conserved, yet the mechanisms by which Rad50 ATP-driven states regulate the sensing, processing and signaling of DNA double-strand breaks are largely unknown. Here we design structure-based mutations in Pyrococcus furiosus Rad50 to alter protein core plasticity and residues undergoing ATP-driven movements within the catalytic domains. With this strategy we identify Rad50 separation-of-function mutants that either promote or destabilize the ATP-bound state. Crystal structures, X-ray scattering, biochemical assays, and functional analyses of mutant PfRad50 complexes show that the ATP-induced ‘closed’ conformation promotes DNA end binding and end tethering, while hydrolysis-induced opening is essential for DNA resection. Reducing the stability of the ATP-bound state impairs DNA repair and Tel1 (ATM) checkpoint signaling in Schizosaccharomyces pombe, double-strand break resection in Saccharomyces cerevisiae, and ATM activation by human Mre11-Rad50-Nbs1 in vitro, supporting the generality of the P. furiosus Rad50 structure-based mutational analyses. These collective results suggest that ATP-dependent Rad50 conformations switch the Mre11-Rad50 complex between DNA tethering, ATM signaling, and 5? strand resection, revealing molecular mechanisms regulating responses to DNA double-strand breaks. PMID:24493214

  13. Assembly and oligomerization of human ATP synthase lacking mitochondrial subunits a and A6L.

    PubMed

    Wittig, Ilka; Meyer, Bjoern; Heide, Heinrich; Steger, Mirco; Bleier, Lea; Wumaier, Zibiernisha; Karas, Michael; Schägger, Hermann

    2010-01-01

    Here we study ATP synthase from human rho0 (rho zero) cells by clear native electrophoresis (CNE or CN-PAGE) and show that ATP synthase is almost fully assembled in spite of the absence of subunits a and A6L. This identifies subunits a and A6L as two of the last subunits to complete the ATP synthase assembly. Minor amounts of dimeric and even tetrameric forms of the large assembly intermediate were preserved under the conditions of CNE, suggesting that it associated further into higher order structures in the mitochondrial membrane. This result was reminiscent to the reduced amounts of dimeric and tetrameric ATP synthase from yeast null mutants of subunits e and g detected by CNE. The dimer/oligomer-stabilizing effects of subunits e/g and a/A6L seem additive in human and yeast cells. The mature IF1 inhibitor was specifically bound to the dimeric/oligomeric forms of ATP synthase and not to the monomer. Conversely, nonprocessed pre-IF1 still containing the mitochondrial targeting sequence was selectively bound to the monomeric assembly intermediate in rho0 cells and not to the dimeric form. This supports previous suggestions that IF1 plays an important role in the dimerization/oligomerization of mammalian ATP synthase and in the regulation of mitochondrial structure and function. PMID:20188060

  14. The bacterial flagellar protein export apparatus processively transports flagellar proteins even with extremely infrequent ATP hydrolysis

    PubMed Central

    Minamino, Tohru; Morimoto, Yusuke V.; Kinoshita, Miki; Aldridge, Phillip D.; Namba, Keiichi

    2014-01-01

    For self-assembly of the bacterial flagellum, a specific protein export apparatus utilizes ATP and proton motive force (PMF) as the energy source to transport component proteins to the distal growing end. The export apparatus consists of a transmembrane PMF-driven export gate and a cytoplasmic ATPase complex composed of FliH, FliI and FliJ. The FliI6FliJ complex is structurally similar to the ?3?3? complex of FOF1-ATPase. FliJ allows the gate to efficiently utilize PMF to drive flagellar protein export but it remains unknown how. Here, we report the role of ATP hydrolysis by the FliI6FliJ complex. The export apparatus processively transported flagellar proteins to grow flagella even with extremely infrequent or no ATP hydrolysis by FliI mutation (E211D and E211Q, respectively). This indicates that the rate of ATP hydrolysis is not at all coupled with the export rate. Deletion of FliI residues 401 to 410 resulted in no flagellar formation although this FliI deletion mutant retained 40% of the ATPase activity, suggesting uncoupling between ATP hydrolysis and activation of the gate. We propose that infrequent ATP hydrolysis by the FliI6FliJ ring is sufficient for gate activation, allowing processive translocation of export substrates for efficient flagellar assembly. PMID:25531309

  15. CaATP as a substrate to investigate the myosin lever arm hypothesis of force generation.

    PubMed Central

    Polosukhina, K; Eden, D; Chinn, M; Highsmith, S

    2000-01-01

    In an effort to test the lever arm model of force generation, the effects of replacing magnesium with calcium as the ATP-chelated divalent cation were determined for several myosin and actomyosin reactions. The isometric force produced by glycerinated muscle fibers when CaATP is the substrate is 20% of the value obtained with MgATP. For myosin subfragment 1 (S1), the degree of lever arm rotation, determined using transient electric birefringence to measure rates of rotational Brownian motion in solution, is not significantly changed when calcium replaces magnesium in an S1-ADP-vanadate complex. Actin activates S1 CaATPase activity, although less than it does MgATPase activity. The increase in actin affinity when S1. CaADP. P(i) is converted to S1. CaADP is somewhat greater than it is for the magnesium case. The ionic strength dependence of actin binding indicates that the change in apparent electrostatic charge at the acto-S1 interface for the S1. CaADP. P(i) to S1. CaADP step is similar to the change when magnesium is bound. In general, CaATP is an inferior substrate compared to MgATP, but all the data are consistent with force production by a lever arm mechanism for both substrates. Possible reasons for the reduced magnitude of force when CaATP is the substrate are discussed. PMID:10692332

  16. ATP-Binding Cassette Proteins: Towards a Computational View of Mechanism

    NASA Astrophysics Data System (ADS)

    Liao, Jielou

    2004-03-01

    Many large machine proteins can generate mechanical force and undergo large-scale conformational changes (LSCC) to perform varying biological tasks in living cells by utilizing ATP. Important examples include ATP-binding cassette (ABC) transporters. They are membrane proteins that couple ATP binding and hydrolysis to the translocation of substrates across membranes [1]. To interpret how the mechanical force generated by ATP binding and hydrolysis is propagated, a coarse-grained ATP-dependent harmonic network model (HNM) [2,3] is applied to the ABC protein, BtuCD. This protein machine transports vitamin B12 across membranes. The analysis shows that subunits of the protein move against each other in a concerted manner. The lowest-frequency modes of the BtuCD protein are found to link the functionally critical domains, and are suggested to be responsible for large-scale ATP-coupled conformational changes. [1] K. P. Locher, A. T. Lee and D. C. Rees. Science 296, 1091-1098 (2002). [2] Atilgan, A. R., S. R. Durell, R. L. Jernigan, M. C. Demirel, O. Keskin, and I. Bahar. Biophys. J. 80, 505-515(2002); M. M Tirion, Phys. Rev. Lett. 77, 1905-1908 (1996). [3] J. -L. Liao and D. N. Beratan, 2003, to be published.

  17. K-ATP channels in dopamine substantia nigra neurons control bursting and novelty-induced exploration

    PubMed Central

    Schiemann, Julia; Schlaudraff, Falk; Klose, Verena; Bingmer, Markus; Seino, Susumu; Magill, Peter J; Zaghloul, Kareem A; Schneider, Gaby; Liss, Birgit; Roeper, Jochen

    2014-01-01

    Phasic activation of the dopamine (DA) midbrain system in response to unexpected reward or novelty is critical for adaptive behavioral strategies. This activation of DA midbrain neurons occurs via a synaptically triggered switch from low-frequency background spiking to transient high-frequency burst firing. We found that, in medial DA neurons of the substantia nigra (SN), activity of ATP-sensitive potassium (K-ATP) channels enabled NMDA-mediated bursting in vitro as well as spontaneous in vivo burst firing in anesthetized mice. Cell-selective silencing of K-ATP channel activity in medial SN DA neurons revealed that their K-ATP channel-gated burst firing was crucial for novelty-dependent exploratory behavior. We also detected a transcriptional upregulation of K-ATP channel and NMDA receptor subunits, as well as high in vivo burst firing, in surviving SN DA neurons from Parkinson’s disease patients, suggesting that burst-gating K-ATP channel function in DA neurons affects phenotypes in both disease and health. PMID:22902720

  18. The uses and abuses of rapid bioluminescence-based ATP assays.

    PubMed

    Shama, G; Malik, D J

    2013-03-01

    Bioluminescence-based ATP testing of solid surfaces has become well established in the food processing industry as part of general hazard analysis and critical control points (HACCP) measures. The rise in healthcare associated infections (HAIs) at the turn of the century focussed attention on the environment as a potential reservoir of the agents responsible for such infections. In response to the need for objective methods of assessing the efficiency of cleaning in healthcare establishments and for rapid methods for detecting the presence of the pathogens responsible for HAIs, it was proposed that ATP testing of environmental surfaces be introduced. We examine the basis behind the assumptions inherent in these proposals. Intracellular ATP levels are shown to vary between microbial taxa and according to environmental conditions. Good correlations between microbial numbers and ATP levels have been obtained under certain specific conditions, but never within healthcare settings. Notwithstanding, ATP testing may still have a role in providing reassurance that cleaning regimes are being carried out satisfactorily. However, ATP results should not be interpreted as surrogate indicators for the presence of microbial pathogens. PMID:22541898

  19. How do taste cells lacking synapses mediate neurotransmission? CALHM1, a voltage-gated ATP channel

    PubMed Central

    Taruno, Akiyuki; Matsumoto, Ichiro; Ma, Zhongming; Marambaud, Philippe; Foskett, J. Kevin

    2014-01-01

    CALHM1 was recently demonstrated to be a voltage-gated ATP-permeable ion channel and to serve as a bona fide conduit for ATP release from sweet-, umami-, and bitter-sensing type II taste cells. Calhm1 is expressed in taste buds exclusively in type II cells and its product has structural and functional similarities with connexins and pannexins, two families of channel protein candidates for ATP release by type II cells. Calhm1 knockout in mice leads to loss of perception of sweet, umami, and bitter compounds and to impaired gustatory nerve responses to these tastants. These new studies validate the concept of ATP as the primary neurotransmitter from type II cells to gustatory neurons. Furthermore, they identify voltage-gated ATP release through CALHM1 as an essential molecular mechanism of ATP release in taste buds. We discuss these new findings, as well as unresolved issues in peripheral taste signaling that we hope will stimulate future research. PMID:24105910

  20. Crystallographic structure of the turbine C-ring from spinach chloroplast F-ATP synthase

    PubMed Central

    Balakrishna, Asha Manikkoth; Seelert, Holger; Marx, Sven-Hendric; Dencher, Norbert A.; Grüber, Gerhard

    2014-01-01

    In eukaryotic and prokaryotic cells, F-ATP synthases provide energy through the synthesis of ATP. The chloroplast F-ATP synthase (CF1FO-ATP synthase) of plants is integrated into the thylakoid membrane via its FO-domain subunits a, b, b’ and c. Subunit c with a stoichiometry of 14 and subunit a form the gate for H+-pumping, enabling the coupling of electrochemical energy with ATP synthesis in the F1 sector. Here we report the crystallization and structure determination of the c14-ring of subunit c of the CF1FO-ATP synthase from spinach chloroplasts. The crystals belonged to space group C2, with unit-cell parameters a=144.420, b=99.295, c=123.51 Å, and ?=104.34° and diffracted to 4.5 Å resolution. Each c-ring contains 14 monomers in the asymmetric unit. The length of the c-ring is 60.32 Å, with an outer ring diameter 52.30 Å and an inner ring width of 40 Å. PMID:24521269

  1. Effect of Inflammatory Mediators on ATP Release of Human Urothelial RT4 Cells

    PubMed Central

    Mansfield, Kylie J.; Hughes, Jessica R.

    2014-01-01

    Inflammation is an important contributor to the aetiology of a number of bladder dysfunctions including interstitial cystitis, painful bladder syndrome, and overactive bladder. The aim of this study was to examine the effects of inflammatory mediators on urothelial ATP release. Human urothelial RT4 cells were exposed to normal buffer or varying concentrations of inflammatory mediators (bradykinin, histamine, and serotonin) in the presence or absence of hypotonic stretch stimuli (1?:?2 dilution of Krebs-Henseleit buffer). Others have demonstrated that bradykinin increased stretch-induced ATP release; however, we observed no change in control or stretch-induced ATP release with bradykinin. Pretreatment of RT4 cells with histamine or serotonin decreased stretch-induced ATP release (P = 0.037, P = 0.040, resp.). Previous studies have demonstrated increased ATP release in response to inflammation utilising whole bladder preparations in contrast to our simple model of cultured urothelial cells. The current study suggests that it is unlikely that there is a direct interaction between the release of inflammatory mediators and increased ATP release, but rather more complex interactions occurring in response to inflammation that lead to increased bladder sensation. PMID:24839598

  2. Synergistic effects of ATP and RNA binding to human DEAD-box protein DDX1

    PubMed Central

    Kellner, Julian N.; Reinstein, Jochen; Meinhart, Anton

    2015-01-01

    RNA helicases of the DEAD-box protein family form the largest group of helicases. The human DEAD-box protein 1 (DDX1) plays an important role in tRNA and mRNA processing, is involved in tumor progression and is also hijacked by several virus families such as HIV-1 for replication and nuclear export. Although important in many cellular processes, the mechanism of DDX1?s enzymatic function is unknown. We have performed equilibrium titrations and transient kinetics to determine affinities for nucleotides and RNA. We find an exceptional tight binding of DDX1 to adenosine diphosphate (ADP), one of the strongest affinities observed for DEAD-box helicases. ADP binds tighter by three orders of magnitude when compared to adenosine triphosphate (ATP), arresting the enzyme in a potential dead-end ADP conformation under physiological conditions. We thus suggest that a nucleotide exchange factor leads to DDX1 recycling. Furthermore, we find a strong cooperativity in binding of RNA and ATP to DDX1 that is also reflected in ATP hydrolysis. We present a model in which either ATP or RNA binding alone can partially shift the equilibrium from an ‘open’ to a ‘closed’-state; this shift appears to be not further pronounced substantially even in the presence of both RNA and ATP as the low rate of ATP hydrolysis does not change. PMID:25690890

  3. Determination of ddATP Levels in Human Immunodeficiency Virus-Infected Patients Treated with Dideoxyinosine†

    PubMed Central

    Le Saint, Cecile; Terreux, Raphael; Duval, Daniele; Durant, Jacques; Ettesse, Helene; Dellamonica, Pierre; Guedj, Roger; Vincent, Jean Pierre; Cupo, Anny

    2004-01-01

    Clinical failures of the highly active antiretroviral therapy could result from inefficient intracellular concentrations of antiviral drugs. The determination of drug contents in target cells of each patient would be useful in clinical investigations and trials. The purpose of this work was to quantify the intracellular concentration of ddATP, the active metabolite of dideoxyinosine (ddI), in peripheral blood mononuclear cells (PBMCs) of human immunodeficiency virus (HIV)-infected patients treated with ddI. We have raised antibodies against ddA-citrate, a stable isostere of ddATP selected on the basis of its structural and electronic analogies with ddATP. The anti-ddA-citrate antibodies recognized ddATP and ddA with nanomolar affinities and cross-reacted neither with any of the nucleotide reverse transcriptase inhibitors used in HIV therapy nor with their phosphorylated metabolites. The three phosphorylated metabolites of ddI (ddAMP, ddADP, and ddATP) were purified by anion exchange chromatography and the amount of each metabolite was determined by radioimmunoassay with or without prior phosphatase treatment. The intracellular levels of the three ddI metabolites were measured both in an in vitro model and in PBMCs of HIV-infected patients under ddI treatment. The possibility to measure intracellular levels of ddATP from small blood samples of HIV-infected patients treated with ddI could be exploited to develop individual therapeutic monitoring. PMID:14742213

  4. Polymorphisms in canine ATP7B: candidate modifier of copper toxicosis in the Bedlington terrier.

    PubMed

    Coronado, Veronica A; O'Neill, Brian; Nanji, Manoj; Cox, Diane W

    2008-08-01

    A COMMD1(MURR1) deletion has been reported as the cause of copper toxicosis (CT) in Bedlington terriers. Recent studies identified Bedlington terriers with copper accumulation without homozygous COMMD1 deletions. Wilson disease in humans is a copper storage disorder similar to CT caused by mutations in ATP7B, and COMMD1 has been shown to interact with the ATP7B protein. ATP7B may act as a modifier in CT, allowing for copper accumulation in Bedlington terriers with one deletion or other variations in COMMD1. In this study, ATP7B was cloned and sequence analysis conducted in a subset of Bedlington terriers from a pedigree that does not show complete association between the COMMD1 deletion and CT. Eleven polymorphisms, two in the coding region, were identified in the Bedlington terrier ATP7B gene. However, these are not unique to the Bedlington terrier and pedigree analysis suggests that ATP7B is not a modifier of COMMD1 in this subset of dogs. PMID:17572118

  5. Crystallographic structure of the turbine c-ring from spinach chloroplast F-ATP synthase.

    PubMed

    Balakrishna, Asha Manikkoth; Seelert, Holger; Marx, Sven-Hendric; Dencher, Norbert A; Grüber, Gerhard

    2014-02-13

    In eukaryotic- and prokaryotic cells F-ATP synthases provide energy through the synthesis of adenosine triphosphate (ATP). The chloroplast F-ATP synthase (CF1FO-ATP synthase) of plants is integrated into the thylakoid membrane via its FO-domain subunits a, b, b' and c. Subunit c with a stoichiometry of 14 and subunit a form the gate for H+-pumping, enabling the coupling of electrochemical energy with ATP synthesis in the F1 sector. Here we report the crystallization and structure determination of the c14-ring of subunit c of the CF1FO-ATP synthase from spinach chloroplasts. The crystals belonged to space group C2, with unit-cell parameters a = 144.420, b = 99.295, c = 123.51 Å, and ? = 104.34º and diffracted to 4.5 Å resolution. Each c-ring contains fourteen monomers in the asymmetric unit. The length of the c-ring is 60.32 Å, with an outer ring diameter 52.30 Å, and an inner ring width of 40 Å. PMID:24521269

  6. CO2/HCO3(-)- and calcium-regulated soluble adenylyl cyclase as a physiological ATP sensor.

    PubMed

    Zippin, Jonathan H; Chen, Yanqiu; Straub, Susanne G; Hess, Kenneth C; Diaz, Ana; Lee, Dana; Tso, Patrick; Holz, George G; Sharp, Geoffrey W G; Levin, Lonny R; Buck, Jochen

    2013-11-15

    The second messenger molecule cAMP is integral for many physiological processes. In mammalian cells, cAMP can be generated from hormone- and G protein-regulated transmembrane adenylyl cyclases or via the widely expressed and structurally and biochemically distinct enzyme soluble adenylyl cyclase (sAC). sAC activity is uniquely stimulated by bicarbonate ions, and in cells, sAC functions as a physiological carbon dioxide, bicarbonate, and pH sensor. sAC activity is also stimulated by calcium, and its affinity for its substrate ATP suggests that it may be sensitive to physiologically relevant fluctuations in intracellular ATP. We demonstrate here that sAC can function as a cellular ATP sensor. In cells, sAC-generated cAMP reflects alterations in intracellular ATP that do not affect transmembrane AC-generated cAMP. In ? cells of the pancreas, glucose metabolism generates ATP, which corresponds to an increase in cAMP, and we show here that sAC is responsible for an ATP-dependent cAMP increase. Glucose metabolism also elicits insulin secretion, and we further show that sAC is necessary for normal glucose-stimulated insulin secretion in vitro and in vivo. PMID:24100033

  7. CO2/HCO3?- and Calcium-regulated Soluble Adenylyl Cyclase as a Physiological ATP Sensor*

    PubMed Central

    Zippin, Jonathan H.; Chen, Yanqiu; Straub, Susanne G.; Hess, Kenneth C.; Diaz, Ana; Lee, Dana; Tso, Patrick; Holz, George G.; Sharp, Geoffrey W. G.; Levin, Lonny R.; Buck, Jochen

    2013-01-01

    The second messenger molecule cAMP is integral for many physiological processes. In mammalian cells, cAMP can be generated from hormone- and G protein-regulated transmembrane adenylyl cyclases or via the widely expressed and structurally and biochemically distinct enzyme soluble adenylyl cyclase (sAC). sAC activity is uniquely stimulated by bicarbonate ions, and in cells, sAC functions as a physiological carbon dioxide, bicarbonate, and pH sensor. sAC activity is also stimulated by calcium, and its affinity for its substrate ATP suggests that it may be sensitive to physiologically relevant fluctuations in intracellular ATP. We demonstrate here that sAC can function as a cellular ATP sensor. In cells, sAC-generated cAMP reflects alterations in intracellular ATP that do not affect transmembrane AC-generated cAMP. In ? cells of the pancreas, glucose metabolism generates ATP, which corresponds to an increase in cAMP, and we show here that sAC is responsible for an ATP-dependent cAMP increase. Glucose metabolism also elicits insulin secretion, and we further show that sAC is necessary for normal glucose-stimulated insulin secretion in vitro and in vivo. PMID:24100033

  8. Catalytic and mechanical cycles in F-ATP synthases. Fourth in the Cycles Review Series.

    PubMed

    Dimroth, Peter; von Ballmoos, Christoph; Meier, Thomas

    2006-03-01

    Cycles have a profound role in cellular life at all levels of organization. Well-known cycles in cell metabolism include the tricarboxylic acid and the urea cycle, in which a specific carrier substrate undergoes a sequence of chemical transformations and is regenerated at the end. Other examples include the interconversions of cofactors, such as NADH or ATP, which are present in the cell in limiting amounts and have to be recycled effectively for metabolism to continue. Every living cell performs a rapid turnover of ATP to ADP to fulfil various energetic demands and effectively regenerates the ATP from ADP in an energy-consuming process. The turnover of the ATP cycle is impressive; a human uses about its body weight in ATP per day. Enzymes perform catalytic reaction cycles in which they undergo several chemical and physical transformations before they are converted back to their original states. The ubiquitous F1F(o) ATP synthase is of particular interest not only because of its biological importance, but also owing to its unique rotational mechanism. Here, we give an overview of the membrane-embedded F(o) sector, particularly with respect to the recent crystal structure of the c ring from Ilyobacter tartaricus, and summarize current hypotheses for the mechanism by which rotation of the c ring is generated. PMID:16607397

  9. Effect of ATP binding and hydrolysis on dynamics of canine parvovirus NS1.

    PubMed

    Niskanen, Einari A; Ihalainen, Teemu O; Kalliolinna, Olli; Häkkinen, Milla M; Vihinen-Ranta, Maija

    2010-05-01

    The replication protein NS1 is essential for genome replication and protein production in parvoviral infection. Many of its functions, including recognition and site-specific nicking of the viral genome, helicase activity, and transactivation of the viral capsid promoter, are dependent on ATP. An ATP-binding pocket resides in the middle of the modular NS1 protein in a superfamily 3 helicase domain. Here we have identified key ATP-binding amino acid residues in canine parvovirus (CPV) NS1 protein and mutated amino acids from the conserved A motif (K406), B motif (E444 and E445), and positively charged region (R508 and R510). All mutations prevented the formation of infectious viruses. When provided in trans, all except the R508A mutation reduced infectivity in a dominant-negative manner, possibly by hindering genome replication. These results suggest that the conserved R510 residue, but not R508, is the arginine finger sensory element of CPV NS1. Moreover, fluorescence recovery after photobleaching (FRAP), complemented by computer simulations, was used to assess the binding properties of mutated fluorescent fusion proteins. These experiments identified ATP-dependent and -independent binding modes for NS1 in living cells. Only the K406M mutant had a single binding site, which was concluded to indicate ATP-independent binding. Furthermore, our data suggest that DNA binding of NS1 is dependent on its ability to both bind and hydrolyze ATP. PMID:20219935

  10. ATP depletion and cell death in the neonatal lamb ductus arteriosus.

    PubMed

    Levin, Max; Goldbarg, Seth; Lindqvist, Anders; Swärd, Karl; Roman, Christine; Liu, Bao Mei; Hultén, Lillemor Mattsson; Borén, Jan; Clyman, Ronald I

    2005-06-01

    Postnatal constriction of the full-term ductus arteriosus produces cell death and remodeling of the ductus wall. Using a bioluminescence imaging technique, we found that after birth, the lamb ductus develops ATP, glucose, and glycogen depletion in addition to hypoxia. In vitro studies showed that cell death correlates best with ATP depletion and is most marked when both glucose and oxygen are severely depleted; in addition, the degree of ATP depletion found in vivo is sufficient to account for the extensive degree of cell death that occurs after birth. Under hypoxic conditions, the immature ductus is more capable of preserving its ATP supply than the mature ductus as a result of increased glucose availability, glycogen stores, and glucose utilization. However, the immature ductus is just as susceptible as the mature ductus to ATP depletion when glucose supplies are restricted. The extensive degree of cell death that occurs in the newborn ductus after birth is associated primarily with ATP depletion. The increased glycolytic capacity of the immature ductus may enable it to tolerate episodes of hypoxia and nutrient shortage, making it more resistant to developing postnatal cell death and permanent closure. PMID:15774827

  11. Photosynthetic regeneration of ATP using a strain of thermophilic blue-green algae

    SciTech Connect

    Sawa, Y.; Kanayama, K.; Ochiai, H.

    1982-02-01

    Photosynthetic ATP accumulation was shown in the presence of exogenous ADP plus ortho-phosphate on illumination to the intact cells of a strain of thermophilic blue-green algae isolated from Matsue hot springs, Mastigocladus sp. Kinetic studies of various effectors on the ATP accumulation proved that the ATP synthesis depends mainly on the cyclic photophosphorylation system around photosystem I (PS-I) in the algal cells. The temperature and pH optima for the accumulation were found at 45 degrees C and pH 7.5. Maximum yield was obtained with light intensity higher than 15 mW/squared cm. Borate ion exerted pronounced enhancement on the ATP synthesis. With a continuous reactor at a flow rate of 1 ml/hour at 45 degrees C and pH 7.5, efficient photoconversion of ADP (2mM, at substrate reservoir) to ATP (1mM, at product outlet) has been maintained for a period of 2.5 days, though the efficiency has decreased in a further 2-day period to the level of 0.5 mM ATP/9.5 h of residence time. (Refs. 24).

  12. Basal Release of ATP: An Autocrine-Paracrine Mechanism for Cell Regulation

    NSDL National Science Digital Library

    Ross Corriden (San Diego; University of California REV)

    2010-01-12

    Virtually every type of eukaryotic cell is regulated by adenosine triphosphate (ATP) and other nucleotides, such as uridine triphosphate (UTP), that the cells release themselves (autocrine signaling) or that are released by neighboring cells (paracrine signaling). Signaling in response to released nucleotides occurs through the activation of plasma membrane–localized P2 receptors: the P2X ion channels and the P2Y heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors. Release of ATP and alteration of cellular function also occur under “basal” conditions. Such basal release of ATP, which can be increased by minimal perturbation of cells through physical or chemical stimuli, influences a wide array of physiological events, including tissue blood flow, ion transport, growth and metastatic potential of malignant cells, endocrine and neuronal activity, neural development, musculoskeletal and renal function, stem cell proliferation, and response to pathogens. This Review, which contains two figures and 216 references, discusses the diverse array of receptors for ATP (and its major hydrolytic product adenosine) and the range of enzymes (including ecto–adenosine triphosphatases and kinases) that regulate extracellular concentrations of ATP, which create a highly versatile and tightly regulated system for the regulation of cell and tissue function by extracellular ATP that is derived from intracellular pools of nucleotides.

  13. Target volume definition for external beam partial breast radiotherapy: Clinical, pathological and technical studies informing current approaches

    Microsoft Academic Search

    Anna M. Kirby; Charlotte E. Coles; John R. Yarnold

    2010-01-01

    Partial breast irradiation (PBI) is currently under investigation in several phase III trials and, following a recent consensus statement, its use off-study may increase despite ongoing uncertainty regarding optimal target volume definition. We review the clinical, pathological and technical evidence for target volume definition in external beam partial breast irradiation (EB-PBI). The optimal method of tumour bed (TB) delineation requires

  14. SEDS experiment design definition

    NASA Technical Reports Server (NTRS)

    Carroll, Joseph A.; Alexander, Charles M.; Oldson, John C.

    1990-01-01

    The Small Expendable-tether Deployment System (SEDS) was developed to design, build, integrate, fly, and safely deploy and release an expendable tether. A suitable concept for an on-orbit test of SEDS was developed. The following tasks were performed: (1) Define experiment objectives and requirements; (2) Define experiment concepts to reach those objectives; (3) Support NASA in experiment concept selection and definition; (4) Perform analyses and tests of SEDS hardware; (5) Refine the selected SEDS experiment concept; and (6) Support interactive SEDS system definition process. Results and conclusions are given.

  15. DEFINITION FOR ASBESTOS.

    USGS Publications Warehouse

    Ross, Malcolm; Kuntze, Richard A.; Clifton, Robert A.

    1984-01-01

    A definition of asbestos is proposed. Under this definition, the term asbestos applies to six naturally occurring minerals exploited commercially for their desirable physical properties, which are in part derived from their asbestiform habit. The six minerals are the serpentine mineral chrysotile and the amphibole minerals grunerite asbestos (also referred to as amosite), riebeckite asbestos (also referred to as crocidolite), anthophyllite asbestos, tremolite asbestos, and actinolite asbestos. Individual mineral particles, however processed and regardless of their mineral name, are not demonstrated to be asbestos if the length-to-width ratio is less than 20:1.

  16. ATP Receptors Gate Microglia Signaling in Neuropathic Pain

    PubMed Central

    Trang, Tuan; Beggs, Simon; Salter, Michael W.

    2013-01-01

    Microglia were described by Pio del Rio-Hortega (1932) as being the ‘third element’ distinct from neurons and astrocytes. Decades after this observation, the function and even the very existence of microglia as a distinct cell type was a topic of intense debate and conjecture. However, considerable advances have been made towards understanding the neurobiology of microglia resulting in a radical shift in our view of them as being passive bystanders that have solely immune and supportive roles, to being active principal players that contribute to central nervous system pathologies caused by disease or following injury. Converging lines of evidence implicate microglia as being essential in the pathogenesis of neuropathic pain, a debilitating chronic pain condition that can occur after peripheral nerve damage caused by disease, infection, or physical injury. A key molecule that modulates microglial activity is ATP, an endogenous ligand of the P2-purinoceptor family consisting of P2X ionotropic and P2Y metabotropic receptors. Microglia express several P2 receptor subtypes, and of these the P2X4, P2X7, and P2Y12 receptor subtypes have been implicated in neuropathic pain. The P2X4 receptor has emerged as the core microglia-neuron signaling pathway: activation of this receptor causes release of brain-derived neurotrophic factor (BDNF) which causes disinhibition of pain-transmission neurons in spinal lamina I. The present review highlights recent advances in understanding the signaling and regulation of P2 receptors expressed in microglia and the implications for microglia-neuron interactions for the management of neuropathic pain. PMID:22116040

  17. ATP citrate lyase knockdown impacts cancer stem cells in vitro

    PubMed Central

    Hanai, J-i; Doro, N; Seth, P; Sukhatme, V P

    2013-01-01

    ATP citrate lyase (ACL) knockdown (KD) causes tumor suppression and induces differentiation. We have previously reported that ACL KD reverses epithelial–mesenchymal transition (EMT) in lung cancer cells. Because EMT is often associated with processes that induce stemness, we hypothesized that ACL KD impacts cancer stem cells. By assessing tumorsphere formation and expression of stem cell markers, we showed this to be the case in A549 cells, which harbor a Ras mutation, and in two other non-small-cell lung cancer cell lines, H1975 and H1650, driven by activating EGFR mutations. Inducible ACL KD had the same effect as stable ACL KD. Similar effects were noted in another well-characterized Ras-induced mammary model system (HMLER). Moreover, treatment with hydroxycitrate phenocopied the effects of ACL KD, suggesting that the enzymatic activity of ACL was critical. Indeed, acetate treatment reversed the ACL KD phenotype. Having previously established that ACL KD impacts signaling through the phosphatidylinositol 3-kinase (PI3K) pathway, not the Ras-mitogen-activated protein kinase (MAPK) pathway, and that EMT can be reversed by PI3K inhibitors, we were surprised to find that stemness in these systems was maintained through Ras-MAPK signaling, and not via PI3K signaling. Snail is a downstream transcription factor impacted by Ras-MAPK signaling and known to promote EMT and stemness. We found that snail expression was reduced by ACL KD. In tumorigenic HMLER cells, ACL overexpression increased snail expression and stemness, both of which were reduced by ACL KD. Furthermore, ACL could not initiate either tumorigenesis or stemness by itself. ACL and snail proteins interacted and ACL expression regulated the transcriptional activity of snail. Finally, ACL KD counteracted stem cell characteristics induced in diverse cell systems driven by activation of pathways outside of Ras-MAPK signaling. Our findings unveil a novel aspect of ACL function, namely its impact on cancer stemness in a broad range of genetically diverse cell types. PMID:23807225

  18. ATP-binding cassette transporters, atherosclerosis, and inflammation.

    PubMed

    Westerterp, Marit; Bochem, Andrea E; Yvan-Charvet, Laurent; Murphy, Andrew J; Wang, Nan; Tall, Alan R

    2014-01-01

    Although recent genome-wide association studies have called into question the causal relationship between high-density lipoprotein (HDL) cholesterol levels and cardiovascular disease, ongoing research in animals and cells has produced increasing evidence that cholesterol efflux pathways mediated by ATP-binding cassette (ABC) transporters and HDL suppress atherosclerosis. These differing perspectives may be reconciled by a modified HDL theory that emphasizes the antiatherogenic role of cholesterol flux pathways, initiated in cells by ABC transporters. ABCA1 and ABCG1 control the proliferation of hematopoietic stem and multipotential progenitor cells in the bone marrow and hematopoietic stem and multipotential progenitor cell mobilization and extramedullary hematopoiesis in the spleen. Thus, activation of cholesterol efflux pathways by HDL infusions or liver X receptor activation results in suppression of hematopoietic stem and multipotential progenitor cell mobilization and extramedullary hematopoiesis, leading to decreased production of monocytes and neutrophils and suppression of atherosclerosis. In addition, macrophage-specific knockout of transporters has confirmed their role in suppression of inflammatory responses in the arterial wall. Recent studies have also shown that ABCG4, a close relative of ABCG1, controls platelet production, atherosclerosis, and thrombosis. ABCG4 is highly expressed in megakaryocyte progenitors, where it promotes cholesterol efflux to HDL and controls the proliferative responses to thrombopoietin. Reconstituted HDL infusions act in an ABCG4-dependent fashion to limit hypercholesterolemia-driven excessive platelet production, thrombosis, and atherogenesis, as occurs in human myeloproliferative syndromes. Activation of ABC transporter-dependent cholesterol efflux pathways in macrophages, hematopoietic stem and multipotential progenitor cells, or platelet progenitors by reconstituted HDL infusion or liver X receptor activation remain promising approaches to the treatment of human atherothrombotic diseases. PMID:24385509

  19. Introducing the human Leigh syndrome mutation T9176G into Saccharomyces cerevisiae mitochondrial DNA leads to severe defects in the incorporation of Atp6p into the ATP synthase and in the mitochondrial morphology.

    PubMed

    Kucharczyk, Roza; Salin, Bénédicte; di Rago, J-P

    2009-08-01

    The Leigh syndrome is a severe neurological disorder that has been associated with mutations affecting the mitochondrial energy transducing system. One of these mutations, T9176G, has been localized in the mitochondrial ATP6 gene encoding the Atp6p (or a) subunit of the ATP synthase. This mutation converts a highly conserved leucine residue into arginine within a presumed trans-membrane alpha-helical segment, at position 217 of Atp6p. The T9176G mutation was previously shown to severely reduce the rate of mitochondrial ATP production in cultured human cells containing high loads of this mutation. However, the underlying mechanism responsible for the impaired ATP production is still unknown. To better understand how T9176G affects the ATP synthase, we have created and analyzed the properties of a yeast strain bearing an equivalent of this mutation. We show that incorporation of Atp6p within the ATP synthase was almost completely prevented in the modified yeast. Based on previous partial biochemical characterization of human T9176G cells, it is likely that this mutation similarly affects the human ATP synthase instead of causing a block in the rotary mechanism of this enzyme as it had been suggested. Interestingly, the T9176G yeast exhibits important anomalies in mitochondrial morphology, an observation which indicates that the pathogenicity of T9176G may not be limited to a bioenergetic deficiency. PMID:19454486

  20. On the Stator of Rotary ATP Synthase: The Binding Strength of Subunit to (R )3 As Determined by Fluorescence Correlation Spectroscopy

    E-print Network

    Junge, Wolfgang

    On the Stator of Rotary ATP Synthase: The Binding Strength of Subunit to (R )3 As Determined; ReVised Manuscript ReceiVed July 20, 1999 ABSTRACT: ATP synthase is conceived as a rotary enzyme2c12. ATP synthase is conceived as a molecular engine (3-5). The hydrolysis of ATP drives

  1. 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: ...

  2. ATP activates non-selective cation channels and calcium release in inner hair cells of the guinea-pig cochlea.

    PubMed Central

    Sugasawa, M; Erostegui, C; Blanchet, C; Dulon, D

    1996-01-01

    1. ATP-evoked currents and Ca2+ signals were simultaneously recorded in isolated inner hair cells (IHC) of guinea-pig cochlea by combining conventional whole-cell or perforated patch clamp recording with indo-1 dual emission microfluorometry. 2. In most IHCs, voltage clamped near resting membrane potential (-40 mV), extracellular ATP evoked a rapid inward current (time constant, 150 ms). This current was concomitant with a slow rise in [Ca2+]i (time constant, 5 s). The ATP-evoked inward currents could be repeated several times with only a small run-down in amplitude (< 10%), while the ATP-evoked Ca2+ responses showed a rapid run-down (> 80% at the third ATP application). 3. The current-voltage relationship of ATP-evoked currents showed a reversal potential at -11 +/- 6 mV (n = 8), suggesting that ATP essentially activated a non-specific cationic conductance. On the contrary, the amplitude of the ATP-evoked Ca2+ responses did not show significant dependence on holding membrane potential. 4. The Ca2+ response showed an apparent Kd for ATP (EC50, 1.8 +/- 0.3 microM; Hill coefficient, 1.0 +/- 0.1) eightfold smaller than for the evoked currents (EC50, 13.7 +/- 3.0 microM; Hill coefficient, 2.0 +/- 0.7). 5. Perfusion with high extracellular Ca2+ solution (10 mM CaCl2) reduced the amplitude of the ATP-evoked currents by 90%, while perfusion with zero Ca2+ solution increased it by more than 100%. However, similar variations in external Ca2+ concentration did not change the amplitude of the ATP-evoked Ca2+ responses. Furthermore, intracellular heparin (1 mg mL-1), a potent inhibitor of InsP3 receptors, did not significantly change the amplitude of ATP-evoked currents but reduced the ATP-evoked Ca2+ response, suggesting again that the latter is related to Ca2+ release from intracellular stores. 6. The results suggested that two types of P2-purinergic receptor are expressed in IHCs: ATP-gated ion channels and ATP-activated metabotropic receptors. At submicromolar ATP concentrations, the metabotropic receptors raising intracellular [Ca2+] would hyperpolarize IHCs via Ca(2+)-sensitive K+ channels. The ATP-gated ion channels activated at higher ATP concentrations would mainly have a depolarizing effect on IHCs. Images Figure 8 PMID:8815205

  3. Cloning and molecular characterization of the atp operon encoding for the F1F0-ATP synthase from a thermoalkaliphilic Bacillus sp. strain TA2.A1.

    PubMed

    Keis, Stefanie; Kaim, Georg; Dimroth, Peter; Cook, Gregory M

    2004-01-01

    The genes encoding the subunits for the F(1)F(0)-ATP synthase from Bacillus sp. strain TA2.A1 were cloned as three overlapping fragments and sequenced. The nine genes were organized in an operon with the gene order atpIBEFHAGDC encoding the i, a, c, b, delta, alpha, gamma, beta, and epsilon subunits, respectively. Northern blot analysis showed a maximum transcript of approximately 7.2 kb, which corresponds to the size of the atp operon and demonstrated that the nine genes are transcribed as a single polycistronic message. The alkaliphilic-specific residues Lys(218) and Gly(245) were conserved in subunit a of strain TA2.A1. Analysis of the C-terminal domain of the epsilon subunit showed several clusters of basic residues which are predicted to form a strong electrostatic interaction with the DELSDED motif in the beta subunit from strain TA2.A1, and may explain the blockage of this enzyme in the ATP hydrolysis direction. PMID:14732496

  4. ATP Synthase and the Actions of Inhibitors Utilized To Study Its Roles in Human Health, Disease, and Other Scientific Areas

    PubMed Central

    Hong, Sangjin; Pedersen, Peter L.

    2008-01-01

    Summary: ATP synthase, a double-motor enzyme, plays various roles in the cell, participating not only in ATP synthesis but in ATP hydrolysis-dependent processes and in the regulation of a proton gradient across some membrane-dependent systems. Recent studies of ATP synthase as a potential molecular target for the treatment of some human diseases have displayed promising results, and this enzyme is now emerging as an attractive molecular target for the development of new therapies for a variety of diseases. Significantly, ATP synthase, because of its complex structure, is inhibited by a number of different inhibitors and provides diverse possibilities in the development of new ATP synthase-directed agents. In this review, we classify over 250 natural and synthetic inhibitors of ATP synthase reported to date and present their inhibitory sites and their known or proposed modes of action. The rich source of ATP synthase inhibitors and their known or purported sites of action presented in this review should provide valuable insights into their applications as potential scaffolds for new therapeutics for human and animal diseases as well as for the discovery of new pesticides and herbicides to help protect the world's food supply. Finally, as ATP synthase is now known to consist of two unique nanomotors involved in making ATP from ADP and Pi, the information provided in this review may greatly assist those investigators entering the emerging field of nanotechnology. PMID:19052322

  5. ATP synthase and the actions of inhibitors utilized to study its roles in human health, disease, and other scientific areas.

    PubMed

    Hong, Sangjin; Pedersen, Peter L

    2008-12-01

    ATP synthase, a double-motor enzyme, plays various roles in the cell, participating not only in ATP synthesis but in ATP hydrolysis-dependent processes and in the regulation of a proton gradient across some membrane-dependent systems. Recent studies of ATP synthase as a potential molecular target for the treatment of some human diseases have displayed promising results, and this enzyme is now emerging as an attractive molecular target for the development of new therapies for a variety of diseases. Significantly, ATP synthase, because of its complex structure, is inhibited by a number of different inhibitors and provides diverse possibilities in the development of new ATP synthase-directed agents. In this review, we classify over 250 natural and synthetic inhibitors of ATP synthase reported to date and present their inhibitory sites and their known or proposed modes of action. The rich source of ATP synthase inhibitors and their known or purported sites of action presented in this review should provide valuable insights into their applications as potential scaffolds for new therapeutics for human and animal diseases as well as for the discovery of new pesticides and herbicides to help protect the world's food supply. Finally, as ATP synthase is now known to consist of two unique nanomotors involved in making ATP from ADP and P(i), the information provided in this review may greatly assist those investigators entering the emerging field of nanotechnology. PMID:19052322

  6. ATP binding to two sites is necessary for dimerization of nucleotide-binding domains of ABC proteins

    PubMed Central

    Zoghbi, Maria E.; Altenberg, Guillermo A.

    2014-01-01

    ATP binding cassette (ABC) transporters have a functional unit formed by two transmembrane domains and two nucleotide binding domains (NBDs). ATP-bound NBDs dimerize in a head-to-tail arrangement, with two nucleotides sandwiched at the dimer interface. Both NBDs contribute residues to each of the two nucleotide-binding sites (NBSs) in the dimer. In previous studies, we showed that the prototypical NBD MJ0796 from M. jannaschii forms ATP-bound dimers that dissociate completely following hydrolysis of one of the two bound ATP molecules. Since hydrolysis of ATP at one NBS is sufficient to drive dimer dissociation, it is unclear why all ABC proteins contain two NBSs. Here, we used luminescence resonance energy transfer (LRET) to study ATP-induced formation of NBD homodimers containing two NBSs competent for ATP binding, and NBD heterodimers with one active NBS (acceptor-labeled) and one binding-defective NBS (donor-labeled). The results showed that binding of two ATP molecules is necessary for NBD dimerization. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dissociation, but two binding sites are required to form the ATP-sandwich NBD dimer necessary for hydrolysis. PMID:24269240

  7. Timely binding of IHF and Fis to DARS2 regulates ATP–DnaA production and replication initiation

    PubMed Central

    Kasho, Kazutoshi; Fujimitsu, Kazuyuki; Matoba, Toshihiro; Oshima, Taku; Katayama, Tsutomu

    2014-01-01

    In Escherichia coli, the ATP-bound form of DnaA (ATP–DnaA) promotes replication initiation. During replication, the bound ATP is hydrolyzed to ADP to yield the ADP-bound form (ADP–DnaA), which is inactive for initiation. The chromosomal site DARS2 facilitates the regeneration of ATP–DnaA by catalyzing nucleotide exchange between free ATP and ADP bound to DnaA. However, the regulatory mechanisms governing this exchange reaction are unclear. Here, using in vitro reconstituted experiments, we show that two nucleoid-associated proteins, IHF and Fis, bind site-specifically to DARS2 to activate coordinately the exchange reaction. The regenerated ATP–DnaA was fully active in replication initiation and underwent DnaA–ATP hydrolysis. ADP–DnaA formed heteromultimeric complexes with IHF and Fis on DARS2, and underwent nucleotide dissociation more efficiently than ATP–DnaA. Consistently, mutant analyses demonstrated that specific binding of IHF and Fis to DARS2 stimulates the formation of ATP–DnaA production, thereby promoting timely initiation. Moreover, we show that IHF–DARS2 binding is temporally regulated during the cell cycle, whereas Fis only binds to DARS2 in exponentially growing cells. These results elucidate the regulation of ATP–DnaA and replication initiation in coordination with the cell cycle and growth phase. PMID:25378325

  8. The Definition of Abstinence

    Microsoft Academic Search

    Angela Nicoletti

    2005-01-01

    A teen's definition of sexual activity most often does not include oral or anal sex. Abstinence only programs vary widely as to how they define sexual behavior and may be contributing to misinformation about STD transmission. Unknown is the extent to which declining teen pregnancy rates are due to non-coital activities replacing vaginal intercourse.

  9. FAMILY MEDICINE* Definition Of

    E-print Network

    Finley Jr., Russell L.

    FAMILY MEDICINE* Definition Of Family medicine is the medical specialty which provides continuing the biological, clinical and behavioral sciences. The scope of family medicine encompasses all ages, both sexes, each organ system and every disease entity. (1986) (2003) Quality Healthcare In Family Medicine Quality

  10. Definition Is it real?

    E-print Network

    Boisvert, Jeff

    declines Oil Discoveries #12;01-4 Important points · No more very large oil fields. Nearly all new oil01-1 · Definition · Is it real? · Consequences MinE 422: Peak Oil Background · How an oil field has that name for a reason, it is difficult oil · Alternatives ­ Natural gas ­ Coal ­ Biofuels

  11. Developmental Education: A Definition.

    ERIC Educational Resources Information Center

    Beckett, Gene

    A discussion is provided of the distinguishing characteristics of developmental education. First, the reasons for the lack of a clear definition are discussed, and the consequences of this ambiguity are highlighted in terms of the existing confusion over the purposes of developmental education. Next, the linguistic and theoretical bases of…

  12. 3 Library Regulations Definitions

    E-print Network

    Mottram, Nigel

    3 Library Regulations Definitions In Regulation 3: 'Library' means the University Library as defined in Regulation 3.1; 'Library staff' means the staff of the University Library; 'Librarian' means the University Librarian and Head of Information Resources Directorate or nominee; `Library Committee' means

  13. Hypoxia is an effective stimulus for vesicular release of ATP from human umbilical vein endothelial cells

    PubMed Central

    Lim To, W.K.; Kumar, P.; Marshall, J.M.

    2015-01-01

    Introduction Hypoxia induces dilatation of the umbilical vein by releasing autocoids from endothelium; prostaglandins (PGs), adenosine and nitric oxide (NO) have been implicated. ATP is vasoactive, thus we tested whether hypoxia releases ATP from primary Human Umbilical Vein Endothelial Cells (HUVEC). Methods HUVEC were grown on inserts under no-flow conditions. ATP was assayed by luciferin–luciferase and visualised by quinacrine labeling. Intracellular Ca2+ ([Ca2+]i) was imaged with Fura-2. Results ATP release occurred constitutively and was increased by hypoxia (PO2: 150–8 mmHg), ?10-fold more from apical, than basolateral surface. Constitutive ATP release was decreased, while hypoxia-induced release was abolished by brefeldin or monensin A, inhibitors of vesicular transport, and LY294002 or Y27632, inhibitors of phosphoinositide 3-kinases (PI3K) and Rho-associated protein kinase (ROCK). ATP release was unaffected by NO donor, but increased by calcium ionophore, by >60-fold from apical, but <25% from basolateral surface. Hypoxia induced a small increase in [Ca2+]i compared with ATP (10 ?M); hypoxia inhibited the ATP response. Quinacrine-ATP fluorescent loci in the perinuclear space, were diminished by hypoxia and monensin, whereas brefeldin A increased fluorescence intensity, consistent with inhibition of anterograde transport. Discussion. Hypoxia within the physiological range releases ATP from HUVEC, particularly from apical/adluminal surfaces by exocytosis, via an increase in [Ca2+]i, PI3K and ROCK, independently of NO. We propose that hypoxia releases ATP at concentrations sufficient to induce umbilical vein dilation via PGs and NO and improve fetal blood flow, but curbs amplification of ATP release by autocrine actions of ATP, so limiting its pro-inflammatory effects. PMID:25956988

  14. Involvement of the cystic fibrosis transmembrane conductance regulator in the acidosis-induced efflux of ATP from rat skeletal muscle

    PubMed Central

    Tu, Jie; Le, Gengyun; Ballard, Heather J

    2010-01-01

    The present study was performed to investigate the effect of acidosis on the efflux of ATP from skeletal muscle. Infusion of lactic acid to the perfused hindlimb muscles of anaesthetised rats produced dose-dependent decreases in pH and increases in the interstitial ATP of extensor digitorum longus (EDL) muscle: 10 mm lactic acid reduced the venous pH from 7.22 ± 0.04 to 6.97 ± 0.02 and increased interstitial ATP from 38 ± 8 to 67 ± 11 nm. The increase in interstitial ATP was well-correlated with the decrease in pH (r2 = 0.93; P < 0.05). Blockade of cellular uptake of lactic acid using ?-cyano-hydroxycinnamic acid abolished the lactic acid-induced ATP release, whilst infusion of sodium lactate failed to depress pH or increase interstitial ATP, suggesting that intracellular pH depression, rather than lactate, stimulated the ATP efflux. Incubation of cultured skeletal myoblasts with 10 mm lactic acid significantly increased the accumulation of ATP in the bathing medium from 0.46 ± 0.06 to 0.76 ± 0.08 ?m, confirming the skeletal muscle cells as the source of the released ATP. Acidosis-induced ATP efflux from the perfused muscle was abolished by CFTRinh-172, a specific inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR), or glibenclamide, an inhibitor of both KATP channels and CFTR, but it was not affected by atractyloside, an inhibitor of the mitochondrial ATP transporter. Silencing of the CFTR gene using an siRNA abolished the acidosis-induced increase in ATP release from cultured myoblasts. CFTR expression on skeletal muscle cells was confirmed using immunostaining in the intact muscle and Western blotting in the cultured cells. These data suggest that depression of the intracellular pH of skeletal muscle cells stimulates ATP efflux, and that CFTR plays an important role in the release mechanism. PMID:20819945

  15. Bacterial Na+ - or H+ -coupled ATP synthases operating at low electrochemical potential.

    PubMed

    Dimroth, Peter; Cook, Gregory M

    2004-01-01

    In certain strictly anaerobic bacteria, the energy for growth is derived entirely from a decarboxylation reaction. A prominent example is Propionigenium modestum, which converts the free energy of the decarboxylation of (S)-methylmalonyl-CoA to propionyl-CoA (DeltaG degrees =-20.6 kJ/mol) into an electrochemical Na(+) ion gradient across the membrane. This energy source is used as a driving force for ATP synthesis by a Na(+)-translocating F(1)F(0) ATP synthase. According to bioenergetic considerations, approximately four decarboxylation events are necessary to support the synthesis of one ATP. This unique feature of using Na(+) instead of H(+) as the coupling ion has made this ATP synthase the paradigm to study the ion pathway across the membrane and its relationship to rotational catalysis. The membrane potential (Deltapsi) is the key driving force to convert ion translocation through the F(0) motor components into torque. The resulting rotation elicits conformational changes at the catalytic sites of the peripheral F(1) domain which are instrumental for ATP synthesis. Alkaliphilic bacteria also face the challenge of synthesizing ATP at a low electrochemical potential, but for entirely different reasons. Here, the low potential is not the result of insufficient energy input from substrate degradation, but of an inverse pH gradient. This is a consequence of the high environmental pH where these bacteria grow and the necessity to keep the intracellular pH in the neutral range. In spite of this unfavorable bioenergetic condition, ATP synthesis in alkaliphilic bacteria is coupled to the proton motive force (DeltamuH(+)) and not to the much higher sodium motive force (DeltamuNa(+)). A peculiar feature of the ATP synthases of alkaliphiles is the specific inhibition of their ATP hydrolysis activity. This inhibition appears to be an essential strategy for survival at high external pH: if the enzyme were to operate as an ATPase, protons would be pumped outwards to counteract the low DeltamuH(+), thus wasting valuable ATP and compromising acidification of the cytoplasm at alkaline pH. PMID:15518831

  16. Population III by Popular Demand - Progress and Previews

    E-print Network

    Timothy C. Beers

    1999-11-10

    I discuss the ongoing search for stars of the Milky Way which have been referred to as members of Population III. Following a discussion of possible definitions for these stars, I consider the reasons why astronomers have undertaken this search, and list some of the numerous astrophysical uses of the extremely metal-poor stars found along the way. I then review survey techniques which have been used in the past, and provide an update on plans for future investigations. Finally, the question of when one might consider the search for Population III Finished is addressed.

  17. The Typically Mitochondrial DNA-encoded ATP6 Subunit of the F1F0ATPase Is Encoded by a Nuclear Gene in Chlamydomonas reinhardtii

    Microsoft Academic Search

    Soledad Funes; Edgar Davidson; M. Gonzalo Claros; Robert van Lis; Xochitl Perez-Martõ ´ nez; Miriam Vazquez-Acevedo; Michael P. King; Diego Gonzalez-Halphen

    2002-01-01

    The atp6 gene, encoding the ATP6 subunit of F1F0- ATP synthase, has thus far been found only as an mtDNA-encoded gene. However, atp6 is absent from mtDNAs of some species, including that of Chlamydomo- nas reinhardtii. Analysis of C. reinhardtii expressed se- quence tags revealed three overlapping sequences that encoded a protein with similarity to ATP6 proteins. PCR and 5-

  18. Persistence of Biomarker ATP and ATP-Generating Capability in Bacterial Cells and Spores Contaminating Spacecraft Materials under Earth Conditions and in a Simulated Martian Environment?

    PubMed Central

    Fajardo-Cavazos, Patricia; Schuerger, Andrew C.; Nicholson, Wayne L.

    2008-01-01

    Most planetary protection research has concentrated on characterizing viable bioloads on spacecraft surfaces, developing techniques for bioload reduction prior to launch, and studying the effects of simulated martian environments on microbial survival. Little research has examined the persistence of biogenic signature molecules on spacecraft materials under simulated martian surface conditions. This study examined how endogenous adenosine-5?-triphosphate (ATP) would persist on aluminum coupons under simulated martian conditions of 7.1 mbar, full-spectrum simulated martian radiation calibrated to 4 W m?2 of UV-C (200 to 280 nm), ?10°C, and a Mars gas mix of CO2 (95.54%), N2 (2.7%), Ar (1.6%), O2 (0.13%), and H2O (0.03%). Cell or spore viabilities of Acinetobacter radioresistens, Bacillus pumilus, and B. subtilis were measured in minutes to hours, while high levels of endogenous ATP were recovered after exposures of up to 21 days. The dominant factor responsible for temporal reductions in viability and loss of ATP was the simulated Mars surface radiation; low pressure, low temperature, and the Mars gas composition exhibited only slight effects. The normal burst of endogenous ATP detected during spore germination in B. pumilus and B. subtilis was reduced by 1 or 2 orders of magnitude following, respectively, 8- or 30-min exposures to simulated martian conditions. The results support the conclusion that endogenous ATP will persist for time periods that are likely to extend beyond the nominal lengths of most surface missions on Mars, and planetary protection protocols prior to launch may require additional rigor to further reduce the presence and abundance of biosignature molecules on spacecraft surfaces. PMID:18567687

  19. Mitochondrial ATP synthase catalytic mechanism: a novel visual comparative structural approach emphasizes pivotal roles for Mg²? and P-loop residues in making ATP.

    PubMed

    Blum, David J; Ko, Young H; Pedersen, Peter L

    2012-02-21

    The mitochondrial ATP synthase (F(o)F(1)) is one of the most abundant, important, and complex enzymes found in animals and humans. In earlier studies, we used the photosensitive phosphate analogue vanadate (V(i)) to study the enzyme's mechanism in the transition state. Significantly, these studies showed that Mg(2+) plays an important role in transition state formation during ATP synthesis. Additionally, in both MgADP·V(i)-F(1) and MgV(i)-F(1) complexes, photoactivation of orthovanadate (V(i)) induced cleavage at the third residue within the P-loop (GGAGVGKT), i.e., ?A158, suggesting its proximity to the ?-phosphate during transition state formation. However, despite our recent release of the F(1)-ATPase structure containing V(i), the structural details regarding the role of Mg(2+) have remained elusive. Therefore, in this study, we sought to improve our understanding of the essential role of Mg(2+) during transition state formation. We utilized Protein Data Bank structural data representing different conformational intermediates of key steps in ATP synthesis to assemble a database of positional relationships between landmark residues of the catalytic site and the bound ligand. Applying novel bioinformatics methods, we combined the resulting interatomic spatial data with an animated model of the catalytic site to visualize the exact nature of the changes in these positional relationships during ATP synthesis. The results of these studies reported here show that the absence of Mg(2+) results in migration of inorganic phosphate (P(i)) from ?A158 to a more medial position in the P-loop binding pocket, thereby disrupting essential placement and orientation of the P(i) needed to form the transition state structure and therefore MgATP. PMID:22243519

  20. The aryl hydrocarbon receptor interacts with ATP5{alpha}1, a subunit of the ATP synthase complex, and modulates mitochondrial function

    SciTech Connect

    Tappenden, Dorothy M.; Lynn, Scott G. [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824-1319 (United States); Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824-1319 (United States); Crawford, Robert B. [Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI 48824-1319 (United States); Lee, KangAe; Vengellur, Ajith [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824-1319 (United States); Kaminski, Norbert E. [Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824-1319 (United States); Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI 48824-1319 (United States); Thomas, Russell S. [The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709 (United States); LaPres, John J., E-mail: lapres@msu.edu [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824-1319 (United States); Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824-1319 (United States); Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, MI 48824-1319 (United States)

    2011-08-01

    Dioxins, including 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), produce a wide range of toxic effects in mammals. Most, if not all, of these toxic effects are regulated by the aryl hydrocarbon receptor (AHR). The AHR is a ligand activated transcription factor that has been shown to interact with numerous proteins capable of influencing the receptor's function. The ability of secondary proteins to alter AHR-mediated transcriptional events, a necessary step for toxicity, led us to determine whether additional interacting proteins could be identified. To this end, we have employed tandem affinity purification (TAP) of the AHR in Hepa1c1c7 cells. TAP of the AHR, followed by mass spectrometry (MS) identified ATP5{alpha}1, a subunit of the ATP synthase complex, as a strong AHR interactor in the absence of ligand. The interaction was lost upon exposure to TCDD. The association was confirmed by co-immunoprecipitation in multiple cell lines. In addition, cell fractionation experiments showed that a fraction of the AHR is found in the mitochondria. To ascribe a potential functional role to the AHR:ATP5{alpha}1 interaction, TCDD was shown to induce a hyperpolarization of the mitochondrial membrane in an AHR-dependent and transcription-independent manner. These results suggest that a fraction of the total cellular AHR pool is localized to the mitochondria and contributes to the organelle's homeostasis. - Highlights: > The AHR interacts with the mitochondrial protein, ATP5{alpha}1. > Cell fractionation experiments show that the AHR can be found in the mitochondria. > TCDD-exposure induces a hyperpolarization of the mitochondrial inner membrane. > The hyperpolarization is AHR-dependent. > The hyperpolarization occurs without altering ATP levels within the cell.

  1. Features of Idebenone and Related Short-Chain Quinones that Rescue ATP Levels under Conditions of Impaired Mitochondrial Complex I

    PubMed Central

    Erb, Michael; Hoffmann-Enger, Barbara; Deppe, Holger; Soeberdt, Michael; Haefeli, Roman H.; Rummey, Christian; Feurer, Achim; Gueven, Nuri

    2012-01-01

    Short-chain quinones have been investigated as therapeutic molecules due to their ability to modulate cellular redox reactions, mitochondrial electron transfer and oxidative stress, which are pathologically altered in many mitochondrial and neuromuscular disorders. Recently, we and others described that certain short-chain quinones are able to bypass a deficiency in complex I by shuttling electrons directly from the cytoplasm to complex III of the mitochondrial respiratory chain to produce ATP. Although this energy rescue activity is highly interesting for the therapy of disorders associated with complex I dysfunction, no structure-activity-relationship has been reported for short-chain quinones so far. Using a panel of 70 quinones, we observed that the capacity for this cellular energy rescue as well as their effect on lipid peroxidation was influenced more by the physicochemical properties (in particular logD) of the whole molecule than the quinone moiety itself. Thus, the observed correlations allow us to explain the differential biological activities and therapeutic potential of short-chain quinones for the therapy of disorders associated with mitochondrial complex I dysfunction and/or oxidative stress. PMID:22558363

  2. APPENDIX C. USEFUL DEFINITE INTEGRALS 1 Useful Definite Integrals

    E-print Network

    Callen, James D.

    APPENDIX C. USEFUL DEFINITE INTEGRALS 1 Appendix C Useful Definite Integrals Definite integrals that often arise in plasma physics are summarized in this appendix. C.1 Integrals Involving A Decaying/) often result in integrals of the form 0 dt tn e-t/ = n+1 0 dx xn e-x (C.1) in which x t/. The most

  3. High-Intensity Interval Training Alters ATP Pathway Flux During Maximal Muscle Contractions in Humans

    PubMed Central

    Larsen, Ryan G.; Maynard, Logan; Kent, Jane A.

    2014-01-01

    Aim High-intensity interval training (HIT) results in potent metabolic adaptations in skeletal muscle, however little is known about the influence of these adaptations on energetics in vivo. We used magnetic resonance spectroscopy to examine the effects of HIT on ATP synthesis from net PCr breakdown (ATPCK), oxidative phosphorylation (ATPOX) and non-oxidative glycolysis (ATPGLY) in vivo in vastus lateralis during a 24-s maximal voluntary contraction (MVC). Methods Eight young men performed 6 sessions of repeated, 30-s “all-out” sprints on a cycle ergometer; measures of muscle energetics were obtained at baseline, and after the first and sixth sessions. Results Training increased peak oxygen consumption (35.8±1.4 to 39.3±1.6 ml·min?1·kg?1, p=0.01) and exercise capacity (217.0±11.0 to 230.5±11.7 W, p=0.04) on the ergometer, with no effects on total ATP production or force-time integral during the MVC. While ATP production by each pathway was unchanged after the first session, 6 sessions increased the relative contribution of ATPOX (from 31±2 to 39±2% of total ATP turnover, p<0.001), and lowered the relative contribution from both ATPCK (49±2 to 44±1%, p=0.004) and ATPGLY (20±2 to 17±1%, p=0.03). Conclusion These alterations to muscle ATP production in vivo indicate that brief, maximal contractions are performed with increased support of oxidative ATP synthesis, and relatively less contribution from anaerobic ATP production following training. These results extend previous reports of molecular and cellular adaptations to HIT and show that 6 training sessions are sufficient to alter in vivo muscle energetics, which likely contributes to increased exercise capacity after short-term HIT. PMID:24612773

  4. Extracellular ATP induces the rapid release of HIV-1 from virus containing compartments of human macrophages.

    PubMed

    Graziano, Francesca; Desdouits, Marion; Garzetti, Livia; Podini, Paola; Alfano, Massimo; Rubartelli, Anna; Furlan, Roberto; Benaroch, Philippe; Poli, Guido

    2015-06-23

    HIV type 1 (HIV-1) infects CD4(+) T lymphocytes and tissue macrophages. Infected macrophages differ from T cells in terms of decreased to absent cytopathicity and for active accumulation of new progeny HIV-1 virions in virus-containing compartments (VCC). For these reasons, infected macrophages are believed to act as "Trojan horses" carrying infectious particles to be released on cell necrosis or functional stimulation. Here we explored the hypothesis that extracellular ATP (eATP) could represent a microenvironmental signal potentially affecting virion release from VCC of infected macrophages. Indeed, eATP triggered the rapid release of infectious HIV-1 from primary human monocyte-derived macrophages (MDM) acutely infected with the CCR5-dependent HIV-1 strain. A similar phenomenon was observed in chronically infected promonocytic U1 cells differentiated to macrophage-like cells (D-U1) by costimulation with phorbol esters and urokinase-type plasminogen activator. Worthy of note, eATP did not cause necrotic, apoptotic, or pyroptotic cell death, and its effect on HIV-1 release was suppressed by Imipramine (an antidepressant agent known to inhibit microvesicle formation by interfering with membrane-associated acid sphingomyelinase). Virion release was not triggered by oxidized ATP, whereas the effect of eATP was inhibited by a specific inhibitor of the P2X7 receptor (P2X7R). Thus, eATP triggered the discharge of virions actively accumulating in VCC of infected macrophages via interaction with the P2X7R in the absence of significant cytopathicity. These findings suggest that the microvesicle pathway and P2X7R could represent exploitable targets for interfering with the VCC-associated reservoir of infectious HIV-1 virions in tissue macrophages. PMID:26056317

  5. ATP requirement of the sodium-dependent magnesium extrusion from human red blood cells.

    PubMed Central

    Frenkel, E J; Graziani, M; Schatzmann, H J

    1989-01-01

    1. Competitive behaviour detectable in the stimulatory action of external sodium (Nao+) and internal magnesium (Mgi2+) corroborates the idea that Nao+-dependent Mg2+ extrusion is a Mgi2+-Nao+ exchange. 2. Mg2+-loaded resealed cells made from metabolically starved cells (with less than 5 mumols/l cells of ATP), show hardly any Nao+-dependent Mg2+ outflow. Incorporation of ATP during lysis-resealing restores this Mg2+ transport. Half-saturation for the effect is reached at an initial ATP concentration of about 150 mumols/l cells. 3. Adenylyl(beta, gamma-methylene) diphosphonate (AMP-PCP) and AMP had no restituting effect, indicating that in order to act ATP must be hydrolysed. 4. Nao+-dependent Mg2+ outflow is not inhibited by vanadate concentrations that completely block the Ca2+ or Na+ pump. Therefore, the Nao+-Mgi2+ exchange does not fall into the class of cation pumps of the E1E2 type. 5. Yet the fact that reversal of the Na+ gradient fails to reverse the direction of the Na+-dependent Mg2+ transport in human red cells (Lüdi & Schatzmann, 1987) and that at equal Na+ concentration inside and outside the rate of Mg2+ transport is still 50% of that at a Na+ concentration difference of approximately 100 mM across the membrane suggests that the Na+ gradient, or the cation gradients in general, are not the only driving forces for Mg2+ movement. The assumption that there is energy input from ATP hydrolysis is compatible with these observations, whereas proposing the action of a protein kinase fails to explain them. 6. It is concluded that the Nao+-Mgi2+ exchange system has an absolute requirement for ATP and that it is more probable that ATP is supplying energy for transport rather than activating transport by protein phosphorylation or simply by binding. PMID:2607436

  6. Platination of the copper transporter ATP7A involved in anticancer drug resistance.

    PubMed

    Calandrini, Vania; Arnesano, Fabio; Galliani, Angela; Nguyen, Trung Hai; Ippoliti, Emiliano; Carloni, Paolo; Natile, Giovanni

    2014-08-21

    The clinical efficacy of the widely used anticancer drug cisplatin is severely limited by the emergence of resistance. This is related to the drug binding to proteins such as the copper influx transporter Ctr1, the copper chaperone Atox1, and the copper pumps ATP7A and ATP7B. While the binding modes of cisplatin to the first two proteins are known, the structural determinants of platinated ATP7A/ATP7B are lacking. Here we investigate the interaction of cisplatin with the first soluble domain of ATP7A. First, we establish by ESI-MS and (1)H, (13)C, and (15)N NMR that, in solution, the adduct is a monomer in which the sulfur atoms of residues Cys19 and Cys22 are cis-coordinated to the [Pt(NH3)2](2+) moiety. Then, we carry out hybrid Car-Parrinello QM/MM simulations and computational spectroscopy calculations on a model adduct based on the NMR structure of the apo protein and featuring the experimentally determined binding mode of the metal ion. These calculations show quantitative agreement with CD spectra and (1)H, (13)C, and (15)N NMR chemical shifts, thus providing a quantitative molecular view of the 3D binding mode of cisplatin to ATP7A. Importantly, the same comparison rules out a variety of alternative models with different coordination modes, that we explored to test the robustness of the computational approach. Using this combined in silico-in vitro approach we provide here for the first time a quantitative 3D atomic view of the platinum binding to the first soluble domain of ATP7A. PMID:24983998

  7. Effect of phenylhydrazine and hydrogen peroxide on ATP-independent proteolysis in bovine heart

    SciTech Connect

    Gecha, O.M.; Fagan, J.M. (Rutgers, The State Univ. of New Jersey, New Brunswick (United States))

    1990-02-26

    Hemoglobin damaged by phenylhydrazine is rapidly degraded in erythroid cells and lysates by an ATP-independent proteolytic system. The authors have found that heart proteins damaged by phenylhydrazine and H{sub 2}O{sub 2} are also rapidly hydrolyzed by an ATP-independent pathway isolated from bovine heart. Soluble bovine cardiac tissue proteins were applied to a DE-52 column equilibrated in Tris (ph 7)/ glycerol (20%) buffer. Proteins which did not bind to the DE-52 resin (F-1) when treated with 1mM phenylhydrazine or 10mM H{sub 2}O{sub 2} and incubated with enzymes which bound to the resin and were eluted with 0.5M NaCl (F-11) were hydrolyzed to amino acids 200-300% more rapidly than untreated F-1 cardiac proteins. This increase was observed in the absence of added ATP and the addition of 5mM ATP did not affect the degradation of oxidant treated F-1 but did cause a 4- to 5-fold increase in the degradation of untreated endogenous heart proteins. When both F-1 and F-11 were treated with 1mM phenylhydrazine an increase of 260% in protein degradation was seen. This suggests that the ATP-independent proteolytic system was not damaged by phenylhydrazine. Myoglobin, a primary component of cardiac F-1, following oxidation with 1mM phenylhydrazine, was more rapidly hydrolyzed (2- to 4-fold) by the ATP-independent proteolytic system in cardiac tissue. These results indicate that cardiac tissue proteins exposed to phenylhydrazine and H{sub 2}O{sub 2} are rapidly degraded by cardiac proteases that do not require ATP.

  8. Subnanometre-resolution structure of the intact Thermus thermophilus H+-driven ATP synthase.

    PubMed

    Lau, Wilson C Y; Rubinstein, John L

    2012-01-12

    Ion-translocating rotary ATPases serve either as ATP synthases, using energy from a transmembrane ion motive force to create the cell's supply of ATP, or as transmembrane ion pumps that are powered by ATP hydrolysis. The members of this family of enzymes each contain two rotary motors: one that couples ion translocation to rotation and one that couples rotation to ATP synthesis or hydrolysis. During ATP synthesis, ion translocation through the membrane-bound region of the complex causes rotation of a central rotor that drives conformational changes and ATP synthesis in the catalytic region of the complex. There are no structural models available for the intact membrane region of any ion-translocating rotary ATPase. Here we present a 9.7?Å resolution map of the H(+)-driven ATP synthase from Thermus thermophilus obtained by electron cryomicroscopy of single particles in ice. The 600-kilodalton complex has an overall subunit composition of A(3)B(3)CDE(2)FG(2)IL(12). The membrane-bound motor consists of a ring of L subunits and the carboxy-terminal region of subunit I, which are equivalent to the c and a subunits of most other rotary ATPases, respectively. The map shows that the ring contains 12 L subunits and that the I subunit has eight transmembrane helices. The L(12) ring and I subunit have a surprisingly small contact area in the middle of the membrane, with helices from the I subunit making contacts with two different L subunits. The transmembrane helices of subunit I form bundles that could serve as half-channels across the membrane, with the first half-channel conducting protons from the periplasm to the L(12) ring and the second half-channel conducting protons from the L(12) ring to the cytoplasm. This structure therefore suggests the mechanism by which a transmembrane proton motive force is converted to rotation in rotary ATPases. PMID:22178924

  9. Hexabromocyclododecane Decreases the Lytic Function and ATP Levels of Human Natural Killer Cells

    PubMed Central

    Hinkson, Natasha C.; Whalen, Margaret M.

    2009-01-01

    This study investigates the effect of Hexabromocyclododecane (HBCD) on the lytic function of human natural killer (NK) cells and on ATP levels in NK cells. NK cells are capable of lysing tumor cells, virally infected cells, and antibody-coated cells. HBCD is a, brominated cyclic alkane used primarily as an additive flame retardant. If HBCD interferes with NK cell function, this could increase risk of tumor development and/or viral infection. NK cells were exposed to various concentrations of HBCD for 24 h, 48 h, and 6 days before determining lytic function and ATP levels. ATP levels and lytic function were also determined in NK cells that were exposed to HBCD for 1 h followed by 24 h, 48 h, and 6 days in HBCD-free media. The results indicated that exposure of NK cells to 10 ?M HBCD for 24 h causes a very significant decrease in both NK cell lytic function and ATP levels (93.5% and 90.5%, respectively). Exposure of NK cells to 10 ?M HBCD for 1 h followed by 24 h in HBCD-free media showed a progressive and persistent loss of lytic function (89.3%) as well as a decrease in ATP levels (46.1%). The results indicate that HBCD exposures decreased lytic function as well as ATP levels. However, a decrease in lytic function was not necessarily accompanied by a similar decrease in ATP. Importantly, these results also indicate that a brief (1 h) exposure to HBCD causes a progressive loss of lytic function over a 6 d period. PMID:19551757

  10. Postnatal constriction, ATP depletion, and cell death in the mature and immature ductus arteriosus.

    PubMed

    Levin, Max; McCurnin, Don; Seidner, Steven R; Yoder, Bradley; Waleh, Nahid; Goldbarg, Seth; Roman, Christine; Liu, Bao Mei; Borén, Jan; Clyman, Ronald I

    2006-02-01

    After birth, constriction of the full-term ductus arteriosus induces oxygen, glucose and ATP depletion, cell death, and anatomic remodeling of the ductus wall. The immature ductus frequently fails to develop the same degree of constriction or anatomic remodeling after birth. In addition, the immature ductus loses its ability to respond to vasoconstrictive agents, like oxygen or indomethacin, with increasing postnatal age. We examined the effects of premature delivery and postnatal constriction on the immature baboon ductus arteriosus. By 6 days after birth, surrogate markers of hypoxia (HIF1alpha/VEGF mRNA) and cell death [dUTP nick-end labeling (TUNEL)-staining] increased, while glucose and ATP concentrations (bioluminescence imaging) decreased in the immature ductus. TUNEL-staining was significantly related to the degree of glucose and ATP depletion. Glucose and ATP depletion were directly related to the degree of ductus constriction; while TUNEL-staining was logarithmically related to the degree of ductus constriction. Extensive cell death (>15% TUNEL-positive cells) occurred only when there was no Doppler flow through the ductus lumen. In contrast, HIF1alpha/VEGF expression and ATP concentrations were significantly altered even when the immature ductus remained open after birth. Decreased ATP concentrations produced decreased oxygen-induced contractile responses in the immature ductus. We hypothesize that ATP depletion in the persistently patent immature newborn ductus is insufficient to induce cell death and remodeling but sufficient to decrease its ability to constrict after birth. This may explain its decreasing contractile response to oxygen, indomethacin, and other contractile agents with increasing postnatal age. PMID:16223843

  11. Review of the operational definition for first-episode psychosiseip

    PubMed Central

    Breitborde, Nicholas J. K.; Srihari, Vinod H.; Woods, Scott W.

    2015-01-01

    Aim Given the growing interest in the study of first-episode psychosis, clinical and research programmes would benefit from a conceptual clarification of how to operationalize ‘first-episode psychosis’. We review the variety of definitions in use and discuss their relative merits with respect to both clinical (e.g. early treatment) and research (e.g. obtaining meaningfully homogeneous populations) agendas. Methods We completed a selective review of the literature to investigate how first-episode psychosis was operationally defined. Results Operational definitions for ‘first-episode psychosis’ fall largely into three categories: (i) first treatment contact; (ii) duration of antipsychotic medication use; and (iii) duration of psychosis. Each definitional category contains a number of underlying assumptions that contribute to the strengths and weaknesses of the definition. Conclusions The term ‘first-episode psychosis’ as used within clinical and research settings is misleading regardless of which operational definition is used. This term is typically used to refer to individuals early in the course of a psychotic illness or treatment rather than individuals who are truly in the midst of a first ‘episode’ of illness. The alternative of ‘recent-onset psychosis’ with related definitions based on ‘duration of psychosis’ is proposed. Based on this review, we provide suggestions with regard to the overarching pragmatic consideration of setting up a clinical service that can attract and assemble a population of early psychosis patients for the related purposes of treatment and research. PMID:22642728

  12. Hyperpolization-activated Ca(2+) channels in guard cell plasma membrane are involved in extracellular ATP-promoted stomatal opening in Vicia faba.

    PubMed

    Wang, Fang; Jia, Juanjuan; Wang, Yufang; Wang, Weixia; Chen, Yuling; Liu, Ting; Shang, Zhonglin

    2014-09-01

    Extracellular ATP (eATP) plays essential roles in plant growth, development, and stress tolerance. Extracellular ATP-regulated stomatal movement of Arabidopsis thaliana has been reported. Here, ATP was found to promote stomatal opening of Vicia faba in a dose-dependent manner. Three weakly hydrolysable ATP analogs (adenosine 5'-O-(3-thio) triphosphate (ATP?S), 3'-O-(4-benzoyl) benzoyl adenosine 5'-triphosphate (Bz-ATP) and 2-methylthio-adenosine 5'-triphosphate (2meATP)) showed similar effects, indicating that ATP acts as a signal molecule rather than an energy charger. ADP promoted stomatal opening, while AMP and adenosine did not affect stomatal movement. An ATP-promoted stomatal opening was blocked by the NADPH oxidase inhibitor diphenylene iodonium (DPI), the reductant dithiothreitol (DTT) or the Ca(2+) channel blockers GdCl3 and LaCl3. A hyperpolarization-activated Ca(2+) channel was detected in plasma membrane of guard cell protoplast. Extracellular ATP and weakly hydrolyzable ATP analogs activated this Ca(2+) channel significantly. Extracellular ATP-promoted Ca(2+) channel activation was markedly inhibited by DPI or DTT. These results indicated that eATP may promote stomatal opening via reactive oxygen species that regulate guard cell plasma membrane Ca(2+) channels. PMID:25014259

  13. Secreted products of a nonmucoid Pseudomonas aeruginosa strain induce two modes of macrophage killing: external-ATP- dependent, P2Z-receptor-mediated necrosis and ATP-independent, caspase-mediated apoptosis

    Microsoft Academic Search

    Olga Zaborina; Neelam Dhiman; Mei Ling Chen; Jan Kostal; Ian Alan Holder; Ananda M. Chakrabarty

    A nonmucoid clinical isolate of Pseudomonas aeruginosa, strain 808, elaborated ATP-dependent and ATP-independent types of cytotoxic factors in the growth medium. These cytotoxic factors, active against macrophages, were secreted during the exponential phase of growth in a complex medium. Commensurate with the appearance of the cytotoxic activities in the cell-free growth medium, several ATP-utilizing enzymic activities, such as adenylate kinase,

  14. The gating of nucleotide-sensitive K + channels in insulin-secreting cells can be modulated by changes in the ratio ATP 4? \\/ADP 3? and by nonhydrolyzable derivatives of both ATP and ADP

    Microsoft Academic Search

    M. J. Dunne; J. A. West-Jordan; R. J. Abraham; R. H. T. Edwards; O. H. Petersen

    1988-01-01

    Summary The31P-NMR technique has been used to assess the intracellular ratios and concentrations of mobile ATP and ADP and the intracellular pH in an insulin-secreting cell line, RINm5F. The single-channel current-recording technique has been used to investigate the effects of changes in the concentrations of ATP and ADP on the gating of nucleotide-dependent K+ channels. Adding ATP to the membrane

  15. Stimulation-Evoked Ca2+ Signals in Astrocytic Processes at Hippocampal CA3–CA1 Synapses of Adult Mice Are Modulated by Glutamate and ATP

    PubMed Central

    Szokol, Karolina; Jensen, Vidar; Enger, Rune; Trivedi, Chintan A.; Hvalby, Øivind; Helm, P. Johannes; Looger, Loren L.; Sprengel, Rolf

    2015-01-01

    To date, it has been difficult to reveal physiological Ca2+ events occurring within the fine astrocytic processes of mature animals. The objective of the study was to explore whether neuronal activity evokes astrocytic Ca2+ signals at glutamatergic synapses of adult mice. We stimulated the Schaffer collateral/commissural fibers in acute hippocampal slices from adult mice transduced with the genetically encoded Ca2+ indicator GCaMP5E driven by the glial fibrillary acidic protein promoter. Two-photon imaging revealed global stimulation-evoked astrocytic Ca2+ signals with distinct latencies, rise rates, and amplitudes in fine processes and somata. Specifically, the Ca2+ signals in the processes were faster and of higher amplitude than those in the somata. A combination of P2 purinergic and group I/II metabotropic glutamate receptor (mGluR) antagonists reduced the amplitude of the Ca2+ transients by 30–40% in both astrocytic compartments. Blockage of the mGluRs alone only modestly reduced the magnitude of the stimulation-evoked Ca2+ signals in processes and failed to affect the somatic Ca2+ response. Local application of group I or I/II mGluR agonists or adenosine triphosphate (ATP) elicited global astrocytic Ca2+ signals that mimicked the stimulation-evoked astrocytic Ca2+ responses. We conclude that stimulation-evoked Ca2+ signals in astrocytic processes at CA3–CA1 synapses of adult mice (1) differ from those in astrocytic somata and (2) are modulated by glutamate and ATP. PMID:25698739

  16. The definition of cross polarization.

    NASA Technical Reports Server (NTRS)

    Ludwig, A. C.

    1973-01-01

    Consideration of three alternate definitions of cross-polarization known to the author. The three definitions are expressed precisely in terms of the same antenna pattern coordinate system, and the relative merits of the various definitions are discussed. One of the definitions is shown to be the proper choice for describing source current polarizations, another is suitable for relating source current distributions to secondary patterns, and the third is the best choice for describing antenna patterns.

  17. A highly selective turn-on ATP fluorescence sensor based on unmodified cysteamine capped CdS quantum dots.

    PubMed

    Tedsana, Wimonsiri; Tuntulani, Thawatchai; Ngeontae, Wittaya

    2013-06-14

    Unmodified cysteamine capped nanocrystalline cadmium sulfide quantum dots (Cys-CdS QDs) were demonstrated as a selective turn-on fluorescence sensor for sensing adenosine-5'-triphosphate (ATP) in aqueous solution for the first time. The fluorescence intensity of the Cys-CdS QDs was significantly enhanced in the presence of ATP. In addition, the fluorescence intensity of the Cys-CdS QDs increased when increasing ATP concentrations. On the other hand, other phosphate metabolites and other tested common anions did not significantly alter the fluorescence intensity of the Cys-CdS QDs. In addition, this sensor showed excellent discrimination of pyrophosphate (PPi) from ATP detection. The proposed sensor could efficiently be used for ATP sensing at very low concentration with LOD of 17 ?M with the linear working concentration range of 20-80 ?M. The feasibility of the proposed sensor for determining ATP in urine samples was also studied, and satisfactory results were obtained. PMID:23726101

  18. Slick (Slo2.1), a rapidly-gating sodium-activated potassium channel inhibited by ATP.

    PubMed

    Bhattacharjee, Arin; Joiner, William J; Wu, Meilin; Yang, Youshan; Sigworth, Fred J; Kaczmarek, Leonard K

    2003-12-17

    Neuronal stressors such as hypoxia and firing of action potentials at very high frequencies cause intracellular Na+ to rise and ATP to be consumed faster than it can be regenerated. We report the cloning of a gene encoding a K+ channel, Slick, and demonstrate that functionally it is a hybrid between two classes of K+ channels, Na+-activated (KNa) and ATP-sensitive (KATP) K+ channels. The Slick channel is activated by intracellular Na+ and Cl- and is inhibited by intracellular ATP. Slick is widely expressed in the CNS and is detected in heart. We identify a consensus ATP binding site near the C terminus of the channel that is required for ATP and its nonhydrolyzable analogs to reduce open probability. The convergence of Na+, Cl-, and ATP sensitivity in one channel may endow Slick with the ability to integrate multiple indicators of the metabolic state of a cell and to adjust electrical activity appropriately. PMID:14684870

  19. Definitions Algebra of complex numbers

    E-print Network

    Lega, Joceline

    Definitions Algebra of complex numbers Polar coordinates form of complex numbers Check your knowledge Review of Complex Numbers Definitions, Algebra of complex numbers, Polar coordinates Review of Complex Numbers #12;Definitions Algebra of complex numbers Polar coordinates form of complex numbers Check

  20. THE DEFINITE INTEGRAL AND COMPUTER

    Microsoft Academic Search

    Dragoslav Herceg

    2009-01-01

    In this paper the results of the learning concept of definite integral and numeric integration with the computer is presented. The tested students attend ?Sabac Chemical Technological college. The aim of this test was to check the student's theoretical, visual and practical knowledge of definite integral. In almost all secondary schools definite integral and its applications are studied. The concept

  1. ATP-sensitive K+ channels and cellular K+ loss in hypoxic and ischaemic mammalian ventricle.

    PubMed Central

    Weiss, J N; Venkatesh, N; Lamp, S T

    1992-01-01

    1. The contribution of ATP-sensitive K+ (K+ATP) channels to the rapid increase in cellular K+ efflux and shortening of action potential duration (APD) during early myocardial ischaemia and hypoxia remains controversial, because for the first 10 min of ischaemia or hypoxia in intact hearts cytosolic [ATP] remains about two orders of magnitude greater than the [ATP] causing half-maximal blockade of K+ATP channels in excised membrane patches. The purpose of this study was to investigate this apparent discrepancy. 2. During substrate-free hypoxia, total, diastolic and systolic unidirectional K+ efflux rates increased by 43, 26 and 103% respectively after 8.3 min in isolated arterially perfused rabbit interventricular septa loaded with 42K+. APD shortened by 39%. From the Goldman-Hodgkin-Katz equation, the relative increases in systolic and diastolic K+ efflux rates were consistent with activation of a voltage-independent K+ conductance. 3. During total global ischaemia, [K+]o measured with intramyocardial valinomycin K(+)-sensitive electrodes increased at a maximal rate of 0.68 mM min-1, which could be explained by a less than 26% increase in unidirectional K+ efflux rate (assuming no change in K+ influx), less than the increase during hypoxia. APD shortened by 23% over 10 min. 4. During hypoxia and ischaemia, cytosolic [ATP] decreased by about one-third from 6.8 +/- 0.5 to 4.3 +/- 0.3 and 4.6 +/- 0.4 mM respectively, and free cytosolic [ADP] increased from 15 to 95 and approximately 63 microM respectively. 5. To estimate the percentage of activation of current through K+ATP channels (IK,ATP) necessary to double the systolic K+ efflux rate (comparable to the increase during hypoxia), K+ efflux during a single simulated action potential was measured by blocking non-K+ currents under control conditions and after IK,ATP was fully activated by metabolic inhibitors. Activation of 0.41 +/- 0.07% of maximal IK,ATP was sufficient to double the systolic K+ efflux rate. The equivalent amount of constant hyperpolarizing current also shortened the APD in the isolated myocytes by 41 +/- 5%, compared to the 39% APD shortening observed during hypoxia in the intact heart. 6. The degree of activation of IK,ATP expected to occur during hypoxia and ischaemia was estimated by characterizing the ATP sensitivity of K+ATP channels in the presence of 2 mM-free Mgi2+ and 0, 10, 100 and 300 microM-ADPi in inside-out membrane patches excised from guinea-pig ventricular myocytes.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:1593462

  2. A single amino acid substitution in CFTR converts ATP to an inhibitory ligand

    PubMed Central

    Lin, Wen-Ying; Jih, Kang-Yang

    2014-01-01

    Cystic fibrosis (CF), one of the most common lethal genetic diseases, is caused by loss-of-function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel that, when phosphorylated, is gated by ATP. The third most common pathogenic mutation, a glycine-to-aspartate mutation at position 551 or G551D, shows a significantly decreased open probability (Po) caused by failure of the mutant channel to respond to ATP. Recently, a CFTR-targeted drug, VX-770 (Ivacaftor), which potentiates G551D-CFTR function in vitro by boosting its Po, has been approved by the FDA to treat CF patients carrying this mutation. Here, we show that, in the presence of VX-770, G551D-CFTR becomes responsive to ATP, albeit with an unusual time course. In marked contrast to wild-type channels, which are stimulated by ATP, sudden removal of ATP in excised inside-out patches elicits an initial increase in macroscopic G551D-CFTR current followed by a slow decrease. Furthermore, decreasing [ATP] from 2 mM to 20 µM resulted in a paradoxical increase in G551D-CFTR current. These results suggest that the two ATP-binding sites in the G551D mutant mediate opposite effects on channel gating. We introduced mutations that specifically alter ATP-binding affinity in either nucleotide-binding domain (NBD1 or NBD2) into the G551D background and determined that this disease-associated mutation converts site 2, formed by the head subdomain of NBD2 and the tail subdomain of NBD1, into an inhibitory site, whereas site 1 remains stimulatory. G551E, but not G551K or G551S, exhibits a similar phenotype, indicating that electrostatic repulsion between the negatively charged side chain of aspartate and the ?-phosphate of ATP accounts for the observed mutational effects. Understanding the molecular mechanism of this gating defect lays a foundation for rational drug design for the treatment of CF. PMID:25225552

  3. The P2Y 1 receptor is an ADP receptor antagonized by ATP and expressed in platelets and megakaryoblastic cells

    Microsoft Academic Search

    Catherine Léon; Béatrice Hechler; Catherine Vial; Claude Leray; Jean-Pierre Cazenave; Christian Gachet

    1997-01-01

    The human P2Y1 purinoceptor has been expressed in Jurkat cells and the effects of HPLC purified nucleotides on calcium movements were measured. The most potent agonist was 2-methylthio-ADP followed by ADP. ATP, Sp-ATP?S and ?,?-methylene-ATP were competitive antagonists. Suramin and PPADS inhibited the effects of ADP. This pharmacological profile is the same as that of the so-called P2T purinoceptor responsible

  4. Effect of extraluminal ATP application on vascular tone and blood flow in skeletal muscle: implications for exercise hyperemia

    PubMed Central

    Al-Khazraji, Baraa K.; Mortensen, Stefan P.; Jackson, Dwayne N.; Ellis, Christopher G.; Hellsten, Ylva

    2013-01-01

    During skeletal muscle contractions, the concentration of ATP increases in muscle interstitial fluid as measured by microdialysis probes. This increase is associated with the magnitude of blood flow, suggesting that interstitial ATP may be important for contraction-induced vasodilation. However, interstitial ATP has solely been described to induce vasoconstriction in skeletal muscle. To examine whether interstitial ATP induces vasodilation in skeletal muscle and to what extent this vasoactive effect is mediated by formation of nitric oxide (NO) and prostanoids, three different experimental models were studied. The rat gluteus maximus skeletal muscle model was used to study changes in local skeletal muscle hemodynamics. Superfused ATP at concentrations found during muscle contractions (1–10 ?M) increased blood flow by up to 400%. In this model, the underlying mechanism was also examined by inhibition of NO and prostanoid formation. Inhibition of these systems abolished the vasodilator effect of ATP. Cell-culture experiments verified ATP-induced formation of NO and prostacyclin in rat skeletal muscle microvascular endothelial cells, and ATP-induced formation of NO in rat skeletal muscle cells. To confirm these findings in humans, ATP was infused into skeletal muscle interstitium of healthy subjects via microdialysis probes and found to increase muscle interstitial concentrations of NO and prostacyclin by ?60% and ?40%, respectively. Collectively, these data suggest that a physiologically relevant elevation in interstitial ATP concentrations increases muscle blood flow, indicating that the contraction-induced increase in skeletal muscle interstitial [ATP] is important for exercise hyperemia. The vasodilator effect of ATP application is mediated by NO and prostanoid formation. PMID:23761642

  5. Maintenance of steelhead trout (Oncorhynchus mykiss) sperm at different in vitro oxygen tensions alters ATP levels and cell functional characteristics

    Microsoft Academic Search

    D. C. Bencic; M. Krisfalusi; J. G. Cloud; R. L. Ingermann

    1999-01-01

    Adenosine triphosphate (ATP) levels in sperm from steelhead trout (Oncorhynchus mykiss) were found to be 12.0 ± 1.4 pmol ATP per 106 sperm cells. Sperm were stored at 0–2 °C for up to 72 h under 100 and 21% O2, and 100% N2. The sperm ATP content of samples maintained under 100 and 21% O2 both decreased to about 70 and 50% of

  6. Toll-Like Receptor-Triggered Calcium Mobilization Protects Mice against Bacterial Infection through Extracellular ATP Release

    PubMed Central

    Ren, Hua; Teng, Yunfei; Tan, Binghe; Zhang, Xiaoyu; Jiang, Wei; Liu, Mingyao; Jiang, Wenzheng

    2014-01-01

    Extracellular ATP (eATP), released as a “danger signal” by injured or stressed cells, plays an important role in the regulation of immune responses, but the relationship between ATP release and innate immune responses is still uncertain. In this study, we demonstrated that ATP was released through Toll-like receptor (TLR)-associated signaling in both Escherichia coli-infected mice and lipopolysaccharide (LPS)- or Pam3CSK4-treated macrophages. This ATP release could be blocked completely only by N-ethylmaleimide (NEM), not by carbenoxolone (CBX), flufenamic acid (FFA), or probenecid, suggesting the key role of exocytosis in this process. Furthermore, LPS-induced ATP release could also be reduced dramatically through suppressing calcium mobilization by use of U73122, caffeine, and thapsigargin (TG). In addition, the secretion of interleukin-1? (IL-1?) and CCL-2 was enhanced significantly by ATP, in a time- and dose-dependent manner. Meanwhile, macrophage-mediated phagocytosis of bacteria was also promoted significantly by ATP stimulation. Furthermore, extracellular ATP reduced the number of invading bacteria and protected mice from peritonitis by activating purinergic receptors. Mechanistically, phosphorylation of AKT and ERK was overtly increased by ATP in antibacterial immune responses. Accordingly, if we blocked the P2X- and P2Y-associated signaling pathway by using suramin and pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid), tetrasodium salt (PPADS), the ATP-enhanced immune response was restrained significantly. Taken together, our findings reveal an internal relationship between danger signals and TLR signaling in innate immune responses, which suggests a potential therapeutic significance of calcium mobilization-mediated ATP release in infectious diseases. PMID:25245808

  7. Cysteine substitution mutants give structural insight and identify ATP binding and activation sites at P2X receptors

    PubMed Central

    Roberts, Jonathan A.; Evans, Richard J.

    2007-01-01

    P2X receptors for extracellular ATP are a distinct family of ligand gated cation channels involved in physiological processes ranging from synaptic transmission to muscle contraction. Common ATP binding motifs are absent from P2X receptors and the extent of the agonist binding site is unclear. We used cysteine scanning mutagenesis, radiolabelled 2-azido ATP binding, and methanethiosulfonate (MTS) compounds, to identify amino acid residues involved in ATP binding and gating of the human P2X1 receptor. The pattern of MTSEA-biotinylation was also used to determine the accessibility of substituted cysteine residues and whether this changed on addition of ATP. Analysis of cysteine substituted mutants of the last 44 amino acid residues (S286-I329) in the extracellular loop before the second transmembrane segment showed that N290, F291, R292 and K309 mutants had reduced ATP potency and 2-azido ATP binding. MTS reagents produced further shifts in ATP potency at these residues suggesting that they are directly involved in ATP binding; the effects were dependent on the charge of the MTS reagent at K309C, one explanation for this is that K309 interacts directly with the negatively charged phosphate of ATP. The remainder of the cysteine substitutions had little or no effect on ATP potency. However at the mutants D316C, G321C, A323C, and I328C MTS reagents did not change ATP potency but modified agonist evoked responses suggesting that this region may contribute to the gating of the channel. PMID:17428985

  8. The assembly of F 1F O-ATP synthase is disrupted upon interference of RNA editing in Trypanosoma brucei

    Microsoft Academic Search

    Hassan Hashimi; Vladislava Benkovi?ová; Petra ?ermáková; De-Hua Lai; Anton Horváth; Julius Lukeš

    2010-01-01

    Throughout eukaryotes, the gene encoding subunit 6 (ATP6) of the F1FO-ATP synthase (complex V) is maintained in mitochondrial (mt) genomes, presumably because of its high hydrophobicity due to its incorporation into the membrane-bound FO moiety. In Trypanosoma species, a mt transcript that undergoes extensive processing by RNA editing has a very low sequence similarity to ATP6 from other organisms. The

  9. A chimeric gene containing the 5? portion of atp6 is associated with cytoplasmic male-sterility of rice

    Microsoft Academic Search

    Koh-ichi Kadowaki; Takeshi Suzuki; Shigeru Kazama

    1990-01-01

    Three ATPase subunit 6 (atp6) genes of rice mitochondria were isolated, one from normal and two from cms-Bo male-sterile cytoplasms, in order to determine whether the extra atp6 gene in cms-Bo rice plays a role in cytoplasmic male-sterility (CMS). The nucleotide sequences of all three genes were determined and analysis showed a chimeric atp6 gene (urf-rmc) as well as a

  10. CT number definition

    NASA Astrophysics Data System (ADS)

    Bryant, J. A.; Drage, N. A.; Richmond, S.

    2012-04-01

    The accuracy of CT number plots has been found lacking in several medical applications. This is of concern since the ability to compare and evaluate results on a reproducible and standard basis is essential to long term development. Apart from the technical limitations arising from the CT scanner and the data treatment, there are fundamental issues with the definition of the Hounsfield number, namely the absence of a standard photon energy and the need to specify the attenuation mechanism for standard measurements. This paper presents calculations to demonstrate the shortcomings of the present definition with a brief discussion. The remedy is straightforward, but probably of long duration as it would require an international agreement.

  11. ATP-dependent activation of an inflammasome in primary gingival epithelial cells infected by Porphyromonas gingivalis

    PubMed Central

    Yilmaz, Özlem; Sater, Ali Abdul; Yao, Luyu; Koutouzis, Theofilos; Pettengill, Matthew; Ojcius, David M.

    2009-01-01

    Summary Production of IL-1? typically requires two-separate signals. The first signal, from a pathogen-associated molecular pattern, promotes intracellular production of immature cytokine. The second signal, derived from a danger signal such as extracellular ATP, results in assembly of an inflammasome, activation of caspase-1, and secretion of mature cytokine. The inflammasome component, Nalp3, plays a nonredundant role in caspase-1 activation in response to ATP binding to P2X7 in macrophages. Gingival epithelial cells (GECs) are an important component of the innate-immune response to periodontal bacteria. We had shown that GECs express a functional P2X7 receptor, but the ability of GECs to secrete IL-1? during infection remained unknown. We find that GECs express a functional Nalp3-inflammasome. Treatment of GECs with LPS or infection with the periodontal pathogen, Porphyromonas gingivalis, induced expression of the il-1? gene and intracellular accumulation of IL-1? protein. However, IL-1? was not secreted unless LPS-treated or infected cells were subsequently stimulated with ATP. Conversely, caspase-1 is activated in GECs following ATP-treatment but not P. gingivalis-infection. Furthermore, depletion of Nalp3 by siRNA abrogated the ability of ATP to induce IL-1? secretion in infected-cells. The Nalp3-inflammasome is therefore a likely to be an important mediator of the inflammatory response in gingival epithelium. PMID:19811501

  12. Activation of the Nlrp1b Inflammasome by Reduction of Cytosolic ATP

    PubMed Central

    Liao, Kuo-Chieh

    2013-01-01

    The efficacy of the innate immune system depends on its ability to mount an appropriate response to diverse infections and damaging agents. Key components of this system are pattern recognition receptors that detect pathogen-associated and damage-associated molecular patterns (PAMPs and DAMPs). Nlrp1b is a pattern recognition receptor that forms a caspase-1 activation platform, known as an inflammasome, upon sensing the proteolytic activity of anthrax lethal toxin. The activation of caspase-1 leads to the release of proinflammatory cytokines that aid in the clearance of the anthrax infection. Here, we demonstrate that Nlrp1b also becomes activated in cells that are subjected to energy stress caused by metabolic inhibitors or by nutrient deprivation. Glucose starvation and hypoxia were used to correlate the level of cytosolic ATP to the degree of inflammasome activation. Because lowering the ratio of cytosolic ATP to AMP activates the main cellular energy sensor, AMP-activated protein kinase (AMPK), we assessed whether AMPK promoted inflammasome activity by using a combination of small interfering RNA (siRNA) and transfection of a dominant negative AMPK subunit. We found that AMPK promoted inflammasome activity, but activation of AMPK in the absence of ATP depletion was not sufficient for caspase-1-mediated pro-interleukin 1? (pro-IL-1?) processing. Finally, we found that mutation of the ATP-binding motif of Nlrp1b caused constitutive activation, suggesting that ATP might inhibit the Nlrp1b inflammasome instead of being required for its assembly. PMID:23230290

  13. Iron transport by proteoliposomes containing mitochondrial F(1)F(0) ATP synthase isolated from rat heart.

    PubMed

    Kim, Misun; Song, Eunsook

    2010-04-01

    In this work, we present evidence of Fe(2+) transport by rat heart mitochondrial F(1)F(0) ATP synthase. Iron uptake by the vesicles containing the enzyme was concentration- and temperature-dependent, with an optimum temperature of 37 degrees C. Both ATP and ADP stimulated iron uptake in a concentration-dependent manner, whereas AMP, AMPPCP, and mADP did not. Inhibitors of the enzyme, oligomycin, and resveratrol similarly blocked iron transport. The iron uptake was confirmed by inhibition using specific antibodies against the alpha, beta, and c subunits of the enzyme. Interestingly, slight transport of common divalent and trivalent metal ions such as Mg(+2), Ca(+2), Mn(+2), Zn(+2), Cu(+2), Fe(+3), and Al(+3) was observed. Moreover, Cu(+2), even in the nM range, inhibited iron uptake and attained maximum inhibition of approximately 56%. Inorganic phosphate (Pi) in the medium exerted an opposite effect depending on the type of adenosine nucleotide, which was suppressed with ATP, but enhanced with ADP. A similarly stimulating effect of ATP and ADP with an inverse effect of Pi suggests that the activity of ATPase and ATP synthase may be associated with iron uptake in a different manner, probably via antiport of H(+). PMID:20100539

  14. The oligomycin-sensitivity conferring protein of mitochondrial ATP synthase: emerging new roles in mitochondrial pathophysiology.

    PubMed

    Antoniel, Manuela; Giorgio, Valentina; Fogolari, Federico; Glick, Gary D; Bernardi, Paolo; Lippe, Giovanna

    2014-01-01

    The oligomycin-sensitivity conferring protein (OSCP) of the mitochondrial F(O)F1 ATP synthase has long been recognized to be essential for the coupling of proton transport to ATP synthesis. Located on top of the catalytic F1 sector, it makes stable contacts with both F1 and the peripheral stalk, ensuring the structural and functional coupling between F(O) and F1, which is disrupted by the antibiotic, oligomycin. Recent data have established that OSCP is the binding target of cyclophilin (CyP) D, a well-characterized inducer of the mitochondrial permeability transition pore (PTP), whose opening can precipitate cell death. CyPD binding affects ATP synthase activity, and most importantly, it decreases the threshold matrix Ca²? required for PTP opening, in striking analogy with benzodiazepine 423, an apoptosis-inducing agent that also binds OSCP. These findings are consistent with the demonstration that dimers of ATP synthase generate Ca²?-dependent currents with features indistinguishable from those of the PTP and suggest that ATP synthase is directly involved in PTP formation, although the underlying mechanism remains to be established. In this scenario, OSCP appears to play a fundamental role, sensing the signal(s) that switches the enzyme of life in a channel able to precipitate cell death. PMID:24786291

  15. Omeprazole, a gastric proton pump inhibitor, inhibits melanogenesis by blocking ATP7A trafficking.

    PubMed

    Matsui, Mary S; Petris, Michael J; Niki, Yoko; Karaman-Jurukovska, Nevena; Muizzuddin, Neelam; Ichihashi, Masamitsu; Yarosh, Daniel B

    2015-03-01

    Omeprazole is a proton pump inhibitor used in the treatment of peptic ulcer disease and gastrosophageal reflux disease and acts by irreversibly blocking ATP4A, a P-type H+/K+ ATPase in gastric parietal cells. We found that omeprazole and its closely related congeners inhibited melanogenesis at micromolar concentrations in B16 mouse melanoma cells, normal human epidermal melanocytes, and in a reconstructed human skin model. Omeprazole topically applied to the skin of UV-irradiated human subjects significantly reduced pigment levels after 3 weeks compared with untreated controls. Omeprazole had no significant inhibitory effect on the activities of purified human tyrosinase or on the mRNA levels of tyrosinase, dopachrome tautomerase, Pmel17, or MITF mRNA levels. Although melanocytes do not express ATP4A, they do express ATP7A, a copper transporting P-type ATPase in the trans-Golgi network that is required for copper acquisition by tyrosinase. ATP7A relocalization from the trans-Golgi network to the plasma membrane in response to elevated copper concentrations in melanocytes was inhibited by omeprazole. Omeprazole treatment increased the proportion of EndoH sensitive tyrosinase, indicating that tyrosinase maturation was impaired. In addition, omeprazole reduced tyrosinase protein abundance in the presence of cycloheximide, suggestive of increased degradation. Our findings are consistent with the hypothesis that omeprazole reduces melanogenesis by inhibiting ATP7A and by enhancing degradation of tyrosinase. PMID:25337692

  16. Molybdate treatment and sulfate starvation decrease ATP and DNA levels in Ferroplasma acidarmanus

    PubMed Central

    Baumler, David J.; Hung, Kai-Foong; Jeong, Kwang Cheol; Kaspar, Charles W.

    2008-01-01

    Sulfate is a primary source of sulfur for most microbes and in some prokaryotes it is used an electron acceptor. The acidophile Ferroplasma acidarmanus (strain fer1) requires a minimum of 150 mM of a sulfate-containing salt for growth. Sulfate is assimilated by F. acidarmanus into proteins and reduced to form the volatile organic sulfur compounds methanethiol and dimethyldisulfide. In the absence of sulfate, cell death occurs by an unknown mechanism. In this study, cell viability and genomic DNA and ATP contents of F. acidarmanus were monitored in response to the absence of sulfate or the presence of sulfate and the sulfate analog molybdate ( MoO42- ). Cellular DNA and ATP contents were monitored as markers of cell viability. The absence of sulfate led to a decrease in viable cell numbers of greater than 7 log10 within 5 days, a > 99% reduction in genomic DNA within 3 days, and a > 60% decrease in ATP within 6 h. Likewise, cells incubated with lost viability (decreased by > 2 log10 in 5 days), extractable genomic DNA (reduction of > 60% in 2 days), and ATP (reduction of > 70 % in 2 hours). These results demonstrate that sulfate deprivation or the presence of molybdate have similar impacts on cell viability and essential biomolecules. Sulfate was coupled to cellular ATP content and maintenance of DNA integrity in F. acidarmanus, a finding that may be applicable to other acidophiles that are typically found in sulfate-rich biotopes. PMID:19054747

  17. Yeast Mitochondrial Interactosome Model: Metabolon Membrane Proteins Complex Involved in the Channeling of ADP/ATP

    PubMed Central

    Clémençon, Benjamin

    2012-01-01

    The existence of a mitochondrial interactosome (MI) has been currently well established in mammalian cells but the exact composition of this super-complex is not precisely known, and its organization seems to be different from that in yeast. One major difference is the absence of mitochondrial creatine kinase (MtCK) in yeast, unlike that described in the organization model of MI, especially in cardiac, skeletal muscle and brain cells. The aim of this review is to provide a detailed description of different partner proteins involved in the synergistic ADP/ATP transport across the mitochondrial membranes in the yeast Saccharomyces cerevisiae and to propose a new mitochondrial interactosome model. The ADP/ATP (Aacp) and inorganic phosphate (PiC) carriers as well as the VDAC (or mitochondrial porin) catalyze the import and export of ADP, ATP and Pi across the mitochondrial membranes. Aacp and PiC, which appear to be associated with the ATP synthase, consist of two nanomotors (F0, F1) under specific conditions and form ATP synthasome. Identification and characterization of such a complex were described for the first time by Pedersen and co-workers in 2003. PMID:22408429

  18. Yeast mitochondrial interactosome model: metabolon membrane proteins complex involved in the channeling of ADP/ATP.

    PubMed

    Clémençon, Benjamin

    2012-01-01

    The existence of a mitochondrial interactosome (MI) has been currently well established in mammalian cells but the exact composition of this super-complex is not precisely known, and its organization seems to be different from that in yeast. One major difference is the absence of mitochondrial creatine kinase (MtCK) in yeast, unlike that described in the organization model of MI, especially in cardiac, skeletal muscle and brain cells. The aim of this review is to provide a detailed description of different partner proteins involved in the synergistic ADP/ATP transport across the mitochondrial membranes in the yeast Saccharomyces cerevisiae and to propose a new mitochondrial interactosome model. The ADP/ATP (Aacp) and inorganic phosphate (PiC) carriers as well as the VDAC (or mitochondrial porin) catalyze the import and export of ADP, ATP and Pi across the mitochondrial membranes. Aacp and PiC, which appear to be associated with the ATP synthase, consist of two nanomotors (F(0), F(1)) under specific conditions and form ATP synthasome. Identification and characterization of such a complex were described for the first time by Pedersen and co-workers in 2003. PMID:22408429

  19. Oxidative stress inhibits vascular K(ATP) channels by S-glutathionylation.

    PubMed

    Yang, Yang; Shi, Weiwei; Cui, Ningren; Wu, Zhongying; Jiang, Chun

    2010-12-01

    The K(ATP) channel is an important player in vascular tone regulation. Its opening and closure lead to vasodilation and vasoconstriction, respectively. Such functions may be disrupted in oxidative stress seen in a variety of cardiovascular diseases, while the underlying mechanism remains unclear. Here, we demonstrated that S-glutathionylation was a modulation mechanism underlying oxidant-mediated vascular K(ATP) channel regulation. An exposure of isolated mesenteric rings to hydrogen peroxide (H(2)O(2)) impaired the K(ATP) channel-mediated vascular dilation. In whole-cell recordings and inside-out patches, H(2)O(2) or diamide caused a strong inhibition of the vascular K(ATP) channel (Kir6.1/SUR2B) in the presence, but not in the absence, of glutathione (GSH). Similar channel inhibition was seen with oxidized glutathione (GSSG) and thiol-modulating reagents. The oxidant-mediated channel inhibition was reversed by the reducing agent dithiothreitol (DTT) and the specific deglutathionylation reagent glutaredoxin-1 (Grx1). Consistent with S-glutathionylation, streptavidin pull-down assays with biotinylated glutathione ethyl ester (BioGEE) showed incorporation of GSH to the Kir6.1 subunit in the presence of H(2)O(2). These results suggest that S-glutathionylation is an important mechanism for the vascular K(ATP) channel modulation in oxidative stress. PMID:20926382

  20. Autocrine regulation of T-cell activation by ATP release and P2X7 receptors.

    PubMed

    Yip, Linda; Woehrle, Tobias; Corriden, Ross; Hirsh, Mark; Chen, Yu; Inoue, Yoshiaki; Ferrari, Vhe; Insel, Paul A; Junger, Wolfgang G

    2009-06-01

    T-cell activation requires the influx of extracellular calcium, although mechanistic details regarding such activation are not fully defined. Here, we show that P2X(7) receptors play a key role in calcium influx and downstream signaling events associated with the activation of T cells. By real-time PCR and immunohistochemistry, we find that Jurkat T cells and human CD4(+) T cells express abundant P2X(7) receptors. We show, using a novel fluorescent microscopy technique, that T-cell receptor (TCR) stimulation triggers the rapid release of ATP (<100 microM). This release of ATP is required for TCR-mediated calcium influx, NFAT activation, and interleukin-2 (IL-2) production. TCR activation up-regulates P2X(7) receptor gene expression. Removal of extracellular ATP by apyrase or alkaline phosphatase treatment, inhibition of ATP release with the maxi-anion channel blocker gadolinium chloride, or siRNA silencing of P2X(7) receptors blocks calcium entry and inhibits T-cell activation. Moreover, lymphocyte activation is impaired in C57BL/6 mice that express poorly functional P2X(7) receptors, compared to control BALB/c mice, which express fully functional P2X(7) receptors. We conclude that ATP release and autocrine, positive feedback through P2X(7) receptors is required for the effective activation of T cells. PMID:19211924