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1

Fe(III).ATP complexes. Models for ferritin and other polynuclear iron complexes with phosphate.  

PubMed

Polynuclear iron complexes of Fe(III) and phosphate occur in seawater and soils and in cells where the iron core of ferritin, the iron storage protein, contains up to 4500 Fe atoms in a complex with an average composition of (FeO.OH)8FeO.OPO3H2. Although phosphate influences the size of the ferritin core and thus the availability of stored iron, little is known about the nature of the Fe(III)-phosphate interaction. In the present study, Fe-phosphate interactions were analyzed in stable complexes of Fe(III).ATP which, in the polynuclear iron form, had phosphate at interior sites. Such Fe(III).ATP complexes are important not only as models but also because they may play a role in intracellular iron transport and in iron toxicity; the complexes were studied by extended x-ray absorption fine structure, EPR, NMR spectroscopy, and measurement of proton release. Mononuclear iron complexes exhibiting a g' = 4.3 EPR signal were formed at Fe:ATP ratios less than or equal to 1:3, and polynuclear iron complexes (Fe greater than or equal to 250, EPR silent at g' = 4.3) were formed at an Fe:ATP ratio of 4:1. No NMR signals due to ATP were observed when Fe was in excess (Fe:ATP = 4:1). Extended x-ray absorption fine structure analysis of the polynuclear Fe(III).ATP complex was able to distinguish an Fe-P distance at 3.27 A in addition to the octahedral O at 1.95 A and 4-5 Fe atoms at 3.36 A. The Fe-O and Fe-Fe distances are the same as in ferritin, and the Fe-P distance is analogous to that in another metal-ATP complex. An observable Fe-P environment in such a large polynuclear iron cluster as the Fe(III).ATP (4:1) complex indicates that the phosphate is distributed throughout rather than merely on the surface, in contrast to earlier models of chelate-stabilized iron clusters. Complexes of Fe(III) and ATP similar to those described here may form in vivo either as normal components of intracellular iron metabolism or during iron excess where the consequent alteration of free nucleotide triphosphate pools could contribute to the observed toxicity of iron. PMID:2989269

Mansour, A N; Thompson, C; Theil, E C; Chasteen, N D; Sayers, D E

1985-07-01

2

Cloning, characterization and mapping of the human ATP5E gene, identification of pseudogene ATP5EP1, and definition of the ATP5E motif.  

PubMed Central

A cDNA encoding the epsilon subunit of human ATP synthase, ATP5E, was isolated from heart, skeletal muscle and spleen cDNA libraries respectively. Its genome structure was characterized as comprising three exons and two introns within a stretch of 5 kb, according to the genomic sequence AL109840. The gene was mapped to human chromosome 20q13.3 between marker D20S173 and 20qter using the radiation hybrid GB4 panel. Northern blot analysis showed that the ATP5E gene was expressed as a single 0.6 kb transcript in all 16 human tissues tested, with a high level present in heart and skeletal muscle. A new conserved motif composed of 24 residues, termed the ATP5E motif [W(R/K)X(5)YX(2)(Y/F)X(3)(C/A)X(4)RX(3)K], was defined on the basis of sequences of ATP synthase epsilon subunits from ten different organisms. In addition, a pseudogene ATP5EP1 was also identified on the basis of genomic sequence AC004066, localized on human chromosome 4q25. By analysing these results combined with the Southern blot patterns of human DNA hybridized with bovine ATP5E cDNA reported previously [Vinas, Powell, Runswick, Iacobazzi and Walker (1990) Biochem. J. 265, 321-326], we provide evidence of yet further homologous sequences (either gene or pseudogene) of ATP5E, in addition to ATP5E and ATP5EP1 in the human genome. PMID:10727396

Tu, Q; Yu, L; Zhang, P; Zhang, M; Zhang, H; Jiang, J; Chen, C; Zhao, S

2000-01-01

3

Cloning, characterization and mapping of the human ATP5E gene, identification of pseudogene ATP5EP1, and definition of the ATP5E motif.  

PubMed

A cDNA encoding the epsilon subunit of human ATP synthase, ATP5E, was isolated from heart, skeletal muscle and spleen cDNA libraries respectively. Its genome structure was characterized as comprising three exons and two introns within a stretch of 5 kb, according to the genomic sequence AL109840. The gene was mapped to human chromosome 20q13.3 between marker D20S173 and 20qter using the radiation hybrid GB4 panel. Northern blot analysis showed that the ATP5E gene was expressed as a single 0.6 kb transcript in all 16 human tissues tested, with a high level present in heart and skeletal muscle. A new conserved motif composed of 24 residues, termed the ATP5E motif [W(R/K)X(5)YX(2)(Y/F)X(3)(C/A)X(4)RX(3)K], was defined on the basis of sequences of ATP synthase epsilon subunits from ten different organisms. In addition, a pseudogene ATP5EP1 was also identified on the basis of genomic sequence AC004066, localized on human chromosome 4q25. By analysing these results combined with the Southern blot patterns of human DNA hybridized with bovine ATP5E cDNA reported previously [Vinas, Powell, Runswick, Iacobazzi and Walker (1990) Biochem. J. 265, 321-326], we provide evidence of yet further homologous sequences (either gene or pseudogene) of ATP5E, in addition to ATP5E and ATP5EP1 in the human genome. PMID:10727396

Tu, Q; Yu, L; Zhang, P; Zhang, M; Zhang, H; Jiang, J; Chen, C; Zhao, S

2000-04-01

4

An ATP-dependent As(III)-glutathione transport system in membrane vesicles of Leishmania tarentolae.  

PubMed Central

Membrane preparations enriched in plasma membrane vesicles prepared from promastigotes of Leishmania tarentolae were shown to accumulate thiolate derivatives of 73As(III). Free arsenite was transported at a low rate, but rapid accumulation was observed after reaction with reduced glutathione (GSH) conditions that favor the formation of As(GS)3. Accumulation required ATP but not electrochemical energy, indicating that As(GS)3 is transported by an ATP-coupled pump. Pentostam, a Sb(V)-containing drug that is one of the first-line therapeutic agents for treatment of leishmaniasis, inhibited uptake after reaction with GSH. Vesicles prepared from a strain in which both copies of the pgpA genes were disrupted accumulated As(GS)3 at wild-type levels, demonstrating that the PgpA protein is not the As(GS)3 pump. These results have important implications for the mechanism of drug resistance in the trypanosomatidae, suggesting that a plasma membrane As(GS)3 pump catalyzes active extrusion of metal thiolates, including the Pentostam-glutathione conjugate. PMID:8700907

Dey, S; Ouellette, M; Lightbody, J; Papadopoulou, B; Rosen, B P

1996-01-01

5

Complexation of curium(III) by adenosine 5?-triphosphate (ATP): A time-resolved laser-induced fluorescence spectroscopy (TRLFS) study  

Microsoft Academic Search

The complex formation of curium(III) with adenosine 5?-triphosphate (ATP) was determined by time-resolved laser-induced fluorescence spectroscopy (TRLFS). The interaction between soluble species of curium(III) with ATP was studied at trace Cm(III) concentrations (3×10?7M). The concentrations of ATP were varied between 6.0×10?7 and 1.5×10?4M in the pH range of 1.5–7.0 using 0.154M NaCl as background electrolyte.Three Cm–ATP species, MpHqLr, could be

Henry Moll; Gerhard Geipel; Gert Bernhard

2005-01-01

6

Regulation of the type III InsP(3) receptor by InsP(3) and ATP.  

PubMed Central

Many hormones and neurotransmitters raise intracellular calcium (Ca(2+)) by generating InsP(3) and activating the inositol 1,4, 5-trisphosphate receptor (InsP(3)R). Multiple isoforms with distinct InsP(3) binding properties () have been identified (). The type III InsP(3)R lacks Ca(2+)-dependent inhibition, a property that makes it ideal for signal initiation (). Regulation of the type III InsP(3)R by InsP(3) and ATP was explored in detail using planar lipid bilayers. In comparison to the type I InsP(3)R, the type III InsP(3)R required a higher concentration of InsP(3) to reach maximal channel activity (EC(50) of 3.2 microM versus 0.5 microM for the types III and I InsP(3)R, respectively). However, the type III InsP(3)R did reach a 2.5-fold higher level of activity. Although activation by InsP(3) was isoform-specific, regulation by ATP was similar for both isoforms. In the presence of 2 microM InsP(3), low ATP concentrations (<6 mM) increased the open probability and mean open time. High ATP concentrations (>6 mM) decreased channel activity. These results illustrate the complex nature of type III InsP(3)R regulation. Enhanced channel activity in the presence of high InsP(3) may be important during periods of prolonged stimulation, whereas allosteric modulation by ATP may help to modulate intracellular Ca(2+) signaling. PMID:10866953

Hagar, R E; Ehrlich, B E

2000-01-01

7

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

PubMed Central

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

2013-01-01

8

National Cholesterol Education Program Adult Treatment Panel III Versus International Diabetic Federation Definition of Metabolic Syndrome, Which One is Associated with Diabetes Mellitus and Coronary Artery Disease?  

PubMed Central

Background: A cluster of risk factors for cardiovascular diseases and type 2 diabetes mellitus, which occur together more often than by chance alone, have been known as the metabolic syndrome. Various definitions have been proposed by different organizations over the past decade. This study was designed to evaluate a new definition of the metabolic syndrome for the prediction of diabetes mellitus among the Iranian population. Methods: This study was carried out in an urban population, aged 20 to 74 years, from Yazd, a city in the center of Iran. The study is a part of the phase I of Yazd Healthy Heart Program, that is, a community-based intervention study for the prevention of cardiovascular disease. The significance level has been defined as P<0.05. Results: Prevalence of the metabolic syndrome by the National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) criteria was 21.3 ± .017%, and by International Diabetes Federation (IDF) criteria it was 30.16 ± .02%. The multivariate analysis showed that the most important relevant factors of diabetes mellitus were: Increased age and metabolic syndrome by both definitions of NCEP and IDF criteria, and also, the most important relevant factors of stable angina were: Increased age, male sex, and metabolic syndrome by only IDF definitions, but the NCEP definition of the metabolic syndrome cannot predict diabetes mellitus independent of age and sex. Conclusion: This study showed that increased age and metabolic syndrome are the most important relevant factors for diabetes mellitus, especially by using the IDF criteria for definition of the metabolic syndrome. PMID:22973485

Rezaianzadeh, Abbas; Namayandeh, Seyedeh-Mahdieh; Sadr, Seyed-Mahmood

2012-01-01

9

Making ATP  

PubMed Central

We present a mesoscopic model for ATP synthesis by F1Fo ATPase. The model combines the existing experimental knowledge of the F1 enzyme into a consistent mathematical model that illuminates how the stages in synthesis are related to the protein structure. For example, the model illuminates how specific interactions between the ?, ?, and ?3?3 subunits couple the Fo motor to events at the catalytic sites. The model also elucidates the origin of ADP inhibition of F1 in its hydrolysis mode. The methodology we develop for constructing the structure-based model should prove useful in modeling other protein motors. PMID:16217018

Xing, Jianhua; Liao, Jung-Chi; Oster, George

2005-01-01

10

Optical ATP Biosensor for Extracellular ATP Measurement  

PubMed Central

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

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

2013-01-01

11

Molecular Structure of ATP  

NSDL National Science Digital Library

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.

2003-05-09

12

Prevalence of the metabolic syndrome in Pudong New Area of Shanghai using three proposed definitions among Chinese adults  

PubMed Central

Background The prevalence of metabolic syndrome (MS) has been increasing in China in recent years. The aim of this study is to estimate and compare the prevalence of MS among Chinese adults in Shanghai, one of the most economic developed areas in China, using definitions proposed by World Health Organization (WHO), National Cholesterol Education Program Adult Treatment Panel (modified ATP III) and International Diabetes Federation (IDF). Methods This cross-sectional study included 5,584 adults at age 20-79 randomly selected from Pudong New Area of Shanghai, China, through a three-stage sampling. All participants were interviewed in-person between April and July of 2008 to collect information on demographic and lifestyle characteristics. At the interview, anthropometry and blood pressure were measured and bio-specimens were collected. Results The prevalence estimates for the MS increased with age for each definition in men and women, but the estimates varied greatly between the definitions and by sex. The prevalence of the MS was higher in men (20.2%) than in women (18.7%) using WHO definition but this sex difference was reversed when using the modified ATP III (28.4% for men vs. 35.1% for women) and the IDF (15.9% for men vs. 26.7% for women) criteria. The most common metabolic disorder in this population was dyslipidaemia, regardless of the definition used. Substantial agreement, estimated using the kappa statistic, was found between the modified ATP III and IDF definition, whereas the lowest agreement was observed between the WHO and ATP III criteria. Conclusions The MS is highly prevalent among Chinese adults in Pudong New Area of Shanghai and the most prevalent component was dyslipidemia. These findings underscore the importance of prevention and control efforts for the MS in this area and the need for a unified predictive definition for the syndrome for use by clinical practitioners and public health agencies. PMID:20459855

2010-01-01

13

Electron transfer precedes ATP hydrolysis during nitrogenase catalysis  

PubMed Central

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 Feox(ADP)2 protein and the reduced MoFe protein. PMID:24062462

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

2013-01-01

14

Assessment of severe malaria in a multicenter, phase III, RTS, S/AS01 malaria candidate vaccine trial: case definition, standardization of data collection and patient care  

PubMed Central

Background An effective malaria vaccine, deployed in conjunction with other malaria interventions, is likely to substantially reduce the malaria burden. Efficacy against severe malaria will be a key driver for decisions on implementation. An initial study of an RTS, S vaccine candidate showed promising efficacy against severe malaria in children in Mozambique. Further evidence of its protective efficacy will be gained in a pivotal, multi-centre, phase III study. This paper describes the case definitions of severe malaria used in this study and the programme for standardized assessment of severe malaria according to the case definition. Methods Case definitions of severe malaria were developed from a literature review and a consensus meeting of expert consultants and the RTS, S Clinical Trial Partnership Committee, in collaboration with the World Health Organization and the Malaria Clinical Trials Alliance. The same groups, with input from an Independent Data Monitoring Committee, developed and implemented a programme for standardized data collection. The case definitions developed reflect the typical presentations of severe malaria in African hospitals. Markers of disease severity were chosen on the basis of their association with poor outcome, occurrence in a significant proportion of cases and on an ability to standardize their measurement across research centres. For the primary case definition, one or more clinical and/or laboratory markers of disease severity have to be present, four major co-morbidities (pneumonia, meningitis, bacteraemia or gastroenteritis with severe dehydration) are excluded, and a Plasmodium falciparum parasite density threshold is introduced, in order to maximize the specificity of the case definition. Secondary case definitions allow inclusion of co-morbidities and/or allow for the presence of parasitaemia at any density. The programmatic implementation of standardized case assessment included a clinical algorithm for evaluating seriously sick children, improvements to care delivery and a robust training and evaluation programme for clinicians. Conclusions The case definition developed for the pivotal phase III RTS, S vaccine study is consistent with WHO recommendations, is locally applicable and appropriately balances sensitivity and specificity in the diagnosis of severe malaria. Processes set up to standardize severe malaria data collection will allow robust assessment of the efficacy of the RTS, S vaccine against severe malaria, strengthen local capacity and benefit patient care for subjects in the trial. Trial registration Clinicaltrials.gov NCT00866619 PMID:21816031

2011-01-01

15

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

SciTech Connect

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

Fernandes, Annemarie T. [Department of Radiation Oncology, University of Pennsylvania, Philadelphia, PA (United States); Mitra, Nandita [Department of Biostatistics and Epidemiology, University of Pennsylvania, Philadelphia, PA (United States); Xanthopoulos, Eric [Department of Radiation Oncology, University of Pennsylvania, Philadelphia, PA (United States); Evans, Tracey; Stevenson, James; Langer, Corey [Department of Medical Oncology, University of Pennsylvania, Philadelphia, PA (United States); Kucharczuk, John C. [Department of Thoracic Surgery, University of Pennsylvania, Philadelphia, PA (United States); Lin, Lilie; Rengan, Ramesh [Department of Radiation Oncology, University of Pennsylvania, Philadelphia, PA (United States)

2012-05-01

16

The role of subunit epsilon in the catalysis and regulation of F OF 1ATP synthase  

Microsoft Academic Search

The regulation of ATP synthase activity is complex and involves several distinct mechanisms. In bacteria and chloroplasts, subunit epsilon plays an important role in this regulation, (i) affecting the efficiency of coupling, (ii) influencing the catalytic pathway, and (iii) selectively inhibiting ATP hydrolysis activity. Several experimental studies indicate that the regulation is achieved through large conformational transitions of the ?-helical

Boris A. Feniouk; Toshiharu Suzuki; Masasuke Yoshida

2006-01-01

17

Optogenetic control of ATP release  

NASA Astrophysics Data System (ADS)

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

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

2013-03-01

18

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

PubMed

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

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

2014-11-01

19

Structure of ATP-Bound Human ATP:Cobalamin Adenosyltransferase  

SciTech Connect

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.

Schubert,H.; Hill, C.

2006-01-01

20

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

SciTech Connect

Purpose: Radiation Therapy Oncology Group protocol 85-31 was designed to evaluate the effectiveness of adjuvant androgen suppression, using goserelin, in unfavorable prognosis carcinoma of the prostate treated with definitive radiotherapy (RT). Methods and Materials: Eligible patients were those with palpable primary tumor extending beyond the prostate (clinical Stage T3) or those with regional lymphatic involvement. Patients who had undergone prostatectomy were eligible if penetration through the prostatic capsule to the margin of resection and/or seminal vesicle involvement was documented histologically. Stratification was based on histologic differentiation, nodal status, acid phosphatase status, and prior prostatectomy. The patients were randomized to either RT and adjuvant goserelin (Arm I) or RT alone followed by observation and application of goserelin at relapse (Arm II). In Arm I, the drug was to be started during the last week of RT and was to be continued indefinitely or until signs of progression. Results: Between 1987 and 1992, when the study was closed, 977 patients were entered: 488 to Arm I and 489 to Arm II. As of July 2003, the median follow-up for all patients was 7.6 years and for living patients was 11 years. At 10 years, the absolute survival rate was significantly greater for the adjuvant arm than for the control arm: 49% vs. 39%, respectively (p = 0.002). The 10-year local failure rate for the adjuvant arm was 23% vs. 38% for the control arm (p <0.0001). The corresponding 10-year rates for the incidence of distant metastases and disease-specific mortality was 24% vs. 39% (p <0.001) and 16% vs. 22% (p = 0.0052), respectively, both in favor of the adjuvant arm. Conclusion: In a population of patients with unfavorable prognosis carcinoma of the prostate, androgen suppression applied as an adjuvant after definitive RT was associated not only with a reduction in disease progression but in a statistically significant improvement in absolute survival. The improvement in survival appeared preferentially in patients with a Gleason score of 7-10.

Pilepich, Miljenko V. [University of California, Los Angeles, School of Medicine, Los Angeles, CA (United States)]. E-mail: mpilepich@mednet.ucla.edu; Winter, Kathryn [Radiation Therapy Oncology Group, Philadelphia, PA (United States); Lawton, Colleen A. [Medical College of Wisconsin, Milwaukee, WI (United States); Krisch, Robert E. [University of Pennsylvania, Philadelphia, PA (United States); Wolkov, Harvey B. [Radiology Associates of Sacramento, Sacramento, CA (United States); Movsas, Benjamin [Fox Chase Cancer Center, Philadelphia, PA (United States); Hug, Eugen B. [Dartmouth Hitchcock Medical Center, Lebanon, NH (United States); Asbell, Sucha O. [Albert Einstein Medical Center, Philadelphia, PA (United States); Grignon, David [Wayne State University, Detroit, MI (United States)

2005-04-01

21

Energy transduction in ATP synthase  

NASA Astrophysics Data System (ADS)

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 Fo 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 Fo is thought to generate a torque which is transmitted to F1 by an asymmetric shaft, the coiled-coil ?-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 protonmotive force biases the rotor's diffusion so that Fo constitutes a rotary motor turning the ? 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.

Elston, Timothy; Wang, Hongyun; Oster, George

1998-01-01

22

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

Microsoft Academic Search

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

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

23

?? sub-complex of thermophilic ATP synthase has the ability to bind ATP  

Microsoft Academic Search

The isolated ? subunit of F1-ATPase from thermophilic Bacillus PS3 (TF1) binds ATP [Y. Kato-Yamada, M. Yoshida, J. Biol. Chem. 278 (2003) 36013]. The obvious question is whether the ATP binding concern with the regulation of ATP synthase activity or not. If so, the ? subunit even in the ATP synthase complex should have the ability to bind ATP. To

Satoshi Iizuka; Shigeyuki Kato; Masasuke Yoshida; Yasuyuki Kato-Yamada

2006-01-01

24

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

PubMed Central

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

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

2001-01-01

25

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

PubMed Central

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 Mg2+, 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 Mg2+ 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 Mg2+, adenylate transport, and phosphate release and supply. PMID:25674099

Igamberdiev, Abir U.; Kleczkowski, Leszek A.

2015-01-01

26

Homeostasis of extracellular ATP in human erythrocytes.  

PubMed

We explored the intra- and extracellular processes governing the kinetics of extracellular ATP (ATPe) in human erythrocytes stimulated with agents that increase cAMP. Using the luciferin-luciferase reaction in off-line luminometry we found both direct adenylyl cyclase activation by forskolin and indirect activation through ?-adrenergic stimulation with isoproterenol-enhanced [ATP]e in a concentration-dependent manner. A mixture (3V) containing a combination of these agents and the phosphodiesterase inhibitor papaverine activated ATP release, leading to a 3-fold increase in [ATP]e, and caused increases in cAMP concentration (3-fold for forskolin + papaverine, and 10-fold for 3V). The pannexin 1 inhibitor carbenoxolone and a pannexin 1 blocking peptide ((10)Panx1) decreased [ATP]e by 75-84%. The residual efflux of ATP resulted from unavoidable mechanical perturbations stimulating a novel, carbenoxolone-insensitive pathway. In real-time luminometry experiments using soluble luciferase, addition of 3V led to an acute increase in [ATP]e to a constant value of ?1 pmol × (10(6) cells)(-1). A similar treatment using a surface attached luciferase (proA-luc) triggered a rapid accumulation of surface ATP levels to a peak concentration of 2.4 pmol × (10(6) cells)(-1), followed by a slower exponential decay (t(½) = 3.7 min) to a constant value of 1.3 pmol × (10(6) cells)(-1). Both for soluble luciferase and proA-luc, ATP efflux was fully blocked by carbenoxolone, pointing to a 3V-induced mechanism of ATP release mediated by pannexin 1. Ecto-ATPase activity was extremely low (?28 fmol × (10(6) cells min)(-1)), but nevertheless physiologically relevant considering the high density of erythrocytes in human blood. PMID:21921036

Montalbetti, Nicolas; Leal Denis, Maria F; Pignataro, Omar P; Kobatake, Eiry; Lazarowski, Eduardo R; Schwarzbaum, Pablo J

2011-11-01

27

Homeostasis of Extracellular ATP in Human Erythrocytes*  

PubMed Central

We explored the intra- and extracellular processes governing the kinetics of extracellular ATP (ATPe) in human erythrocytes stimulated with agents that increase cAMP. Using the luciferin-luciferase reaction in off-line luminometry we found both direct adenylyl cyclase activation by forskolin and indirect activation through ?-adrenergic stimulation with isoproterenol-enhanced [ATP]e in a concentration-dependent manner. A mixture (3V) containing a combination of these agents and the phosphodiesterase inhibitor papaverine activated ATP release, leading to a 3-fold increase in [ATP]e, and caused increases in cAMP concentration (3-fold for forskolin + papaverine, and 10-fold for 3V). The pannexin 1 inhibitor carbenoxolone and a pannexin 1 blocking peptide (10Panx1) decreased [ATP]e by 75–84%. The residual efflux of ATP resulted from unavoidable mechanical perturbations stimulating a novel, carbenoxolone-insensitive pathway. In real-time luminometry experiments using soluble luciferase, addition of 3V led to an acute increase in [ATP]e to a constant value of ?1 pmol × (106 cells)?1. A similar treatment using a surface attached luciferase (proA-luc) triggered a rapid accumulation of surface ATP levels to a peak concentration of 2.4 pmol × (106 cells)?1, followed by a slower exponential decay (t½ = 3.7 min) to a constant value of 1.3 pmol × (106 cells)?1. Both for soluble luciferase and proA-luc, ATP efflux was fully blocked by carbenoxolone, pointing to a 3V-induced mechanism of ATP release mediated by pannexin 1. Ecto-ATPase activity was extremely low (?28 fmol × (106 cells min)?1), but nevertheless physiologically relevant considering the high density of erythrocytes in human blood. PMID:21921036

Montalbetti, Nicolas; Leal Denis, Maria F.; Pignataro, Omar P.; Kobatake, Eiry; Lazarowski, Eduardo R.; Schwarzbaum, Pablo J.

2011-01-01

28

Methylene ATP analogs as modulators of extracellular ATP metabolism and accumulation  

PubMed Central

Transient accumulation of extracellular ATP reflects both release of ATP from intracellular stores and altered rates of ATP metabolism by ecto-enzymes. Ecto-nucleoside triphosphate diphosphohydrolases (eNTPDases) and ecto-nucleotide pyrophosphatases (eNPPs) degrade ATP, while ecto-nucleotide diphosphokinases (eNDPKs) synthesize ATP from ambient ADP. Although the methylene ATP analogs ??-meATP and ??-meATP are widely used as metabolically stable tools for the analysis of purinergic signaling, their specific effects on eNTPDase, eNPP, and eNDPK activities have not been defined. This study compared the actions of these analogs on extracellular ATP metabolism by human 1321N1 astrocytes, rat PC12 pheochomocytoma cells, and rat C6 glioma cells. Both analogs significantly reduced clearance of extracellular ATP by 1321N1 cells that express both eNTPDases and eNPPs, as well as by C6 cells that exclusively express eNPPs. In contrast, both analogs were much less efficacious in inhibiting ATP clearance by PC12 cells that predominantly express eNTPDases. ??-meATP, but not ??-meATP, was effectively hydrolyzed by the 1321N1 and C6 cells; PC12 cells did not significantly degrade this analog. ??-meATP, but not ??-meATP, acted as a substrate for purified yeast NDPK to generate ATP via trans-phosphorylation of ADP. ??-meATP also acted as substrate for the eNDPK activities expressed by 1321N1, PC12, and C6 cells and thereby induced extracellular ATP accumulation in the presence of ambient or exogenously added ADP. These results indicate that methylene ATP analogs exert complex and cell-specific effects on extracellular ATP metabolism that can significantly modify interpretation of studies that use these reagents as probes of purinergic signal transduction in intact tissues. PMID:15210578

Joseph, Sheldon M; Pifer, Matthew A; Przybylski, Ronald J; Dubyak, George R

2004-01-01

29

Communication Definitions... general definition  

E-print Network

design Natural selection will favour individuals that produce effective signals Of maximum effectivenessCommunication Definitions... general definition "the process of conveying information from a sender

Jones, Ian L.

30

Resilience: Theory and Applications Contents .........................................................................................................................................iii  

E-print Network

#12;#12;#12;#12;Resilience: Theory and Applications iii Contents Contents................................................................................................................................... 9 2 Definition and Properties of Resilience.................................................................................. 11 2.1 Defining Resilience

Kemner, Ken

31

Genetics Home Reference: Mucolipidosis III alpha/beta  

MedlinePLUS

... literature OMIM Genetic disorder catalog Conditions > Mucolipidosis III alpha/beta On this page: Description Genetic changes Inheritance ... definitions Reviewed October 2014 What is mucolipidosis III alpha/beta? Mucolipidosis III alpha/beta is a slowly ...

32

ATP Synthase Complex of Plasmodium falciparum  

PubMed Central

The rotary nanomotor ATP synthase is a central player in the bioenergetics of most organisms. Yet the role of ATP synthase in malaria parasites has remained unclear, as blood stages of Plasmodium falciparum appear to derive ATP largely through glycolysis. Also, genes for essential subunits of the FO sector of the complex could not be detected in the parasite genomes. Here, we have used molecular genetic and immunological tools to investigate the localization, complex formation, and functional significance of predicted ATP synthase subunits in P. falciparum. We generated transgenic P. falciparum lines expressing seven epitope-tagged canonical ATP synthase subunits, revealing localization of all but one of the subunits to the mitochondrion. Blue native gel electrophoresis of P. falciparum mitochondrial membranes suggested the molecular mass of the ATP synthase complex to be greater than 1 million daltons. This size is consistent with the complex being assembled as a dimer in a manner similar to the complexes observed in other eukaryotic organisms. This observation also suggests the presence of previously unknown subunits in addition to the canonical subunits in P. falciparum ATP synthase complex. Our attempts to disrupt genes encoding ? and ? subunits were unsuccessful, suggesting an essential role played by the ATP synthase complex in blood stages of P. falciparum. These studies suggest that, despite some unconventional features and its minimal contribution to ATP synthesis, P. falciparum ATP synthase is localized to the parasite mitochondrion, assembled as a large dimeric complex, and is likely essential for parasite survival. PMID:21984828

Nina, Praveen Balabaskaran; Morrisey, Joanne M.; Ganesan, Suresh M.; Ke, Hangjun; Pershing, April M.; Mather, Michael W.; Vaidya, Akhil B.

2011-01-01

33

Enhanced efficiency of ATP hydrolysis during nitrogenase catalysis utilizing reductants that form the all-ferrous redox state of the Fe protein.  

PubMed

The amount of MgATP hydrolyzed per pair of electrons transferred (ATP/2e) during nitrogenase catalysis (1.0 atm N(2), 30 degrees C) using titanium(III) citrate (Ti(III)) as reductant was measured and compared to the same reaction using dithionite (DT). ATP/2e values near 2.0 for Ti(III) and 5.0 for DT indicate that nitrogenase has a much lower ATP requirement using Ti(III) as reductant. Using reduced Azotobacter vinelandii flavoprotein (AvFlpH(2)), a possible in vivo nitrogenase reductant, ATP/2e values near 2.0 were also observed. When the reaction was conducted using Ti(III) under N(2), 5% CO in N(2), Ar, 5% CO in Ar, or acetylene, ATP/2e values near 2.0 were also observed. With Ti(III) as reductant, ATP/2e values near 2.0 were measured as a function of temperature, Fe:MoFe protein ratio, and MoFe:Fe protein ratio, in contrast to measured values of 4.0-25 when DT is used under the same conditions. Both Ti(III) and AvFlpH(2) are capable of forming the [Fe(4)S(4)](0) cluster state of the Fe protein whereas DT is not, suggesting that ATP/2e values near 2.0 arise from operation of the [Fe(4)S(4)](2+)/[Fe(4)S(4)](0) redox couple with hydrolysis of only 2 ATPs per pair of electrons transferred. Additional experiments showed that ATP/2e values near 2. 0 correlated with slower rates of product formation and that faster rates of product formation produced ATP/2e values near 5.0. ATP/2e values of 5.0 are consistent with the operation of the [Fe(4)S(4)](2+)/[Fe(4)S(4)](1+) redox couple while ATP/2e values of 2.0 could arise from operation of the [Fe(4)S(4)](2+)/[Fe(4)S(4)](0) redox couple. These results suggest that two distinct Fe protein redox couples may be functional during nitrogenase catalysis and that the efficiency of ATP utilization depends on which of these redox couples is dominant. PMID:10572002

Erickson, J A; Nyborg, A C; Johnson, J L; Truscott, S M; Gunn, A; Nordmeyer, F R; Watt, G D

1999-10-26

34

Evidence for an ATP-sensitive K+ channel in mitoplasts isolated from Trypanosoma cruzi and Crithidia fasciculata.  

PubMed

Mammalian mitochondria, as well as rat, plant and Caenorhabditis elegans mitochondria, possess an ATP-sensitive K+ channel (mitoK(ATP)) that has been pharmacologically characterised. Opening of mitoK(ATP) and the subsequent K+ entry into the matrix was shown to have three effects on mitochondria physiology: (i) an increase in matrix volume (swelling), (ii) an acceleration of respiration, and (iii) an increase in reactive oxygen species (ROS) production. These effects on mitochondria bioenergetics have been shown to be part of distinct intracellular signalling pathways, to protect against cell death and to modulate gene transcription. To date, such a channel or its activity has not been described in trypanosomatids. In the present study, we show pharmacological evidence for the presence of a mitoK(ATP) in trypanosomatids. Cells were incubated in a hypotonic medium followed by mild detergent exposure to isolate mitoplasts from Trypanosoma cruzi and Crithidia fasciculata. Mitoplasts swelled when incubated in KCl medium due to respiration-driven K+ entry into the matrix. Swelling was sensitive to the presence of ATP when the mitoplast suspension was incubated in K+ -containing, but not in K+ -free, medium. The ATP inhibition of swelling was reversed by the mitoK(ATP) agonist diazoxide and the diazoxide-induced swelling was inhibited by the mitoK(ATP) blockers 5-hydroxydecanoate (5HD) or glibenclamide. Similar to mammalian and rat mitochondria, trypanosomatid mitoK(ATP) activity was modulated by the general protein kinase C (PKC) agonist phorbol 12-myristate 13-acetate (PMA) and antagonist chelerythrine. As expected, the potassium ionophore valinomycin could also reverse the ATP-inhibited state but this reversal was not sensitive to 5HD or glibenclamide. Dose response curves for ATP, diazoxide and 5HD are presented. These results provide strong evidence for the presence of an ATP-sensitive K+ in trypanosomatid mitochondria. PMID:19504755

Costa, Alexandre D T; Krieger, Marco A

2009-07-15

35

A Parametric Study of IR and UV Optical Designs for Neutral Particle Beam (NPB) Acquisition, Tracking, and Pointing (ATP) Applications  

NASA Astrophysics Data System (ADS)

Several optical configurations were investigated for acquisition, tracking, and pointing (ATP) systems. These configurations were to be employed for ATPing targets that were to be interrogated by neutral particle beams (NPBs). NPBs delineate real targets from replica or false targets. This particular optical design investigation was for the NPB Concepts and Requirements Definitions Study (CARDS). The proposed NPB CARDS ATP consisted "of a coarse and a precision-passive IR acquisition and tracking sensor coupled to a precision-active UV tracker that referenced boresight, range, and target motion to an NPB-pointing control system."1

Wilkerson, Gary W.; Pitalo, Stephen K.

1990-01-01

36

Profiling Protein Kinases and Other ATP Binding Proteins in Arabidopsis Using Acyl-ATP Probes*  

PubMed Central

Many protein activities are driven by ATP binding and hydrolysis. Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using acyl-ATP (AcATP)1 probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Gel-based profiling using biotinylated AcATP showed that labeling is dependent on pH and divalent ions and can be competed by nucleotides. The vast majority of these AcATP-labeled proteins are known ATP binding proteins. Our search for labeled peptides upon in-gel digest led to the discovery that the biotin moiety of the labeled peptides is oxidized. The in-gel analysis displayed kinase domains of two receptor-like kinases (RLKs) at a lower than expected molecular weight, indicating that these RLKs lost the extracellular domain, possibly as a result of receptor shedding. Analysis of modified peptides using a gel-free platform identified 242 different labeling sites for AcATP in the Arabidopsis proteome. Examination of each individual labeling site revealed a preference of labeling in ATP binding pockets for a broad diversity of ATP binding proteins. Of these, 24 labeled peptides were from a diverse range of protein kinases, including RLKs, mitogen-activated protein kinases, and calcium-dependent kinases. A significant portion of the labeling sites could not be assigned to known nucleotide binding sites. However, the fact that labeling could be competed with ATP indicates that these labeling sites might represent previously uncharacterized nucleotide binding sites. A plot of spectral counts against expression levels illustrates the high specificity of AcATP probes for protein kinases and known ATP binding proteins. This work introduces profiling of ATP binding activities of a large diversity of proteins in plant proteomes. The data have been deposited in ProteomeXchange with the identifier PXD000188. PMID:23722185

Villamor, Joji Grace; Kaschani, Farnusch; Colby, Tom; Oeljeklaus, Julian; Zhao, David; Kaiser, Markus; Patricelli, Matthew P.; van der Hoorn, Renier A. L.

2013-01-01

37

Catalytic strategy used by the myosin motor to hydrolyze ATP  

PubMed Central

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

Kiani, Farooq Ahmad; Fischer, Stefan

2014-01-01

38

NADH supplementation decreases pinacidil-primed IK(ATP) in ventricular cardiomyocytes by increasing intracellular ATP  

PubMed Central

The aim of this study was to investigate the effect of nicotinamide-adenine dinucleotide (NADH) supplementation on the metabolic condition of isolated guinea-pig ventricular cardiomyocytes. The pinacidil-primed ATP-dependent potassium current IK(ATP) was used as an indicator of subsarcolemmal ATP concentration and intracellular adenine nucleotide contents were measured. Membrane currents were studied using the patch-clamp technique in the whole-cell recording mode at 36–37°C. Adenine nucleotides were determined by HPLC. Under physiological conditions (4.3 mM ATP in the pipette solution, ATPi) IK(ATP) did not contribute to basal electrical activity. The ATP-dependent potassium (K(ATP)) channel opener pinacidil activated IK(ATP) dependent on [ATP]i showing a significantly more pronounced activation at lower (1 mM) [ATP]i. Supplementation of cardiomyocytes with 300 ?g ml?1 NADH (4–6 h) resulted in a significantly reduced IK(ATP) activation by pinacidil compared to control cells. The current density was 13.8±3.78 (n=6) versus 28.9±3.38 pA pF?1 (n=19; P<0.05). Equimolar amounts of the related compounds nicotinamide and NAD+ did not achieve a similar effect like NADH. Measurement of adenine nucleotides by HPLC revealed a significant increase in intracellular ATP (NADH supplementation: 45.6±1.88 nmol mg?1 protein versus control: 35.4±2.57 nmol mg?1 protein, P<0.000005). These data show that supplementation of guinea-pig ventricular cardiomyocytes with NADH results in a decreased activation of IK(ATP) by pinacidil compared to control myocytes, indicating a higher subsarcolemmal ATP concentration. Analysis of intracellular adenine nucleotides by HPLC confirmed the significant increase in ATP. PMID:12812998

Pelzmann, Brigitte; Hallström, Seth; Schaffer, Peter; Lang, Petra; Nadlinger, Karl; Birkmayer, George D; Vrecko, Karoline; Reibnegger, Gilbert; Koidl, Bernd

2003-01-01

39

Imaging exocytosis of ATP-containing vesicles with TIRF microscopy in lung epithelial A549 cells.  

PubMed

Nucleotide release constitutes the first step of the purinergic signaling cascade, but its underlying mechanisms remain incompletely understood. In alveolar A549 cells much of the experimental data is consistent with Ca(2+)-regulated vesicular exocytosis, but definitive evidence for such a release mechanism is missing, and alternative pathways have been proposed. In this study, we examined ATP secretion from A549 cells by total internal reflection fluorescence microscopy to directly visualize ATP-loaded vesicles and their fusion with the plasma membrane. A549 cells were labeled with quinacrine or Bodipy-ATP, fluorescent markers of intracellular ATP storage sites, and time-lapse imaging of vesicles present in the evanescent field was undertaken. Under basal conditions, individual vesicles showed occasional quasi-instantaneous loss of fluorescence, as expected from spontaneous vesicle fusion with the plasma membrane and dispersal of its fluorescent cargo. Hypo-osmotic stress stimulation (osmolality reduction from 316 to 160 mOsm) resulted in a transient, several-fold increment of exocytotic event frequency. Lowering the temperature from 37°C to 20°C dramatically diminished the fraction of vesicles that underwent exocytosis during the 2-min stimulation, from ~40% to ?1%, respectively. Parallel ATP efflux experiments with luciferase bioluminescence assay revealed that pharmacological interference with vesicular transport (brefeldin, monensin), or disruption of the cytoskeleton (nocodazole, cytochalasin), significantly suppressed ATP release (by up to ~80%), whereas it was completely blocked by N-ethylmaleimide. Collectively, our data demonstrate that regulated exocytosis of ATP-loaded vesicles likely constitutes a major pathway of hypotonic stress-induced ATP secretion from A549 cells. PMID:21881960

Akopova, Irina; Tatur, Sabina; Grygorczyk, Mariusz; Luchowski, Rafa?; Gryczynski, Ignacy; Gryczynski, Zygmunt; Borejdo, Julian; Grygorczyk, Ryszard

2012-03-01

40

ATP-triggered anticancer drug delivery  

NASA Astrophysics Data System (ADS)

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

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

2014-03-01

41

ATP2B4/A0114  

NSDL National Science Digital Library

Plasma membrane calcium-transporting ATPase 4 (ATP2B4), also known as A0114, is a multi-pass membrane protein, which catalyzes the hydrolysis of adenosine triphosphate (ATP) coupled with the transport of calcium out of the cell.

2009-04-14

42

Glucose and the ATP paradox in yeast.  

PubMed Central

A sustained decrease in the intracellular ATP concentration has been observed when extra glucose was added to yeast cells growing aerobically under glucose limitation. Because glucose degradation is the main source of ATP-derived free energy, this is a counter-intuitive phenomenon, which cannot be attributed to transient ATP consumption in the initial steps of glycolysis. We present a core model for aerobic growth in which glucose supplies carbon, as well as free energy, for biosynthesis. With Metabolic Control Analysis and numerical simulations, we demonstrate that the decrease in the ATP concentration can be reproduced if the biosynthetic route is more strongly activated by carbon substrates than is the catabolic (ATP-producing) route. PMID:11085955

Somsen, O J; Hoeben, M A; Esgalhado, E; Snoep, J L; Visser, D; van der Heijden, R T; Heijnen, J J; Westerhoff, H V

2000-01-01

43

A Slow ATP-induced Conformational Change Limits the Rate of DNA Binding but Not the Rate of ? Clamp Binding by the Escherichia coli ? Complex Clamp Loader*  

PubMed Central

In Escherichia coli, the ? complex clamp loader loads the ?-sliding clamp onto DNA. The ? clamp tethers DNA polymerase III to DNA and enhances the efficiency of replication by increasing the processivity of DNA synthesis. In the presence of ATP, ? complex binds ? and DNA to form a ternary complex. Binding to primed template DNA triggers ? complex to hydrolyze ATP and release the clamp onto DNA. Here, we investigated the kinetics of forming a ternary complex by measuring rates of ? complex binding ? and DNA. A fluorescence intensity-based ? binding assay was developed in which the fluorescence of pyrene covalently attached to ? increases when bound by ? complex. Using this assay, an association rate constant of 2.3 × 107 m?1 s?1 for ? complex binding ? was determined. The rate of ? binding was the same in experiments in which ? complex was preincubated with ATP before adding ? or added directly to ? and ATP. In contrast, when ? complex is preincubated with ATP, DNA binding is faster than when ? complex is added to DNA and ATP at the same time. Slow DNA binding in the absence of ATP preincubation is the result of a rate-limiting ATP-induced conformational change. Our results strongly suggest that the ATP-induced conformational changes that promote ? binding and DNA binding differ. The slow ATP-induced conformational change that precedes DNA binding may provide a kinetic preference for ? complex to bind ? before DNA during the clamp loading reaction cycle. PMID:19759003

Thompson, Jennifer A.; Paschall, Christopher O.; O'Donnell, Mike; Bloom, Linda B.

2009-01-01

44

ATP Photosynthetic vesicles for light-driven bioprocesses  

Microsoft Academic Search

We prepared ATP photosynthetic vesicles from inside-out membranes of Escherichia coli cells that express delta-rhodopsin (a novel light-driven H+ transporter) and TF0F1-ATP synthase (a thermo-stable ATP synthase). These vesicles showed light-dependent ATP synthesis. Furthermore, coupling the\\u000a ATP photosynthetic vesicles with an ATP-hydrolyzing hexokinase enabled light-dependent glucose consumption. The ATP photosynthetic\\u000a vesicles indicate their potential to applied to light-driven ATP-regenerating bioprocess

Kiyotaka Y. Hara; Rie Suzuki; Toshiharu Suzuki; Masasuke Yoshida; Kuniki Kino

2011-01-01

45

Target-induced structure switching of hairpin aptamers for label-free and sensitive fluorescent detection of ATP via exonuclease-catalyzed target recycling amplification.  

PubMed

In this work, we described the development of a new label-free, simple and sensitive fluorescent ATP sensing platform based on exonuclease III (Exo III)-catalyzed target recycling (ECTR) amplification and SYBR Green I indicator. The hairpin aptamer probes underwent conformational structure switching and re-configuration in the presence of ATP, which led to catalytic cleavage of the re-configured aptamers by Exo III to release ATP and to initiate the ECTR process. Such ECTR process resulted in the digestion of a significant number of the hairpin aptamer probes, leading to much less intercalation of SYBR Green I to the hairpin stems and drastic suppression of the fluorescence emission for sensitive ATP detection down to the low nanomolar level. Due to the highly specific affinity bindings between aptamers and ATP, the developed method exhibited excellent selectivity toward ATP against other analogous molecules. Besides, our ATP sensing approach used un-modified aptamer probes and could be performed in a "mix-and-detect" fashion in homogenous solutions. All these distinct advantages of the developed method thus made it hold great potential for the development of simple and robust sensing strategies for the detection of other small molecules. PMID:23974161

Xu, Yunying; Xu, Jin; Xiang, Yun; Yuan, Ruo; Chai, Yaqin

2014-01-15

46

Translocation of platinum anticancer drugs by human copper ATPases ATP7A and ATP7B.  

PubMed

Cisplatin, carboplatin, and oxaliplatin are widely used anticancer drugs. Their efficacy is strongly reduced by development of cell resistance. Down-regulation of CTR1 and up-regulation of the Cu-ATPases, ATP7A and ATP7B, have been associated to augmented drug resistance. To gain information on translocation of Pt drugs by human Cu-ATPases, we performed electrical measurements on the COS-1 cell microsomal fraction, enriched with recombinant ATP7A, ATP7B, and selected mutants, and adsorbed on a solid supported membrane. The experimental results indicate that Pt drugs activate Cu-ATPases and undergo ATP-dependent translocation in a fashion similar to that of Cu. We then used NMR spectroscopy and ESI-MS to determine the binding mode of these drugs to the first N-terminal metal-binding domain of ATP7A (Mnk1). PMID:24375922

Tadini-Buoninsegni, Francesco; Bartolommei, Gianluca; Moncelli, Maria Rosa; Inesi, Giuseppe; Galliani, Angela; Sinisi, Marilù; Losacco, Maurizio; Natile, Giovanni; Arnesano, Fabio

2014-01-27

47

ATP-driven stepwise rotation of FoF1ATP synthase  

Microsoft Academic Search

FoF1-ATP synthase (FoF1) is a motor enzyme that couples ATP synthesis\\/hydrolysis with a transmembrane proton translocation. F1, a water-soluble ATPase portion of FoF1, rotates by repeating ATP-waiting dwell, 80° substep rotation, catalytic dwell, and 40°-substep rotation. Compared with F1, rotation of FoF1 has yet been poorly understood, and, here, we analyzed ATP-driven rotations of FoF1. Rotation was probed with an

Hiroshi Ueno; Toshiharu Suzuki; Kazuhiko Kinosita Jr.; Masasuke Yoshida

2005-01-01

48

Cell-free expression and assembly of ATP synthase.  

PubMed

Cell-free (CF) expression technologies have emerged as promising methods for the production of individual membrane proteins of different types and origin. However, many membrane proteins need to be integrated in complex assemblies by interaction with soluble and membrane-integrated subunits in order to adopt stable and functionally folded structures. The production of complete molecular machines by CF expression as advancement of the production of only individual subunits would open a variety of new possibilities to study their assembly mechanisms, function, or composition. We demonstrate the successful CF formation of large molecular complexes consisting of both membrane-integrated and soluble subunits by expression of the atp operon from Caldalkalibacillus thermarum strain TA2.A1 using Escherichia coli extracts. The operon comprises nine open reading frames, and the 542-kDa F(1)F(o)-ATP synthase complex is composed of 9 soluble and 16 membrane-embedded proteins in the stoichiometry ?(3)?(3)???ab(2)c(13). Complete assembly into the functional complex was accomplished in all three typically used CF expression modes by (i) solubilizing initial precipitates, (ii) cotranslational insertion into detergent micelles or (iii) cotranslational insertion into preformed liposomes. The presence of all eight subunits, as well as specific enzyme activity and inhibition of the complex, was confirmed by biochemical analyses, freeze-fracture electron microscopy, and immunogold labeling. Further, single-particle analysis demonstrates that the structure and subunit organization of the CF and the reference in vivo expressed ATP synthase complexes are identical. This work establishes the production of highly complex molecular machines in defined environments either as proteomicelles or as proteoliposomes as a new application of CF expression systems. PMID:21925509

Matthies, Doreen; Haberstock, Stefan; Joos, Friederike; Dötsch, Volker; Vonck, Janet; Bernhard, Frank; Meier, Thomas

2011-10-28

49

Allosteric Effects of RuvA Protein, ATP, and DNA on RuvB Protein-Mediated ATP Hydrolysis  

E-print Network

Allosteric Effects of RuvA Protein, ATP, and DNA on RuvB Protein-Mediated ATP Hydrolysis Paul E ABSTRACT: A detailed characterization of RuvB protein-mediated ATP hydrolysis in the presence of RuvA protein has provided (a) the steady-state kinetic parameters of ATP hydrolysis within a RuvAB complex

Cox, Michael M.

50

Acknowledgments.............................................................................................................................. iii  

E-print Network

Guide includes updates from the Military Services, Defense Agencies, and other organizations, as well as changes in the May 2003 DoD 5000 series. Inputs were collated and finalized by DAU Program Director for Test and Evaluation Dr. John Claxton. Freelance writer Christina Cavoli and Defense AT&L editor-in-chief Collie Johnson edited the final document. Freelance designer and cartoonist Jim Elmore designed the cover, and final desktop publishing and layout was accomplished by Bartlett Communications and DAU Visual Information Specialist Deborah Gonzalez. iii iv FOREWORD This book is one of many technical management educational guides written from a Department of Defense perspective; i.e., non-Service specific. They are intended primarily for use in courses at Defense Acquisition University and secondarily as a desk reference for program and project management personnel. These guidebooks are written for current and potential acquisition management personnel who are familiar with basic terms and definitions employed in program offices. The guidebooks are designed to assist government and industry personnel in executing their management responsibilities

unknown authors

2005-01-01

51

ATP as effector of inorganic pyrophosphatase of Escherichia coli. Identification of the binding site for ATP.  

PubMed

The interaction of Escherichia coli inorganic pyrophosphatase (E-PPase) with effector ATP has been studied. The E-PPase has been chemically modified with the dialdehyde derivative of ATP. It has been established that in the experiment only one molecule of effector ATP is bound to each subunit of the hexameric enzyme. Tryptic digestion of the adenylated protein followed by isolation of a modified peptide by HPLC and its mass-spectrometric identification has showed that it is an amino group of Lys146 that undergoes modification. Molecular docking of ATP to E-PPase indicates that the binding site for effector ATP is located in a cluster of positively charged amino acid residues proposed earlier on the basis of site-directed mutagenesis to participate in binding of effector pyrophosphate. Molecular docking also reveals several other amino acid residues probably involved in the interaction with effectors. PMID:17309442

Rodina, E V; Vorobyeva, N N; Kurilova, S A; Belenikin, M S; Fedorova, N V; Nazarova, T I

2007-01-01

52

Inhibition of ATP Synthase by Chlorinated Adenosine Analogue  

PubMed Central

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

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

2009-01-01

53

ATP-driven stepwise rotation of FoF1-ATP synthase  

PubMed Central

FoF1-ATP synthase (FoF1) is a motor enzyme that couples ATP synthesis/hydrolysis with a transmembrane proton translocation. F1, a water-soluble ATPase portion of FoF1, rotates by repeating ATP-waiting dwell, 80° substep rotation, catalytic dwell, and 40°-substep rotation. Compared with F1, rotation of FoF1 has yet been poorly understood, and, here, we analyzed ATP-driven rotations of FoF1. Rotation was probed with an 80-nm bead attached to the ring of c subunits in the immobilized FoF1 and recorded with a submillisecond fast camera. The rotation rates at various ATP concentrations obeyed the curve defined by a Km of ?30 ?M and a Vmax of ?350 revolutions per second (at 37°C). At low ATP, ATP-waiting dwell was seen and the kon-ATP was estimated to be 3.6 × 107 M-1·s-1. At high ATP, fast, poorly defined stepwise motions were observed that probably reflect the catalytic dwells. When a slowly hydrolyzable substrate, adenosine 5?-[?-thio]triphosphate, was used, the catalytic dwells consisting of two events were seen more clearly at the angular position of ?80°. The rotational behavior of FoF1 resembles that of F1. This finding indicates that “friction” in Fo motor is negligible during the ATP-driven rotation. Tributyltin chloride, a specific inhibitor of proton translocation, slowed the rotation rate by 96%. However, dwells at clearly defined angular positions were not observed under these conditions, indicating that inhibition by tributyltin chloride is complex. PMID:15668386

Ueno, Hiroshi; Suzuki, Toshiharu; Kinosita, Kazuhiko; Yoshida, Masasuke

2005-01-01

54

The atpIBEXF operon coding for the F0 sector of the ATP synthase from the purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus  

Microsoft Academic Search

The atpIBEXF operon coding for the F0 sector of the ATP synthase from Rhodobacter capsulatus was cloned and sequenced. The genes for the five subunits were present in the order: atpI (subunit I), atpB (subunit a), atpE (subunit c), atpX (subunit b?), and atpF (subunit b). The transcription initiation site was defined by primer-extension analysis. A duplicated and divergent copy

Roberto Borghese; Paola Turina; Luca Lambertini; Bruno Andrea Melandri

1998-01-01

55

The Structural Basis of ATP as an Allosteric Modulator  

PubMed Central

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

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

2014-01-01

56

Mitochondrial ATP 6 and 8 polymorphisms in irritable bowel syndrome with diarrhea  

PubMed Central

AIM: To investigate mitochondrial ATP 6 and 8 polymorphisms in the colon and ileum of patients with irritable bowel syndrome with diarrhea (IBS-D). METHODS: Twenty-eight patients fulfilling the Rome III criteria for IBS-D and 28 healthy subjects were investigated. All study participants underwent screening colonoscopy and mucosal biopsies were obtained from the colon and/or terminal ileum. Genomic DNA was extracted from specimens based on standard protocols. Mitochondrial ATP (MT-ATP) 6 and 8 genes in specimens were polymerase chain reaction amplified and sequenced. Sequencing data were analyzed via Variant Reporter™ Software and compared with the reference sequence from Genbank (accession No. NC_012920) to indicate possible polymorphisms. The protocol was registered at www.clinicaltrials.gov as NCT01028898. RESULTS: Twenty-five polymorphic sites of MT-ATP 6 and 8 genes were detected and 12 of them were missense mutations. A median of two polymorphic sites in MT-ATP genes was found in colon specimens of controls while a median of three polymorphic sites was noted in patients with IBS-D (Mann-Whitney test, P = 0.012). The variants of the colon and ileum specimens from the same subjects were identical in all but one case. Symptom duration in IBS was not found to be a significant factor associated with the mtDNA polymorphism (Spearman correlation, P = 0.592). The mitochondrial DNA change at 8860 was present in all cases of both groups. The frequency of the 8701 polymorphism was found to be the second most frequent; however, no statistical difference was noted between the groups (?2 test, P = 0.584). CONCLUSION: Patients with IBS-D have a higher incidence of MT-ATP 6 and 8 polymorphisms than healthy subjects, implying that the mtDNA polymorphism may play a role in IBS-D. PMID:23840124

Wang, Wei-Feng; Li, Xin; Guo, Ming-Zhou; Chen, Jian-De; Yang, Yun-Sheng; Peng, Li-Hua; Wang, Yong-Hua; Zhang, Chun-Yan; Li, Hui-Hui

2013-01-01

57

Torque Generation and Utilization in Motor Enzyme F0F1-ATP Synthase  

PubMed Central

ATP synthase (F0F1) is made of two motors, a proton-driven motor (F0) and an ATP-driven motor (F1), connected by a common rotary shaft, and catalyzes proton flow-driven ATP synthesis and ATP-driven proton pumping. In F1, the central ? subunit rotates inside the ?3?3 ring. Here we report structural features of F1 responsible for torque generation and the catalytic ability of the low-torque F0F1. (i) Deletion of one or two turns in the ?-helix in the C-terminal domain of catalytic ? subunit at the rotor/stator contact region generates mutant F1s, termed F1(1/2)s, that rotate with about half of the normal torque. This helix would support the helix-loop-helix structure acting as a solid “pushrod” to push the rotor ? subunit, but the short helix in F1(1/2)s would fail to accomplish this task. (ii) Three different half-torque F0F1(1/2)s were purified and reconstituted into proteoliposomes. They carry out ATP-driven proton pumping and build up the same small transmembrane ?pH, indicating that the final ?pH is directly related to the amount of torque. (iii) The half-torque F0F1(1/2)s can catalyze ATP synthesis, although slowly. The rate of synthesis varies widely among the three F0F1(1/2)s, which suggests that the rate reflects subtle conformational variations of individual mutants. PMID:22128167

Usukura, Eiji; Suzuki, Toshiharu; Furuike, Shou; Soga, Naoki; Saita, Ei-ichiro; Hisabori, Toru; Kinosita, Kazuhiko; Yoshida, Masasuke

2012-01-01

58

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

PubMed

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

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

2013-04-01

59

Glucose is required to maintain high ATP-levels for the energy-utilizing steps during PDT-induced apoptosis.  

PubMed

Photodynamic therapy (PDT) may trigger apoptosis or necrosis in cancer cells. Several steps in the induction and execution of apoptosis require high amounts of adenosine-5'-triphosphate (ATP). Because the mitochondrial membrane potential (delta psi) decreases early in apoptosis, we raised the question about the mechanisms of maintaining a sufficiently high ATP level. We therefore monitored delta psi and the intracellular ATP level of apoptotic human epidermoid carcinoma cells (A431) after photodynamic treatment with aluminum (III) phthalocyanine tetrasulfonate. A maximum of caspase-3-like activity and nuclear fragmentation was found at fluences of about 4 J cm(-2). Under these conditions apoptotic cells reduced delta psi rapidly, while the ATP level remained high for 4-6 h after treatment for cells supplied with glucose. To analyze the contribution of glycolysis to the energy supply during apoptosis, experiments were carried out with cells deprived of glucose. These cells showed a rapid drop of ATP content and neither caspase activation nor nuclear fragmentation could be detected. We conclude that the use of glucose as a source of ATP is obligatory for the execution of PDT-induced apoptosis. PMID:12511053

Oberdanner, Christian Benno; Kiesslich, Tobias; Krammer, Barbara; Plaetzer, Kristjan

2002-12-01

60

77 FR 39626 - Further Definition of “Swap Dealer,” “Security-Based Swap Dealer,” “Major Swap Participant...  

Federal Register 2010, 2011, 2012, 2013

...in the third column, correct paragraph (hhh)(6)(iii)(B)(2) to read as follows: Sec. 1.3 Definitions. * * * * * (hhh) * * * (6) * * * (iii) * * * (B...the amount calculated under paragraph (hhh)(6)(iii)(B)(1) of this...

2012-07-05

61

Electric Field Driven Torque in ATP Synthase  

PubMed Central

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

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

2013-01-01

62

ATP-independent contractile proteins from plants  

Microsoft Academic Search

Emerging technologies are creating increasing interest in smart materials that may serve as actuators in micro- and nanodevices. Mechanically active polymers currently studied include a variety of materials. ATP-driven motor proteins, the actuators of living cells, possess promising characteristics, but their dependence on strictly defined chemical environments can be disadvantagous. Natural proteins that deform reversibly by entropic mechanisms might serve

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

2003-01-01

63

A reusable prepositioned ATP reaction chamber  

NASA Technical Reports Server (NTRS)

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

Hoffman, D. G.

1972-01-01

64

Historical review: ATP as a neurotransmitter  

E-print Network

- cellular actions of ATP and adenosine on the heart and coronary blood vessels. The published follow-up signalling is now recognized to be involved in a wide range of activities of the nervous system, including neuroprotection, central control of autonomic functions, neural­glial interactions, control of vessel tone

Burnstock, Geoffrey

65

Mechanically driven ATP synthesis by F1ATPase  

Microsoft Academic Search

ATP, the main biological energy currency, is synthesized from ADP and inorganic phosphate by ATP synthase in an energy-requiring reaction. The F1 portion of ATP synthase, also known as F1-ATPase, functions as a rotary molecular motor: in vitro its gamma-subunit rotates against the surrounding alpha3beta3 subunits, hydrolysing ATP in three separate catalytic sites on the beta-subunits. It is widely believed

Hiroyasu Itoh; Akira Takahashi; Kengo Adachi; Hiroyuki Noji; Ryohei Yasuda; Masasuke Yoshida; Kazuhiko Kinosita

2004-01-01

66

Translocation of Platinum Anticancer Drugs by Human Copper ATPases ATP7A and ATP7B**  

PubMed Central

Cisplatin, carboplatin, and oxaliplatin are widely used anticancer drugs. Their efficacy is strongly reduced by development of cell resistance, a phenomenon not entirely understood, with contribution of drug detoxification, defective accumulation, and efflux from the cell. Down-regulation of CTR1, responsible for Cu uptake by the cell, and up-regulation of the Cu-ATPases, ATP7A and ATP7B, which accept Cu from the cytosolic chaperone Atox1 and transfer the metal ion into the secretory pathway where it is incorporated into cuproenzymes, have been associated to augmented drug resistance. To gain information on translocation of Pt drugs by human Cu-ATPases, we performed electrical measurements on COS-1 cell microsomal fraction, enriched with recombinant ATP7A, ATP7B, and selected mutants, adsorbed on a solid supported membrane (SSM). The experimental results demonstrate that Pt drugs activate Cu-ATPases and undergo ATP-dependent translocation with a mechanism identical to that of Cu. We then used NMR spectroscopy and ESI-MS to determine the binding mode of these drugs to the first N-terminal metal binding domain of ATP7A (Mnk1). PMID:24375922

Tadini-Buoninsegni, Francesco; Bartolommei, Gianluca; Moncelli, Maria Rosa; Inesi, Giuseppe; Galliani, Angela; Sinisi, Marilù; Losacco, Maurizio; Natile, Giovanni; Arnesano, Fabio

2014-01-01

67

Renal Cell-to-Cell Communication via Extracellular ATP  

NSDL National Science Digital Library

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.

MD Peter Komlosi (University of Alabama at Birmingham Departments of Medicine and Physiology, Division of Nephrology); Attila Fintha (University of Alabama Departments of Medicine and Physiology, Division of Nephrology); PhD P. Darwin Bell (University of Alabama Departments of Medicine and Physiology, Division of Nephrology)

2005-04-01

68

ATP Hydrolysis Stimulates Large Length Fluctuations in Single Actin Filaments  

E-print Network

ATP Hydrolysis Stimulates Large Length Fluctuations in Single Actin Filaments Evgeny B. Stukalin is investigated theoretically using a stochastic model that takes into account the hydrolysis of ATP filaments. It is found that the ATP hydrolysis has a strong effect on dynamic properties of single actin

69

Physiological levels of ATP Negatively Regulate Proteasome Function  

PubMed Central

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

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

70

43 CFR 10000.3 - Definitions.  

...Interior Regulations Relating to Public Lands (Continued) UTAH RECLAMATION MITIGATION AND CONSERVATION COMMISSION ORGANIZATION...FUNCTIONS § 10000.3 Definitions. Act refers to the Central Utah Project Completion Act, Titles II, III, IV, V, and VI of...

2014-10-01

71

43 CFR 10000.3 - Definitions.  

Code of Federal Regulations, 2013 CFR

...Interior Regulations Relating to Public Lands (Continued) UTAH RECLAMATION MITIGATION AND CONSERVATION COMMISSION ORGANIZATION...FUNCTIONS § 10000.3 Definitions. Act refers to the Central Utah Project Completion Act, Titles II, III, IV, V, and VI of...

2013-10-01

72

43 CFR 10000.3 - Definitions.  

Code of Federal Regulations, 2011 CFR

...Interior Regulations Relating to Public Lands (Continued) UTAH RECLAMATION MITIGATION AND CONSERVATION COMMISSION ORGANIZATION...FUNCTIONS § 10000.3 Definitions. Act refers to the Central Utah Project Completion Act, Titles II, III, IV, V, and VI of...

2011-10-01

73

43 CFR 10000.3 - Definitions.  

Code of Federal Regulations, 2012 CFR

...Interior Regulations Relating to Public Lands (Continued) UTAH RECLAMATION MITIGATION AND CONSERVATION COMMISSION ORGANIZATION...FUNCTIONS § 10000.3 Definitions. Act refers to the Central Utah Project Completion Act, Titles II, III, IV, V, and VI of...

2012-10-01

74

Molecular analysis of Wilson patients: direct sequencing and MLPA analysis in the ATP7B gene and Atox1 and COMMD1 gene analysis.  

PubMed

ATP7B mutations result in Cu storage in the liver and brain in Wilson disease (WD). Atox1 and COMMD1 were found to interact with ATP7B and involved in copper transport in the hepatocyte. To understand the molecular etiology of WD, we analyzed ATP7B, Atox1 and COMMD1 genes. Direct sequencing of (i) ATP7B gene was performed in 112 WD patients to identify the spectrum of disease-causing mutations in the French population, (ii) Atox1 gene was performed to study the known polymorphism 5'UTR-99T>C in 78 WD patients with two ATP7B mutations and (iii) COMMD1 gene was performed to detect the nucleotide change c.492GAT>GAC. MLPA (Multiplex Ligation-dependent Probe Amplification) analysis was performed in WD patients presenting only one ATP7B mutation. Among our 112 WD unrelated patients, 83 different ATP7B gene mutations were identified, 27 of which were novel. Two ATP7B mutations were identified in 98 WD cases, and one mutation was identified in 14 cases. In two of these 14 WD patients, we identified the deletion of exon 4 of the ATP7B gene by MLPA technique. In 78 selected patients of the cohort with two mutations in ATP7B, we have examined genotype-phenotype correlation between the detected changes in Atox1 and COMMD1 genes, and the presentation of the WD patients. Based on the data of this study, no major role can be attributed to Atox1 and COMMD in the pathophysiology or clinical variation of WD. PMID:22677543

Bost, Muriel; Piguet-Lacroix, Guénaelle; Parant, François; Wilson, C M R

2012-06-01

75

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

NASA Technical Reports Server (NTRS)

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

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

1974-01-01

76

ATP-binding cassette transporters in liver.  

PubMed

The human ATP-binding cassette (ABC) superfamily consists of 48 members with 14 of them identified in normal human liver at the protein level. Most of the ABC members act as ATP dependent efflux transport systems. In the liver, ABC transporters are involved in diverse physiological processes including export of cholesterol, bile salts, and metabolic endproducts. Consequently, impaired ABC transporter function is involved in inherited diseases like sitosterolemia, hyperbilirubinemia, or cholestasis. Furthermore, altered expression of some of the hepatic ABCs have been associated with primary liver tumors. This review gives a short overview about the function of hepatic ABCs. Special focus is addressed on the localization and ontogenesis of ABC transporters in the human liver. In addition, their expression pattern in primary liver tumors is discussed. PMID:24105869

Wlcek, Katrin; Stieger, Bruno

2014-01-01

77

Space shuttle (ATP configuration) abort staging investigation  

NASA Technical Reports Server (NTRS)

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.

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

1973-01-01

78

IMPLICATION OF ATP RECEPTORS IN BRAIN FUNCTIONS  

Microsoft Academic Search

The possible implication of P2-purinoceptors in brain functions is reviewed. Involvement of P2-purinoceptors in memory and learning (Section 2) is suggested by ATP release from hippocampal slices [Wieraszko et al. (1989)Brain Res. 485, 244–250], induction of fast synaptic currents in cultured hippocampal neurons [Inoue et al. (1992a)Neurosci. Lett. 134, 294–299] and long-lasting enhancement of the population spikes [Wieraszko and Seyfried

KAZUHIDE INOUE; SCHUICHI KOIZUMI; SHINYA UENO

1996-01-01

79

Yeast ADP/ATP Carrier Isoform 2  

PubMed Central

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

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

2011-01-01

80

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

PubMed

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

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

2014-06-01

81

ATP promotes extracellular matrix biosynthesis of intervertebral disc cells.  

PubMed

We have recently found a high accumulation of extracellular adenosine triphosphate (ATP) in the center of healthy porcine intervertebral discs (IVD). Since ATP is a powerful extracellular signaling molecule, extracellular ATP accumulation might regulate biological activities in the IVD. The objective of this study was therefore to investigate the effects of extracellular ATP on the extracellular matrix (ECM) biosynthesis of porcine IVD cells isolated from two distinct anatomical regions: the annulus fibrosus (AF) and nucleus pulposus (NP). ATP treatment significantly promotes ECM deposition and corresponding gene expression (aggrecan and type II collagen) by both cell types in three-dimensional agarose culture. A significant increase in ECM accumulation has been found in AF cells at a lower ATP treatment level (20 ?M) compared with NP cells (100 ?M), indicating that AF cells are more sensitive to extracellular ATP than NP cells. NP cells also exhibit higher ECM accumulation and intracellular ATP than AF cells under control and treatment conditions, suggesting that NP cells are intrinsically more metabolically active. Moreover, ATP treatment also augments the intracellular ATP level in NP and AF cells. Our findings suggest that extracellular ATP not only promotes ECM biosynthesis via a molecular pathway, but also increases energy supply to fuel that process. PMID:25407524

Gonzales, Silvia; Wang, Chong; Levene, Howard; Cheung, Herman S; Huang, Chun-Yuh Charles

2015-02-01

82

Identification of a plant receptor for extracellular ATP.  

PubMed

Extracellular adenosine 5'-triphosphate (ATP) is an essential signaling molecule that is perceived in mammals by plasma membrane P2-type purinoceptors. Similar ATP receptors do not exist in plants, although extracellular ATP has been shown to play critical roles in plant growth, development, and stress responses. Here, we identify an ATP-insensitive Arabidopsis mutant, dorn1 (Does not Respond to Nucleotides 1), defective in lectin receptor kinase I.9 (Arabidopsis Information Resource accession code At5g60300). DORN1 binds ATP with high affinity (dissociation constant of 45.7 ± 3.1 nanomolar) and is required for ATP-induced calcium response, mitogen-activated protein kinase activation, and gene expression. Ectopic expression of DORN1 increased the plant response to physical wounding. We propose that DORN1 is essential for perception of extracellular ATP and likely plays a variety of roles in plant stress resistance. PMID:24436418

Choi, Jeongmin; Tanaka, Kiwamu; Cao, Yangrong; Qi, Yue; Qiu, Jing; Liang, Yan; Lee, Sang Yeol; Stacey, Gary

2014-01-17

83

Loss of LRPPRC causes ATP synthase deficiency  

PubMed Central

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

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

2014-01-01

84

Loss of LRPPRC causes ATP synthase deficiency.  

PubMed

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

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

2014-05-15

85

77 FR 40478 - Removal of Category IIIa, IIIb, and IIIc Definitions; Confirmation of Effective Date and Response...  

Federal Register 2010, 2011, 2012, 2013

...In that document, the FAA...remove the definitions of Category...and IIIc definitions from 14 CFR...operational documents such as advisory...the sub- definitions, CAT III...Rulemaking Documents An...

2012-07-10

86

Efficient ATP synthesis by thermophilic Bacillus FoF1-ATP synthase  

PubMed Central

FoF1-ATP synthase (FoF1) synthesizes ATP in the F1 portion when protons flow through Fo to rotate the shaft common to F1 and Fo. Rotary synthesis in isolated F1 alone has been shown by applying external torque to F1 of thermophilic origin. Proton-driven ATP synthesis by thermophilic Bacillus PS3 FoF1 (TFoF1), however, has so far been poor in vitro, of the order of 1 s?1 or less, hampering reliable characterization. Here, by using a mutant TFoF1 lacking an inhibitory segment of the ?-subunit, we have developed highly reproducible, simple procedures for the preparation of active proteoliposomes and for kinetic analysis of ATP synthesis, which was driven by acid–base transition and K+-diffusion potential. The synthesis activity reached ? 16 s?1 at 30 °C with a Q10 temperature coefficient of 3–4 between 10 and 30 °C, suggesting a high level of activity at the physiological temperature of ? 60 °C. The Michaelis–Menten constants for the substrates ADP and inorganic phosphate were 13 ?m and 0.55 mm, respectively, which are an order of magnitude lower than previous estimates and are suited to efficient ATP synthesis. PMID:21605343

Soga, Naoki; Kinosita, Kazuhiko; Yoshida, Masasuke; Suzuki, Toshiharu

2011-01-01

87

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

SciTech Connect

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

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

2009-01-01

88

Mitochondrial ATP-Pi exchange complex and the site of uncoupling of oxidative phosphorylation.  

PubMed

Five enzyme complexes, which are concerned with electron transport and oxidative phosphorylation, have been isolated from beef heart mitochondria. Enzyme complexes I, II, III and IV are the electron transfer complexes discovered in 1961. Complex V is an energy-conserving complex. It catalyzes ATP-Pi exchange and ATP hydrolysis. The exchange reaction is sensitive to uncouplers, rutamycin, valinomycin plus K-+, dicyclorexylcarboditmide, arsenate, azide, and adenylyl imidodiphosphate. It is also specific for ATP; ITP, GTP and UTP are essentially ineffective. Studies with the photoaffinity labeling uncoupler, 2-azido-4-nitrophenol (NPA), have shown that the mitochondrial uncoupler-binding sites are located exclusively in complex V. Complexes I, III and IV, which carry the three coupling sites of the respiratory chain, had negligible capacity for the binding of NPA, whereas the uncoupler-binding capacity of complex V appeared to be increased two- to threefold as compared to mitochondria. Complexes I, II, III, IV and V are obtained from the same batch of mitochondria by a simple fractionation procedure, which employs cholate, deoxycholate, ammonium acetate and ammonium sulfate. Studies with NPA have shown that mitochondria contain per milligram protein about 0.6 nmole of uniformly reacting uncoupler binding site. All of the uncouplers tested appeared to interact competitively with this site. Photoaffinity labeling with tritiated NPA has shown that a major portion of NPA binds to a polypeptide of molecular weight between 26,000 and 30,000. Other studies on the mechanism of uncoupling have shown that picrate is a membrane-impermeable uncoupler. It cannot uncouple mitochondria. However, it is an effective uncoupler of ATP synthesis and ATP-induced transhydrogenation or reverse electron transfer when used in conjunction with sonicated submitochondrial particles, which have an inside-out orientation of the inner membrane with respect to the medium. In these particles, picrate binds to the same uncoupler-binding site as NPA and other uncouplers. However, unlike the membrane-permeable uncouplers, picrate is a poor protonophore. It has a very small effect on the proton permeability of phosphorylating submitochondrial vesicles, even at two to three times the concentration needed for complete uncoupling. The increase in the proton permeability of submitochondrial vesicles caused by such high concentrations of picrate (500 mum) can be achieved with approximately 5 mum 2,4-dinitrophenol. At this concentration, dinitrophenol results in only about 20% uncoupling. PMID:1093889

Hatefi, Y; Hanstein, W G; Galante, Y; Stiggall, D L

1975-07-01

89

Effect of ATP binding cassette/multidrug resistance proteins on ATP efflux of Saccharomyces cerevisiae.  

PubMed

Multidrug resistance (MDR) in mammalian tumors or tissues is often associated with the overexpression of the putative drug efflux pump P-glycoprotein (Pgp). One theory concerning the mechanism of Pgp activity is that efflux of ATP is coupled to drug efflux. Evidence in support of this theory has been observed in mammalian cells. Recently, the STS1 gene, which is a multidrug resistance gene related to the mammalian Pgp's, has been characterized in S. cerevisiae. Also, the mouse mdr3 Pgp has been functionally expressed in yeast cells. Therefore, it was of interest to determine whether the expression of these proteins affected ATP efflux from yeast. Although both genes were shown to confer MDR, thus confirming functional expression, the endogenous glucose-dependent, drug-stimulated ATP efflux activity of yeast was not affected by expression of STS1, and was decreased by the expression of mouse mdr3. PMID:9020050

Boyum, R; Guidotti, G

1997-01-01

90

Kinetic mechanism of the dimeric ATP sulfurylase from plants.  

PubMed

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

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

2013-01-01

91

ATP independent type IB topoisomerase of Leishmania donovani is stimulated by ATP: an insight into the functional mechanism.  

PubMed

Most type IB topoisomerases do not require ATP and Mg(2+) for activity. However, as shown previously for vaccinia topoisomerase I, we demonstrate that ATP stimulates the relaxation activity of the unusual heterodimeric type IB topoisomerase from Leishmania donovani (LdTOP1L/S) in the absence of Mg(2+). The stimulation is independent of ATP hydrolysis but requires salt as a co-activator. ATP binds to LdTOP1L/S and increases its rate of strand rotation. Docking studies indicate that the amino acid residues His93, Tyr95, Arg188 and Arg190 of the large subunit may be involved in ATP binding. Site directed mutagenesis of these four residues individually to alanine and subsequent relaxation assays reveal that the R190A mutant topoisomerase is unable to exhibit ATP-mediated stimulation in the absence of Mg(2+). However, the ATP-independent relaxation activities of all the four mutant enzymes remain unaffected. Additionally, we provide evidence that ATP binds LdTOP1L/S and modulates the activity of the otherwise ATP-independent enzyme. This study establishes ATP as an activator of LdTOP1L/S in the absence of Mg(2+). PMID:21186185

Sengupta, Souvik; Ganguly, Agneyo; Roy, Amit; Bosedasgupta, Somdeb; D'Annessa, Ilda; Desideri, Alessandro; Majumder, Hemanta K

2011-04-01

92

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

PubMed Central

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

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

2012-01-01

93

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

PubMed Central

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

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

2013-01-01

94

Single molecule thermodynamics of ATP synthesis by F$_1$-ATPase  

E-print Network

F$_\\mathrm{o}$F$_1$-ATP synthase is a factory for synthesizing ATP in virtually all cells. Its core machinery is the subcomplex F$_1$-motor (F$_1$-ATPase) and performs the reversible mechanochemical coupling. Isolated F$_1$-motor hydrolyzes ATP, which is accompanied by unidirectional rotation of its central $\\gamma$-shaft. When a strong opposing torque is imposed, the $\\gamma$-shaft rotates in the opposite direction and drives the F$_1$-motor to synthesize ATP. This mechanical-to-chemical free-energy transduction is the final and central step of the multistep cellular ATP-synthetic pathway. Here, we determined the amount of mechanical work exploited by the F$_1$-motor to synthesize an ATP molecule during forced rotations using methodology combining a nonequilibrium theory and single molecule measurements of responses to external torque. We found that the internal dissipation of the motor is negligible even during rotations far from a quasistatic process.

Shoichi Toyabe; Eiro Muneyuki

2015-01-16

95

Single molecule thermodynamics of ATP synthesis by F1-ATPase  

NASA Astrophysics Data System (ADS)

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

Toyabe, Shoichi; Muneyuki, Eiro

2015-01-01

96

Snapshots of the maltose transporter during ATP hydrolysis  

SciTech Connect

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

Oldham, Michael L.; Chen, Jue (Purdue)

2011-12-05

97

Modification and application of a soil ATP determination method  

Microsoft Academic Search

Accurate estimation of microbial adenosine 5?-triphosphate (ATP) is a pre-requisite to quantify the impact of varying environments on microbial activity of soil. We investigated the effectiveness of a high efficiency soil ATP determination method (PA) [Webster, J.J., Hampton, G.J., Leach, F.R., 1984. ATP in soil: a new extractant and extraction procedure. Soil Biology & Biochemistry 4, 335–342] in 10 Ontario

Guang Wen; R. P. Voroney; D. Curtin; J. J. Schoenau; P. Y. Qian; S. Inanaga

2005-01-01

98

Therapeutic Targeting of ATP7B in Ovarian Carcinoma  

PubMed Central

Purpose Resistance to platinum chemotherapy remains a significant problem in ovarian carcinoma. Here, we examined the biological mechanisms and therapeutic potential of targeting a critical platinum resistance gene, ATP7B, using both in vitro and in vivo models. Experimental Design Expression of ATP7A and ATP7B was examined in ovarian cancer cell lines by real-time reverse transcription-PCR and Western blot analysis. ATP7A and ATP7B gene silencing was achieved with targeted small interfering RNA (siRNA) and its effects on cell viability and DNA adduct formation were examined. For in vivo therapy experiments, siRNA was incorporated into the neutral nanoliposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). Results ATP7A and ATP7B genes were expressed at higher levels in platinum-resistant cells compared with sensitive cells; however, only differences in ATP7B reached statistical significance. ATP7A gene silencing had no significant effect on the sensitivity of resistant cells to cisplatin, but ATP7B silencing resulted in 2.5-fold reduction of cisplatin IC50 levels and increased DNA adduct formation in cisplatin-resistant cells (A2780-CP20 and RMG2). Cisplatin was found to bind to the NH2-terminal copper-binding domain of ATP7B, which might be a contributing factor to cisplatin resistance. For in vivo therapy experiments, ATP7B siRNA was incorporated into DOPC and was highly effective in reducing tumor growth in combination with cisplatin (70-88% reduction in both models compared with controls). This reduction in tumor growth was accompanied by reduced proliferation, increased tumor cell apoptosis, and reduced angiogenesis. Conclusion These data provide a new understanding of cisplatin resistance in cancer cells and may have implications for therapeutic reversal of drug resistance. PMID:19470734

Mangala, Lingegowda S.; Zuzel, Vesna; Schmandt, Rosemarie; Leshane, Erik S.; Halder, Jyotsna B.; Armaiz-Pena, Guillermo N.; Spannuth, Whitney A.; Tanaka, Takemi; Shahzad, Mian M.K.; Lin, Yvonne G.; Nick, Alpa M.; Danes, Christopher G.; Lee, Jeong-Won; Jennings, Nicholas B.; Vivas-Mejia, Pablo E.; Wolf, Judith K.; Coleman, Robert L.; Siddik, Zahid H.; Lopez-Berestein, Gabriel; Lutsenko, Svetlana; Sood, Anil K.

2009-01-01

99

ATP7B detoxifies silver in ciliated airway epithelial cells  

SciTech Connect

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.

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

100

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

PubMed

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

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

101

Application of luciferase assay for ATP to antimicrobial drug susceptibility  

NASA Technical Reports Server (NTRS)

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.

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

1977-01-01

102

Imaging changes in the cytosolic ATP-to-ADP ratio  

PubMed Central

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

Tantama, Mathew; Yellen, Gary

2015-01-01

103

Diversity and regulation of ATP sulfurylase in photosynthetic organisms.  

PubMed

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

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

2014-01-01

104

Rows of ATP Synthase Dimers in Native Mitochondrial Inner Membranes  

PubMed Central

The ATP synthase is a nanometric rotary machine that uses a transmembrane electrochemical gradient to form ATP. The structures of most components of the ATP synthase are known, and their organization has been elucidated. However, the supramolecular assembly of ATP synthases in biological membranes remains unknown. Here we show with submolecular resolution the organization of ATP synthases in the yeast mitochondrial inner membranes. The atomic force microscopy images we have obtained show how these molecules form dimers with characteristic 15 nm distance between the axes of their rotors through stereospecific interactions of the membrane embedded portions of their stators. A different interaction surface is responsible for the formation of rows of dimers. Such an organization elucidates the role of the ATP synthase in mitochondrial morphology. Some dimers have a different morphology with 10 nm stalk-to-stalk distance, in line with ATP synthases that are accessible to IF1 inhibition. Rotation torque compensation within ATP synthase dimers stabilizes the ATP synthase structure, in particular the stator-rotor interaction. PMID:17557793

Buzhynskyy, Nikolay; Sens, Pierre; Prima, Valerie; Sturgis, James N.; Scheuring, Simon

2007-01-01

105

Diversity and regulation of ATP sulfurylase in photosynthetic organisms  

PubMed Central

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

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

2014-01-01

106

SAGE III  

SAGE III Data and Information The Stratospheric Aerosol and Gas ... Guide Documents:  Project Guide Data Products User's Guide  (PDF) Relevant Documents:  ... Additional Info:  Data Format: HDF-EOS or Big Endian/IEEE Binary SCAR-B Block:  ...

2014-12-04

107

ATP Synthase with Its ? Subunit Reduced to the N-terminal Helix Can Still Catalyze ATP Synthesis*  

PubMed Central

ATP synthase uses a unique rotary mechanism to couple ATP synthesis and hydrolysis to transmembrane proton translocation. As part of the synthesis mechanism, the torque of the rotor has to be converted into conformational rearrangements of the catalytic binding sites on the stator to allow synthesis and release of ATP. The ? subunit of the rotor, which plays a central role in the energy conversion, consists of two long helices inside the central cavity of the stator cylinder plus a globular portion outside the cylinder. Here, we show that the N-terminal helix alone is able to fulfill the function of full-length ? in ATP synthesis as long as it connects to the rest of the rotor. This connection can occur via the ? subunit. No direct contact between ? and the c ring seems to be required. In addition, the results indicate that the ? subunit of the rotor exists in two different conformations during ATP synthesis and ATP hydrolysis. PMID:19636076

Mnatsakanyan, Nelli; Hook, Jonathon A.; Quisenberry, Leah; Weber, Joachim

2009-01-01

108

Molecular switch of F 0 F 1 ATP synthase, G-protein, and other ATP-driven enzymes  

Microsoft Academic Search

Exchange-out of amide tritium from labeled ?-subunit of ?3?3? complex of F0F1-ATP synthase was not accelerated by ATP, suggesting that hemagglutinin-type transition of coiled-coil structure did not occur in ?-subunit. Local topology of nucleotide binding site and “switch II” region of G-protein ? resemble those of F1-? subunit and other proteins which catalyze ATP-triggered reactions. Probably, binding of nucleotide to

Hiroyuki Noji; Toyoki Amano; Masasuke Yoshida I

1996-01-01

109

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

NASA Technical Reports Server (NTRS)

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

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

1996-01-01

110

Mitochondrial ATP synthasome: three-dimensional structure by electron microscopy of the ATP synthase in complex formation with carriers for Pi and ADP/ATP.  

PubMed

The terminal steps involved in making ATP in mitochondria require an ATP synthase (F(0)F(1)) comprised of two motors, a phosphate carrier (PIC), and an adenine nucleotide carrier (ANC). Under mild conditions, these entities sub-fractionate as an ATP synthase/PIC/ANC complex or "ATP synthasome" (Ko, Y.H., Delannoy, M, Hullihen, J., Chiu, W., and Pedersen, P.L. (2003) J. Biol. Chem. 278, 12305-12309). As a first step toward obtaining three-dimensional information about this large complex or "metabolon" and the locations of PIC and ANC therein, we dispersed ATP synthasomes into single complexes and visualized negatively stained images by electron microscopy (EM) that showed clearly the classical headpiece, central stalk, and basepiece. Parallel immuno-EM studies revealed the presence of PIC and ANC located non-centrally in the basepiece, and other studies implicated an ATP synthase/PIC/ANC stoichiometry near 1:1:1. Single ATP synthasome images (7506) were boxed, and, using EMAN software, a three-dimensional model was obtained at a resolution of 23 A. Significantly, the basepiece is oblong and contains two domains, the larger of which connects to the central stalk, whereas the smaller appears as an extension. Docking studies with known structures together with the immuno-EM studies suggest that PIC or ANC may be located in the smaller domain, whereas the other transporter resides nearby in the larger domain. Collectively, these finding support a mechanism in which the entry of the substrates ADP and P(i) into mitochondria, the synthesis of ATP on F(1), and the release and exit of ATP are very localized and highly coordinated events. PMID:15166242

Chen, Chen; Ko, Young; Delannoy, Michael; Ludtke, Steven J; Chiu, Wah; Pedersen, Peter L

2004-07-23

111

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

PubMed

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

Yi, Ling; Kaler, Stephen

2014-05-01

112

ATP is released from guinea pig ureter epithelium on distension  

E-print Network

School, London NW3 2PF, United Kingdom; and 2 Department of Pharmacology, School of Medicine, University such as the rat pulmonary vascular bed (25) and rabbit and guinea pig coronary beds (49, 51). ATP is also released from both human and rabbit red blood cells (RBCs) in response to mechanical deforma- tion (48). The ATP

Burnstock, Geoffrey

113

Extracellular ATP drives systemic inflammation, tissue damage and mortality  

PubMed Central

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

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

2014-01-01

114

Synphilin-1 Binds ATP and Regulates Intracellular Energy Status  

PubMed Central

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

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

2014-01-01

115

ATP synthase — a marvellous rotary engine of the cell  

Microsoft Academic Search

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.

Masasuke Yoshida; Eiro Muneyuki; Toru Hisabori

2001-01-01

116

A common topology of proteins catalyzing ATP-triggered reactions  

Microsoft Academic Search

A protein fold, six parallel ? strands surrounding the central ? helix, is likely to be a common structure in protein families known to have a typical set of nucleotide binding consensus sequenced motifs A and B and to catalyze ATP-triggered reactions. According to this ATP-triggered protein fold, the conserved Glu (or Asp), which acts as a general base to

Masasuke Yoshida; Toyoki Amano

1995-01-01

117

[Treatment of supraventricular paroxysmal tachycardia by intravenous administration of ATP].  

PubMed

The results of treatment of supraventricular paroxysmal tachycardia by the intravenous administration of ATP in a dose of 10-30 mg make it possible to consider ATP as an agent of choice for relieving supraventricular paroxysmal tachycardia under conditions of emergency medical aid. PMID:2664342

Nesterov, Iu I; Kiselev, A G; Suvorova, L I; Trushina, T S; Bo?ko, V I

1989-03-01

118

Rapid antibiotic susceptibility tests on Enterobacteriaceae by ATP bioluminescence  

Microsoft Academic Search

Summary. The susceptibility of 76 clinical isolates of Enterobacteriaceae to ampicillin, piperacillin and gentamicin was assessed by ATP bioluminescence in a 4-h test. For most organisms tested (Escherichia, Klebsiella, Enterobacter and Serratia), there was good correlation with traditional MIC values estimated on 18-h cultures. However strains of Proteus mirabilis showed false resistance to the p-lactam agents with the ATP method;

P. F. Wheat; J. G. M. Hastings; R. C. Spencer

1988-01-01

119

The Dark Side of Extracellular ATP in Kidney Diseases.  

PubMed

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

Solini, Anna; Usuelli, Vera; Fiorina, Paolo

2014-12-01

120

Modelling the ATP production in mitochondria  

E-print Network

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 ...

Saa, Alberto

2012-01-01

121

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

PubMed Central

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

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

2004-01-01

122

Extracellular ATP stimulates volume decrease in Necturus red blood cells.  

PubMed

This study examined whether extracellular ATP stimulates regulatory volume decrease (RVD) in Necturus maculosus (mudpuppy) red blood cells (RBCs). The hemolytic index (a measure of osmotic fragility) decreased with extracellular ATP (50 microM). In contrast, the ATP scavenger hexokinase (2.5 U/ml, 1 mM glucose) increased osmotic fragility. In addition, the ATP-dependent K+ channel antagonist glibenclamide (100 microM) increased the hemolytic index, and this inhibition was reversed with ATP (50 microM). We also measured cell volume recovery in response to hypotonic shock electronically with a Coulter counter. Extracellular ATP (50 microM) enhanced cell volume decrease in a hypotonic (0.5x) Ringer solution. In contrast, hexokinase (2.5 U/ml) and apyrase (an ATP diphosphohydrolase, 2.5 U/ml) inhibited cell volume recovery. The inhibitory effect of hexokinase was reversed with the Ca2+ ionophore A-23187 (1 microM); it also was reversed with the cationophore gramicidin (5 microM in a choline-Ringer solution), indicating that ATP was linked to K+ efflux. In addition, glibenclamide (100 microM) and gadolinium (10 microM) inhibited cell volume decrease, and the effect of these agents was reversed with ATP (50 microM) and A-23187 (1 microM). Using the whole cell patch-clamp technique, we found that ATP (50 microM) stimulated a whole cell current under isosmotic conditions. In addition, apyrase (2.5 U/ml), glibenclamide (100 microM), and gadolinium (10 microM) inhibited whole cell currents that were activated during hypotonic swelling. The inhibitory effect of apyrase was reversed with the nonhydrolyzable analog adenosine 5'-O-(3-thiotriphosphate) (50 microM), and the effect of glibenclamide or gadolinium was reversed with ATP (50 microM). Finally, anionic whole cell currents were activated with hypotonic swelling when ATP was the only significant charge carrier, suggesting that increases in cell volume led to ATP efflux through a conductive pathway. Taken together, these results indicate that extracellular ATP stimulated cell volume decrease via a Ca2+-dependent step that led to K+ efflux. PMID:10484335

Light, D B; Capes, T L; Gronau, R T; Adler, M R

1999-09-01

123

Performance and Specificity of the Covalently Linked Immunomagnetic Separation-ATP Method for Rapid Detection and Enumeration of Enterococci in Coastal Environments  

PubMed Central

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

Zimmer-Faust, Amity G.; Thulsiraj, Vanessa; Ferguson, Donna

2014-01-01

124

Mechanism of ATP loss in nonoxidative contracting muscle  

NSDL National Science Digital Library

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 3ÃÂ4 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.

2011-03-01

125

Emerging enzymes for ATP regeneration in biocatalytic processes.  

PubMed

Adenosine-5'-triphosphate-dependent enzyme catalysed reactions are widespread in nature. Consequently, the enzymes involved have an intrinsic potential for use in syntheses of high value products. Although regeneration systems for ATP starting from adenosine-5'-diphosphate are available, certain limitations exist for both in vitro and in vivo applications requiring ATP regeneration from adenosine-5'-monophosphate, or adenosine. Following a short overview of the chemical and thermodynamic background, this Minireview focuses on emerging enzymes and methodologies for ATP regeneration. A large range of as yet unexploited reactions will be accessible with new, powerful, multistep ATP regeneration systems that use cheap phosphate donors and provide high longevity, compatibility, and robustness under process conditions. Their potential might go far beyond the direct use of ATP in enzymatic reactions; enzyme discovery, and engineering, as well as immobilisation strategies, will help to realise such systems. PMID:25619338

Andexer, Jennifer N; Richter, Michael

2015-02-01

126

Radioprotective effects of ATP in human blood ex vivo  

SciTech Connect

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

Swennen, Els L.R. [Department of Epidemiology, NUTRIM Maastricht University, P.O. Box 616, 6200 MD Maastricht (Netherlands); Department of Pharmacology and Toxicology, NUTRIM Maastricht University, P.O. Box 616, 6200 MD Maastricht (Netherlands)], E-mail: e.swennen@farmaco.unimaas.nl; Dagnelie, Pieter C. [Department of Epidemiology, NUTRIM Maastricht University, P.O. Box 616, 6200 MD Maastricht (Netherlands); Van den Beucken, Twan [Department of Radiation Oncology, Maastricht Radiation oncology (Maastro) Lab, GROW, Maastricht University, P.O. Box 616, 6200 MD Maastricht (Netherlands); Bast, Aalt [Department of Pharmacology and Toxicology, NUTRIM Maastricht University, P.O. Box 616, 6200 MD Maastricht (Netherlands)

2008-03-07

127

Cooperative, ATP-dependent association of the nucleotide binding cassettes during the catalytic cycle of ATP-binding cassette transporters.  

PubMed

ATP-binding cassette (ABC) transporters harvest the energy present in cellular ATP to drive the translocation of a structurally diverse set of solutes across the membrane barriers of eubacteria, archaebacteria, and eukaryotes. The positively cooperative ATPase activity (Hill coefficient, 1.7) of a model soluble cassette of known structure, MJ0796, from Methanococcus jannaschii indicates that at least two binding sites participate in the catalytic reaction. Mutation of the catalytic base in MJ0796, E171Q, produced a cassette that can bind but not efficiently hydrolyze ATP. The equivalent mutation (E179Q) in a homologous cassette, MJ1267, had an identical effect. Both mutant cassettes formed dimers in the presence of ATP but not ADP, indicating that the energy of ATP binding is first coupled to the transport cycle through a domain association reaction. The non-hydrolyzable nucleotides adenosine 5'-(beta,gamma-imino)triphosphate and adenosine 5'-3-O-(thio)triphosphate were poor analogues of ATP in terms of their ability to promote dimerization. Moreover, inclusion of MgCl2, substitution of KCl for NaCl, or alterations in the polarity of the side chain at the catalytic base all weakened the ATP-dependent dimer, suggesting that electrostatic interactions are critical for the association reaction. Thus, upon hydrolysis of bound ATP and the release of product, both electrostatic and conformational changes drive the cassettes apart, providing a second opportunity to couple free energy changes to the transport reaction. PMID:11964392

Moody, Jonathan E; Millen, Linda; Binns, Derk; Hunt, John F; Thomas, Philip J

2002-06-14

128

Differential expression of ATP7A, ATP7B and CTR1 in adult rat dorsal root ganglion tissue  

PubMed Central

Background ATP7A, ATP7B and CTR1 are metal transporting proteins that control the cellular disposition of copper and platinum drugs, but their expression in dorsal root ganglion (DRG) tissue and their role in platinum-induced neurotoxicity are unknown. To investigate the DRG expression of ATP7A, ATP7B and CTR1, lumbar DRG and reference tissues were collected for real time quantitative PCR, RT-PCR, immunohistochemistry and Western blot analysis from healthy control adult rats or from animals treated with intraperitoneal oxaliplatin (1.85 mg/kg) or drug vehicle twice weekly for 8 weeks. Results In DRG tissue from healthy control animals, ATP7A mRNA was clearly detectable at levels similar to those found in the brain and spinal cord, and intense ATP7A immunoreactivity was localised to the cytoplasm of cell bodies of smaller DRG neurons without staining of satellite cells, nerve fibres or co-localisation with phosphorylated heavy neurofilament subunit (pNF-H). High levels of CTR1 mRNA were detected in all tissues from healthy control animals, and strong CTR1 immunoreactivity was associated with plasma membranes and vesicular cytoplasmic structures of the cell bodies of larger-sized DRG neurons without co-localization with ATP7A. DRG neurons with strong expression of ATP7A or CTR1 had distinct cell body size profiles with minimal overlap between them. Oxaliplatin treatment did not alter the size profile of strongly ATP7A-immunoreactive neurons but significantly reduced the size profile of strongly CTR1-immunoreactive neurons. ATP7B mRNA was barely detectable, and no specific immunoreactivity for ATP7B was found, in DRG tissue from healthy control animals. Conclusions In conclusion, adult rat DRG tissue exhibits a specific pattern of expression of copper transporters with distinct subsets of peripheral sensory neurons intensely expressing either ATP7A or CTR1, but not both or ATP7B. The neuron subtype-specific and largely non-overlapping distribution of ATP7A and CTR1 within rat DRG tissue may be required to support the potentially differing cuproenzyme requirements of distinct subsets of sensory neurons, and could influence the transport and neurotoxicity of oxaliplatin. PMID:20836889

2010-01-01

129

LANDVIEW III  

EPA Science Inventory

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

130

Welding III.  

ERIC Educational Resources Information Center

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

Allegheny County Community Coll., Pittsburgh, PA.

131

Genetics Home Reference: Congenital disorder of glycosylation type IIi  

MedlinePLUS

... to speak. Other features of CDG IIi include short stature, an unusually small head size (microcephaly), and distinctive ... neurological ; oligosaccharides ; protein ; recessive ; sensorineural ; sensorineural hearing loss ; short stature ; stature ; syndrome You may find definitions for these ...

132

ATP release and purinergic signaling in NLRP3 inflammasome activation  

PubMed Central

The NLRP3 inflammasome is a protein complex involved in IL-1? and IL-18 processing that senses pathogen- and danger-associated molecular patterns (PAMPs and DAMPs). One step- or two step-models have been proposed to explain the tight regulation of IL-1? production during inflammation. Moreover, cellular stimulation triggers adenosine triphosphate (ATP) release and subsequent activation of purinergic receptors at the cell surface. Importantly some studies have reported roles for extracellular ATP, in NLRP3 inflammasome activation in response to PAMPs and DAMPs. In this mini review, we will discuss the link between active ATP release, purinergic signaling and NLRP3 inflammasome activation. We will focus on the role of autocrine or paracrine ATP export in particle-induced NLRP3 inflammasome activation and discuss how particle activators are competent to induce maturation and secretion of IL-1? through a process that involves, as a first event, extracellular release of endogenous ATP through hemichannel opening, and as a second event, signaling through purinergic receptors that trigger NLRP3 inflammasome activation. Finally, we will review the evidence for ATP as a key pro-inflammatory mediator released by dying cells. In particular we will discuss how cancer cells dying via autophagy trigger ATP-dependent NLRP3 inflammasome activation in the macrophages engulfing them, eliciting an immunogenic response against tumors. PMID:23316199

Gombault, Aurélie; Baron, Ludivine; Couillin, Isabelle

2013-01-01

133

ATP and potassium ions: a deadly combination for astrocytes.  

PubMed

The ATP release channel Pannexin1 (Panx1) is self-regulated, i.e. the permeant ATP inhibits the channel from the extracellular space. The affinity of the ATP binding site is lower than that of the purinergic P2X7 receptor allowing a transient activation of Panx1 by ATP through P2X7R. Here we show that the inhibition of Panx1 by ATP is abrogated by increased extracellular potassium ion concentration ([K(+)]o) in a dose-dependent manner. Since increased [K(+)]o is also a stimulus for Panx1 channels, it can be expected that a combination of ATP and increased [K(+)]o would be deadly for cells. Indeed, astrocytes did not survive exposure to these combined stimuli. The death mechanism, although involving P2X7R, does not appear to strictly follow a pyroptotic pathway. Instead, caspase-3 was activated, a process inhibited by Panx1 inhibitors. These data suggest that Panx1 plays an early role in the cell death signaling pathway involving ATP and K(+) ions. Additionally, Panx1 may play a second role once cells are committed to apoptosis, since Panx1 is also a substrate of caspase-3. PMID:24694658

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

2014-01-01

134

ATP and potassium ions: a deadly combination for astrocytes  

NASA Astrophysics Data System (ADS)

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

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

2014-04-01

135

Altered responsiveness to extracellular ATP enhances acetaminophen hepatotoxicity  

PubMed Central

Background Adenosine triphosphate (ATP) is secreted from hepatocytes under physiological conditions and plays an important role in liver biology through the activation of P2 receptors. Conversely, higher extracellular ATP concentrations, as observed during necrosis, trigger inflammatory responses that contribute to the progression of liver injury. Impaired calcium (Ca2+) homeostasis is a hallmark of acetaminophen (APAP)-induced hepatotoxicity, and since ATP induces mobilization of the intracellular Ca2+ stocks, we evaluated if the release of ATP during APAP-induced necrosis could directly contribute to hepatocyte death. Results APAP overdose resulted in liver necrosis, massive neutrophil infiltration and large non-perfused areas, as well as remote lung inflammation. In the liver, these effects were significantly abrogated after ATP metabolism by apyrase or P2X receptors blockage, but none of the treatments prevented remote lung inflammation, suggesting a confined local contribution of purinergic signaling into liver environment. In vitro, APAP administration to primary mouse hepatocytes and also HepG2 cells caused cell death in a dose-dependent manner. Interestingly, exposure of HepG2 cells to APAP elicited significant release of ATP to the supernatant in levels that were high enough to promote direct cytotoxicity to healthy primary hepatocytes or HepG2 cells. In agreement to our in vivo results, apyrase treatment or blockage of P2 receptors reduced APAP cytotoxicity. Likewise, ATP exposure caused significant higher intracellular Ca2+ signal in APAP-treated primary hepatocytes, which was reproduced in HepG2 cells. Quantitative real time PCR showed that APAP-challenged HepG2 cells expressed higher levels of several purinergic receptors, which may explain the hypersensitivity to extracellular ATP. This phenotype was confirmed in humans analyzing liver biopsies from patients diagnosed with acute hepatic failure. Conclusion We suggest that under pathological conditions, ATP may act not only an immune system activator, but also as a paracrine direct cytotoxic DAMP through the dysregulation of Ca2+ homeostasis. PMID:23384127

2013-01-01

136

ATP13A2 regulates mitochondrial bioenergetics through macroautophagy  

PubMed Central

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

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

2012-01-01

137

A new system for ATP-metric immunoanalysis.  

PubMed

Treatment of amino-group-containing antigens with adenosine-5'-trimetaphosphate results in their chemical modification by -pppA residues. An immunoanalytical system is proposed based upon competition of these ATP-labelled antigens with those of the sample for immobilized antibodies. Mild acidic treatment of complexes of ATP-labelled antigens with immobilized antibodies results in quantitative liberation of intact ATP. The latter may be determined by the ultrosenstive bioluminescent techniques based upon emission of light with firefly luciferase. The validity of the system has been studied with two clinically important antigens, thyroxine and myoglobin. PMID:6313423

Grachev, M A; Dobrikov, M I; Knorre, V D; Pressman, E K; Roschke, V V; Shishkin, G V

1983-10-17

138

Twisting and subunit rotation in single FOF1-ATP synthase  

PubMed Central

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

Sielaff, Hendrik; Börsch, Michael

2013-01-01

139

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

PubMed

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

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

2015-01-20

140

Inhibitors of the 5-lipoxygenase arachidonic acid pathway induce ATP release and ATP-dependent organic cation transport in macrophages.  

PubMed

We have previously described that arachidonic acid (AA)-5-lipoxygenase (5-LO) metabolism inhibitors such as NDGA and MK886, inhibit cell death by apoptosis, but not by necrosis, induced by extracellular ATP (ATPe) binding to P2X7 receptors in macrophages. ATPe binding to P2X7 also induces large cationic and anionic organic molecules uptake in these cells, a process that involves at least two distinct transport mechanisms: one for cations and another for anions. Here we show that inhibitors of the AA-5-LO pathway do not inhibit P2X7 receptors, as judged by the maintenance of the ATPe-induced uptake of fluorescent anionic dyes. In addition, we describe two new transport phenomena induced by these inhibitors in macrophages: a cation-selective uptake of fluorescent dyes and the release of ATP. The cation uptake requires secreted ATPe, but, differently from the P2X7/ATPe-induced phenomena, it is also present in macrophages derived from mice deficient in the P2X7 gene. Inhibitors of phospholipase A2 and of the AA-cyclooxygenase pathway did not induce the cation uptake. The uptake of non-organic cations was investigated by measuring the free intracellular Ca(2+) concentration ([Ca(2+)]i) by Fura-2 fluorescence. NDGA, but not MK886, induced an increase in [Ca(2+)]i. Chelating Ca(2+) ions in the extracellular medium suppressed the intracellular Ca(2+) signal without interfering in the uptake of cationic dyes. We conclude that inhibitors of the AA-5-LO pathway do not block P2X7 receptors, trigger the release of ATP, and induce an ATP-dependent uptake of organic cations by a Ca(2+)- and P2X7-independent transport mechanism in macrophages. PMID:24743022

da Silva-Souza, Hercules Antônio; Lira, Maria Nathalia de; Costa-Junior, Helio Miranda; da Cruz, Cristiane Monteiro; Vasconcellos, Jorge Silvio Silva; Mendes, Anderson Nogueira; Pimenta-Reis, Gabriela; Alvarez, Cora Lilia; Faccioli, Lucia Helena; Serezani, Carlos Henrique; Schachter, Julieta; Persechini, Pedro Muanis

2014-07-01

141

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

PubMed

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

Kirby, Brett S; Hanna, Gabi; Hendargo, Hansford C; McMahon, Timothy J

2014-12-15

142

Biochemistry 1990, 29, 1671-1683 1611 Inhibition of RecA Protein Promoted ATP Hydrolysis. 2. Longitudinal Assembly  

E-print Network

Biochemistry 1990, 29, 1671-1683 1611 Inhibition of RecA Protein Promoted ATP Hydrolysis. 2 of the new ADP/ATP ratio. This phase is manifested by a lag in ATP hydrolysis when ATP is added to preformed ADP filaments, and by a burst in ATP hydrolysis in all other cases. More than 15 ATPs are hydrolyzed

Cox, Michael M.

143

A unified, long-term, high-latitude stratospheric aerosol and cloud database using SAM II, SAGE II, and POAM II\\/III data: Algorithm description, database definition, and climatology  

Microsoft Academic Search

A 22 year, high-latitude, stratospheric aerosol and cloud database has been formed in a “unified” manner by combining the Stratospheric Aerosol Measurement (SAM) II, Stratospheric Aerosol and Gas Measurement (SAGE) II, Polar Ozone and Aerosol Measurement (POAM) II, and POAM III 1 ?m aerosol extinction profiles. The database is “unified” in that it embodies similar aerosol extinction measurements, uses a

Michael Fromm; Jerome Alfred; Michael Pitts

2003-01-01

144

A unified, long-term, high-latitude stratospheric aerosol and cloud database using SAM II, SAGE II, and POAM II\\/III data: Algorithm description, database definition, and climatology  

Microsoft Academic Search

A 22 year, high-latitude, stratospheric aerosol and cloud database has been formed in a ``unified'' manner by combining the Stratospheric Aerosol Measurement (SAM) II, Stratospheric Aerosol and Gas Measurement (SAGE) II, Polar Ozone and Aerosol Measurement (POAM) II, and POAM III 1 mum aerosol extinction profiles. The database is ``unified'' in that it embodies similar aerosol extinction measurements, uses a

Michael Fromm; Jerome Alfred; Michael Pitts

2003-01-01

145

RESEARCH PAPER Role of ATP and related purines in inhibitory  

E-print Network

physiological saline solution at 37°C and gassed with 95% O2 and 5% CO2 for isometric force recordings. Key relaxations. At basal tension, EFS- and ATP-induced contractions were resistant to desensitization or blockade

Burnstock, Geoffrey

146

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

147

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

148

Highly Divergent Mitochondrial ATP Synthase Complexes in Tetrahymena thermophila  

PubMed Central

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

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

149

Argininosuccinate synthetase: a stereochemical study using chiral ATP analogs  

E-print Network

ARGININOSIJCCINATF. SYNTHETASE: A STEREOCHEMICAL STUDY USING CHIRAL ATP ANALOGS A Thesis bv TAMARA LOUISE CHAPMAN NESS Submitted to the Graduate College of Texas A6M University in partial fulfillment of the requirements for the degree of.... MASTER OF SCIENCE December 1984 Major Subject: Chemistry ARGININOSUCCINATE SYNTHETASE: A STEREOCHEMICAL STUDY USING CHIRAL ATP ANALOGS A Thesis by TAMARA LOUISE CHAPMAN HESS Approved as to style and content by: Dr. Frank Raushel (Chairman...

Hess, Tamara Louise Chapman

2012-06-07

150

Portable ATP Luminometry for Evaluating Salmon Roe Processing Facilities  

Microsoft Academic Search

Sanitation conditions in two salmon roe processing operations were evaluated by adenosine triphosphate (ATP) assays using a portable luminometer and single-use reaction swabs. The bioluminescent reaction provided real-time testing (< 1 min) when compared to standard microbiological analyses (48-72 hr). The ATP assay was quicker and more sensitive than a rapid protein assay (10 min) in detecting presence of biological

Brian H. Himelbloom; Susan M. Vitt; Chuck Crapo

2004-01-01

151

Clinical application of adenosine and ATP for pain control  

Microsoft Academic Search

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

Masakazu Hayashida; Ken-ichi Fukuda; Atsuo Fukunaga

2005-01-01

152

The ATP-binding cassette family: a structural perspective  

Microsoft Academic Search

The ATP-binding cassette family is one of the largest groupings of membrane proteins, moving allocrites across lipid membranes,\\u000a using energy from ATP. In bacteria, they reside in the inner membrane and are involved in both uptake and export. In eukaryotes,\\u000a these transporters reside in the cell’s internal membranes as well as in the plasma membrane and are unidirectional—out of\\u000a the

Veronica Kos; Robert Curtis Ford

2009-01-01

153

ATP-regulated K+ channels in cardiac muscle  

Microsoft Academic Search

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,

A. Noma

1983-01-01

154

Facilitation and depression of ATP and noradrenaline release from sympathetic nerves of rat tail artery  

PubMed Central

Excitatory junction currents (EJCs) were used to measure ATP release; noradrenaline (NA) oxidation currents and fractional overflow of labelled NA, [3H]NA, were used to monitor the release of endogenous and exogenous NA, respectively, from post-ganglionic sympathetic nerves of rat tail artery. During nerve stimulation with 100 pulses at 5-20 Hz the EJCs initially grew in size (maximally by 23%, at 2–10 Hz), and then depressed, maximally by 68% at 20 Hz. The peak amplitude of NA oxidation currents in response to nerve stimulation with 100 pulses at 2–20 Hz grew in size with frequency, while the area was independent of frequency and roughly constant. The size of the NA oxidation currents evoked by nerve stimulation with 4–100 pulses at 20 Hz grew linearly with train length between pulses 4–16. Between pulses 20–100 there was a train length-dependent depression of the signal. Fractional overflow of [3H]NA in response to nerve stimulation with 5–100 pulses at 20 Hz behaved similarly to the EJCs. It initially grew roughly linearly between pulses 5–25, and then showed a dramatic depression similar to that of the EJCs. The ?2-adrenoceptor antagonists rauwolscine and yohimbine increased the overflow of [3H]NA and the amplitude of NA oxidation currents, but not that of the EJCs. It is concluded that during high-frequency stimulation (i) the release of ATP and NA is first briefly facilitated then markedly depressed, (ii) facilitation and depression of the two transmitters are similar in magnitude and time course, and (iii) ?2-adrenoceptor antagonists differentially modify EJCs and the NA signals. The results obtained in the absence of drugs are compatible with the hypothesis that ATP and NA are released in parallel, while the effects of ?2-adrenoceptor antagonists seem to suggest dissociated release. PMID:10050018

Msghina, Mussie; Gonon, François; Stjärne, Lennart

1999-01-01

155

ATP signaling in brain: release, excitotoxicity and potential therapeutic targets.  

PubMed

Adenosine 5'-triphosphate (ATP) is released as a genuine co-transmitter, or as a principal purinergic neurotransmitter, in an exocytotic and non-exocytotic manner. It activates ionotropic (P2X) and metabotropic (P2Y) receptors which mediate a plethora of functions in the brain. In particular, P2X7 receptor (P2X7R) are expressed in all brain cells and its activation can form a large pore allowing the passage of organic cations, the leakage of metabolites of up to 900 Da and the release of ATP itself. In turn, pannexins (Panx) are a family of proteins forming hemichannels that can release ATP. In this review, we summarize the progress in the understanding of the mechanisms of ATP release both in physiological and pathophysiological stages. We also provide data suggesting that P2X7R and pannexin 1 (Panx1) may form a large pore in cortical neurons as assessed by electrophysiology. Finally, the participation of calcium homeostasis modulator 1 is also suggested, another non-selective ion channel that can release ATP, and that could play a role in ischemic events, together with P2X7 and Panx1 during excitotoxicity by ATP. PMID:25096398

Cisneros-Mejorado, Abraham; Pérez-Samartín, Alberto; Gottlieb, Miroslav; Matute, Carlos

2015-01-01

156

Reaction Dynamics of ATP Hydrolysis Catalyzed by P-Glycoprotein  

PubMed Central

P-glycoprotein (P-gp) is a member of the ABC transporter family that confers drug resistance to many tumors by catalyzing their efflux, and it is a major component of drug–drug interactions. P-gp couples drug efflux with ATP hydrolysis by coordinating conformational changes in the drug binding sites with the hydrolysis of ATP and release of ADP. To understand the relative rates of the chemical step for hydrolysis and the conformational changes that follow it, we exploited isotope exchange methods to determine the extent to which the ATP hydrolysis step is reversible. With ?18O4-labeled ATP, no positional isotope exchange is detectable at the bridging ?-phosphorus–O??-phosphorus bond. Furthermore, the phosphate derived from hydrolysis includes a constant ratio of three 18O/two 18O/one 18O that reflects the isotopic composition of the starting ATP in multiple experiments. Thus, H2O-exchange with HPO42– (Pi) was negligible, suggesting that a [P-gp·ADP·Pi] is not long-lived. This further demonstrates that the hydrolysis is essentially irreversible in the active site. These mechanistic details of ATP hydrolysis are consistent with a very fast conformational change immediately following, or concomitant with, hydrolysis of the ?-phosphate linkage that ensures a high commitment to catalysis in both drug-free and drug-bound states. PMID:24506763

2015-01-01

157

A molecular switch in SecA protein couples ATP hydrolysis to protein translocation  

E-print Network

A molecular switch in SecA protein couples ATP hydrolysis to protein translocation SpyridoulaA dimers. NBD1 is suffi- cient for single rounds of SecA ATP hydrolysis. Multi- ple ATP turnovers at NBD1. This intramolecular regulator of ATP hydrolysis (IRA) mediates N-/C-domain binding and acts as a molecular switch

Economou, Tassos

158

Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP  

PubMed Central

Dicer is a multi-domain RNase III-related endonuclease responsible for processing double-stranded RNA (dsRNA) to small interfering RNAs (siRNAs) during a process of RNA interference (RNAi). It also catalyses excision of the regulatory microRNAs from their precursors. In this work, we describe the purification and properties of a recombinant human Dicer. The protein cleaves dsRNAs into ?22 nucleotide siRNAs. Accumulation of processing intermediates of discrete sizes, and experiments performed with substrates containing modified ends, indicate that Dicer preferentially cleaves dsRNAs at their termini. Binding of the enzyme to the substrate can be uncoupled from the cleavage step by omitting Mg2+ or performing the reaction at 4°C. Activity of the recombinant Dicer, and of the endogenous protein present in mammalian cell extracts, is stimulated by limited proteolysis, and the proteolysed enzyme becomes active at 4°C. Cleavage of dsRNA by purifed Dicer and the endogenous enzyme is ATP independent. Additional experiments suggest that if ATP participates in the Dicer reaction in mammalian cells, it might be involved in product release needed for the multiple turnover of the enzyme. PMID:12411505

Zhang, Haidi; Kolb, Fabrice A.; Brondani, Vincent; Billy, Eric; Filipowicz, Witold

2002-01-01

159

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

NASA Astrophysics Data System (ADS)

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

Waxman, Lloyd; Goldberg, Alfred L.

1982-08-01

160

Definition Chemistry  

E-print Network

1 · Definition · Chemistry · Factors · Mitigation MinE 422 Acid Rock Drainage Online `Gard Guide is a great source of information Terminology · acid rock drainage (ARD) · saline drainage (SD) · acid mine or acid and metalliferous drainage (AMD) · mining influenced water (MIW) · neutral mine drainage (NMD

Boisvert, Jeff

161

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

Microsoft Academic Search

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

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

2011-01-01

162

Regulation of yeast acetohydroxyacid synthase by valine and ATP.  

PubMed Central

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

Pang, S S; Duggleby, R G

2001-01-01

163

The effect of alpha, beta-methylene ATP on the depolarization evoked by noradrenaline (gamma-adrenoceptor response) and ATP in the immature rat basilar artery.  

PubMed Central

Depolarizations evoked by noradrenaline that were resistant to alpha- and beta-adrenoceptor antagonists were recorded in the rat basilar artery. These gamma-adrenoceptor-mediated responses and the depolarizations to adenosine triphosphate (ATP) were blocked by pretreatment of the tissue with alpha, beta-methylene ATP. These data are discussed with respect to the selectivity of alpha, beta-methylene ATP. PMID:3011173

Byrne, N. G.; Large, W. A.

1986-01-01

164

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

ATP-synthase of Rhodobacter capsulatus: coupling of proton £ow through FH to reactions in FI under. Proton transfer through ATP-synthase (measured by electrochromic carotenoid bandshift and by p the amount of protons translocated by FHFI and the ATP yield decreased with the flash number from an apparent

Steinhoff, Heinz-Jürgen

165

Title. ATP7B copper-regulated traffic and association with the tight junctions: copper excretion into Short title. ATP7B and copper excretion by liver  

E-print Network

1 Title. ATP7B copper-regulated traffic and association with the tight junctions: copper excretion into the bile Short title. ATP7B and copper excretion by liver Authors. Sonia Hernandez*§ , Yo Tsuchiya manuscript Gastroenterology 2008;134(4):1215-23 #12;2 Abstract The copper transporter ATP7B plays a central

Boyer, Edmond

166

Skeletal muscle ATP kinetics are impaired in frail mice.  

PubMed

The interleukin-10 knockout mouse (IL10(tm/tm)) has been proposed as a model for human frailty, a geriatric syndrome characterized by skeletal muscle (SM) weakness, because it develops an age-related decline in SM strength compared to control (C57BL/6J) mice. Compromised energy metabolism and energy deprivation appear to play a central role in muscle weakness in metabolic myopathies and muscular dystrophies. Nonetheless, it is not known whether SM energy metabolism is altered in frailty. A combination of in vivo (31)P nuclear magnetic resonance experiments and biochemical assays was used to measure high-energy phosphate concentrations, the rate of ATP synthesis via creatine kinase (CK), the primary energy reserve reaction in SM, as well as the unidirectional rates of ATP synthesis from inorganic phosphate (Pi) in hind limb SM of 92-week-old control (n = 7) and IL10(tm/tm) (n = 6) mice. SM Phosphocreatine (20.2 ± 2.3 vs. 16.8 ± 2.3 ?mol/g, control vs. IL10(tm/tm), p < 0.05), ATP flux via CK (5.0 ± 0.9 vs. 3.1 ± 1.1 ?mol/g/s, p < 0.01), ATP synthesis from inorganic phosphate (Pi ? ATP) (0.58 ± 0.3 vs. 0.26 ± 0.2 ?mol/g/s, p < 0.05) and the free energy released from ATP hydrolysis (?G ?ATP) were significantly lower and [Pi] (2.8 ± 1.0 vs. 5.3 ± 2.0 ?mol/g, control vs. IL10(tm/tm), p < 0.05) markedly higher in IL10(tm/tm) than in control mice. These observations demonstrate that, despite normal in vitro metabolic enzyme activities, in vivo SM ATP kinetics, high-energy phosphate levels and energy release from ATP hydrolysis are reduced and inorganic phosphate is elevated in a murine model of frailty. These observations do not prove, but are consistent with the premise, that energetic abnormalities may contribute metabolically to SM weakness in this geriatric syndrome. PMID:23695949

Akki, Ashwin; Yang, Huanle; Gupta, Ashish; Chacko, Vadappuram P; Yano, Toshiyuki; Leppo, Michelle K; Steenbergen, Charles; Walston, Jeremy; Weiss, Robert G

2014-02-01

167

Heat shock protein 70 (Hsp70) inhibits oxidative phosphorylation and compensates ATP balance through enhanced glycolytic activity  

PubMed Central

To address possible effects of heat shock protein 70 (Hsp70) on energy metabolism, we established a cell line expressing different levels of Hsp70 and evaluated changes in glucose and lactate metabolites, as well as ATP levels accordingly. In addition, activities of enzymes involved in glycolysis [phosphofructokinase (PFK) and lactate dehydrogenase (LDH)], Krebs cycle [citric synthase (CS)], and oxidative phosphorylation {NADH dehydrogenase [complex I (CI)] and ubiquinol:cytochrome-c reductase [complex III (CIII)]} were analyzed. The results show that both glucose consumption and lactate excretion were elevated significantly in cells expressing increased levels of Hsp70. Simultaneously, the activities of glycolytic enzymes PFK and LDH were increased markedly in cells overexpressing Hsp70. Activities of enzymes CI and CIII, both involved in oxidative phosphorylation, decreased upon increased expression of Hsp70. These findings were supported by nonsignificant reductions of CS activities in cells that overexpressed Hsp70, whereas intracellular ATP levels remained constant over a wide range of Hsp70 expression. In conclusion, overexpression of Hsp70 in HeLa cells results in downregulation of oxidative phosphorylation, in particular, multiprotein CIII, the main source of reactive oxygen species. In exchange, upregulation of the glycolytic pathway compensates for the homeostasis of cellular ATP supply. PMID:23042904

Wang, Liangli; Schumann, Uwe; Prokopchuk, Olga; Steinacker, Jürgen M.

2012-01-01

168

Structure of Dimeric F1F0-ATP Synthase*  

PubMed Central

The structure of the dimeric ATP synthase from yeast mitochondria was analyzed by transmission electron microscopy and single particle image analysis. In addition to the previously reported side views of the dimer, top view and intermediate projections served to resolve the arrangement of the rotary c10 ring and the other stator subunits at the F0-F0 dimeric interface. A three-dimensional reconstruction of the complex was calculated from a data set of 9960 molecular images at a resolution of 27 ?. The structural model of the dimeric ATP synthase shows the two monomers arranged at an angle of ?45°, consistent with our earlier analysis of the ATP synthase from bovine heart mitochondria (Minauro-Sanmiguel, F., Wilkens, S., and Garcia, J. J. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 12356–12358). In the ATP synthase dimer, the two peripheral stalks are located near the F1-F1 interface but are turned away from each other so that they are not in contact. Based on the three-dimensional reconstruction, a model of how dimeric ATP synthase assembles to form the higher order oligomeric structures that are required for mitochondrial cristae biogenesis is discussed. PMID:20833715

Couoh-Cardel, Sergio J.; Uribe-Carvajal, Salvador; Wilkens, Stephan; García-Trejo, José J.

2010-01-01

169

Capture and quality control mechanisms for ATP binding  

PubMed Central

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

Li, Li; Martinis, Susan A.

2013-01-01

170

ATP P2X3 receptors and neuronal sensitization  

PubMed Central

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

Fabbretti, Elsa

2013-01-01

171

Phenomenological analysis of ATP dependence of motor protein  

E-print Network

In this study, through phenomenological comparison of the velocity-force data of processive motor proteins, including conventional kinesin, cytoplasmic dynein and myosin V, we found that, the ratio between motor velocities of two different ATP concentrations is almost invariant for any substall, superstall or negative external loads. Therefore, the velocity of motor can be well approximated by a Michaelis-Menten like formula $V=\\atp k(F)L/(\\atp +K_M)$, with $L$ the step size, and $k(F)$ the external load $F$ dependent rate of one mechanochemical cycle of motor motion in saturated ATP solution. The difference of Michaelis-Menten constant $K_M$ for substall, superstall and negative external load indicates, the ATP molecule affinity of motor head for these three cases are different, though the expression of $k(F)$ as a function of $F$ might be unchanged for any external load $F$. Verifications of this Michaelis-Menten like formula has also been done by fitting to the recent experimental data.

Yunxin Zhang

2011-08-09

172

Calcium regulation of tension redevelopment kinetics with 2-deoxy-ATP or low [ATP] in rabbit skeletal muscle.  

PubMed Central

The correlation of acto-myosin ATPase rate with tension redevelopment kinetics (k(tr)) was determined during Ca(+2)-activated contractions of demembranated rabbit psoas muscle fibers; the ATPase rate was either increased or decreased relative to control by substitution of ATP (5.0 mM) with 2-deoxy-ATP (dATP) (5.0 mM) or by lowering [ATP] to 0.5 mM, respectively. The activation dependence of k(tr) and unloaded shortening velocity (Vu) was measured with each substrate. With 5.0 mM ATP, Vu depended linearly on tension (P), whereas k(tr) exhibited a nonlinear dependence on P, being relatively independent of P at submaximum levels and rising steeply at P > 0.6-0.7 of maximum tension (Po). With dATP, Vu was 25% greater than control at Po and was elevated at all P > 0.15Po, whereas Po was unchanged. Furthermore, the Ca(+2) sensitivity of both k(tr) and P increased, such that the dependence of k(tr) on P was not significantly different from control, despite an elevation of Vu and maximal k(tr). In contrast, lowering [ATP] caused a slight (8%) elevation of Po, no change in the Ca(+2) sensitivity of P, and a decrease in Vu at all P. Moreover, k(tr) was decreased relative to control at P > 0.75Po, but was elevated at P < 0.75Po. These data demonstrate that the cross-bridge cycling rate dominates k(tr) at maximum but not submaximum levels of Ca(2+) activation. PMID:9545059

Regnier, M; Martyn, D A; Chase, P B

1998-01-01

173

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)

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.

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

2014-06-01

174

NPY mediates ATP-induced neuroproliferation in adult mouse olfactory epithelium  

PubMed Central

In the CNS, ATP is released upon injury and promotes neuroproliferation via purinergic receptors. In the olfactory epithelium, ATP promotes the synthesis and release of neurotrophic factor NPY in neonates and induces neuroproliferation in neonatal and adult mice. We tested the hypothesis that NPY is involved in ATP-induced neuroproliferation in adult mice olfactory epithelium. Intranasal instillation of ATP significantly increased protein levels and number of NPY+ cells. Pre-intranasal instillation of purinergic receptor antagonist PPADS significantly reduced ATP-induced upregulation of NPY. Intranasal instillation of NPY-Y1 receptor antagonist BIBP3226 following ATP instillation significantly inhibited the ATP-induced increase in BrdU incorporation, suggesting that NPY is released after ATP instillation and activates Y1 receptors to promote neuroproliferation. These data indicate that ATP initiates neuroproliferation via NPY upregulation, NPY release, and Y1 receptor activation, and suggests that the olfactory epithelium is good model to study neuroregenerative mechanisms in the CNS. PMID:20211262

Jia, Cuihong; Hegg, Colleen Cosgrove

2010-01-01

175

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

PubMed

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

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

2012-01-01

176

Thermal activation and ATP dependence of the cytoskeleton remodeling dynamics  

NASA Astrophysics Data System (ADS)

The cytoskeleton (CSK) is a nonequilibrium polymer network that uses hydrolyzable sources of free energy such as adenosine triphosphate (ATP) to remodel its internal structure. As in inert nonequilibrium soft materials, CSK remodeling has been associated with structural rearrangements driven by energy-activated processes. We carry out particle tracking and traction microscopy measurements of alveolar epithelial cells at various temperatures and ATP concentrations. We provide the first experimental evidence that the remodeling dynamics of the CSK is driven by structural rearrangements over free-energy barriers induced by thermally activated forces mediated by ATP. The measured activation energy of these forces is ˜40kBTr ( kB being the Boltzmann constant and Tr being the room temperature). Our experiments provide clues to understand the analogy between the dynamics of the living CSK and that of inert nonequilibrium soft materials.

Sunyer, R.; Ritort, F.; Farré, R.; Navajas, D.

2009-05-01

177

Release of Adenosine and ATP During Ischemia and Epilepsy  

PubMed Central

Eighty years ago Drury & Szent-Györgyi described the actions of adenosine, AMP (adenylic acid) and ATP (pyrophosphoric or diphosphoric ester of adenylic acid) on the mammalian cardiovascular system, skeletal muscle, intestinal and urinary systems. Since then considerable insight has been gleaned on the means by which these compounds act, not least of which in the distinction between the two broad classes of their respective receptors, with their many subtypes, and the ensuing diversity in cellular consequences their activation invokes. These myriad actions are of course predicated on the release of the purines into the extracellular milieu, but, surprisingly, there is still considerable ambiguity as to how this occurs in various physiological and pathophysiological conditions. In this review we summarise the release of ATP and adenosine during seizures and cerebral ischemia and discuss mechanisms by which the purines adenosine and ATP may be released from cells in the CNS under these conditions. PMID:20190959

Dale, Nicholas; Frenguelli, Bruno G

2009-01-01

178

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

PubMed Central

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

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

179

Involvement of the phosphatidylinositol kinase pathway in augmentation of ATP-sensitive K(+) channel currents by hypo-osmotic stress in rat ventricular myocytes.  

PubMed

The objective of this study was to investigate the mechanisms of increase in the efficacy of ATP-sensitive K(+) channel (KATP) openings by hypo-osmotic stress. The whole-cell KATP currents (IK,ATP) stimulated by 100 ?mol/L pinacidil, a K(+) channel opening drug, were significantly augmented during hypo-osmotic stress (189 mOsmol/L) compared with normal conditions (303 mOsmol/L). The EC50 and Emax value for pinacidil-activated IK,ATP (measured at 0 mV) was 154 ?mol/L and 844 pA, respectively, in normal solution and 16.6 ?mol/L and 1266 pA, respectively, in hypo-osmotic solution. Augmentation of IK,ATP during hypo-osmotic stress was attenuated by wortmannin (50 ?mol/L), an inhibitor of phosphatidylinositol 3- and 4-kinases, but not by (i) phalloidin (30 ?mol/L), an actin filament stabilizer, (ii) the absence of Ca(2+) from the internal and external solutions, and (iii) the presence of creatine phosphate (3 mmol/L), which affects creatine kinase regulation of the KATP channels. In the single-channel recordings, an inside-out patch was made after approximately 5 min exposure of the myocyte to hypo-osmotic solution. However, the IC50 value for ATP under such conditions was not different from that obtained in normal osmotic solution. In conclusion, hypo-osmotic stress could augment cardiac IK,ATP through intracellular mechanisms involving the phosphatidylinositol kinase pathway. PMID:23984989

Mitsuyama, Hirofumi; Yokoshiki, Hisashi; Irie, Yuki; Watanabe, Masaya; Mizukami, Kazuya; Tsutsui, Hiroyuki

2013-09-01

180

The giant cardiac membrane patch method: stimulation of outward Na(+)-Ca2+ exchange current by MgATP.  

PubMed Central

1. A giant patch method was used to study the stimulatory effect of cytoplasmic MgATP on outward Na(+)-Ca2+ exchange current in inside-out cardiac membrane patches (1-10 G omega seals with 14-24 microns pipette tip diameters) excised from guinea-pig, rabbit and mouse myocytes. 2. To establish the validity of the method with respect to structure, bleb formation was examined with electron microscopy and with confocal fluorescence light microscopy. The blebs, which form as the sarcolemma detaches, excluded intracellular organelles and transverse tubules. The blebbed cells contained normal sarcomeres, sarcoplasmic reticulum, triads and diads. 3. To further establish the validity of the method for ion transport studies, measurements of Na(+)-K+ pump currents and charge movements are described briefly which demonstrate (i) free access to the cytoplasmic membrane side, (ii) MgATP dependence comparable to reconstituted pump (Kd, 94 microns), (iii) fast, rigorous concentration control and (iv) Na(+)-K+ pump densities in the range of whole-cell densities. 4. Stimulation of outward Na(+)-Ca2+ exchange current by MgATP attenuated exchange current decay during step increments of cytoplasmic sodium, shifted the secondary activation of outward exchange current by cytoplasmic calcium to lower free calcium concentrations and, particularly in mouse cardiac sarcolemma, induced cytoplasmic calcium-independent current. 5. Upon removal of MgATP the stimulatory effect usually decayed with a t50 (half-time) of about 3 min. However, the reversal took place much more rapidly (t50, 5-20 s) in patches from individual guinea-pig and rabbit myocyte batches. When decay was rapid, secondary activation by cytoplasmic calcium was shifted to higher free cytoplasmic calcium concentrations (Kd, 10-65 microns-free calcium). 6. With repeated applications of MgATP the rate and magnitude of the stimulatory effect progressively decreased. 7. The Kd for MgATP of the initial rate of stimulation of outward exchange current was 3 mM or greater. When decay was rapid, the steady-state dependence of exchange current on MgATP also had a Kd of 3 mM or greater. 8. Stimulation of Na(+)-Ca2+ exchange current by MgATP occurred in the absence of cytoplasmic calcium with 9 mM-EGTA. 9. The stimulatory effect of 2 mM-MgATP was not inhibited by up to 200 microM of the protein kinase inhibitor 1-(5-isoquinoline sulphonyl)-2-methylpiperazine (H7), or by peptide inhibitors of cyclic AMP-dependent protein kinase, protein kinase C and calcium-calmodulin-dependent protein kinase II.(ABSTRACT TRUNCATED AT 400 WORDS) Images Fig. 14 Fig. 15 Fig. 16 Fig. 17 Fig. 18 PMID:1335502

Collins, A; Somlyo, A V; Hilgemann, D W

1992-01-01

181

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

NSDL National Science Digital Library

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.

Alain Trautmann (France; Universit預aris Descartes REV)

2009-02-03

182

Adenosine 5'-triphosphate (ATP) inhibits schwann cell demyelination during Wallerian degeneration.  

PubMed

Adenosine 5'-triphosphate (ATP) is implicated in intercellular communication as a neurotransmitter in the peripheral nervous system. In addition, ATP is known as lysosomal exocytosis activator. In this study, we investigated the role of extracellular ATP on demyelination during Wallerian degeneration (WD) using ex vivo and in vivo nerve degeneration models. We found that extracellular ATP inhibited myelin fragmentation and axonal degradation during WD. Furthermore, metformin and chlorpromazine, lysosomal exocytosis antagonists blocked the effect of ATP on the inhibition of demyelination. Thus, these findings indicate that ATP-induced-lysosomal exocytosis may be involved in demyelination during WD. PMID:24363123

Shin, Youn Ho; Chung, Hyung-Joo; Park, Chan; Jung, Junyang; Jeong, Na Young

2014-04-01

183

Characterization of ATP Alternations in an Alzheimer’s Transgenic Mouse Model  

PubMed Central

Mitochondrial impairment as evidenced by decline in adenosine 5?-triphosphate (ATP) is associated with oxidative stress in Alzheimer’s neuropathology and suggests that mitochondria may fail to maintain cellular energy, through reduced ATP production in neurons. To gain insights into the ATP characteristics of Alzheimer’s disease transgenic (Tg) mice, we investigated ATP contents in the brain and whole blood of Tg mice at three ages (1-, 5- and 24-month-old). Overall, our results demonstrate that tissue ATP contents in Tg mice are significantly reduced, suggesting a decrease of tissue ATP production and mitochondrial dysfunction. PMID:25261448

Zhang, Cheng; Rissman, Robert A.; Feng, June

2014-01-01

184

ATP sequestration by a synthetic ATP-binding protein leads to novel phenotypic changes in Escherichia coli.  

PubMed

Artificial proteins that bind key metabolites with high affinity and specificity hold great promise as new tools in synthetic biology, but little has been done to create such molecules and examine their effects on living cells. Experiments of this kind have the potential to expand our understanding of cellular systems, as certain phenotypes may be physically realistic but not yet observed in nature. Here, we examine the physiology and morphology of a population of Escherichia coli as they respond to a genetically encoded, non-biological ATP-binding protein. Unlike natural ATP-dependent proteins, which transiently bind ATP during metabolic transformations, the synthetic protein DX depletes the concentration of intracellular ATP and ADP by a mechanism of protein-mediated ligand sequestration. The resulting ATP/ADP imbalance leads to an adaptive response in which a large population of bacilli cells transition to a filamentous state with dense lipid structures that segregate the cells into compartmentalized units. A wide range of biochemical and microscopy techniques extensively characterized these novel lipid structures, which we have termed endoliposomes. We show that endoliposomes adopt well-defined box-like structures that span the full width of the cell but exclude the synthetic protein DX. We further show that prolonged DX exposure causes a large fraction of the population to enter a viable-but-non-culturable state that is not easily reversed. Both phenotypes correlate with strong intracellular changes in ATP and ADP concentration. We suggest that artificial proteins, such as DX, could be used to control and regulate specific targets in metabolic pathways. PMID:23181457

Korch, Shaleen B; Stomel, Joshua M; León, Megan A; Hamada, Matt A; Stevenson, Christine R; Simpson, Brent W; Gujulla, Sunil K; Chaput, John C

2013-02-15

185

Negative feedback of extracellular ADP on ATP release in goldfish hepatocytes: a theoretical study.  

PubMed

A mathematical model was built to account for the kinetic of extracellular ATP (ATPe) and extracellular ADP (ADPe) concentrations from goldfish hepatocytes exposed to hypotonicity. The model was based on previous experimental results on the time course of ATPe accumulation, ectoATPase activity, and cell viability [Pafundo et al., 2008]. The kinetic of ATPe is controlled by a lytic ATP flux, a non-lytic ATP flux, and ecto-ATPase activity, whereas ADPe kinetic is governed by a lytic ADP flux and both ecto-ATPase and ecto-ADPase activities. Non-lytic ATPe efflux was included as a diffusion equation modulated by ATPe activation (positive feedback) and ADPe inhibition (negative feedback). The model yielded physically meaningful and stable steady-state solutions, was able to fit the experimental time evolution of ATPe and simulated the concomitant kinetic of ADPe. According to the model during the first minute of hypotonicity the concentration of ATPe is mainly governed by both lytic and non-lytic ATP efflux, with almost no contribution from ecto-ATPase activity. Later on, ecto-ATPase activity becomes important in defining the time dependent decay of ATPe levels. ADPe inhibition of the non-lytic ATP efflux was strong, whereas ATPe activation was minimal. Finally, the model was able to predict the consequences of partial inhibition of ecto-ATPase activity on the ATPe kinetic, thus emulating the exposure of goldfish cells to hypotonic medium in the presence of the ATP analog AMP-PCP. The model predicts this analog to both inhibit ectoATPase activity and increase non-lytic ATP release. PMID:20303983

Chara, Osvaldo; Pafundo, Diego E; Schwarzbaum, Pablo J

2010-06-21

186

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

PubMed Central

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

1989-01-01

187

Extracellular ATP Induces the Accumulation of Superoxide via NADPH Oxidases in Arabidopsis1  

PubMed Central

Extracellular ATP can serve as a signaling agent in animal cells, and, as suggested by recent reports, may also do so in plant cells. In animal cells it induces the production of reactive oxygen species through the mediation of NADPH oxidase. Similarly, here we report that in leaves of Arabidopsis (Arabidopsis thaliana), applied ATP, but not AMP or phosphate, induces the accumulation of superoxide (O2?) in a biphasic, dose-dependent manner, with a threshold at 500 nm ATP. This effect did not require ATP hydrolysis for it was mimicked by ATP?S. ATP also induced increased levels of Arabidopsis respiratory burst oxidase homolog D (AtrbohD) mRNA, but ATP-treated plants that had disrupted AtrbohD and AtrbohF genes did not accumulate O2?, indicating that NADPH oxidases are responsible for the induced O2? accumulation. Inhibitors of mammalian P2-type ATP receptors abolished ATP-induced O2? production, suggesting that the ATP effects may be mediated through P2-like receptors in plants. Cytosolic Ca2+ and calmodulin are likely to help transduce the ATP responses, as they do in animal cells, because a Ca2+ channel blocker, a Ca2+ chelator, and calmodulin antagonist all reduced ATP-induced O2? accumulation. Furthermore, ATP treatment enhanced the expression of genes that are induced by wounds and other stresses. The ATP measured at wound sites averaged 40 ?m, well above the level needed to induce O2? accumulation and gene expression changes. Transgenic plants overexpressing an apyrase gene had reduced O2? production in response to applied ATP and wounding. Together, these data suggest a possible role for extracellular ATP as a signal potentially in wound and stress responses. PMID:16428598

Song, Charlotte J.; Steinebrunner, Iris; Wang, Xuanzhi; Stout, Stephen C.; Roux, Stanley J.

2006-01-01

188

Evidence that Na+/H+ exchanger 1 is an ATP-binding protein.  

PubMed

Na(+)/H(+) exchanger (NHE) 1 is a member of the solute carrier superfamily, which regulates intracellular ionic homeostasis. NHE1 is known to require cellular ATP for its activity, despite there being no requirement for energy input from ATP hydrolysis. In this study, we investigated whether NHE1 is an ATP-binding protein. We designed a baculovirus vector carrying both epitope-tagged NHE1 and its cytosolic subunit CHP1, and expressed the functional NHE1-CHP1 complex on the surface of Sf9 insect cells. Using the purified complex protein consisting of NHE1 and CHP1 from Sf9 cells, we examined a photoaffinity labeling reaction with 8-azido-ATP-biotin. UV irradiation promoted the incorporation of 8-azido-ATP into NHE1, but not into CHP1, with an apparent Kd of 29.1 µM in the presence of Mg(2+). The nonlabeled nucleotides ATP, GTP, TTP and CTP all inhibited this crosslinking. However, ATP had the strongest inhibitory effect, with an apparent inhibition constant (IC50) for ATP of 2.2 mM, close to the ATP concentration giving the half-maximal activation of NHE1 activity. Importantly, crosslinking was more strongly inhibited by ATP than by ADP, suggesting that ATP is dissociated from NHE1 upon ATP hydrolysis. Limited proteolysis with thrombin and deletion mutant analysis revealed that the 8-azido-ATP-binding site is within the C-terminal cytoplasmic domain of NHE1. Equilibrium dialysis with NHE1-derived peptides provided evidence that ATP directly binds to the proximal cytoplasmic region (Gly542-Pro598), which is critical for ATP-dependent regulation of NHE1. These findings suggest that NHE1 is an ATP-binding transporter. Thus, ATP may serve as a direct activator of NHE1. PMID:23331996

Shimada-Shimizu, Naoko; Hisamitsu, Takashi; Nakamura, Tomoe Y; Wakabayashi, Shigeo

2013-03-01

189

ATP 3-20.16 Mobile Gun System Platoon  

E-print Network

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

US Army Corps of Engineers

190

ATP binding turns plant cryptochrome into an efficient natural photoswitch.  

PubMed

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

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

2014-01-01

191

ATP Binding Turns Plant Cryptochrome Into an Efficient Natural Photoswitch  

NASA Astrophysics Data System (ADS)

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

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

2014-06-01

192

Treatment of heterotopic ossification through remote ATP hydrolysis.  

PubMed

Heterotopic ossification (HO) is the pathologic development of ectopic bone in soft tissues because of a local or systemic inflammatory insult, such as burn injury or trauma. In HO, mesenchymal stem cells (MSCs) are inappropriately activated to undergo osteogenic differentiation. Through the correlation of in vitro assays and in vivo studies (dorsal scald burn with Achilles tenotomy), we have shown that burn injury enhances the osteogenic potential of MSCs and causes ectopic endochondral heterotopic bone formation and functional contractures through bone morphogenetic protein-mediated canonical SMAD signaling. We further demonstrated a prevention strategy for HO through adenosine triphosphate (ATP) hydrolysis at the burn site using apyrase. Burn site apyrase treatment decreased ATP, increased adenosine 3',5'-monophosphate, and decreased phosphorylation of SMAD1/5/8 in MSCs in vitro. This ATP hydrolysis also decreased HO formation and mitigated functional impairment in vivo. Similarly, selective inhibition of SMAD1/5/8 phosphorylation with LDN-193189 decreased HO formation and increased range of motion at the injury site in our burn model in vivo. Our results suggest that burn injury-exacerbated HO formation can be treated through therapeutics that target burn site ATP hydrolysis and modulation of SMAD1/5/8 phosphorylation. PMID:25253675

Peterson, Jonathan R; De La Rosa, Sara; Eboda, Oluwatobi; Cilwa, Katherine E; Agarwal, Shailesh; Buchman, Steven R; Cederna, Paul S; Xi, Chuanwu; Morris, Michael D; Herndon, David N; Xiao, Wenzhong; Tompkins, Ronald G; Krebsbach, Paul H; Wang, Stewart C; Levi, Benjamin

2014-09-24

193

ATP-induced noncooperative thermal unfolding of hen lysozyme  

SciTech Connect

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.

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

194

Detection of ATP and NADH: A Bioluminescent Experience.  

ERIC Educational Resources Information Center

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

Selig, Ted C.; And Others

1984-01-01

195

Cyclodextrin-based microcapsules as bioreactors for ATP biosynthesis.  

PubMed

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

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

2013-09-01

196

ATP hydrolysis stimulates large length fluctuations in single actin filaments  

E-print Network

Polymerization dynamics of single actin filaments is investigated theoretically using a stochastic model that takes into account the hydrolysis of ATP-actin subunits, the geometry of actin filament tips, the lateral interactions between the monomers as well as the processes at both ends of the polymer. Exact analytical expressions are obtained for a mean growth velocity and for dispersion in length fluctuations. It is found that the ATP hydrolysis has a strong effect on dynamic properties of single actin filaments. At high concentrations of free actin monomers the mean size of unhydrolyzed ATP-cap is very large, and the dynamics is governed by association/dissociation of ATP-actin subunits. However, at low concentrations the size of the cap becomes finite, and the dissociation of ADP-actin subunits makes a significant contribution to overall dynamics. Actin filament length fluctuations reach the maximum at the boundary between two dynamic regimes, and this boundary is always larger than the critical concentration. Random and vectorial mechanisms of hydrolysis are compared, and it is found that they predict qualitatively similar dynamic properties. The possibility of attachment and detachment of oligomers is also discussed. Our theoretical approach is successfully applied to analyze the latest experiments on the growth and length fluctuations of individual actin filaments.

Evgeny B. Stukalin; Anatoly B. Kolomeisky

2005-07-27

197

ATP6V0D1/A0403  

NSDL National Science Digital Library

ATPase, proton-transporting, lysosomal V0 subunit D1 (ATP6V0D1, also known as A0403) is responsible for the acidification of endosomes, lysosomes, and other intracellular organelles in eukaryotic cells, thus providing most of the energy required for ...

2009-04-14

198

Extracellular ATP signaling in plants Kiwamu Tanaka1  

E-print Network

Extracellular ATP signaling in plants Kiwamu Tanaka1 , Simon Gilroy2 , Alan M. Jones3 and Gary- naling in animal cells appear to be lacking in plant cells, although some cellular responses are similar in animal and plant cells. This comparison will aid our overall understanding of plant physiology and also

Jones, Alan M.

199

ATP Binding Turns Plant Cryptochrome Into an Efficient Natural Photoswitch  

PubMed Central

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

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

2014-01-01

200

A unified, long-term, high-latitude stratospheric aerosol and cloud database using SAM II, SAGE II, and POAM II/III data: Algorithm description, database definition, and climatology  

NASA Astrophysics Data System (ADS)

A 22 year, high-latitude, stratospheric aerosol and cloud database has been formed in a "unified" manner by combining the Stratospheric Aerosol Measurement (SAM) II, Stratospheric Aerosol and Gas Measurement (SAGE) II, Polar Ozone and Aerosol Measurement (POAM) II, and POAM III 1 ?m aerosol extinction profiles. The database is "unified" in that it embodies similar aerosol extinction measurements, uses a single meteorological data set, and employs a single algorithm for calculating background extinction and cloud detection thresholds. Latitude is constrained to poleward of 45° in each hemisphere. The Unified cloud detection algorithm and database are designed for the straightforward addition of new data when other compatible data sets (e.g., SAGE III) become available. "Unified" cloud detection is similar to, but a refinement of, earlier attempts to identify polar stratospheric clouds (PSCs) with SAM II and POAM II data. The Unified algorithm is instrument-independent and circumvents fundamental cloud detection pitfalls. The database contains over 73,000 (36,000) polar vortex-region profiles in the Antarctic (Arctic) and over 21,000 (2000) PSC observations. An introductory climatology of Unified "background" extinction is presented. It is seen that volcanic effects dominate the evolution of outside-vortex background extinctions, but perturbations apparently not related to volcanoes are seen as well. Interannual variations of background extinction inside the austral vortex are seen to be nearly decoupled from volcanic effects, while in the Arctic, inside-vortex extinctions show a considerable volcanic influence. An analysis of long-term PSC sighting is presented. Midwinter (July and January) PSC and clear-sky measurements at 20 km, in a fixed temperature range, are used for computing PSC probability. The grand average PSC probability calculated this way is nearly identical between hemispheres. In the Antarctic the interannual PSC probability pattern is distinctly cyclic but is convoluted by volcanic perturbations in background aerosol. In the Arctic the PSC probability has much less temporal coherence than in the Antarctic but is similarly impacted by volcanic background increases. An explanation for the variation in PSC probabilities, in terms of interannual differences in denitrification, is discussed. Finally, a statistical analysis of tropopause height in relation to PSC formation is also presented. PSC observations are seen to be strongly associated with elevated tropopause heights, indicating that tropospheric, synoptic-scale flow perturbations are the primary forcing mechanism for Arctic PSC formation, as evidenced in this long-term satellite record.

Fromm, Michael; Alfred, Jerome; Pitts, Michael

2003-06-01

201

Activation of ATP Binding for the Autophosphorylation of DosS, a Mycobacterium tuberculosis Histidine Kinase Lacking an ATP Lid Motif*  

PubMed Central

The sensor histidine kinases of Mycobacterium tuberculosis, DosS and DosT, are responsible for sensing hypoxic conditions and consist of sensor and kinase cores responsible for accepting signals and phosphorylation activity, respectively. The kinase core contains a dimerization and histidine phosphate-accepting (DHp) domain and an ATP binding domain (ABD). The 13 histidine kinase genes of M. tuberculosis can be grouped based on the presence or absence of the ATP lid motif and F box (elements known to play roles in ATP binding) in their ABDs; DosS and DosT have ABDs lacking both these elements, and the crystal structures of their ABDs indicated that they were unsuitable for ATP binding, as a short loop covers the putative ATP binding site. Although the ABD alone cannot bind ATP, the kinase core is functional in autophosphorylation. Appropriate spatial arrangement of the ABD and DHp domain within the kinase core is required for both autophosphorylation and ATP binding. An ionic interaction between Arg440 in the DHp domain and Glu537 in the short loop of the ABD is available and may open the ATP binding site, by repositioning the short loop away from the site. Mutations at Arg440 and Glu537 reduce autophosphorylation activity. Unlike other histidine kinases containing an ATP lid, which protects bound ATP, DosS is unable to accept ATP until the ABD is properly positioned relative to the histidine; this may prevent unexpected ATP reactions. ATP binding can, therefore, function as a control mechanism for histidine kinase activity. PMID:23486471

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

2013-01-01

202

Novel ATP6V1B1 and ATP6V0A4 mutations in autosomal recessive distal renal tubular acidosis with new evidence for hearing loss  

PubMed Central

Autosomal recessive distal renal tubular acidosis (rdRTA) is characterised by severe hyperchloraemic metabolic acidosis in childhood, hypokalaemia, decreased urinary calcium solubility, and impaired bone physiology and growth. Two types of rdRTA have been differentiated by the presence or absence of sensorineural hearing loss, but appear otherwise clinically similar. Recently, we identified mutations in genes encoding two different subunits of the renal ?-intercalated cell's apical H+-ATPase that cause rdRTA. Defects in the B1 subunit gene ATP6V1B1, and the a4 subunit gene ATP6V0A4, cause rdRTA with deafness and with preserved hearing, respectively. We have investigated 26 new rdRTA kindreds, of which 23 are consanguineous. Linkage analysis of seven novel SNPs and five polymorphic markers in, and tightly linked to, ATP6V1B1 and ATP6V0A4 suggested that four families do not link to either locus, providing strong evidence for additional genetic heterogeneity. In ATP6V1B1, one novel and five previously reported mutations were found in 10 kindreds. In 12 ATP6V0A4 kindreds, seven of 10 mutations were novel. A further nine novel ATP6V0A4 mutations were found in "sporadic" cases. The previously reported association between ATP6V1B1 defects and severe hearing loss in childhood was maintained. However, several patients with ATP6V0A4 mutations have developed hearing loss, usually in young adulthood. We show here that ATP6V0A4 is expressed within the human inner ear. These findings provide further evidence for genetic heterogeneity in rdRTA, extend the spectrum of disease causing mutations in ATP6V1B1 and ATP6V0A4, and show ATP6V0A4 expression within the cochlea for the first time. PMID:12414817

Stover, E; Borthwick, K; Bavalia, C; Eady, N; Fritz, D; Rungroj, N; Giersch, A; Morton, C; Axon, P; Akil, I; Al-Sabban, E; Baguley, D; Bianca, S; Bakkaloglu, A; Bircan, Z; Chauveau, D; Clermont, M; Guala, A; Hulton, S; Kroes, H; Li, V; Mir, S; Mocan, H; Nayir, A; Ozen, S; Rodriguez, S; Sanjad, S; Tasic, V; Taylor, C; Topaloglu, R; Smith, A; Karet, F

2002-01-01

203

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

E-print Network

in the signaling steps that link biotic stresses to plant defense responses and growth changes. Plant cells release, Texas 78712 Plant cells release ATP into their extracellular matrix as they grow, and extracellular ATP

Webb, Lauren J.

204

Regulation of Extracellular ATP in Human Erythrocytes Infected with Plasmodium falciparum  

PubMed Central

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

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

205

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

PubMed

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

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

206

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

Microsoft Academic Search

Mitochondrial translation of the Saccharomyces cerevisiae Atp6p subunit of F1–F0 ATP synthase is regulated by the F1 ATPase. Here we show normal expression of Atp6p in HeLa cells depleted of the F1 ? subunit. Instead of being translationally down-regulated, HeLa cells lacking F1 degrade Atp6p, thereby preventing proton leakage across the inner membrane. Mammalian mitochondria also differ in the way

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

2011-01-01

207

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

Microsoft Academic Search

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

Antti Nykänen; Benjamin Haley; Phillip D. Zamore

2001-01-01

208

HtrA2 deficiency causes mitochondrial uncoupling through the F1F0-ATP synthase and consequent ATP depletion  

PubMed Central

Loss of the mitochondrial protease HtrA2 (Omi) in mice leads to mitochondrial dysfunction, neurodegeneration and premature death, but the mechanism underlying this pathology remains unclear. Using primary cultures from wild-type and HtrA2-knockout mice, we find that HtrA2 deficiency significantly reduces mitochondrial membrane potential in a range of cell types. This depolarisation was found to result from mitochondrial uncoupling, as mitochondrial respiration was increased in HtrA2-deficient cells and respiratory control ratio was dramatically reduced. HtrA2-knockout cells exhibit increased proton translocation through the ATP synthase, in combination with decreased ATP production and truncation of the F1 ?-subunit, suggesting the ATP synthase as the source of the proton leak. Uncoupling in the HtrA2-deficient mice is accompanied by altered breathing pattern and, on a cellular level, ATP depletion and vulnerability to chemical ischaemia. We propose that this vulnerability may ultimately cause the neurodegeneration observed in these mice. PMID:22739987

Plun-Favreau, H; Burchell, V S; Holmström, K M; Yao, Z; Deas, E; Cain, K; Fedele, V; Moisoi, N; Campanella, M; Miguel Martins, L; Wood, N W; Gourine, A V; Abramov, A Y

2012-01-01

209

ATPase-Independent Type-III Protein Secretion in Salmonella enterica  

PubMed Central

Type-III protein secretion systems are utilized by gram-negative pathogens to secrete building blocks of the bacterial flagellum, virulence effectors from the cytoplasm into host cells, and structural subunits of the needle complex. The flagellar type-III secretion apparatus utilizes both the energy of the proton motive force and ATP hydrolysis to energize substrate unfolding and translocation. We report formation of functional flagella in the absence of type-III ATPase activity by mutations that increased the proton motive force and flagellar substrate levels. We additionally show that increased proton motive force bypassed the requirement of the Salmonella pathogenicity island 1 virulence-associated type-III ATPase for secretion. Our data support a role for type-III ATPases in enhancing secretion efficiency under limited secretion substrate concentrations and reveal the dispensability of ATPase activity in the type-III protein export process. PMID:25393010

Erhardt, Marc; Mertens, Max E.; Fabiani, Florian D.; Hughes, Kelly T.

2014-01-01

210

A Novel Neuronal P,, ATP Receptor Ion Channel with Widespread Distribution in the Brain  

Microsoft Academic Search

There is strong evidence that ATP acts as an excitatory neuro- transmitter in the periphery, yet little is known about fast central ATP-mediated transmission. We report here the molecular cloning of a novel neuronal ionotropic ATP receptor of the P,, subtype (P& isolated from rat brain. This central P,, channel subunit has significant amino acid homology with two recently cloned

Ali Haghighi; Ellis Cooper

1996-01-01

211

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

PubMed Central

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

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

212

Organized Unidirectional Waves of ATP Hydrolysis within a RecA Filament  

E-print Network

Organized Unidirectional Waves of ATP Hydrolysis within a RecA Filament Julia M. Cox, Oleg V on opposite filament ends, with disassembly requiring ATP hydrolysis. When filaments form on duplex DNA, Rec. RecA filament state was monitored with a coupled spectrophotometric assay for ATP hydrolysis

Cox, Michael M.

213

ATP Hydrolysis Enhances RNA Recognition and Antiviral Signal Transduction by the Innate Immune Sensor,  

E-print Network

ATP Hydrolysis Enhances RNA Recognition and Antiviral Signal Transduction by the Innate Immune receptor required for innate antiviral signaling. Results: LGP2 uses ATP hydrolysis to diversify RNA infec- tion and initiate antiviral signal transduction cascades. The ATP hydrolysis activity of LGP2

Myong, Sua

214

RecA-mediated SOS induction requires an extended filament conformation but no ATP hydrolysis  

E-print Network

RecA-mediated SOS induction requires an extended filament conformation but no ATP hydrolysis induction in vivo after UV treatment. Thus, SOS induction does not require ATP hydrolysis by the Rec an improved reagent for studies of the function of ATP hydrolysis by RecA in vivo and in vitro. Introduction

Cox, Michael M.

215

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

216

Inhibition of the ATPase activity of Escherichia coli ATP synthase by magnesium fluoride  

E-print Network

Inhibition of the ATPase activity of Escherichia coli ATP synthase by magnesium fluoride Zulfiqar activity of Escherichia coli ATP synthase by magnesium fluoride (MgFx) was studied. Wild-type F1-ATPase synthesis mechanism; Magnesium fluoride; ATPase inhibition; Transition state analog 1. Introduction ATP

Zulfiqar Ahmad

217

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

PubMed

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

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

2014-01-01

218

Role of the P-Type ATPases, ATP7A and ATP7B in brain copper homeostasis  

PubMed Central

Over the past two decades there have been significant advances in our understanding of copper homeostasis and the pathological consequences of copper dysregulation. Cumulative evidence is revealing a complex regulatory network of proteins and pathways that maintain copper homeostasis. The recognition of copper dysregulation as a key pathological feature in prominent neurodegenerative disorders such as Alzheimer’s, Parkinson’s, and prion diseases has led to increased research focus on the mechanisms controlling copper homeostasis in the brain. The copper-transporting P-type ATPases (copper-ATPases), ATP7A and ATP7B, are critical components of the copper regulatory network. Our understanding of the biochemistry and cell biology of these complex proteins has grown significantly since their discovery in 1993. They are large polytopic transmembrane proteins with six copper-binding motifs within the cytoplasmic N-terminal domain, eight transmembrane domains, and highly conserved catalytic domains. These proteins catalyze ATP-dependent copper transport across cell membranes for the metallation of many essential cuproenzymes, as well as for the removal of excess cellular copper to prevent copper toxicity. A key functional aspect of these copper transporters is their copper-responsive trafficking between the trans-Golgi network and the cell periphery. ATP7A- and ATP7B-deficiency, due to genetic mutation, underlie the inherited copper transport disorders, Menkes and Wilson diseases, respectively. Their importance in maintaining brain copper homeostasis is underscored by the severe neuropathological deficits in these disorders. Herein we will review and update our current knowledge of these copper transporters in the brain and the central nervous system, their distribution and regulation, their role in normal brain copper homeostasis, and how their absence or dysfunction contributes to disturbances in copper homeostasis and neurodegeneration. PMID:23986700

Telianidis, Jonathon; Hung, Ya Hui; Materia, Stephanie; Fontaine, Sharon La

2013-01-01

219

cDNA sequence, gene structure, and chromosomal localization of the human ATP-sensitive potassium channel, uK{sub ATP}-1, gene (KCNJ8)  

SciTech Connect

ATP-sensitive K{sup +} (KATP) channels play a crucial role in coupling metabolic energy to the membrane potential of cells. Recently, we have isolated a KATP channel cDNA (uK{sub ATP}-1) that is expressed ubiquitously in rat tissues including pancreatic islets, pituitary, skeletal muscle, and heart. Here, we report cloning of the human cDNA and gene encoding uK{sub ATP}-1. Human uK{sub ATP}-1 is a protein of 424 amino acids exhibiting 98% identity with rat uK{sub ATP}-1. The human gene encoding uK{sub ATP}-1, designated KCNJ8, is approximately 9.7 kb in length and is composed of three exons. KCNJ8 was mapped to chromosome 12p11.23 using fluorescence in situ hybridization. 14 refs., 2 figs., 1 tab.

Inagaki, Nobuya; Seino, Susumu [Chiba Univ. School of Medicine, Chiba (Japan); Inazawa, Johji [Kyoto Prefectural Univ. of Medicine (Japan)

1995-11-01

220

Thermophilic ATP synthase has a decamer c-ring: Indication of noninteger 10:3 H+\\/ATP ratio and permissive elastic coupling  

Microsoft Academic Search

In a rotary motor FoF1-ATP synthase that couples H+ transport with ATP synthesis\\/hydrolysis, it is thought that an Foc subunit oligomer ring (c-ring) in the membrane rotates as protons pass through Fo and a 120° rotation produces one ATP at F1. Despite several structural studies, the copy number of Foc subunits in the c-ring has not been determined for any

Noriyo Mitome; Toshiharu Suzuki; Shigehiko Hayashi; Masasuke Yoshida

2004-01-01

221

Genetic Variation in ATP5O Is Associated with Skeletal Muscle ATP50 mRNA Expression and Glucose Uptake in Young Twins  

Microsoft Academic Search

BackgroundImpaired oxidative capacity of skeletal muscle mitochondria contribute to insulin resistance and type 2 diabetes (T2D). Furthermore, mRNA expression of genes involved in oxidative phosphorylation, including ATP5O, is reduced in skeletal muscle from T2D patients. Our aims were to investigate mechanisms regulating ATP5O expression in skeletal muscle and association with glucose metabolism, and the relationship between ATP5O single nucleotide polymorphisms

Tina Rönn; Pernille Poulsen; Tiinamaija Tuomi; Bo Isomaa; Leif Groop; Allan Vaag; Charlotte Ling; Maurizio Capogrossi

2009-01-01

222

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

SciTech Connect

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

Shertzer, Howard G. [Department of Environmental Health and Center for Environmental Genetics, University of Cincinnati Medical Center, P.O. Box 670056 Cincinnati, OH 45267-0056 (United States)]. E-mail: shertzhg@ucmail.uc.edu; Genter, Mary Beth [Department of Environmental Health and Center for Environmental Genetics, University of Cincinnati Medical Center, P.O. Box 670056 Cincinnati, OH 45267-0056 (United States); Shen, Dongxiao [Department of Environmental Health and Center for Environmental Genetics, University of Cincinnati Medical Center, P.O. Box 670056 Cincinnati, OH 45267-0056 (United States); Nebert, Daniel W. [Department of Environmental Health and Center for Environmental Genetics, University of Cincinnati Medical Center, P.O. Box 670056 Cincinnati, OH 45267-0056 (United States); Chen, Ying [Department of Environmental Health and Center for Environmental Genetics, University of Cincinnati Medical Center, P.O. Box 670056 Cincinnati, OH 45267-0056 (United States); Dalton, Timothy P. [Department of Environmental Health and Center for Environmental Genetics, University of Cincinnati Medical Center, P.O. Box 670056 Cincinnati, OH 45267-0056 (United States)

2006-12-15

223

Oxa1 Directly Interacts with Atp9 and Mediates Its Assembly into the Mitochondrial F1Fo-ATP Synthase Complex  

PubMed Central

The yeast Oxa1 protein is involved in the biogenesis of the mitochondrial oxidative phosphorylation (OXPHOS) machinery. The involvement of Oxa1 in the assembly of the cytochrome oxidase (COX) complex, where it facilitates the cotranslational membrane insertion of mitochondrially encoded COX subunits, is well documented. In this study we have addressed the role of Oxa1, and its sequence-related protein Cox18/Oxa2, in the biogenesis of the F1Fo-ATP synthase complex. We demonstrate that Oxa1, but not Cox18/Oxa2, directly supports the assembly of the membrane embedded Fo-sector of the ATP synthase. Oxa1 was found to physically interact with newly synthesized mitochondrially encoded Atp9 protein in a posttranslational manner and in a manner that is not dependent on the C-terminal, matrix-localized region of Oxa1. The stable manner of the Atp9-Oxa1 interaction is in contrast to the cotranslational and transient interaction previously observed for the mitochondrially encoded COX subunits with Oxa1. In the absence of Oxa1, Atp9 was observed to assemble into an oligomeric complex containing F1-subunits, but its further assembly with subunit 6 (Atp6) of the Fo-sector was perturbed. We propose that by directly interacting with newly synthesized Atp9 in a posttranslational manner, Oxa1 is required to maintain the assembly competence of the Atp9-F1-subcomplex for its association with Atp6. PMID:17344477

Jia, Lixia; Dienhart, Mary K.

2007-01-01

224

Pathogenic VCP mutations induce mitochondrial uncoupling and reduced ATP levels.  

PubMed

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

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

2013-04-10

225

Students' interdisciplinary reasoning about "high-energy bonds" and ATP  

NSDL National Science Digital Library

Students' sometimes contradictory ideas about ATP (adenosine triphosphate) and the nature of chemical bonds have been studied in the biology and chemistry education literatures, but these topics are rarely part of the introductory physics curriculum. We present qualitative data from an introductory physics course for undergraduate biology majors that seeks to build greater interdisciplinary coherence and therefore includes these topics. In these data, students grapple with the apparent contradiction between the energy released when the phosphate bond in ATP is broken and the idea that an energy input is required to break a bond. We see that students' perceptions of how each scientific discipline bounds the system of interest can influence how they justify their reasoning about a topic that crosses disciplines. This has consequences for a vision of interdisciplinary education that respects disciplinary perspectives while bringing them into interaction in ways that demonstrate consistency amongst the perspectives

Dreyfus, Benjamin W.; Geller, Benjamin D.; Sawtelle, Vashti; Svoboda, Julia; Turpen, Chandra; Redish, Edward F.

2013-09-07

226

ATP-dependent reorganization of human sperm nuclear chromatin.  

PubMed

Chromosomes in terminally differentiated mammalian spermatozoa are extensively condensed by protamines but a small proportion of histones remain. We examined the primary organization of somatic-type chromatin in lysolecithin-permeabilized human sperm nuclei and report that nucleosomes are closely packed with a periodicity of approximately 150 bp. Incubation of nuclei in the presence of exogenous Mg2+ and ATP induced chromatin reorganization leading to an increase in spacing of the nucleosomes to approximately 190 bp. This ATP-dependent chromatin rearrangement involved phosphorylation of both protamine and histone H2a. Increase in linker length between nucleosomes correlated with the phosphorylation of H2aX, the major H2a variant in human spermatozoa, predominantly at the C-terminal end. Chromatin reorganization was independent of detectable nuclear dispersion, which is an early chromosomal event in male pronuclear formation during fertilization. PMID:7769017

Banerjee, S; Smallwood, A; Hultén, M

1995-02-01

227

Statistical Mechanics Analysis of ATP Binding to a Multisubunit Enzyme  

NASA Astrophysics Data System (ADS)

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.

Zhang, Yun-Xin

2014-10-01

228

ATP Binding Cassette A1 Transporter Function and Tangier Disease  

Microsoft Academic Search

\\u000a The ATP binding cassette A1 (ABCA1) transporter facilitates the efflux of free cholesterol and phospholipid onto high density\\u000a lipoprotein (HDL) particles, mainly very small discoidal precursor HDL particles, known as prebeta 1 HDL. Once these particles\\u000a pick up these lipids, they are converted to small discoidal alpha 4 migrating HDL and with cholesterol esterification mature\\u000a into larger spherical alpha 3

Ernst J. Schaefer; H. Bryan Brewer

229

Inhibition of Escherichia coli ATP synthase by amphibian antimicrobial peptides.  

PubMed

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

Laughlin, Thomas F; Ahmad, Zulfiqar

2010-04-01

230

Internal Motions in Myosin Head: Effect of ADP and ATP  

Microsoft Academic Search

Internal flexibility of myosin heads in glycerinated muscle fibres in the presence of MgADP plus orthovanadate and after addition of Ca-ATP was studied using an isothiocyanate-based spin label attached to the reactive sulfhydryl sites of myosin. The spin labels were immobilized on the microsecond time scale and exhibited significant orientational order in rigor. In AM+.ADP.Vistate a smaller fraction of ordered

Joseph Belagyi; Dénes Lo?rinczy

1996-01-01

231

An intersubunit signaling network coordinates ATP hydrolysis by m-AAA proteases  

PubMed Central

Summary Ring-shaped AAA+ ATPases control a variety of cellular processes by substrate unfolding and remodeling of macromolecular structures. However, how ATP hydrolysis within AAA+ rings is regulated and coupled to mechanical work is poorly understood. Here, we demonstrate coordinated ATP hydrolysis within m-AAA protease ring complexes, conserved AAA+ machines in the inner membrane of mitochondria. ATP binding to one AAA subunit inhibits ATP hydrolysis by the neighboring subunit leading to coordinated rather than stochastic ATP hydrolysis within the AAA ring. Unbiased genetic screens define an intersubunit signaling pathway involving conserved AAA motifs and reveal an intimate coupling of ATPase activities to central AAA pore loops. Coordinated ATP hydrolysis between adjacent subunits is required for membrane dislocation of substrates but not for substrate processing. These findings provide new insight how AAA+ proteins convert energy derived from ATP hydrolysis into mechanical work. PMID:19748354

Augustin, Steffen; Gerdes, Florian; Lee, Sukyeong; Tsai, Francis T.F.; Langer, Thomas; Tatsuta, Takashi

2009-01-01

232

Overview of photo-induced therapy for ATP production  

NASA Astrophysics Data System (ADS)

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

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

2012-10-01

233

48 CFR 52.234-1 - Industrial Resources Developed Under Defense Production Act Title III.  

Code of Federal Regulations, 2010 CFR

...Clauses 52.234-1 Industrial Resources Developed Under...the following clause: Industrial Resources Developed Under...Definitions. Title III industrial resource means materials, services, processes, or manufacturing...

2010-10-01

234

48 CFR 52.234-1 - Industrial Resources Developed Under Defense Production Act Title III.  

Code of Federal Regulations, 2011 CFR

...Clauses 52.234-1 Industrial Resources Developed Under...the following clause: Industrial Resources Developed Under...Definitions. Title III industrial resource means materials, services, processes, or manufacturing...

2011-10-01

235

[Cloning and expression of atp6 and atp9 genes from ramie (Boehmeria nivea (L.) Gaud.) and their relationship with cytoplasmic male sterility].  

PubMed

The atp6 and apt9 gene fragments associated with cytoplasmic male sterility (CMS) were cloned from the mitochondrial DNA of a ramie (Boehmeria nivea (L.) Gaud.) cytoplasmic male sterile line and its maintainer and restorer lines using PCR and degenerated primer strategy. The primers were designed according to the reserved sequences in the encoding region of mitochondrial genes atp6 and atp9 of some dicotyledons from GenBank. These fragments did not have complete encoding region but showed the homology of 94% and 85% with atp6 and atp9 genes from the referred dicotyledons in GenBank. The complete atp6 and atp9 genes including the complete open reading frames were cloned by means of amplifying the 3' and 5'end unknown sequences of these gene fragments using DNA Walking method. The atp6 gene showed no difference among ramie male sterile line, maintainer and restorer lines at mtDNA sequence, transcription and translation control and protein level. However, compared to the maintainer and restorer lines, the atp9 gene of the male sterile line was different and deletion in several bases at the 3' end of the encoding region. An abnormally high expression of atp9 gene in the male sterile line at the budding stage and full-bloom stage was analyzed by RT-PCR analysis. These results indicated that the variation in DNA sequence and/or abnormality in expression of atp9 gene in the male sterile line maybe closely related to ramie CMS. PMID:19073559

Duan, Ji-Qiang; DU, Guang-Hui; Li, Jian-Yong; Liang, Xue-Ni; Liu, Fei-Hu

2008-11-01

236

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

PubMed Central

ATP Binding Cassette (ABC) transporters couple the binding and hydrolysis of ATP to the transport of substrate molecules across the membrane. The mechanism by which ATP binding and/or hydrolysis drives the conformational changes associated with substrate transport has not yet been characterized fully. Here, changes in the conformation of the ABC export protein P-glycoprotein on ATP binding are examined in a series of molecular dynamics simulations. When one molecule of ATP is placed at the ATP binding site associated with each of the two nucleotide binding domains (NBDs), the membrane-embedded P-glycoprotein crystal structure adopts two distinct metastable conformations. In one, each ATP molecule interacts primarily with the Walker A motif of the corresponding NBD. In the other, the ATP molecules interacts with both Walker A motif of one NBD and the Signature motif of the opposite NBD inducing the partial dimerization of the NBDs. This interaction is more extensive in one of the two ATP binding site, leading to an asymmetric structure. The overall conformation of the transmembrane domains is not altered in either of these metastable states, indicating that the conformational changes associated with ATP binding observed in the simulations in the absence of substrate do not lead to the outward-facing conformation and thus would be insufficient in themselves to drive transport. Nevertheless, the metastable intermediate ATP-bound conformations observed are compatible with a wide range of experimental cross-linking data demonstrating the simulations do capture physiologically important conformations. Analysis of the interaction between ATP and its cofactor Mg2+ with each NBD indicates that the coordination of ATP and Mg2+ differs between the two NBDs. The role structural asymmetry may play in ATP binding and hydrolysis is discussed. Furthermore, we demonstrate that our results are not heavily influenced by the crystal structure chosen for initiation of the simulations. PMID:24632881

O’Mara, Megan L.; Mark, Alan E.

2014-01-01

237

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

PubMed Central

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

2013-01-01

238

Different sulfonylurea and ATP sensitivity characterizes the juvenile and the adult form of K ATP channel complex of rat skeletal muscle  

Microsoft Academic Search

We have described here the changes of the biophysical and pharmacological properties of the sarcolemmal ATP-sensitive K+ channels (KATP) of rat skeletal muscle fibres, occurring from an early postnatal period (5 days) to adulthood (210 days). The age-dependent changes of the mean current of the KATP channel (channel activity) and the effects of the blockers, ATP and glybenclamide, were examined

Domenico Tricarico; Rosanna Petruzzi; Diana Conte Camerino

1997-01-01

239

Supramolecular interaction between water-soluble cali[4]xarene and ATP—the catalysis of calix[4]arene for hydrolysis of ATP  

NASA Astrophysics Data System (ADS)

The catalysis effect of water-soluble calix[4]arene C[4] (calix[4]arene-5,11,17,23-tetrasulfonate) on hydrolysis of ATP in aqueous solution was studied by HPLC. Using laser photolysis and pulse radiolysis, the supramolecular interaction between water-soluble calix[4]arene and ATP was investigated.

Tian-Ming, Yao; Zhi-Feng, Ye; Li, Wang; Jin-Ying, Gu; Si-De, Yao; Xian-Fa, Shi

2002-12-01

240

Supramolecular interaction between water-soluble calix[4]arene and ATP--the catalysis of calix[4]arene for hydrolysis of ATP.  

PubMed

The catalysis effect of water-soluble calix[4]arene C[4] (calix[4]arene-5,11,17,23-tetrasulfonate) on hydrolysis of ATP in aqueous solution was studied by HPLC. Using laser photolysis and pulse radiolysis, the supramolecular interaction between water-soluble calix[4]arene and ATP was investigated. PMID:12511088

Tian-Ming, Yao; Zhi-Feng, Ye; Li, Wang; Jin-Ying, Gu; Si-De, Yao; Xian-Fa, Shi

2002-12-01

241

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

PubMed Central

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

Omidbakhsh, Navid; Ahmadpour, Faraz; Kenny, Nicole

2014-01-01

242

Exponential ATP amplification through simultaneous regeneration from AMP and pyrophosphate for luminescence detection of bacteria.  

PubMed

Bacteria monitoring is essential for many industrial manufacturing processes, particularly those involving in food, biopharmaceuticals, and semiconductor production. Firefly luciferase ATP luminescence assay is a rapid and simple bacteria detection method. However, the detection limit of this assay for Escherichia coli is approximately 10(4) colony-forming units (CFU), which is insufficient for many applications. This study aims to improve the assay sensitivity by simultaneous conversion of PP(i) and AMP, two products of the luciferase reaction, back to ATP to form two chain-reaction loops. Because each consumed ATP continuously produces two new ATP molecules, this approach can achieve exponential amplification of ATP. Two consecutive enzyme reactions were employed to regenerate AMP into ATP: adenylate kinase converting AMP into ADP using UTP as the energy source, and acetate kinase catalyzing acetyl phosphate and ADP into ATP. The PP(i)-recycling loop was completed using ATP sulfurylase and adenosine 5' phosphosulfate. The modification maintains good quantification linearity in the ATP luminescence assay and greatly increases its bacteria detection sensitivity. This improved method can detect bacteria concentrations of fewer than 10 CFU. This exponential ATP amplification assay will benefit bacteria monitoring in public health and manufacturing processes that require high-quality water. PMID:21810404

Lee, Hui-Ju; Ho, Min-Rong; Tseng, Chih-Sian; Hsu, Ching-Yi; Huang, Meng-Shun; Peng, Hwei-Ling; Chang, Hwan-You

2011-11-01

243

A microfluidic in situ analyzer for ATP quantification in ocean environments.  

PubMed

We have developed and tested a functionally integrated in situ analyzer, the IISA-ATP system, for microbial activity assays based on a quantitative determination of the total (particulate and dissolved) ATP in ocean environments. The IISA-ATP utilizes a PDMS-glass hybrid microfluidic device as its core functional element, which can perform cell lysis and total ATP quantification by a luciferin-luciferase bioluminescence assay in situ. Transparent heaters and a temperature sensor fabricated on a glass substrate provide temperature control. As a result of the evaluation using the microfluidic device with ATP standard solutions, the bioluminescence intensity was linearly correlated with 2 × 10(-12) to 2 × 10(-8) M of ATP. A detection limit of 1.1 × 10(-11) M was determined using the completed IISA-ATP system, which includes a miniature pumping module and a control module. As a result of the evaluation using the environmental seawater sample collected from Tokyo Bay, Japan, 2.7 × 10(-10) M of total ATP was successfully determined in the laboratory by the IISA-ATP. The system was operated at a shallow submarine hot spring area in Okinawa, Japan for an in situ trial. The result shows the system was successfully operated in situ and the total ATP was determined to be 3.4 × 10(-10) M. PMID:21879105

Fukuba, Tatsuhiro; Aoki, Yusuke; Fukuzawa, Noriyuki; Yamamoto, Takatoki; Kyo, Masanori; Fujii, Teruo

2011-10-21

244

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

PubMed Central

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

Kaler, Stephen G.

2014-01-01

245

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

PubMed Central

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

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

2013-01-01

246

Conformational Transitions of Subunit ? in ATP Synthase from Thermophilic Bacillus PS3  

PubMed Central

Abstract Subunit ? of bacterial and chloroplast FOF1-ATP synthase is responsible for inhibition of ATPase activity. In Bacillus PS3 enzyme, subunit ? can adopt two conformations. In the “extended”, inhibitory conformation, its two C-terminal ?-helices are stretched along subunit ?. In the “contracted”, noninhibitory conformation, these helices form a hairpin. The transition of subunit ? from an extended to a contracted state was studied in ATP synthase incorporated in Bacillus PS3 membranes at 59°C. Fluorescence energy resonance transfer between fluorophores introduced in the C-terminus of subunit ? and in the N-terminus of subunit ? was used to follow the conformational transition in real time. It was found that ATP induced the conformational transition from the extended to the contracted state (half-maximum transition extent at 140 ?M ATP). ADP could neither prevent nor reverse the ATP-induced conformational change, but it did slow it down. Acid residues in the DELSEED region of subunit ? were found to stabilize the extended conformation of ?. Binding of ATP directly to ? was not essential for the ATP-induced conformational change. The ATP concentration necessary for the half-maximal transition (140 ?M) suggests that subunit ? probably adopts the extended state and strongly inhibits ATP hydrolysis only when the intracellular ATP level drops significantly below the normal value. PMID:20141757

Feniouk, Boris A.; Kato-Yamada, Yasuyuki; Yoshida, Masasuke; Suzuki, Toshiharu

2010-01-01

247

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

PubMed Central

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

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

2012-01-01

248

Reciprocating-flow ATP amplification system for increasing the number of amplification cycles.  

PubMed

We constructed a novel ATP amplification reactor using a reciprocating-flow system to increase the number of ATP amplification cycles without an increase in backpressure. We previously reported a continuous-flow ATP amplification system that effectively and quantitatively amplified ATP and increased the sensitivity of a quantitative bioluminescence assay. However, it was difficult to increase the number of amplification cycles due to backpressure in the system. Because addition of immobilized adenylate kinase (ADK) and pyruvate kinase (PK) columns increased backpressure, the maximum number of ATP amplification cycles within column durability was only 4. In this study, ATP amplification was performed using a reciprocating-flow system, and 10 cycles of ATP amplification could be achieved without an increase in backpressure. As a result, ATP was amplified more than 100-fold after 10 cycles of reciprocating flow. The gradient of ATP amplification was approximately 1.76(N). The backpressure on the columns was 0.03 MPa in 1-10 ATP amplification cycles, and no increases in backpressure were observed. PMID:19699705

Satoh, Tetsuya; Tsuruta, Kosuke; Shinoda, Yasuharu; Hirota, Ryuichi; Noda, Kenichi; Kuroda, Akio; Murakami, Yuji

2009-12-15

249

Air-Stimulated ATP Release from Keratinocytes Occurs through Connexin Hemichannels  

PubMed Central

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

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

2013-01-01

250

Extracellular ATP raises cytosolic calcium and activates basolateral chloride conductance in Necturus proximal tubule  

PubMed Central

Extracellular nucleotides modulate ionic transport mechanisms in various epithelia. In the present study, we investigated the effects of extracellular ATP on the intracellular free Ca2+ concentration ([Ca2+]i) and electrophysiological properties of Necturus maculosus proximal convoluted tubule (PCT). ATP raised [Ca2+]i in microdissected fura-2-loaded PCTs (half-maximal effect, ?15 ?mol l?1 ATP). The initial ATP-induced changes in [Ca2+]i were not blunted by the removal of external Ca2+ nor by the presence of Ca2+ channel blockers, but were abolished by thapsigargin and suramin. The sequence for the potency of various agonists on [Ca2+]i was 2-methylthioATP (2MeSATP) = ADP = ATP ? UTP, 2?,3?-O-(4-benzoilbenzoil) ATP (BzATP), ?,?-methylene ATP (AMPCPP), adenosine. In vivo electrophysiological measurements showed that 100 ?mol l?1 peritubular ATP added to a Ringer solution reduced the basolateral cell membrane potential (Vm) and increased the cell membrane input conductance. In a low Cl? solution, this ATP-induced depolarization was enhanced. These effects were inhibited by 1 mmol l?1SITS, consistent with the activation of a basolateral Cl? conductance. The ATP-induced change in Vm was reproduced by ADP but not by UTP or adenosine, and was prevented by suramin. The ATP-induced membrane depolarization was not influenced by thapsigargin, BAPTA AM, or staurosporin and was not reproduced by manoeuvres increasing [Ca2+]i or intracellular cAMP content. We conclude that, in Necturus PCT, a P2y receptor mobilizes Ca2+ mainly from intracellular pools and increases a basolateral Cl? conductance, GCl. The activation of GCl occurs by a mechanism which is not related either to an increase in [Ca2+]i or cAMP content, or to PKC activation. PMID:9706002

Bouyer, Patrice; Paulais, Marc; Cougnon, Marc; Hulin, Philippe; Anagnostopoulos, Takis; Planelles, Gabrielle

1998-01-01

251

Molecular characterization and serological reactivity of a vacuolar ATP synthase subunit ?-like protein from Clonorchis sinensis.  

PubMed

The vacuolar ATPase enzyme complex (V-ATPase) pumps protons across membranes, energized by hydrolysis of ATP. Extensive investigations on structural and biochemical features of these molecules have implied their importance in the physiological process. In this study, a full-length sequence encoding a vacuolar ATP synthase subunit ?-like protein of Clonorchis sinensis (CsATP-?) was isolated from our cDNA library. The hypothetical 226 amino acid sequence shared 76% identity with ATP-? proteins of Schistosoma japonicum and above 55% identity with ATP-? proteins from human and other eukaryotes. Characteristic Asp??? amino acid residues and seven B-cell epitopes were predicted in this sequence. The complete coding sequence of the gene was expressed in Escherichia coli. Recombinant CsATP-? (rCsATP-?) protein could be probed by anti-rCsATP-? rat serum and C.sinensis-infected human serum in Western blotting experiment, indicating that it is an antigen of strong antigenicity. The high level of antibody titers (1:204,800) showed that CsATP-? has a powerful immunogenicity. Both the increased level and the change trend of IgG1/IgG2a subtypes in serum showed that the rCsATP-? can induce strong combined Th1/Th2 immune responses in rats and stimulate the immune response changes to the dominant Th2 from Th1 along with long time infection. The results of immunoblot and immunolocalization demonstrated that CsATP-? was consecutively expressed at various developmental stages of the parasite, which was supported by real-time PCR analysis. In immunohistochemistry, CsATP-? was localized on the intestine, vitellarium, and testicle of an adult worm and excretory bladder of metacercaria, implying that CsATP-? may relate to energy intake and metabolism. This fundamental study would contribute to further researches that are related to growth and development and immunomodulation of C. sinensis. PMID:24535733

Lv, Xiaoli; Huang, Lisi; Chen, Wenjun; Wang, Xiaoyun; Huang, Yan; Deng, Chuanhuan; Sun, Jiufeng; Tian, Yanli; Mao, Qiang; Lei, Huali; Yu, Xinbing

2014-04-01

252

Bacterial RTX toxins allow acute ATP release from human erythrocytes directly through the toxin pore.  

PubMed

ATP is as an extracellular signaling molecule able to amplify the cell lysis inflicted by certain bacterial toxins including the two RTX toxins ?-hemolysin (HlyA) from Escherichia coli and leukotoxin A (LtxA) from Aggregatibacter actinomycetemcomitans. Inhibition of P2X receptors completely blocks the RTX toxin-induced hemolysis over a larger concentration range. It is, however, at present not known how the ATP that provides the amplification is released from the attacked cells. Here we show that both HlyA and LtxA trigger acute release of ATP from human erythrocytes that preceded and were not caused by cell lysis. This early ATP release did not occur via previously described ATP-release pathways in the erythrocyte. Both HlyA and LtxA were capable of triggering ATP release in the presence of the pannexin 1 blockers carbenoxolone and probenecid, and the HlyA-induced ATP release was found to be similar in erythrocytes from pannexin 1 wild type and knock-out mice. Moreover, the voltage-dependent anion channel antagonist TRO19622 had no effect on ATP release by either of the toxins. Finally, we showed that both HlyA and LtxA were able to release ATP from ATP-loaded lipid (1-palmitoyl-2-oleoyl-phosphatidylcholine) vesicles devoid of any erythrocyte channels or transporters. Again we were able to show that this happened in a non-lytic fashion, using calcein-containing vesicles as controls. These data show that both toxins incorporate into lipid vesicles and allow ATP to be released. We suggest that both toxins cause acute ATP release by letting ATP pass the toxin pores in both human erythrocytes and artificial membranes. PMID:24860098

Skals, Marianne; Bjaelde, Randi G; Reinholdt, Jesper; Poulsen, Knud; Vad, Brian S; Otzen, Daniel E; Leipziger, Jens; Praetorius, Helle A

2014-07-01

253

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

PubMed

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

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

2014-02-01

254

Elevated Pressure Triggers a Physiological Release of ATP from the Retina: Possible Role for Pannexin Hemichannels  

PubMed Central

Increased hydrostatic pressure can damage neurons, although the mechanisms linking pressure to neurochemical imbalance or cell injury are not fully established. Throughout the body, mechanical perturbations such as shear stress, cell stretching, or changes in pressure can lead to excessive release of ATP. It is thus possible that increased pressure across neural tissues triggers an elevated release of ATP into extracellular space. As stimulation of the P2X7 receptor for ATP on retinal ganglion cells leads to elevation of intracellular calcium and excitotoxic death, we asked whether increased levels of extracellular ATP accompanied an elevation in pressure across the retina. The hydrostatic pressure surrounding bovine retinal eyecups was increased and the ATP content of the vitreal compartment adjacent to the retina was determined. A step increase of only 20 mmHg induced a three-fold increase in the vitreal ATP concentration. The ATP levels correlated closely with the degree of pressure increase over 20–100 mmHg range. The increase was transient at lower pressures but sustained at higher pressures. The rise in vitreal ATP was the same regardless of whether nitrogen or air was used to increase pressure, implying changes in oxygen partial pressure did not contribute. Lactate dehydrogenase activity was not affected by pressure, ruling out a substantial contribution from cell lysis. The ATP increase was largely inhibited by either 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) or carbenoxolone (CBX). While this is consistent with physiological release of ATP through pannexins hemichannels, a contribution from anion channels, vesicular release or other mechanisms cannot be ruled out. In conclusion, a step elevation in pressure leads to a physiologic increase in the levels of extracellular ATP bathing retinal neurons. This excess extracellular ATP may link increased pressure to the death of ganglion cells in acute glaucoma, and suggests a role for ATP in the neuronal damage accompanying increased intracranial pressure. PMID:18822352

Reigada, David; Lu, Wennan; Zhang, May; Mitchell, Claire H.

2008-01-01

255

Phosphate binding and ATP-binding sites coexist in Na+/K(+)-transporting ATPase, as demonstrated by the inactivating MgPO4 complex analogue Co(NH3)4PO4.  

PubMed

Tetrammine cobalt(III) phosphate [Co(NH3)4PO4] inactivates Na+/K(+)-ATPase in the E2 conformational state, dependent on time and concentration, according to Eqn (1): Co(NH3)4PO4 + E2 Kd in equilibrium E2.Co(NH3)4PO4k2----E'2.Co(NH3)4PO4. The inactivation rate constant k2 for the formation of a stable E'2.Co(NH3)4PO4 at 37 degrees C was 0.057 min-1; the dissociation constant, Kd = 300 microM. The activation energy for the inactivation process was 149 kJ/mol. ATP and the uncleavable adenosine 5'-[beta, gamma-methylene]triphosphate competed with Co(NH3)4PO4 for its binding site with Ks = 0.41 mM and 5 mM, respectively. MgPO4 competed with Co(NH3)4PO4 linearly, with Ks = 50 microM, as did phosphate (Ks = 16 mM) and Mg2+ (Ks = 160 microM). It is concluded that the MgPO4 analogue binds to the MgPO4-binding subsite of the low-affinity ATP-binding site (of the E2 conformation). Also, Na+ (Ks = 860 microM) protected the enzyme against inactivation in a competitive manner. From the intersecting (slope and intercept linear) noncompetitive effect of Na+ against the inactivation by Co(NH3)4PO4, apparent affinities of K+ for the free enzyme of 41 microM, and for the E.Co(NH3)4PO4 complex of 720 microM, were calculated. Binding of Co(NH3)4PO4 to the enzyme inactivated Na+/K(+)-ATPase and K(+)-activated phosphatase, and, moreover, prevented the occlusion of 86Rb+; however, the activity of the Na(+)-ATPase, the phosphorylation capacity of the high-affinity ATP-binding site and the ATP/ADP-exchange reaction remained unchanged. With Co(NH3)432PO4 a binding capacity of 135 pmol unit enzyme was found. Phosphorylation and complete inactivation of the enzyme with Co(NH3)432PO4 or the 32P-labelled tetramminecobalt ATP ([gamma-32P]Co(NH3)4ATP) at the low-affinity ATP-binding site, allowed (independent of the purity of the Na+/K(+)-ATPase preparation) a further incorporation of radioactivity from 32P-labelled tetraaquachromium(III) ATP ([gamma-32P]CrATP) to the high-affinity ATP-binding site with unchanged phosphorylation capacity. However, inactivation and phosphorylation of Na+/K(+)-ATPase by [gamma-32P]CrATP prevented the binding of Co(NH3)4 32PO4 or [gamma-32P]Co(NH3)4ATP to the enzyme. [gamma-32P]CO(NH3)4ATP and Co(NH3)432PO4 are mutually exclusive. The data are consistent with the assumption of a cooperation of catalytic subunits within an (alpha,beta)2-diprotomer, which change their interactions during the Na+/K(+)-pumping process. Our findings seem not to support a symmetrical Repke and Stein model of enzyme action. PMID:1847680

Buxbaum, E; Schoner, W

1991-01-30

256

The progressive ankylosis gene product ANK regulates extracellular ATP levels in primary articular chondrocytes  

PubMed Central

Introduction Extracellular ATP (eATP) is released by articular chondrocytes under physiological and pathological conditions. High eATP levels cause pathologic calcification, damage cartilage, and mediate pain. We recently showed that stable over-expression of the progressive ankylosis gene product, ANK, increased chondrocyte eATP levels, but the mechanisms of this effect remained unexplored. The purpose of this work was to further investigate mechanisms of eATP efflux in primary articular chondrocytes and to better define the role of ANK in this process. Methods We measured eATP levels using a bioluminescence-based assay in adult porcine articular chondrocyte media with or without a 10 minute exposure to hypotonic stress. siRNAs for known ATP membrane transporters and pharmacologic inhibitors of ATP egress pathways were used to identify participants involved in chondrocyte eATP release. Results eATP levels increased after exposure to hypotonic media in a calcium-dependent manner in monolayer and 3-dimensional agarose gel cultures (p < 0.001). A potent transient receptor potential vanilloid 4 (TRPV4) agonist mimicked the effects of hypotonic media. ANK siRNA suppressed basal (p < 0.01) and hypotonically-stressed (p < 0.001) ATP levels. This effect was not mediated by altered extracellular pyrophosphate (ePPi) levels, and was mimicked by the ANK inhibitor, probenecid (p < 0.001). The P2X7/4 receptor inhibitor Brilliant Blue G also suppressed eATP efflux induced by hypotonic media (p < 0.001), while ivermectin, a P2X4 receptor stimulant, increased eATP levels (p < 0.001). Pharmacologic inhibitors of hemichannels, maxianion channels and other volume-sensitive eATP efflux pathways did not suppress eATP levels. Conclusions These findings implicate ANK and P2X7/4 receptors in chondrocyte eATP efflux. Understanding the mechanisms of eATP efflux may result in novel therapies for calcium crystal arthritis and osteoarthritis. PMID:24286344

2013-01-01

257

Extracellular ATP promotes stomatal opening of Arabidopsis thaliana through heterotrimeric G protein ? subunit and reactive oxygen species.  

PubMed

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

Hao, Li-Hua; Wang, Wei-Xia; Chen, Chen; Wang, Yu-Fang; Liu, Ting; Li, Xia; Shang, Zhong-Lin

2012-07-01

258

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

PubMed Central

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

Fields, R. Douglas

2011-01-01

259

Structure guided simulations illuminate the mechanism of ATP transport through VDAC1  

PubMed Central

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

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

2014-01-01

260

Definitions of dwelling  

E-print Network

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 ...

Olgyay, Victor W. (Victor Wayne)

1986-01-01

261

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

PubMed Central

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

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

2010-01-01

262

Functional characterization of a gene encoding a fourth ATP sulfurylase isoform from Arabidopsis thaliana  

Microsoft Academic Search

ATP sulfurylase (ATP: sulfate adenylyl transferase, EC 2.7.7.4), the first enzyme of the sulfate assimilation pathway, is present in the chloroplast and cytosol of plants. In Arabidopsis thaliana cDNA cloning revealed the existence of three ATP sulfurylase isoforms (APS1, -2, and -3) all of which appear to be localized in plastids. In the present study the cytosolic isoform was sought

Yves Hatzfeld; Sangman Lee; Minsang Lee; Thomas Leustek; Kazuki Saito

2000-01-01

263

ATP hydrolysis is required for DEAD-box protein recycling but not for duplex unwinding  

Microsoft Academic Search

DEAD-box proteins, the largest helicase family, catalyze ATP-dependent remodeling of RNA-protein complexes and the unwinding of RNA duplexes. Because DEAD-box proteins hydrolyze ATP in an RNA-dependent fashion, the energy provided by ATP hydrolysis is commonly assumed to drive the energetically unfavorable duplex unwinding. Here, we show efficient unwinding of stable duplexes by several DEAD-box proteins in the presence of the

Fei Liu; Andrea Putnam; Eckhard Jankowsky

2008-01-01

264

ATP release in human kidney cortex and its mitogenic effects in visceral glomerular epithelial cells  

Microsoft Academic Search

ATP release in human kidney cortex and its mitogenic effects in visceral glomerular epithelial cells.BackgroundIn chronic renal failure the sympathetic nervous system is activated. Sympathetic cotransmitters released within the kidney may contribute to the progression of renal disease through receptor-mediated proliferative mechanisms.MethodsIn human renal cortex electrical stimulation induced adenosine 5?-triphosphate (ATP; luciferin-luciferase-assay) and norepinephrine (HPLC) release was measured. ATP release

Oliver Vonend; Vitus Oberhauser; Ivar Von Kügelgen; Thomas W. Apel; Kerstin Amann; Eberhard Ritz; Lars Christian Rump

2002-01-01

265

Bioluminescent assay of microbial ATP in postmortem tissues for the estimation of postmortem interval  

Microsoft Academic Search

Summary  To study the relationship between changes of microbial ATP in four kinds of murine tissues and the postmortem interval (PMI),\\u000a healthy SD rats were sacrificed and their muscles, livers, spleens and kidneys were sampled at different postmortem intervals.\\u000a The concentration of microbial ATP was detected using bioluminescent assay and the data was statistically analyzed. The concentration\\u000a of microbial ATP in

Qian Liu; Qing Sun; Yan Liu; Lan Zhou; Na Zheng; Liang Liu

2009-01-01

266

Redox Regulation of Mitochondrial ATP synthase: Implications for Cardiac Resynchronization Therapy  

PubMed Central

Rationale Cardiac resynchronization therapy (CRT) is an effective clinical treatment for heart failure patients with conduction delay, impaired contraction and energetics. Our recent studies have revealed that mitochondrial post-translational modifications (PTM) may contribute to its benefits, motivating the present study of the oxidative regulation of mitochondrial ATP synthase. Objectives Here, we tested whether CRT alteration of ATP synthase function is linked to cysteine (Cys) oxidative PTM (Ox-PTM) of specific ATP synthase subunits. Methods and Results Canine left ventricular myocardium was collected under conditions to preserve Ox-PTM from control, dyssynchronous heart failure (DHF) or hearts that have undergone CRT. In-gel ATPase activity showed that CRT increased ATPase activity by ~2 fold (p<0.05) over DHF, approaching control levels and this effect was recapitulated with a reducing agent. ATP synthase function and three Ox-PTM: disulfide bond, S-glutathionylation and S-nitrosation were assessed. ATP synthase from DHF hearts contained two novel disulfide bonds, between ATP synthase alpha subunits themselves and between alpha and gamma subunits, both of which were decreased in CRT hearts (4.38±0.13 and 4.23±0.36-fold, respectively, p<0.01). S-glutathionylation of ATP synthase alpha subunit occurred in DHF hearts and was reversed by CRT (1.56±0.16-fold, p<0.04). In contrast, S-nitrosation of ATP synthase alpha subunit in DHF hearts was lower than in CRT hearts (1.53±0.19-fold, p<0.05). All modifications occurred at ATP synthase alpha subunit Cys294 and Cys to Ser mutation indicated that this residue is critical for ATP synthase function. Conclusions A selective Cys in ATP synthase alpha subunit is targeted by multiple Ox-PTM suggesting that this Cys residue may act as a redox sensor modulating ATP synthase function. PMID:21817160

Wang, Sheng-Bing; Foster, D. Brian; Rucker, Jasma; O’Rourke, Brian; Kass, David A.; Van Eyk, Jennifer E.

2012-01-01

267

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

SciTech Connect

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.

Masker, W.E.

1982-07-01

268

Docked granules, the exocytic burst, and the need for ATP hydrolysis in endocrine cells  

Microsoft Academic Search

Ca2+-triggered exocytosis was studied in single rat melanotrophs and bovine chromaffin cells by capacitance measurements. Sustained exocytosis required MgATP, but even in the absence of MgATP, Ca2+ could trigger exocytosis of 2700 granules in a typical melanotroph and of 840 granules in a chromaffin cell. Granules undergoing ATP-independent exocytosis were similar in number to those appearing docked to the plasmalemma

T. D. Parsons; J. R. Coorssen; H. Horstmann; W. Almers

1995-01-01

269

Potentiation of inhibitory synaptic transmission by extracellular ATP in rat suprachiasmatic nuclei.  

PubMed

The hypothalamic suprachiasmatic nuclei (SCN), the circadian master clock in mammals, releases ATP in a rhythm, but the role of extracellular ATP in the SCN is still unknown. In this study, we examined the expression and function of ATP-gated P2X receptors (P2XRs) in the SCN neurons of slices isolated from the brain of 16- to 20-day-old rats. Quantitative RT-PCR showed that the SCN contains mRNA for P2X 1-7 receptors and several G-protein-coupled P2Y receptors. Among the P2XR subunits, the P2X2 > P2X7 > P2X4 mRNAs were the most abundant. Whole-cell patch-clamp recordings from SCN neurons revealed that extracellular ATP application increased the frequency of spontaneous GABAergic IPSCs without changes in their amplitudes. The effect of ATP appears to be mediated by presynaptic P2X2Rs because ATP?S and 2MeS-ATP mimics, while the P2XR antagonist PPADS blocks, the observed enhancement of the frequency of GABA currents. There were significant differences between two SCN regions in that the effect of ATP was higher in the ventrolateral subdivision, which is densely innervated from outside the SCN. Little evidence was found for the presence of P2XR channels in somata of SCN neurons as P2X2R immunoreactivity colocalized with synapsin and ATP-induced current was observed in only 7% of cells. In fura-2 AM-loaded slices, BzATP as well as ADP stimulated intracellular Ca(2+) increase, indicating that the SCN cells express functional P2X7 and P2Y receptors. Our data suggest that ATP activates presynaptic P2X2Rs to regulate inhibitory synaptic transmission within the SCN and that this effect varies between regions. PMID:23637193

Bhattacharya, Anirban; Vavra, Vojtech; Svobodova, Irena; Bendova, Zdena; Vereb, Gyorgy; Zemkova, Hana

2013-05-01

270

A definition of dyslexia  

Microsoft Academic Search

This paper elaborates on the components of a working definition of developmental dyslexia. It follows the general format of\\u000a a paper by Lyon published in Annals of Dyslexia in 1995, which elaborated on a working definition proposed in 1994 (Lyon,\\u000a 1995). The current definition agreed on by the work group updates and expands on the working definition from 1994.

G. Reid Lyon; Sally E. Shaywitz; Bennett A. Shaywitz

2003-01-01

271

Functional production of the Na+ F1F(O) ATP synthase from Acetobacterium woodii in Escherichia coli requires the native AtpI.  

PubMed

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

Brandt, Karsten; Müller, Daniel B; Hoffmann, Jan; Hübert, Christine; Brutschy, Bernd; Deckers-Hebestreit, Gabriele; Müller, Volker

2013-02-01

272

Effect of ATP Sulfurylase Overexpression in Bright Yellow 2 Tobacco Cells1  

PubMed Central

To determine if the ATP sulfurylase reaction is a regulatory step for the SO42?-assimilation pathway in plants, an Arabidopsis thaliana ATP sulfurylase cDNA, APS2, was fused to the 35S promoter of the cauliflower mosaic virus and introduced by Agrobacterium tumefaciens-mediated transformation into isolated Bright Yellow 2 tobacco (Nicotiana tabacum) cells. The ATP sulfurylase activity in transgenic cells was 8-fold that in control cells, and was correlated with the expression of a specific polypeptide revealed by western analysis using an anti-ATP sulfurylase antibody. The molecular mass of this polypeptide agreed with that for the overexpressed mature protein. ATP sulfurylase overexpression had no effect on [35S]SO42? influx or ATP sulfurylase activity regulation by S availability, except that ATP sulfurylase activity variations in response to S starvation in transgenic cells were 8 times higher than in the wild type. There were also no differences in cell growth or sensitivity to SeO42? (a toxic SO42? analog) between transgenic and wild-type cells. We propose that in Bright Yellow 2 tobacco cells, the ATP sulfurylase derepression by S deficiency may involve a posttranscriptional mechanism, and that the ATP sulfurylase abundance is not limiting for cell metabolism. PMID:9536047

Hatzfeld, Yves; Cathala, Nicole; Grignon, Claude; Davidian, Jean-Claude

1998-01-01

273

Mechanisms of ATP release and signalling in the blood vessel wall  

PubMed Central

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

Lohman, Alexander W.; Billaud, Marie; Isakson, Brant E.

2012-01-01

274

Red blood cell ATP/ADP & nitric oxide: The best vasodilators in diabetic patients  

PubMed Central

Background Diabetes mellitus is a group of metabolic diseases characterized by high blood sugar (glucose) levels that result from defects in insulin secretion, or action, or both. Inspired by previous report the release of ATP from RBCs, which may participate in vessel dilation by stimulating NO production in the endothelium through purinergic receptor signaling and so, the aim of this study is to clearly determined relationship between RBC ATP/ADP ratio with nitric oxide. Methods The ATP/ADP ratio of erythrocytes among four groups of normal individuals (young & middle age), athletes’ subjects and diabetic patients were compared and the relationship between ATP/ADP ratio and NO level of plasma was determined with AVOVA test and bioluminescence method. Results ATP/ADP level in four groups normal (young & middle age), athletes, diabetes] are measured and analyzed with ANOVA test that show a significant difference between groups (P-value < 0.001). A significant positive correlation was found between RBC ATP/ADP content (r = 0.705; P < 0.001). Plasma NO content is also analyzed with ANOVA test which shows a significant difference between groups. Conclusion In this study, a positive relationship between RBC ATP/ADP ratio and NO was found. Based on the obtained result, higher RBC ATP/ADP content may control the ratio of plasma NO in different individuals, also this results show that ATP can activate endothelial cells in NO production and is a main factor in releasing of NO from endothelial cells. PMID:23497445

2012-01-01

275

Role of connexin 32 hemichannels in the release of ATP from peripheral nerves.  

PubMed

Extracellular purines elicit strong signals in the nervous system. Adenosine-5'-triphosphate (ATP) does not spontaneously cross the plasma membrane, and nervous cells secrete ATP by exocytosis or through plasma membrane proteins such as connexin hemichannels. Using a combination of imaging, luminescence and electrophysiological techniques, we explored the possibility that Connexin 32 (Cx32), expressed in Schwann cells (SCs) myelinating the peripheral nervous system could be an important source of ATP in peripheral nerves. We triggered the release of ATP in vivo from mice sciatic nerves by electrical stimulation and from cultured SCs by high extracellular potassium concentration-evoked depolarization. No ATP was detected in the extracellular media after treatment of the sciatic nerve with Octanol or Carbenoxolone, and ATP release was significantly inhibited after silencing Cx32 from SCs cultures. We investigated the permeability of Cx32 to ATP by expressing Cx32 hemichannels in Xenopus laevis oocytes. We found that ATP release is coupled to the inward tail current generated after the activation of Cx32 hemichannels by depolarization pulses, and it is sensitive to low extracellular calcium concentrations. Moreover, we found altered ATP release in mutated Cx32 hemichannels related to the X-linked form of Charcot-Marie-Tooth disease, suggesting that purinergic-mediated signaling in peripheral nerves could underlie the physiopathology of this neuropathy. PMID:24123415

Nualart-Marti, Anna; del Molino, Ezequiel Mas; Grandes, Xènia; Bahima, Laia; Martin-Satué, Mireia; Puchal, Rafel; Fasciani, Ilaria; González-Nieto, Daniel; Ziganshin, Bulat; Llobet, Artur; Barrio, Luis C; Solsona, Carles

2013-12-01

276

Testing for violations of microscopic reversibility in ATP-sensitive potassium channel gating.  

PubMed

In pancreatic beta cells, insulin secretion is tightly controlled by the cells' metabolic state via the ATP-sensitive potassium (KATP) channel. ATP is a key mediator in this signaling process, where its role as an inhibitor of KATP channels has been extensively studied. Since the channel contains an ATPase as an accessory subunit, the possibility that ATP hydrolysis mediates KATP channel opening has also been proposed. However, a rigorous test of coupling between ATP hydrolysis and channel gating has not previously been performed. In the present work, we examine whether KATP channel gating obeys detailed balance in order to determine whether ATP hydrolysis is strongly coupled to the gating of the KATP channel. Single-channel records were obtained from inside-out patches of transiently transfected HEK-293 cells. Channel activity in membrane patches with exactly one channel shows no violations of microscopic reversibility. Although KATP channel gating shows long closed times on the time scale where ATP hydrolysis takes place, the time symmetry of channel gating indicates that it is not tightly coupled to ATP hydrolysis. This lack of coupling suggests that channel gating operates close to equilibrium; although detailed balance is not expected to hold for ATP hydrolysis, it still does so in channel gating. On the basis of these results, the function of the ATPase active site in channel gating may be to sense nucleotides by differential binding of ATP and ADP, rather than to drive a thermodynamically unfavorable conformational change. PMID:18661924

Choi, Kee-Hyun; Tantama, Mathew; Licht, Stuart

2008-08-21

277

Measurement of ATP-Induced Membrane Potential Changes in IVD cells.  

PubMed

Extracellular adenosine-5'-triphosphate (ATP) triggers biological responses in a wide variety of cells and tissues and activates signaling cascades that affect cell membrane potential and excitability. It has been demonstrated that compressive loading promotes ATP production and release by intervertebral disc (IVD) cells, while a high level of extracellular ATP accumulates in the nucleus pulposus (NP) of the IVD. In this study, a noninvasive system was developed to measure ATP-induced changes in the membrane potential of porcine IVD cells using the potential sensitive dye di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS).The responses of NP and annulus fibrosus (AF) cells to ATP were examined in monolayer and 3-dimensional cultures. It was found that the pattern and magnitude of membrane potential change in IVD cells induced by extracellular ATP depended on cell type, culture condition, and ATP dose. In addition, gene expression of P2X4 purinergic receptor was found in both cell types. Inhibition of the ATP-induced response by pyridoxalphosphate-6-azophenyl-2', 4'-disulfonate (PPADS), a non-competitive inhibitor of P2 receptors, suggests that ATP may modulate the biological activities of IVD cells via P2 purinergic receptors. PMID:25386223

Gonzales, Silvia; Rodriguez, Brittany; Barrera, Carlos; Huang, Chun-Yuh Charles

2014-12-01

278

ATP-responsive controlled release system using aptamer-functionalized mesoporous silica nanoparticles.  

PubMed

Adenosine-5'-triphosphate (ATP) is a multifunctional nucleotide, which plays a vital role in many biological processes, including muscle contraction, cells functioning, synthesis and degradation of important cellular compounds, and membrane transport. Thus, the development of ATP-responsive controlled release system for bioorganism application is very significative. Here, an original and facile ATP-responsive controlled release system consisting of mesoporous silica nanoparticles (MSN) functionalized with an aptamer as cap has been designed. In this system, the ATP aptamer was first hybridized with arm single-stranded DNA1 (arm ssDNA1) and arm single-stranded DNA2 (arm ssDNA2) to form the sandwich-type DNA structure and then grafted onto the MSN surface through click chemistry approach, resulting in blockage of pores and inhibition of guest molecules release. In the presence of ATP, the ATP aptamer combined with ATP and got away from the pore, leaving the arm ssDNA1 and ssDNA2 on the surface of MSN. The guest molecules can be released because single-stranded DNA is flexible. The release of the guest molecules from this system then can be triggered by the addition of ATP. As a proof-of-principle, Ru(bipy)(3)(2+) was selected as the guest molecules, and the ATP-responsive loading and release of Ru(bipy)(3)(2+) have been investigated. The results demonstrate that the system had excellent loading efficiency (215.0 ?mol g(-1) SiO(2)) and the dye release percentage can reach 83.2% after treatment with 20 mM ATP for 7 h. Moreover, the ATP-responsive behavior shows high selectivity with ATP analogues. However, the leakage of Ru(bipy)(3)(2+) molecule is neglectable if ATP was not added, indicating an excellent capping efficiency. Interestingly, this system can respond not only to the commercial ATP but also to the ATP extracted from living cells. By the way, this system is also relatively stable in mouse serum solution at 37 °C. This proof of concept might promote the application of ATP-responsive devices and can also provide an idea to design various target-responsive systems using other aptamers as cap. PMID:22889263

He, Xiaoxiao; Zhao, Yingxiang; He, Dinggeng; Wang, Kemin; Xu, Fengzhou; Tang, Jinlu

2012-09-01

279

One Rotary Mechanism for F1-ATPase over ATP Concentrations from Millimolar down to Nanomolar  

PubMed Central

F1-ATPase is a rotary molecular motor in which the central ?-subunit rotates inside a cylinder made of ?3?3-subunits. The rotation is driven by ATP hydrolysis in three catalytic sites on the ?-subunits. How many of the three catalytic sites are filled with a nucleotide during the course of rotation is an important yet unsettled question. Here we inquire whether F1 rotates at extremely low ATP concentrations where the site occupancy is expected to be low. We observed under an optical microscope rotation of individual F1 molecules that carried a bead duplex on the ?-subunit. Time-averaged rotation rate was proportional to the ATP concentration down to 200 pM, giving an apparent rate constant for ATP binding of 2 × 107 M?1s?1. A similar rate constant characterized bulk ATP hydrolysis in solution, which obeyed a simple Michaelis-Menten scheme between 6 mM and 60 nM ATP. F1 produced the same torque of ?40 pN·nm at 2 mM, 60 nM, and 2 nM ATP. These results point to one rotary mechanism governing the entire range of nanomolar to millimolar ATP, although a switchover between two mechanisms cannot be dismissed. Below 1 nM ATP, we observed less regular rotations, indicative of the appearance of another reaction scheme. PMID:15626703

Sakaki, Naoyoshi; Shimo-Kon, Rieko; Adachi, Kengo; Itoh, Hiroyasu; Furuike, Shou; Muneyuki, Eiro; Yoshida, Masasuke; Kinosita, Kazuhiko

2005-01-01

280

Peroxisomal ATP Import Is Essential for Seedling Development in Arabidopsis thaliana[W  

PubMed Central

Several recent proteomic studies of plant peroxisomes indicate that the peroxisomal matrix harbors multiple ATP-dependent enzymes and chaperones. However, it is unknown whether plant peroxisomes are able to produce ATP by substrate-level phosphorylation or whether external ATP fuels the energy-dependent reactions within peroxisomes. The existence of transport proteins that supply plant peroxisomes with energy for fatty acid oxidation and other ATP-dependent processes has not previously been demonstrated. Here, we describe two Arabidopsis thaliana genes that encode peroxisomal adenine nucleotide carriers, PNC1 and PNC2. Both proteins, when fused to enhanced yellow fluorescent protein, are targeted to peroxisomes. Complementation of a yeast mutant deficient in peroxisomal ATP import and in vitro transport assays using recombinant transporter proteins revealed that PNC1 and PNC2 catalyze the counterexchange of ATP with ADP or AMP. Transgenic Arabidopsis lines repressing both PNC genes were generated using ethanol-inducible RNA interference. A detailed analysis of these plants showed that an impaired peroxisomal ATP import inhibits fatty acid breakdown during early seedling growth and other ?-oxidation reactions, such as auxin biosynthesis. We show conclusively that PNC1 and PNC2 are essential for supplying peroxisomes with ATP, indicating that no other ATP generating systems exist inside plant peroxisomes. PMID:19073763

Linka, Nicole; Theodoulou, Frederica L.; Haslam, Richard P.; Linka, Marc; Napier, Jonathan A.; Neuhaus, H. Ekkehard; Weber, Andreas P.M.

2008-01-01

281

Mechanism of an ATP-independent Protein Disaggregase  

PubMed Central

The ability of molecular chaperones to overcome the misfolding and aggregation of proteins is essential for the maintenance of proper protein homeostasis in all cells. Thus far, the best studied disaggregase systems are the Clp/Hsp100 family of “ATPases associated with various cellular activities” (AAA+) ATPases, which use mechanical forces powered by ATP hydrolysis to remodel protein aggregates. An alternative system to disassemble large protein aggregates is provided by the 38-kDa subunit of the chloroplast signal recognition particle (cpSRP43), which uses binding energy with its substrate proteins to drive disaggregation. The mechanism of this novel chaperone remains unclear. Here, molecular genetics and structure-activity analyses show that the action of cpSRP43 can be dissected into two steps with distinct molecular requirements: (i) initial recognition, during which cpSRP43 binds specifically to a recognition motif displayed on the surface of the aggregate; and (ii) aggregate remodeling, during which highly adaptable binding interactions of cpSRP43 with hydrophobic transmembrane domains of the substrate protein compete with the packing interactions within the aggregate. This establishes a useful framework to understand the molecular mechanism by which binding interactions from a molecular chaperone can be used to overcome protein aggregates in the absence of external energy input from ATP. PMID:23519468

Jaru-Ampornpan, Peera; Liang, Fu-Cheng; Nisthal, Alex; Nguyen, Thang X.; Wang, Pengcheng; Shen, Kuang; Mayo, Steven L.; Shan, Shu-ou

2013-01-01

282

Mechanosensitive ATP Release Maintains Proper Mucus Hydration of Airways  

PubMed Central

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

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

2013-01-01

283

Catalytic mechanism of bacteriophage T4 Rad50 ATP hydrolysis.  

PubMed

Spontaneous double-strand breaks (DSBs) are one of the most deleterious forms of DNA damage, and their improper repair can lead to cellular dysfunction. The Mre11 and Rad50 proteins, a nuclease and an ATPase, respectively, form a well-conserved complex that is involved in the initial processing of DSBs. Here we examine the kinetic and catalytic mechanism of ATP hydrolysis by T4 Rad50 (gp46) in the presence and absence of Mre11 (gp47) and DNA. Single-turnover and pre-steady state kinetics on the wild-type protein indicate that the rate-limiting step for Rad50, the MR complex, and the MR-DNA complex is either chemistry or a conformational change prior to catalysis. Pre-steady state product release kinetics, coupled with viscosity steady state kinetics, also supports that the binding of DNA to the MR complex does not alter the rate-limiting step. The lack of a positive deuterium solvent isotope effect for the wild type and several active site mutants, combined with pH-rate profiles, implies that chemistry is rate-limiting and the ATPase mechanism proceeds via an asymmetric, dissociative-like transition state. Mutation of the Walker A/B and H-loop residues also affects the allosteric communication between Rad50 active sites, suggesting possible routes for cooperativity between the ATP active sites. PMID:25137526

Herdendorf, Timothy J; Nelson, Scott W

2014-09-01

284

Efficacy and Limitations of an ATP-Based Monitoring System  

PubMed Central

Monitoring of sanitation is an essential function of laboratory animal facilities. The purpose of the current study was to assess the ability of an ATP-based system to detect microbes and organic contaminants. Serial dilutions of Escherichia coli, Staphylococcus aureus, Toxocara canis eggs, Toxoplasma gondii tachyzoites, epithelial cells, and rodent blood, urine, and feces were analyzed according to the manufacturer's recommendations. The limit of E. coli detection was 104 organisms; sonication of E. coli significantly improved detection, indicating incomplete bacterial lysis in the detection system. Detection of S. aureus was significantly greater than that of E. coli with a limit of detection of 102; sonication did not alter results. In contrast, detection of T. canis, T. gondii, RBC, and epithelial cells was robust and ranged from 2 T. canis eggs to 10 epithelial cells. Urine was weakly detected, with a limit of detection at 1:10 dilution. Detection of all cell types except epithelia had a strong linear correlation to total cell number. In addition, our data demonstrate that the efficacy of the detection system can be affected adversely by residual disinfectants and that sample-bearing swabs are stable for more than 7 h after swabbing. These data demonstrate that this ATP based system sensitively detects pure cells and organic contaminants with a strong degree of linear predictability. A limitation of the system is its inability to detect gram-negative bacteria efficiently because of incomplete cell lysis. PMID:20353694

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

2010-01-01

285

Fragments of ATP synthase mediate plant perception of insect attack  

PubMed Central

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

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

2006-01-01

286

The a subunit asymmetry dictates the two opposite rotation directions in the synthesis and hydrolysis of ATP by the mitochondrial ATP synthase.  

PubMed

The main and best known role of the mitochondrial ATP synthase is to synthesize ATP by exploiting the transmembrane electrochemical gradient of protons and their downhill movement. However, under different conditions, the same enzyme can also switch to the opposite function of ATP hydrolysis and exploits its energy to pump protons against their gradient and energize the membrane. The change in functionality is linked to the change of direction of rotation of the two matched sectors of this unique complex, namely the hydrophilic F1, which performs the catalysis, and the hydrophobic membrane-embedded FO, which channels protons. Accordingly, viewed from the matrix side, ATP synthesis is driven by counterclockwise rotation and ATP hydrolysis by clockwise rotation of the FO rotor which is transmitted to F1. ATP dissipation through this mechanism features some diseases such as myocardial ischemia. Increasing evidence shoulders the hypothesis that the asymmetry of the a subunit of FO and particularly the steric arrangement of the two inner semi-channels for protons, play a key role in conferring to the coupled bi-functional complex the ability to reverse rotation by switching from ATP synthesis to ATP hydrolysis and vice versa. Accordingly, the conserved steric arrangement of the chiral a subunit of FO yields the same direction of rotation for all the ATP synthases. According to this hypothesis, the a subunit chirality imposes the direction of rotation of the rotor according to the proton gradient across the membrane. It seems likely that the direction of rotation of the membrane-embedded c-ring, which is adjacent to the a-subunit and acts as a rotor, may be under multiple control, being rotation essential to make the whole enzyme machinery work. However, the asymmetric features of the a subunit would make it the master regulator, thus directly determining which of the two functions, ATP production or ATP dissipation, will be performed. The handedness of a subunit should be considered in drug design to counteract tissue damage under all pathological conditions linked to functional impairment of ATP synthase. PMID:25497387

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

2015-01-01

287

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

PubMed Central

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

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

1996-01-01

288

Activation and labelling of the purified skeletal muscle ryanodine receptor by an oxidized ATP analogue.  

PubMed Central

We have tested the periodate-oxidized ATP analogue 2',3'-dialdehyde adenosine triphosphate (oATP) as a ligand for the skeletal muscle ryanodine receptor/Ca(2+)-release channel. Ca2+ efflux from passively loaded heavy sarcoplasmic reticulum vesicles of skeletal muscle is biphasic. oATP stimulates the initial phase of Ca2+ release in a concentration-dependent manner (EC50 160 microM), and the efflux proceeds with a half-time in the range 100-200 ms. This oATP-modulated initial rapid Ca2+ release was specifically inhibited by millimolar concentrations of Mg2+ and micromolar concentrations of Ruthenium Red, indicating that the effect of oATP was mediated via the ryanodine receptor. The purified Ca(2+)-release channel was incorporated into planar lipid bilayers, and single-channel recordings were carried out to verify a direct interaction of oATP with the ryanodine receptor. Addition of oATP to the cytoplasmic side activated the channel with an EC50 of 76 microM, which is roughly 30-fold higher than the apparent affinity of ATP. The oATP-induced increase in the open probability of the ryanodine receptor displays a steep concentration-response curve with a Hill coefficient of approximately 2, which suggests a co-operativity of the ATP binding sites in the tetrameric protein. oATP binds to the ryanodine receptor in a quasi-irreversible manner via Schiff base formation between the aldehyde groups of oATP and amino groups in the nucleotide binding pocket. This allows for the covalent specific incorporation of [alpha-32P]oATP by borhydride reduction. A typical adenine nucleotide binding site cannot be identified in the primary sequence of the ryanodine receptor. Our results demonstrate that oATP can be used to probe the structure and function of the nucleotide binding pocket of the ryanodine receptor and presumably of other ATP-regulated ion channels. Images Figure 5 PMID:7755553

Hohenegger, M; Herrmann-Frank, A; Richter, M; Lehmann-Horn, F

1995-01-01

289

Sources of intravascular ATP during exercise in humans: critical role for skeletal muscle perfusion.  

PubMed

Exercise hyperaemia is regulated by several factors, and one factor known to increase with exercise that evokes a powerful vasomotor action is extracellular ATP. The origin of ATP detected in plasma from exercising muscle of humans is, however, a matter of debate, and ATP has been suggested to arise from sympathetic nerves, blood sources (e.g. erythrocytes), endothelial cells and skeletal myocytes, among others. Therefore, we tested the hypothesis that acute augmentation of sympathetic nervous system activity (SNA) results in elevated plasma ATP draining skeletal muscle, and that SNA superimposition during exercise increases ATP more than exercise alone. We showed that increased SNA via -40 mmHg lower body negative pressure (LBNP) at rest did not increase plasma ATP (51±8 nmol l(-1) at rest versus 58±7 nmol l(-1) with LBNP), nor did it increase [ATP] above levels observed during rhythmic hand-grip exercise (79±11 nmol l(-1) with exercise alone versus 71±8 nmol l(-1) with LBNP). Next, we tested the hypothesis that active perfusion of skeletal muscle is essential to observe increased plasma ATP during exercise. We showed that complete obstruction of blood flow to contracting muscle abolished exercise-mediated increases in plasma ATP (from 90±19 to 49±12 nmol l(-1)), and that cessation of blood flow prior to exercise completely inhibited the typical rise in ATP (3 versus 61%, obstructed versus intact perfusion). The lack of change in ATP during occlusion occurred in the face of continued muscular work and elevated SNA, indicating that the rise of intravascular ATP did not result from these extravascular sources. Our collective observations indicated that the elevation in extracellular ATP observed in blood during exercise was unlikely to originate from sympathetic nerves or the contacting muscle itself, but rather was dependent on intact skeletal muscle perfusion. We conclude that an intravascular source for ATP is essential, which indicates an important role for blood sources (e.g. red blood cells) in augmenting and maintaining elevated plasma ATP during exercise. PMID:23315195

Kirby, Brett S; Crecelius, Anne R; Richards, Jennifer C; Dinenno, Frank A

2013-05-01

290

Acute inhibition of the betaine transporter by ATP and adenosine in renal MDCK cells  

PubMed Central

Extracellular ATP interacts with purinergic P2 receptors to regulate a range of physiological responses, including downregulation of transport activity in the nephron. ATP is released from cells by mechanical stimuli such as cell volume changes, and autocrine signaling by extracellular ATP could occur in renal medullary cells during diuresis. This was tested in Madin-Darby canine kidney (MDCK) cells, a model used frequently to study P1 and P2 receptor activity. ATP was released within 1 min after transfer from 500 to 300 mosmol/kgH2O medium. A 30-min incubation with ATP produced dose-dependent inhibition (0.01–0.10 mM) of the renal betaine/GABA transporter (BGT1) with little effect on other osmolyte transporters. Inhibition was reproduced by specific agonists for P2X (?,?-methylene-ATP) and P2Y (UTP) receptors. Adenosine, the final product of ATP hydrolysis, also inhibited BGT1 but not taurine transport. Inhibition by ATP and adenosine was blocked by pertussis toxin and A73122, suggesting involvement of inhibitory G protein and PLC in postreceptor signaling. Both ATP and adenosine (0.1 mM) produced rapid increases in intracellular Ca2+, due to the mobilization of intracellular Ca2+ stores and Ca2+ influx. Blocking these Ca2+ increases with BAPTA-AM also blocked the action of ATP and adenosine on BGT1 transport. Finally, immunohistochemical studies indicated that inhibition of BGT1 transport may be due to endocytic accumulation of BGT1 proteins from the plasma membrane. We conclude that ATP and adenosine, through stimulation of PLC and intracellular Ca2+, may be rapidly acting regulators of BGT1 transport especially in response to a fall in extracellular osmolarity. PMID:18448594

Kempson, Stephen A.; Edwards, Jason M.; Osborn, Alyssa; Sturek, Michael

2008-01-01

291

The thermodynamic H+/ATP ratios of the H+-ATPsynthases from chloroplasts and Escherichia coli  

PubMed Central

The H+/ATP ratio is an important parameter for the energy balance of all cells and for the coupling mechanism between proton transport and ATP synthesis. A straightforward interpretation of rotational catalysis predicts that the H+/ATP coincides with the ratio of the c-subunits to ?-subunits, implying that, for the chloroplast and Escherichia coli ATPsynthases, numbers of 4.7 and 3.3 are expected. Here, the energetics described by the chemiosmotic theory was used to determine the H+/ATP ratio for the two enzymes. The isolated complexes were reconstituted into liposomes, and parallel measurements were performed under identical conditions. The internal phase of the liposomes was equilibrated with the acidic medium during reconstitution, allowing to measure the internal pH with a glass electrode. An acid–base transition was carried out and the initial rates of ATP synthesis or ATP hydrolysis were measured with luciferin/luciferase as a function of ?pH at constant Q = [ATP]/([ADP][Pi]). From the shift of the equilibrium ?pH as a function of Q the standard Gibbs free energy for phosphorylation, ?Gp0?; and the H+/ATP ratio were determined. It resulted ?Gp0? = 38 ± 3 kJ·mol?1 and H+/ATP = 4.0 ± 0.2 for the chloroplast and H+/ATP = 4.0 ± 0.3 for the E. coli enzyme, indicating that the thermodynamic H+/ATP ratio is the same for both enzymes and that it is different from the subunit stoichiometric ratio. PMID:18316723

Steigmiller, Stefan; Turina, Paola; Gräber, Peter

2008-01-01

292

Pyridoxal 5'-phosphate is an ATP-receptor antagonist in freshly isolated rat cardiomyocytes.  

PubMed

Although extracellular ATP is considered to exert a positive inotropic action on the myocardium through purinoceptors, very little information is available regarding interventions which may modify the actions of ATP on the heart. We report here that pyridoxal 5'-phosphate (PLP), an active form of vitamin B6, shows antagonism towards ATP-induced positive inotropic effect in isolated perfused rat hearts, ATP-induced increase in [Ca2+] in freshly isolated adult cardiomyocytes and ATP-binding in cardiac sarcolemma; ED50 for PLP in each of these cases varied from 10-15 microM. PLP (5-50 microM) was observed to antagonize the positive inotropic effect of ATP but did not modify the action of isoproterenol in the isolated perfused heart. Preincubation of cardiomyocytes with 1-50 microM PLP prevented the ATP-induced increase in [Ca2+]i in a concentration-dependent manner but showed no effect on the KCl-induced increase in [Ca2+]i. Creatine phosphate and Na2HPO4 as well as vitamin B6-related compounds, such as pyridoxine, pyridoxal, 4-deoxypyridoxine and isonicotinic acid hydrazide showed no effect on the ATP-induced increase in [Ca2+]i in cardiomyocytes. Furthermore, different concentrations of PLP (1-50 microM) were shown to inhibit the specific ATP gamma S binding at both the high and low affinity sites in the cardiac sarcolemmal membrane; adrenoceptor and Ca2+-channel inhibitors did not affect the ATP-binding. It is concluded that PLP may antagonize the actions of ATP on the heart in a selective manner and both pyridoxal and phosphate moieties are essential for its action. Furthermore, it is suggested that PLP may serve as a valuable tool for monitoring the role of purinoceptors in cellular function. PMID:10336844

Wang, X; Dakshinamurti, K; Musat, S; Dhalla, N S

1999-05-01

293

Do ATP and UTP involve cGMP in positive inotropism on rat atria?  

PubMed

ATP and UTP induced a dual inotropic effect in rat left atria: first a decrease and then an increase in contractile tension were observed. PPADS, an antagonist of P2X receptors, inhibited positive inotropism induced by ATP and alpha,beta-meATP. Chiefly, we investigated intracellular mechanisms responsible for the positive inotropism. We tested cromakalim and glibenclamide, an activator and an inhibitor, respectively, of ATP-sensitive K(+) channels. These compounds did not influence the effects of ATP. IBMX, a phosphodiesterase inhibitor, and H-7, an inhibitor of protein kinase C and cAMP-dependent protein kinase, did not modify the inotropic effects of ATP. Instead, H-8, an inhibitor of cAMP- and cGMP-dependent protein kinases, strongly inhibited the positive effects of both ATP and UTP, suggesting the possible involvement of cGMP in the inotropism. Also, LY 83583, an inhibitor of cGMP production, reduced positive inotropism by alpha,beta-meATP, ATP and UTP. Moreover, 8-Br-cGMP (50 microM), a stable analogue of cGMP, inhibited positive inotropism by all nucleotides. Lastly, we determined intracellular cGMP levels by RIA; the cyclic nucleotide increased during positive inotropism induced by ATP and UTP. The results regarding positive inotropism suggest that: (a) ATP acts through P2X receptors, while UTP may act by P2X, but also through PPADS-insensitive receptors; and (b) changes in intracellular cGMP concentration are involved in this inotropic effect. PMID:11239839

Froldi, G; Ragazzi, E; Caparrotta, L

2001-02-01

294

ATP secretion in the male reproductive tract: essential role of CFTR  

PubMed Central

Extracellular ATP is essential for the function of the epididymis and spermatozoa, but ATP release in the epididymis remains uncharacterized. We investigated here whether epithelial cells release ATP into the lumen of the epididymis, and we examined the role of the cystic fibrosis transmembrane conductance regulator (CFTR), a Cl? and HCO3? conducting ion channel known to be associated with male fertility, in this process. Immunofluorescence labelling of mouse cauda epididymidis showed expression of CFTR in principal cells but not in other epithelial cells. CFTR mRNA was not detectable in clear cells isolated by fluorescence-activated cell sorting (FACS) from B1-EGFP mice, which express enhanced green fluorescent protein (EGFP) exclusively in these cells in the epididymis. ATP release was detected from the mouse epididymal principal cell line (DC2) and increased by adrenaline and forskolin. Inhibition of CFTR with CFTRinh172 and transfection with CFTR-specific siRNAs in DC2 cells reduced basal and forskolin-activated ATP release. CFTR-dependent ATP release was also observed in primary cultures of mouse epididymal epithelial cells. In addition, steady-state ATP release was detected in vivo in mice, by measuring ATP concentration in a solution perfused through the lumen of the cauda epididymidis tubule and collected by cannulation of the vas deferens. Luminal CFTRinh172 reduced the ATP concentration detected in the perfusate. This study shows that CFTR is involved in the regulation of ATP release from principal cells in the cauda epididymidis. Given that mutations in CFTR are a leading cause of male infertility, we propose that defective ATP signalling in the epididymis might contribute to dysfunction of the male reproductive tract associated with these mutations. PMID:22711960

Ruan, Ye Chun; Shum, Winnie W C; Belleannée, Clémence; Da Silva, Nicolas; Breton, Sylvie

2012-01-01

295

Phase II study of imatinib mesylate and hydroxyurea for recurrent grade III malignant gliomas  

Microsoft Academic Search

Purpose  Recent reports demonstrate the activity of imatinib mesylate, an ATP-mimetic, tyrosine kinase inhibitor, plus hydroxyurea,\\u000a a ribonucleotide reductase inhibitor, in patients with recurrent glioblastoma multiforme. We performed the current phase 2\\u000a study to evaluate this regimen among patients with recurrent WHO grade III malignant glioma (MG).\\u000a \\u000a \\u000a \\u000a Patients and method  Patients with grade III MG at any recurrence, received imatinib mesylate plus

Annick Desjardins; Jennifer A. Quinn; James J. Vredenburgh; Sith Sathornsumetee; Allan H. Friedman; James E. Herndon; Roger E. McLendon; James M. Provenzale; Jeremy N. Rich; John H. Sampson; Sridharan Gururangan; Jeannette M. Dowell; August Salvado; Henry S. Friedman; David A. Reardon

2007-01-01

296

Speicherring DORIS III DORIS III Betrieb 2000  

E-print Network

, in der unter anderem alle Triplett-Kammern und alle Kicker-Magnete erneuert werden sollen- genommen davon sind nur die Kammern der drei Injektions-Tripletts, da bisher von den vier Kicker- 203 #12;DORIS III Magneten, die ausgetauscht werden sollten, nur der Kicker für die vertikale Anregung eingebaut

297

Three-Dimensional Structures Reveal Multiple ADP/ATP Binding Modes  

SciTech Connect

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.

C Simmons; C Magee; D Smith; L Lauman; J Chaput; J Allen

2011-12-31

298

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

E-print Network

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

Grosell, Martin

299

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

300

Supporting Information Selective ATP-competitive inhibitors of TOR suppress rapamycin insensitive  

E-print Network

Supporting Information Selective ATP-competitive inhibitors of TOR suppress rapamycin insensitive. Yeast strain SH121 and SH121 harboring pRS314[HA TOR2] plasmid were obtained from Yoshinori Ohsumi,000rpm for 1 min. One µl of supernatant was used as a template for PCR. The ATP pocket region in TOR1

Sabatini, David M.

301

Cation Transport Coupled to ATP Hydrolysis by the (Na, K)-ATPase: An Integrated, Animated Model  

ERIC Educational Resources Information Center

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…

Leone, Francisco A.; Furriel, Rosa P. M.; McNamara, John C.; Horisberger, Jean D.; Borin, Ivana A.

2010-01-01

302

An ATP-responsive smart gate fabricated with a graphene oxide-aptamer-nanochannel architecture.  

PubMed

Here, we report a graphene oxide-aptamer-nanochannel architecture for the fabrication of a novel stimuli-responsive gate. The gate is switched OFF in the absence of ATP, and is switched ON when ATP is present. The concept we proposed may contribute to a versatile platform for the development of stimuli-responsive gates. PMID:25406894

Zhu, Xiaoli; Zhang, Bin; Ye, Zonghuang; Shi, Hai; Shen, Yalan; Li, Genxi

2015-01-14

303

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

PubMed

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

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

1980-06-01

304

ATP/ADP ratio, the missed connection between mitochondria and the Warburg effect.  

PubMed

Non-proliferating cells generate the bulk of cellular ATP by fully oxidizing respiratory substrates in mitochondria. Respiratory substrates cross the mitochondrial outer membrane through only one channel, the voltage dependent anion channel (VDAC). Once in the matrix, respiratory substrates are oxidized in the tricarboxylic acid cycle to generate mostly NADH that is further oxidized in the respiratory chain to generate a proton motive force comprised mainly of membrane potential (??) to synthesize ATP. Mitochondrial ?? then drives the release of ATP(4-) from the matrix in exchange for ADP(3-) in the cytosol via the adenine nucleotide translocator (ANT) located in the mitochondrial inner membrane. Thus, mitochondrial function in non-proliferating cells drives a high cytosolic ATP/ADP ratio, essential to inhibit glycolysis. By contrast, the bioenergetics of the Warburg phenotype of proliferating cells is characterized by enhanced aerobic glycolysis and the suppression of mitochondrial metabolism. Suppressed mitochondrial function leads to lower production of mitochondrial ATP and hence lower cytosolic ATP/ADP ratios that favor enhanced glycolysis. Thus, the cytosolic ATP/ADP ratio is a key feature that determines if cell metabolism is predominantly oxidative or glycolytic. Here, we describe two novel mechanisms to explain the suppression of mitochondrial metabolism in cancer cells: the relative closure of VDAC by free tubulin and the inactivation of ANT. Both mechanisms contribute to low ATP/ADP ratios that activate glycolysis. PMID:25229666

Maldonado, Eduardo N; Lemasters, John J

2014-11-01

305

ORIGINAL ARTICLE An in vivo model of melanoma: treatment with ATP  

E-print Network

by methods of cancer treatment such as surgery, radiotherapy and chemo- therapy. Despite significant advances for the use of ATP as a treatment for melanoma. Keywords Melanoma . Cancer. Purinergic signalling . ATP Introduction Malignant melanoma is a very aggressive cancer that originates from melanocytes, the pigment

Burnstock, Geoffrey

306

ATP hydrolysis pathways and their contributions to pial arteriolar dilation in rats  

PubMed Central

ATP is thought to be released to the extracellular compartment by neurons and astrocytes during neural activation. We examined whether ATP exerts its effect of promoting pial arteriolar dilation (PAD) directly or upon conversion (via ecto-nucleotidase action) to AMP and adenosine. Blockade of extracellular direct ATP to AMP conversion, with ARL-67156, significantly reduced sciatic nerve stimulation-evoked PADs by 68%. We then monitored PADs during suffusions of ATP, ADP, AMP, and adenosine in the presence and absence of the following: 1) the ecto-5?-nucleotidase inhibitor ?,?-methylene adenosine 5?-diphosphate (AOPCP), 2) the A2 receptor blocker ZM 241385, 3) the ADP P2Y1 receptor antagonist MRS 2179, and 4) ARL-67156. Vasodilations induced by 1 and 10 ?M, but not 100 ?M, ATP were markedly attenuated by ZM 241385, AOPCP, and ARL-67156. Substantial loss of reactivity to 100 ?M ATP required coapplications of ZM 241385 and MRS 2179. Dilations induced by ADP were blocked by MRS 2179 but were not affected by either ZM 241385 or AOPCP. AMP-elicited dilation was partially inhibited by AOPCP and completely abolished by ZM 241385. Collectively, these and previous results indicate that extracellular ATP-derived adenosine and AMP, via A2 receptors, play key roles in neural activation-evoked PAD. However, at high extracellular ATP levels, some conversion to ADP may occur and contribute to PAD through P2Y1 activation. PMID:21803949

Vetri, Francesco; Mao, Lizhen; Paisansathan, Chanannait; Pelligrino, Dale A.

2011-01-01

307

ATP Hydrolysis in Water -A Density Functional Study J. Akola and R. O. Jones*  

E-print Network

ATP Hydrolysis in Water - A Density Functional Study J. Akola and R. O. Jones* Institut fu¨r Festko-dependent hydrolysis reaction. Two paths for ATP hydrolysis in water with Mg2+ are studied here using the density) in the triphosphate tail of the molecule as an energy-rich bond that releases energy upon hydrolysis due

308

Role of ATP-Hydrolysis in the Dynamics of a Single Actin Filament Padinhateeri Ranjith,  

E-print Network

Role of ATP-Hydrolysis in the Dynamics of a Single Actin Filament Padinhateeri Ranjith, * Kirone, and ATP hydrolysis of subunits either according to the vectorial mechanism or to the random mechanism. In a previous work, we developed a model for a single actin or microtubule filament where hydrolysis occurred

Lacoste, David

309

Autism Post-Mortem Neuroinformatic Resource: The Autism Tissue Program (ATP) Informatics Portal  

ERIC Educational Resources Information Center

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…

Brimacombe, Michael B.; Pickett, Richard; Pickett, Jane

2007-01-01

310

ATP Synthesis, Mitochondrial Function, and Steroid Biosynthesis in Rodent Primary and Tumor Leydig Cells1  

PubMed Central

Previous studies in MA-10 tumor Leydig cells demonstrated that disruption of the mitochondrial electron-transport chain (ETC), membrane potential (??m), or ATP synthesis independently inhibited steroidogenesis. In contrast, studies of primary Leydig cells indicated that the ETC, ??m, and ATP synthesis cooperatively affected steroidogenesis. These results suggest significant differences between the two systems and call into question the extent to which results from tumor Leydig cells relate to primary cells. Thus, to further understand the similarities and differences between the two systems as well as the impact of ATP disruption on steroidogenesis, we performed comparative studies of MA-10 and primary Leydig cells under similar conditions of mitochondrial disruption. We show that mitochondrial ATP synthesis is critical for steroidogenesis in both primary and tumor Leydig cells. However, in striking contrast to primary cells, perturbation of ??m in MA-10 cells did not substantially decrease cellular ATP content, a perplexing finding because ??m powers the mitochondrial ATP synthase. Further studies revealed that a significant proportion of cellular ATP in MA-10 cells derives from glycolysis. In contrast, primary cells appear to be almost completely dependent on mitochondrial respiration for their energy provision. Inhibitor studies also suggested that the MA-10 ETC is impaired. This work underscores the importance of mitochondrial ATP for hormone-stimulated steroid production in both MA-10 and primary Leydig cells while indicating that caution must be exercised in extrapolating data from tumor cells to primary tissue. PMID:21228212

Midzak, Andrew S.; Chen, Haolin; Aon, Miguel A.; Papadopoulos, Vassilios; Zirkin, Barry R.

2011-01-01

311

Effects of luciferin concentration on the quantitative assay of ATP using crude luciferase preparations  

Microsoft Academic Search

The reaction kinetics of crude firefly lantern extracts with and without added synthetic luciferin were examined. The addition of exogenous luciferin to the reaction mixture resulted in an apparent increase in net light emission per unit of ATP in solution. This additional reactivity (up to 1000-fold) enables the detection of subpicogram levels of ATP. The effects of enzyme preparation, dilution,

D. M. Karl; O. Holm-Hansen

1976-01-01

312

Human Copper-Transporting ATPase ATP7B (The Wilson's Disease Protein): Biochemical Properties and Regulation  

Microsoft Academic Search

Wilson's disease protein (WNDP) is a product of a gene ATP7B that is mutated in patients with Wilson's disease, a severe genetic disorder with hepatic and neurological manifestations caused by accumulation of copper in the liver and brain. In a cell, WNDP transports copper across various cell membranes using energy of ATP-hydrolysis. Copper regulates WNDP at several levels, modulating its

Svetlana Lutsenko; Roman G. Efremov; Ruslan Tsivkovskii; Joel M. Walker

2002-01-01

313

Structure and Evolutionary Analysis of a Non-biological ATP-binding Protein  

E-print Network

Structure and Evolutionary Analysis of a Non-biological ATP-binding Protein Sheref S. Mansy1 op- timization of a non-biological protein derived from a library of random amino acid sequences sequence into a stably folded, high affinity ATP binding protein structure. While the evolutionarily

Heller, Eric

314

Multiple turnovers of DNAzyme for amplified detection of ATP and reduced thiol in cell homogenates.  

PubMed

A conveniently amplified DNAzyme-based fluorescence strategy was designed for highly sensitive detection of ATP or reduced thiol based on the introduction of an ATP aptamer or a disulfide bond in the bioconjugates of magnetic nanoparticles (MNP) and polystyrene microsphere-DNAzyme complexes (PSM-DNAzyme). PMID:25429374

Guo, Yingshu; Liu, Jia; Yang, Guangxu; Sun, Xiaofeng; Chen, Hong-Yuan; Xu, Jing-Juan

2015-01-18

315

Bioenergetic Pattern of Turtle Brain and Resistance to Profound Loss of Mitochondrial ATP Generation  

Microsoft Academic Search

The adaptations in the freshwater turtle that permit survival despite prolonged loss of mitochondrial ATP generation were investigated by comparing the bioenergetics of turtle brain slices with rat brain slices. Aerobic turtle brain shows no significant difference in basal levels of total ATP generation compared to rat brain; levels in turtle brain and rat brain were 18.4 ± 2.8 (SD)

Eugene D. Robin; Norman Lewiston; Andrew Newman; Lawrence M. Simon; James Theodore

1979-01-01

316

Integration of Motor Proteins – Towards an ATP Fueled Soft Actuator  

PubMed Central

We present a soft bio-machine constructed from biological motors (actin/myosin). We have found that chemically cross-linked polymer-actin complex gel filaments can move on myosin coated surfaces with a velocity as high as that of native F-actin, by coupling to ATP hydrolysis. Additionally, it is shown that the velocity of polymer-actin complex gel depends on the species of polycations binding to the F-actins. Since the design of functional actuators of well-defined size and morphology is important, the structural behavior of polymer-actin complexes has been investigated. Our results show that the morphology and growth size of polymer-actin complex can be controlled by changes in the electrostatic interactions between F-actins and polycations. Our results indicate that bio actuators with desired shapes can be created by using a polymer-actin complex. PMID:19325826

Kakugo, Akira; Shikinaka, Kazuhiro; Gong, Jian Ping

2008-01-01

317

NASA ATP Force Measurement Technology Capability Strategic Plan  

NASA Technical Reports Server (NTRS)

The Aeronautics Test Program (ATP) within the National Aeronautics and Space Administration (NASA) Aeronautics Research Mission Directorate (ARMD) initiated a strategic planning effort to re-vitalize the force measurement capability within NASA. The team responsible for developing the plan included members from three NASA Centers (Langley, Ames and Glenn) as well as members from the Air Force s Arnold Engineering and Development Center (AEDC). After visiting and discussing force measurement needs and current capabilities at each participating facility as well as selected force measurement companies, a strategic plan was developed to guide future NASA investments. This paper will provide the details of the strategic plan and include asset management, organization and technology research and development investment priorities as well as efforts to date.

Rhew, Ray D.

2008-01-01

318

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

PubMed Central

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 AUGMet1 to produce plastid-targeted ATPS2 pre-proteins or at AUGMet52 or AUGMet58 within the transit peptide to have ATPS2 stay in cytosol. Translational initiation of ATPS2 at AUGMet52 or AUGMet58 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 AUGMet52 and AUGMet58 significantly reduced the firefly luciferase activity, while AUGMet52 was a relatively preferred site for the alternative translational initiation. The activity of luciferase fusion protein starting at AUGMet52 or AUGMet58 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

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

2015-01-01

319

Epochs I,II Epochs III-V  

E-print Network

Epochs I,II Epochs III-V Epochs VI-VIII A History of Algebraic Geometry Rob Easton February 26, 2010 Rob Easton A History of Algebraic Geometry #12;Epochs I,II Epochs III-V Epochs VI-VIII References., 1985. Rob Easton A History of Algebraic Geometry #12;Epochs I,II Epochs III-V Epochs VI-VIII Jean

Easton, Robert W.

320

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

PubMed

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

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

321

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

PubMed Central

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

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

322

Au(III)-assisted core-shell iron oxide@poly(o-phenylenediamine) nanostructures for ultrasensitive electrochemical aptasensors based on DNase I-catalyzed target recycling.  

PubMed

A redox-active Au(III)-assisted core-shell iron oxide@poly(o-phenylenediamine) nanostructure was designed as a sensing platform for ultrasensitive electrochemical detection of small molecules (ATP, used as a model here) by coupling with DNase I-catalyzed target recycling. PMID:22286177

Liu, Bingqian; Cui, Yuling; Tang, Dianping; Yang, Huanghao; Chen, Guonan

2012-03-01

323

Swelling-Induced, Cftr-Independent Atp Release from a Human Epithelial Cell Line  

PubMed Central

To examine a possible relation between the swelling-induced ATP release pathway and the volume-sensitive Cl? channel, we measured the extracellular concentration of ATP released upon osmotic swelling and whole-cell volume-sensitive Cl? currents in a human epithelial cell line, Intestine 407, which lacks expression of cystic fibrosis transmembrane conductance regulator (CFTR). Significant release of ATP was observed within several minutes after a hypotonic challenge (56–80% osmolality) by the luciferin/luciferase assay. A carboxylate analogue Cl? channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate, suppressed ATP release in a concentration-dependent manner with a half-maximal inhibition concentration of 6.3 ?M. However, swelling-induced ATP release was not affected by a stilbene-derivative Cl? channel blocker, 4-acetamido-4?-isothiocyanostilbene at 100 ?M. Glibenclamide (500 ?M) and arachidonic acid (100 ?M), which are known to block volume-sensitive outwardly rectifying (VSOR) Cl? channels, were also ineffective in inhibiting the swelling-induced ATP release. Gd3+, a putative blocker of stretch-activated channels, inhibited swelling-induced ATP release in a concentration-dependent manner, whereas the trivalent lanthanide failed to inhibit VSOR Cl? currents. Upon osmotic swelling, the local ATP concentration in the immediate vicinity of the cell surface was found to reach ?13 ?M by a biosensor technique using P2X2 receptors expressed in PC12 cells. We have raised antibodies that inhibit swelling-induced ATP release from Intestine 407 cells. Earlier treatment with the antibodies almost completely suppressed swelling-induced ATP release, whereas the activity of VSOR Cl? channel was not affected by pretreatment with the antibodies. Taking the above results together, the following conclusions were reached: first, in a CFTR-lacking human epithelial cell line, osmotic swelling induces ATP release and increases the cell surface ATP concentration over 10 ?M, which is high enough to stimulate purinergic receptors; second, the pathway of ATP release is distinct from the pore of the volume-sensitive outwardly rectifying Cl? channel; and third, the ATP release is not a prerequisite to activation of the Cl? channel. PMID:10498671

Hazama, Akihiro; Shimizu, Takahiro; Ando-Akatsuka, Yuhko; Hayashi, Seiji; Tanaka, Shoko; Maeno, Emi; Okada, Yasunobu

1999-01-01

324

Kinetics of extracellular ATP hydrolysis by microvascular endothelial cells from rat heart.  

PubMed Central

We have characterized the ectonucleotidases that catalyse the reaction sequence ATP-->ADP-->AMP-->adenosine on microvascular endothelial cells cultured from the rat heart. Computer simulation and data fitting of progress of reaction curves showed that depletion of substrate at the cell surface dominates the regulation of the rate of hydrolysis of ATP when it is presented to the cells. Preferential delivery of AMP by ADPase to 5'-nucleotidase makes a significant contribution to the regulation of adenosine production from ATP or ADP. By contrast, we found no evidence for the preferential delivery of ADP from ATPase to ADPase. Feed-forward inhibition of AMP hydrolysis by ADP and/or ATP also modulated the rate of adenosine production. The properties of the ectonucleotidases on rat heart microvascular cells are such that adenosine is produced at a steady rate over a wide range of ATP concentrations. Images Scheme 1 PMID:8948425

Meghji, P; Pearson, J D; Slakey, L L

1995-01-01

325

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

PubMed

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

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

2014-12-01

326

Effect of adenosine 5'triphosphate (ATP) on the isolated rectum of the rainbow lizard Agama agama.  

PubMed

ATP but not adenosine contracts lizard isolated rectal muscles. Theophylline, quinidine, nifedipine and lack of Ca2+ inhibited ATP contractions, but dipyridamole, indomethacin, atropine, D-tubocurarine, phentolamine, L-propranolol, cimetidine, mepyramine and methysergide did not significantly modify the contractions. ATP inhibited Ca2+-induced contractions in Na+-free, high K+ media. In normal Ringer, it relaxed carbachol-contracted muscles and inhibited KCl contractions. The results suggest that ATP contractions are dependent upon external Ca2+, and are probably associated with extracellular Ca+ influx. The contractions appear to have occurred independently of receptor activation and may have been mediated by a depolarising action of the nucleotide. The relaxant effect of ATP possibly occurred as a consequence of inhibition of Ca2+ influx into carbachol or K+-depolarised muscles. PMID:4018537

Savage, A O; Atanga, G K

1985-01-01

327

Electron cryomicroscopy of acto-myosin-S1 during steady-state ATP hydrolysis.  

PubMed Central

The structure of the complex of actin and myosin subfragment-1 (S1) during steady-state ATP hydrolysis has been examined by electron microscopy. This complex is normally dissociated by ATP in vitro but was stabilized here by low ionic strength. Optimal conditions for attachment were established by light-scattering experiments that showed that approximately 70% of S1 could be bound in the presence of ATP. Micrographs of the unstained complex in vitreous water suggest that S1 attaches to actin in a variety of configurations in ATP; this contrasts with the single attached configuration seen in the presence of ADP. The data are therefore compatible with the idea that a change in attached configuration of the myosin cross-bridge is the origin of muscle force. In control experiments where ATP was allowed to hydrolyze completely the binding of the S1 seemed cooperative. Images FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 PMID:8061205

Walker, M; White, H; Belknap, B; Trinick, J

1994-01-01

328

ATP: a mediator for HCl-induced TRPV1 activation in esophageal mucosa  

PubMed Central

In esophageal mucosa, HCl causes TRPV1-mediated release of calcitonin gene-related peptide (CGRP) and substance P (SP) from submucosal neurons and of platelet-activating factor (PAF) from epithelial cells. CGRP and SP release was unaffected by PAF antagonists but reduced by the purinergic antagonist suramin. ATP caused CGRP and SP release from esophageal mucosa, confirming a role of ATP in the release. The human esophageal epithelial cell line HET-1A was used to identify epithelial cells as the site of ATP release. HCl caused ATP release from HET-1A, which was reduced by the TRPV1 antagonist 5-iodoresiniferatoxin. Real-time PCR demonstrated the presence of mRNA for several P2X and P2Y purinergic receptors in epithelial cells. HCl also increased activity of lyso-PAF acetyl-CoA transferase (lyso-PAF AT), the enzyme responsible for production of PAF. The increase was blocked by suramin. ATP caused a similar increase, confirming ATP as a mediator for the TRPV1-induced increase in enzyme activity. Repeated exposure of HET-1A cells to HCl over 2 days caused upregulation of mRNA and protein expression for lyso-PAF AT. Suramin blocked this response. Repeated exposure to ATP caused a similar mRNA increase, confirming ATP as a mediator for upregulation of the enzyme. Thus, HCl-induced activation of TRPV1 causes ATP release from esophageal epithelial cells that causes release of CGRP and SP from esophageal submucosal neurons and activation of lyso-PAF AT, the enzyme responsible for the production of PAF in epithelial cells. Repeated application of HCl or of ATP causes upregulation of lyso-PAF AT in epithelial cells. PMID:21960521

Ma, Jie; Altomare, Annamaria; Rieder, Florian; Behar, Jose; Biancani, Piero

2011-01-01

329

ATP appears to act via different receptors in terminals vs. somata of the Hypothalamic Neurohypophysial System  

PubMed Central

ATP-induced ionic currents were investigated in isolated terminals and somata of the Hypothalamic Neurohypophysial System (HNS). Both terminals and somata showed inward rectification of the ATP-induced currents and reversal near 0 mV. In terminals, ATP dose-dependently evoked an inactivating, inward current. However, in hypothalamic somata ATP evoked a very slowly inactivating, inward current with a higher density, and different dose dependence; EC50 of 50 ?M in somata vs. 9.6 ?M in terminals. The ATP induced currents, in both the HNS terminals and somata, were highly and reversibly inhibited by suramin, suggesting the involvement of a P2X receptor. However, the suramin inhibition was significantly different in the two HNS compartments: IC50 of 3.6 ?M in somata vs 11.6 ?M in terminals. Also, both HNS compartments show significantly different responses to the purinergic receptor agonists ATP-?-S and Benzoyl-benzoyl-ATP. Finally, there was an initial desensitization to ATP upon successive stimulations in the terminals which was not observed in the somata. These differences in EC50, inactivation, desensitization, and agonist sensitivity in terminals vs. somata indicate that different P2X receptors mediate the responses in these two compartments of HNS neurons. Previous work has revealed mRNA transcripts for multiple purinergic receptors in micropunches of the hypothalamus. In the HNS terminals, the P2X purinergic receptor types P2X2, 3, 4, and 7 but not 6 have been shown to exist in AVP terminals. Immonohistochemistry now indicates that P2X4R is only present in AVP terminals and that the P2X7R is found in both AVP and OT terminals and somata. We speculate that these differences in receptor types reflects the specific function of endogenous ATP in the terminals vs. somata of these CNS neurons. PMID:22340013

Knott, Thomas K.; Hussy, Nicolas; Cuadra, Adolfo E.; Lee, Ryan H.; Ortiz-Miranda, Sonia; Custer, Edward E.; Lemos, José R.

2012-01-01

330

Intracellular ATP does not inhibit Slo2.1 K+ channels  

PubMed Central

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

Garg, Priyanka; Sanguinetti, Michael C.

2014-01-01

331

A stable ATP binding to the nucleotide binding domain is important for reliable gating cycle in an ABC transporter CFTR.  

PubMed

Cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, a member of ABC transporter superfamily, gates following ATP-dependent conformational changes of the nucleotide binding domains (NBD). Reflecting the hundreds of milliseconds duration of the channel open state corresponding to the dimerization of two NBDs, macroscopic WT-CFTR currents usually showed a fast, single exponential relaxation upon removal of cytoplasmic ATP. Mutations of tyrosine1219, a residue critical for ATP binding in second NBD (NBD2), induced a significant slow phase in the current relaxation, suggesting that weakening ATP binding affinity at NBD2 increases the probability of the stable open state. The slow phase was effectively diminished by a higher affinity ATP analogue. These data suggest that a stable binding of ATP to NBD2 is required for normal CFTR gating cycle, andthat the instability of ATP binding frequently halts the gating cycle in the open state presumably through a failure of ATP hydrolysis at NBD2. PMID:20628841

Shimizu, Hiroyasu; Yu, Ying-Chun; Kono, Koichi; Kubota, Takahiro; Yasui, Masato; Li, Min; Hwang, Tzyh-Chang; Sohma, Yoshiro

2010-09-01

332

Structure of dimeric mitochondrial ATP synthase: Novel F0 bridging features and the structural basis of mitochondrial cristae biogenesis  

Microsoft Academic Search

The F1F0-ATP synthase exists as a dimer in mitochondria, where it is essential for the biogenesis of the inner membrane cristae. How two ATP synthase complexes dimerize to promote cristae formation is unknown. Here we resolved the structure of the dimeric F1F0 ATP synthase complex isolated from bovine heart mitochondria by transmission electron microscopy. The structure of the ATP synthase

Fernando Minauro-Sanmiguel; Stephan Wilkens; José J. García

2005-01-01

333

A sensitive, simple assay of mitochondrial ATP synthesis of cultured mammalian cells suitable for high-throughput analysis  

Microsoft Academic Search

A new assay has been developed to measure mitochondrial ATP synthesis of cultured mammalian cells. Cells in a microplate are exposed to streptolysin O to make plasma membranes permeable without damaging mitochondrial function and ATP synthesis is monitored by luciferase. Addition of inhibitors of FoF1-ATP synthase (FoF1), respiratory chain, TCA cycle and ATP\\/ADP translocator, as well as knockdown of ?-subunit

Makoto Fujikawa; Masasuke Yoshida

2010-01-01

334

ATP-binding sites in brain p97/VCP (valosin-containing protein), a multifunctional AAA ATPase.  

PubMed Central

VCP (valosin-containing protein) or p97 is a member of the AAA family (ATPases associated with a variety of cellular activities family), a diverse group of proteins sharing a key conserved AAA module containing duplicate putative ATP-binding sites. Although the functions of the AAA family are related to their putative ATP-binding sites, the binding of ATP to these sites has not yet been demonstrated. In the present study, the ATP-binding site(s) of brain VCP was characterized using the photoreactive ATP analogue, BzATP [3'- O -(4-benzoylbenzoyl)ATP]. Photo-activation of Bz-[alpha-(32)P]ATP resulted in its covalent binding to a 97-kDa purified soluble or membrane-associated protein, identified by amino acid sequencing as VCP. Bz-[alpha-(32)P]ATP covalently bound to the purified homo-hexameric VCP with an apparent high affinity (74-111 nM). A molar stoichiometry of 2.23+/-0.14 BzATP bound per homo-hexameric VCP (n =6) was determined using different methods for analysis of radiolabelling and protein determination. Nucleotides inhibited the binding of Bz-[alpha-(32)P]ATP to VCP with the following efficiency: BzATP>ATP>ADP>>adenosine 5'-[beta,gamma-imido]triphosphate>or=adenosine 5'-[beta,gamma-methylene]triphosphate, whereas AMP, GTP and CTP were ineffective. VCP was observed to possess very low ATPase activity, with nucleotide specificity similar to that for BzATP binding. Conformational changes induced by an alternating site mechanism for ATP binding are suggested as a molecular mechanism for coupling ATP binding to the diverse activities of the AAA family. PMID:12747802

Zalk, Ran; Shoshan-Barmatz, Varda

2003-01-01

335

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

PubMed Central

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

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

1990-01-01

336

Evidence that ATP acts at two sites to evoke contraction in the rat isolated tail artery  

PubMed Central

The site(s) at which P2-receptor agonists act to evoke contractions of the rat isolated tail artery was studied by use of P2-receptor antagonists and the extracellular ATPase inhibitor 6-N,N-diethyl-D-?,?-dibromomethyleneATP (ARL 67156).Suramin (1??M–1?mM) and pyridoxalphosphate-6-azophenyl-2?,4?-disulphonic acid (PPADS) (0.3–300??M) inhibited contractions evoked by equi-effective concentrations of ?,?-methyleneATP (?,?-meATP) (5??M), 2-methylthioATP (2-meSATP) (100??M) and adenosine 5?-triphosphate (ATP) (1?mM) in a concentration-dependent manner. Responses to ?,?-meATP and 2-meSATP were abolished, but approximately one third of the peak response to ATP was resistant to suramin and PPADS.Contractions evoked by uridine 5?-triphosphate (UTP) (1?mM) were slightly inhibited by suramin (100 and 300??M) and potentiated by PPADS (300??M).Desensitization of the P2X1-receptor by ?,?-meATP abolished contractions evoked by 2-meSATP (100??M) and reduced those to ATP (1?mM) and UTP (1?mM) to 15±3% and 68±4% of control.Responses to ?,?-meATP (5??M) and 2-meSATP (100??M) were abolished when tissues were bathed in nominally calcium-free solution, while the peak contractions to ATP (1?mM) and UTP (1?mM) were reduced to 24±6% and 61±13%, respectively, of their control response.ARL 67156 (3–100??M) potentiated contractions elicited by UTP (1?mM), but inhibited responses to ?,?-meATP (5??M), 2-meSATP (100??M) and ATP (1?mM) in a concentration-dependent manner.These results suggest that two populations of P2-receptors are present in the rat tail artery; ligand-gated P2X1-receptors and G-protein-coupled P2Y-receptors. PMID:9630336

McLaren, G J; Burke, K S; Buchanan, K J; Sneddon, P; Kennedy, C

1998-01-01

337

Fading and rebound of currents induced by ATP in PC12 cells.  

PubMed Central

1. Patch clamp recording (whole cell configuration) was used to study the action of ATP on rat phaeochromocytoma (PC12) cells usually held at -70 mV and rapidly superfused with buffered saline. ATP (0.5, 1 or 5 mM), applied from micropipettes by pressure application with brief (< or = 50 ms) pulses, induced inward currents with rapid onset and decay. ADP and alpha, beta-methylene ATP were ineffective. 2. ATP (5 mM) applied with pulses > 200 ms long elicited a complex current response characterized by a rapid peak which faded and was followed by a strong current rebound (lasting several s) as soon as the application was terminated. This type of response was readily replicated as long as ATP applications were spaced at 2-3 min intervals. The amplitude of peak and rebound currents was dependent on the length of pressure pulse and was similarly depressed by bath application of a threshold dose (25 microM) of ATP. Rapid fading and rebound of ATP-induced membrane currents were also observed when the Y-tube method was used for applying this agonist. 3. The reversal potential for peak and rebound currents was the same while the time constant values for peak fading and rebound onset were insensitive to changes in membrane potential between -70 and -40 mV. When ATP was applied to a cell clamped at depolarized potential, no current was observed but rapid return of the membrane potential to -70 mV immediately at the end of ATP application was associated with a large rebound current. 4. Brief (20 ms) application of ATP during the onset of the rebound current strongly and transiently suppressed it. The same application performed during the gradual decay of the rebound wave elicited a transient inward current which was much smaller and shorter than the one observed when the cell was in its resting state. Application of 2 s ATP pulses at 20 s intervals equally reduced the initial peak and rebound currents which recovered at the same rate. 5. The present data are interpreted according to a scheme which suggests two types of ATP receptor desensitization. The first one (D1) would be characterized by fast kinetics and low agonist affinity; rapid recovery from D1 would then be manifested as current rebound presumably due to receptor reactivation. The second desensitized state (D2) has slow kinetics and high affinity for the agonist: it is therefore typically seen with sustained application of a low dose of ATP. It is proposed that desensitization and its recovery can influence the time course of membrane responses mediated by purinoceptors. PMID:8922757

Giniatullin, R.; Khiroug, L.; Talantova, M.; Nistri, A.

1996-01-01

338

Crystal structure of the R-protein of the multisubunit ATP-dependent restriction endonuclease NgoAVII  

PubMed Central

The restriction endonuclease (REase) NgoAVII is composed of two proteins, R.NgoAVII and N.NgoAVII, and shares features of both Type II restriction enzymes and Type I/III ATP-dependent restriction enzymes (see accompanying paper Zaremba et al., 2014). Here we present crystal structures of the R.NgoAVII apo-protein and the R.NgoAVII C-terminal domain bound to a specific DNA. R.NgoAVII is composed of two domains: an N-terminal nucleolytic PLD domain; and a C-terminal B3-like DNA-binding domain identified previously in BfiI and EcoRII REases, and in plant transcription factors. Structural comparison of the B3-like domains of R.NgoAVII, EcoRII, BfiI and the plant transcription factors revealed a conserved DNA-binding surface comprised of N- and C-arms that together grip the DNA. The C-arms of R.NgoAVII, EcoRII, BfiI and plant B3 domains are similar in size, but the R.NgoAVII N-arm which makes the majority of the contacts to the target site is much longer. The overall structures of R.NgoAVII and BfiI are similar; however, whilst BfiI has stand-alone catalytic activity, R.NgoAVII requires an auxiliary cognate N.NgoAVII protein and ATP hydrolysis in order to cleave DNA at the target site. The structures we present will help formulate future experiments to explore the molecular mechanisms of intersubunit crosstalk that control DNA cleavage by R.NgoAVII and related endonucleases. PMID:25429979

Tamulaitiene, Giedre; Silanskas, Arunas; Grazulis, Saulius; Zaremba, Mindaugas; Siksnys, Virginijus

2014-01-01

339

Serine Biosynthesis with One Carbon Catabolism and the Glycine Cleavage System Represents a Novel Pathway for ATP Generation  

Microsoft Academic Search

Previous experimental evidence indicates that some cancer cells have an alternative glycolysis pathway with net zero ATP production, implying that upregulation of glycolysis in these cells may not be related to the generation of ATP. Here we use a genome-scale model of human cell metabolism to investigate the potential metabolic alterations in cells using net zero ATP glycolysis. We uncover

Alexei Vazquez; Elke K. Markert; Zoltán N. Oltvai

2011-01-01

340

A Two-site Kinetic Mechanism for ATP Binding and Hydrolysis by E. coli Rep Helicase Dimer Bound to a  

E-print Network

A Two-site Kinetic Mechanism for ATP Binding and Hydrolysis by E. coli Rep Helicase Dimer Bound that are coupled to ATP binding and hydrolysis. We have investi- gated the kinetic mechanism of ATP binding and hydrolysis by a proposed intermediate in Rep-catalyzed DNA unwinding, the Rep ``P2S'' dimer (formed

Lohman, Timothy M.

341

Rotation of the ? Subunit in F 1 ATPase; Evidence That ATP Synthase Is a Rotary Motor Enzyme  

Microsoft Academic Search

ATP-dependent, azide-sensitive rotation of the ? subunit relative to the a3ß3 hexagonal ring of ATP synthase was observed with a single molecule imaging system. Thus, ATP synthase is a rotary motor enzyme, the first ever found.

Ryohei Yasuda; Hiroyuki Noji; Kazuhiko Kinosita; Fumihiro Motojima; Masasuke Yoshida

1997-01-01

342

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

E-print Network

RecA as a Motor Protein TESTING MODELS FOR THE ROLE OF ATP HYDROLYSIS IN DNA STRAND EXCHANGE, Madison, Wisconsin 53706 ATP hydrolysis (by RecA protein) fundamentally al- ters the properties of RecA protein-mediated DNA strand exchange reactions. ATP hydrolysis renders DNA strand exchange unidirectional

Cox, Michael M.

343

Tandem function of nucleotide binding domains confers competence to sulfonylurea receptor in gating ATP-sensitive K+ channels.  

PubMed

Fundamental to the metabolic sensor function of ATP-sensitive K(+) (K(ATP)) channels is the sulfonylurea receptor. This ATP-binding cassette protein, which contains nucleotide binding domains (NBD1 and NBD2) with conserved Walker motifs, regulates the ATP sensitivity of the pore-forming Kir6.2 subunit. Although NBD2 hydrolyzes ATP, a property essential in K(ATP) channel gating, the role of NBD1, which has limited catalytic activity, if at all, remains less understood. Here, we provide functional evidence that cooperative interaction, rather than the independent contribution of each NBD, is critical for K(ATP) channel regulation. Gating of cardiac K(ATP) channels by distinct conformations in the NBD2 ATPase cycle, induced by gamma-phosphate analogs, was disrupted by point mutation not only of the Walker motif in NBD2 but also in NBD1. Cooling membrane patches to decelerate the intrinsic ATPase activity counteracted ATP-induced K(ATP) channel inhibition, an effect that mimicked stabilization of the MgADP-bound posthydrolytic state at NBD2 by the gamma-phosphate analog orthovanadate. Temperature-induced channel activation was abolished by mutations that either prevent stabilization of MgADP at NBD2 or ATP at NBD1. These findings provide a paradigm of K(ATP) channel gating based on integration of both NBDs into a functional unit within the multimeric channel complex. PMID:11825892

Zingman, Leonid V; Hodgson, Denice M; Bienengraeber, Martin; Karger, Amy B; Kathmann, Eva C; Alekseev, Alexey E; Terzic, Andre

2002-04-19

344

Time-resolved measurements of intracellular ATP in the yeast Saccharomyces cerevisiae using a new type of nanobiosensor.  

PubMed

Adenosine 5'-triphosphate is a universal molecule in all living cells, where it functions in bioenergetics and cell signaling. To understand how the concentration of ATP is regulated by cell metabolism and in turn how it regulates the activities of enzymes in the cell it would be beneficial if we could measure ATP concentration in the intact cell in real time. Using a novel aptamer-based ATP nanosensor, which can readily monitor intracellular ATP in eukaryotic cells with a time resolution of seconds, we have performed the first on-line measurements of the intracellular concentration of ATP in the yeast Saccharomyces cerevisiae. These ATP measurements show that the ATP concentration in the yeast cell is not stationary. In addition to an oscillating ATP concentration, we also observe that the concentration is high in the starved cells and starts to decrease when glycolysis is induced. The decrease in ATP concentration is shown to be caused by the activity of membrane-bound ATPases such as the mitochondrial F(0)F(1) ATPase-hydrolyzing ATP and the plasma membrane ATPase (PMA1). The activity of these two ATPases are under strict control by the glucose concentration in the cell. Finally, the measurements of intracellular ATP suggest that 2-deoxyglucose (2-DG) may have more complex function than just a catabolic block. Surprisingly, addition of 2-DG induces only a moderate decline in ATP. Furthermore, our results suggest that 2-DG may inhibit the activation of PMA1 after addition of glucose. PMID:20880841

Ozalp, Veli C; Pedersen, Tina R; Nielsen, Lise J; Olsen, Lars F

2010-11-26

345

7 CFR 3300.4 - Definitions.  

Code of Federal Regulations, 2011 CFR

...means the Agreement on the International Carriage of Perishable...foodstuffs are carried. International carriage means transportation...test in an approved testing laboratory, and can thereby serve...ATP. U.S. ATP testing laboratory means a facility in...

2011-01-01

346

7 CFR 3300.4 - Definitions.  

...means the Agreement on the International Carriage of Perishable...foodstuffs are carried. International carriage means transportation...test in an approved testing laboratory, and can thereby serve...ATP. U.S. ATP testing laboratory means a facility in...

2014-01-01

347

7 CFR 3300.4 - Definitions.  

Code of Federal Regulations, 2012 CFR

...means the Agreement on the International Carriage of Perishable...foodstuffs are carried. International carriage means transportation...test in an approved testing laboratory, and can thereby serve...ATP. U.S. ATP testing laboratory means a facility in...

2012-01-01

348

7 CFR 3300.4 - Definitions.  

Code of Federal Regulations, 2013 CFR

...means the Agreement on the International Carriage of Perishable...foodstuffs are carried. International carriage means transportation...test in an approved testing laboratory, and can thereby serve...ATP. U.S. ATP testing laboratory means a facility in...

2013-01-01

349

7 CFR 3300.4 - Definitions.  

Code of Federal Regulations, 2010 CFR

...means the Agreement on the International Carriage of Perishable...foodstuffs are carried. International carriage means transportation...test in an approved testing laboratory, and can thereby serve...ATP. U.S. ATP testing laboratory means a facility in...

2010-01-01

350

A Kinetic Assay of Mitochondrial ATP-ADP Exchange Rate in Permeabilized Cells  

PubMed Central

We have previously described a method to measure ADP-ATP exchange rates in isolated mitochondria by recording the changes in free extramitochondrial [Mg2+] reported by a Mg2+-sensitive fluorescent indicator, exploiting the differential affinity of ADP and ATP to Mg2+. In this manuscript we describe a modification of this method suited for following ADP-ATP exchange rates in environments with competing reactions that interconvert adenine nucleotides, such as in permeabilized cells that harbor phosphorylases and kinases, ion pumps exhibiting substantial ATPase activity and myosin ATPase activity. Here we report that addition of BeF3? and Na3VO4 to media containing digitonin-permeabilized cells inhibit all ATP-ADP utilizing reactions, except the ANT-mediated mitochondrial ATP-ADP exchange. An advantage of this assay is that mitochondria that may have been also permeabilized by digitonin do not contribute to ATP consumption by the exposed F1Fo-ATPase, due to its sensitivity to BeF3? and Na3VO4. With this assay, ADP-ATP exchange rate mediated by the ANT in permeabilized cells is measured for the entire range of mitochondrial membrane potential titrated by stepwise additions of an uncoupler, and expressed as a function of citrate synthase activity per total amount of protein. PMID:20691655

Kawamata, Hibiki; Starkov, Anatoly A; Manfredi, Giovanni; Chinopoulos, Christos

2010-01-01

351

Cellular ATP levels and nitrogenase switchoff upon oxygen stress in chemostat cultures of Azotobacter vinelandii.  

PubMed Central

When Azotobacter vinelandii, growing diazotrophically in chemostat culture, was subjected to sudden increases in the ambient oxygen concentration (oxygen stress), nitrogenase activity was switched off and cellular ATP pools decreased at rates depending on the stress level. Following a fast decrease, the ATP pool approached a lower level. When the stress was released, these effects were reversed. The reversible decrease of the ATP pool upon oxygen stress could also be observed with cultures assimilating ammonium and, at the same time, fixing dinitrogen because of growth at a high C/N ratio but not with cultures growing only at the expense of ammonium. When strains OP and UW136 of A. vinelandii were subjected to long-term increases in ambient oxygen, the sizes of cellular ATP pools eventually started to increase to the level before stress and diazotrophic growth resumed. The cytochrome d-deficient mutant MK5 of A. vinelandii, however, impaired in aerotolerant diazotrophic growth, was unable to recover from stress on the basis of its ATP pool. The results suggest that adaptation to higher ambient oxygen depends on increased ATP synthesis requiring increased electron flow through the entire respiratory chain, which is possible only in combination with the more active, yet possibly uncoupled, branch terminated by cytochrome d. It is proposed that the decrease of the cellular ATP level under oxygen stress resulted from the increased energy and electron donor requirement of nitrogenase in reacting with oxygen. PMID:7665517

Linkerhägner, K; Oelze, J

1995-01-01

352

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

SciTech Connect

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.

Hattori, Motoyuki; Gouaux, Eric (Oregon HSU)

2012-10-24

353

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

PubMed Central

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

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

2012-01-01

354

Role of mitochondrial and sarcolemmal K(ATP) channels in ischemic preconditioning of the canine heart.  

PubMed

We tested whether mitochondrial or sarcolemmal ATP-sensitive K(+) (K(ATP)) channels play a key role in ischemic preconditioning (IP) in canine hearts. In open-chest beagle dogs, the left anterior descending artery was occluded four times for 5 min each with 5-min intervals of reperfusion (IP), occluded for 90 min, and reperfused for 6 h. IP as well as cromakalim and nicorandil (nonspecific K(ATP) channel openers) markedly limited infarct size (6.3 +/- 1.2, 8.9 +/- 1.9, and 7.2 +/- 1.6%, respectively) compared with the control group (40.9 +/- 4.1%). A selective mitochondrial K(ATP) channel blocker, 5-hydroxydecanoate, partially blunted the limitation of infarct size in the animals subjected to IP and those treated with cromakalim and nicorandil (21.6 +/- 3.8, 25.1 +/- 4.6, and 19.8 +/- 5.2%, respectively). A nonspecific K(ATP) channel blocker, glibenclamide, completely abolished the effect of IP (38.5 +/- 6.2%). Intracoronary or intravenous administration of a mitochondria-selective K(ATP) channel opener, diazoxide, at >100 micromol/l could only partially decrease infarct size (19.5 +/- 4.3 and 20.1 +/- 4.4%, respectively). In conclusion, mitochondrial and sarcolemmal K(ATP) channels independently play an important role in the limitation of infarct size by IP in the canine heart. PMID:11123240

Sanada, S; Kitakaze, M; Asanuma, H; Harada, K; Ogita, H; Node, K; Takashima, S; Sakata, Y; Asakura, M; Shinozaki, Y; Mori, H; Kuzuya, T; Hori, M

2001-01-01

355

Response of ATP sulfurylase and serine acetyltransferase towards cadmium in hyperaccumulator Sedum alfredii Hance*  

PubMed Central

We studied the responses of the activities of adenosine-triphosphate (ATP) sulfurylase (ATPS) and serine acetyltransferase (SAT) to cadmium (Cd) levels and treatment time in hyperaccumulating ecotype (HE) Sedum alfredii Hance, as compared with its non-hyperaccumulating ecotype (NHE). The results show that plant growth was inhibited in NHE but promoted in HE when exposed to high Cd level. Cd concentrations in leaves and shoots rapidly increased in HE rather than in NHE, and they became much higher in HE than in NHE along with increasing treatment time and Cd supply levels. ATPS activity was higher in HE than in NHE in all Cd treatments, and increased with increasing Cd supply levels in both HE and NHE when exposed to Cd treatment within 8 h. However, a marked difference of ATPS activity between HE and NHE was found with Cd treatment for 168 h, where ATPS activity increased in HE but decreased in NHE. Similarly, SAT activity was higher in HE than in NHE at all Cd treatments, but was more sensitive in NHE than in HE. Both ATPS and SAT activities in NHE leaves tended to decrease with increasing treatment time after 8 h at all Cd levels. The results reveal the different responses in sulfur assimilation enzymes and Cd accumulation between HE and NHE. With increasing Cd stress, the activities of sulfur assimilation enzymes (ATPS and SAT) were induced in HE, which may contribute to Cd accumulation in the hyperaccumulator Sedum alfredii Hance. PMID:19353742

Guo, Wei-dong; Liang, Jun; Yang, Xiao-e; Chao, Yue-en; Feng, Ying

2009-01-01

356

Wilson Disease Protein ATP7B Utilizes Lysosomal Exocytosis to Maintain Copper Homeostasis  

PubMed Central

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

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

357

Liver ATP Synthesis Is Lower and Relates to Insulin Sensitivity in Patients With Type 2 Diabetes  

PubMed Central

OBJECTIVE Steatosis associates with insulin resistance and may even predict type 2 diabetes and cardiovascular complications. Because muscular insulin resistance relates to myocellular fat deposition and disturbed energy metabolism, we hypothesized that reduced hepatic ATP turnover (fATP) underlies insulin resistance and elevated hepatocellular lipid (HCL) contents. RESEARCH DESIGN AND METHODS We measured hepatic fATP using 31P magnetic resonance spectroscopy in patients with type 2 diabetes and age- and body mass–matched controls. Peripheral (M and M/I) and hepatic (suppression of endogenous glucose production) insulin sensitivity were assessed with euglycemic-hyperinsulinemic clamps. RESULTS Diabetic individuals had 29% and 28% lower peripheral and hepatic insulin sensitivity as well as 42% reduced fATP than controls. After adjusting for HCL, fATP correlated positively with peripheral and hepatic insulin sensitivity but negatively with waist circumference, BMI, and fasting plasma glucose. Multiple regression analysis identified waist circumference as an independent predictor of fATP and inorganic phosphate (PI) concentrations, explaining 65% (P = 0.001) and 56% (P = 0.003) of the variations. Hepatocellular PI primarily determined the alterations in fATP. CONCLUSIONS In patients with type 2 diabetes, insulin resistance relates to perturbed hepatic energy metabolism, which is at least partly accounted for by fat depots. PMID:21216854

Schmid, Albrecht Ingo; Szendroedi, Julia; Chmelik, Marek; Krššák, Martin; Moser, Ewald; Roden, Michael

2011-01-01

358

Extracellular ATP inhibits Schwann cell dedifferentiation and proliferation in an ex vivo model of Wallerian degeneration  

SciTech Connect

Highlights: Black-Right-Pointing-Pointer ATP-treated sciatic explants shows the decreased expression of p75NGFR. Black-Right-Pointing-Pointer Extracellular ATP inhibits the expression of phospho-ERK1/2. Black-Right-Pointing-Pointer Lysosomal exocytosis is involved in Schwann cell dedifferentiation. Black-Right-Pointing-Pointer Extracellular ATP blocks Schwann cell proliferation in sciatic explants. -- Abstract: After nerve injury, Schwann cells proliferate and revert to a phenotype that supports nerve regeneration. This phenotype-changing process can be viewed as Schwann cell dedifferentiation. Here, we investigated the role of extracellular ATP in Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Using several markers of Schwann cell dedifferentiation and proliferation in sciatic explants, we found that extracellular ATP inhibits Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Furthermore, the blockage of lysosomal exocytosis in ATP-treated sciatic explants is sufficient to induce Schwann cell dedifferentiation. Together, these findings suggest that ATP-induced lysosomal exocytosis may be involved in Schwann cell dedifferentiation.

Shin, Youn Ho; Lee, Seo Jin [Department of Anatomy, College of Medicine, Kyung Hee University, Heogi-Dong 1, Dongdaemun-Gu, Seoul 130-701 (Korea, Republic of)] [Department of Anatomy, College of Medicine, Kyung Hee University, Heogi-Dong 1, Dongdaemun-Gu, Seoul 130-701 (Korea, Republic of); Jung, Junyang, E-mail: jjung@khu.ac.kr [Department of Anatomy, College of Medicine, Kyung Hee University, Heogi-Dong 1, Dongdaemun-Gu, Seoul 130-701 (Korea, Republic of)] [Department of Anatomy, College of Medicine, Kyung Hee University, Heogi-Dong 1, Dongdaemun-Gu, Seoul 130-701 (Korea, Republic of)

2013-01-11

359

Evidence for Ca2+-Regulated ATP Release in Gastrointestinal Stromal Tumors  

PubMed Central

Gastrointestinal stromal tumors (GISTs) are thought to originate from the electrically active pacemaker cells of the gastrointestinal tract. Despite the presence of synaptic-like vesicles and proteins involved in cell secretion it remains unclear whether GIST cells possess regulated release mechanisms. The GIST tumor cell line GIST882 was used as a model cell system, and stimulus-release coupling was investigated by confocal microscopy of cytoplasmic free Ca2+ concentration ([Ca2+]i), flow cytometry, and luminometric measurements of extracellular ATP. We demonstrate that GIST cells have an intact intracellular Ca2+-signaling pathway that regulates ATP release. Cell viability and cell membrane integrity was preserved, excluding ATP leakage due to cell death and suggesting active ATP release. The stimulus-secretion signal transduction is at least partly dependent on Ca2+ influx since exclusion of extracellular Ca2+ diminishes the ATP release. We conclude that measurements of ATP release in GISTs may be a useful tool for dissecting the signal transduction pathway, mapping exocytotic components, and possibly for the development and evaluation of drugs. Additionally, release of ATP from GISTs may have importance for tumor tissue homeostasis and immune surveillance escape. PMID:23499741

Berglund, Erik; Berglund, David; Akcakaya, Pinar; Ghaderi, Mehran; Daré, Elisabetta; Berggren, Per-Olof; Köhler, Martin; Aspinwall, Craig A.; Lui, Weng-Onn; Zedenius, Jan; Larsson, Catharina; Bränström, Robert

2013-01-01

360

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

PubMed Central

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

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

2015-01-01

361

VARIABLY MODULATED GATING OF THE 26S PROTEASOME BY ATP AND POLYUBIQUITIN  

PubMed Central

SYNOPSIS The 26S proteasome is a 2,500,000-dalton protease complex that degrades polyubiquitylated proteins by a mechanism that requires ATP hydrolysis. It also degrades short non-ubiquitylated peptides and certain unstructured proteins by an energy-independent mechanism that requires bound ATP to maintain its component subcomplexes, the 20S proteasome and PA700, in a functionally assembled state. Proteolysis of both types of substrates requires PA700-induced opening of reversible gates at substrate access pores of the 20S proteasome. Here we demonstrate that the rate of peptide substrate hydrolysis, a functional monitor of gate opening, is regulated variably by multiple effectors. ATP?S and other nonhydrolyzable ATP analogs increased peptide substrate hydrolysis by intact 26S proteasome. Thus, nucleotides that maintained 26S proteasome structure but did not support ATP hydrolysis or the degradation of polyubiquitylated proteins promoted enhanced rates of peptide hydrolysis. Polyubiquitin and a peptoid that binds selectively to a single ATPase subunit of PA700 also increased rates of peptide hydrolysis but had disparate effects on rates of ATP hydrolysis. The effect of polyubiquitin was specific for ubiquitin-ubiquitin linkages that supported proteolysis of protein substrates. These results indicate that gating of the 26S proteasome is not a simple two-state process but can be variably modulated. Our results suggest that modulated gating of the proteasome may be an important element of the mechanism of proteolysis of polyubiquitylated proteins. PMID:19435460

Li, Xiaohua; DeMartino, George N.

2010-01-01

362

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

PubMed

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

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

2015-01-01

363

Identification of an Arabidopsis plasma membrane-located ATP transporter important for anther development.  

PubMed

ATP acts as an extracellular signal molecule in plants. However, the nature of the mechanisms that export this compound into the apoplast are under debate. We identified the protein PM-ANT1 as a candidate transporter able to mediate ATP export. PM-ANT1 joins the mitochondrial carrier family, lacks an N-terminal amino acid extension required for organelle localization, and locates to the plasma membrane. Recombinant PM-ANT1 transports ATP, and the gene is substantially expressed in mature pollen grains. Artificial microRNA (amiRNA) mutants show reduced silique length and less seeds per silique but increased seed weight associated with unchanged pollen viability. Anthers from amiRNA mutants exhibited a normal early development, but stomium breakage is inhibited, leading to impaired anther dehiscence. This results in reduced self-pollination and thus decreased fertilization efficiency. amiRNA pollen grains showed increased intracellular ATP levels but decreased extracellular ATP levels. The latter effects are in line with transport properties of recombinant PM-ANT1, supporting in planta that functional PM-ANT1 resides in the plasma membrane and concur with the PM-ANT1 expression pattern. We assume that PM-ANT1 contributes to ATP export during pollen maturation. ATP export may serve as an extracellular signal required for anther dehiscence and is a novel factor critical for pollination and autogamy. PMID:21540435

Rieder, Benjamin; Neuhaus, H Ekkehard

2011-05-01

364

Selective Migration of Subpopulations of Bone Marrow Cells along an SDF-1? and ATP Gradient  

PubMed Central

Both stem cell chemokine stromal cell-derived factor-1? (SDF-1?) and extracellular nucleotides such as adenosine triphosphate (ATP) are increased in ischemic myocardium. Since ATP has been reported to influence cell migration, we analysed the migratory response of bone marrow cells towards a combination of SDF-1 and ATP. Total nucleated cells (BM-TNCs) were isolated from bone marrow of cardiac surgery patients. Migration assays were performed in vitro. Subsequently, migrated cells were subjected to multicolor flow cytometric analysis of CD133, CD34, CD117, CD184, CD309, and CD14 expression. BM-TNCs migrated significantly towards a combination of SDF-1 and ATP. The proportions of CD34+ cells as well as subpopulations coexpressing multiple stem cell markers were selectively enhanced after migration towards SDF-1 or SDF-1 + ATP. After spontaneous migration, significantly fewer stem cells and CD184+ cells were detected. Direct incubation with SDF-1 led to a reduction of CD184+ but not stem cell marker-positive cells, while incubation with ATP significantly increased CD14+ percentage. In summary, we found that while a combination of SDF-1 and ATP elicited strong migration of BM-TNCs in vitro, only SDF-1 was responsible for selective attraction of hematopoietic stem cells. Meanwhile, spontaneous migration of stem cells was lower compared to BM-TNCs or monocytes. PMID:25610653

Laupheimer, Michael; Skorska, Anna; Große, Jana; Tiedemann, Gudrun; Steinhoff, Gustav; David, Robert; Lux, Cornelia A.

2014-01-01

365

Kinetic and thermodynamic control of ATP synthesis by sarcoplasmic reticulum adenosinetriphosphatase.  

PubMed

Several experimental parameters, critical to the analysis of ATP synthesis by sarcoplasmic reticulum ATPase, were determined experimentally. 1) The phosphorylated enzyme intermediate obtained with acetylphosphate in the presence of a Ca2+ gradient was shown to be entirely ADP sensitive but quite stable in the absence of added ADP. On the contrary, the phosphoenzyme obtained with ATP is unstable due to the ADP formed during the phosphoryl transfer reaction. For this reason, addition of ADP to [32P]phosphoenzyme obtained with [32P]acetylphosphate provides the simplest conditions for kinetic studies on [gamma-32P]ATP synthesis. 2) The dissociation rate constant of newly synthesized ATP (in the reverse direction of the ATPase cycle) was measured experimentally and found to be 16 s-1. This value agrees well with the dissociation rate constant determined for adenyl-5'-yl imidodiphosphate bound to this enzyme. 3) ATP synthesis observed in the absence of a Ca2+ gradient was shown to be a kinetic overshoot due to ligand-induced perturbation of a limited number of partial reactions and occurring before equilibration of the entire system. Most of the ATP formed under these conditions was subsequently hydrolyzed as the overall equilibrium was reached. 4) Based on these and other (previously characterized) parameters, satisfactory simulations of single and multiple cycle ATP synthesis, in the presence and in the absence of a Ca2+ gradient, were obtained. PMID:2958449

Teruel, J A; Kurzmack, M; Inesi, G

1987-09-25

366

Gating of the CFTR Cl- channel by ATP-driven nucleotide-binding domain dimerisation.  

PubMed

The cystic fibrosis transmembrane conductance regulator (CFTR) plays a fundamental role in fluid and electrolyte transport across epithelial tissues. Based on its structure, function and regulation, CFTR is an ATP-binding cassette (ABC) transporter. These transporters are assembled from two membrane-spanning domains (MSDs) and two nucleotide-binding domains (NBDs). In the vast majority of ABC transporters, the NBDs form a common engine that utilises the energy of ATP hydrolysis to pump a wide spectrum of substrates through diverse transmembrane pathways formed by the MSDs. By contrast, in CFTR the MSDs form a pathway for passive anion flow that is gated by cycles of ATP binding and hydrolysis by the NBDs. Here, we consider how the interaction of ATP with two ATP-binding sites, formed by the NBDs, powers conformational changes in CFTR structure to gate the channel pore. We explore how conserved sequences from both NBDs form ATP-binding sites at the interface of an NBD dimer and highlight the distinct roles that each binding site plays during the gating cycle. Knowledge of how ATP gates the CFTR Cl- channel is critical for understanding CFTR's physiological role, its malfunction in disease and the mechanism of action of small molecules that modulate CFTR channel gating. PMID:19332488

Hwang, Tzyh-Chang; Sheppard, David N

2009-05-15

367

Definitions of Entomological Terms  

NSDL National Science Digital Library

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.

0000-00-00

368

Food Allergies DEFINITIONS  

E-print Network

are a digestive system response and are much more common than food allergies. Food Allergy vs. Food Intolerance's digestive system or when a person is unable to properly digest a food. SYMPTOMS: Symptoms of an AllergicFood Allergies DEFINITIONS: Definition of a Food Allergy: Immune system response to a food

Maxwell, Bruce D.

369

Functional characterization of missense mutations in ATP7B: Wilson disease mutation or normal variant?  

PubMed Central

Wilson disease is an autosomal recessive disorder of copper transport that causes hepatic and/or neurological disease resulting from copper accumulation in the liver and brain. The protein defective in this disorder is a putative copper-transporting P-type ATPase, ATP7B. More than 100 mutations have been identified in the ATP7B gene of patients with Wilson disease. To determine the effect of Wilson disease missense mutations on ATP7B function, we have developed a yeast complementation assay based on the ability of ATP7B to complement the high-affinity iron-uptake deficiency of the yeast mutant ccc2. We characterized missense mutations found in the predicted membrane-spanning segments of ATP7B. Ten mutations have been made in the ATP7B cDNA by site-directed mutagenesis: five Wilson disease missense mutations, two mutations originally classified as possible disease-causing mutations, two putative ATP7B normal variants, and mutation of the cysteine-proline-cysteine (CPC) motif conserved in heavy-metal-transporting P-type ATPases. All seven putative Wilson disease mutants tested were able to at least partially complement ccc2 mutant yeast, indicating that they retain some ability to transport copper. One mutation was a temperature-sensitive mutation that was able to complement ccc2 mutant yeast at 30 degreesC but was unable to complement at 37 degreesC. Mutation of the CPC motif resulted in a nonfunctional protein, which demonstrates that this motif is essential for copper transport by ATP7B. Of the two putative ATP7B normal variants tested, one resulted in a nonfunctional protein, which suggests that it is a disease-causing mutation. PMID:9837819

Forbes, J R; Cox, D W

1998-01-01

370

Critical roles of interdomain interactions for modulatory ATP binding to sarcoplasmic reticulum Ca2+-ATPase.  

PubMed

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

Clausen, Johannes D; Holdensen, Anne Nyholm; Andersen, Jens Peter

2014-10-17

371

Extracellular ATP activates MAPK and ROS signaling during injury response in the fungus Trichoderma atroviride  

PubMed Central

The response to mechanical damage is crucial for the survival of multicellular organisms, enabling their adaptation to hostile environments. Trichoderma atroviride, a filamentous fungus of great importance in the biological control of plant diseases, responds to mechanical damage by activating regenerative processes and asexual reproduction (conidiation). During this response, reactive oxygen species (ROS) are produced by the NADPH oxidase complex. To understand the underlying early signaling events, we evaluated molecules such as extracellular ATP (eATP) and Ca2+ that are known to trigger wound-induced responses in plants and animals. Concretely, we investigated the activation of mitogen-activated protein kinase (MAPK) pathways by eATP, Ca2+, and ROS. Indeed, application of exogenous ATP and Ca2+ triggered conidiation. Furthermore, eATP promoted the Nox1-dependent production of ROS and activated a MAPK pathway. Mutants in the MAPK-encoding genes tmk1 and tmk3 were affected in wound-induced conidiation, and phosphorylation of both Tmk1 and Tmk3 was triggered by eATP. We conclude that in this fungus, eATP acts as a damage-associated molecular pattern (DAMP). Our data indicate the existence of an eATP receptor and suggest that in fungi, eATP triggers pathways that converge to regulate asexual reproduction genes that are required for injury-induced conidiation. By contrast, Ca2+ is more likely to act as a downstream second messenger. The early steps of mechanical damage response in T. atroviride share conserved elements with those known from plants and animals. PMID:25484887

Medina-Castellanos, Elizabeth; Esquivel-Naranjo, Edgardo U.; Heil, Martin; Herrera-Estrella, Alfredo

2014-01-01

372

Inflammation Promotes Airway Epithelial ATP Release via Calcium-Dependent Vesicular Pathways  

PubMed Central

ATP in airway surface liquid (ASL) controls mucociliary clearance functions via the activation of airway epithelial purinergic receptors. However, abnormally elevated ATP levels have been reported in inflamed airways, suggesting that excessive ATP in ASL contributes to airway inflammation. Despite these observations, little is known about the mechanisms of ATP accumulation in the ASL covering inflamed airways. In this study, links between cystic fibrosis (CF)–associated airway inflammation and airway epithelial ATP release were investigated. Primary human bronchial epithelial (HBE) cells isolated from CF lungs exhibited enhanced IL-8 secretion after 6 to 11 days, but not 28 to 35 days, in culture, compared with normal HBE cells. Hypotonic cell swelling–promoted ATP release was increased in 6- to 11-day-old CF HBE cells compared with non-CF HBE cells, but returned to normal values after 28 to 35 days in culture. The exposure of non-CF HBE cells to airway secretions isolated from CF lungs, namely, sterile supernatants of mucopurulent material (SMM), also caused enhanced IL-8 secretion and increased ATP release. The SMM-induced increase in ATP release was sensitive to Ca2+ chelation and vesicle trafficking/exocytosis inhibitors, but not to pannexin inhibition. Transcript levels of the vesicular nucleotide transporter, but not pannexin 1, were up-regulated after SMM exposure. SMM-treated cultures displayed increased basal mucin secretion, but mucin secretion was not enhanced in response to hypotonic challenge after the exposure of cells to either vehicle or SMM. We propose that CF airway inflammation up-regulates the capacity of airway epithelia to release ATP via Ca2+-dependent vesicular mechanisms not associated with mucin granule secretion. PMID:23763446

Okada, Seiko F.; Ribeiro, Carla M. P.; Sesma, Juliana I.; Seminario-Vidal, Lucia; Abdullah, Lubna H.; van Heusden, Catharina; Lazarowski, Eduardo R.

2013-01-01

373

Mitochondrial inefficiencies and anoxic ATP hydrolysis capacities in diabetic rat heart.  

PubMed

As ~80% of diabetic patients die from heart failure, an understanding of diabetic cardiomyopathy is crucial. Mitochondria occupy 35-40% of the mammalian cardiomyocyte volume and supply 95% of the heart's ATP, and diabetic heart mitochondria show impaired structure, arrangement, and function. We predict that bioenergetic inefficiencies are present in diabetic heart mitochondria; therefore, we explored mitochondrial proton and electron handling by linking oxygen flux to steady-state ATP synthesis, reactive oxygen species (ROS) production, and mitochondrial membrane potential (??) within rat heart tissues. Sprague-Dawley rats were injected with streptozotocin (STZ, 55 mg/kg) to induce type 1 diabetes or an equivalent volume of saline (control, n = 12) and fed standard rat chow for 8 wk. By coupling high-resolution respirometers with purpose-built fluorometers, we followed Magnesium Green (ATP synthesis), Amplex UltraRed (ROS production), and safranin-O (??). Relative to control rats, the mass-specific respiration of STZ-diabetic hearts was depressed in oxidative phosphorylation (OXPHOS) states. Steady-state ATP synthesis capacity was almost one-third lower in STZ-diabetic heart, which, relative to oxygen flux, equates to an estimated 12% depression in OXPHOS efficiency. However, with anoxic transition, STZ-diabetic and control heart tissues showed similar ATP hydrolysis capacities through reversal of the F1F0-ATP synthase. STZ-diabetic cardiac mitochondria also produced more net ROS relative to oxygen flux (ROS/O) in OXPHOS. While ?? did not differ between groups, the time to develop ?? with the onset of OXPHOS was protracted in STZ-diabetic mitochondria. ROS/O is higher in lifelike OXPHOS states, and potential delays in the time to develop ?? may delay ATP synthesis with interbeat fluctuations in ADP concentrations. Whereas diabetic cardiac mitochondria produce less ATP in normoxia, they consume as much ATP in anoxic infarct-like states. PMID:24920675

Pham, Toan; Loiselle, Denis; Power, Amelia; Hickey, Anthony J R

2014-09-15

374

Thermophilic ATP synthase has a decamer c-ring: Indication of noninteger 10:3 H+/ATP ratio and permissive elastic coupling  

PubMed Central

In a rotary motor FoF1-ATP synthase that couples H+ transport with ATP synthesis/hydrolysis, it is thought that an Foc subunit oligomer ring (c-ring) in the membrane rotates as protons pass through Fo and a 120° rotation produces one ATP at F1. Despite several structural studies, the copy number of Foc subunits in the c-ring has not been determined for any functional FoF1. Here, we have generated and isolated thermophilic Bacillus FoF1, each containing genetically fused 2-mer–14-mer c (c2–c14). Among them, FoF1 containing c2, c5, or c10 showed ATP-synthesis and other activities. When F1 was removed, Fo containing c10 worked as an H+ channel but Fos containing c9, c11 or c12 did not. Thus, the c-ring of functional FoF1 of this organism is a decamer. The inevitable consequence of this finding is noninteger ratios of rotation step sizes of F1/Fo (120°/36°) and of H+/ATP (10:3). This step-mismatch necessitates elastic twisting of the rotor shaft (and/or the side stalk) during rotation and permissive coupling between unit rotations by H+ transport at Fo and elementary events in catalysis at F1. PMID:15302927

Mitome, Noriyo; Suzuki, Toshiharu; Hayashi, Shigehiko; Yoshida, Masasuke

2004-01-01

375

The definition of cross polarization  

Microsoft Academic Search

There are at least three different definitions of cross polarization used in the literature. The alternative definitions are discussed with respect to several applications, and the definition which corresponds to one standard measurement practice is proposed as the best choice.

A. Ludwig

1973-01-01

376

ATP molecule ATP molecule  

E-print Network

carbon atoms are grey, hydrogen white, oxygen red, nitrogen blue, and phospho- rus orange. The situation and much more, but this approach is restricted to systems containing few atoms. The description

377

31P NMR Methods for the Direct Determination of ADP in the Presence of ATP  

NASA Astrophysics Data System (ADS)

A new method is presented for the direct measurement of the amount of ADP in the presence of ATP by selectively eliminating the ?- and ?-ATP signals. The method is compared with other methods, both experimentally and theoretically, using the product-operator formalism. An analysis of the effect of B1inhomogeneity on the efficiency of the methods is also given. Experimental results obtained using adiabatic pulses to compensate for such effects are shown. The accuracy of this new method is demonstrated by measuring various ADP concentrations in a series of solutions containing ADP and ATP.

Ben-Bashat, Dafna; Shinar, Hadassah; Navon, Gil

378

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

PubMed Central

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

Corriden, Ross; Insel, Paul A.

2011-01-01

379

Effects of sulphuric compounds on the ATP content of the peat moss Sphagnum fuscum  

SciTech Connect

Luminometric determination of ATP in the photosynthetic tissues of the peat moss Sphagnum fuscum proved to be a suitable technique in studying the effects of bisulphite and sulphate on the metabolism of the mosses. The method has the advantage that it is rapid and easy to perform, and that the results are reliable and equal with those obtained by using other techniques. Bisulphite (HSO/sub 3//sup -/) caused marked reductions in the ATP contents at the 1 mM level, and the 5 mM level was clearly detrimental to the energy metabolism of the mosses. In contrast, sulphate (SO/sub 4//sup 2 -/) increased the ATP contents markedly.

Aulio, K.

1984-01-01

380

Structural mechanism of the ATP-induced dissociation of rigor myosin from actin  

PubMed Central

Myosin is a true nanomachine, which produces mechanical force from ATP hydrolysis by cyclically interacting with actin filaments in a four-step cycle. The principle underlying each step is that structural changes in separate regions of the protein must be mechanically coupled. The step in which myosin dissociates from tightly bound actin (the rigor state) is triggered by the 30 ? distant binding of ATP. Large conformational differences between the crystal structures make it difficult to perceive the coupling mechanism. Energetically accessible transition pathways computed at atomic detail reveal a simple coupling mechanism for the reciprocal binding of ATP and actin. PMID:21518908

Kühner, Sebastian; Fischer, Stefan

2011-01-01

381

Capillary electrophoresis with laser-induced fluorescence detection for ATP quantification in spermatozoa and oocytes  

Microsoft Academic Search

We describe a new capillary electrophoresis laser-induced fluorescence (CE-LIF) method for the quantification of adenosine\\u000a 5?-triphosphate (ATP) in spermatozoa and oocytes. The optimization of the precapillary derivatization reaction between ATP\\u000a and 4,4-difluoro-5,7-dimethyl-4-bora-3a,4adiaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) has been described. BODIPY-ATP conjugate was analysed\\u000a in an uncoated fused silica capillary of 75 ?m ID and 50 cm effective length

Angelo Zinellu; Valeria Pasciu; Salvatore Sotgia; Bastianina Scanu; Fiammetta Berlinguer; Giovanni Leoni; Sara Succu; Ignazio Cossu; Eraldo Sanna Passino; Salvatore Naitana; Luca Deiana; Ciriaco Carru

2010-01-01

382

Estradiol inhibits atp-induced intracellular calcium concentration increase in dorsal root ganglia neurons.  

PubMed

Estrogen has been implicated in modulation of pain processing. Although this modulation occurs within the CNS, estrogen may also act on primary afferent neurons whose cell bodies are located within the dorsal root ganglia (DRG). Primary cultures of rat DRG neurons were loaded with Fura-2 and tested for ATP-induced changes in intracellular calcium concentration ([Ca(2+)](i)) by fluorescent ratio imaging. ATP, an algesic agent, induces [Ca(2+)](i) changes via activation of purinergic 2X (P2X) type receptors and voltage-gated Ca(2+) channels (VGCC). ATP (10 microM) caused increased [Ca(2+)](i) transients (226.6+/-16.7 nM, n = 42) in 53% of small to medium DRG neurons. A 5-min incubation with 17 beta-estradiol (100 nM) inhibited ATP-induced [Ca(2+)](i) (164+/-14.6 nM, P<0.05) in 85% of the ATP-responsive DRG neurons, whereas the inactive isomer 17 alpha-estradiol had no effect. Both the mixed agonist/antagonist tamoxifen (1 microM) and specific estrogen receptor antagonist ICI 182780 (1 microM) blocked the estradiol inhibition of ATP-induced [Ca(2+)](i) transients. Estradiol coupled to bovine serum albumin, which does not diffuse through the plasma membrane, blocked ATP-induced [Ca(2+)](i), suggesting that estradiol acts at a membrane-associated estrogen receptor. Attenuation of [Ca(2+)](i) transients was mediated by estrogen action on VGCC. Nifedipine (10 microM), an L-type VGCC antagonist mimicked the effect of estrogen and when co-administered did not increase the estradiol inhibition of ATP-induced [Ca(2+)](i) transients. N- and P-type VGCC antagonists omega-conotoxin GVIA (1 microM) and omega-agatoxin IVA (100 nM), attenuated the ATP-induced [Ca(2+)](i) transients. Co-administration of these blockers with estrogen induced a further decrease of the ATP-induced [Ca(2+)](i) flux. Together, these results suggest that although ATP stimulation of P2X receptors activates L-, N-, and P-type VGCC, estradiol primarily blocks L-type VGCC. The estradiol regulation of this ATP-induced [Ca(2+)](i) transients suggests a mechanism through which estradiol may modulate nociceptive signaling in the peripheral nervous system. PMID:12732239

Chaban, V V; Mayer, E A; Ennes, H S; Micevych, P E

2003-01-01

383

Topography of the yeast ATP synthase F0 sector.  

PubMed

The interaction between the hydrophilic C-terminal part of subunit 4 (subunit b) and OSCP, which are two components of the connecting stalk of the yeast ATP synthase, was shown after reconstitution of the two over-expressed proteins and by the two-hybrid method. The organization of a part of the F0 sector was studied by the use of mutants containing cysteine residues in a loop connecting the two N-terminal postulated membrane-spanning segments. Labelling of the mutated subunits 4 by a maleimide fluorescent probe revealed that the sulfhydryl groups were modified upon incubation of intact mitochondria. In addition, non-permeant maleimide reagents labeled subunit 4D54C, thus showing a location of this residue in the intermembrane space. Cross-linking experiments revealed the proximity of subunits 4 and f. In addition, a disulfide bridge between subunit 4D54C and subunit 6 was evidenced, thus demonstrating near-neighbor relationships of the two subunits and a location of the N-terminal part of the mitochondrially-encoded subunit 6 in the intermembrane space. PMID:9893937

Velours, J; Spannagel, C; Chaignepain, S; Vaillier, J; Arselin, G; Graves, P V; Velours, G; Camougrand, N

1998-10-01

384

Redox regulation of the rotation of F(1)-ATP synthase.  

PubMed

In F(1)-ATPase, the smallest known motor enzyme, unidirectional rotation of the central axis subunit gamma is coupled to ATP hydrolysis. In the present study, we report the redox switching of the rotation of this enzyme. For this purpose, the switch region from the gamma subunit of the redox-sensitive chloroplast F(1)-ATPase was introduced into the bacterial F(1)-ATPase. The ATPase activity of the obtained complex was increased up to 3-fold upon reduction (Bald, D., Noji, H., Stumpp, M. T., Yoshida, M. & Hisabori, T. (2000) J. Biol. Chem. 275, 12757-12762). Here, we successfully observed the modulation of rotation of gamma in this chimeric complex by changes in the redox conditions. In addition we revealed that the suppressed enzymatic activity of the oxidized F(1)-ATPase complex was characterized by more frequent long pauses in the rotation of the gamma subunit. These findings obtained by the single molecule analysis therefore provide new insights into the mechanisms of enzyme regulation. PMID:11518700

Bald, D; Noji, H; Yoshida, M; Hirono-Hara, Y; Hisabori, T

2001-10-26

385

Adenylation enzyme characterization using ?-18O4-ATP pyrophosphate exchange  

PubMed Central

SUMMARY We present here a rapid, highly sensitive nonradioactive assay for adenylation enzyme selectivity determination and characterization. This method measures the isotopic back exchange of unlabeled pyrophosphate into ?–18O4-labeled ATP via MALDI-TOFMS, ESI-LC/MS or ESI-LC/MS/MS and is demonstrated for both nonribosomal (TycA, ValA) and ribosomal synthetases (TrpRS, LysRS) of known specificity. This low volume (6?L) method detects as little as 0.01% (600 fmol) exchange, comparable in sensitivity to previously reported radioactive assays and readily adaptable to kinetics measurements and high throughput analysis of a wide spectrum of synthetases. Finally, a previously uncharacterized A-T didomain from anthramycin biosynthesis in the thermophile S. refuinius was demonstrated to selectively activate 4-methyl-3-hydroxyanthranilic acid at 47 °C, providing biochemical evidence for a new aromatic ?–amino acid activating adenylation domain and the first functional analysis of the anthramycin biosynthetic gene cluster. PMID:19477411

Phelan, Vanessa V.; Du, Yu; McLean, John A.; Bachmann, Brian O.

2009-01-01

386

The bizarre pharmacology of the ATP release channel pannexin1.  

PubMed

Pannexins were originally thought to represent a second and redundant family of gap junction proteins in addition to the well characterized connexins. However, it is now evident that pannexins function as unapposed membrane channels and the major role of Panx1 is that of an ATP release channel. Despite the contrasting functional roles, connexins, innexins and pannexins share pharmacological properties. Most gap junction blockers also attenuate the function of Panx1, including carbenoxolone, mefloquine and flufenamic acid. However, in contrast to connexin based gap junction channels, Panx1 channel activity can be attenuated by several groups of drugs hitherto considered very specific for other proteins. The drugs affecting Panx1 channels include several transport inhibitors, chloride channel blockers, mitochondrial inhibitors, P2X7 receptor ligands, inflammasome inhibitors and malaria drugs. These observations indicate that Panx1 may play an extended role in a wider spectrum of physiological functions. Alternatively, Panx1 may share structural domains with other proteins, not readily revealed by sequence alignments. This article is part of the Special Issue Section entitled 'Current Pharmacology of Gap Junction Channels and Hemichannels'. PMID:23499662

Dahl, Gerhard; Qiu, Feng; Wang, Junjie

2013-12-01

387

ATP-Binding Cassette Efflux Transporters in Human Placenta  

PubMed Central

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

Ni, Zhanglin; Mao, Qingcheng

2010-01-01

388

ATP-dependent nucleosome unwrapping catalyzed by human RAD51  

PubMed Central

Double-strand breaks (DSB) occur in chromatin following replication fork collapse and chemical or physical damage [Symington and Gautier (Double-strand break end resection and repair pathway choice. Annu. Rev. Genet. 2011;45:247–271.)] and may be repaired by homologous recombination (HR) and non-homologous end-joining. Nucleosomes are the fundamental units of chromatin and must be remodeled during DSB repair by HR [Andrews and Luger (Nucleosome structure(s) and stability: variations on a theme. Annu. Rev. Biophys. 2011;40:99–117.)]. Physical initiation of HR requires RAD51, which forms a nucleoprotein filament (NPF) that catalyzes homologous pairing and strand exchange (recombinase) between DNAs that ultimately bridges the DSB gap [San Filippo, Sung and Klein. (Mechanism of eukaryotic HR. Annu. Rev. Biochem. 2008;77:229–257.)]. RAD51 forms an NPF on single-stranded DNA and double-stranded DNA (dsDNA). Although the single-stranded DNA NPF is essential for recombinase initiation, the role of the dsDNA NPF is less clear. Here, we demonstrate that the human RAD51 (HsRAD51) dsDNA NPF disassembles nucleosomes by unwrapping the DNA from the core histones. HsRAD51 that has been constitutively or biochemically activated for recombinase functions displays significantly reduced nucleosome disassembly activity. These results suggest that HsRAD51 can perform ATP hydrolysis-dependent nucleosome disassembly in addition to its recombinase functions. PMID:23757189

North, Justin A.; Amunugama, Ravindra; Klajner, Marcelina; Bruns, Aaron N.; Poirier, Michael G.; Fishel, Richard

2013-01-01

389

An exonuclease I-based label-free fluorometric aptasensor for adenosine triphosphate (ATP) detection with a wide concentration range.  

PubMed

A novel aptamer-based label-free assay for sensitive and selective detection of ATP was developed. This assay employs a new aptamer/fluorescent probe system that shows resistance to exonuclease I (Exo I) digestion upon binding to ATP molecules. In the absence of ATP, the complex between the ATP-binding aptamer (ATP-aptamer) and a DNA binding dye, berberine, is digested upon the addition of exonuclease I, leading to the release of berberine into solution and consequently, quenched berberine fluorescence. In the presence of ATP, the ATP-binding aptamer folds into a G-quadruplex structure that is resistant to Exo I digestion. Accordingly, berberine is protected in the G-quadruplex structure and high fluorescence intensity is observed. As such, based on the fluorescence signal change, a label-free fluorescence assay for ATP was developed. Factors affecting the analysis of ATP including the concentration of ATP-binding aptamer, reaction time, temperature and the concentration of Exo I were comprehensively investigated. Under optimal conditions, the fluorescence intensity of the sensing system displayed a response for ATP in a wide range up to 17.5 mM with a detection limit of 140 nM. PMID:25113049

Wei, Yanli; Chen, Yanxia; Li, Huanhuan; Shuang, Shaomin; Dong, Chuan; Wang, Gufeng

2015-01-15

390

Conformation-selective ATP-competitive inhibitors control regulatory interactions and noncatalytic functions of mitogen-activated protein kinases.  

PubMed

Most potent protein kinase inhibitors act by competing with ATP to block the phosphotransferase activity of their targets. However, emerging evidence demonstrates that ATP-competitive inhibitors can affect kinase interactions and functions in ways beyond blocking catalytic activity. Here, we show that stabilizing alternative ATP-binding site conformations of the mitogen-activated protein kinases (MAPKs) p38? and Erk2 with ATP-competitive inhibitors differentially, and in some cases divergently, modulates the abilities of these kinases to interact with upstream activators and deactivating phosphatases. Conformation-selective ligands are also able to modulate Erk2's ability to allosterically activate the MAPK phosphatase DUSP6, highlighting how ATP-competitive ligands can control noncatalytic kinase functions. Overall, these studies underscore the relationship between the ATP-binding and regulatory sites of MAPKs and provide insight into how ATP-competitive ligands can be designed to confer graded control over protein kinase function. PMID:24704509

Hari, Sanjay B; Merritt, Ethan A; Maly, Dustin J

2014-05-22

391

15 CFR 295.11 - Technical and educational services for ATP recipients.  

Code of Federal Regulations, 2010 CFR

...PROGRAMS ADVANCED TECHNOLOGY PROGRAM General § 295.11 Technical and educational services for ATP recipients...Under the Federal Technology Transfer Act of 1986...Institute of Standards and Technology. (c) From time...and undertake other educational activities to...

2010-01-01

392

Neuronal adenosine release, and not astrocytic ATP release, mediates feedback inhibition of excitatory activity  

PubMed Central

Adenosine is a potent anticonvulsant acting on excitatory synapses through A1 receptors. Cellular release of ATP, and its subsequent extracellular enzymatic degradation to adenosine, could provide a powerful mechanism for astrocytes to control the activity of neural networks during high-intensity activity. Despite adenosine's importance, the cellular source of adenosine remains unclear. We report here that multiple enzymes degrade extracellular ATP in brain tissue, whereas only Nt5e degrades AMP to adenosine. However, endogenous A1 receptor activation during cortical seizures in vivo or heterosynaptic depression in situ is independent of Nt5e activity, and activation of astrocytic ATP release via Ca2+ photolysis does not trigger synaptic depression. In contrast, selective activation of postsynaptic CA1 neurons leads to release of adenosine and synaptic depression. This study shows that adenosine-mediated synaptic depression is not a consequence of astrocytic ATP release, but is instead an autonomic feedback mechanism that suppresses excitatory transmission during prolonged activity. PMID:22421436

Lovatt, Ditte; Xu, Qiwu; Liu, Wei; Takano, Takahiro; Smith, Nathan A.; Schnermann, Jurgen; Tieu, Kim; Nedergaard, Maiken

2012-01-01

393

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

PubMed Central

Summary Mitochondrial translation of the Saccharomyces cerevisiae Atp6p subunit of F1-F0 ATP synthase is regulated by the F1 ATPase. Here we show normal expression of Atp6p in HeLa cells depleted of the F1 ? subunit. Instead of being translationally down-regulated, HeLa cells lacking F1 degrade Atp6p, thereby preventing proton leakage across the inner membrane. Mammalian mitochondria also differ in the way they minimize the harmful effect of unassembled F1 ? subunit. While yeast mutants lacking ? subunit have stable aggregated F1 ? subunit in the mitochondrial matrix, the human ? subunit is completely degraded in cells deficient in F1 ? 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

Rak, Malgorzata; McStay, Gavin P.; Fujikawa, Makoto; Yoshida, Masasuke; Manfredi, Giovanni; Tzagoloff1, Alexander

2011-01-01

394

Structural evaluation of the DNA aptamer for ATP DH25.42 by AFM.  

PubMed

Atomic force microscopy (AFM) can dynamically detect the adhesion or affinity force between a sample surface and a cantilever. This feature could be used to analyze bio-molecular interactions between a DNA aptamer and a target molecule. In this study, the binding force between adenosine triphosphate (ATP) and the anti-ATP DNA aptamer DH25.42, based on structural changes was measured using AFM. In addition, the relationship between the cations in the binding buffer, and the affinity and structure formation of the DNA aptamer was also evaluated using AFM and circular dichroism (CD) spectrum analysis. As a result, the specific force between DH25.42 and ATP could be measured by AFM. Moreover, it was suggested that Mg(2+) in the binding buffer was critical to the binding function of DH25.42 to ATP. PMID:24588754

Miyachi, Yusuke; Ogino, Chiaki; Kondo, Akihiko

2014-01-01

395

Cofactor Strap regulates oxidative phosphorylation and mitochondrial p53 activity through ATP synthase.  

PubMed

Metabolic reprogramming is a hallmark of cancer cells. Strap (stress-responsive activator of p300) is a novel TPR motif OB-fold protein that contributes to p53 transcriptional activation. We show here that, in addition to its established transcriptional role, Strap is localised at mitochondria where one of its key interaction partners is ATP synthase. Significantly, the interaction between Strap and ATP synthase downregulates mitochondrial ATP production. Under glucose-limiting conditions, cancer cells are sensitised by mitochondrial Strap to apoptosis, which is rescued by supplementing cells with an extracellular source of ATP. Furthermore, Strap augments the apoptotic effects of mitochondrial p53. These findings define Strap as a dual regulator of cellular reprogramming: first as a nuclear transcription cofactor and second in the direct regulation of mitochondrial respiration. PMID:25168243

Maniam, S; Coutts, A S; Stratford, M R; McGouran, J; Kessler, B; La Thangue, N B

2015-01-01

396

Laboratory procedures manual for the firefly luciferase assay for adenosine triphosphate (ATP)  

NASA Technical Reports Server (NTRS)

A manual on the procedures and instruments developed for the adenosine triphosphate (ATP) luciferase assay is presented. Data cover, laboratory maintenance, maintenance of bacterial cultures, bacteria measurement, reagents, luciferase procedures, and determination of microbal susceptibility to antibiotics.

Chappelle, E. W.; Picciolo, G. L.; Curtis, C. A.; Knust, E. A.; Nibley, D. A.; Vance, R. B.

1975-01-01

397

Dependence of thermodynamic efficiency of proton pumps on frequency of oscillatory concentration of ATP.  

PubMed Central

In order to evaluate the utilization of variable ATP concentration produced by an oscillatory reaction (as in anaerobic glycolysis), we analyze the thermodynamic efficiency of power output of a cyclic, ATP-driven proton pump found in the plasma membrane of plant cells. The model used includes the coupling of potassium and calcium ion transport. Oscillations in the concentration of ATP can lead to either increases or decreases in efficiency compared to that at constant ATP concentration, with corresponding decreases and increases in dissipation in the irreversible processes of the proton pump, depending on the frequency of the oscillations. Variations of imposed frequencies induce, in the periodic response, variations of phase shifts between the components of the total membrane current, which consist of the pump's proton current and the currents of potassium and calcium ions. Increases in efficiency are attained when the phase shifts are such that maxima (or minima) in the proton pump current and membrane potential occur simultaneously. PMID:3025871

Schell, M; Kundu, K; Ross, J

1987-01-01

398

SIRT4 regulates ATP homeostasis and mediates a retrograde signaling via AMPK  

PubMed Central

Efficient coupling of cellular energy production to metabolic demand is crucial to maintain organismal homeostasis. Here, we report that the mitochondrial Sirtuin Sirt4 regulates mitochondrial ATP homeostasis. We find that Sirt4 affects mitochondrial uncoupling via the adenine nucleotide translocator 2 (ANT2). Loss of Sirt4 expression leads to decreased cellular ATP levels in vitro and in vivo while Sirt4 overexpression is associated with increased ATP levels. Further, we provide evidence that lack of Sirt4 activates a retrograde signaling response from the mitochondria to the nucleus that includes AMPK, PGC1?, key regulators of ?-oxidation such as Acetyl-CoA carboxylase, and components of the mitochondrial respiratory machinery. This study highlights the ability of Sirt4 to regulate ATP levels via ANT2 and a feedback loop involving AMPK. PMID:24296486

Banerjee, Kushal Kr; George, Suji; Lin, Wei; Deota, Shaunak; Saha, Asish K.; Nakamura, Ken; Gut, Philipp

2013-01-01

399

Effects of a supplement designed to increase ATP levels on muscle strength, power output, and endurance  

E-print Network

Background: The present study examined the acute effects of a nutritional supplement intended to improve adenosine triphosphate (ATP) concentrations on vertical jump height, isometric strength of the leg extensors, leg ...

Herda, Trent J.; Ryan, Eric D.; Stout, Jeffrey R.; Cramer, Joel T.

2008-01-01

400

Mitochondrial protein sorting as a therapeutic target for ATP synthase disorders  

PubMed Central

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

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

401

The Rome III Classification of Dyspepsia: Will It Help Research?  

Microsoft Academic Search

A major change in the Rome III criteria relates to the condition previously called functional dyspepsia (FD). Rome I and Rome II defined FD as pain or discomfort centered in the upper abdomen without a definite structural or biochemical explanation. The condition was further sub-classified into ulcer-like or dysmotility-like dyspepsia. However, subsequent studies failed to show that single-symptoms are present

Nicholas J. Talley; Kevin Ruff; Xuan Jiang; Hye Kyung Jung

2008-01-01

402

Fusion Power Demonstration III  

SciTech Connect

This is the third in the series of reports covering the Fusion Power Demonstration (FPD) design study. This volume considers the FPD-III configuration that incorporates an octopole end plug. As compared with the quadrupole end-plugged designs of FPD-I and FPD-II, this octopole configuration reduces the number of end cell magnets and shortens the minimum ignition length of the central cell. The end-cell plasma length is also reduced, which in turn reduces the size and cost of the end cell magnets and shielding. As a contiuation in the series of documents covering the FPD, this report does not stand alone as a design description of FPD-III. Design details of FPD-III subsystems that do not differ significantly from those of the FPD-II configuration are not duplicated in this report.

Lee, J.D. (ed.)

1985-07-01

403

Effect of chemical hypoxia on intracellular ATP and cytosolic Mg2+ levels.  

PubMed

Intracellular magnesium is intimately associated with adenosine triphosphate (ATP) concentrations and energy utilization. We determined adenine nucleotide concentrations (ATP, adenosine diphosphate, and adenosine triphosphate) and the associated changes in intracellular free Mg2+ ([Mg2+]i) by high-performance liquid chromatography and fluorescent methods, respectively. Various mitochondrial inhibitors were used to deplete intracellular ATP and alter energy charge in epithelial cells. The opossum kidney (OK) cell line was used as a prototypic renal epithelial cell. These agents markedly deplete intracellular ATP levels with modest changes in [Mg2+]i and [Ca2+]i. Because these agents have disparate actions, it is likely that these changes were due to alterations in ATP rather than to selective drug effects. Cyanide resulted in a rapid (within 2 minutes) fall in ATP from 25.85 to 10.58 nmol/mg protein or about 3 mmol/L, whereas [Mg2+]i increased gradually (10 minutes), from 513 +/- 7 to 1096 +/- 105 mumol/L [Ca2+]i increased from 109 +/- 12 to 153 +/- 10 nmol/L within 20 seconds, then returned to basal concentrations. The changes in ATP, Mg2+, and Ca2+ were not altered by removing external Na+o, adding ruthenium red, or treating with vanadate. Antimycin diminished ATP levels in a manner similar to the effect of cyanide, but by contrast [Mg2+]i decreased to 436 +/- 13 mumol/L and [Ca2+]i transiently increased. These studies indicate that we are able to distinguish Mg2+ movements from those of Ca2+ by fluorescent techniques and suggest that intracellular regulation of [Mg2+]i is distinctive from that of [Ca2+]i. Oligomycin resulted in marked and rapid falls in [ATP]i with disproportionate increases in [Mg2+]i. The response of magnesium-depleted cells (basal [Mg2+]i, 231 +/- 10 mumol/L) after inhibitor-induced energy depletion was similar to that of control cells. These studies suggest that large changes in intracellular ATP levels do not markedly alter intracellular [Mg2+]i control and, in turn, that intracellular free Mg2+ is not a limiting factor in ATP metabolism after energy depletion with chemical hypoxia. PMID:8409702

Li, H Y; Dai, L J; Quamme, G A

1993-09-01

404

Extracellular ATP and P2Y Receptor Activation Induce a Proinflammatory Host Response in the Human Urinary Tract?  

PubMed Central

Extracellular ATP can be released by many cell types under conditions of cellular stress and signals through activation of purinergic receptors. Bladder uroepithelial cells grown in vitro have previously been shown to release ATP in response to stretch. In the present study, we investigated ATP release from uroepithelial cells infected with bacteria and the effect of ATP on the host cell proinflammatory interleukin 8 (IL-8) response. The human kidney epithelial cell line A498 and the human uroepithelial cell line UROtsa were grown in culture and stimulated by the uropathogenic Escherichia coli (UPEC) IA2 strain or the stable ATP analogue ATP-?-S. ATP and IL-8 levels were measured in cell culture medium with a luciferin-luciferase assay and enzyme-linked immunosorbent assay (ELISA), respectively. The results showed that UPEC infection of uroepithelial cells for 1 h significantly increased (P < 0.01) the extracellular ATP levels. ATP-?-S (10 and 100 ?M) stimulated release of IL-8 from UROtsa and A498 cells after 6 and 24 h. Experiments with different purinoceptor agonists suggested that P2Y receptors, and not P2X receptors, were responsible for the ATP-?-S-induced IL-8 release. The potency profile further suggested involvement of P2Y1, P2Y2, and/or P2Y11 receptors, and reverse transcription-PCR (RT-PCR) studies confirmed that the cells expressed these receptors. The amount of IL-8 released increased 12-fold in UPEC-infected cells, and apyrase, an enzyme that degrades ATP, reduced this increase by approximately 50%. The present study suggests that enhanced ATP release and P2Y receptor activation during urinary tract infection may represent a novel, non-TLR4-mediated mechanism for production of proinflammatory IL-8 in human urinary tract epithelial cells. PMID:20515921

Säve, Susanne; Persson, Katarina

2010-01-01

405

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

PubMed Central

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

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

2010-01-01

406