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1

JIS Definition Identified More Malaysian Adults with Metabolic Syndrome Compared to the NCEP-ATP III and IDF Criteria  

PubMed Central

Metabolic syndrome (MetS) is a steering force for the cardiovascular diseases epidemic in Asia. This study aimed to compare the prevalence of MetS in Malaysian adults using NCEP-ATP III, IDF, and JIS definitions, identify the demographic factors associated with MetS, and determine the level of agreement between these definitions. The analytic sample consisted of 8,836 adults aged ?30 years recruited at baseline in 2007–2011 from the Cardiovascular Risk Prevention Study (CRisPS), an ongoing, prospective cohort study involving 18 urban and 22 rural communities in Malaysia. JIS definition gave the highest overall prevalence (43.4%) compared to NCEP-ATP III (26.5%) and IDF (37.4%), P < 0.001. Indians had significantly higher age-adjusted prevalence compared to other ethnic groups across all MetS definitions (30.1% by NCEP-ATP III, 50.8% by IDF, and 56.5% by JIS). The likelihood of having MetS amongst the rural and urban populations was similar across all definitions. A high level of agreement between the IDF and JIS was observed (Kappa index = 0.867), while there was a lower level of agreement between the IDF and NCEP-ATP III (Kappa index = 0.580). JIS definition identified more Malaysian adults with MetS and therefore should be recommended as the preferred diagnostic criterion. PMID:24175300

Daher, Aqil Mohammad; Noor Khan Nor-Ashikin, Mohamed; Mat-Nasir, Nafiza; Keat Ng, Kien; Ambigga, Krishnapillai S.; Ariffin, Farnaza; Yasin Mazapuspavina, Md; Abdul-Razak, Suraya; Abdul-Hamid, Hasidah; Abd-Majid, Fadhlina; Abu-Bakar, Najmin; Nawawi, Hapizah; Yusoff, Khalid

2013-01-01

2

Inactivation and phosphorylation of sarcoplasmic reticulum Ca(2+)-ATPase by Mg.ATP analogues Rh(III)-ATP and Co(III)-ATP.  

PubMed

The interaction of sarcoplasmic reticulum Ca(2+)-ATPase with the Mg.ATP analogues Rh(H2O)4ATP and Co(NH3)4ATP have been examined. Co(NH3)4ATP slowly inactivates Ca(2+)-ATPase in a first order process, with a rate constant of 1.13 x 10(-3) s-1 and an apparent inactivation constant, KI, of 32 mM. Rh(H2O)4ATP likewise inactivates sarcoplasmic reticulum Ca(2+)-ATPase, but the plot of reciprocal apparent inactivation rate constants versus 1/[Rh(H2O)4ATP] is biphasic. The chi-intercepts of this plot yield apparent inactivation constants for the inhibition of Ca(2+)-ATPase by Rh(H2O)4ATP of KI1 = 30 microM and KI2 = 221 microM. The corresponding values of k2, the maximal first-order rate constant for inhibition in these two phases, are 1.16 and 2.19 x 10(-4)s-1. Tridentate Rh(H2O)3ATP also inhibits Ca(2+)-ATPase, but only after much longer incubation times. Ca(2+)-ATPase inactivation is accompanied by incorporation of radioactivity from gamma-32P into an acid-precipitable enzyme. Both processes were dependent on the presence of Ca2+ ions and were quenched by excess ATP. The first-order rate constant for inactivation of Ca(2+)-dependent ATPase activity in this experiment was 2.19 x 10(-4)s-1, and the first-order rate constant for Ca(2+)-dependent E-P formation was 2.07 x 10(-4)s-1, in excellent agreement with the value for inactivation. A linear relationship is observed between ATPase inactivation and E-P formation. Moreover, atomic absorption analysis demonstrates that the phosphorylation of Ca(2+)-ATPase by Rh(H2O)4ATP is accompanied by incorporation and tight binding of rhodium, with a stoichiometry of one rhodium incorporated per ATPase molecule phosphorylated. The characteristics of ATPase inactivation and phosphorylation (i.e., Ca2+ dependence, ATP competition, agreement of rate constants, and stoichiometric rhodium incorporation) suggest that Rh(H2O)4ATP is binding to the catalytic nucleotide site on Ca(2+)-ATPase and producing a highly stable, phosphorylated intermediate. PMID:1533500

Kuntzweiler, T A; Grisham, C M

1992-05-15

3

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

4

Quantifying the Effect of Applying the NCEP ATP III Criteria in a Managed Care Population Treated With Statin Therapy  

Microsoft Academic Search

OBJECTIVE: Revised treatment goals suggested by the third report of the National Cholesterol Education Program (NCEP) Adult Treatment Panel (ATP III) represent a challenge to both physicians and the health care industry. We sought to quantify the impact of these changes in a large managed care population being treated with statin therapy. METHODS: Using data collected from a retrospective chart

BRIAN J. QUILLIAM; H. ED PEREZ; VICKIE ANDROS; PETER JONES

2004-01-01

5

Cellular ATP synthesis mediated by type III sodium-dependent phosphate transporter Pit-1 is critical to chondrogenesis.  

PubMed

Disturbed endochondral ossification in X-linked hypophosphatemia indicates an involvement of P(i) in chondrogenesis. We studied the role of the sodium-dependent P(i) cotransporters (NPT), which are a widely recognized regulator of cellular P(i) homeostasis, and the downstream events in chondrogenesis using Hyp mice, the murine homolog of human X-linked hypophosphatemia. Hyp mice showed reduced apoptosis and mineralization in hypertrophic cartilage. Hyp chondrocytes in culture displayed decreased apoptosis and mineralization compared with WT chondrocytes, whereas glycosaminoglycan synthesis, an early event in chondrogenesis, was not altered. Expression of the type III NPT Pit-1 and P(i) uptake were diminished, and intracellular ATP levels were also reduced in parallel with decreased caspase-9 and caspase-3 activity in Hyp chondrocytes. The competitive NPT inhibitor phosphonoformic acid and ATP synthesis inhibitor 3-bromopyruvate disturbed endochondral ossification with reduced apoptosis in vivo and suppressed apoptosis and mineralization in conjunction with reduced P(i) uptake and ATP synthesis in WT chondrocytes. Overexpression of Pit-1 in Hyp chondrocytes reversed P(i) uptake and ATP synthesis and restored apoptosis and mineralization. Our results suggest that cellular ATP synthesis consequent to P(i) uptake via Pit-1 plays an important role in chondrocyte apoptosis and mineralization, and that chondrogenesis is ATP-dependent. PMID:21075853

Sugita, Atsushi; Kawai, Shinji; Hayashibara, Tetsuyuki; Amano, Atsuo; Ooshima, Takashi; Michigami, Toshimi; Yoshikawa, Hideki; Yoneda, Toshiyuki

2011-01-28

6

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

PubMed Central

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

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

2012-01-01

7

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

Microsoft Academic Search

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

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

2009-01-01

8

Complexation of bisphosphonates with Ytterbium(III): Application of phosphate and ATP detection assay based on Yb3+-pyrocatechol violet  

PubMed Central

The coordination chemistry of bisphosphonates with Yb3+ was investigated to evaluate the potential of the UV-Vis based detection method using the Yb3+-pyrocatechol complexation reaction as a sensor for bisphosphonates. The complexation chemistry of Yb3+ with phosphate and ATP analogs was previously described (E. Gaidamauskas et al J. Biol. Inorg. Chem. 13 (2008) 1291-1299), and we here study the complexation chemistry of bisphosphonates in this system. The spectrophotometric assay yields direct evidence for formation of a 4:3 metal to ligand complex at neutral pH. Direct evidence for Yb3+ : methylenebis(phosphonate) complexes with 1:1 and 1:2 stoichiometry was also obtained by potentiometry at acidic and basic pH. Direct evidence for complex formation was obtained using 1H NMR spectroscopy although the stoichiometry was not accessed at neutral pH. Our results suggest that the spectroscopic observation of the YbPV complex can be used to conveniently measure concentrations of bisphosphonates down to 2-3 ?M . PMID:19850352

Gaidamauskas, Ernestas; Parker, Helen; Kashemirov, Boris A.; Holder, Alvin A.; Saejueng, Kanokkarn; McKenna, Charles E.; Crans, Debbie C.

2009-01-01

9

Prevalence and cardiovascular disease risk differences for erectile dysfunction patients by three metabolic syndrome definitions  

Microsoft Academic Search

There is growing evidence of a link between ED, metabolic syndrome (MS) and cardiovascular disease (CVD). The study was to explore the prevalence of MS using three different definitions (World Health Organization (WHO), International Diabetes Foundation (IDF) and Adult Treatment Panel III (ATP III)), and to compare the association of CVD in ED outpatients using these definitions. This study enrolled

S-T Chang; C-M Chu; K-L Pan; Y-S Lin; P-C Wang; J-J Shee; C-S Chen

2011-01-01

10

Utility of the modified ATP III defined metabolic syndrome and severe obesity as predictors of insulin resistance in overweight children and adolescents: a cross-sectional study  

PubMed Central

Background The rising prevalence of obesity and metabolic syndrome (MetS) has received increased attention since both place individuals at risk for Type II diabetes and cardiovascular disease. Insulin resistance (IR) has been implicated in the pathogenesis of obesity and MetS in both children and adults and is a known independent cardiovascular risk factor. However measures of IR are not routinely performed in children while MetS or severe obesity when present, are considered as clinical markers for IR. Objective The study was undertaken to assess the utility of ATPIII defined metabolic syndrome (MetS) and severe obesity as predictors of insulin resistance (IR) in a group of 576 overweight children and adolescents attending a pediatric obesity clinic in Brooklyn. Methods Inclusion criteria were children ages 3–19, and body mass index > 95th percentile for age. MetS was defined using ATP III criteria, modified for age. IR was defined as upper tertile of homeostasis model assessment (HOMA) within 3 age groups (3–8, n = 122; 9–11, n = 164; 12–19, n = 290). Sensitivity, specificity, positive predictive values and odds ratios (OR) with 95% confidence intervals (CI) were calculated within age groups for predicting IR using MetS and severe obesity respectively. Results MetS was present in 45%, 48% and 42% of the respective age groups and significantly predicted IR only in the oldest group (OR = 2.0, 95% CI 1.2, 3.4; p = .006). Sensitivities were <55%; specificities <63% and positive predictive values ? 42% in all groups. Severe obesity was significantly associated with IR in both the 9–11 (p = .002) and 12–18 (p = .01) groups but positive predictive values were nonetheless ? 51% for all groups. Conclusion The expression of IR in overweight children and adolescents is heterogeneous and MetS or severe obesity may not be sufficiently sensitive and specific indicators of insulin resistance. In addition to screening for MetS in overweight children markers for IR should be routinely performed. Further research is needed to establish threshold values of insulin measures in overweight children who may be at greater associated risk of adverse outcomes whether or not MetS is present. PMID:17300718

Dhuper, Sarita; Cohen, Hillel W; Daniel, Josephine; Gumidyala, Padmasree; Agarwalla, Vipin; St Victor, Rosemarie; Dhuper, Sunil

2007-01-01

11

The concept of major depression. III. Concurrent validity of six competing operational definitions for the clinical ICD-9 diagnosis.  

PubMed

The comparative validity of six operational diagnoses of major depression was evaluated in 600 psychiatric inpatients using the independently assessed clinical ICD-9 diagnoses as a yardstick. Agreement with, and positive predictive value for the ICD-9 categories of pure (endogenous and psychogenic) depression served as validation criteria; sensitivity of major depression diagnoses for detecting ICD-9 bipolar depressions was additionally used for examining the adequacy of width, time and exclusion criteria of the competing operational definitions. Three essential results were found. First, the "old" diagnostic definitions of RDC and FDC are superior to all newer definitions because they define the time criteria and the schizophrenic exclusion criteria more adequately than, for example, both DSM-III and DSM-III-R definition. Secondly, the current ICD-10 definition of 1989 ("mild", "moderate" or "severe" depression) comes closer to the concurrent validity of RDC and FDC than DSM-III, DSM-III-R and the previous ICD-10 definition of 1987. Thirdly, using the criterion of identifying a high proportion of ICD-9 bipolar depressions, all six competing diagnostic systems are too restrictive. Evaluations of predictive and criterion-related validity will be needed to substantiate these findings. PMID:1829002

Philipp, M; Maier, W; Delmo, C D

1991-01-01

12

Prevalence and Determinants of Metabolic Syndrome According to Three Definitions in Middle-Aged Chinese Men  

PubMed Central

Abstract Background The prevalence of metabolic syndrome has varied markedly between different studies because of the lack of internationally agreed-upon criteria to define the condition. We estimated the prevalence and lifestyle risk factors of metabolic syndrome according to three definitions of metabolic syndrome in urban Chinese men participating in the Shanghai Men's Health Study (SMHS). Methods In this cross-sectional study, 3988 middle-aged, urban Chinese men 40–74 years of age who were free of type 2 diabetes at baseline provided fasting blood samples, anthropometric measurements, and information on lifestyle factors and disease history. Results The three definitions of metabolic syndrome used in this report are from the International Diabetes Federation (IDF), the U.S. Third Report of the National Cholesterol Education Program, Adult Treatment Panel (ATP III), and a modified version of the ATP III criteria for Asian populations (ATP III–modified criteria). The prevalence of metabolic syndrome was 18.63%, 18.36%, and 29.34% according to IDF, ATP III, and ATP III–modified criteria, respectively. Agreement between the IDF and ATP III criteria was moderate (??=?0.43), whereas agreement between ATP III–modified and the IDF and ATP III criteria was good (??=?0.71 and 0.70, respectively). Physical activity was associated with a lower prevalence of metabolic syndrome, whereas drinking more than three drinks per day was associated with a higher risk of metabolic syndrome, regardless of the criteria employed. The association between smoking and the prevalence of metabolic syndrome in this population failed to reach significance. Conclusions Results from this representative sample of middle-aged, urban Chinese men show that metabolic syndrome is highly prevalent in this population. Our data support the hypothesis that physical activity decreases the risk of developing metabolic syndrome and that high alcohol consumption increases risk. PMID:19032041

Xiang, Yong-Bing; Yang, Gong; Cai, Qiuyin; Fazio, Sergio; Linton, MacRae F.; Elasy, Tom; Xu, Wang-Hong; Li, Honglan; Cai, Hui; Gao, Yu-Tang; Zheng, Wei; Shu, Xiao Ou

2009-01-01

13

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

14

Adenosine and ATP Receptors  

Microsoft Academic Search

Adenosine and ATP, via P1 and P2 receptors respectively, can modulate pain transmission under physiological, inflammatory,\\u000a and neuropathic pain conditions. Such influences reflect peripheral and central actions and effects on neurons as well as\\u000a other cell types. In general, adenosine A1 receptors produce inhibitory effects on pain in a number of preclinical models\\u000a and are a focus of attention. In

J. Sawynok

15

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

Microsoft Academic Search

Purpose: This study was designed to assess the 3-year experience with superselective intraarterial chemotherapy prior to definitive treatment for stages III and IV squamous cell carcinomas of the mouth. Methods: Twenty-two patients prospectively received superselective intraarterial chemotherapy using relatively low-dose cisplatin via a transfemoral approach. The locations of the tumors were the tongue (n= 12), gingiva (n= 5), buccal mucosa

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

1999-01-01

16

Roles for Helicases as ATP-Dependent Molecular Switches.  

PubMed

On the basis of the familial name, a "helicase" might be expected to have an enzymatic activity that unwinds duplex polynucleotides to form single strands. A more encompassing taxonomy that captures alternative enzymatic roles has defined helicases as a sub-class of molecular motors that move directionally and processively along nucleic acids, the so-called "translocases". However, even this definition may be limiting in capturing the full scope of helicase mechanism and activity. Discussed here is another, alternative view of helicases-as machines which couple NTP-binding and hydrolysis to changes in protein conformation to resolve stable nucleoprotein assembly states. This "molecular switch" role differs from the classical view of helicases as molecular motors in that only a single catalytic NTPase cycle may be involved. This is illustrated using results obtained with the DEAD-box family of RNA helicases and with a model bacterial system, the ATP-dependent Type III restriction-modification enzymes. Further examples are discussed and illustrate the wide-ranging examples of molecular switches in genome metabolism. PMID:23161014

Szczelkun, Mark D

2013-01-01

17

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

18

Stages III and IV squamous cell carcinoma of the mouth: Three-year experience with superselective intraarterial chemotherapy using cisplatin prior to definitive treatment  

Microsoft Academic Search

Purpose: This study was designed to assess the 3-year experience with superselective intraarterial chemotherapy prior to definitive\\u000a treatment for stages III and IV squamous cell carcinomas of the mouth.\\u000a \\u000a \\u000a Methods: Twenty-two patients prospectively received superselective intraarterial chemotherapy using relatively low-dose cisplatin\\u000a via a transfemoral approach. The locations of the tumors were the tongue (n=12), gingiva (n=5), buccal mucosa (n=2), hard

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

1999-01-01

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

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

PubMed Central

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

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

2012-01-01

21

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

Microsoft Academic Search

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

Miljenko V.. Pilepich; Kathryn Winter; Colleen A. Lawton; Robert E. Krisch; Harvey B. Wolkov; Benjamin Movsas; Eugen B. Hug; Sucha O. Asbell; David Grignon

2005-01-01

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

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

PubMed Central

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

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

2012-01-01

24

Short cationic antimicrobial peptides interact with ATP.  

PubMed

The mode of action of short, nonhelical antimicrobial peptides is still not well understood. Here we show that these peptides interact with ATP and directly inhibit the actions of certain ATP-dependent enzymes, such as firefly luciferase, DnaK, and DNA polymerase. ?-Helical and planar or circular antimicrobial peptides did not show such interaction with ATP. PMID:20660668

Hilpert, Kai; McLeod, Brett; Yu, Jessie; Elliott, Melissa R; Rautenbach, Marina; Ruden, Serge; Bürck, Jochen; Muhle-Goll, Claudia; Ulrich, Anne S; Keller, Sandro; Hancock, Robert E W

2010-10-01

25

ATP metabolism in skeletal muscle arterioles  

PubMed Central

Abstract The purpose of this study was to investigate the metabolism of Adenosine triphosphate (ATP) in skeletal muscle resistance arterioles and to determine whether this metabolism is altered during the rapid growth phase of the rat. We attempted to quantify ATP metabolism in gastrocnemius first?order arterioles from 8?, 10?, and 12?week?old rats. We measured ATP metabolism using an ATPase/GTPase assay with whole vessel segments as well as using a real?time adenosine biosensor following electric field stimulation. Our first method of measuring ATP metabolism allowed us to measure the amount of free phosphate produced with ATP as a substrate. When ecto?nucleotidase activity was inhibited by ARL67156, pyridoxal phosphate?6?azophenly?2?, 4??disulfonic acid (PPADS), or suramin prior to adding ATP, we found that the rate of phosphate production was significantly reduced by 27%, 21%, and 22%, respectively (P < 0.05). Our second method of measuring ATP metabolism allowed us to measure the amount of adenosine produced following electric field stimulation of the arteriole with and without nucleotidase inhibitors. Surprisingly, we found that adenosine overflow was not attenuated by nucleotidase inhibitors. We concluded that ecto?phosphodieterase/phyrophophatase (E?NPP), ecto?diadenosine polyphosphatase (ApnA), NTPDase1 and 2, and E5NT may be present on the gastrocnemius 1A arteriole and do play a role in ATP metabolism. Between the ages of 8 weeks and 12 weeks, however, overall ATP metabolism may not change. PMID:24744886

Stone, Audrey J.; Evanson, Kirk W.; Kluess, Heidi A.

2014-01-01

26

ATP metabolism in skeletal muscle arterioles.  

PubMed

The purpose of this study was to investigate the metabolism of Adenosine triphosphate (ATP) in skeletal muscle resistance arterioles and to determine whether this metabolism is altered during the rapid growth phase of the rat. We attempted to quantify ATP metabolism in gastrocnemius first-order arterioles from 8-, 10-, and 12-week-old rats. We measured ATP metabolism using an ATPase/GTPase assay with whole vessel segments as well as using a real-time adenosine biosensor following electric field stimulation. Our first method of measuring ATP metabolism allowed us to measure the amount of free phosphate produced with ATP as a substrate. When ecto-nucleotidase activity was inhibited by ARL67156, pyridoxal phosphate-6-azophenly-2', 4'-disulfonic acid (PPADS), or suramin prior to adding ATP, we found that the rate of phosphate production was significantly reduced by 27%, 21%, and 22%, respectively (P < 0.05). Our second method of measuring ATP metabolism allowed us to measure the amount of adenosine produced following electric field stimulation of the arteriole with and without nucleotidase inhibitors. Surprisingly, we found that adenosine overflow was not attenuated by nucleotidase inhibitors. We concluded that ecto-phosphodieterase/phyrophophatase (E-NPP), ecto-diadenosine polyphosphatase (ApnA), NTPDase1 and 2, and E5NT may be present on the gastrocnemius 1A arteriole and do play a role in ATP metabolism. Between the ages of 8 weeks and 12 weeks, however, overall ATP metabolism may not change. PMID:24744886

Stone, Audrey J; Evanson, Kirk W; Kluess, Heidi A

2014-01-01

27

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

SciTech Connect

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

Hirai, Toshinori; Korogi, Yukunori; Hamatake, Satoshi; Nishimura, Ryuichi; Baba, Yuji; Takahashi, Mutsumasa [Department of Radiology, Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto 860 (Japan); Uji, Yasuyoshi; Taen, Akira [Department of Oral and Maxillofacial Surgery, Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto 860 (Japan)

1999-05-15

28

Conformational dynamics of ATP/Mg:ATP in motor proteins via data mining and molecular simulation  

NASA Astrophysics Data System (ADS)

The conformational diversity of ATP/Mg:ATP in motor proteins was investigated using molecular dynamics and data mining. Adenosine triphosphate (ATP) conformations were found to be constrained mostly by inter cavity motifs in the motor proteins. It is demonstrated that ATP favors extended conformations in the tight pockets of motor proteins such as F1-ATPase and actin whereas compact structures are favored in motor proteins such as RNA polymerase and DNA helicase. The incorporation of Mg2+ leads to increased flexibility of ATP molecules. The differences in the conformational dynamics of ATP/Mg:ATP in various motor proteins was quantified by the radius of gyration. The relationship between the simulation results and those obtained by data mining of motor proteins available in the protein data bank is analyzed. The data mining analysis of motor proteins supports the conformational diversity of the phosphate group of ATP obtained computationally.

Bojovschi, A.; Liu, Ming S.; Sadus, Richard J.

2012-08-01

29

Genetics Home Reference: Glycogen storage disease type III  

MedlinePLUS

... Research studies OMIM Genetic disorder catalog Conditions > Glycogen storage disease type III On this page: Description Genetic ... Glossary definitions Reviewed September 2010 What is glycogen storage disease type III? Glycogen storage disease type III ( ...

30

January 2007 Economic Impact of ATP's  

E-print Network

, and services from ATP supported projects) · Impacts (long term impacts on U.S. industry, society, and economyJanuary 2007 Economic Impact of ATP's Contributions to DNA Diagnostics Technologies GCR 06 Assessment Office (EAO) has performed rigorous and multifaceted evaluations to assess the impact

31

Steganography Ben Lee III  

E-print Network

Steganography Ben Lee III Long Truong ECE 478 - Spring 2002 Definition Steganography: (from Greek it contains encrypted data #12;Introduction Computer Steganography: two principles ­ Digitized images in image color or sound quality In modern digital steganography, data is first encrypted by the usual

32

ATP-dependent nucleosome remodeling.  

PubMed

It has been a long-standing challenge to decipher the principles that enable cells to both organize their genomes into compact chromatin and ensure that the genetic information remains accessible to regulatory factors and enzymes within the confines of the nucleus. The discovery of nucleosome remodeling activities that utilize the energy of ATP to render nucleosomal DNA accessible has been a great leap forward. In vitro, these enzymes weaken the tight wrapping of DNA around the histone octamers, thereby facilitating the sliding of histone octamers to neighboring DNA segments, their displacement to unlinked DNA, and the accumulation of patches of accessible DNA on the surface of nucleosomes. It is presumed that the collective action of these enzymes endows chromatin with dynamic properties that govern all nuclear functions dealing with chromatin as a substrate. The diverse set of ATPases that qualify as the molecular motors of the nucleosome remodeling process have a common history and are part of a superfamily. The physiological context of their remodeling action builds on the association with a wide range of other proteins to form distinct complexes for nucleosome remodeling. This review summarizes the recent progress in our understanding of the mechanisms underlying the nucleosome remodeling reaction, the targeting of remodeling machines to selected sites in chromatin, and their integration into complex regulatory schemes. PMID:12045097

Becker, Peter B; Hörz, Wolfram

2002-01-01

33

Pharmacokinetics of intravenous ATP in cancer patients  

Microsoft Academic Search

Objective: To characterise the pharmacokinetics of adenosine 5?-triphosphate (ATP) in patients with lung cancer after i.v. administration\\u000a of different ATP dosages.\\u000a \\u000a \\u000a \\u000a Methods: Twenty-eight patients received a total of 176 i.v. ATP courses of 30?h. Fifty-two infusions were given as low-dose infusions\\u000a of 25–40??g kg?1 min?1, 47 as middle-dose infusions of 45–60??g kg?1 min?1 and 77 as high-dose infusions of 65–75??g

H. J. Agteresch; P. C. Dagnelie; T. Rietveld; J. W. O. van den Berg; A. H. J. Danser; J. H. P. Wilson

2000-01-01

34

An RNA motif that binds ATP  

NASA Technical Reports Server (NTRS)

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

Sassanfar, M.; Szostak, J. W.

1993-01-01

35

ATP’s impact on accelerating development and commercialization of advanced technology  

Microsoft Academic Search

One of ATP’s legislated mandates is to accelerate industry’s development and commercialization of new technologies. This survey\\u000a of 28 projects funded in 1991 found that ATP helped cut technology development cycle time by 50% in most cases. Slightly more\\u000a than half of the interviewees provided quantitative estimates of the economic value of reducing cycle time by a single year.\\u000a Most

Frances Jean Laidlaw

1998-01-01

36

Tau binds ATP and induces its aggregation.  

PubMed

Tau is a microtubule-associated protein mainly found in neurons. The protein is associated with process of microtubule assembly, which plays an important role in intracellular transport and cell structure of the neuron. Tauopathies are a group of neurodegenerative diseases specifically associated with tau abnormalities. While a well-defined mechanism remains unknown, most facts point to tau as a prominent culprit in neurodegeneration. In most cases of Tauopathies, aggregates of hyperphosphorylated tau have been found. Two proposals are present when discussing tau toxicity, one being the aggregation of tau proteins and the other points toward a conformational change within the protein. Previous work we carried out showed tau hyperphosphorylation promotes tau to behave abnormally resulting in microtubule assembly disruption as well as a breakdown in tau self-assembly. We found that tau's N-terminal region has a putative site for ATP/GTP binding. In this paper we demonstrate that tau is able to bind ATP and not GTP, that this binding induces tau self-assembly into filaments. At 1 mM ATP the filaments are 4-7 nm in width, whereas at 10 mM ATP the filaments appeared to establish lateral interaction, bundling and twisting, forming filaments that resembled the Paired Helical Filaments (PHF) isolated from Alzheimer disease brain. ATP-induced self-assembly is not energy dependent because the nonhydrolysable analogue of the ATP induces the same assembly. PMID:24258797

Farid, Mina; Corbo, Christopher P; Alonso, Alejandra Del C

2014-02-01

37

Flavonoid diosmetin increases ATP levels in kidney cells and relieves ATP depleting effect of ochratoxin A.  

PubMed

Diosmetin (DIOS) is a flavone aglycone commonly occurring in citrus species and olive leaves, in addition it is one of the active ingredients of some medications. Based on both in vitro and in vivo studies several beneficial effects are attributed to DIOS but the biochemical background of its action seems to be complex and it has not been completely explored yet. Previous investigations suggest that most of the flavonoid aglycones have negative effect on ATP synthesis in a dose dependent manner. In our study 17 flavonoids were tested and interestingly DIOS caused a significant elevation of intracellular ATP levels after 6- and 12-h incubation in MDCK kidney cells. In order to understand the mechanism of action, intracellular ATP and protein levels, ATP/ADP ratio, cell viability and ROS levels were determined after DIOS treatment. In addition, impacts of different enzyme inhibitors and effect of DIOS on isolated rat liver mitochondria were also tested. Finally, the influence of DIOS on the ATP depleting effect of the mycotoxin, ochratoxin A was also investigated. Our major conclusions are the followings: DIOS increases intracellular ATP levels both in kidney and in liver cells. Inhibition of glycolysis or citric acid cycle does not decrease the observed effect. DIOS-induced elevation of ATP levels is completely abolished by the inhibition of ATP synthase. DIOS is able to completely reverse the ATP-depleting effect of the mycotoxin, ochratoxin A. Most probably the DIOS-induced impact on ATP system does not originate from the antioxidant property of DIOS. Based on our findings DIOS may be promising agent to positively influence ATP depletion caused by some metabolic poisons. PMID:24556581

Poór, Miklós; Veres, Balázs; Jakus, Péter B; Antus, Csenge; Montskó, Gergely; Zrínyi, Zita; Vladimir-Kneževi?, Sanda; Petrik, József; K?szegi, Tamás

2014-03-01

38

ATP Synthesis in the Extremely Halophilic Bacteria  

NASA Technical Reports Server (NTRS)

The proton-translocating ATPases are multimeric enzymes that carry out a multitude of essential functions. Their origin and evolution represent a seminal event in the early evolution of life. Amino acid sequences of the two largest subunits from archaeal ATPases (A-ATPases), vacuolar ATPases (V-ATPases), and FOF1-ATP syntheses (FATPases) suggest these ATPases evolved from an ancestral vacuolar-like ATP syntheses. A necessary consequence of this notion is that the A-ATPases are ATP syntheses. With the possible exception of the A-ATPase from Halobacterium salinarium. no A-ATPase has been demonstrated to synthesize ATP. The evidence for this case is dubious since ATP synthesis occurs only when conditions are distinctively unphysiological. We demonstrated that ATP synthesis in H.saccharovorum is inconsistent with the operation of an A-type ATPase. In order to determine if this phenomenon was unique to H. saccharovorum, ATP synthesis was examined in various extremely halophilic bacteria with the goal of ascertaining if it resembled what occurred in a. saccharovorum, or was consistent with the operation of an A-type ATPase. A-, V-, and F-type ATPases respond singularly to certain inhibitors. Therefore, the effect of these inhibitors on ATP synthesis in several extreme halophiles was determined. Inhibitors that either blocked or collapsed proton-gradients inhibited the steady state synthesis of ATP thus verifying that synthesis took place at the expense of a proton gradient. Azide, an inhibitor of F-ATPases inhibited ATP synthesis. Since the arginine-dependent synthesis of ATP, which occurs by way of substrate-level phosphorylation, was unaffected by azide, it was unlikely that azide acted as an "uncoupler." N -ethylmaleimide and nitrate, which inhibit V- and A-ATPases, either did not inhibit ATP synthesis or resulted in higher steady-state levels of ATP. These results suggest there are two types of proton-motive ATPases in the extreme halophiles (and presumably in other Archaea). One, the V-like enzyme which, provides protons that are subsequently used for solute translocation. The other ATPase is the familiar and ubiquitous F-ATPase that functions as a reversible proton pump and is the ATP Synthase in the extreme halophiles. Thus, while the suggested evolution of the proton -translocating ATPases accounts for the relationship among these ATPases, this scheme does not account for the presence of F-ATPases in the Archaea. Discounting lateral gene transfer, perhaps an F-type ATPase evolved before the eucaryal-archaeal and bacterial bifurcation. The presence of V-type ATPases in the Bacterial Domain is consistent with this suggestion. Finally, it is of interest to note that if an F-type ATPase appeared before the bifurcation, an endosymbiotic event need not be invoked to explain the presence of F-ATPases in the Eucarya.

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

1994-01-01

39

ATP8B1 is essential for maintaining normal hearing  

PubMed Central

ATP8B1 deficiency is caused by autosomal recessive mutations in ATP8B1, which encodes the putative phospatidylserine flippase ATP8B1 (formerly called FIC1). ATP8B1 deficiency is primarily characterized by cholestasis, but extrahepatic symptoms are also found. Because patients sometimes report reduced hearing capability, we investigated the role of ATP8B1 in auditory function. Here we show that ATP8B1/Atp8b1 deficiency, both in patients and in Atp8b1G308V/G308V mutant mice, causes hearing loss, associated with progressive degeneration of cochlear hair cells. Atp8b1 is specifically localized in the stereocilia of these hair cells. This indicates that the mechanosensory function and integrity of the cochlear hair cells is critically dependent on ATP8B1 activity, possibly through maintaining lipid asymmetry in the cellular membranes of stereocilia. PMID:19478059

Stapelbroek, Janneke M.; Peters, Theo A.; van Beurden, Denis H. A.; Curfs, Jo H. A. J.; Joosten, Anneke; Beynon, Andy J.; van Leeuwen, Bibian M.; van der Velden, Lieke M.; Bull, Laura; Oude Elferink, Ronald P.; van Zanten, Bert A.; Klomp, Leo W. J.; Houwen, Roderick H. J.

2009-01-01

40

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

PubMed

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

Kaim, G; Dimroth, P

1999-08-01

41

Assembly of the rotor component of yeast mitochondrial ATP synthase is enhanced when atp9p is supplied by atp9p-cox6p complexes.  

PubMed

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

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

2014-11-01

42

BRET-linked ATP assay with luciferase.  

PubMed

Taking advantage of BRET, a mutant firefly luciferase with higher pH- and thermo-stability than the wild-type could be coupled with the red-emitting fluorescent protein of mCherry in both a fused and unfused format. The BRET pair allows >40% of the light emitted to be red shifted over 600 nm to the mCherry acceptor wavelength. Taking the expected quantum yield for mCherry (0.22), a good fit to predicted light transfer is shown, with no other losses. Two measurements are considered for ATP determination: (a) a ratiometric technique for ATP measurement using both donor and acceptor emission intensities, making the calibration slope independent of protein concentration in a broad range. This measurement was limited by the BRET efficiency and the low quantum yield of the mCherry acceptor, but this detection limit might be improved with other fluorescent proteins with higher quantum yield. The fused BRET pair also resulted in a small increase in the BRET ratio. (b) An ATP dependent shift in the wavelength maximum using just the acceptor mCherry emission was also proposed for ATP determination. This did not require a high BRET efficiency and only uses emission above 600 nm to obtain the acceptor emission maximum, but not its intensity; it is independent of protein concentration across a broad range. This offers a novel and robust method for determination of ATP between 10(-11) to 10(-5) M with an easy baseline calibration with ATP concentration >10(-4) M. PMID:24845959

Borghei, Golnaz; Hall, Elizabeth A H

2014-09-01

43

ATP overflow in skeletal muscle 1A arterioles  

PubMed Central

The purpose of this study was to investigate the sources of ATP in the 1A arteriole, and to investigate age-related changes in ATP overflow. Arterioles (1A) from the red portion of the gastrocnemius muscle were isolated, cannulated and pressurized in a microvessel chamber with field stimulation electrodes. ATP overflow was determined using probes specific for ATP and null probes that were constructed similar to the ATP probes, but did not contain the enzyme coating. ATP concentrations were determined using a normal curve (0.78 to 25 ?mol l?1 ATP). ATP overflow occurred in two phases. Phase one began in the first 20 s following stimulation and phase two started 35 s after field stimulation. Tetrodotoxin, a potent neurotoxin that blocks action potential generation in nerves, abolished both phases of ATP overflow. ?1-Receptor blockade resulted in a small decrease in ATP overflow in phase two, but endothelial removal resulted in an increase in ATP overflow. ATP overflow was lowest in 6-month-old rats and highest in 12- and 2-month-old rats (P < 0.05). ATP overflow measured via biosensors was of neural origin with a small contribution from the vascular smooth muscle. The endothelium seems to play an important role in attenuating ATP overflow in 1A arterioles. PMID:20566660

Kluess, Heidi A; Stone, Audrey J; Evanson, Kirk W

2010-01-01

44

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

45

A large conformational change in the putative ATP pyrophosphatase PF0828 induced by ATP binding.  

PubMed

ATP pyrophosphatases (ATP PPases) are widely distributed in archaea and eukaryotes. They share an HUP domain at the N-terminus with a conserved PP-motif that interacts with the phosphates of ATP. The PF0828 protein from Pyrococcus furiosus is a member of the ATP PPase superfamily and it also has a 100-residue C-terminal extension that contains a strictly conserved EGG(E/D)xE(T/S) motif, which has been named the EGT-motif. Here, crystal structures of PF0828 alone and in complex with ATP or AMP are reported. The HUP domain contains a central five-stranded ?-sheet that is surrounded by four helices, as in other related structures. The C-terminal extension forms a separate domain, named the EGT domain, which makes tight interactions with the HUP domain, bringing the EGT-motif near to the PP-motif and defining the putative active site of PF0828. Both motifs interact with the phosphate groups of ATP. A loop in the HUP domain undergoes a large conformational change to recognize the adenine base of ATP. In solution and in the crystal PF0828 is a dimer formed by the side-by-side arrangement of the HUP domains of the two monomers. The putative active site is located far from the dimer interface. PMID:22102225

Forouhar, Farhad; Saadat, Nabila; Hussain, Munif; Seetharaman, Jayaraman; Lee, Insun; Janjua, Haleema; Xiao, Rong; Shastry, Ritu; Acton, Thomas B; Montelione, Gaetano T; Tong, Liang

2011-11-01

46

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

PubMed Central

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

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

2013-01-01

47

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

PubMed

The spontaneous action potential (AP) firing rate of sinoatrial node cells (SANCs) involves high-throughput signaling via Ca(2+)-calmodulin activated adenylyl cyclases (AC), cAMP-mediated protein kinase A (PKA), and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII)-dependent phosphorylation of SR Ca(2+) cycling and surface membrane ion channel proteins. When the throughput of this signaling increases, e.g., in response to ?-adrenergic receptor activation, the resultant increase in spontaneous AP firing rate increases the demand for ATP. We hypothesized that an increase of ATP production to match the increased ATP demand is achieved via a direct effect of increased mitochondrial Ca(2+) (Ca(2+)m) and an indirect effect via enhanced Ca(2+)-cAMP/PKA-CaMKII signaling to mitochondria. To increase ATP demand, single isolated rabbit SANCs were superfused by physiological saline at 35 ± 0.5°C with isoproterenol, or by phosphodiesterase or protein phosphatase inhibition. We measured cytosolic and mitochondrial Ca(2+) and flavoprotein fluorescence in single SANC, and we measured cAMP, ATP, and O? consumption in SANC suspensions. Although the increase in spontaneous AP firing rate was accompanied by an increase in O? consumption, the ATP level and flavoprotein fluorescence remained constant, indicating that ATP production had increased. Both Ca(2+)m and cAMP increased concurrently with the increase in AP firing rate. When Ca(2+)m was reduced by Ru360, the increase in spontaneous AP firing rate in response to isoproterenol was reduced by 25%. Thus, both an increase in Ca(2+)m and an increase in Ca(2+) activated cAMP-PKA-CaMKII signaling regulate the increase in ATP supply to meet ATP demand above the basal level. PMID:23604710

Yaniv, Yael; Spurgeon, Harold A; Ziman, Bruce D; Lyashkov, Alexey E; Lakatta, Edward G

2013-06-01

48

Identification, expression, and functional analyses of a thylakoid ATP/ADP carrier from Arabidopsis.  

PubMed

In plants the chloroplast thylakoid membrane is the site of light-dependent photosynthetic reactions coupled to ATP synthesis. The ability of the plant cell to build and alter this membrane system is essential for efficient photosynthesis. A nucleotide translocator homologous to the bovine mitochondrial ADP/ATP carrier (AAC) was previously found in spinach thylakoids. Here we have identified and characterized a thylakoid ATP/ADP carrier (TAAC) from Arabidopsis.(i) Sequence homology with the bovine AAC and the prediction of chloroplast transit peptides indicated a putative carrier encoded by the At5g01500 gene, as a TAAC. (ii) Transiently expressed TAAC-green fluorescent protein fusion construct was targeted to the chloroplast. Western blotting using a peptide-specific antibody together with immunogold electron microscopy revealed a major location of TAAC in the thylakoid membrane. Previous proteomic analyses identified this protein in chloroplast envelope preparations. (iii) Recombinant TAAC protein specifically imports ATP in exchange for ADP across the cytoplasmic membrane of Escherichia coli. Studies on isolated thylakoids from Arabidopsis confirmed these observations. (iv) The lack of TAAC in an Arabidopsis T-DNA insertion mutant caused a 30-40% reduction in the thylakoid ATP transport and metabolism. (v) TAAC is readily expressed in dark-grown Arabidopsis seedlings, and its level remains stable throughout the greening process. Its expression is highest in developing green tissues and in leaves undergoing senescence or abiotic stress. We propose that the TAAC protein supplies ATP for energy-dependent reactions during thylakoid biogenesis and turnover in plants. PMID:17261580

Thuswaldner, Sophie; Lagerstedt, Jens O; Rojas-Stütz, Marc; Bouhidel, Karim; Der, Christophe; Leborgne-Castel, Nathalie; Mishra, Arti; Marty, Francis; Schoefs, Benoît; Adamska, Iwona; Persson, Bengt L; Spetea, Cornelia

2007-03-23

49

Electric field driven torque in ATP synthase.  

PubMed

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

50

Homocysteine modifies extracellular ATP availability in macrophages.  

PubMed

Increased levels of plasma homocysteine (hyperhomocysteinemia-HHcy) are associated to the development of coronary artery disease (CAD), peripheral vascular disease and thrombosis. In addition, recent studies have shown that inflammation, probably mediated by macrophages, mediates the pathogenesis associated to high levels of homocysteine (Hcy). In the present study, we evaluated the Hcy effects in the ATP hydrolysis and its breakdown products in murine macrophages. The results showed that micromolar concentrations of Hcy increased the ATP, ADP and AMP hydrolysis. Additionally, our results show decreased inosine levels in the extracellular milieu of Hcy-exposed macrophages. The increasing in ATP, ADP and AMP hydrolysis are not explained by increased transcription or protein expression of NTPDases and ecto-5'-nucleotidase (ecto-5'-NT/CD73) enzymes. Moreover, the formation of reactive oxygen species did not interfere in the Hcy effects, which suggest that Hcy or Hcy metabolites act directly on the modulation of NTPDases and ecto-5'-NT/CD73 activities. In conclusion, Hcy induces the rapid breakdown of ATP, ADP and AMP to adenosine (ADO), which is classically known as an anti-inflammatory response in immune cells. However, by the action of these enzymes, the extracellular adenosine generated during Hcy treatment probably is uptaken into the cells, as evidenced by the decreased in inosine formation, and thus collaborating to the inflammatory complications associates to HHcy. PMID:24055814

Zanin, Rafael Fernandes; Bergamin, Letícia Scussel; Braganhol, Elizandra; Sévigny, Jean; de Souza Wyse, Angela Terezinha; Battastini, Ana Maria Oliveira

2013-12-01

51

Mitochondrial DNA & Oxidative Phosphorylation ATP SYNTHASE  

E-print Network

# RNAi #12;Ethidium bromide - DNA intercalating dye EtBr inserts itself between the bases of the DNAMitochondrial DNA & Oxidative Phosphorylation 2 proteins ATP SYNTHASE COMPLEX IV COMPLEX IIICOMPLEX II COMPLEX I 1 protein 3 proteins Human mitochondrial DNA 7 proteins #12;Replication Organisation

Schnaufer, Achim

52

The concept of major depression. I. Descriptive comparison of six competing operational definitions including ICD-10 and DSM-III-R.  

PubMed

All operationalized diagnostic systems contain a diagnostic category, which corresponds to the concept of major depression. Yet, these corresponding definitions are not identical. Up to now, no comprehensive comparisons of the competing diagnoses have been published. We will therefore present a series of studies, describing six different operational definitions of major depression according to their content and construction and empirically comparing them in large inpatient and outpatient samples. This first paper presents a descriptive comparison of the definitions given in the Feighner Diagnostic Criteria, the Research Diagnostic Criteria, the Diagnostic and Statistical Manual of Mental Disorders, third edition and third edition, revised, and in two developmental drafts of the ICD-10 diagnostic criteria for research (draft April 1987-I87; draft April 1989-I89). The descriptive comparison will demonstrate that there are many similarities, especially concerning the symptom-criteria of major depression. Classificatory relevance could only be assumed for those differences found for cut-offs, for time criteria and especially for exclusion criteria. Whether these differences are negligible and whether patients classified by different diagnostic systems are really comparable will be examined in subsequent publications. PMID:1829000

Philipp, M; Maier, W; Delmo, C D

1991-01-01

53

ADP and ATP binding to noncatalytic sites of thiol-modulated chloroplast ATP synthase  

Microsoft Academic Search

A modified ‘cold chase’ technique was used to study tight [14C]ADP and [14C]ATP binding to noncatalytic sites of chloroplast ATP synthase (CF0F1). The binding was very low in the dark and sharply increased with light intensity. Dissociation of labeled nucleotides incorporated into noncatalytic sites of CF0F1 or CF1 reconstituted with EDTA-treated thylakoid membranes was also found to be light-dependent. Time

Alexander N. Malyan

2006-01-01

54

[ATP-insulin conjugates and their use for immunofactor analysis].  

PubMed

A method resulting in ATP-insulin conjugates by covalent binding of ATP modified at C(6) amino group of the adenine residue with insulin was developed. The modified ATP was bound to insulin by means of metha-p-toluene sulfonate-N-cyclohezyl Nf [2-morpholinyl(4)ethyl]-carbodiimide. The ATP analogs and ATP-insulin conjugates possess the coenzyme activity in a reaction of luciferin oxidation by luciferase from the fireflies Luciola mingrelica. the catalytic properties of soluble and immobilize on CNBR-activated. Sepharose enzymes in reactions with native ATR, its modified derivatives and ATP--insulin conjugates were compared. The bioluminescence reaction involving ATP--insulin conjugate is inhibited by antibodies against insulin. This effect can form a basis for insulin detection in solution, which is based on competitive binding of free and antibody-labelled ATP--insulin conjugates. PMID:7018595

Gavrilova, E M; Kiseleva, N I; Egorov, A M; Berezin, I V

1981-02-01

55

ATP-dependent Mitochondrial Porphyrin Importer ABCB6 Protects against Phenylhydrazine Toxicity*  

PubMed Central

Abcb6 is a mammalian mitochondrial ATP-binding cassette (ABC) transporter that regulates de novo porphyrin synthesis. In previous studies, haploinsufficient (Abcb6+/?) embryonic stem cells showed impaired porphyrin synthesis. Unexpectedly, Abcb6?/? mice derived from these stem cells appeared phenotypically normal. We hypothesized that other ATP-dependent and/or -independent mechanisms conserve porphyrins. Here, we demonstrate that Abcb6?/? mice lack mitochondrial ATP-driven import of coproporphyrin III. Gene expression analysis revealed that loss of Abcb6 results in up-regulation of compensatory porphyrin and iron pathways, associated with elevated protoporphyrin IX (PPIX). Phenylhydrazine-induced stress caused higher mortality in Abcb6?/? mice, possibly because of sustained elevation of PPIX and an inability to convert PPIX to heme despite elevated ferrochelatase levels. Therefore, Abcb6 is the sole ATP-dependent porphyrin importer, and loss of Abcb6 produces up-regulation of heme and iron pathways necessary for normal development. However, under extreme demand for porphyrins (e.g. phenylhydrazine stress), these adaptations appear inadequate, which suggests that under these conditions Abcb6 is important for optimal survival. PMID:22294697

Ulrich, Dagny L.; Lynch, John; Wang, Yao; Fukuda, Yu; Nachagari, Deepa; Du, Guoqing; Sun, Daxi; Fan, Yiping; Tsurkan, Lyudmila; Potter, Philip M.; Rehg, Jerold E.; Schuetz, John D.

2012-01-01

56

Molecular Analysis of an ATP-Dependent Anion Pump  

Microsoft Academic Search

The plasmid-borne arsenical resistance operon encodes an ATP-driven oxyanion pump for the extrusion of the oxyanions arsenite, antimonite and arsenate from bacterial cells. The catalytic component of the pump, the 63 kDa ArsA protein, hydrolyses ATP in the presence of its anionic substrate antimonite (SbO^-_2). The ATP analogue 5'-p-fluorosulphonylbenzoyladenosine was used to modify the ATP binding site(s) of the ArsA

B. P. Rosen; C.-M. Hsu; C. E. Karkaria; J. B. Owolabi; L. S. Tisa

1990-01-01

57

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

58

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

E-print Network

in a DNA-stimulated manner (Shiba et al., 1991; Mu¨ller et al., 1993b; Mitchell & West, 1994; MarrioneAllosteric 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 Ruv

Cox, Michael M.

59

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

Clemencon, Benjamin; Rey, Martial; Trezeguet, Veronique; Forest, Eric; Pelosi, Ludovic

2011-01-01

60

Autism post-mortem neuroinformatic resource: the autism tissue program (ATP) informatics portal.  

PubMed

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 sorting of existing cases based on donor ante-mortem history as well as agonal states and post-mortem tissue conditions. Phase II of development established administrative tracking of registrants intending to donate, as well as management of tissue requests and the awarding and tracking of tissue. Phase III is the ongoing assimilation of data sets derived from research on a core group of donors with searchable access by investigators. PMID:16933088

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

2007-03-01

61

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

62

Efficient ATP synthesis by thermophilic Bacillus FoF1-ATP synthase.  

PubMed

F(o)F(1)-ATP synthase (F(o)F(1)) synthesizes ATP in the F(1) portion when protons flow through F(o) to rotate the shaft common to F(1) and F(o). Rotary synthesis in isolated F(1) alone has been shown by applying external torque to F(1) of thermophilic origin. Proton-driven ATP synthesis by thermophilic Bacillus PS3 F(o)F(1) (TF(o)F(1)), 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 TF(o)F(1) 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 Q(10) 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

2011-08-01

63

Hypoxia stimulates vesicular ATP release from rat osteoblasts.  

PubMed

Many neuronal and non-neuronal cell types release ATP in a controlled manner. After release, extracellular ATP (or, following hydrolysis, ADP) acts on cells in a paracrine manner via P2 receptors. Extracellular nucleotides are now thought to play an important role in the regulation of bone cell function. ATP (and ADP), acting via the P2Y(1) receptor, stimulate osteoclast formation and activity, whilst P2Y(2) receptor stimulation by ATP (or UTP) inhibits bone mineralization by osteoblasts. We found that rat calvarial osteoblasts released ATP constitutively, in a differentiation-dependent manner, with mature, bone-forming osteoblasts releasing up to sevenfold more ATP than undifferentiated, proliferating cells. The inhibitors of vesicular exocytosis, monensin, and N-ethylmaleimide (1-1,000 microM) inhibited basal ATP release by up to 99%. The presence of granular ATP-filled vesicles within the osteoblast cytoplasm was demonstrated by quinacrine staining. Exposure to hypoxia (2% O(2)) for up to 3 min increased ATP release from osteoblasts up to 2.5-fold without affecting cell viability. Peak concentrations of ATP released into culture medium were >1 microM, which equates with concentrations known to exert significant effects on osteoblast and osteoclast function. Monensin and N-ethylmaleimide (100 microM) attenuated the hypoxia-induced ATP release by up to 80%. Depletion of quinacrine-stained vesicles was also apparent after hypoxic stimulation, indicating that ATP release had taken place. These data suggest that vesicular exocytosis is a key mediator of ATP release from osteoblasts, in biologically significant amounts. Moreover, increased extracellular ATP levels following acute exposure to low O(2) could influence local purinergic signaling and affect the balance between bone formation and bone resorption. PMID:19259945

Orriss, Isabel R; Knight, Gillian E; Utting, Jennifer C; Taylor, Sarah E B; Burnstock, Geoffrey; Arnett, Timothy R

2009-07-01

64

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

65

Structural Basis of GLUT1 Inhibition by Cytoplasmic ATP  

PubMed Central

Cytoplasmic ATP inhibits human erythrocyte glucose transport protein (GLUT1)–mediated glucose transport in human red blood cells by reducing net glucose transport but not exchange glucose transport (Cloherty, E.K., D.L. Diamond, K.S. Heard, and A. Carruthers. 1996. Biochemistry. 35:13231–13239). We investigated the mechanism of ATP regulation of GLUT1 by identifying GLUT1 domains that undergo significant conformational change upon GLUT1–ATP interaction. ATP (but not GTP) protects GLUT1 against tryptic digestion. Immunoblot analysis indicates that ATP protection extends across multiple GLUT1 domains. Peptide-directed antibody binding to full-length GLUT1 is reduced by ATP at two specific locations: exofacial loop 7–8 and the cytoplasmic C terminus. C-terminal antibody binding to wild-type GLUT1 expressed in HEK cells is inhibited by ATP but binding of the same antibody to a GLUT1–GLUT4 chimera in which loop 6–7 of GLUT1 is substituted with loop 6–7 of GLUT4 is unaffected. ATP reduces GLUT1 lysine covalent modification by sulfo-NHS-LC-biotin by 40%. AMP is without effect on lysine accessibility but antagonizes ATP inhibition of lysine modification. Tandem electrospray ionization mass spectrometry analysis indicates that ATP reduces covalent modification of lysine residues 245, 255, 256, and 477, whereas labeling at lysine residues 225, 229, and 230 is unchanged. Exogenous, intracellular GLUT1 C-terminal peptide mimics ATP modulation of transport whereas C-terminal peptide-directed IgGs inhibit ATP modulation of glucose transport. These findings suggest that transport regulation involves ATP-dependent conformational changes in (or interactions between) the GLUT1 C terminus and the C-terminal half of GLUT1 cytoplasmic loop 6–7. PMID:17635959

Blodgett, David M.; De Zutter, Julie K.; Levine, Kara B.; Karim, Pusha; Carruthers, Anthony

2007-01-01

66

ZEPLIN-III  

SciTech Connect

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

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

2009-09-08

67

Yeast Cells Depleted in Atp14p Fail to Assemble Atp6p within the ATP Synthase and Exhibit Altered Mitochondrial Cristae Morphology*  

PubMed Central

Within the yeast mitochondrial ATP synthase, subunit h is a small nuclear encoded protein belonging to the so-called “peripheral stalk” that connects the enzyme catalytic F1 component to the mitochondrial inner membrane. This study examines the role of subunit h in ATP synthase function and assembly using a regulatable, doxycycline-repressible subunit h gene to overcome the strong instability of the mtDNA previously observed in strains lacking the native subunit h gene. Yeast cells expressing less than 3% of subunit h, but still containing intact mitochondrial genomes, grew poorly on respiratory substrates because of a major impairment of ATP synthesis originating from the ATP synthase, whereas the respiratory chain complexes were not affected. The lack of ATP synthesis in the subunit h-depleted (?h) mitochondria was attributed to defects in the assembly/stability of the ATP synthase. A main feature of ?h-mitochondria was a very low content (<6%) in the mitochondrially encoded Atp6p subunit, an essential component of the enzyme proton channel, which was in large part because of a slowing down in translation. Interestingly, depletion of subunit h resulted in dramatic changes in mitochondrial cristae morphology, which further supports the existence of a link between the ATP synthase and the folding/biogenesis of the inner mitochondrial membrane. PMID:18252710

Goyon, Vanessa; Fronzes, Remi; Salin, Benedicte; di-Rago, Jean-Paul; Velours, Jean; Brethes, Daniel

2008-01-01

68

Are rod outer segment ATP-ase and ATP-synthase activity expression of the same protein?  

PubMed

Vertebrate retinal rod outer segments (OS) consist of a stack of disks surrounded by the plasma membrane, where phototransduction takes place. Energetic metabolism in rod OS remains obscure. Literature described a so-called Mg(2+)-dependent ATPase activity, while our previous results demonstrated the presence of oxidative phosphorylation (OXPHOS) in OS, sustained by an ATP synthetic activity. Here we propose that the OS ATPase and ATP synthase are the expression of the same protein, i.e., of F1Fo-ATP synthase. Imaging on bovine retinal sections showed that some OXPHOS proteins are expressed in the OS. Biochemical data on bovine purified rod OS, characterized for purity, show an ATP synthase activity, inhibited by classical F1Fo-ATP synthase inhibitors. Moreover, OS possess a pH-dependent ATP hydrolysis, inhibited by pH values below 7, suggestive of the functioning of the inhibitor of F1 (IF1) protein. WB confirmed the presence of IF1 in OS, substantiating the expression of F1Fo ATP synthase in OS. Data suggest that the OS F1Fo ATP synthase is able to hydrolyze or synthesize ATP, depending on in vitro or in vivo conditions and that the role of IF1 would be pivotal in the prevention of the reversal of ATP synthase in OS, for example during hypoxia, granting photoreceptor survival. PMID:23568658

Calzia, Daniela; Candiani, Simona; Garbarino, Greta; Caicci, Federico; Ravera, Silvia; Bruschi, Maurizio; Manni, Lucia; Morelli, Alessandro; Traverso, Carlo Enrico; Candiano, Giovanni; Tacchetti, Carlo; Panfoli, Isabella

2013-07-01

69

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

PubMed

F(0)F(1)-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 F(0)F(1)s 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]·[P(i)]), 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

2012-07-10

70

The Binding Mode of ATP Revealed by the Solution Structure of the N-domain of Human ATP7A*  

PubMed Central

We report the solution NMR structures of the N-domain of the Menkes protein (ATP7A) in the ATP-free and ATP-bound forms. The structures consist of a twisted antiparallel six-stranded ?-sheet flanked by two pairs of ?-helices. A protein loop of 50 amino acids located between ?3 and ?4 is disordered and mobile on the subnanosecond time scale. ATP binds with an affinity constant of (1.2 ± 0.1) × 104 m?1 and exchanges with a rate of the order of 1 × 103 s?1. The ATP-binding cavity is considerably affected by the presence of the ligand, resulting in a more compact conformation in the ATP-bound than in the ATP-free form. This structural variation is due to the movement of the ?1-?2 and ?2-?3 loops, both of which are highly conserved in copper(I)-transporting PIB-type ATPases. The present structure reveals a characteristic binding mode of ATP within the protein scaffold of the copper(I)-transporting PIB-type ATPases with respect to the other P-type ATPases. In particular, the binding cavity contains mainly hydrophobic aliphatic residues, which are involved in van der Waal's interactions with the adenine ring of ATP, and a Glu side chain, which forms a crucial hydrogen bond to the amino group of ATP. PMID:19917612

Banci, Lucia; Bertini, Ivano; Cantini, Francesca; Inagaki, Sayaka; Migliardi, Manuele; Rosato, Antonio

2010-01-01

71

Characterization of ATP transport into chromaffin granule ghosts. Synergy of ATP and serotonin accumulation in chromaffin granule ghosts.  

PubMed

ATP is an excitatory neurotransmitter that is stored and cosecreted with catecholamines from cells of the adrenal medulla. While the transport of catecholamines into chromaffin granule ghosts has been extensively characterized, there is little information on the mechanism of ATP transport into these structures. Here we show that ATP transport is driven by the electrical component of the electrochemical proton gradient created by the chromaffin granule membrane H+-ATPase, and that the accumulated nucleotide is released from the vesicles by inhibition of the H+-ATPase. GTP and UTP are also substrates for this transporter, distinguishing it from the mitochondrial ADP/ATP exchanger. Accumulation of ADP and ATP (rather than exchange with intravesicular ATP) is demonstrated by high pressure liquid chromatography measurements. The anion transport inhibitor 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (Ki = 27 microM) inhibits ATP transport, while atractyloside, the inhibitor of the mitochondrial ATP/ADP exchanger, is a very poor inhibitor. Finally, we have demonstrated a synergy between the accumulation of ATP and that of serotonin (i.e. more of each solute accumulates when the two are accumulated together), supporting the view that there is an interaction between serotonin and ATP that reduces their effective concentration within the ghosts. PMID:8663306

Bankston, L A; Guidotti, G

1996-07-19

72

Subunit structure of ATP synthase from Chloroflexus aurantiacus.  

PubMed

An ATP synthase has been isolated from green nonsulfur photosynthetic bacterium Chloroflexus aurantiacus, a representative of a lower branch of eubacteria. The enzyme, reconstituted with the bacterial lipids into proteoliposomes, is shown to catalyze [32P]Pi-ATP exchange (at a rate of 180 nmol [32P]ATP/min/mg). The ATP synthase is composed of nine polypeptide species (60, 50, 33, 19, 16.5, 15.5, 14.5, 13, and 8 kDa as determined by urea-SDS-PAGE). The catalytic part of the ATP synthase (which is detached by chloroform treatment) contains the first four polypeptides. In the intact ATP synthase the 14.5 and 13 kDa polypeptides are connected by disulfide bonds to form a heterodimer of 25 kDa. PMID:8243672

Yanyushin, M F

1993-11-29

73

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

74

Synaptic Co-Release of ATP and GABA  

Microsoft Academic Search

\\u000a Over the last 30 years, adenosine 5’-triphosphate (ATP) has been clearly established as a cotransmitter with noradrenaline\\u000a and acetylcholine in the peripheral nervous system. More recently, ATP was also identified as a cotransmitter in the central\\u000a nervous system. In neuronal cultures from postnatal rat spinal cord dorsal horn or embryonic chick and postnatal mouse lateral\\u000a hypothalamus, ATP was surprisingly found

S. Hugel; Y. H. Jo; R. Schlichter

75

Role of macula densa adenosine triphosphate (ATP) in tubuloglomerular feedback  

Microsoft Academic Search

Role of macula densa adenosine triphosphate (ATP) in tubuloglomerular feedback.BackgroundRecent studies have shown that adenosine triphosphate (ATP) is liberated from macula densa cells in response to increased tubular NaCl in vitro. We tested the hypothesis that increased NaCl in the macula densa stimulates the release of ATP, resulting in extracellular formation of adenosine which is involved in signal transmission of

YILIN REN; JEFFREY L GARVIN; RUISHENG LIU; OSCAR A CARRETERO

2004-01-01

76

Hypoxia stimulates vesicular ATP release from rat osteoblasts  

Microsoft Academic Search

Many neuronal and non-neuronal cell types release ATP in a controlled manner. After release, extracellular ATP (or, following hydrolysis, ADP) acts on cells in a paracrine manner via P2 receptors. Extracellular nucleotides are now thought to play an important role in the regulation of bone cell function. ATP (and ADP), acting via the P2Y1 receptor, stimulate osteoclast formation and activity,

Isabel R. Orriss; Gillian E. Knight; Jennifer C. Utting; Sarah E. B. Taylor; Geoffrey Burnstock; Timothy R. Arnett

2009-01-01

77

Oxygen reduction and optimum production of ATP in photosynthesis  

Microsoft Academic Search

THE accepted pathway of CO2 fixation in plant photosynthesis requires that the photosynthetic light reactions produce ATP and reduced pyridine nucleotide (NADPH) in the molar ratio 3:2 (ref. 1). Early studies of photosynthetic phosphorylation suggested that non-cyclic electron transport could produce only equimolar amounts of ATP and NADPH, and the source of the extra ATP was presumed to be cyclic

J. F. Allen

1975-01-01

78

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

79

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

80

Binding of ATP by pertussis toxin and isolated toxin subunits  

SciTech Connect

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

Hausman, S.Z.; Manclark, C.R.; Burns, D.L. (Center for Biologics Evaluation and Research, Bethesda, MD (USA))

1990-07-03

81

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.

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

2014-01-01

82

Theoretical studies of the ATP hydrolysis mechanism of myosin.  

PubMed Central

The ATP hydrolysis mechanism of myosin was studied using quantum chemical (QM) and molecular dynamics calculations. The initial model compound for QM calculations was constructed on the basis of the energy-minimized structure of the myosin(S1dc)-ATP complex, which was determined by molecular mechanics calculations. The result of QM calculations suggested that the ATP hydrolysis mechanism of myosin consists of a single elementary reaction in which a water molecule nucleophilically attacked gamma-phosphorus of ATP. In addition, we performed molecular dynamics simulations of the initial and final states of the ATP hydrolysis reaction, that is, the myosin-ATP and myosin-ADP.Pi complexes. These calculations revealed roles of several amino acid residues (Lys185, Thr186, Ser237, Arg238, and Glu459) in the ATPase pocket. Lys185 maintains the conformation of beta- and gamma-phosphate groups of ATP by forming the hydrogen bonds. Thr186 and Ser237 are coordinated to a Mg(2+) ion, which interacts with the phosphates of ATP and therefore contributes to the stabilization of the ATP structure. Arg238 and Glu459, which consisted of the gate of the ATPase pocket, retain the water molecule acting on the hydrolysis at the appropriate position for initiating the hydrolysis. PMID:11606291

Okimoto, N; Yamanaka, K; Ueno, J; Hata, M; Hoshino, T; Tsuda, M

2001-01-01

83

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

84

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

PubMed Central

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; Antunez, C; Ramirez-Lorca, R; DeStefano, A L; Gonzalez-Perez, A; Gayan, J; Lopez-Arrieta, J; Ikram, M A; Hernandez, I; Marin, J; Galan, J J; Bis, J C; Mauleon, A; Rosende-Roca, M; Moreno-Rey, C; Gudnasson, V; Moron, F J; Velasco, J; Carrasco, J M; Alegret, M; Espinosa, A; Vinyes, G; Lafuente, A; Vargas, L; Fitzpatrick, A L; Launer, L J; Saez, M E; Vazquez, E; Becker, J T; Lopez, O L; Serrano-Rios, M; Tarraga, L; van Duijn, C M; Real, L M; Seshadri, S; Ruiz, A

2014-01-01

85

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

86

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

87

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

88

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

PubMed Central

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

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

2014-01-01

89

October 2006 ATP and the U.S. Innovation  

E-print Network

, and services from ATP supported projects) · Impacts (long term impacts on U.S. industry, society, and economy Assessment Office (EAO) has performed rigorous and multifaceted evaluations to assess the impact economic impact of ATP projects. · Special Surveys, including the Survey of Applicants and the Survey

90

Myosin Motors: The Chemical Restraints Imposed by ATP  

E-print Network

2 Myosin Motors: The Chemical Restraints Imposed by ATP I. Rayment and J. Allingham University organization of linear molecular motors seen today with a focus on myosin. This chapter is written to ask@biochem.wisc.edu I. Rayment and J. Allingham: Myosin Motors: The Chemical Restraints Imposed by ATP, Lect. Notes Phys

Rayment, Ivan

91

The effect of polyhydroxyphenols on brain ATP in the mouse  

Microsoft Academic Search

The penetration of pyrogallol and catechol into the brains of mice after an intraperitoneal injection has been studied together with their effects on locomotor activity and on the concentration of cerebral ATP. A 60 mg\\/kg dose of catechol produced convulsive activity together with a decrease in concentration of brain ATP. The time courses of these effects were similar, and closely

A. Angel; R. N. Lemon; K. J. Rogers; P. Banks

1969-01-01

92

Methods to monitor and compare mitochondrial and glycolytic ATP production.  

PubMed

ATP is commonly considered as the main energy unit of the cell and participates in a variety of cellular processes. Thus, intracellular ATP concentrations rapidly vary in response to a wide variety of stimuli, including nutrients, hormones, cytotoxic agents, and hypoxia. Such alterations not necessarily affect cytosolic and mitochondrial ATP to similar extents. From an oncological perspective, this is particularly relevant in the course of tumor progression as well as in the response of cancer cells to therapy. In normal cells, mitochondrial oxidative phosphorylation (OXPHOS) is the predominant source of ATP. Conversely, many cancer cells exhibit an increased flux through glycolysis irrespective of oxygen tension. Assessing the relative contribution of glycolysis and OXPHOS to intracellular ATP production is fundamental not only for obtaining further insights into the peculiarities and complexities of oncometabolism but also for developing therapeutic and diagnostic tools. Several techniques have been developed to measure intracellular ATP levels including enzymatic methods based on hexokinase, glucose-6-phosphate dehydrogenase, and firefly luciferase. Here, we summarize conventional methods for measuring intracellular ATP levels and we provide a detailed protocol based on cytosol- and mitochondrion-targeted variants of firefly luciferase to determine the relative contribution of glycolysis and OXPHOS to ATP synthesis. PMID:24862273

Patergnani, Simone; Baldassari, Federica; De Marchi, Elena; Karkucinska-Wieckowska, Agnieszka; Wieckowski, Mariusz R; Pinton, Paolo

2014-01-01

93

Stabilization of amorphous calcium phosphate by Mg and ATP  

Microsoft Academic Search

Summary A synergistic effect has been demonstrated when magnesium and adenosine triphosphate (ATP) are used together in solution to delay the conversion of a slurry of amorphous calcium phosphate (ACP) to crystalline hydroxyapatite (HA). Conversion is delayed in some instances more than 10 times as long as with either ATP or Mg alone. In all experiments conversion did not begin

N. C. Blumenthal; F. Betts; A. S. Posner

1977-01-01

94

Renal epithelial cells can release ATP by vesicular fusion  

PubMed Central

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

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

2013-01-01

95

ATP is released from guinea pig ureter epithelium on distension  

E-print Network

was released from epithelial cells during distension as mechani- cal removal of the urothelium blocked release exocytosis ATP IS RAPIDLY RELEASED from vascular endothelial cells during periods of increased flow, shear from both human and rabbit red blood cells (RBCs) in response to mechanical deforma- tion (48). The ATP

Burnstock, Geoffrey

96

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

PubMed

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

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

2014-10-01

97

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

PubMed Central

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

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

2014-01-01

98

In Vivo Function of Hsp90 Is Dependent on ATP Binding and ATP Hydrolysis  

Microsoft Academic Search

Heat shock protein 90 (Hsp90), an abundant molecular chaperone in the eukaryotic cytosol, is in- volved in the folding of a set of cell regulatory proteins and in the re-folding of stress-denatured polypeptides. The basic mechanism of action of Hsp90 is not yet un- derstood. In particular, it has been debated whether Hsp90 function is ATP dependent. A recent crystal

Wolfgang M. J. Obermann; Holger Sondermann; Alicia A. Russo; Nikola P. Pavletich; F. Ulrich Hartl

1998-01-01

99

The wheat chloroplast gene for CF0 subunit I of ATP synthase contains a large intron  

PubMed Central

The gene for CF0 subunit I of ATP synthase has been located in wheat chloroplast DNA, between the genes for CF0 subunit III and ? subunit of CF1. Nucleotide sequencing and analysis of RNA-DNA hybrids indicated that the gene is interrupted by an 823-bp intron which has boundaries similar to those previously described for the introns in protein-coding chloroplast genes of Euglena gracilis. The deduced amino acid sequence of CF0 subunit I indicates a polypeptide of 183 amino acid residues. However, N-terminal amino acid sequencing of the mature spinach CF0 subunit I suggests that the protein is synthesised with a N-terminal extension of 17 amino acid residues and is processed to give a protein of mol. wt. 19 001 of 166 amino acids residues. The mature CF0 subunit I shows similarities in primary and predicted secondary structure to F0 subunit b of Escherichia coli ATP synthase. A major transcript of 3.3 kb containing sequences from the genes for CF0 subunit III, subunit I and CF1 subunit ? has been observed by RNA-DNA hybridisation. ImagesFig. 2.Fig. 4.Fig. 5.Fig. 6. PMID:16453616

Bird, C. R.; Koller, B.; Auffret, A. D.; Huttly, A. K.; Howe, C. J.; Dyer, T. A.; Gray, J. C.

1985-01-01

100

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

101

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.

102

Electrophysiological effects of extracellular ATP on Necturus gallbladder epithelium  

PubMed Central

The effects of addition of ATP to the mucosal bathing solution on transepithelial, apical, and basolateral membrane voltages and resistances in Necturus gallbladder epithelium were determined. Mucosal ATP (100 microM) caused a rapid hyperpolarization of both apical (Vmc) and basolateral (Vcs) cell membrane voltages (delta Vm = 18 +/- 1 mV), a fall in transepithelial resistance (Rt) from 142 +/- 8 to 122 +/- 7 omega.cm2, and a decrease in fractional apical membrane resistance (fRa) from 0.93 +/- 0.02 to 0.83 +/- 0.03. The rapid initial hyperpolarization of Vmc and Vcs was followed by a slower depolarization of cell membrane voltages and a lumen-negative change in transepithelial voltage (Vms). This phase also included an additional decrease in fRa. Removal of the ATP caused a further depolarization of membrane voltages followed by a hyperpolarization and then a return to control values. fRa fell to a minimum after removal of ATP and then returned to control values as the cell membrane voltages repolarized. Similar responses could be elicited by ADP but not by adenosine. The results of two-point cable experiments revealed that ATP induced an initial increase in cell membrane conductance followed by a decrease. Transient elevations of mucosal solution [K+] induced a larger depolarization of Vmc and Vcs during exposure to ATP than under control conditions. Reduction of mucosal solution [Cl-] induced a slow hyperpolarization of Vmc and Vcs before exposure to ATP and a rapid depolarization during exposure to ATP. We conclude that ATP4- is the active agent and that it causes a concentration-dependent increase in apical and basolateral membrane K+ permeability. In addition, an apical membrane electrodiffusive Cl- permeability is activated by ATP4-. PMID:1713948

1991-01-01

103

Electrophysiological effects of extracellular ATP on Necturus gallbladder epithelium.  

PubMed

The effects of addition of ATP to the mucosal bathing solution on transepithelial, apical, and basolateral membrane voltages and resistances in Necturus gallbladder epithelium were determined. Mucosal ATP (100 microM) caused a rapid hyperpolarization of both apical (Vmc) and basolateral (Vcs) cell membrane voltages (delta Vm = 18 +/- 1 mV), a fall in transepithelial resistance (Rt) from 142 +/- 8 to 122 +/- 7 omega.cm2, and a decrease in fractional apical membrane resistance (fRa) from 0.93 +/- 0.02 to 0.83 +/- 0.03. The rapid initial hyperpolarization of Vmc and Vcs was followed by a slower depolarization of cell membrane voltages and a lumen-negative change in transepithelial voltage (Vms). This phase also included an additional decrease in fRa. Removal of the ATP caused a further depolarization of membrane voltages followed by a hyperpolarization and then a return to control values. fRa fell to a minimum after removal of ATP and then returned to control values as the cell membrane voltages repolarized. Similar responses could be elicited by ADP but not by adenosine. The results of two-point cable experiments revealed that ATP induced an initial increase in cell membrane conductance followed by a decrease. Transient elevations of mucosal solution [K+] induced a larger depolarization of Vmc and Vcs during exposure to ATP than under control conditions. Reduction of mucosal solution [Cl-] induced a slow hyperpolarization of Vmc and Vcs before exposure to ATP and a rapid depolarization during exposure to ATP. We conclude that ATP4- is the active agent and that it causes a concentration-dependent increase in apical and basolateral membrane K+ permeability. In addition, an apical membrane electrodiffusive Cl- permeability is activated by ATP4-. PMID:1713948

Cotton, C U; Reuss, L

1991-05-01

104

ATP binding to the ? subunit of thermophilic ATP synthase is crucial for efficient coupling of ATPase and H+ pump activities.  

PubMed

ATP binding to the ? subunit of F1-ATPase, a soluble subcomplex of TFoF1 (FoF1-ATPase synthase from the thermophilic Bacillus strain PS3), affects the regulation of F1-ATPase activity by stabilizing the compact, ATPase-active, form of the ? subunit [Kato, S., Yoshida, M. and Kato-Yamada, Y. (2007) J. Biol. Chem. 282, 37618-37623]. In the present study, we report how ATP binding to the ? subunit affects ATPase and H+ pumping activities in the holoenzyme TFoF1. Wild-type TFoF1 showed significant H+ pumping activity when ATP was used as the substrate. However, GTP, which bound poorly to the ? subunit, did not support efficient H+ pumping. Addition of small amounts of ATP to the GTP substrate restored coupling between GTPase and H+ pumping activities. Similar uncoupling was observed when TFoF1 contained an ATP-binding-deficient ? subunit, even with ATP as a substrate. Further analysis suggested that the compact conformation of the ? subunit induced by ATP binding was required to couple ATPase and H+ pumping activities in TFoF1 unless the ? subunit was in its extended-state conformation. The present study reveals a novel role of the ? subunit as an ATP-sensitive regulator of the coupling of ATPase and H+ pumping activities of TFoF1. PMID:21510843

Kadoya, Fumitaka; Kato, Shigeyuki; Watanabe, Kei; Kato-Yamada, Yasuyuki

2011-07-01

105

Lab III -1 LABORATORY III  

E-print Network

down the principles of Physics that you will use to solve the problem. (Hint: Think about Newton's laws the effect of forces on the motion of objects. In the first problem, you will investigate the effects by saying a system is in "equilibrium." · Write down the force law for a frictional force. #12;Lab III - 2

Minnesota, University of

106

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

107

A1Ao-ATP Synthase of Methanobrevibacter ruminantium Couples Sodium Ions for ATP Synthesis under Physiological Conditions*  

PubMed Central

An unresolved question in the bioenergetics of methanogenic archaea is how the generation of proton-motive and sodium-motive forces during methane production is used to synthesize ATP by the membrane-bound A1Ao-ATP synthase, with both proton- and sodium-coupled enzymes being reported in methanogens. To address this question, we investigated the biochemical characteristics of the A1Ao-ATP synthase (MbbrA1Ao) of Methanobrevibacter ruminantium M1, a predominant methanogen in the rumen. Growth of M. ruminantium M1 was inhibited by protonophores and sodium ionophores, demonstrating that both ion gradients were essential for growth. To study the role of these ions in ATP synthesis, the ahaHIKECFABD operon encoding the MbbrA1Ao was expressed in Escherichia coli strain DK8 (?atp) and purified yielding a 9-subunit protein with an SDS-stable c oligomer. Analysis of the c subunit amino acid sequence revealed that it consisted of four transmembrane helices, and each hairpin displayed a complete Na+-binding signature made up of identical amino acid residues. The purified MbbrA1Ao was stimulated by sodium ions, and Na+ provided pH-dependent protection against inhibition by dicyclohexylcarbodiimide but not tributyltin chloride. ATP synthesis in inverted membrane vesicles lacking sodium ions was driven by a membrane potential that was sensitive to cyanide m-chlorophenylhydrazone but not to monensin. ATP synthesis could not be driven by a chemical gradient of sodium ions unless a membrane potential was imposed. ATP synthesis under these conditions was sensitive to monensin but not cyanide m-chlorophenylhydrazone. These data suggest that the M. ruminantium M1 A1Ao-ATP synthase exhibits all the properties of a sodium-coupled enzyme, but it is also able to use protons to drive ATP synthesis under conditions that favor proton coupling, such as low pH and low levels of sodium ions. PMID:21953465

McMillan, Duncan G. G.; Ferguson, Scott A.; Dey, Debjit; Schröder, Katja; Aung, Htin Lin; Carbone, Vincenzo; Attwood, Graeme T.; Ronimus, Ron S.; Meier, Thomas; Janssen, Peter H.; Cook, Gregory M.

2011-01-01

108

Communication Definitions... general definition  

E-print Network

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

Jones, Ian L.

109

Dimers of mitochondrial ATP synthase form the permeability transition pore  

PubMed Central

Here we define the molecular nature of the mitochondrial permeability transition pore (PTP), a key effector of cell death. The PTP is regulated by matrix cyclophilin D (CyPD), which also binds the lateral stalk of the FOF1 ATP synthase. We show that CyPD binds the oligomycin sensitivity-conferring protein subunit of the enzyme at the same site as the ATP synthase inhibitor benzodiazepine 423 (Bz-423), that Bz-423 sensitizes the PTP to Ca2+ like CyPD itself, and that decreasing oligomycin sensitivity-conferring protein expression by RNAi increases the sensitivity of the PTP to Ca2+. Purified dimers of the ATP synthase, which did not contain voltage-dependent anion channel or adenine nucleotide translocator, were reconstituted into lipid bilayers. In the presence of Ca2+, addition of Bz-423 triggered opening of a channel with currents that were typical of the mitochondrial megachannel, which is the PTP electrophysiological equivalent. Channel openings were inhibited by the ATP synthase inhibitor AMP-PNP (?-imino ATP, a nonhydrolyzable ATP analog) and Mg2+/ADP. These results indicate that the PTP forms from dimers of the ATP synthase. PMID:23530243

Giorgio, Valentina; von Stockum, Sophia; Antoniel, Manuela; Fabbro, Astrid; Fogolari, Federico; Forte, Michael; Glick, Gary D.; Petronilli, Valeria; Zoratti, Mario; Szabo, Ildiko; Lippe, Giovanna; Bernardi, Paolo

2013-01-01

110

Synthesis and fluorescence characteristics of ATP-based FRET probes.  

PubMed

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

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

2013-12-28

111

ATP and potassium ions: a deadly combination for astrocytes  

PubMed Central

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

112

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

113

Vesicular Nucleotide Transporter-Mediated ATP Release Regulates Insulin Secretion  

PubMed Central

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

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

2013-01-01

114

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

115

A Small Aptamer with Strong and Specific Recognition of the Triphosphate of ATP  

E-print Network

Chemicals and Materials Adenosine 5-monophosphate and adenosine 5-triphosphate (linked via a 9-atom isourea-triphosphate (- methylene ATP), - methyleneadenosine 5-triphosphate (- methylene ATP), adenosine 5-(--imido

Heller, Eric

116

Regulation of ATP production by mitochondrial Ca2+  

PubMed Central

Stimulation of mitochondrial oxidative metabolism by Ca2+ is now generally recognised as important for the control of cellular ATP homeostasis. Here, we review the mechanisms through which Ca2+ regulates mitochondrial ATP synthesis. We focus on cardiac myocytes and pancreatic ?-cells, where tight control of this process is likely to play an important role in the response to rapid changes in workload and to nutrient stimulation, respectively. We also describe a novel approach for imaging the Ca2+-dependent regulation of ATP levels dynamically in single cells. PMID:22502861

Tarasov, Andrei I.; Griffiths, Elinor J.; Rutter, Guy A.

2012-01-01

117

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; Borsch, Michael

2013-01-01

118

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

119

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

SciTech Connect

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

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

1996-12-01

120

Functional identification of ATP-driven Ca2+ pump in the peribacteroid membrane of broad bean root nodules.  

PubMed

A Ca2+ indicator arsenazo III was used to demonstrate calcium uptake activity of symbiosomes and the peribacteroid membrane (PBM) vesicles isolated from broad bean root nodules and placed in the medium containing ATP and Mg2+ ions. This process was shown to be rapidly stopped by vanadate, completely reversed in the presence of the calcium ionophore A23187 but insensitive to agents abolishing electrical potential or pH difference across the PBM. The presence of an endogenous calcium pool within isolated symbiosomes and bacteroids was detected using a Ca2+ indicator chlortetracycline. These results prove a primary active transport of Ca2+ through the PBM of legume root nodules and provide the first functional identification of an ATP-driven Ca2+-pump, most likely Mg2+-dependent Ca2+-translocating ATPase, in this membrane. PMID:10218580

Andreev, I M; Dubrovo, P N; Krylova, V V; Izmailov, S F

1999-03-19

121

Distinct neurological disorders with ATP1A3 mutations.  

PubMed

Genetic research has shown that mutations that modify the protein-coding sequence of ATP1A3, the gene encoding the ?3 subunit of Na(+)/K(+)-ATPase, cause both rapid-onset dystonia parkinsonism and alternating hemiplegia of childhood. These discoveries link two clinically distinct neurological diseases to the same gene, however, ATP1A3 mutations are, with one exception, disease-specific. Although the exact mechanism of how these mutations lead to disease is still unknown, much knowledge has been gained about functional consequences of ATP1A3 mutations using a range of in-vitro and animal model systems, and the role of Na(+)/K(+)-ATPases in the brain. Researchers and clinicians are attempting to further characterise neurological manifestations associated with mutations in ATP1A3, and to build on the existing molecular knowledge to understand how specific mutations can lead to different diseases. PMID:24739246

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

2014-05-01

122

Possible ATP release through lysosomal exocytosis from primary sensory neurons.  

PubMed

The adenosine triphosphate (ATP) plays important roles under physiological and pathological conditions such as traumatic brain injury, neuroinflammation and neuropathic pain. In the present study, we set out to study the role of lysosomal vesicles on ATP release from the dorsal root ganglion neurons. We found that the lysosomal vesicles, which contain the quinacrine-positive fluorescence and express the vesicular nucleotide transporter (VNUT), were localized within the soma and growth cone of the cultured dorsal root ganglion neurons. In addition, the number of the quinacrine staining was decreased by application of lysosomal exocytosis activators, and this decrease was suppressed by the metformin and vacuolin-1, which suppressed lysosomal exocytosis. Thus, these findings suggest that ATP release via the lysosomal exocytosis may be one of the pathways for ATP release in response to stimulation. PMID:23237805

Jung, Junyang; Shin, Youn Ho; Konishi, Hiroyuki; Lee, Seo Jin; Kiyama, Hiroshi

2013-01-11

123

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

124

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

125

ATP and ADP hydrolysis in fish, chicken and rat synaptosomes  

Microsoft Academic Search

Ecto-enzymes capable of hydrolyzing ATP and ADP (NTPDase) are present in the central nervous system of various species. In the present investigation we studied the synaptosomal NTPDase (ATP diphosphohydrolase, apyrase, E.C. 3.6.1.5) from fish, chicken and rats under different conditions and in the presence of several classical inhibitors. The cation concentration required for maximal activity was 0.5 mM for fish,

Maria Rosa Chitolina Schetinger; Vânia Lúcia Pimentel Vieira; Vera Maria Morsch; Daniela Balz

2001-01-01

126

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

127

Allopurinol Acutely Increases ATP Energy Delivery in Failing Human Hearts  

PubMed Central

Objectives We tested the hypothesis that acute administration of the XO inhibitor, allopurinol, improves cardiac high-energy phosphate concentrations in human HF and increases the rate of ATP synthesis through creatine kinase (CK), the primary myocardial energy reserve. Background Studies of patients and animal models implicate impaired myocardial high-energy phosphate availability in heart failure (HF). The xanthine oxidase (XO) reaction is a critical terminal step in ATP and purine degradation and an important source of reactive oxygen species. Thus, XO inhibition is a potentially attractive means to improve energy metabolism in the failing human heart. Methods We randomized 16 patients with non-ischemic cardiomyopathy in a double-blind fashion to allopurinol (300 mg IV) or placebo infusion, 4:1, the latter for purposes of blinding only. The myocardial concentrations of adenosine triphosphate (ATP) and creatine phosphate (PCr) and the rate of ATP synthesis through CK (CK flux) were determined by 31P magnetic resonance spectroscopy. Results Allopurinol infusion increased mean cardiac PCr/ATP and [PCr] by ~11% (P < 0.02), and mean CKs flux by 39% (2.07 ± 1.27 ?mol/g/s to 2.87 ± 1.82 ?mol/g/s, p < 0.007). Calculated cytosolic [ADP] declined while the free energy of ATP hydrolysis (?G~ATP) increased with allopurinol. The increased CK flux was disproportionate to substrate changes, indicating increased CK enzyme activity. Conclusions Intravenous administration of the XO inhibitor, allopurinol, acutely improves the relative and absolute concentrations of myocardial high-energy phosphates and ATP flux through CK in the failing human heart, offering direct evidence that myofibrillar CK energy delivery can be pharmaceutically-augmented in the failing human heart. PMID:22361399

Hirsch, Glenn A.; Bottomley, Paul A.; Gerstenblith, Gary; Weiss, Robert G.

2013-01-01

128

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

129

50 CFR 230.2 - Definitions.  

Code of Federal Regulations, 2013 CFR

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

2013-10-01

130

The rate of production of uric acid by hepatocytes is a sensitive index of compromised cell ATP homeostasis.  

PubMed

Plasma levels of uric acid, the final product of purine degradation in humans, are elevated in metabolic syndrome and are strongly associated with insulin resistance and nonalcoholic fatty liver disease (NAFLD). Hepatic and blood levels of purine metabolites (inosine, hypoxanthine, and xanthine) are also altered in pathophysiological states. We optimized a rat hepatocyte model to test the hypothesis that the production of uric acid by hepatocytes is a potential marker of compromised homeostasis of hepatocellular inorganic phosphate (Pi) and/or ATP. The basal rate of uric acid production from endogenous substrates in rat hepatocytes was comparable to that in human liver and was <10% of the maximum rate with saturating concentrations of purine substrates. It was marginally (~20%) decreased by insulin and increased by glucagon but was stimulated more than twofold by substrates (fructose and glycerol) that lower both cell ATP and Pi, and by inhibitors of mitochondrial respiration (complexes I, III, and V) that lower ATP but raise cell Pi. Clearance of inosine and its degradation to uric acid were also inhibited by cell Pi depletion. Analysis of gene expression in NAFLD biopsies showed an association between mRNA expression of GCKR, the glucokinase regulatory protein that is functionally linked to uric acid production, and mRNA expression of the phosphate transporters encoded by SLC17A1/3. Uric acid production by hepatocytes is a very sensitive index of ATP depletion irrespective of whether cell Pi is lowered or raised. This suggests that raised plasma uric acid may be a marker of compromised hepatic ATP homeostasis. PMID:24045866

Petrie, John L; Patman, Gillian L; Sinha, Ishita; Alexander, Thomas D; Reeves, Helen L; Agius, Loranne

2013-11-15

131

In silico exploration of the fructose-6-phosphate phosphorylation step in glycolysis: genomic evidence of the coexistence of an atypical ATP-dependent along with a PPi-dependent phosphofructokinase in Propionibacterium freudenreichii subsp. shermanii.  

PubMed

We performed a detailed bioinformatic study of the catalytic step of fructose-6-phosphate phosphorylation in glycolysis based on the raw genomic draft of Propionibacterium freudenreichii subsp. shermanii (P. shermanii) ATCC9614 [Meurice et al., 2004]. Our results provide the first in silico evidence of the coexistence of genes coding for an ATP-dependent phosphofructokinase (ATP-PFK) and a PPi-dependent phosphofructokinase (PPi-PFK), whereas the fructose-1,6-bisphosphatase (FBP) and ADP-dependent phosphofructokinase (ADP-PFK) are absent. The deduced amino acid sequence corresponding to the PPi-PFK (AJ508922) shares 100% similarity with the already characterised propionibacterial protein (P29495; Ladror et al., 1991]. The unexpected ATP-PFK gene (AJ509827) encodes a protein of 373 aa which is highly similar (50% positive residues) along at least 95% of its sequence length to different well-characterised ATP-PFKs. The characteristic PROSITE pattern important for the enzyme function of ATP-PFKs (PS00433) was conserved in the putative ATP-PFK sequence: 8 out of 9 amino acid residues. According to the recent evolutionary study of PFK proteins with different phosphate donors [Bapteste et al., 2003], the propionibacterial ATP-PFK harbours a G104-K124 residue combination, which strongly suggested that this enzyme belongs to the group of atypical ATP-PFKs. According to our phylogenetic analyses the amino acid sequence of the ATP-PFK is clustered with the atypical ATP-PFKs from group III of the Siebers classification [Siebers et al., 1998], whereas the expected PPi-PFK protein is closer to the PPi-PFKs from clade P [Müller et al., 2001]. The possible significance of the co-existence of these two PFKs and their importance for the regulation of glycolytic pathway flux in P. shermanii is discussed. PMID:15507000

Meurice, Guillaume; Deborde, Catherine; Jacob, Daniel; Falentin, Hélène; Boyaval, Patrick; Dimova, Diliana

2004-01-01

132

Cardiolipin affects the supramolecular organization of ATP synthase in mitochondria.  

PubMed

F(1)F(0) ATP synthase forms dimers that tend to assemble into large supramolecular structures. We show that the presence of cardiolipin is critical for the degree of oligomerization and the degree of order in these ATP synthase assemblies. This conclusion was drawn from the statistical analysis of cryoelectron tomograms of cristae vesicles isolated from Drosophila flight-muscle mitochondria, which are very rich in ATP synthase. Our study included a wild-type control, a cardiolipin synthase mutant with nearly complete loss of cardiolipin, and a tafazzin mutant with reduced cardiolipin levels. In the wild-type, the high-curvature edge of crista vesicles was densely populated with ATP synthase molecules that were typically organized in one or two rows of dimers. In both mutants, the density of ATP synthase was reduced at the high-curvature zone despite unchanged expression levels. Compared to the wild-type, dimer rows were less extended in the mutants and there was more scatter in the orientation of dimers. These data suggest that cardiolipin promotes the ribbonlike assembly of ATP synthase dimers and thus affects lateral organization and morphology of the crista membrane. PMID:21539786

Acehan, Devrim; Malhotra, Ashim; Xu, Yang; Ren, Mindong; Stokes, David L; Schlame, Michael

2011-05-01

133

A genetically encoded fluorescent reporter of ATP/ADP ratio  

PubMed Central

A fluorescent sensor of adenylate nucleotides was constructed by combining a circularly permuted variant of green fluorescent protein with a bacterial regulatory protein, GlnK1, from Methanococcus jannaschii. The affinity for Mg-ATP is below 100 nM, as seen for the other members of the bacterial PII regulator family – a surprisingly high affinity given normal intracellular [ATP] in the millimolar range. ADP binds to the same site, competing with Mg-ATP but producing a smaller change in fluorescence. With normal physiological concentrations of ATP and ADP, the binding site is saturated, but competition between the two substrates causes the sensor to behave as a nearly ideal reporter of the ATP/ADP concentration ratio. This principle for sensing the ratio of two analytes by competition at a high affinity site probably underlies the normal functioning of PII regulatory proteins. The engineered sensor, Perceval, can be used to monitor the ATP/ADP ratio during live cell imaging. PMID:19122669

Berg, Jim; Hung, Yin Pun; Yellen, Gary

2008-01-01

134

Structural basis for the ATP-induced isomerization of kinesin.  

PubMed

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

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

2013-06-12

135

Reaction dynamics of ATP hydrolysis catalyzed by P-glycoprotein.  

PubMed

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 ?(18)O4-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 (18)O/two (18)O/one (18)O that reflects the isotopic composition of the starting ATP in multiple experiments. Thus, H2O-exchange with HPO4(2-) (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

Scian, Michele; Acchione, Mauro; Li, Mavis; Atkins, William M

2014-02-18

136

Characterization of domain interfaces in monomeric and dimeric ATP synthase.  

PubMed

We disassembled monomeric and dimeric yeast ATP synthase under mild conditions to identify labile proteins and transiently stable subcomplexes that had not been observed before. Specific removal of subunits alpha, beta, oligomycin sensitivity conferring protein (OSCP), and h disrupted the ATP synthase at the gamma-alpha(3)beta(3) rotor-stator interface. Loss of two F(1)-parts from dimeric ATP synthase led to the isolation of a dimeric subcomplex containing membrane and peripheral stalk proteins thus identifying the membrane/peripheral stalk sectors immediately as the dimerizing parts of ATP synthase. Almost all subunit a was found associated with a ring of 10 c-subunits in two-dimensional blue native/SDS gels. We therefore postulate that c10a1-complex is a stable structure in resting ATP synthase until the entry of protons induces a breaking of interactions and stepwise rotation of the c-ring relative to the a-subunit in the catalytic mechanism. Dimeric subunit a was identified in SDS gels in association with two c10-rings suggesting that a c10a2c10-complex may constitute an important part of the monomer-monomer interface in dimeric ATP synthase that seems to be further tightened by subunits b, i, e, g, and h. In contrast to the monomer-monomer interface, the interface between dimers in higher oligomeric structures remains largely unknown. However, we could show that the natural inhibitor protein Inh1 is not required for oligomerization. PMID:18245802

Wittig, Ilka; Velours, Jean; Stuart, Rosemary; Schägger, Hermann

2008-05-01

137

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

PubMed

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

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

2012-01-01

138

A Convenient One-Step Extraction of Cellular ATP Using Boiling Water for the Luciferin–Luciferase Assay of ATP  

Microsoft Academic Search

Cellular ATP is commonly determined as production of bioluminescence using a luciferin–luciferase reaction system. Before the measurement of bioluminescence, cellular ATP must first be extracted. Two commonly used extraction methods are: (1) Tris–borate buffer (pH 9.2) coupled with a heating process (to inactivate ATPase) and (2) perchloric acid followed by neutralization. However, we found that both Tris–borate buffer and perchloric

Nae-Cherng Yang; Wai-Meng Ho; Yu-Hsuan Chen; Miao-Lin Hu

2002-01-01

139

A new approach to time-resolved studies of ATP-requiring biological systems; laser flash photolysis of caged ATP.  

PubMed Central

2-Nitrobenzyl derivatives have been used for several years as photolabile protecting groups in synthetic organic chemistry. Recently, P3-1-(2-nitro phenylethyladenosine 5'-triphosphate "caged ATP" was synthesized and its photolysis was shown to generate ATP in situ. This and related reactions have great potential for structural and kinetic studies of both intact and soluble biological systems and it is thus important to define the kinetic characteristics of the photolytic reaction. Caged ATP (2.5 mM) was photolyzed at 347 nm by a single 30-nsec pulse from a frequency-doubled ruby laser of 25 mJ energy to generate 500 microM ATP. The kinetics of the overall reaction were determined by monitoring the kinetics of ATP-induced dissociation of actomyosin, a reaction of known kinetic characteristics. Release of 500 microM ATP was found to be controlled by a process having a rate constant of 2.2 X 10(9) [H+] sec-1 at 22 degrees C at pH 5.8-9.5, which corresponds to 220 sec-1 at pH 7. This process is believed to be the breakdown of an aci-nitro compound, which was identified on the basis of its spectral properties and the photochromicity of related 2-nitrobenzyl compounds. Images PMID:6938971

McCray, J A; Herbette, L; Kihara, T; Trentham, D R

1980-01-01

140

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

141

Molecular mechanisms of ATP secretion during immunogenic cell death.  

PubMed

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

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

2014-01-01

142

ATP-DEPENDENT SUGAR TRANSPORT COMPLEXITY IN HUMAN ERYTHROCYTES  

PubMed Central

Human erythrocyte glucose sugar transport was examined in resealed red cell ghosts under equilibrium exchange conditions ([sugar]intracellular = [sugar]extracellular). Exchange 3-O-methylglucose (3MG) import and export are monophasic in the absence of cytoplasmic ATP but are biphasic when ATP is present. Biphasic exchange is observed as the rapid filling of a large compartment (66% cell volume) followed by the slow filling of the remaining cytoplasmic space. Biphasic exchange at 20 mM 3MG eliminates the possibility that the rapid exchange phase represents ATP-dependent 3MG binding to the glucose transport protein (GLUT1; cellular [GLUT1] ? 20 ?M). Immunofluorescence activated cell sorting analysis shows that biphasic exchange does not result from heterogeneity in cell size or GLUT1 content. Nucleoside transporter mediated uridine exchange proceeds as rapidly as 3MG exchange but is monoexponential regardless of cytoplasmic [ATP]. This eliminates cellular heterogeneity or an ATP-dependent, nonspecific intracellular diffusion barrier as causes of biphasic exchange. Red cell ghost 3MG and uridine equilibrium volumes (130 fL) are unaffected by ATP. GLUT1 intrinsic activity is unchanged during rapid and slow phases of 3MG exchange. Two models for biphasic sugar transport are presented in which 3MG must overcome a sugar-specific, physical (diffusional) or chemical (isomerization) barrier to equilibrate with cell water. Partial transport inhibition using cytochalasin B or maltose depresses both rapid and slow phases of transport thereby eliminating the physical barrier hypothesis. We propose that biphasic 3MG transport results from ATP-dependent, differential transport of 3MG anomers in which Vmax/Km(app) for ?-3MG exchange transport is 19-fold greater than Vmax/Km(app) for ?-3MG transport. PMID:16928769

Leitch, Jeffry; Carruthers, Anthony

2014-01-01

143

New soluble ATP-dependent protease, Ti, in Escherichia coli that is distinct from protease La  

Microsoft Academic Search

E. coli must contain other ATP-requiring proteolytic systems in addition to protease La (the lon gene product). A new ATP-dependent protease was purified from lon cells which lack protease La, as shown by immuno-blotting. This enzyme hydrolyzes (TH)casein to acid-soluble products in the presence of ATP (or dATP) and MgS . Nonhydrolyzable ATP analogs, other nucleoside triphosphates and AMP can

C. H. Chung; B. J. Hwang; W. J. Park; A. L. Goldberg

1987-01-01

144

Nucleotide binding to noncatalytic sites is essential for ATP-dependent stimulation and ADP-dependent inactivation of the chloroplast ATP synthase  

Microsoft Academic Search

Light-dependent binding of labeled ADP and ATP to noncatalytic sites of chloroplast ATP synthase and the effect of light-exposed\\u000a thylakoid membrane preincubation with ADP or ATP on ATPase activity were studied. ADP binding during the preincubation was\\u000a shown to inactivate the chloroplast ATPase, whereas ATP binding caused its activation. The rate and equilibrium constants\\u000a of ATPase inactivation and activation were

Alexander N. Malyan

2010-01-01

145

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

Microsoft Academic Search

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

Marcel D. Waldinger; Dave H. Schweitzer

2006-01-01

146

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

147

Expanding the clinical phenotypes of MT-ATP6 mutations.  

PubMed

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

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

2014-12-01

148

ATP and ADP hydrolysis in cell membranes from rat myometrium.  

PubMed

Extracellular nucleotides affect female reproductive functions, fertilization, and pregnancy. The aim of this study was to investigate biochemical characteristics of ATP and ADP hydrolysis and identify E-NTPDases in myometrial cell membranes from Wistar albino rats. The apparent K (m) values were 506.4 ± 62.1 and 638.8 ± 31.3 ?M, with a calculated V (max) (app) of 3,973.0 ± 279.5 and 2,853.9 ± 79.8 nmol/min/mg for ATP and ADP, respectively. The enzyme activity described here has common properties characteristic for NTPDases: divalent cation dependence; alkaline pH optimum for both substrates, insensitivity to some of classical ATPase inhibitors (ouabain, oligomycine, theophylline, levamisole) and significant inhibition by suramine and high concentration of sodium azides (5 mM). According to similar apparent K(m) values for both substrates, the ATP/ADP hydrolysis ratio, and Chevillard competition plot, NTPDase1 is dominant ATP/ADP hydrolyzing enzyme in myometrial cell membranes. RT-PCR analysis revealed expression of three members of ectonucleoside triphosphate diphosphohydrolase family (NTPDase 1, 2, and 8) in rat uterus. These findings may further elucidate the role of NTPDases and ATP in reproductive physiology. PMID:22956447

Miloševi?, Maja; Petrovi?, Snježana; Veli?kovi?, Nataša; Grkovi?, Ivana; Ignjatovi?, Marija; Horvat, Anica

2012-12-01

149

ATP synthase: from single molecule to human bioenergetics  

PubMed Central

ATP synthase (FoF1) consists of an ATP-driven motor (F1) and a H+-driven motor (Fo), which rotate in opposite directions. FoF1 reconstituted into a lipid membrane is capable of ATP synthesis driven by H+ flux. As the basic structures of F1 (?3?3???) and Fo (ab2c10) are ubiquitous, stable thermophilic FoF1 (TFoF1) has been used to elucidate molecular mechanisms, while human F1Fo (HF1Fo) has been used to study biomedical significance. Among F1s, only thermophilic F1 (TF1) can be analyzed simultaneously by reconstitution, crystallography, mutagenesis and nanotechnology for torque-driven ATP synthesis using elastic coupling mechanisms. In contrast to the single operon of TFoF1, HFoF1 is encoded by both nuclear DNA with introns and mitochondrial DNA. The regulatory mechanism, tissue specificity and physiopathology of HFoF1 were elucidated by proteomics, RNA interference, cytoplasts and transgenic mice. The ATP synthesized daily by HFoF1 is in the order of tens of kilograms, and is primarily controlled by the brain in response to fluctuations in activity. PMID:20689227

KAGAWA, Yasuo

2010-01-01

150

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

151

Mechanism of Feedback Allosteric Inhibition of ATP Phosphoribosyltransferase  

PubMed Central

MtATP-phosphoribosyltransferase catalyzes the first and committed step in l-histidine biosynthesis in Mycobacterium tuberculosis and is therefore subjected to allosteric feedback regulation. Because of its essentiality, this enzyme is being studied as a potential target for novel anti-infectives. To understand the basis for its regulation, we characterized the allosteric inhibition using gel filtration, steady-state and pre-steady-state kinetics, and the pH dependence of inhibition and binding. Gel filtration experiments indicate that MtATP-phosphoribosyltransferase is a hexamer in solution, in the presence or absence of l-histidine. Steady-state kinetic studies demonstrate that l-histidine inhibition is uncompetitive versus ATP and noncompetitive versus PRPP. At pH values close to neutrality, a Kii value of 4 ?M was obtained for l-histidine. Pre-steady-state kinetic experiments indicate that chemistry is not rate-limiting for the overall reaction and that l-histidine inhibition is caused by trapping the enzyme in an inactive conformation. The pH dependence of binding, obtained by nuclear magnetic resonance, indicates that l-histidine binds better as the neutral ?-amino group. The pH dependence of inhibition (Kii), on the contrary, indicates that l-histidine better inhibits MtATP-phosphoribosytransferase with a neutral imidazole and an ionized ?-amino group. These results are combined into a model that accounts for the allosteric inhibition of MtATP-phosphoribosyltransferase. PMID:22989207

2012-01-01

152

Cardiac Metabolism in Heart Failure - Implications beyond ATP production  

PubMed Central

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

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

2013-01-01

153

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

154

NIF Title III engineering plan  

SciTech Connect

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

Deis, G

1998-06-01

155

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

PubMed Central

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

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

2013-01-01

156

ATP-activated oligomerization as a mechanism for apoptosis regulation: fold and mechanism prediction for CED-4.  

PubMed

Fold recognition algorithm FFAS (Rychlewski et al., Protein Sci, 2000;9:232-241) was used to match the nucleotide-binding adaptor shared by APAF-1, certain R gene products and CED-4 (NB-ARC domain) to the structure of the D2 domain of N-ethylemaleimide-Sensitive Fusion Protein and the delta; subunit of clamp loader of DNA polymerase III. The predicted structure consists of the p-loop ATP-binding domain, followed by two alpha-helical domains that regulate the oligomerization process. This prediction suggests a detailed molecular mechanism for the "induced proximity" hypothesis (Salvesen and Dixit, Proc Natl Acad Sci USA 1999;96:10964-10967) for CED3/caspase-9 activation by CED4/APAF-1 complex. According to this model, the ATP binding acts as a trigger in CED-4 oligomerization and the helical domain immediately following the ATP-binding domain provides additional mechanisms for regulation of the oligomerization process. This model explains most of known experimental data about CED-4-mediated caspase activation and, at the same time, suggest experiments that could test this hypothesis. PMID:10737940

Jaroszewski, L; Rychlewski, L; Reed, J C; Godzik, A

2000-05-15

157

Diverse Functional Properties of Wilson Disease ATP7B Variants  

PubMed Central

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

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

2012-01-01

158

Mechanism of ATP turnover inhibition in the EJC  

PubMed Central

The exon junction complex (EJC) is deposited onto spliced mRNAs and is involved in many aspects of mRNA function. We have recently reconstituted and solved the crystal structure of the EJC core made of MAGOH, Y14, the most conserved portion of MLN51, and the DEAD-box ATPase eIF4AIII bound to RNA in the presence of an ATP analog. The heterodimer MAGOH/Y14 inhibits ATP turnover by eIF4AIII, thereby trapping the EJC core onto RNA, but the exact mechanism behind this remains unclear. Here, we present the crystal structure of the EJC core bound to ADP-AIF3, the first structure of a DEAD-box helicase in the transition-mimicking state during ATP hydrolysis. It reveals a dissociative transition state geometry and suggests that the locking of the EJC onto the RNA by MAGOH/Y14 is not caused by preventing ATP hydrolysis. We further show that ATP can be hydrolyzed inside the EJC, demonstrating that MAGOH/Y14 acts by locking the conformation of the EJC, so that the release of inorganic phosphate, ADP, and RNA is prevented. Unifying features of ATP hydrolysis are revealed by comparison of our structure with the EJC–ADPNP structure and other helicases. The reconstitution of a transition state mimicking complex is not limited to the EJC and eIF4AIII as we were also able to reconstitute the complex Dbp5–RNA–ADP–AlF3, suggesting that the use of ADP–AlF3 may be a valuable tool for examining DEAD-box ATPases in general. PMID:19033377

Nielsen, Klaus H.; Chamieh, Hala; Andersen, Christian B.F.; Fredslund, Folmer; Hamborg, Kristiane; Le Hir, Herve; Andersen, Gregers R.

2009-01-01

159

ATP-Sensitive K+ Channels: Paradigm Lost, Paradigm Regained  

NSDL National Science Digital Library

Access to the article is free, however registration and sign-in are required. In an update of a previous Perspective [L. H. Philipson and D. F. Steiner, Science 268, 372 (1995)], Philipson discusses the molecular basis for Ksubscript ATP, a potassium current that mediates glucose regulation of insulin secretion. A prediction in the previous Perspective is borne out by a paper in this issue of Science (Inagaki et al., p. 1166), which reports the cloning of a new inward rectifier potassium channel and shows that Ksubscript ATP is formed when this channel combines with the sulfonylurea receptor.

Louis H. Philipson (University of Chicago;Department of Medicine and is on the Committee on Cell Physiology)

1995-11-17

160

Processing mechanics of alternate twist ply (ATP) yarn technology  

NASA Astrophysics Data System (ADS)

Ply yarns are important in many textile manufacturing processes and various applications. The primary process used for producing ply yarns is cabling. The speed of cabling is limited to about 35m/min. With the world's increasing demands of ply yarn supply, cabling is incompatible with today's demand activated manufacturing strategies. The Alternate Twist Ply (ATP) yarn technology is a relatively new process for producing ply yarns with improved productivity and flexibility. This technology involves self plying of twisted singles yarn to produce ply yarn. The ATP process can run more than ten times faster than cabling. To implement the ATP process to produce ply yarns there are major quality issues; uniform Twist Profile and yarn Twist Efficiency. The goal of this thesis is to improve these issues through process modeling based on understanding the physics and processing mechanics of the ATP yarn system. In our study we determine the main parameters that control the yarn twist profile. Process modeling of the yarn twist across different process zones was done. A computational model was designed to predict the process parameters required to achieve a square wave twist profile. Twist efficiency, a measure of yarn torsional stability and bulk, is determined by the ratio of ply yarn twist to singles yarn twist. Response Surface Methodology was used to develop the processing window that can reproduce ATP yarns with high twist efficiency. Equilibrium conditions of tensions and torques acting on the yarns at the self ply point were analyzed and determined the pathway for achieving higher twist efficiency. Mechanistic modeling relating equilibrium conditions to the twist efficiency was developed. A static tester was designed to zoom into the self ply zone of the ATP yarn. A computer controlled, prototypic ATP machine was constructed and confirmed the mechanistic model results. Optimum parameters achieving maximum twist efficiency were determined in this study. The successful results of this work have led to the filing of a US patent disclosing the method for producing ATP yarns with high yarn twist efficiency using a high convergence angle at the self ply point together with applying ply torque.

Elkhamy, Donia Said

161

Guanylyl cyclase is an ATP sensor coupling nitric oxide signaling to cell metabolism  

PubMed Central

Defending cellular integrity against disturbances in intracellular concentrations of ATP ([ATP]i) is predicated on coordinating the selection of substrates and their flux through metabolic pathways (metabolic signaling), ATP transfer from sites of production to utilization (energetic signaling), and the regulation of processes consuming energy (cell signaling). Whereas NO and its receptor, soluble guanylyl cyclase (sGC), are emerging as key mediators coordinating ATP supply and demand, mechanisms coupling this pathway with metabolic and energetic signaling remain undefined. Here, we demonstrate that sGC is a nucleotide sensor whose responsiveness to NO is regulated by [ATP]i. Indeed, ATP inhibits purified sGC with a Ki predicting >60% inhibition of NO signaling in cells maintaining physiological [nucleotide]i. ATP inhibits sGC by interacting with a regulatory site that prefers ATP > GTP. Moreover, alterations in [ATP]i, by permeabilization and nucleotide clamping or inhibition of mitochondrial ATP synthase, regulate NO signaling by sGC. Thus, [ATP]i serves as a “gain control” for NO signaling by sGC. At homeostatic [ATP]i, NO activation of sGC is repressed, whereas insults that reduce [ATP]i, derepress sGC and amplify responses to NO. Hence, sGC forms a key synapse integrating metabolic, energetic, and cell signaling, wherein ATP is the transmitter, allosteric inhibition the coupling mechanism, and regulated accumulation of cGMP the response. PMID:14684830

Ruiz-Stewart, I.; Tiyyagura, S. R.; Lin, J. E.; Kazerounian, S.; Pitari, G. M.; Schulz, S.; Martin, E.; Murad, F.; Waldman, S. A.

2004-01-01

162

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

PubMed Central

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; Calderon, Julio; del Valle, Luis J; Talledo, Miguel; Ramirez, Pablo

2012-01-01

163

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

PubMed

Metal complexes of pyridoxine mono hydrochloride (vitamin B6) are prepared using La(III), Ce(III), Sm(III) and Y(III). The resulting complexes are investigated. Some physical properties, conductivity, analytical data and the composition of the four pyridoxine complexes are discussed. The elemental analysis shows that the formed complexes of La(III), Ce(III), Sm(III) and Y(III) with pyridoxine are of 1:2 (metal:PN) molar ratio. All the synthesized complexes are brown in color and possess high melting points. These complexes are partially soluble in hot methanol, dimethylsulfoxide and dimethylformamide and insoluble in water and some other organic solvents. Elemental analysis data, spectroscopic (IR, UV-vis. and florescence), effective magnetic moment in Bohr magnetons and the proton NMR suggest the structures. However, definite particle size is determined by invoking the X-ray powder diffraction and scanning electron microscopy data. The results obtained suggested that pyridoxine reacted with metal ions as a bidentate ligand through its phenolate oxygen and the oxygen of the adjacent group at the 4'-position. The molar conductance measurements proved that the pyridoxine complexes are electrolytic in nature. The kinetic and thermodynamic parameters such as: Ea, ?H(*), ?S(*) and ?G(*) were estimated from the DTG curves. The antibacterial evaluation of the pyridoxine and their complexes were also performed against some gram positive, negative bacteria as well as fungi. PMID:24632173

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

2014-06-01

164

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

165

Paracrine stimulation of P2X7 receptor by ATP activates a proliferative pathway in ovarian carcinoma cells.  

PubMed

P2X7 is a purinergic receptor-channel; its activation by ATP elicits a broad set of cellular actions, from apoptosis to signals for survival. Here, P2X7 expression and function was studied in human ovarian carcinoma (OCA) cells, and biopsies from non-cancerous and cancer patients were analyzed by immunohistochemistry. Ovarian surface epithelium in healthy tissue expressed P2X7 at a high level that was maintained throughout the cancer. The cell lines SKOV-3 and CAOV-3 were used to investigate P2X7 functions in OCA. In SKOV-3 cells, selective stimulation of P2X7 by 2'(3')-O-(4-benzoylbenzoyl) adenosine-5'-triphosphate (BzATP) induced a dose-dependent increase of intracellular Ca(2+) concentration ([Ca(2+)](i)) but not cell death. Instead, BzATP increased the levels of phosphorylated ERK and AKT (pERK and pAKT), with an EC(50) of 44?±?2 and 1.27?±?0.5??M, respectively; 10??M BzATP evoked a maximum effect within 15?min that lasted for 120?min. Interestingly, basal levels of pERK and pAKT were decreased in the presence of apyrase in the medium, strongly suggesting an endogenous, ATP-mediated phenomenon. Accordingly: (i) mechanically stimulated cells generated a [Ca(2+)](i) increase that was abolished by apyrase; (ii) apyrase induced a decrease in culture viability, as measured by the MTS assay for mitochondrial activity; and (iii) incubation with 10??M AZ10606120, a specific P2X7 antagonist and transfection with the dominant negative P2X7 mutant E496A, both reduced cell viability to 70.1?±?8.9% and to 76.5?±?5%, respectively, of control cultures. These observations suggested that P2X7 activity was auto-induced through ATP efflux; this increased pERK and pAKT levels that generated a positive feedback on cell viability. PMID:24913779

Vázquez-Cuevas, Francisco G; Martínez-Ramírez, Angélica S; Robles-Martínez, Leticia; Garay, Edith; García-Carrancá, Alejandro; Pérez-Montiel, Delia; Castañeda-García, Carolina; Arellano, Rogelio O

2014-11-01

166

Elucidation of the ATP7B N-Domain Mg2+-ATP Coordination Site and Its Allosteric Regulation  

PubMed Central

The diagnostic of orphan genetic disease is often a puzzling task as less attention is paid to the elucidation of the pathophysiology of these rare disorders at the molecular level. We present here a multidisciplinary approach using molecular modeling tools and surface plasmonic resonance to study the function of the ATP7B protein, which is impaired in the Wilson disease. Experimentally validated in silico models allow the elucidation in the Nucleotide binding domain (N-domain) of the Mg2+-ATP coordination site and answer to the controversial role of the Mg2+ ion in the nucleotide binding process. The analysis of protein motions revealed a substantial effect on a long flexible loop branched to the N-domain protein core. We demonstrated the capacity of the loop to disrupt the interaction between Mg2+-ATP complex and the N-domain and propose a role for this loop in the allosteric regulation of the nucleotide binding process. PMID:22046264

Hercend, Claude; Bauvais, Cyril; Bollot, Guillaume; Delacotte, Nicolas; Chappuis, Philippe; Woimant, France; Launay, Jean-Marie; Manivet, Philippe

2011-01-01

167

Prevalence of metabolic syndrome in a cohort of Chinese schoolchildren: comparison of two definitions and assessment of adipokines as components by factor analysis  

PubMed Central

Background Although attention to metabolic syndrome (MetS) in children has increased, there is still no universally accepted definition and its pathogenesis remains unclear. Our aim was to compare the current definitions of childhood MetS in a Chinese cohort and to examine the clustering pattern of MetS risk factors, particularly inclusion of leptin and adiponectin as additional components. Methods 3373 schoolchildren aged 6 to 18 years were recruited. Anthropometric and biochemical parameters and adipokines were measured. MetS was identified using both the International Diabetes Federation (IDF) and a modified Adult Treatment Panel III (ATP III) definitions. Exploratory factor analysis was performed to establish grouping of metabolic characteristics. Results For children ?10 years, the prevalence of MetS was 14.3% in the obese group and 3.7% in the overweight group according to the new IDF definition, and 32.3% in the obese group and 8.4% in the overweight group according to the modified ATPIII definition. Frequency of hypertriglyceridemia, low high-density lipoprotein cholesterol (HDL-C), impaired fasting glucose, elevated blood pressure, and central obesity according to the new IDF definition was 16.7%, 20.7%, 15.8%, 25.5% and 75.5% in obese boys and 14.7%, 24.0%, 12.0%, 11.0% and 89.0% in obese girls, respectively. Metabolic abnormalities in children under 10 years of age were also noted. Using factor analysis on eight conventional variables led to the extraction of 3 factors. Waist circumference (WC) provided a connection between two factors in boys and all three factors in girls, suggesting its central role in the clustering of metabolic risk factors. Addition of leptin and adiponectin also led to the extraction of 3 factors, with leptin providing a connection between two factors in girls. When using WC, mean arterial pressure, triglyceride/HDL-C ratio, HOMA-IR and leptin/adiponectin ratio as variables, a single-factor model was extracted. WC had the biggest factor loading, followed by leptin/adiponectin ratio. Conclusions MetS was highly prevalent amongst obese children and adolescents in this cohort, regardless of the definition used. Central obesity is the key player in the clustering of metabolic risk factors in children, supporting the new IDF definition. Moreover, our findings suggest that a common factor may underlie MetS. Leptin/adiponectin ratio as a possible component of MetS deserves further consideration. PMID:23514611

2013-01-01

168

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

169

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

PubMed

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

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

2013-11-01

170

Pharmacological properties of ATP-sensitive purinergic receptors expressed in human G292 osteoblastic cells  

Microsoft Academic Search

We characterized the pharmacological properties of P2 receptors expressed in G292 osteoblastic cells by studying the responses or changes in intracellular Ca2+ level to P2 receptor agonists, antagonists and modulators. ATP induced robust responses in a concentration-dependent manner with EC50 of 0.5±0.07 ?M. While ?,?-methylene-ATP (??meATP) and 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP) were ineffective, ADP mimicked the action of ATP with EC50 of

Dong-Liang Li; Xing Liu; Rong Xia; Claire Ross; Xuebin Yang; Lin-Hua Jiang

2009-01-01

171

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

172

Volume-dependent ATP-conductive large-conductance anion channel as a pathway for swelling-induced ATP release.  

PubMed

In mouse mammary C127i cells, during whole-cell clamp, osmotic cell swelling activated an anion channel current, when the phloretin-sensitive, volume-activated outwardly rectifying Cl(-) channel was eliminated. This current exhibited time-dependent inactivation at positive and negative voltages greater than around +/-25 mV. The whole-cell current was selective for anions and sensitive to Gd(3)+. In on-cell patches, single-channel events appeared with a lag period of approximately 15 min after a hypotonic challenge. Under isotonic conditions, cell-attached patches were silent, but patch excision led to activation of currents that consisted of multiple large-conductance unitary steps. The current displayed voltage- and time-dependent inactivation similar to that of whole-cell current. Voltage-dependent activation profile was bell-shaped with the maximum open probability at -20 to 0 mV. The channel in inside-out patches had the unitary conductance of approximately 400 pS, a linear current-voltage relationship, and anion selectivity. The outward (but not inward) single-channel conductance was suppressed by extracellular ATP with an IC(50) of 12.3 mM and an electric distance (delta) of 0.47, whereas the inward (but not outward) conductance was inhibited by intracellular ATP with an IC(50) of 12.9 mM and delta of 0.40. Despite the open channel block by ATP, the channel was ATP-conductive with P(ATP)/P(Cl) of 0.09. The single-channel activity was sensitive to Gd(3)+, SITS, and NPPB, but insensitive to phloretin, niflumic acid, and glibenclamide. The same pharmacological pattern was found in swelling-induced ATP release. Thus, it is concluded that the volume- and voltage-dependent ATP-conductive large-conductance anion channel serves as a conductive pathway for the swelling-induced ATP release in C127i cells. PMID:11524456

Sabirov, R Z; Dutta, A K; Okada, Y

2001-09-01

173

2'(3')-O-[N- [2- [3- [5-fluoresceinyl] thioureido] ethyl] carbamoyl] adenosine 5'-triphosphate and its Cr(H2O)4 and Co(NH3)4 complex derivatives are new fluorescent tools for labelling ATP binding sites of Na+/K+-ATPase.  

PubMed

2'(3')-O-[N- [2- [3- [5-fluoresceinyl] thioureido] ethyl] carbamoyl] adenosine 5'-triphosphate (FEDA-ATP), a spectroscopic tool used for studying skeletal muscle myosin ATPase subfragment 1, was applied to Na+/K+-ATPase (EC 3.6.1.37). In contrast to the myosin subfragment, we found that FEDA-ATP is not a substrate for Na+/K+-ATPase. On the other hand, FEDA-ATP showed an affinity for both the low (E2, Kd=200 microM) and the high (E1, Kd=22 microM) affinity ATP-binding sites. When the microscopic affinities of FEDA-ATP were used for calculating the macroscopic affinity in the overall reaction according to Ki=(KdE1*KdE2)1/2, the experimentally measured inhibition constant of 66 microM was obtained. To evoke irreversible binding inhibitors, FEDA-ATP was transferred in its chromium(III) and cobalt(III) complex analogs, which are suitable tools for labelling the ATP binding sites of Na+/K+-ATPase in a specific way. PMID:9728479

Linnertz, H; Lastres Becker, I; Krumscheid, R; Amler, E; Thoenges, D; Schoner, W

1997-01-01

174

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

175

Quantum and classical dynamics simulations of ATP hydrolysis in solution  

PubMed Central

ATP hydrolysis is a key reaction in living cells that drives many cellular processes. The reaction, which involves gamma phosphate cleavage from ATP, converting it to ADP, has been suggested to occur via an associative or dissociative mechanism dependent upon the surrounding environment. Prior quantum chemical studies suffered from short simulation timescales failing to capture free energy contributions due to relaxation of the surrounding aqueous environment. We have developed a highly parallelized QM/MM implementation in the NAMD and OpenAtom simulation packages, using the dual grid, dual length scale method for combined plane-wave and Eular exponential spline-based QM/MM simulations. This approach, using message-driven parallel quantum and classical dynamics, permits sufficient timescale simulations for quantum chemical events such as ATP hydrolysis, and is found to accurately and reliably include the free energy contributions of solvent relaxation to hydrolysis. In this paper we describe the application of the dual grid, dual length plane-wave-based QM/MM method to study both the associative and dissociative mechanisms of ATP hydrolysis, accounting for the free energy contribution from solvent relaxation, as well as for the key role of Mg2+ in the reaction. PMID:23293550

Harrison, Christopher B.; Schulten, Klaus

2012-01-01

176

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

177

Methods to determine the status of mitochondrial ATP synthase assembly.  

PubMed

The adenosine triphosphate (ATP) synthase (F1-F0 complex) of the mitochondrial inner membrane is responsible for making nearly all of the ATP utilized by eukaryotic organisms. The enzyme is an oligomer of more than 20 different subunits, 14 of which are essential for its catalytic activity. The other subunits function in the regulation and structure of the complex. Subunits essential for catalytic activity make up the proton pore, the bulk of the F1 headpiece, and the two stalks that physically and functionally couple the catalytic and proton-translocating activities of the ATP synthase. Saccharomyces cerevisiae provides an excellent model system for studying mutations that affect assembly of the complex because of the ability of this organism to survive on the ATP produced from fermentation in the absence of mitochondrial respiration or oxidative phosphorylation. Studies of such mutants have been instrumental in identifying novel molecular chaperones that act at discrete steps of F1-F0 assembly. Here, we describe some experimental approaches useful in assessing the status of F1-F0 assembly. PMID:18314739

Ackerman, Sharon H; Tzagoloff, Alexander

2007-01-01

178

Intertwined translational regulations set uneven stoichiometry of chloroplast ATP synthase  

E-print Network

Intertwined translational regulations set uneven stoichiometry of chloroplast ATP synthase subunits mechanism ensur- ing this unique stoichiometry, required for the functional assembly of the chloroplast translation of the chloroplast-encoded subunit b, which in turn stimulates the expression of the chloroplast

179

Abiogenic Photophosphorylation of ADP to ATP Sensitized by Flavoproteinoid Microspheres  

NASA Astrophysics Data System (ADS)

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

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

2008-06-01

180

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

181

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

182

Updated 19 March 2009 PHD TOPICS AVAILABLE AT NICTA, ATP  

E-print Network

Updated 19 March 2009 PHD TOPICS AVAILABLE AT NICTA, ATP To contact any staff listed below by email staff is available on our people page http://www.nicta.com.au/people PhD Topics in Making Sense of Data and environmental factors. Extensive research is needed to investigate the best methods to capture and represent

Heiser, Gernot

183

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

184

IV ATP potentiates midazolam sedation as assessed by bispectral index.  

PubMed

In this study, by measuring bispectral index (BIS), we tested the hypothesis that intravenous adenosine 5'-triphosphate (ATP) infusion would deepen the level of midazolam-induced sedation. Ten healthy volunteers underwent 2 experiments with at least 2 weeks' interval: immediately after intravenous bolus administration of midazolam (0.04 mg/kg), they received continuous infusion of either ATP infusion (100 ?g/kg/min) or placebo (saline) for 40 minutes in a double-blind, randomized, crossover manner. Changes in BIS values and responsiveness to verbal command as well as cardiorespiratory variables were observed throughout the study periods. Administration of midazolam alone reduced BIS value from control: 97 ± 1 to 68 ± 18 at 25 minutes, which was accompanied by significant cardiopulmonary depressant effects, while maintaining responsiveness to verbal command (consciousness) throughout the study period. Coadministration of ATP with midazolam further reduced BIS value to 51 ± 13, associated with complete loss of consciousness without adverse effect on the cardiorespiratory systems. We conclude that the addition of ATP infusion to midazolam significantly enhances midazolam sedation without disturbing cardiorespiratory functions. PMID:25191981

Sakurai, Satoru; Fukunaga, Atsuo; Ichinohe, Tatsuya; Kaneko, Yuzuru

2014-01-01

185

Developmental aspects of amperometric ATP biosensors based on entrapped enzymes.  

PubMed

A novel concept for a dual-enzyme-based microbiosensor for the detection of adenosine-5'-triphosphate (ATP) was developed. The employed enzymes pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) and hexokinase were entrapped, using pH-shift-induced precipitation of electrodeposition paint (EDP) at platinum microelectrodes (diameter of 25 microm). PQQ-GDH is known showing a superior activity for glucose conversion at the relevant conditions (low oxygen concentration) for ATP detection in targeted biomedical studies. For immobilizing the two enzymes PQQ-GDH and hexokinase, the deposition conditions of EDP Resydrol AY498w/35WA were adapted to ensure high immobilization rates. Prior to ATP sensing, the conversion of glucose, which is the co-substrate for both enzymatic reactions, was optimized. Optimization was targeted towards ATP measurements in biomedical environments by optimizing the PQQ-GDH sensor for glucose. Therefore, different mediators were tested regarding their electron transfer rate and their compatibility with the enzyme: free-diffusing N-methylphenazonium methyl sulfate (PMS) and ferrocenemethanol, and an immobilized chromium hexacyanoferrate layer at platinum electrode. Free-diffusing ferrocenemethanol reveals high sensitivity towards glucose of 1.5 +/- 0.4 nA/mM. In a next step, hexokinase was co-entrapped in the polymer film resulting in a sensitivity of up to 290 pA/microM. PMID:19779927

Weber, Cornelia; Gauda, Estelle; Mizaikoff, Boris; Kranz, Christine

2009-11-01

186

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

187

Processing mechanics of alternate twist ply (ATP) yarn technology  

Microsoft Academic Search

Ply yarns are important in many textile manufacturing processes and various applications. The primary process used for producing ply yarns is cabling. The speed of cabling is limited to about 35m\\/min. With the world's increasing demands of ply yarn supply, cabling is incompatible with today's demand activated manufacturing strategies. The Alternate Twist Ply (ATP) yarn technology is a relatively new

Donia Said Elkhamy

2007-01-01

188

Extracellular ATP signaling in plants Kiwamu Tanaka1  

E-print Network

]. Scientific interest in extracellular ATP accelerated after the first purinoceptor was cloned to stimulate closure of the venus flytrap [4], endonuclease activity in excised oat leaves [5], and potassium male sterility due to abortion of pollen germination [20]. Interestingly, complemented lines

Jones, Alan M.

189

Modification of the E1ATP binding site of Na+/K(+)-ATPase by the chromium complex of adenosine 5'-[beta,gamma-methylene]triphosphate blocks the overall reaction but not the partial activities of the E2 conformation.  

PubMed

The chromium complex of adenosine 5'-[beta,gamma-methylene]triphosphate, Cr(H2O)4AdoPP[CH2]P, inactivates Na+/K(+)-ATPase from pig kidney at 37 degrees C with an inactivation velocity constant of 7.1 x 10(-3) min-1 by binding to the high-affinity ATP site (E1ATP site). The dissociation constant (Kd) of the analogue at this site is 26 microM, and of ATP 0.8 microM. Inactivation of the overall reaction of Na+/K(+)-ATPase by Cr(H2O)4AdoPP[CH2]P did not alter the activities of the E2 conformational state such as K(+)-activated p-nitrophenylphosphatase, 86Rb+ occlusion and [3H]ouabain binding by the 'backdoor' phosphorylation. However, [3H]ouabain binding via the forwards reaction from E1ATP in the presence of Na+ + Mg2+ is inhibited. K(+)-activated p-nitrophenylphosphatase activity of the Cr(H2O)4AdoPP[CH2]P-inactivated enzyme decreases when an MgATP analogue, the tetraammine cobalt complex of ATP, Co(NH3)4ATP, is used additionally to inactivate the E2ATP site. The enzyme activity of K(+)-activated phosphatase is also lost if the beta,gamma-bidentate chromium(III) complex of ATP, Cr(H2O)4ATP, which may form a stable E1-chromo-phosphointermediate, is used for the inactivation of Na+/K(+)-ATPase. We conclude that the phenomenon of a blockade of the overall reaction of Na+/K(+)-ATPase by the formation of a stable E1.CrAdoPP[CH2]P complex, leading thereby to a loss of the partial activities of the E1 conformation, but not of the E2 conformation, is consistent with the postulate of an (alpha beta)2 diprotomeric nature of the sodium pump. The observation, moreover, that treatment of the sodium pump with Cr(H2O)4ATP but not with Cr(H2O)4AdoPP[CH2]P leads to an inactivation of K(+)-activated phosphatase seems to indicate that the formation of a E1-phosphointermediate affects the E2ATP site. PMID:8386635

Hamer, E; Schoner, W

1993-04-15

190

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

NASA Technical Reports Server (NTRS)

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

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

2003-01-01

191

P2Y Receptor Modulation of ATP Release in the Urothelium  

PubMed Central

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

Mansfield, Kylie J.; Hughes, Jessica R.

2014-01-01

192

Chap. III : structures  

E-print Network

Chap. III : Les objets structur´es Laurent Poinsot Plan Chap. III : Les objets structur´es Laurent Poinsot 25 septembre 2009 #12;Chap. III : Les objets structur´es Laurent Poinsot Plan Plan 1 Les objets structur´es Introduction S´equences Listes Ensembles Cha^ines de caract`eres #12;Chap. III : Les objets

Poinsot, Laurent

193

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

194

Metabolomic Analysis of Differential Changes in Metabolites during ATP Oscillations in Chondrogenesis  

PubMed Central

Prechondrogenic condensation is a critical step for skeletal pattern formation. Recent studies reported that ATP oscillations play an essential role in prechondrogenic condensation. However, the molecular mechanism to underlie ATP oscillations remains poorly understood. In the present study, it was investigated how changes in metabolites are implicated in ATP oscillations during chondrogenesis by using capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS). CE-TOF-MS detected 93 cationic and 109 anionic compounds derived from known metabolic pathways. 15 cationic and 18 anionic compounds revealed significant change between peak and trough of ATP oscillations. These results implicate that glycolysis, mitochondrial respiration and uronic acid pathway oscillate in phase with ATP oscillations, while PPRP and nucleotides synthesis pathways oscillate in antiphase with ATP oscillations. This suggests that the ATP-producing glycolysis and mitochondrial respiration oscillate in antiphase with the ATP-consuming PPRP/nucleotide synthesis pathway during chondrogenesis. PMID:23878799

Ohmiya, Yoshihiro

2013-01-01

195

Bringing Definitions into High Definition  

ERIC Educational Resources Information Center

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

Mason, John

2010-01-01

196

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 Sa Pinheiro, Ana Acacia; Silva, Leandro de Souza; Verstraeten, Sandra Viviana; Persechini, Pedro Muanis; Schwarzbaum, Pablo Julio

2014-01-01

197

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

Microsoft Academic Search

The degradation of proteins in bacteria requires metabolism energy. One important enzyme in this process is protease La, a soluble ATP-dependent protease encoded by the lon gene. However, lon mutants that lack a functional protease La still show some ATP-dependent protein breakdown. The authors have reported an ATP-stimulated endoproteolytic activity associated with the inner membrane of E. coli. This ATP-stimulated

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

1986-01-01

198

Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La  

Microsoft Academic Search

The energy requirement for protein breakdown in Escherichia coli has generally been attributed to the ATP-dependence of protease La, the lon gene product. The authors have partially purified another ATP-dependent protease from lon⁻ cells that lack protease La (as shown by immunoblotting). This enzyme hydrolyzes (³H)methyl-casein to acid-soluble products in the presence of ATP and Mg\\/sup 2 +\\/. ATP hydrolysis

B. J. Hwang; W. J. Park; C. H. Chung; A. L. Goldberg

1987-01-01

199

Escherichia coli Contains a Soluble ATP-Dependent Protease (Ti) Distinct from Protease La  

Microsoft Academic Search

The energy requirement for protein breakdown in Escherichia coli has generally been attributed to the ATP-dependence of protease La, the lon gene product. We have partially purified another ATP-dependent protease from lon- cells that lack protease La (as shown by immunoblotting). This enzyme hydrolyzes [3H]methyl-casein to acid-soluble products in the presence of ATP and Mg2+. ATP hydrolysis appears necessary for

Byung Joon Hwang; Woo Jin Park; Chin Ha Chung; Alfred L. Goldberg

1987-01-01

200

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

201

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

202

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

Microsoft Academic Search

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 mu M) and are affected similarly by various inhibitors, activators,

Lloyd Waxman; Alfred L. Goldberg

1982-01-01

203

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

204

Simultaneous Measurements of Action Potential Duration and Intracellular ATP in Isolated Ferret Hearts Exposed to Cyanide  

Microsoft Academic Search

Shortening of the cardiac action potential during ischemia and anoxia is likely to contribute to the decline in contractility that occurs under such conditions. It has been hypothesized that a decrease in the intracellular ATP concentration ((ATP),) underlies the changes hi the action potential. The recently discovered potassium channel activated at low ATP concentrations might provide the link between action

A. C. Elliott; G. L. Smith; D. G. Allen

2010-01-01

205

“Wages of Fear”: transient threefold decrease in intracellular ATP level imposes apoptosis  

Microsoft Academic Search

In HeLa cells, complete inhibition of oxidative phosphorylation by oligomycin, myxothiazol or FCCP combined with partial inhibition of glycolysis by DOG resulted in a steady threefold decrease in the intracellular ATP level. The ATP level recovers when the DOG-containing medium was replaced by that with high glucose. In 48 h after a transient (3 h) [ATP] lowering followed by recovery

Denis S Izyumov; Armine V Avetisyan; Olga Yu Pletjushkina; Dmitrii V Sakharov; Karel W Wirtz; Boris V Chernyak; Vladimir P Skulachev

2004-01-01

206

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

207

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

PubMed

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

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

2014-09-24

208

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

PubMed

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

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

2014-10-01

209

The HflB protease of Escherichia coli degrades its inhibitor lambda cIII.  

PubMed Central

The cIII protein of bacteriophage lambda is known to protect two regulatory proteins from degradation by the essential Escherichia coli protease HflB (also known as FtsH), viz., the lambda cII protein and the host heat shock sigma factor sigma32. lambda cIII, itself an unstable protein, is partially stabilized when the HflB concentration is decreased, and its half-life is decreased when HflB is overproduced, strongly suggesting that it is degraded by HflB in vivo. The in vivo degradation of lambda cIII (unlike that of sigma32) does not require the molecular chaperone DnaK. Furthermore, the half-life of lambda cIII is not affected by depletion of the endogenous ATP pool, suggesting that lambda cIII degradation is ATP independent (unlike that of lambda cII and sigma32). The lambda cIII protein, which is predicted to contain a 22-amino-acid amphipathic helix, is associated with the membrane, and nonlethal overproduction of lambda cIII makes cells hypersensitive to the detergent sodium dodecyl sulfate. This could reflect a direct lambda cIII-membrane interaction or an indirect association via the membrane-bound HflB protein, which is known to be involved in the assembly of certain periplasmic and outer membrane proteins. PMID:8990286

Herman, C; Thevenet, D; D'Ari, R; Bouloc, P

1997-01-01

210

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

211

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

PubMed Central

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

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

2004-01-01

212

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

213

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

NASA Astrophysics Data System (ADS)

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

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

2011-03-01

214

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

215

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

NASA Astrophysics Data System (ADS)

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.

2013-01-01

216

Somatic ATP1A1, ATP2B3, and KCNJ5 mutations in aldosterone-producing adenomas.  

PubMed

Aldosterone-producing adenomas (APAs) cause a sporadic form of primary aldosteronism and somatic mutations in the KCNJ5 gene, which encodes the G-protein-activated inward rectifier K(+) channel 4, GIRK4, account for ?40% of APAs. Additional somatic APA mutations were identified recently in 2 other genes, ATP1A1 and ATP2B3, encoding Na(+)/K(+)-ATPase 1 and Ca(2+)-ATPase 3, respectively, at a combined prevalence of 6.8%. We have screened 112 APAs for mutations in known hotspots for genetic alterations associated with primary aldosteronism. Somatic mutations in ATP1A1, ATP2B3, and KCNJ5 were present in 6.3%, 0.9%, and 39.3% of APAs, respectively, and included 2 novel mutations (Na(+)/K(+)-ATPase p.Gly99Arg and GIRK4 p.Trp126Arg). CYP11B2 gene expression was higher in APAs harboring ATP1A1 and ATP2B3 mutations compared with those without these or KCNJ5 mutations. Overexpression of Na(+)/K(+)-ATPase p.Gly99Arg and GIRK4 p.Trp126Arg in HAC15 adrenal cells resulted in upregulation of CYP11B2 gene expression and its transcriptional regulator NR4A2. Structural modeling of the Na(+)/K(+)-ATPase showed that the Gly99Arg substitution most likely interferes with the gateway to the ion binding pocket. In vitro functional assays demonstrated that Gly99Arg displays severely impaired ATPase activity, a reduced apparent affinity for Na(+) activation of phosphorylation and K(+) inhibition of phosphorylation that indicate decreased Na(+) and K(+) binding, respectively. Moreover, whole cell patch-clamp studies established that overexpression of Na(+)/K(+)-ATPase Gly99Arg causes membrane voltage depolarization. In conclusion, somatic mutations are common in APAs that result in an increase in CYP11B2 gene expression and may account for the dysregulated aldosterone production in a subset of patients with sporadic primary aldosteronism. PMID:24082052

Williams, Tracy Ann; Monticone, Silvia; Schack, Vivien R; Stindl, Julia; Burrello, Jacopo; Buffolo, Fabrizio; Annaratone, Laura; Castellano, Isabella; Beuschlein, Felix; Reincke, Martin; Lucatello, Barbara; Ronconi, Vanessa; Fallo, Francesco; Bernini, Giampaolo; Maccario, Mauro; Giacchetti, Gilberta; Veglio, Franco; Warth, Richard; Vilsen, Bente; Mulatero, Paolo

2014-01-01

217

Phosphocreatine and ATP regulation in the hypoxic developing rat brain  

Microsoft Academic Search

Decreased brain ATP and phosphocreatine (PCr) concentrations and intracellular pH were compared in hypoxic 4-, 10–11, and 24–25-day-old rats. Surface coil 31P-nuclear magnetic resonance (NMR) spectra were acquired in vivo every minute before, during, and after 7 min of breathing 4% O2. At all ages PCr decreased rapidly. At the two younger ages, the nucleoside triphosphate signal was still 80–85%

Miles Tsuji; Elizabeth Allred; Frances Jensen; David Holtzman

1995-01-01

218

Protons, the thylakoid membrane, and the chloroplast ATP synthase  

SciTech Connect

According to the chemiosmotic theory, proton pumps and ATP synthases are coupled by lateral proton flow through aqueous phases. Three long-standing challenges to this concept were examined in the light of experiments carried out with thylakoids: (1) Nearest neighbor interaction between pumps and ATP synthases. Considering the large distances between photosystem II and CFoCF1, in stacked thylakoids this is a priori absent. (2) Enhanced proton diffusion along the surface of the membrane. This could not be substantiated for the outer side of the thylakoid membrane. Even for the interface between pure lipid and water, two laboratories have reported the absence of enhanced diffusion. (3) Localized proton ducts in the membrane. Intramembrane domains that can transiently trap protons do exist in thylakoid membranes, but because of their limited storage capacity for protons, they probably do not matter for photophosphorylation under continuous light. Seemingly in favor of localized proton ducts is the failure of a supposedly permeant buffer to enhance the onset lag of photophosphorylation. However, it was found that failure of some buffers and the ability of others in this respect were correlated with their failure/ability to quench pH transients in the thylakoid lumen, as predicted by the chemiosmotic theory. It was shown that the chemiosmotic concept is a fair approximation, even for narrow aqueous phases, as in stacked thylakoids. These are approximately isopotential, and protons are taken in by the ATP synthase straight from the lumen. The molecular mechanism by which F0F1 ATPases couple proton flow to ATP synthesis is still unknown. The threefold structural symmetry of the headpiece that, probably, finds a corollary in the channel portion of these enzymes appeals to the common wisdom that structural symmetry causes functional symmetry.

Junge, W. (Universitaet Osnabrueck, Osnabrueck (Germany, F.R.))

1989-01-01

219

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

220

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.

221

ATP-regenerating system in the cilia of Paramecium caudatum.  

PubMed

The energy supply for eukaryotic ciliary and flagellar movement is thought to be maintained by ATP-regenerating enzymes such as adenylate kinase, creatine kinase and arginine kinase. In this study, the energy-supplying system for the ciliary movement of Paramecium caudatum was examined. Arginine kinase and adenylate kinase activities were detected in the cilia. To demonstrate that phosphoarginine satisfactorily supplies high-energy phosphate compounds into the narrow ciliary space, we prepared an intact ciliated cortical sheet from live Paramecium caudatum. These cortical sheets, with an intact ciliary membrane, produced a half-closed system in which each cilium was covered with a ciliary membrane with an opening to the cell body. Ciliary beating on the intact cortical sheets was induced by perfusing not only ATP but also ADP. Addition of phosphoarginine (0.2 mmol l(-1)) increased the beat frequency. A further increase in beat frequency was observed in 0.4 mmol l(-1) phosphoarginine, and this was enhanced when the cilia were reactivated with relatively low concentrations of ATP. We have demonstrated that phosphoarginine supplies energy as a 'phosphagen' for ciliary beating in Paramecium caudatum, suggesting that phosphoarginine functions not only as a reservoir of energy but also as a transporter of energy in these continuously energy-consuming circumstances. http://www.biologists.com/JEB/movies/jeb3123.html PMID:11222125

Noguchi, M; Sawada, T; Akazawa, T

2001-03-01

222

Curcumin Attenuates Cr(VI)-Induced Ascites and Changes in the Activity of Aconitase and F1 F0 ATPase and the ATP Content in Rat Liver Mitochondria.  

PubMed

Occupational and environmental exposure to potassium dichromate (K2 Cr2 O7 ), a hexavalent chromium compound, can result in liver damage associated with oxidative stress and mitochondrial dysfunction. The purpose of this study was to evaluate the effect of the antioxidant curcumin (400 mg/kg b.w.) on the K2 Cr2 O7 -induced injury, with special emphasis on ascitic fluid accumulation and oxidative phosphorylation mitochondrial enzymes and the adenosine triphosphate (ATP) levels in isolated mitochondria from livers of rats treated with K2 Cr2 O7 (15 mg/kg b.w.). Thus, curcumin attenuated the ascites generation, prevented the decrease in the activities of aconitase and F1 F0 ATPase, and maintained the ATP levels. The activity of complex II was not completely reestablished by curcumin, whereas complexes III and IV activities were unchanged. PMID:25130536

García-Niño, Wylly Ramsés; Zazueta, Cecilia; Tapia, Edilia; Pedraza-Chaverri, José

2014-11-01

223

Hemolysis is a primary ATP-release mechanism in human erythrocytes  

PubMed Central

The hypothesis that regulated ATP release from red blood cells (RBCs) contributes to nitric oxide-dependent control of local blood flow has sparked much interest in underlying release mechanisms. Several stimuli, including shear stress and hypoxia, have been found to induce significant RBC ATP release attributed to activation of ATP-conducting channels. In the present study, we first evaluated different experimental approaches investigating stimulated RBC ATP release and quantifying hemolysis. We then measured ATP and free hemoglobin in each and every RBC supernatant sample to directly assess the contribution of hemolysis to ATP release. Hypotonic shock, shear stress, and hypoxia, but not cyclic adenosine monophosphate agonists, significantly enhanced ATP release. It tightly correlated, however, with free hemoglobin in RBC supernatants, indicating that lysis was responsible for most, if not all, ATP release. Luminescence ATP imaging combined with simultaneous infrared cell imaging showed that ATP was released exclusively from lysing cells with no contribution from intact cells. In summary, with all stimuli tested, we found no evidence of regulated ATP release from intact RBCs other than by cell lysis. Such a release mechanism might be physiologically relevant in vivo, eg, during exercise and hypoxia where intravascular hemolysis, predominantly of senescent cells, is augmented. PMID:25097178

Sikora, Jacek; Orlov, Sergei N.; Furuya, Kishio

2014-01-01

224

Continuous-flow ATP amplification system for increasing the sensitivity of quantitative bioluminescence assay.  

PubMed

We constructed a novel ATP amplification reactor using a continuous-flow system, and this allowed us to increase the sensitivity of a quantitative bioluminescence assay by controlling the number of ATP amplification cycles. We previously developed a bioluminescence assay coupled with ATP amplification using a batch system. However, it was difficult to control the number of amplification cycles. In this study, ATP amplification was performed using a continuous-flow system, and significant linear correlations between amplified luminescence and initial ATP concentration were observed. When performing four cycles of continuous-flow ATP amplification, the gradient of amplification was 1.87(N). Whereas the lower quantifiable level was 500 pM without amplification, values as low as 50 pM ATP could be measured after amplification. The sensitivity thus increased 10-fold, with further improvements expected with additional amplification cycles. The continuous-flow system thus effectively increased the sensitivity of the quantitative bioluminescence assay. PMID:18492480

Satoh, Tetsuya; Shinoda, Yasuharu; Alexandrov, Maxym; Kuroda, Akio; Murakami, Yuji

2008-08-01

225

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

PubMed Central

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

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

2012-01-01

226

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

227

Arithmetic, Geometry Calculus III  

E-print Network

students. Calculus III goes by various names: Vector Calculus, Multivariable Calculus, CalculusArithmetic, Geometry and Calculus III by W. Stephen Wilson Department of Mathematics Johns to approach the problem. In both the fall of 2007 and the fall of 2008, I taught Calculus III to over 200

Wilson, W. Stephen

228

Chap. III : structures  

E-print Network

Chap. III : Les objets structur´es Laurent Poinsot Plan Chap. III : Les objets structur´es Laurent Poinsot 25 septembre 2009 #12;Chap. III : Les objets structur´es Laurent Poinsot Plan Plan 1 Les objets structur´es Introduction S´equences Listes Ensembles Cha^ines de caract`eres 2 Quelques exercices #12;Chap

Poinsot, Laurent

229

Biosynthesis of the class III lantipeptide catenulipeptin.  

PubMed

Lantipeptides are ribosomally synthesized and posttranslationally modified peptides containing lanthionine and/or labionin structures. In this study, a novel class III lantipeptide termed catenulipeptin was discovered from Catenulispora acidiphila DSM 44928, and its biosynthesis was reconstituted in vitro. The multifunctional enzyme AciKC catalyzes both dehydration and cyclization of its peptide substrate AciA and installs two labionin structures in catenulipeptin. AciKC shows promiscuity with respect to cosubstrate and accepts all four NTPs. The C-terminal domain of AciKC is responsible for the labionin formation in catenulipeptin. The cyclase activity of AciKC requires the leader peptide of AciA substrate but does not require ATP or Zn(2+). Mutagenesis studies suggest that the labionin cyclization may proceed in a C-to-N-terminal direction. Catenulipeptin partially restores aerial hyphae growth when applied to surfactin-treated Streptomyces coelicolor. PMID:22725258

Wang, Huan; van der Donk, Wilfred A

2012-09-21

230

Selective and ATP-driven transport of ions across supported membranes into nanoporous carriers using gramicidin A and ATP synthase.  

PubMed

We report a robust and versatile membrane protein based system for selective uptake and release of ions from nanoporous particles sealed with ion-tight lipid bilayers of various compositions that is driven by the addition of ATP or a chemical potential gradient. We have successfully incorporated both a passive ion channel-type peptide (gramicidin A) and a more complex primary sodium ion transporter (ATP synthase) into the supported lipid bilayers on solid nanoporous silica particles. Protein-mediated controlled release/uptake of sodium ions across the ion-tight lipid bilayer seal from or into the nanoporous silica carrier was imaged in real time using a confocal laser scanning microscope and the intensity changes were quantified. ATP-driven transport of sodium ions across the supported lipid bilayer against a chemical gradient was demonstrated. The possibility of designing durable carriers with tight lipid membranes, containing membrane proteins for selective ion uptake and release, offers new possibilities for functional studies of single or cascading membrane protein systems and could also be used as biomimetic microreactors for controlled synthesis of inorganic multicomponent materials. PMID:23321853

Oliynyk, Vitaliy; Mille, Christian; Ng, Jovice B S; von Ballmoos, Christoph; Corkery, Robert W; Bergström, Lennart

2013-02-28

231

Contributions of citrate in redox potential maintenance and ATP production: metabolic pathways and their regulation in Lactobacillus panis PM1.  

PubMed

Lactobacillus panis PM1 belongs to the group III heterofermentative lactobacilli and can utilize various NADH-reoxidizing routes (e.g., citrate, glycerol, and oxygen) according to environmental conditions. In this study, we investigated the ability of L. panis PM1 to produce succinate, acetate, and lactate via citrate utilization. Possible pathways, as well as regulation, for citrate metabolism were examined on the basis of the genome sequence data and metabolic profiles of L. panis PM1. The presence of citrate led to the up-regulation, at the transcriptional level, of the genes encoding for citrate lyase, malate dehydrogenase, and malic enzyme of the citrate pathways by 10- to 120-fold. The transcriptional regulator of the dha operon coding for glycerol dehydratase of L. panis PM1 repressed the expression of the citrate lyase gene (10-fold). Metabolite analyses indicated that the transcriptional enhancement by citrate stimulated succinate yield. Citrate metabolism contributed to energy production by providing a major alternate pathway for NAD(+) regeneration and allowed acetyl phosphate to yield acetate/ATP instead of ethanol/NAD(+). Additionally, a branching pathway from oxaloacetate to pyruvate increased the pool of lactate, which was then used to produce ATP during stationary phase. However, the redirection of NADH-to-citrate utilization resulted in stress caused by end-products (i.e., succinate and acetate). This stress reduced succinate production by up to 50 % but did not cause significant changes at transcriptional level. Overall, citrate utilization was beneficial for the growth of L. panis PM1 by providing a NAD(+) regeneration route and producing extra ATP. PMID:23912115

Kang, Tae Sun; Korber, Darren R; Tanaka, Takuji

2013-10-01

232

Hydration of lanthanoids(III) and actinoids(III): an experimental/theoretical saga.  

PubMed

The latest experimental and theoretical studies on structural and dynamical properties of lanthanoid(III) and actinoid(III) ions in water have been reviewed. In the last years, most of the issues about lanthanoid(III) hydration have been resolved combining X-ray absorption experiments and different theoretical methods. Since 2008 an effort has been made to treat the entire series thus obtaining coherent sets of experimental and theoretical results that were lately put together in such a way that it was possible to derive new basic properties, such as effective ionic radii, across the series. While for the hydration of lanthanoids(III) many experiments and simulations have been reported, the hydration of actinoids(III) was less investigated. There are some experiments performed by different research groups and few simulations that we discuss in this review. Currently, there are enough results that it is possible to gain some understanding of the hydration behavior of lanthanoids(III) and actinoids(III). The ultimate goal of this review is to provide clues on the analogies and differences between the two series. These aspects are connected to several issues: 1) technological: the separation of these elements that is necessary for recycling and stocking of nuclear waste, 2) practical: because experiments on actinoids need particular care, the definition of possible analogies will give the possibility to use the correct lanthanoid when the information on a specific actinoid is needed, 3) fundamental: related to chemical similarities between the two series. PMID:22893476

D'Angelo, Paola; Spezia, Riccardo

2012-09-01

233

Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold.  

PubMed Central

The alpha- and beta-subunits of membrane-bound ATP synthase complex bind ATP and ADP: beta contributes to catalytic sites, and alpha may be involved in regulation of ATP synthase activity. The sequences of beta-subunits are highly conserved in Escherichia coli and bovine mitochondria. Also alpha and beta are weakly homologous to each other throughout most of their amino acid sequences, suggesting that they have common functions in catalysis. Related sequences in both alpha and beta and in other enzymes that bind ATP or ADP in catalysis, notably myosin, phosphofructokinase, and adenylate kinase, help to identify regions contributing to an adenine nucleotide binding fold in both ATP synthase subunits. PMID:6329717

Walker, J E; Saraste, M; Runswick, M J; Gay, N J

1982-01-01

234

Studies on the beef heart mitochondrial F/sub 1/-ATPase with the photoaffinity label BzATP  

SciTech Connect

The photoaffinity analog of ATP, 3'-O-(4-benzoyl) benzoyl ATP (BzATP), was used in kinetics and binding studies to investigate the mechanism of the beef heart mitochondrial F/sup 1/-ATPase. New methods were developed for the synthesis and purification of non-radioactive BzATP, /sup 3/H-BzATP, and ..gamma..-/sup 32/P-BzATP, and the molar absorption coefficient for BzATP was determined. Experimental conditions for photolysis and binding studies were defined in which the stability of both BzATP and F/sub 1/ was maintained. Initial experiments examined the kinetic interactions between F/sub 1/ and BzATP. In the absence of actinic illumination, BzATP was a slow substrate for the enzyme and behaved as a classical competitive inhibitor versus ATP. Under photolytic conditions, BzATP inactivated F/sub 1/ with pseudo first-order kinetics, and the photoinactivation reaction showed rate saturation suggesting specific, reversible binding of BzATP to F/sub 1/ prior to covalent bond formation. ATP protected against F/sub 1/ photoinactivation and F/sub 1/ preparations partially modified covalently yielded the same K/sub m/ for ATP as unmodified enzyme preparations. These results strongly suggested that BzATP was bound to catalytic sites on the enzyme.

Ackerman, S.H.

1987-01-01

235

Single point mutations in ATP synthase compensate for mitochondrial genome loss in trypanosomes  

PubMed Central

Viability of the tsetse fly-transmitted African trypanosome Trypanosoma brucei depends on maintenance and expression of its kinetoplast (kDNA), the mitochondrial genome of this parasite and a putative target for veterinary and human antitrypanosomatid drugs. However, the closely related animal pathogens T. evansi and T. equiperdum are transmitted independently of tsetse flies and survive without a functional kinetoplast for reasons that have remained unclear. Here, we provide definitive evidence that single amino acid changes in the nuclearly encoded F1FO–ATPase subunit ? can compensate for complete physical loss of kDNA in these parasites. Our results provide insight into the molecular mechanism of compensation for kDNA loss by showing FO-independent generation of the mitochondrial membrane potential with increased dependence on the ADP/ATP carrier. Our findings also suggest that, in the pathogenic bloodstream stage of T. brucei, the huge and energetically demanding apparatus required for kDNA maintenance and expression serves the production of a single F1FO–ATPase subunit. These results have important implications for drug discovery and our understanding of the evolution of these parasites. PMID:23959897

Dean, Samuel; Gould, Matthew K.; Dewar, Caroline E.; Schnaufer, Achim C.

2013-01-01

236

Single point mutations in ATP synthase compensate for mitochondrial genome loss in trypanosomes.  

PubMed

Viability of the tsetse fly-transmitted African trypanosome Trypanosoma brucei depends on maintenance and expression of its kinetoplast (kDNA), the mitochondrial genome of this parasite and a putative target for veterinary and human antitrypanosomatid drugs. However, the closely related animal pathogens T. evansi and T. equiperdum are transmitted independently of tsetse flies and survive without a functional kinetoplast for reasons that have remained unclear. Here, we provide definitive evidence that single amino acid changes in the nuclearly encoded F1FO-ATPase subunit ? can compensate for complete physical loss of kDNA in these parasites. Our results provide insight into the molecular mechanism of compensation for kDNA loss by showing FO-independent generation of the mitochondrial membrane potential with increased dependence on the ADP/ATP carrier. Our findings also suggest that, in the pathogenic bloodstream stage of T. brucei, the huge and energetically demanding apparatus required for kDNA maintenance and expression serves the production of a single F1FO-ATPase subunit. These results have important implications for drug discovery and our understanding of the evolution of these parasites. PMID:23959897

Dean, Samuel; Gould, Matthew K; Dewar, Caroline E; Schnaufer, Achim C

2013-09-01

237

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

238

Modeling the effects of hypoxia on ATP turnover in exercising muscle  

NASA Technical Reports Server (NTRS)

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

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

1992-01-01

239

Copper Directs ATP7B to the Apical Domain of Hepatic Cells via Basolateral Endosomes.  

PubMed

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

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

2014-12-01

240

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

SciTech Connect

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

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

1986-05-01

241

Conformational transitions of subunit epsilon in ATP synthase from thermophilic Bacillus PS3.  

PubMed

Subunit epsilon of bacterial and chloroplast F(O)F(1)-ATP synthase is responsible for inhibition of ATPase activity. In Bacillus PS3 enzyme, subunit epsilon can adopt two conformations. In the "extended", inhibitory conformation, its two C-terminal alpha-helices are stretched along subunit gamma. In the "contracted", noninhibitory conformation, these helices form a hairpin. The transition of subunit epsilon from an extended to a contracted state was studied in ATP synthase incorporated in Bacillus PS3 membranes at 59 degrees C. Fluorescence energy resonance transfer between fluorophores introduced in the C-terminus of subunit epsilon and in the N-terminus of subunit gamma 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 microM 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 beta were found to stabilize the extended conformation of epsilon. Binding of ATP directly to epsilon was not essential for the ATP-induced conformational change. The ATP concentration necessary for the half-maximal transition (140 microM) suggests that subunit epsilon 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

2010-02-01

242

Structural basis of PP2A activation by PTPA, an ATP-dependent activation chaperone  

PubMed Central

Proper activation of protein phosphatase 2A (PP2A) catalytic subunit is central for the complex PP2A regulation and is crucial for broad aspects of cellular function. The crystal structure of PP2A bound to PP2A phosphatase activator (PTPA) and ATP?S reveals that PTPA makes broad contacts with the structural elements surrounding the PP2A active site and the adenine moiety of ATP. PTPA-binding stabilizes the protein fold of apo-PP2A required for activation, and orients ATP phosphoryl groups to bind directly to the PP2A active site. This allows ATP to modulate the metal-binding preferences of the PP2A active site and utilize the PP2A active site for ATP hydrolysis. In vitro, ATP selectively and drastically enhances binding of endogenous catalytic metal ions, which requires ATP hydrolysis and is crucial for acquisition of pSer/Thr-specific phosphatase activity. Furthermore, both PP2A- and ATP-binding are required for PTPA function in cell proliferation and survival. Our results suggest novel mechanisms of PTPA in PP2A activation with structural economy and a unique ATP-binding pocket that could potentially serve as a specific therapeutic target. PMID:24100351

Guo, Feng; Stanevich, Vitali; Wlodarchak, Nathan; Sengupta, Rituparna; Jiang, Li; Satyshur, Kenneth A; Xing, Yongna

2014-01-01

243

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

244

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

245

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

246

Vps4 Stimulatory Element of the Cofactor Vta1 Contacts the ATPase Vps4 ?7 and ?9 to Stimulate ATP Hydrolysis.  

PubMed

The endosomal sorting complexes required for transport (ESCRTs) function in a variety of membrane remodeling processes including multivesicular body sorting, abscission during cytokinesis, budding of enveloped viruses, and repair of the plasma membrane. Vps4 ATPase activity modulates ESCRT function and is itself modulated by its cofactor Vta1 and its substrate ESCRT-III. The carboxyl-terminal Vta1/SBP-1/Lip5 (VSL) domain of Vta1 binds to the Vps4 ?-domain to promote Vps4 oligomerization-dependent ATP hydrolysis. Additionally, the Vps4 stimulatory element (VSE) of Vta1 contributes to enhancing Vps4 oligomer ATP hydrolysis. The VSE is also required for Vta1-dependent stimulation of Vps4 by ESCRT-III subunits. However, the manner by which the Vta1 VSE contributes to Vps4 activation is unknown. Existing structural data were used to generate a model of the Vta1 VSE in complex with Vps4. This model implicated residues within the small ATPase associated with various activities (AAA) domain, specifically ?-helices 7 and 9, as relevant contact sites. Rational generation of Vps4 mutants defective for VSE-mediated stimulation, as well as intergenic compensatory mutations, support the validity of this model. These findings have uncovered the Vps4 surface responsible for coordinating ESCRT-III-stimulated Vta1 input during ESCRT function and identified a novel mechanism of Vps4 stimulation. PMID:25164817

Davies, Brian A; Norgan, Andrew P; Payne, Johanna A; Schulz, Mary E; Nichols, Micah D; Tan, Jason A; Xu, Zhaohui; Katzmann, David J

2014-10-10

247

PROTECTED VETERAN DEFINITIONS TITLE DEFINITION  

E-print Network

if the veteran has been determined to have a serious employment handicap). Disabled Veteran Veteran who servedPROTECTED VETERAN DEFINITIONS TITLE DEFINITION Veteran of the Vietnam Era Veteran of the U because of a service connected disability. "Vietnam era veteran" also includes any veteran of the U

Tipple, Brett

248

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

249

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

250

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

PubMed Central

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

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

2013-01-01

251

ATP-sensitive K+ channels in the kidney.  

PubMed

ATP-sensitive K+ channels (KATP channels) form a link between the metabolic state of the cell and the permeability of the cell membrane for K+ which, in turn, is a major determinant of cell membrane potential. KATP channels are found in many different cell types. Their regulation by ATP and other nucleotides and their modulation by other cellular factors such as pH and kinase activity varies widely and is fine-tuned for the function that these channels have to fulfill. In most excitable tissues they are closed and open when cell metabolism is impaired; thereby the cell is clamped in the resting state which saves ATP and helps to preserve the structural integrity of the cell. There are, however, notable exceptions from this rule; in pancreatic beta-cells, certain neurons and some vascular beds, these channels are open during the normal functioning of the cell. In the renal tubular system, KATP channels are found in the proximal tubule, the thick ascending limb of Henle's loop and the cortical collecting duct. Under physiological conditions, these channels have a high open probability and play an important role in the reabsorption of electrolytes and solutes as well as in K+ homeostasis. The physiological role of their nucleotide sensitivity is not entirely clear; one consequence is the coupling of channel activity to the activity of the Na-K-ATPase (pump-leak coupling), resulting in coordinated vectorial transport. In ischemia, however, the reduced ATP/ADP ratio would increase the open probability of the KATP channels independently from pump activity; this is particularly dangerous in the proximal tubule, where 60 to 70% of the glomerular ultrafiltrate is reabsorbed. The pharmacology of KATP channels is well developed including the sulphonylureas as standard blockers and the structurally heterogeneous family of channel openers. Blockers and openers, exemplified by glibenclamide and levcromakalim, show a wide spectrum of affinities towards the different types of KATP channels. Recent cloning efforts have solved the mystery about the structure of the channel: the KATP channels in the pancreatic beta-cell and in the principal cell of the renal cortical collecting duct are heteromultimers, composed of an inwardly rectifying K+ channel and sulphonylurea binding subunit(s) with unknown stoichiometry. The proteins making up the KATP channel in these two cell types are different (though homologous), explaining the physiological and pharmacological differences between these channel subtypes. PMID:8878050

Quast, U

1996-01-01

252

ATP-induced vasodilation in human skeletal muscle  

PubMed Central

The purine nucleotide adenosine-5?-triphosphate (ATP) exerts pronounced effects on the cardiovascular system. The mechanism of action of the vasodilator response to ATP in humans has not been elucidated yet. The proposed endothelium-derived relaxing factors (EDRFs) were studied in a series of experiments, using the perfused forearm technique. Adenosine 5?-triphosphate (0.2, 0.6, 6 and 20 nmol dl?1 forearm volume min?1) evoked a dose-dependent forearm vasodilator response, which could not be inhibited by separate infusion of the nonselective COX inhibitor indomethacin (5 ?g dl?1 min?1, n=10), the blocker of Na+/K+-ATPase ouabain (0.2 ?g dl?1 min?1, n=8), the blocker of KCa channels tetraethylammonium chloride (TEA, 0.1 ?g dl?1 min?1, n=10), nor by the KATP-channel blocker glibenclamide (2 ?g dl?1 min?1, n=10). All blockers, except glibenclamide, caused a significant increase in baseline vascular tone. The obtained results might be due to compensatory actions of unblocked EDRFs. Combined infusion of TEA, indomethacin and L-NMMA (n=6) significantly increased the baseline forearm vascular resistance. The ATP-induced relative decreases in forearm vascular resistance were 48±5, 67±3, 88±2, and 92±2% in the absence and 23±7, 62±4, 89±2, and 93±1% in the presence of the combination of TEA, indomethacin and L-NMMA (P<0.05, repeated-measures ANOVA, n=6). A similar inhibition was obtained for sodium nitroprusside (SNP, P<0.05 repeated-measures ANOVA, n=6), indicating a nonspecific interaction due to the blocker-induced vasoconstriction. ATP-induced vasodilation in the human forearm cannot be inhibited by separate infusion of indomethacin, ouabain, glibenclamide or TEA, or by a combined infusion of TEA, indomethacin, and L-NMMA. Endothelium-independent mechanisms and involvement of unblocked EDRFs, such as CO, might play a role, and call for further studies. PMID:14769779

van Ginneken, E E M; Meijer, P; Verkaik, N; Smits, P; Rongen, G A

2004-01-01

253

Arrangement of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1  

PubMed Central

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

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

2011-01-01

254

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;Université Âaris Descartes REV)

2009-02-03

255

Na,K-dependent adenosine triphosphate phosphohydrolase: activation of the phosphatase reaction by ATP analogs.  

PubMed

The effect of N1-substituted analogs of ATP on the hydrolysis of umbelliferone phosphate by Na,K-ATPase has demonstrated: analogs having a negatively charged substituent (N1-oxy- or N1-carbo-methoxy-ATP) and capable of accepting H+ induce an activation similar to that of ATP; N1-methoxy-ATP, containing an uncharged substituent, does not affect the phosphatase reaction at low concentration and inhibits it at higher concentration. It has been assumed that ATP binding to Na,K-ATPase induces formation of a hydrogen bond between the nitrogen atom at the first position of the purine base and appropriate amino acid of active centre, with a subsequent attachment of H+ to ATP, thus facilitating the transition of Na,K-ATPase from the K+- to the Na+-form. PMID:6090214

Boldyrev, A A; Lopina, O D; Gulyaev, N N; Baranova, L A; Severin, E S

1984-10-01

256

Molecular Cloning and Characterization of ATP-Phosphoribosyl Transferase from Arabidopsis, a Key Enzyme in the Histidine Biosynthetic Pathway  

PubMed Central

We have characterized two isoforms of ATP-phosphoribosyl transferase (ATP-PRT) from Arabidopsis (AtATP-PRT1 [accession no. AB025251] and AtATP-PRT2), catalyzing the first step of the pathway of hisidine (His) biosynthesis. The primary structures deduced from AtATP-PRT1 and AtATP-PRT2 cDNAs share an overall amino acid identity of 74.6% and contain N-terminal chloroplast transit peptide sequences. DNA-blot analyses indicated that the ATP-PRTs in Arabidopsis are encoded by two separate genes with a closely similar gene structural organization. Both gene transcripts were detected throughout development, and protein-blot analysis revealed predominant accumulation of the AtATP-PRT proteins in Arabidopsis leaves. The His auxotrophy of a his1 mutant of Saccharomyces cerevisiae was suppressed by the transformation with AtATP-PRT1 and AtATP-PRT2 cDNAs, indicating that both isoforms are functionally active ATP-PRT enzymes. The Km values for ATP and phosphoribosyl pyrophosphate of the recombinant AtATP-PRT proteins were comparable to those of the native ATP-PRTs from higher plants and bacteria. It was demonstrated that the recombinant AtATP-PRTs were inhibited by l-His (50% inhibition of initial activity = 40–320 ?m), suggesting that His biosynthesis was regulated in plants through feedback inhibition by l-His. PMID:10712555

Ohta, Daisaku; Fujimori, Ko; Mizutani, Masaharu; Nakayama, Yumiko; Kunpaisal-Hashimoto, Rosarin; Munzer, Silvia; Kozaki, Akiko

2000-01-01

257

Stepwise rotation of the ?-subunit of EFoF1-ATP synthase during ATP synthesis: a single-molecule FRET approach  

NASA Astrophysics Data System (ADS)

FoF1-ATP synthases couple proton translocation with the synthesis of ATP using two rotary motors within the enzyme. To monitor inter-subunit movements during catalysis, we selectively attached two fluorophores to the F1 part, sulforhodamine B at one of three ?-subunits and Cy5 at the ?-subunit. Reassembly with Fo parts embedded in liposomes yielded functional holoenzymes. Fluorescence resonance energy transfer (FRET) was investigated in photon bursts of freely diffusing liposomes with reconstituted ATP synthases using a confocal set-up for single-molecule detection. Incubation with AMPPNP resulted in stable intensity ratios within a burst and three different FRET efficiencies. Upon ATP addition, a repeating sequence of three distinct FRET efficiencies was observed, indicating the stepwise movement of the ?-subunit during ATP hydrolysis. With this single-molecule FRET approach we detected a stepwise rotation of the ?-subunit under conditions for ATP synthesis (i.e. energization of the proteoliposomes by an acid-base-transition). The direction of rotation is opposite to the direction observed during ATP hydrolysis.

Borsch, Michael; Diez, Manuel; Zimmermann, Boris; Trost, Matthias; Steigmiller, Stefan; Graber, Peter

2003-06-01

258

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

259

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

260

[ATP-dependent FtsH and Lon chloroplast proteases].  

PubMed

Arabidopsis thaliana proteome contains 667 proteases; some tens of them are chloroplast-targeted proteins, encoded by genes orthologous to the ones coding for bacterial proteolytic enzymes. It is thought that chloroplast proteases are involved in chloroplasts' proteins turnover and quality control (maturation of nucleus-encoded proteins and removal of nonfunctional ones). Some ATP-dependent chloroplast proteases belonging to FtsH family (especially FtsH2 and FtsH5) are considered to be involved in numerous aspects of chloroplast and whole plant maintenance under non-stressing as well as stressing conditions. This notion is supported by severe phenotype appearance of mutants deficient in these proteases. In contrast to seemingly high physiological importance of chloroplast members of FtsH protease family, only a few individual proteins have been identified so far as their physiological targets (i.e. Lhcb1, Lhcb3, PsbA and Rieske protein). Our knowledge regarding structure and molecular mechanisms of these enzymes' action is limited when compared with what is known about FtsHs of bacterial origin. Equally limited is the knowledge about ATP-dependent Lon4 protease being the single known chloroplast-targeted ortholog of Lon protease of Escherichia coli. PMID:21735825

Baranek, Ma?gorzata; Grabsztunowicz, Magda; Sikora, Bogna; Jackowski, Grzegorz

2011-01-01

261

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

262

Performance of the Third 50 Completed ATP Projects  

E-print Network

AND TECHNOLOGY William Jeffrey, Director ADVANCED TECHNOLOGY PROGRAM Marc Stanley, Director ADVANCED TECHNOLOGY PROGRAM #12;NATIONAL INSTITUTE OF STANDARDS AND TECHNOLOGY Advanced Technology Program, Economic.......................................................................................................... 117 ADVANCED TECHNOLOGY PROGRAM iii #12;iv #12;ACKNOWLEDGEMENTS We are pleased to announce

263

Performance of the Third 50 Completed ATP Projects  

E-print Network

INSTITUTE OF STANDARDS AND TECHNOLOGY William Jeffrey, Director ADVANCED TECHNOLOGY PROGRAM Marc Stanley, Director ADVANCED TECHNOLOGY PROGRAM #12;NATIONAL INSTITUTE OF STANDARDS AND TECHNOLOGY Advanced Technology.......................................................................................................... 107 ADVANCED TECHNOLOGY PROGRAM iii #12;iv #12;ACKNOWLEDGEMENTS We are pleased to announce

264

Extracellular ATP Activates a P2 Receptor in Necturus Erythrocytes During Hypotonic Swelling  

Microsoft Academic Search

.   We recently reported that ATP is released from Necturus erythrocytes via a conductive pathway during hypotonic swelling and that extracellular ATP potentiates regulatory volume\\u000a decrease (RVD). This study was designed to determine whether extracellular ATP exerts its effect via a purinoceptor. This\\u000a was accomplished using three different experimental approaches: 1) hemolysis studies to examine osmotic fragility, 2) a Coulter

D. B. Light; P. K. Dahlstrom; R. T. Gronau; N. L. Baumann

2001-01-01

265

Pharmacological properties of ATP-sensitive purinergic receptors expressed in human G292 osteoblastic cells.  

PubMed

We characterized the pharmacological properties of P2 receptors expressed in G292 osteoblastic cells by studying the responses or changes in intracellular Ca(2+) level to P2 receptor agonists, antagonists and modulators. ATP induced robust responses in a concentration-dependent manner with EC(50) of 0.5+/-0.07 microM. While alpha,beta-methylene-ATP (alphabetameATP) and 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP) were ineffective, ADP mimicked the action of ATP with EC(50) of 0.7+/-0.2 microM. UTP and UDP also evoked responses with EC(50) of 2.0+/-0.4 microM and 0.5+/-0.1 microM respectively, but their responses were much smaller, resulting in an order of the response magnitude: ATP~ADP>UTP~UDP. The responses evoked by ATP and ADP were blocked by pyridoxal-5'-phosphate-6-azophenyl-2,4,-disulfonate (PPADS) with IC(50) of 3.0+/-0.05 microM and 5.0+/-0.4 microM respectively, but not by suramin up to 30 microM. ATP-evoked responses were insensitive to inhibition by trinitrophenyl-ATP (TNP-ATP) and brilliant blue G. ADP-evoked responses were significantly inhibited by 2'-deoxy-N(6)-methyladenosine-3',5'-biphosphate (MRS2179) and 2-chloro-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate (MRS2279) with IC(50) of 48+/-1.9 microM and 7.7+/-0.9 microM respectively. Taken together, these results provide strong evidence for functional expression of ATP-sensitive P2Y receptors and particularly P2Y(1)-like receptor in G292 cells. PMID:19577559

Li, Dong-Liang; Liu, Xing; Xia, Rong; Ross, Claire; Yang, Xuebin; Jiang, Lin-Hua

2009-09-01

266

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

Microsoft Academic Search

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

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

1990-01-01

267

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

PubMed Central

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

1989-01-01

268

Analysis of the mechanism of ATP stimulation of calf thymus DNA alpha-polymerase.  

PubMed

Biochemical kinetic analyses of the ATP stimulation of the A2 form of calf DNA alpha-polymerase show that when DNA or primer termini are the variable substrates, maximum reaction velocity is independent of ATP concentration. When dNTP concentration is the variable substrate, the apparent Km is invariant with ATP. Such results indicate that the increase in the synthetic rate caused by ATP results from an improvement in synthesis initiation at primer termini. The effect of ATP on the DNA binding affinity of alpha-A2-polymerase was examined by using column chromatography. Passage of the polymerase through native DNA-cellulose at 70 mM ionic strength resulted in 40% binding of the enzyme. In the presence of 4 mM ATP, binding increased to 80%. In both cases, the bound polymerase could be eluted by a 370 mM ionic strength wash. An elution profile similar to that observed in the absence of ATP was obtained with 0.1 mM ATP, 4 mM GTP, or 4 mM each of the nonhydrolyzable ATP analogues adenyl-5'-yl imidodiphosphate or adenosine 5'-O-(3-thiotriphosphate). These results suggest that hydrolysis of the gamma-phosphate occurs at millimolar levels of ATP and leads to a higher affinity of polymerase for DNA. To distinguish the effects of ATP on RNA priming from those on DNA synthesis, products synthesized processively by alpha-A2-polymerase were sized by gel filtration. Results indicate that essentially all products made on a gapped fd replicative form template in the presence of four dNTPs and 4 mM ATP result from the extension of preexisting DNA primers.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:6487601

Lawton, K G; Wierowski, J V; Schechter, S; Hilf, R; Bambara, R A

1984-09-11

269

Periodate-oxidized ATP modulates macrophage functions during infection with Leishmania amazonensis.  

PubMed

Previously, we showed that treating macrophages with ATP impairs the intracellular growth of Leishmania amazonensis, and that the P2X7 purinergic receptor is overexpressed during leishmaniasis. In the present study, we directly evaluated the effect of periodate-oxidized ATP (oATP) on parasite control in Leishmania-infected macrophages. We found that oATP impaired the attachment/entrance of L. amazonensis promastigotes to C57BL/6 mouse macrophages in a P2X7 receptor-independent manner, as macrophages from P2X7(-/-) mice were similarly affected. Although oATP directly inhibited the growth of axenic promastigotes in culture, promoted rapid ultrastructural alterations, and impaired Leishmania internalization by macrophages, it did not affect intracellular parasite multiplication. Upon infection, phagosomal acidification was diminished in oATP-treated macrophages, accompanied by reduced endosomal proteolysis. Likewise, MHC class II molecules expression and ectoATPase activity was decreased by oATP added to macrophages at the time of parasite infection. These inhibitory effects were not due to a cytotoxic effect, as no additional release of lactate dehydrogenase was detected in culture supernatants. Moreover, the capacity of macrophages to produce nitric oxide and reactive oxygen species was not affected by the presence of oATP during infection. We conclude that oATP directly affects extracellular parasite integrity and macrophage functioning. PMID:24804957

Figliuolo, V R; Chaves, S P; Santoro, G F; Coutinho, C M L M; Meyer-Fernandes, J R; Rossi-Bergmann, B; Coutinho-Silva, R

2014-07-01

270

Intracellular ATP Levels are a Pivotal Determinant of Chemoresistance in Colon Cancer Cells  

PubMed Central

Altered metabolism in cancer cells is suspected to contribute to chemoresistance but the precise mechanisms are unclear. Here we show that intracellular ATP levels are a core determinant in the development of acquired cross-drug resistance of human colon cancer cells that harbor different genetic backgrounds. Drug-resistant cells were characterized by defective mitochondrial ATP production, elevated aerobic glycolysis, higher absolute levels of intracellular ATP and enhanced HIF-1?-mediated signaling. Interestingly, direct delivery of ATP into cross-chemoresistant cells destabilized HIF-1? and inhibited glycolysis. Thus, drug-resistant cells exhibit a greater “ATP debt” defined as the extra amount of ATP needed to maintain homeostasis of survival pathways under genotoxic stress. Direct delivery of ATP was sufficient to render drug-sensitive cells drug resistant. Conversely, depleting ATP by cell treatment with an inhibitor of glycolysis, 3-bromopyruvate, was sufficient to sensitize cells cross-resistant to multiple chemotherapeutic drugs. In revealing intracellular ATP levels are a core determinant of chemoresistance in colon cancer cells, our findings may offer a foundation for new improvements to colon cancer treatment. PMID:22084398

Zhou, Yunfei; Tozzi, Federico; Chen, Jinyu; Fan, Fan; Xia, Ling; Wang, Jinrong; Gao, Guang; Zhang, Aijun; Xia, Xuefeng; Brasher, Heather; Widger, William; Ellis, Lee M; Weihua, Zhang

2013-01-01

271

New soluble ATP-dependent protease, Ti, in Escherichia coli that is distinct from protease La  

SciTech Connect

E. coli must contain other ATP-requiring proteolytic systems in addition to protease La (the lon gene product). A new ATP-dependent protease was purified from lon cells which lack protease La, as shown by immuno-blotting. This enzyme hydrolyzes (TH)casein to acid-soluble products in the presence of ATP (or dATP) and MgS . Nonhydrolyzable ATP analogs, other nucleoside triphosphates and AMP can not replace ATP. Therefore, ATP hydrolysis appears necessary for proteolysis. The enzyme appears to be a serine protease, but also contains essential thiol residues. Unlike protease La, it is not inhibited by vanadate, heparin, or the defective R9 subunit of protease La. On gel filtration, this enzyme has an apparent Mr of 340,000 and is comprised of two components of 190,000D and 130,000D, which can be separated by phosphocellulose chromatography. By themselves, these components do not show ATP-dependent proteolysis, but when mixed, full activity is restored. These finding and similar ones of Maurizi and Gottesman indicate that E. coli contain two soluble ATP-dependent proteases, which function by different mechanisms. This new enzyme may contribute to the rapid breakdown of abnormal polypeptides or of normal proteins during starvation. The authors propose to name it protease Ti.

Chung, C.H.; Hwang, B.J.; Park, W.J.; Goldberg, A.L.

1987-05-01

272

ATP induces mild hypothermia in rats but has a strikingly detrimental impact on focal cerebral ischemia.  

PubMed

Ischemic stroke is a devastating condition lacking effective therapies. A promising approach to attenuate ischemic injury is mild hypothermia. Recent studies show that adenosine nucleotides can induce hypothermia in mice. The purpose of the present study was to test the hypothesis that adenosine 5'-triphosphate (ATP) induces mild hypothermia in rats and reduces ischemic brain injury. We found that intraperitoneal injections of ATP decreased core body temperature in a dose-dependent manner; the dose appropriate for mild hypothermia was 2 g/kg. When ATP-induced hypothermia was applied to stroke induced by middle cerebral artery occlusion, however, a neuroprotective effect was not observed. Instead, the infarct volume grew even larger in ATP-treated rats. This was accompanied by an increased rate of seizure events, hemorrhagic transformation, and higher mortality. Continuous monitoring of physiologic parameters revealed that ATP reduced heartbeat rate and blood pressure. ATP also increased blood glucose, accompanied by severe acidosis and hypocalcemia. Western blotting showed that ATP decreased levels of both phospho-Akt and total-Akt in the cortex. Our results reveal that, despite inducing hypothermia, ATP is not appropriate for protecting the brain against stroke. Instead, we show for the first time that ATP treatment is associated with exaggerated ischemic outcomes and dangerous systemic side effects. PMID:23072747

Zhang, Meijuan; Li, Wenjin; Niu, Guangming; Leak, Rehana K; Chen, Jun; Zhang, Feng

2013-01-01

273

Differentiation of hematopoietic stem cell and myeloid populations by ATP is modulated by cytokines  

PubMed Central

Extracellular nucleotides are emerging as important regulators of inflammation, cell proliferation and differentiation in a variety of tissues, including the hematopoietic system. In this study, the role of ATP was investigated during murine hematopoiesis. ATP was able to reduce the percentage of hematopoietic stem cells (HSCs), common myeloid progenitors and granulocyte–macrophage progenitors (GMPs), whereas differentiation into megakaryocyte–erythroid progenitors was not affected. In addition, in vivo administration of ATP to mice reduced the number of GMPs, but increased the number of Gr-1+Mac-1+ myeloid cells. ATP also induced an increased proliferation rate and reduced Notch expression in HSCs and impaired HSC-mediated bone marrow reconstitution in sublethally irradiated mice. Moreover, the effects elicited by ATP were inhibited by suramin, a P2 receptor antagonist, and BAPTA, an intracellular Ca2+ chelator. We further investigated whether the presence of cytokines might modulate the observed ATP-induced differentiation. Treatment of cells with cytokines (stem cell factor, interleukin-3 and granulocyte–monocyte colony stimulator factor) before ATP stimulation led to reduced ATP-dependent differentiation in long-term bone marrow cultures, thereby restoring the ability of HSCs to reconstitute hematopoiesis. Thus, our data suggest that ATP induces the differentiation of murine HSCs into the myeloid lineage and that this effect can be modulated by cytokines. PMID:21633388

Barbosa, C M V; Leon, C M M P; Nogueira-Pedro, A; Wasinsk, F; Araujo, R C; Miranda, A; Ferreira, A T; Paredes-Gamero, E J

2011-01-01

274

Studies of the C-terminal Region of the Gamma Subunit of the Chloroplast ATP Synthase  

E-print Network

) ---- the Energy Carrier B. The ATP Synthase 1. General Structure and Subunit Stoichiometry of the ATP Synthases 2. The Functional Core of the ATP Synthase ---- the F 1 Enzyme 3. Crystal Structures of the ATP Synthase F 1 Enzymes 4. The Binding Change... xiii xvi 1 1 1 2 5 6 10 14 14 15 18 ix 4. Nucleotide Binding Sites on CF 1 and Inter-site Cooperativity D. Focus of this Research Chapter Two. Mutational Studies of the C-terminal Region of the Gamma Subunit...

He, Feng

2008-06-18

275

PharmGKB Submission Update: IV. PMT Submissions of Genetic Variations in ATP-  

E-print Network

published on December 1, 2005 Category: genotype Project: Pharmacogenetics of Membrane Transporters Table 1 IDs. Pharmacogenetic Significance: Genetic variation in the ATP-binding cassette (ABC) family

Sali, Andrej

276

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

PubMed

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

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

2014-10-01

277

Intracellular ATP levels are a pivotal determinant of chemoresistance in colon cancer cells.  

PubMed

Altered metabolism in cancer cells is suspected to contribute to chemoresistance, but the precise mechanisms are unclear. Here, we show that intracellular ATP levels are a core determinant in the development of acquired cross-drug resistance of human colon cancer cells that harbor different genetic backgrounds. Drug-resistant cells were characterized by defective mitochondrial ATP production, elevated aerobic glycolysis, higher absolute levels of intracellular ATP, and enhanced HIF-1?-mediated signaling. Interestingly, direct delivery of ATP into cross-chemoresistant cells destabilized HIF-1? and inhibited glycolysis. Thus, drug-resistant cells exhibit a greater "ATP debt" defined as the extra amount of ATP needed to maintain homeostasis of survival pathways under genotoxic stress. Direct delivery of ATP was sufficient to render drug-sensitive cells drug resistant. Conversely, depleting ATP by cell treatment with an inhibitor of glycolysis, 3-bromopyruvate, was sufficient to sensitize cells cross-resistant to multiple chemotherapeutic drugs. In revealing that intracellular ATP levels are a core determinant of chemoresistance in colon cancer cells, our findings may offer a foundation for new improvements to colon cancer treatment. PMID:22084398

Zhou, Yunfei; Tozzi, Federico; Chen, Jinyu; Fan, Fan; Xia, Ling; Wang, Jinrong; Gao, Guang; Zhang, Aijun; Xia, Xuefeng; Brasher, Heather; Widger, William; Ellis, Lee M; Weihua, Zhang

2012-01-01

278

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

279

Multiscale approach to link red blood cell dynamics, shear viscosity, and ATP release  

PubMed Central

RBCs are known to release ATP, which acts as a signaling molecule to cause dilation of blood vessels. A reduction in the release of ATP from RBCs has been linked to diseases such as type II diabetes and cystic fibrosis. Furthermore, reduced deformation of RBCs has been correlated with myocardial infarction and coronary heart disease. Because ATP release has been linked to cell deformation, we undertook a multiscale approach to understand the links between single RBC dynamics, ATP release, and macroscopic viscosity all at physiological shear rates. Our experimental approach included microfluidics, ATP measurements using a bioluminescent reaction, and rheology. Using microfluidics technology with high-speed imaging, we visualize the deformation and dynamics of single cells, which are known to undergo motions such as tumbling, swinging, tanktreading, and deformation. We report that shear thinning is not due to cellular deformation as previously believed, but rather it is due to the tumbling-to-tanktreading transition. In addition, our results indicate that ATP release is constant at shear stresses below a threshold (3 Pa), whereas above the threshold ATP release is increased and accompanied by large cellular deformations. Finally, performing experiments with well-known inhibitors, we show that the Pannexin 1 hemichannel is the main avenue for ATP release both above and below the threshold, whereas, the cystic fibrosis transmembrane conductance regulator only contributes to deformation-dependent ATP release above the stress threshold. PMID:21690355

Forsyth, Alison M.; Wan, Jiandi; Owrutsky, Philip D.; Abkarian, Manouk; Stone, Howard A.

2011-01-01

280

Activation of neuronal NMDA receptors triggers transient ATP-mediated microglial process outgrowth.  

PubMed

Microglia are morphologically dynamic cells that rapidly extend their processes in response to various stimuli including extracellular ATP. In this study, we tested the hypothesis that stimulation of neuronal NMDARs trigger ATP release leading to communication with microglia. We used acute mouse hippocampal brain slices and two-photon laser scanning microscopy to study microglial dynamics and developed a novel protocol for fixation and immunolabeling of microglia processes. Similar to direct topical ATP application in vivo, short multiple applications of NMDA triggered transient microglia process outgrowth that was reversible and repeatable indicating that this was not due to excitotoxic damage. Stimulation of NMDAR was required as NMDAR antagonists, but not blockers of AMPA/kainate receptors or voltage-gated sodium channels, prevented microglial outgrowth. We report that ATP release, secondary to NMDAR activation, was the key mediator of this neuron-microglia communication as both blocking purinergic receptors and inhibiting hydrolysis of ATP to prevent locally generated gradients abolished outgrowth. Pharmacological and genetic analyses showed that the NMDA-triggered microglia process extension was independent of Pannexin 1, the ATP releasing channels, ATP release from astrocytes via connexins, and nitric oxide generation. Finally, using whole-cell patch clamping we demonstrate that activation of dendritic NMDAR on single neurons is sufficient to trigger microglia process outgrowth. Our results suggest that dendritic neuronal NMDAR activation triggers ATP release via a Pannexin 1-independent manner that induces outgrowth of microglia processes. This represents a novel uncharacterized form of neuron-microglial communication mediated by ATP. PMID:25100586

Dissing-Olesen, Lasse; LeDue, Jeffrey M; Rungta, Ravi L; Hefendehl, Jasmin K; Choi, Hyun B; MacVicar, Brian A

2014-08-01

281

Iridium(III) azuliporphyrins.  

PubMed

Azuliporphyrins were reacted with [Ir(COD)Cl](2) in refluxing o- or p-xylene to give novel iridium(III) derivatives that regioselectively incorporated an oxidized solvent molecule. The iridium(III) is inserted within the porphyrinoid macrocycle and possesses an additional apical acyl unit. PMID:23111425

Lash, Timothy D; Pokharel, Komal; Zeller, Matthias; Ferrence, Gregory M

2012-12-14

282

SUPERSTARS III: K-2.  

ERIC Educational Resources Information Center

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

North Carolina State Dept. of Public Education, Raleigh.

283

CITY III: Small Scale.  

ERIC Educational Resources Information Center

CITY III is a computer-assisted simulation game in which participants make decisions affecting the economic, governmental, and social conditions of a simulated urban area. In one of its five city options (called Lothian) the CITY III simulation model focuses on a micro-view of an urban area by reducing the level of aggregation. The population is…

Envirometrics, Inc., Washington, DC.

284

Glyoxylate lowers metabolic ATP in human platelets without altering adenylate energy charge or aggregation.  

PubMed

Human blood platelets adhere to exposed collagen at the site of vascular injury, initiating a signaling cascade leading to fibrinogen activation, secretion of granules and aggregation, thus producing a stable thrombus. All these steps require metabolic ATP. In this study we have labeled the metabolic pool of ATP with nucleotides, treated platelets with various inhibitors and have monitored their ability to be activated. Incubating platelets with glyoxylate dramatically reduced the ATP level without a change in the adenylate energy charge (AEC). This reduction of ATP did not affect ADP-induced primary or secondary aggregation, whereas glyoxal, methyl glyoxal, or the combination of antimycin plus deoxyglucose reduced both ATP and AEC and inhibited aggregation. The reduction of ATP by glyoxylate was almost quantitatively matched by an increase in hypoxanthine without elevation of ADP. AMP, IMP or inosine, acetoacetate, aspartate, or glutamate had no effect on glyoxylate-induced breakdown of ATP, while pyruvate stopped the ATP reduction fast and efficiently. Glyoxylate also lowered the citrate content. The glyoxylate-induced breakdown of ATP coincided with an increase in fructose-1,6-bisphosphate, indicating that the phosphofructokinase reaction was the main ATP-consuming step. Glyoxylate was a substrate for lactate dehydrogenase although with a Km almost 100 times higher than pyruvate. We suggest that glyoxylate primarily competes with pyruvate in the pyruvate dehydrogenase reaction, thus lowering the citrate concentration, which in turn activates phosphofructokinase. Clearly, lowering of ATP in the cytosol by more than 50% does not affect platelet aggregation provided that the AEC is not reduced. PMID:23488475

Dangelmaier, Carol A; Holmsen, Holm

2014-01-01

285

Urinary ATP May Be a Dynamic Biomarker of Detrusor Overactivity in Women with Overactive Bladder Syndrome  

PubMed Central

Background Nowadays, there is a considerable bulk of evidence showing that ATP has a prominent role in the regulation of human urinary bladder function and in the pathophysiology of detrusor overactivity. ATP mediates nonadrenergic-noncholinergic detrusor contractions in overactive bladders. In vitro studies have demonstrated that uroepithelial cells and cholinergic nerves from overactive human bladder samples (OAB) release more ATP than controls. Here, we compared the urinary ATP concentration in samples collected non-invasively from OAB women with detrusor overactivity and age-matched controls. Methods Patients with neurologic diseases, history of malignancy, urinary tract infections or renal impairment (creatinine clearance <70 ml/min) were excluded. All patients completed a 3-day voiding diary, a 24 h urine collection and blood sampling to evaluate creatinine clearance. Urine samples collected during voluntary voids were immediately freeze-preserved for ATP determination by the luciferin-luciferase bioluminescence assay; for comparison purposes, samples were also tested for urinary nerve growth factor (NGF) by ELISA. Results The urinary content of ATP, but not of NGF, normalized to patients’ urine creatinine levels (ATP/Cr) or urinary volume (ATP.Vol) were significantly (P<0.05) higher in OAB women with detrusor overactivity (n?=?34) than in healthy controls (n?=?30). Significant differences between the two groups were still observed by boosting urinary ATP/Cr content after water intake, but these were not detected for NGF/Cr. In OAB patients, urinary ATP/Cr levels correlated inversely with mean voided volumes determined in a 3-day voiding diary. Conclusion A high area under the receiver operator characteristics (ROC) curve (0.741; 95% CI 0.62–0.86; P<0.001) is consistent with urinary ATP/Cr being a highly sensitive dynamic biomarker for assessing detrusor overactivity in women with OAB syndrome. PMID:23741373

Oliveira, Olga; Ferreira, Sonia; Reis, Maria Julia; Oliveira, Jose Carlos; Correia-de-Sa, Paulo

2013-01-01

286

Strain Background Modifies Phenotypes in the ATP8B1-Deficient Mouse  

PubMed Central

Background Mutations in ATP8B1 (FIC1) underlie cases of cholestatic disease, ranging from chronic and progressive (progressive familial intrahepatic cholestasis) to intermittent (benign recurrent intrahepatic cholestasis). The ATP8B1-deficient mouse serves as an animal model of human ATP8B1 deficiency. Methodology/Principal Findings We investigated the effect of genetic background on phenotypes of ATP8B1-deficient and wild-type mice, using C57Bl/6 (B6), 129, and (B6-129) F1 strain backgrounds. B6 background resulted in greater abnormalities in ATP8B1-deficient mice than did 129 and/or F1 background. ATP8B1-deficient pups of B6 background gained less weight. In adult ATP8B1-deficient mice at baseline, those of B6 background had lower serum cholesterol levels, higher serum alkaline phosphatase levels, and larger livers. After challenge with cholate-supplemented diet, these mice exhibited higher serum alkaline phosphatase and bilirubin levels, greater weight loss and larger livers. ATP8B1-deficient phenotypes in mice of F1 and 129 backgrounds are usually similar, suggesting that susceptibility to manifestations of ATP8B1 deficiency may be recessive. We also detected differences in hepatobiliary phenotypes between wild-type mice of differing strains. Conclusions/Significance Our results indicate that the ATP8B1-deficient mouse in a B6 background may be a better model of human ATP8B1 deficiency and highlight the importance of informed background strain selection for mouse models of liver disease. PMID:20126555

Vargas, Julie C.; Xu, Hongmei; Groen, Annamiek; Paulusma, Coen C.; Grenert, James P.; Pawlikowska, Ludmila; Sen, Saunak; Elferink, Ronald P. J. Oude; Bull, Laura N.

2010-01-01

287

Mechanical Modulation of ATP-binding Affinity of V1-ATPase*  

PubMed Central

V1-ATPase is a rotary motor protein that rotates the central shaft in a counterclockwise direction hydrolyzing ATP. Although the ATP-binding process is suggested to be the most critical reaction step for torque generation in F1-ATPase (the closest relative of V1-ATPase evolutionarily), the role of ATP binding for V1-ATPase in torque generation has remained unclear. In the present study, we performed single-molecule manipulation experiments on V1-ATPase from Thermus thermophilus to investigate how the ATP-binding process is modulated upon rotation of the rotary shaft. When V1-ATPase showed an ATP-waiting pause, it was stalled at a target angle and then released. Based on the response of the V1-ATPase released, the ATP-binding probability was determined at individual stall angles. It was observed that the rate constant of ATP binding (kon) was exponentially accelerated with forward rotation, whereas the rate constant of ATP release (koff) was exponentially reduced. The angle dependence of the koff of V1-ATPase was significantly smaller than that of F1-ATPase, suggesting that the ATP-binding process is not the major torque-generating step in V1-ATPase. When V1-ATPase was stalled at the mean binding angle to restrict rotary Brownian motion, kon was evidently slower than that determined from free rotation, showing the reaction rate enhancement by conformational fluctuation. It was also suggested that shaft of V1-ATPase should be rotated at least 277° in a clockwise direction for efficient release of ATP under ATP-synthesis conditions. PMID:23155048

Tirtom, Naciye Esma; Okuno, Daichi; Nakano, Masahiro; Yokoyama, Ken; Noji, Hiroyuki

2013-01-01

288

Extracellular ATP induces ion fluxes and inhibits growth of Friend erythroleukemia cells.  

PubMed

Extracellular ATP (1 mM) inhibited the growth of Friend virus-infected murine erythroleukemia cells (MEL cells) but had no effect on dimethyl sulfoxide-induced differentiation. ATP (1 mM) also caused changes in the permeability of MEL cells to ions. There was an increased influx of 45Ca2+ from a basal level of 5 pmol/min to 18 pmol/min/10(6) cells to achieve a 2-fold increase in steady-state Ca2+ as measured at isotopic equilibration. Ca2+ influx was blocked by diisothiocyanostilbene disulfonate (DIDS), an inhibitor of anion transport. ATP also stimulated Cl- uptake, and this flux was inhibited by DIDS. The ratio of ATP stimulated Cl- to Ca2+ uptake was 1.6:1. K+ and Na+ influx were also stimulated by ATP, but phosphate uptake was inhibited; the Na+ influx dissipated the Na+ gradient and thus inhibited nutrient uptake. ATP-stimulated K+ influx was ouabain inhibitable; however, the total cellular K+ decreased due to an ATP-stimulated ouabain-resistant K+ efflux. Na+ influx and Ca2+ influx occurred by separate independent routes, since Na+ influx was not inhibited by DIDS. The effects observed were specific for ATP *K1/2 MgATP = 0.7 mM) since AMP, GTP, adenosine, and the slowly hydrolyzable ATP analogue adenyl-5'-yl imidodiphosphate were without effect. The major ionic changes in the cell were a decrease in K+ and increase in Na+; cytoplasmic pH and free Ca2+ did not change appreciably. These ATP-induced changes in ion flux are considered to be responsible for growth inhibition. PMID:6594338

Chahwala, S B; Cantley, L C

1984-11-25

289

Comparison of DSM-III and DSM-III-R diagnoses for prepubertal children: changes in prevalence and validity.  

PubMed

A structured and reliable diagnostic procedure based on a revised version of the Diagnostic Interview Schedule for Children for children, parents, and teachers was used to assign both DSM-III and DSM-III-R diagnoses to 177 outpatient boys aged 7 to 12 years. Compared to their DSM-III counterparts, DSM-III-R oppositional defiant disorder was 25.5% less prevalent, DSM-III-R dysthymia was 37.8% less prevalent, and DSM-III-R conduct disorder (CD) was 44.3% less prevalent. However, DSM-III-R attention deficit hyperactivity disorder was 14.4% more prevalent than DSM-III attention deficit disorder with hyperactivity. The two definitions of CD were compared to exemplify an empirical approach to diagnostic validation. The DSM-III-R diagnosis of CD appears to be more valid as it is more strongly associated with police contacts, school suspensions, and history of antisocial personality disorder in the biological father, but both CD diagnoses are associated with family histories of criminal convictions. PMID:2387798

Lahey, B B; Loeber, R; Stouthamer-Loeber, M; Christ, M A; Green, S; Russo, M F; Frick, P J; Dulcan, M

1990-07-01

290

Development of ATP-competitive mTOR Inhibitors  

PubMed Central

The mTOR mediated signaling transduction pathway has been observed to be deregulated in a wide variety of cancer and metabolic diseases. Despite extensive clinical development efforts, the well-known allosteric mTOR inhibitor rapamycin and structurally related rapalogs have failed to show significant single-agent anti-tumor efficacy in most types of cancer. This limited clinical success maybe due to the inability of the rapalogs to maintain a complete blockade mTOR mediated signaling. Therefore numerous efforts have been initiated to develop ATP-competitive mTOR inhibitors that would block both mTORC1 and mTORC2 complex activity. Here we describe our experimental approaches to develop Torin1 using a medium throughput cell-based screening assay and structure-guided drug design. PMID:22125084

Liu, Qingsong; Kang, Seong A.; Thoreen, Carson C.; Hur, Wooyoung; Wang, Jinhua; Chang, Jae Won; Markhard, Andrew; Zhang, Jianming; Sim, Taebo; Sabatini, David M.; Gray, Nathanael S.

2014-01-01

291

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

292

Kinetics of ATP hydrolysis by the F 1ATPase from Bacillus PS3: a reappraisal of the effects of ATP and Mg 2+  

Microsoft Academic Search

ATPase activity of the F1-ATPase from the thermophilic Bacillus PS3 (TF1) was measured as a function of ATP concentration at three different magnesium ion concentrations. A high-performance chromatographic method was used to determine directly ADP concentration in the reaction medium and to measure the steady-state rate of its appearance. Multiphasic curves of ATPase activity versus ATP concentration were obtained, with

Stéphane Pezennec; Gérard Berger; Sandra Andrianambinintsoa; Nicolas Radziszewski; Guy Girault; Jean Michel Galmiche; Edmund Bäuerlein

1995-01-01

293

ATP Excites Interneurons and Astrocytes to Increase Synaptic Inhibition in Neuronal Networks  

Microsoft Academic Search

We investigated the role of extracellular ATP at astrocytes and inhibitory GABAergic interneurons in the stratum radiatum area of the mouse hippocampus. We show that exogenously applied ATP increased astrocyte intracellular Ca 2 levels and depolarized all calbindin- and calretinin-positive interneurons in the stratum radiatum region of mouse hippocampus, leading to action potential firing and enhanced synaptic inhibition onto the

David N. Bowser; Baljit S. Khakh

2004-01-01

294

ORIGINAL INVESTIGATION Further characterization of ATP6V0A2-related autosomal  

E-print Network

ORIGINAL INVESTIGATION Further characterization of ATP6V0A2-related autosomal recessive cutis laxa Autosomal recessive cutis laxa (ARCL) syn- dromes are phenotypically overlapping, but genetically heterogeneous disorders. Mutations in the ATP6V0A2 gene were found to underlie both, autosomal recessive cutis

295

Characterisation of an ATP diphosphohydrolase (Apyrase, EC 3.6.1.5) activity in Trichomonas vaginalis  

E-print Network

kinase, did not inhibit the enzyme activity. The enzyme has apparent Km (Michaelis Constant) values of 49 to the activity of a group of ecto- enzymes, the ectonucleotidases, which includes ecto-ATP diphosphohydrolaseCharacterisation of an ATP diphosphohydrolase (Apyrase, EC 3.6.1.5) activity in Trichomonas

Eizirik, Eduardo

296

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

297

A ratiometric fluorescent probe with unexpected high selectivity for ATP and its application in cell imaging.  

PubMed

Naphthalimide-rhodamine compound (NR) is developed as a ratiometric fluorescent probe for ATP detection based on the FRET mechanism. It shows an unexpected high selectivity for ATP over other anions, especially organic phosphate anions, due to simultaneous interactions of two recognition sites, which benefits fluorescence imaging in living cells. PMID:25350832

Tang, Jia-Liang; Li, Chun-Yan; Li, Yong-Fei; Zou, Chun-Xiang

2014-12-18

298

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

NSDL National Science Digital Library

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

C. Dahout-Gonzalez (UMR 5092 CEA-CNRS-Université Joseph Fourier Département de Réponse et Dynamique Cellulaires); H. Nury (UMR 5075 CEA-CNRS-Université Joseph Fourier); V. Trézéguet (UMR 5095 CNRS-Université Bordeaux 2 Institut de Biochimie et Génétique Cellulaires); G. J.-M. Lauquin (UMR 5095 CNRS-Université Bordeaux 2 Institut de Biochimie et Génétique Cellulaires); E. Pebay-Peyroula (UMR 5075 CEA-CNRS-Université Joseph Fourier); G. Brandolin (UMR 5092 CEA-CNRS-Université Joseph Fourier Département de Réponse et Dynamique Cellulaires)

2006-08-01

299

Transient increase of ATP as a response to temperature up-shift in Escherichia coli  

Microsoft Academic Search

SUMMARY: BACKGROUND: Escherichia coli induces the heat shock response to a temperature up-shift which is connected to the synthesis of a characteristic set of proteins, including ATP dependent chaperones and proteases. Therefore the balance of the nucleotide pool is important for the adaptation and continuous function of the cell. Whereas it has been observed in eukaryotic cells, that the ATP

Jaakko Soini; Christina Falschlehner; Christina Mayer; Daniela Böhm; Stefan Weinel; Johanna Panula; Antti Vasala; Peter Neubauer

2005-01-01

300

Structure of the Bis(Mg2+ )-ATP-Oxalate Complex of the Rabbit Muscle Pyruvate  

E-print Network

Structure of the Bis(Mg2+ )-ATP-Oxalate Complex of the Rabbit Muscle Pyruvate Kinase at 2.1 Ã? as a complex with MgIIATP, oxalate, Mg2+, and either K+ or Na+. Crystals with either Na+ or K+ belong of enzymic complexes of ATP and pyruvate, which require extended incubation periods. In contrast, oxalate

Rayment, Ivan

301

An ATP-driven proton pump in brush-border membranes from rat renal cortex  

Microsoft Academic Search

Summary The rate of ATP hydrolysis in ATP-preloaded plasma membrane vesicles derived from the luminal membrane of renal cortical tubules, and the rate of H+ secretion out of the same vesicles were investigated. Both were inhibited at low temperature, by the action of filipin, an antibiotic that complexes with cholesterol in plasma membranes, and by the action of blockers of

E. Kinne-Saffran; R. Beauwens; R. Kinne

1982-01-01

302

Keratinocyte ATP Release Assay for Testing Skin-Irritating Potentials of Structurally Diverse Chemicals  

Microsoft Academic Search

Irritant dermatitis represents innate inflammatory responses to toxic chemicals. We have reported recently that ATP released from chemically injured keratinocytes may serve as a causative mediator for irritant dermatitis. In this study, we examined whether ATP release from keratinocytes would serve as a reliable readout for predicting skin irritating potentials of structurally diverse compounds. A vast majority (19\\/20) of the

Norikatsu Mizumoto; Mark E. Mummert; David Shalhevet; Akira Takashima

2003-01-01

303

Novel 30 kDa protein possessing ATP-binding and chaperone activities.  

PubMed Central

A 30 kDa protein was purified from pig liver cytosol by using ATP-Sepharose and Green A column chromatography. The partial amino acid sequences of the protein (95 amino acid residues) had no similarity with any proteins recorded in data banks. The protein was able to form a stable complex with unfolded dihydrofolate reductase (DHFR). The spontaneous refolding of chemically denatured DHFR was arrested by the 30 kDa protein. This inhibition presumably results from the formation of a stable complex between the 30 kDa protein and DHFR. Bound DHFR could be released from the protein with ATP. The protein also showed protease resistance in an ATP-dependent manner. Incubation of the 30 kDa protein with 5 mM ATP resulted in its resistance to V8 protease or to trypsin treated with 1-chloro-4-phenyl-3-L-toluene-p-sulphonamidobutan-2-one. Divalent cations enhanced the ATP-protection effect. CD analysis of the 30 kDa protein showed that ATP induced an increase in the beta-pleated sheet content and a decrease in the alpha-helix content of the 30 kDa protein. These results suggest that the 30 kDa protein, a novel cytosolic protein, might have an affinity for ATP, a chaperonin activity, and and an ATP-protection effect against some proteases in vivo. PMID:9291133

Itoh, H; Tashima, Y

1997-01-01

304

ATP Synthase Is Responsible for Maintaining Mitochondrial Membrane Potential in Bloodstream Form Trypanosoma brucei  

Microsoft Academic Search

Received 25 July 2005\\/Accepted 4 November 2005 The mitochondrion of Trypanosoma brucei bloodstream form maintains a membrane potential, although it lacks cytochromes and several Krebs cycle enzymes. At this stage, the ATP synthase is present at reduced, although significant, levels. To test whether the ATP synthase at this stage is important for maintaining the mitochondrial membrane potential, we used RNA

Silvia V. Brown; Paul Hosking; Jinlei Li; Noreen Williams

2006-01-01

305

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

306

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

PubMed Central

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

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

2004-01-01

307

ATP-dependent regulation of nuclear Ca2 levels in plant cells  

E-print Network

ATP-dependent regulation of nuclear Ca2 levels in plant cells Tom D. Bunney, Peter J. Shaw, Peter A stimuli. Due to the large size of nuclear pores, it has generally been assumed that intranuclear Ca2 in [Ca2+ ] occurs in the nuclear periphery. The occurrence of ATP-dependent Ca2+ uptake in plant nuclei

Shaw, Peter

308

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

309

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

310

Extracellular Ca2+ regulates the stimulus-elicited ATP release from urothelium.  

PubMed

Accumulating evidence shows that the epithelial cells in urinary bladder (urothelium) serve as a sensory organ in micturition and/or in nociception pathway by releasing ATP in response to mechanical and/or chemical stimuli. Here, we compared the effects of capsaicin, acetylcholine, and prostaglandin E(2) receptor EP1 agonist (ONO-DI-004) on the urothelial ATP release in primary cultured mouse urothelial cells in low Ca(2+) medium. All of these chemicals induced a gradual ATP release from urothelium, implying that the downstream Ca(2+) release from endoplasmic reticulum could trigger the ATP release. Consistent with this suggestion, blockade of inositol 1,4,5-triphosphate receptor reduced the distention-induced ATP release from urothelial tissues. The distention-induced ATP release was not affected by tetrodotoxin. However, an increase in extracellular Ca(2+) diminished both chemical- and distention-induced ATP release from urothelium. Thus raising the extracellular Ca(2+) concentration was found to inhibit stimulation-evoked ATP urothelial release. PMID:19525154

Matsumoto-Miyai, Kazumasa; Kagase, Ai; Murakawa, Yuki; Momota, Yoshiharu; Kawatani, Masahito

2009-10-01

311

Modulation of Hippocampal Glutamatergic Transmission by ATP Is Dependent on Adenosine A1 Receptors  

Microsoft Academic Search

Excitatory glutamatergic synapses in the hippocampal CA1 region of rats are potently inhibited by purines, including aden- osine, ATP, and ATP analogs. Adenosine A1 receptors are known to mediate at least part of the response to adenine nucleotides, either because adenine nucleotides activate A1 receptors directly, or activate them secondarily upon the nu- cleotides' conversion to adenosine. In the present

SUSAN A. MASINO; LIHONG DIAO; PETER ILLES; NANCY R. ZAHNISER; GAYNOR A. LARSON; ORN JOHANSSON; BERTIL B. FREDHOLM; THOMAS V. DUNWIDDIE

2002-01-01

312

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

E-print Network

for the fact that Mg2+ binds to ATP more readily than to adenosine 5-diphosphate (¨rperforschung, Forschungszentrum Ju¨lich, D-52425 Ju¨lich, Germany ReceiVed: June 2, 2003; In Final Form: August 11, 2003 Adenosine and their derivatives are present in mammalian cells, the most abundant being adenosine 5-triphosphate ATP (Scheme 1

313

Extracellular ATP Functions as an Endogenous External Metabolite Regulating Plant Cell Viability  

Microsoft Academic Search

ATP is a vital molecule used by living organisms as a universal source of energy required to drive the cogwheels of intra- cellular biochemical reactions necessary for growth and development. Animal cells release ATP to the extracellular milieu, where it functions as the primary signaling cue at the epicenter of a diverse range of physiological processes. Although recent findings revealed

Stephen Chivas; Bongani K. Ndimba; William J. Simon; Keith Lindsey; Antoni R. Slabas

2005-01-01

314

One- and two-dimensional 31P spin-echo studies of myocardial ATP and phosphocreatine  

NASA Astrophysics Data System (ADS)

31P spin-spin coupling constants and natural linewidths were studied in isolated perfused rat hearts. The values of the coupling constants are equal to those of Mg 2+-ATP in solution. The linewidth measurements suggest that the myocardial ATP is exchanging between a free and a bound site in the cytosol.

Turner, Christopher J.; Garlick, Pamela B.

315

Visualized discrimination of ATP from ADP and AMP through collapse of supramolecular gels.  

PubMed

A supramolecular gel was fabricated through mixing of a cationic gelator with methyl orange. The addition of ATP into the gel caused a distinct gel-collapse, whereas ADP and AMP preserved the gel formation. This observation provided a simple visualized way to discriminate ATP from AMP and ADP. PMID:25205284

Yang, Dong; Liu, Changxia; Zhang, Li; Liu, Minghua

2014-10-28

316

Structural, biochemical and genetic characterization of dissimilatory ATP sulfurylase from Allochromatium vinosum.  

PubMed

ATP sulfurylase (ATPS) catalyzes a key reaction in the global sulfur cycle by reversibly converting inorganic sulfate (SO4 (2-)) with ATP to adenosine 5'-phosphosulfate (APS) and pyrophosphate (PPi). In this work we report on the sat encoded dissimilatory ATP sulfurylase from the sulfur-oxidizing purple sulfur bacterium Allochromatium vinosum. In this organism, the sat gene is located in one operon and co-transcribed with the aprMBA genes for membrane-bound APS reductase. Like APS reductase, Sat is dispensible for growth on reduced sulfur compounds due to the presence of an alternate, so far unidentified sulfite-oxidizing pathway in A. vinosum. Sulfate assimilation also proceeds independently of Sat by a separate pathway involving a cysDN-encoded assimilatory ATP sulfurylase. We produced the purple bacterial sat-encoded ATP sulfurylase as a recombinant protein in E. coli, determined crucial kinetic parameters and obtained a crystal structure in an open state with a ligand-free active site. By comparison with several known structures of the ATPS-APS complex in the closed state a scenario about substrate-induced conformational changes was worked out. Despite different kinetic properties ATPS involved in sulfur-oxidizing and sulfate-reducing processes are not distinguishable on a structural level presumably due to the interference between functional and evolutionary processes. PMID:24073218

Parey, Kristian; Demmer, Ulrike; Warkentin, Eberhard; Wynen, Astrid; Ermler, Ulrich; Dahl, Christiane

2013-01-01

317

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

318

Hydrolysis and synthesis of ATP by membrane-bound ATPase from a motile Streptococcus.  

PubMed

ATPase was detected in the membranes of a motile Streptococcus. Maximal enzymic activity was observed at pH 8 and ATP/Mg2+ ratio of 2. Mn2+ and Ca2+ could replace Mg2+ to some extent. Besides ATP, GTP and ITP were substrates. The enzyme was inhibited by N,N'-dicyclohexylcarbodiimide but not by sodium azide, uncouplers or bathophenanthroline. An electrochemical gradient of protons, which was artificially imposed across the membranes of Streptococcus cells by manipulation of either the K+ diffusion potential or the transmembrane pH gradient, led to ATP synthesis. ATP synthesis was abolished by proton conductors, an inhibitor of the ATPase or an increase in the extracellular K+ concentration. A comparison between the phosphate potential and the electrochemical proton gradient showed that the data found are in agreement with a stoichiometry of 2 protons translocated per molecule ATP synthesized. PMID:31147

van der Drift, C; Janssen, D B; van Wezenbeek, P M

1978-10-01

319

DNA uptake in competent Streptococcus pneumoniae requires ATP and is regulated by cytoplasmic pH.  

PubMed

DNA uptake in competent Streptococcus pneumoniae was strongly dependent on intracellular pH. Ionophore treatments that either acidified or alkalinized the cytoplasm reduced DNA transport. This indicates that the optimum pH for DNA uptake corresponds to the intracellular pH of competent bacteria which is 8.3 +/- 0.2. In addition, the ATP pool of the bacteria appeared to be a critical parameter in the process. The pattern of inhibition by arsenate, when the culture was treated at different steps of the competence cycle, suggested firstly, that a threshold ATP level was required to trigger transport and secondly, an ATP requirement for the process itself. This may indicate an ATP involvement in the activation of an uptake machinery functioning at the expense of ATP. PMID:2612880

Clavé, C; Trombe, M C

1989-11-01

320

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

321

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

PubMed

Erythrocytes have been implicated as controllers of vascular caliber by virtue of their ability to release the vasodilator ATP in response to local physiological and pharmacological stimuli. The regulated release of ATP from erythrocytes requires activation of a signaling pathway involving G proteins (G(i) or G(s)), adenylyl cyclase, protein kinase A, and the cystic fibrosis transmembrane conductance regulator as well as a final conduit through which this highly charged anion exits the cell. Although pannexin 1 has been shown to be the final conduit for ATP release from human erythrocytes in response to reduced oxygen tension, it does not participate in transport of ATP following stimulation of the prostacyclin (IP) receptor in these cells, which suggests that an additional protein must be involved. Using antibodies directed against voltage-dependent anion channel (VDAC)1, we confirm that this protein is present in human erythrocyte membranes. To address the role of VDAC in ATP release, two structurally dissimilar VDAC inhibitors, Bcl-x(L) BH4(4-23) and TRO19622, were used. In response to the IP receptor agonists, iloprost and UT-15C, ATP release was inhibited by both VDAC inhibitors although neither iloprost-induced cAMP accumulation nor total intracellular ATP concentration were altered. Together, these findings support the hypothesis that VDAC is the ATP conduit in the IP receptor-mediated signaling pathway in human erythrocytes. In addition, neither the pannexin inhibitor carbenoxolone nor Bcl-x(L) BH4(4-23) attenuated ATP release in response to incubation of erythrocytes with the ?-adrenergic receptor agonist isoproterenol, suggesting the presence of yet another channel for ATP release from human erythrocytes. PMID:22159995

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

2012-02-01

322

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

323

Definitions of \\  

Microsoft Academic Search

Dissimilarity-based compound selection involves identifying a database subset in which the constituent compounds are as dissimilar to each other as possible, thus ensuring coverage of the full range of structural diversity in the original database. This paper provides a quantitative comparison of four different definitions of dissimilarity. Experiments with three different measures of diversity demonstrate that the effectiveness of the

John D. Holliday; Peter Willett

1996-01-01

324

Caesar Cipher III  

NSDL National Science Digital Library

Decode encrypted messages to determine the form for an affine cipher, and practice your reasoning and arithmetic skills. Input your guesses for the multiplier and constant. Caesar Cipher III is one of the Interactivate assessment explorers.

325

ATP binding cassette transporter G1 (ABCG1) is an intracellular sterol transporter  

PubMed Central

Four members of the mammalian ATP binding cassette (ABC) transporter G subfamily are thought to be involved in transmembrane (TM) transport of sterols. The residues responsible for this transport are unknown. The mechanism of action of ABCG1 is controversial and it has been proposed to act at the plasma membrane to facilitate the efflux of cellular sterols to exogenous high-density lipoprotein (HDL). Here we show that ABCG1 function is dependent on localization to intracellular endosomes. Importantly, localization to the endosome pathway distinguishes ABCG1 and/or ABCG4 from all other mammalian members of this superfamily, including other sterol transporters. We have identified critical residues within the TM domains of ABCG1 that are both essential for sterol transport and conserved in some other members of the ABCG subfamily and/or the insulin-induced gene 2 (INSIG-2). Our conclusions are based on studies in which (i) biotinylation of peritoneal macrophages showed that endogenous ABCG1 is intracellular and undetectable at the cell surface, (ii) a chimeric protein containing the TM of ABCG1 and the cytoplasmic domains of the nonsterol transporter ABCG2 is both targeted to endosomes and functional, and (iii) ABCG1 colocalizes with multiple proteins that mark late endosomes and recycling endosomes. Mutagenesis studies identify critical residues in the TM domains that are important for ABCG1 to alter sterol efflux, induce sterol regulatory element binding protein-2 (SREBP-2) processing, and selectively attenuate the oxysterol-mediated repression of SREBP-2 processing. Our data demonstrate that ABCG1 is an intracellular sterol transporter that localizes to endocytic vesicles to facilitate the redistribution of specific intracellular sterols away from the endoplasmic reticulum (ER). PMID:22095132

Tarling, Elizabeth J.; Edwards, Peter A.

2011-01-01

326

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

PubMed

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

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

2006-01-01

327

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

328

A membrane-bound archaeal Lon protease displays ATP-independent proteolytic activity towards unfolded proteins and ATP-dependent activity for folded proteins.  

PubMed

In contrast to the eucaryal 26S proteasome and the bacterial ATP-dependent proteases, little is known about the energy-dependent proteolysis in members of the third domain, Archae. We cloned a gene homologous to ATP-dependent Lon protease from a hyperthermophilic archaeon and observed the unique properties of the archaeal Lon. Lon from Thermococcus kodakaraensis KOD1 (Lon(Tk)) is a 70-kDa protein with an N-terminal ATPase domain belonging to the AAA(+) superfamily and a C-terminal protease domain including a putative catalytic triad. Interestingly, a secondary structure prediction suggested the presence of two transmembrane helices within the ATPase domain and Western blot analysis using specific antiserum against the recombinant protein clearly indicated that Lon(Tk) was actually a membrane-bound protein. The recombinant Lon(Tk) possessed thermostable ATPase activity and peptide cleavage activity toward fluorogenic peptides with optimum temperatures of 95 and 70 degrees C, respectively. Unlike the enzyme from Escherichia coli, we found that Lon(Tk) showed higher peptide cleavage activity in the absence of ATP than it did in the presence of ATP. When three kinds of proteins with different thermostabilities were examined as substrates, it was found that Lon(Tk) required ATP for degradation of folded proteins, probably due to a chaperone-like function of the ATPase domain, along with ATP hydrolysis. In contrast, Lon(Tk) degraded unfolded proteins in an ATP-independent manner, suggesting a mode of action in Lon(Tk) different from that of its bacterial counterpart. PMID:12057965

Fukui, Toshiaki; Eguchi, Tomohiro; Atomi, Haruyuki; Imanaka, Tadayuki

2002-07-01

329

ATP-competitive inhibitors of mTOR: an update.  

PubMed

mTOR (mammalian target of rapamycin) is a serine-threonine kinase belonging to the PI3K/Akt/mTOR signalling pathway that is involved in several cell functions, including growth, proliferation, apoptosis and autophagy. mTOR hyperactivation has been detected in several human cancers, thus representing, together with its upstream effectors, an important target for cancer therapy. mTOR exists in two different complexes in cells, mTORC1 and mTORC2 which could both be targeted by potential anticancer agents. Rapamycin, the selective and allosteric inhibitor of mTOR, inhibits the enzyme in mTORC1, but not in mTORC2. In the last few years a number of mTOR ATP-competitive inhibitors has been reported acting on mTOR in both complexes and possessing a more complete anticancer activity in comparison with that of rapamycin and its derivatives. mTOR shares high sequence homology in the hinge-region with PI3K that is a lipid kinase upstream to mTOR in the same signaling pathway; for this reason some compounds originally developed as PI3K inhibitors later showed to also target mTOR. As indicated by preclinical and clinical studies, compounds acting on more than one target could result in a better biological response and in enhanced therapeutic potential and also dual PI3K/mTOR inhibitors result of great interest as potential antitumor agents. This review mainly reports the recently discovered mTOR ATP-competitive inhibitors in terms of medicinal chemistry, classified by their chemical structures, focusing on SAR and modelling studies that led to the discovery of very potent and selective agents, such as AZD-8055, OSI-027 and INK128, already entered clinical trials, or WYE-132, Torin1 and others in preclinical studies. Also some examples of dual PI3K/mTOR inhibitors, including PI-103, GNE477, WJD008 and GSK2126458 are reported together with their biological and clinical data. PMID:21651476

Schenone, S; Brullo, C; Musumeci, F; Radi, M; Botta, M

2011-01-01

330

Type III restriction-modification enzymes: a historical perspective.  

PubMed

Restriction endonucleases interact with DNA at specific sites leading to cleavage of DNA. Bacterial DNA is protected from restriction endonuclease cleavage by modifying the DNA using a DNA methyltransferase. Based on their molecular structure, sequence recognition, cleavage position and cofactor requirements, restriction-modification (R-M) systems are classified into four groups. Type III R-M enzymes need to interact with two separate unmethylated DNA sequences in inversely repeated head-to-head orientations for efficient cleavage to occur at a defined location (25-27 bp downstream of one of the recognition sites). Like the Type I R-M enzymes, Type III R-M enzymes possess a sequence-specific ATPase activity for DNA cleavage. ATP hydrolysis is required for the long-distance communication between the sites before cleavage. Different models, based on 1D diffusion and/or 3D-DNA looping, exist to explain how the long-distance interaction between the two recognition sites takes place. Type III R-M systems are found in most sequenced bacteria. Genome sequencing of many pathogenic bacteria also shows the presence of a number of phase-variable Type III R-M systems, which play a role in virulence. A growing number of these enzymes are being subjected to biochemical and genetic studies, which, when combined with ongoing structural analyses, promise to provide details for mechanisms of DNA recognition and catalysis. PMID:23863841

Rao, Desirazu N; Dryden, David T F; Bheemanaik, Shivakumara

2014-01-01

331

Type III restriction-modification enzymes: a historical perspective  

PubMed Central

Restriction endonucleases interact with DNA at specific sites leading to cleavage of DNA. Bacterial DNA is protected from restriction endonuclease cleavage by modifying the DNA using a DNA methyltransferase. Based on their molecular structure, sequence recognition, cleavage position and cofactor requirements, restriction–modification (R–M) systems are classified into four groups. Type III R–M enzymes need to interact with two separate unmethylated DNA sequences in inversely repeated head-to-head orientations for efficient cleavage to occur at a defined location (25–27 bp downstream of one of the recognition sites). Like the Type I R–M enzymes, Type III R–M enzymes possess a sequence-specific ATPase activity for DNA cleavage. ATP hydrolysis is required for the long-distance communication between the sites before cleavage. Different models, based on 1D diffusion and/or 3D-DNA looping, exist to explain how the long-distance interaction between the two recognition sites takes place. Type III R–M systems are found in most sequenced bacteria. Genome sequencing of many pathogenic bacteria also shows the presence of a number of phase-variable Type III R–M systems, which play a role in virulence. A growing number of these enzymes are being subjected to biochemical and genetic studies, which, when combined with ongoing structural analyses, promise to provide details for mechanisms of DNA recognition and catalysis. PMID:23863841

Rao, Desirazu N.; Dryden, David T. F.; Bheemanaik, Shivakumara

2014-01-01

332

Ecto-nucleoside triphosphate diphosphohydrolase 2 modulates local ATP-induced calcium signaling in human HaCaT keratinocytes.  

PubMed

Keratinocytes are the major building blocks of the human epidermis. In many physiological and pathophysiological conditions, keratinocytes release adenosine triphosphate (ATP) as an autocrine/paracrine mediator that regulates cell proliferation, differentiation, and migration. ATP receptors have been identified in various epidermal cell types; therefore, extracellular ATP homeostasis likely determines its long-term, trophic effects on skin health. We investigated the possibility that human keratinocytes express surface-located enzymes that modulate ATP concentration, as well as the corresponding receptor activation, in the pericellular microenvironment. We observed that the human keratinocyte cell line HaCaT released ATP and hydrolyzed extracellular ATP. Interestingly, ATP hydrolysis resulted in adenosine diphosphate (ADP) accumulation in the extracellular space. Pharmacological inhibition by ARL 67156 or gene silencing of the endogenous ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) isoform 2 resulted in a 25% reduction in both ATP hydrolysis and ADP formation. Using intracellular calcium as a reporter, we found that although NTPDase2 hydrolyzed ATP and generated sustainable ADP levels, only ATP contributed to increased intracellular calcium via P2Y2 receptor activation. Furthermore, knocking down NTPDase2 potentiated the nanomolar ATP-induced intracellular calcium increase, suggesting that NTPDase2 globally attenuates nucleotide concentration in the pericellular microenvironment as well as locally shields receptors in the vicinity from being activated by extracellular ATP. Our findings reveal an important role of human keratinocyte NTPDase2 in modulating nucleotide signaling in the extracellular milieu of human epidermis. PMID:23536768

Ho, Chia-Lin; Yang, Chih-Yung; Lin, Wen-Jie; Lin, Chi-Hung

2013-01-01

333

Ecto-Nucleoside Triphosphate Diphosphohydrolase 2 Modulates Local ATP-Induced Calcium Signaling in Human HaCaT Keratinocytes  

PubMed Central

Keratinocytes are the major building blocks of the human epidermis. In many physiological and pathophysiological conditions, keratinocytes release adenosine triphosphate (ATP) as an autocrine/paracrine mediator that regulates cell proliferation, differentiation, and migration. ATP receptors have been identified in various epidermal cell types; therefore, extracellular ATP homeostasis likely determines its long-term, trophic effects on skin health. We investigated the possibility that human keratinocytes express surface-located enzymes that modulate ATP concentration, as well as the corresponding receptor activation, in the pericellular microenvironment. We observed that the human keratinocyte cell line HaCaT released ATP and hydrolyzed extracellular ATP. Interestingly, ATP hydrolysis resulted in adenosine diphosphate (ADP) accumulation in the extracellular space. Pharmacological inhibition by ARL 67156 or gene silencing of the endogenous ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) isoform 2 resulted in a 25% reduction in both ATP hydrolysis and ADP formation. Using intracellular calcium as a reporter, we found that although NTPDase2 hydrolyzed ATP and generated sustainable ADP levels, only ATP contributed to increased intracellular calcium via P2Y2 receptor activation. Furthermore, knocking down NTPDase2 potentiated the nanomolar ATP-induced intracellular calcium increase, suggesting that NTPDase2 globally attenuates nucleotide concentration in the pericellular microenvironment as well as locally shields receptors in the vicinity from being activated by extracellular ATP. Our findings reveal an important role of human keratinocyte NTPDase2 in modulating nucleotide signaling in the extracellular milieu of human epidermis. PMID:23536768

Ho, Chia-Lin; Yang, Chih-Yung; Lin, Wen-Jie; Lin, Chi-Hung

2013-01-01

334

Rate Constants for the Reactions of ATP-and ADP-Actin with the Ends of Actin Filaments  

E-print Network

and dissociation of ATP-actin are constant. This shows that the nucleotide composition at or near the end nine different rate constants: the association and dissociation rate constants for ATP-actin and ADPRate Constants for the Reactions of ATP- and ADP-Actin with the Ends of Actin Filaments Thomas D

335

Time-resolved Measurements of Intracellular ATP in the Yeast Saccharomyces cerevisiae using a New Type of Nanobiosensor*  

PubMed Central

Adenosine 5?-triphosphate is a universal molecule in all living cells, where it functions in bioenergetics and cell signaling. To understand how the concentration of ATP is regulated by cell metabolism and in turn how it regulates the activities of enzymes in the cell it would be beneficial if we could measure ATP concentration in the intact cell in real time. Using a novel aptamer-based ATP nanosensor, which can readily monitor intracellular ATP in eukaryotic cells with a time resolution of seconds, we have performed the first on-line measurements of the intracellular concentration of ATP in the yeast Saccharomyces cerevisiae. These ATP measurements show that the ATP concentration in the yeast cell is not stationary. In addition to an oscillating ATP concentration, we also observe that the concentration is high in the starved cells and starts to decrease when glycolysis is induced. The decrease in ATP concentration is shown to be caused by the activity of membrane-bound ATPases such as the mitochondrial F0F1 ATPase-hydrolyzing ATP and the plasma membrane ATPase (PMA1). The activity of these two ATPases are under strict control by the glucose concentration in the cell. Finally, the measurements of intracellular ATP suggest that 2-deoxyglucose (2-DG) may have more complex function than just a catabolic block. Surprisingly, addition of 2-DG induces only a moderate decline in ATP. Furthermore, our results suggest that 2-DG may inhibit the activation of PMA1 after addition of glucose. PMID:20880841

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

2010-01-01

336

Salvicine functions as novel topoisomerase II poison by binding to ATP pocket.  

PubMed

Salvicine, a structurally modified diterpenoid quinone derived from Salvia prionitis, is a nonintercalative topoisomerase II (topo II) poison. The compound possesses potent in vitro and in vivo antitumor activity with a broad spectrum of anti-multidrug resistance activity and is currently in phase II clinical trials. To elucidate the distinct antitumor properties of salvicine and obtain valuable structural information of salvicine-topo II interactions, we characterized the effects of salvicine on human topo IIalpha (htopo IIalpha), including possible binding sites and molecular interactions. The enzymatic assays disclosed that salvicine mainly inhibits the catalytic activity with weak DNA cleavage action, in contrast to the classic topo II poison etoposide (VP16). Molecular modeling studies predicted that salvicine binds to the ATP pocket in the ATPase domain and superimposes on the phosphate and ribose groups. In a surface plasmon resonance binding assay, salvicine exhibited higher affinity for the ATPase domain of htopo IIalpha than ATP and ADP. Competitive inhibition tests demonstrated that ATP competitively and dose-dependently blocked the interactions between salvicine and ATPase domain of htopo IIalpha. The data illustrate that salvicine shares a common binding site with ATP and functions as an ATP competitor. To our knowledge, this is the first report to identify an ATP-binding pocket as the structural binding motif for a nonintercalative eukaryotic topo II poison. These findings collectively support the potential value of an ATP competitor of htopo IIalpha in tumor chemotherapy. PMID:16914642

Hu, Chao-Xin; Zuo, Zhi-Li; Xiong, Bing; Ma, Jin-Gui; Geng, Mei-Yu; Lin, Li-Ping; Jiang, Hua-Liang; Ding, Jian

2006-11-01

337

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

338

Wilson disease protein ATP7B utilizes lysosomal exocytosis to maintain copper homeostasis.  

PubMed

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

2014-06-23

339

ATP monitoring technology for microbial growth control in potable water systems  

NASA Astrophysics Data System (ADS)

ATP (Adenosine Triphosphate) is the primary energy transfer molecule present in all living biological cells on Earth. ATP cannot be produced or maintained by anything but a living organism, and as such, its measurement is a direct indication of biological activity. The main advantage of ATP as a biological indicator is the speed of the analysis - from collecting the sample to obtaining the result, only minutes are required. The technology to measure ATP is already widely utilized to verify disinfection efficacy in the food industry and is also commonly applied in industrial water processes such as cooling water systems to monitor microbial growth and biocide applications. Research has indicated that ATP measurement technology can also play a key role in such important industries as potable water distribution and biological wastewater treatment. As will be detailed in this paper, LuminUltra Technologies has developed and applied ATP measurement technologies designed for any water type, and as such can provide a method to rapidly and accurately determine the level of biological activity in drinking water supplies. Because of its speed and specificity to biological activity, ATP measurement can play a key role in defending against failing drinking water quality, including those encountered during routine operation and also bioterrorism.

Whalen, Patrick A.; Whalen, Philip J.; Cairns, James E.

2006-05-01

340

Exercise sensitizes skeletal muscle to extracellular ATP for IL-6 expression in mice.  

PubMed

Active skeletal muscle synthesizes and releases interleukin-6 (IL-6), which plays important roles in the organism's adaptation to exercise. Autocrine/paracrine ATP signaling has been shown to modulate IL-6 expression. The aim of this study was to determine whether a period of physical activity modifies the ATP-induced IL-6 expression. BalbC mice were either subject to 5 weeks voluntary wheel running (VA) or kept sedentary (SED). Flexor digitorum brevis muscles were dissected, stimulated with different ATP concentrations (0-100 ?M) and IL-6 mRNA levels were measured using qPCR. ATP evoked a concentration-dependent rise in IL-6 mRNA in both SED and VA mice. VA mice however, had significantly higher ATP sensitivity (pD2 pharmacological values: VA=5.58±0.02 vs. SED=4.95±0.04, p<0.05). Interestingly, in VA mice we observed a positive correlation between the level of physical activity and the IL-6 mRNA increase following fiber stimulation with 10??M ATP. In addition, there were lower P2Y2- and higher P2Y14-receptor mRNA levels in skeletal muscles of VA compared to SED mice, showing plasticity of nucleotide receptors with exercise. These results suggest that exercise increases skeletal muscle ATP sensitivity, a response dependent on the level of physical activity performed. This could have an important role in the mechanisms controlling skeletal muscle adaptation to exercise and training. PMID:24022572

Fernández-Verdejo, R; Casas, M; Galgani, J E; Jaimovich, E; Buvinic, S

2014-04-01

341

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; Dare, Elisabetta; Berggren, Per-Olof; Kohler, Martin; Aspinwall, Craig A.; Lui, Weng-Onn; Zedenius, Jan; Larsson, Catharina; Branstrom, Robert

2013-01-01

342

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

343

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

344

A kinetic assay of mitochondrial ADP-ATP exchange rate in permeabilized cells.  

PubMed

We previously described a method to measure ADP-ATP exchange rates in isolated mitochondria by recording the changes in free extramitochondrial [Mg(2+)] reported by an Mg(2+)-sensitive fluorescent indicator, exploiting the differential affinity of ADP and ATP to Mg(2+). In the current article, 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 the addition of BeF(3)(-) and sodium orthovanadate (Na(3)VO(4)) to medium containing digitonin-permeabilized cells inhibits all ADP-ATP-using reactions except the adenine nucleotide translocase (ANT)-mediated mitochondrial ADP-ATP 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 F(1)F(o)-ATPase due to its sensitivity to BeF(3)(-) and Na(3)VO(4). 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

2010-12-01

345

Rho GTPase signaling regulates tight junction assembly and protects tight junctions during ATP depletion.  

PubMed

Tight junctions control paracellular permeability and cell polarity. Rho GTPase regulates tight junction assembly, and ATP depletion of Madin-Darby canine kidney (MDCK) cells (an in vitro model of renal ischemia) disrupts tight junctions. The relationship between Rho GTPase signaling and ATP depletion was examined. Rho inhibition resulted in decreased localization of zonula occludens-1 (ZO-1) and occludin at cell junctions; conversely, constitutive Rho signaling caused an accumulation of ZO-1 and occludin at cell junctions. Inhibiting Rho before ATP depletion resulted in more extensive loss of junctional components between transfected cells than control junctions, whereas cells expressing activated Rho better maintained junctions during ATP depletion than control cells. ATP depletion and Rho signaling altered phosphorylation signaling mechanisms. ZO-1 and occludin exhibited rapid decreases in phosphoamino acid content following ATP depletion, which was restored on recovery. Expression of Rho mutant proteins in MDCK cells also altered levels of occludin serine/threonine phosphorylation, indicating that occludin is a target for Rho signaling. We conclude that Rho GTPase signaling induces posttranslational effects on tight junction components. Our data also demonstrate that activating Rho signaling protects tight junctions from damage during ATP depletion. PMID:9730964

Gopalakrishnan, S; Raman, N; Atkinson, S J; Marrs, J A

1998-09-01

346

Thiol modulation of the chloroplast ATP synthase is dependent on the energization of thylakoid membranes.  

PubMed

Thiol modulation of the chloroplast ATP synthase ? subunit has been recognized as an important regulatory system for the activation of ATP hydrolysis activity, although the physiological significance of this regulation system remains poorly characterized. Since the membrane potential required by this enzyme to initiate ATP synthesis for the reduced enzyme is lower than that needed for the oxidized form, reduction of this enzyme was interpreted as effective regulation for efficient photophosphorylation. However, no concrete evidence has been obtained to date relating to the timing and mode of chloroplast ATP synthase reduction and oxidation in green plants. In this study, thorough analysis of the redox state of regulatory cysteines of the chloroplast ATP synthase ? subunit in intact chloroplasts and leaves shows that thiol modulation of this enzyme is pivotal in prohibiting futile ATP hydrolysis activity in the dark. However, the physiological importance of efficient ATP synthesis driven by the reduced enzyme in the light could not be demonstrated. In addition, we investigated the significance of the electrochemical proton gradient in reducing the ? subunit by the reduced form of thioredoxin in chloroplasts, providing strong insights into the molecular mechanisms underlying the formation and reduction of the disulfide bond on the ? subunit in vivo. PMID:22362842

Konno, Hiroki; Nakane, Takeshi; Yoshida, Masasuke; Ueoka-Nakanishi, Hanayo; Hara, Satoshi; Hisabori, Toru

2012-04-01

347

Role of the epsilon subunit of thermophilic F1-ATPase as a sensor for ATP.  

PubMed

The epsilon subunit of F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)) has been shown to bind ATP. The precise nature of the regulatory role of ATP binding to the epsilon subunit remains to be determined. To address this question, 11 mutants of the epsilon subunit were prepared, in which one of the basic or acidic residues was substituted with alanine. ATP binding to these mutants was tested by gel-filtration chromatography. Among them, four mutants that showed no ATP binding were selected and reconstituted with the alpha(3)beta(3)gamma complex of TF(1). The ATPase activity of the resulting alpha(3)beta(3)gammaepsilon complexes was measured, and the extent of inhibition by the mutant epsilon subunits was compared in each case. With one exception, weaker binding of ATP correlated with greater inhibition of ATPase activity. These results clearly indicate that ATP binding to the epsilon subunit plays a regulatory role and that ATP binding may stabilize the ATPase-active form of TF(1) by fixing the epsilon subunit into the folded conformation. PMID:17933866

Kato, Shigeyuki; Yoshida, Masasuke; Kato-Yamada, Yasuyuki

2007-12-28

348

Identification of an Arabidopsis Plasma Membrane-Located ATP Transporter Important for Anther Development[W  

PubMed Central

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

2011-01-01

349

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

350

Impairment of vesicular ATP release affects glucose metabolism and increases insulin sensitivity  

PubMed Central

Neuroendocrine cells store ATP in secretory granules and release it along with hormones that may trigger a variety of cellular responses in a process called purinergic chemical transmission. Although the vesicular nucleotide transporter (VNUT) has been shown to be involved in vesicular storage and release of ATP, its physiological relevance in vivo is far less well understood. In Vnut knockout (Vnut?/?) mice, we found that the loss of functional VNUT in adrenal chromaffin granules and insulin granules in the islets of Langerhans led to several significant effects. Vesicular ATP accumulation and depolarization-dependent ATP release were absent in the chromaffin granules of Vnut?/? mice. Glucose-responsive ATP release was also absent in pancreatic ?-cells in Vnut?/? mice, while glucose-responsive insulin secretion was enhanced to a greater extent than that in wild-type tissue. Vnut?/? mice exhibited improved glucose tolerance and low blood glucose upon fasting due to increased insulin sensitivity. These results demonstrated an essential role of VNUT in vesicular storage and release of ATP in neuroendocrine cells in vivo and suggest that vesicular ATP and/or its degradation products act as feedback regulators in catecholamine and insulin secretion, thereby regulating blood glucose homeostasis. PMID:25331291

Sakamoto, Shohei; Miyaji, Takaaki; Hiasa, Miki; Ichikawa, Reiko; Uematsu, Akira; Iwatsuki, Ken; Shibata, Atsushi; Uneyama, Hisayuki; Takayanagi, Ryoichi; Yamamoto, Akitsugu; Omote, Hiroshi; Nomura, Masatoshi; Moriyama, Yoshinori

2014-01-01

351

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

PubMed

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

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

2014-02-01

352

ATP release and Ca2+ signalling by human bronchial epithelial cells following Alternaria aeroallergen exposure  

PubMed Central

Exposure of human bronchial epithelial (HBE) cells from normal and asthmatic subjects to extracts from Alternaria alternata evoked a rapid and sustained release of ATP with greater efficacy observed in epithelial cells from asthmatic patients. Previously, Alternaria allergens were shown to produce a sustained increase in intracellular Ca2+ concentration ([Ca2+]i) that was dependent on the coordinated activation of specific purinergic receptor (P2Y2 and P2X7) subtypes. In the present study, pretreatment with a cell-permeable Ca2+-chelating compound (BAPTA-AM) significantly inhibited ATP release, indicating dependency on [Ca2+]i. Alternaria-evoked ATP release exhibited a greater peak response and a slightly lower EC50 value in cells obtained from asthmatic donors compared to normal control cells. Furthermore, the maximum increase in [Ca2+]i resulting from Alternaria treatment was greater in cells from asthmatic patients compared to normal subjects. The vesicle transport inhibitor brefeldin A and BAPTA-AM significantly blocked Alternaria-stimulated incorporation of fluorescent lipid (FM1-43)-labelled vesicles into the plasma membrane and ATP release. In addition, inhibiting uptake of ATP into exocytotic vesicles with bafilomycin also reduced ATP release comparable to the effects of brefeldin A and BAPTA-AM. These results indicate that an important mechanism for Alternaria-induced ATP release is Ca2+ dependent and involves exocytosis of ATP. Serine and cysteine protease inhibitors also reduced Alternaria-induced ATP release; however, the sustained increase in [Ca2+]i typically observed following Alternaria exposure appeared to be independent of protease-activated receptor (PAR2) stimulation. PMID:23858006

O'Grady, Scott M; Patil, Nandadavi; Melkamu, Tamene; Maniak, Peter J; Lancto, Cheryl; Kita, Hirohito

2013-01-01

353

ATP is a potent stimulator of the activation and formation of rodent osteoclasts.  

PubMed

1. There is increasing evidence that extracellular ATP acts directly on bone cells via P2 receptors. In normal rat osteoclasts, ATP activates both non-selective cation channels and Ca2+-dependent K+ channels. In this study we investigated the action of ATP on the formation of osteoclasts and on the ultimate function of these cells, namely resorption pit formation. 2. We found that ATP stimulated resorption pit formation up to 5.6-fold when osteoclast-containing bone cell populations from neonatal rats were cultured for 26 h on ivory discs, with a maximum effect occurring at relatively low concentrations (0.2-2 microM). The stimulatory effect of ATP was amplified greatly when osteoclasts were activated by culture in acidified media (pH 6.9-7.0). Pit formation by acid-activated osteoclasts in the absence of ATP was inhibited by apyrase, an ecto-ATPase and by suramin, an antagonist of P2 receptors. 3. Over the same concentration range at which rat osteoclast activation occurred (0.2-2 microM), ATP also enhanced osteoclast formation in 10 day mouse marrow cultures, by up to 3.3-fold, with corresponding increases in resorption pit formation. Higher concentrations of ATP (20-200 microM) reduced or blocked osteoclast formation. Adenosine, a P1 receptor agonist, was without effect on either osteoclast activation or formation. 4. These results suggest that low levels of extracellular ATP may play a fundamental role in modulating both the resorptive function and formation of mammalian osteoclasts. PMID:9706025

Morrison, M S; Turin, L; King, B F; Burnstock, G; Arnett, T R

1998-09-01

354

Inhibition of store-operated Ca2+ entry by extracellular ATP in rat brown adipocytes  

PubMed Central

Modulation of intracellular free Ca2+ concentration ([Ca2+]i) by extracellular ATP was investigated in cultured adult rat brown adipocytes using the fluorescent Ca2+ indicator fura-2.Bath application of ATP in micromolar concentrations caused a large increase in [Ca2+]i in cells previously stimulated with noradrenaline. This ATP-induced [Ca2+]i increase exhibited a monotonic decline to near the resting levels within approximately 2 min, even in the continued presence of the agonist.The magnitude and time course of the [Ca2+]i increase in response to ATP were not significantly affected by removal of extracellular Ca2+, suggesting that a mobilization of intracellular Ca2+ primarily contributes to the increase.The [Ca2+]i increase in response to ATP was sensitive to inhibition by suramin, suggesting the involvement of P2 purinoceptors in the response.Thapsigargin (100 nm) evoked a gradual and irreversible increase in [Ca2+]i which was entirely dependent upon extracellular Ca2+, providing functional evidence for the expression of store-operated Ca2+ entry in these brown adipocytes.Extracellular ATP at a concentration of 10 ?m depressed this thapsigargin (100 nm)-induced [Ca2+]i increase by 92 ± 3 % (n= 8 cells), strongly suggesting that ATP inhibits an influx of Ca2+ across the plasma membrane through the store-operated pathway. Bath application of phorbol 12-myristate 13-acetate (PMA, 5 ?m) did not affect the thapsigargin-induced [Ca2+]i increase, indicating that the inhibitory action of ATP is not mediated by activation of protein kinase C (PKC).These results indicate that extracellular ATP not only mobilizes Ca2+ from the intracellular stores but also exerts a potent inhibitory effect on the store-operated Ca2+ entry process in adult rat brown adipocytes. PMID:10601492

Omatsu-Kanbe, Mariko; Matsuura, Hiroshi

1999-01-01

355

Imaging and characterization of stretch-induced ATP release from alveolar A549 cells  

PubMed Central

Mechano-transduction at cellular and tissue levels often involves ATP release and activation of the purinergic signalling cascade. In the lungs, stretch is an important physical stimulus but its impact on ATP release, the underlying release mechanisms and transduction pathways are poorly understood. Here, we investigated the effect of unidirectional stretch on ATP release from human alveolar A549 cells by real-time luciferin–luciferase bioluminescence imaging coupled with simultaneous infrared imaging, to monitor the extent of cell stretch and to identify ATP releasing cells. In subconfluent (<90%) cell cultures, single 1 s stretch (10–40%)-induced transient ATP release from a small fraction (?1.5%) of cells that grew in number dose-dependently with increasing extent of stretch. ATP concentration in the proximity (?150 ?m) of releasing cells often exceeded 10 ?m, sufficient for autocrine/paracrine purinoreceptor stimulation of neighbouring cells. ATP release responses were insensitive to the putative ATP channel blockers carbenoxolone and 5-nitro-2-(3-phenylpropyl-amino) benzoic acid, but were inhibited by N-ethylmaleimide and bafilomycin. In confluent cell cultures, the maximal fraction of responding cells dropped to <0.2%, but was enhanced several-fold in the wound/scratch area after it was repopulated by new cells during the healing process. Fluo8 fluorescence experiments revealed two types of stretch-induced intracellular Ca2+ responses, rapid sustained Ca2+ elevations in a limited number of cells and delayed secondary responses in neighbouring cells, seen as Ca2+ waves whose propagation was consistent with extracellular diffusion of released ATP. Our experiments revealed that a single >10% stretch was sufficient to initiate intercellular purinergic signalling in alveolar cells, which may contribute to the regulation of surfactant secretion and wound healing. PMID:23247110

Grygorczyk, Ryszard; Furuya, Kishio; Sokabe, Masahiro

2013-01-01

356

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

357

The role of intracellular pH in cell growth arrest induced by ATP.  

PubMed

In this study, we investigated ionic mechanisms involved in growth arrest induced by extracellular ATP in androgen-independent prostate cancer cells. Extracellular ATP reversibly induced a rapid and sustained intracellular pH (pH(i)) decrease from 7.41 to 7.11. Inhibition of Ca(2+) influx, lowering extracellular Ca(2+), and buffering cytoplasmic Ca(2+) inhibited ATP-induced acidification, thereby demonstrating that acidification is a consequence of Ca(2+) entry. We show that ATP induced reuptake of Ca(2+) by the mitochondria and a transient depolarization of the inner mitochondrial membrane. ATP-induced acidification was reduced after the dissipation of the mitochondrial proton gradient by rotenone and carbonyl cyanide p-trifluoromethoxyphenylhydrazone, after inhibition of Ca(2+) uptake into the mitochondria by ruthenium red, and after inhibition of the F(0)F(1)-ATPase with oligomycin. ATP-induced acidification was not induced by either stimulation of the Cl(-)/HCO(3)(-) exchanger or inhibition of the Na(+)/H(+) exchanger. In addition, intracellular acidification, induced by an ammonium prepulse method, reduced the amount of releasable Ca(2+) from the endoplasmic reticulum, assessed by measuring change in cytosolic Ca(2+) induced by thapsigargin or ATP in a Ca(2+)-free medium. This latter finding reveals cross talk between pH(i) and Ca(2+) homeostasis in which the Ca(2+)-induced intracellular acidification can in turn regulate the amount of Ca(2+) that can be released from the endoplasmic reticulum. Furthermore, pH(i) decrease was capable of reducing cell growth. Taken together, our results suggest that ATP-induced acidification in DU-145 cells results from specific effect of mitochondrial function and is one of the major mechanisms leading to growth arrest induced by ATP. PMID:15355852

Humez, Sandrine; Monet, Michaël; van Coppenolle, Fabien; Delcourt, Philippe; Prevarskaya, Natalia

2004-12-01

358

Use of ATP analogs to inhibit HIV-1 transcription  

PubMed Central

Human immunodeficiency virus type 1 (HIV-1) is the etiological agent of AIDS. Chronic persistent infection is an important reason for the presence of “latent cell populations” even after Anti Retroviral Therapy (ART). We have analyzed the effect of ATP analogs in inhibiting cdk9/T1 complex in infected cells. A third generation drug named CR8#13 is an effective inhibitor of Tat activated transcription. Following drug treatment, we observed a decreased loading of cdk9 onto the HIV-1 DNA. We found multiple novel cdk9/T1 complexes present in infected and uninfected cells with one complex being unique to infected cells. This complex is sensitive to CR8#13 in kinase assays. Treatment of PBMC with CR8#13 does not kill infected cells as compared to Flavopiridol. Interestingly, there is a difference in sensitivity of various clades to these analogs. Collectively, these results point to targeting novel complexes for inhibition of cellular proteins that are unique to infected cells. PMID:22771113

Narayanan, Aarthi; Sampey, Gavin; Van Duyne, Rachel; Guendel, Irene; Kehn-Hall, Kylene; Roman, Jessica; Currer, Robert; Galons, Herve; Oumata, Nassima; Joseph, Benoit; Meijer, Laurent; Caputi, Massimo; Nekhai, Sergei; Kashanchi, Fatah

2012-01-01

359

Compartmentation of ATP:Citrate Lyase in Plants1  

PubMed Central

Extracts prepared from young leaves of Pea (Pisum sativum), tobacco (Nicotiana tabacum), rape (Brassica napus), and spinach (Spinacia oleracea) all contained ATP:citrate lyase (ACL) activity, which was most active in rape leaflets (130 nmol min?1 g fresh weight). In rape and spinach, ACL activity was predominantly localized in the plastids (between about 78% and 90% of the total activity), whereas in pea and tobacco, distribution was mainly cytosolic (about 85% and 78%, respectively, of the total). These distributions were calculated from the relative distributions of plastid and cytosol marker enzymes. Cross-reactivity between plant and rat ACL antibody was carried out by immunoblot analysis and, in rape and spinach, showed that a 120-kD protein, presumably indicating homomeric ACL proteins, was present in both cytosolic and plastidic fractions. In pea, two cross-reacting proteins were detected, the major material being in the cytosol fraction. Therefore, ACL occurs both in the cytosol and plastids of higher plants, but the distribution of activity changes according to the species. The plastidic ACL is proposed to function for the supply of acetyl-coenzyme A for lipid biosynthesis de novo, whereas the cytosolic ACL may provide acetyl-coenzyme A for the mevalonate pathway or fatty acid elongation. PMID:10759519

Rangasamy, Dhandapani; Ratledge, Colin

2000-01-01

360

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

361

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

362

ATP-mediated mineralization of MC3T3-E1 osteoblast cultures.  

PubMed

While bone is hypomineralized in hypophosphatemia patients and in tissue-nonspecific alkaline phosphatase (Tnsalp)-deficient mice, the extensive mineralization that nevertheless occurs suggests involvement of other phosphatases in providing phosphate ions for mineral deposition. Although the source of phosphate liberated by these phosphatases is unknown, pyrophosphate, ATP, pyridoxal-5'-phosphate (PLP) and phoshoethanolamine (PEA) are likely candidates. In this study, we have induced mineralization of MC3T3-E1 osteoblast cultures using ATP, and have investigated potential phosphatases involved in this mineralization process. MC3T3-E1 osteoblasts were cultured for 12 days and treated either with beta-glycerophosphate (betaGP) or ATP. Matrix and mineral deposition was examined by biochemical, cytochemical, ultrastructural and X-ray microanalytical methods. ATP added at levels of 4-5 mM resulted in mineral deposition similar to that following conventional treatment with betaGP. Collagen levels were similarly normal in ATP-mineralized cultures and transmission electron microscopy and X-ray microanalysis confirmed hydroxyapatite mineral deposition along the collagen fibrils in the ECM. Phosphate release from 4 mM ATP into the medium was rapid and resulted in approximately twice the phosphate levels than after release from 10 mM betaGP. ATP treatment did not affect mineralization by altering the expression of mineral-regulating genes such as Enpp1, Ank, and Mgp, nor phosphatase genes indicating that ATP induces mineralization by serving as a phosphate source for mineral deposition. Levamisole, an inhibitor of TNSALP, completely blocked mineralization in betaGP-treated cultures, but had minor effects on ATP-mediated mineralization, indicating that other phosphatases such as plasma membrane Ca2+ transport ATPase 1 (PMCA1) and transglutaminase 2 (TG2) are contributing to ATP hydrolysis. To examine their involvement in ATP-mediated mineralization, the inhibitors cystamine (TG2 inhibitor) and ortho-vanadate (PMCA inhibitor) were added to the cultures - both inhibitors significantly reduced mineralization whereas suppression of the phosphate release by ortho-vanadate was minor comparing to other two inhibitors. The contribution of PMCA1 to mineralization may occur through pumping of calcium towards calcification sites and TG2 can likely act as an ATPase in the ECM. Unlike the GTPase activity of TG2, its ATPase function was resistant to calcium, demonstrating the potential for participation in ATP hydrolysis and mineral deposition within the ECM at elevated calcium concentrations. PMID:17669706

Nakano, Yukiko; Addison, William N; Kaartinen, Mari T

2007-10-01

363

ATP-activated inward current and calcium-permeable channels in rat macrophage plasma membranes.  

PubMed Central

1. To study mechanisms of receptor-operated Ca2+ influx in non-excitable cells, membrane currents of rat peritoneal macrophages were recorded using whole-cell cell-attached and outside-out configurations of the patch clamp technique. Under whole-cell recording conditions, ATP applied in micromolar concentrations elicited an inward current response when the bath solution contained Ba2+, Ca2+ or Na+ as the only permeant cations. 2. Increasing the Mg2+ concentration had an inhibitory effect on the ATP-induced inward current indicating that the active form of ATP responsible for the cation entry is ATP4-. The nucleotide potency order was ATP > ATP gamma S > ADP. UTP was completely ineffective (n = 19). The data obtained are consistent with the ATP receptor being of the P2Z type. 3. The macrophage plasma membrane was impermeable to Tris+ during the ATP-induced current at ATP4- concentrations varying from 0.07 to 500 microM. At higher concentrations, ATP produced a large inward steady-state current, which could be attributed to membrane permeabilization. 4. Activity of single channels was recorded when ATP was applied to the external surface of the patch membrane both in cell-attached and outside-out experiments. A specific property of the channels appeared to be the existence of at least four conductance sublevels. With 105 mM Ba2+ as the permeant cation, the conductance sublevels were 3.5, 7, 10 and 15 pS. With 10 mM Ca2+ the sublevel conductances were equal to 4, 9, 13 and 17 pS. 5. The unitary conductance estimated from the whole-cell current noise analysis (3.5-4.5 pS for 105 mM Ba2+) was significantly lower than that obtained from single channel measurements at the main (3rd) current level, but it was very close to the conductance of the minimum (1st) level. 6. Extrapolated reversal potential values estimated from current-voltage curves for predominant conductance levels were equal to +40 and +26 mV for 105 mM Ba2+ and 10 mM Ca2+, respectively. The permeability ratios fell in the sequence: PCa:PBa:PK = 71.:29:1. Thus, ATP-activated channels in the macrophage membrane are rather selective for divalent vs. monovalent cations, with the predominant permeability being for Ca2+. PMID:7473200

Naumov, A P; Kaznacheyeva, E V; Kiselyov, K I; Kuryshev, Y A; Mamin, A G; Mozhayeva, G N

1995-01-01

364

Development of a Generator to Power ATP-Driven Molecular Motors  

SciTech Connect

Here, we report a maximum ATP synthesis rate of 193 nmol/min/mg for thermophilic F{sub 1}F{sub 0}. This rate is somewhat lower than the previously observed maximum rate of 500-700 nmol/min/mg (Pitard et al., 1996). However, ATP synthesis rates vary considerably with experimental conditions, and our observed rates compare favorably with the wide range of rates (40-700 nmol/min/mg) observed by these authors. Future research will focus on maximizing the ATP synthesis rate by adjusting environmental conditions, including the lipid and cholesterol composition of the proteoliposomes.

Montemagno, Carlo

2002-10-23

365

Defining the Pathogenesis of the Human Atp12p W94R Mutation Using a Saccharomyces cerevisiae Yeast Model*  

PubMed Central

Studies in yeast have shown that a deficiency in Atp12p prevents assembly of the extrinsic domain (F1) of complex V and renders cells unable to make ATP through oxidative phosphorylation. De Meirleir et al. (De Meirleir, L., Seneca, S., Lissens, W., De Clercq, I., Eyskens, F., Gerlo, E., Smet, J., and Van Coster, R. (2004) J. Med. Genet. 41, 120–124) have reported that a homozygous missense mutation in the gene for human Atp12p (HuAtp12p), which replaces Trp-94 with Arg, was linked to the death of a 14-month-old patient. We have investigated the impact of the pathogenic W94R mutation on Atp12p structure/function. Plasmid-borne wild type human Atp12p rescues the respiratory defect of a yeast ATP12 deletion mutant (?atp12). The W94R mutation alters the protein at the most highly conserved position in the Pfam sequence and renders HuAtp12p insoluble in the background of ?atp12. In contrast, the yeast protein harboring the corresponding mutation, ScAtp12p(W103R), is soluble in the background of ?atp12 but not in the background of ?atp12?fmc1, a strain that also lacks Fmc1p. Fmc1p is a yeast mitochondrial protein not found in higher eukaryotes. Tryptophan 94 (human) or 103 (yeast) is located in a positively charged region of Atp12p, and hence its mutation to arginine does not alter significantly the electrostatic properties of the protein. Instead, we provide evidence that the primary effect of the substitution is on the dynamic properties of Atp12p. PMID:19933271

Meulemans, Ann; Seneca, Sara; Pribyl, Thomas; Smet, Joel; Alderweirldt, Valerie; Waeytens, Anouk; Lissens, Willy; Van Coster, Rudy; De Meirleir, Linda; di Rago, Jean-Paul; Gatti, Domenico L.; Ackerman, Sharon H.

2010-01-01

366

Interaction of purine nucleotides with inert paramagnetic Cr(III) probes evaluated by NMR relaxation effects. Molecular mechanics calculations on Cr(III) and Co(III) polyphosphate complexes.  

PubMed

The 1H NMR relaxation effects produced by paramagnetic Cr(III) complexes on nucleoside 5'-mono- and -triphosphates in D2O solution at pH' = 3 were measured. The paramagnetic probes were [Cr(III)(H2O)6]3+, [Cr(III)(H2O)3(HATP)], [Cr(III)(H2O)3(HCTP)] and [Cr(III)(H2O)3(UTP)-, while the matrix nucleotides (0.1 M) were H2AMP, HIMP-, and H2ATP2-. For the aromatic base protons, the ratios of the transverse to longitudinal paramagnetic relaxation rates (R2p/R1p) for the [Cr(III)(H2O)6]3+/H2ATP2-, [Cr(III)(H2O)3(HATP)]/H2ATP2-, [Cr(III)(H2O)3(HCTP)]/H2ATP2 and [Cr(III)(H2O)3(UTP)]-/H2ATP2 systems were below 2.33 so the dipolar term predominates. For a given nucleotide, R1p for the purine H(8) signal was larger than for the H(2) signal with the [Cr(III)(H2O)6]3+ probe, while R1p for the H(2) signal was larger with all the other Cr(III) probes. Molecular mechanics computations on the [Cr(III)(H2O)4(HPP)(alpha,beta)], [Cr(III)(NH3)4(HPP)(alpha,beta)], [Co(III)(NH3)3(H2PPP)(alpha,beta,gamma)] and [Co(III)(NH3)4(HPP)(alpha,beta)] complexes gave calculated energy-minimized geometries in good agreement with those reported in crystal structures. The molecular mechanics force constants found were then used to calculate the geometry of the inner sphere [Cr(III)(H2O)6]3+ and [Cr(III)(H2O)3(HATP)(alpha,beta,gamma)] complexes as well as the structures of the outer sphere [Cr(III)(H2O)6]3(+)-(H2AMP) and [Cr(III)(H2O)6]-(HIMP)- species. The gas-phase structure of the [Cr(III)(H2O)3(HATP)(alpha,beta,gamma)] complex shows the existence of a hydrogen bond interaction between a water ligand and the adenine N(7)(O...N = 2.82 A). The structure is also stabilized by intramolecular hydrogen bonds involving the -O(2')H group and the adenine N(3) (O...N = 2.80 A) as well as phosphate oxygen atoms and a water molecule (O...O = 2.47 A). The metal center has an almost regular octahedral coordination geometry. The structures of the two outer-sphere species reveal that the phosphate group interacts strongly with the hexa-aquochromium probe. In both complexes, the nucleotides have a similar "anti" conformation around the N(9)-C(1') glycosidic bond. However, a very important difference characterizes the two structures. For the (HIMP)- complex, strong hydrogen bond interactions exist between one and two water ligands and the inosine N(7) and O(6) atoms, respectively (O...O = 2.63 A; O...N = 2.72, 2.70 A). For the H2AMP complex, the [Cr(III)(H2O)6]3+ cation does not interact with N(7) since it is far from the purine system. Hydrogen bonds occur between water ligands and phosphate oxygens. The Cr-H(8) and Cr-H(2) distances revealed by the energy-minimized geometries for the two outer sphere species were used to calculate the R1p values for the H(8) and H(2) signals for comparison with the observed R1p values: 0.92(c), 1.04(ob) (H(8)) and 0.06(c), 0.35(ob) (H(2)) for H2AMP; and 3.76(c), 4.53(ob) (H(8)) and 0.16(c), 0.77(ob) s-1 (H(2)) for HIMP-.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2310520

Cini, R; Giorgi, G; Laschi, F; Rossi, C; Marzilli, L G

1990-02-01

367

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

SciTech Connect

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

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

2005-07-21

368

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

369

Structure of the 26S proteasome with ATP-?S bound provides insights into the mechanism of nucleotide-dependent substrate translocation  

PubMed Central

The 26S proteasome is a 2.5-MDa, ATP-dependent multisubunit proteolytic complex that processively destroys proteins carrying a degradation signal. The proteasomal ATPase heterohexamer is a key module of the 19S regulatory particle; it unfolds substrates and translocates them into the 20S core particle where degradation takes place. We used cryoelectron microscopy single-particle analysis to obtain insights into the structural changes of 26S proteasome upon the binding and hydrolysis of ATP. The ATPase ring adopts at least two distinct helical staircase conformations dependent on the nucleotide state. The transition from the conformation observed in the presence of ATP to the predominant conformation in the presence of ATP-?S induces a sliding motion of the ATPase ring over the 20S core particle ring leading to an alignment of the translocation channels of the ATPase and the core particle gate, a conformational state likely to facilitate substrate translocation. Two types of intersubunit modules formed by the large ATPase domain of one ATPase subunit and the small ATPase domain of its neighbor exist. They resemble the contacts observed in the crystal structures of ClpX and proteasome-activating nucleotidase, respectively. The ClpX-like contacts are positioned consecutively and give rise to helical shape in the hexamer, whereas the proteasome-activating nucleotidase-like contact is required to close the ring. Conformational switching between these forms allows adopting different helical conformations in different nucleotide states. We postulate that ATP hydrolysis by the regulatory particle ATPase (Rpt) 5 subunit initiates a cascade of conformational changes, leading to pulling of the substrate, which is primarily executed by Rpt1, Rpt2, and Rpt6. PMID:23589842

Sledz, Pawel; Unverdorben, Pia; Beck, Florian; Pfeifer, Gunter; Schweitzer, Andreas; Forster, Friedrich; Baumeister, Wolfgang

2013-01-01

370

The evolution of A-, F-, and V-type ATP synthases and ATPases: reversals in function and changes in the H+/ATP coupling ratio.  

PubMed

Members of the FoF1, AoA1 and VoV1 family of ATP synthases and ATPases have undergone at least two reversals in primary function. The first was from a progenitor proton-pumping ATPase to a proton-driven ATP synthase. The second involved transforming the synthase back into a proton-pumping ATPase. As proposed earlier [FEBS Lett. 259 (1990) 227], these reversals required changes in the H+/ATP coupling ratio from an optimal value of about 2 for an ATPase function to about 4 for an ATP synthase function. The doubling of the ratio that occurred at the ATPase-to-Synthase transition was accomplished by duplicating the gene that encodes the nucleotide-binding catalytic subunits followed by loss of function in one of the genes. The halving of the ratio that occurred at the Synthase-to-ATPase transition was achieved by a duplication/fusion of the gene that encodes the proton-binding transporter subunits, followed by a loss of function in one half of the double-sized protein. These events allowed conservation of quaternary structure, while maintaining a sufficient driving force to sustain an adequate phosphorylation potential or electrochemical gradient. Here, we describe intermediate evolutionary steps and a fine-tuning of the H+/ATP coupling ratio to optimize synthase function in response to different environments. In addition, we propose a third reversal of function, from an ATPase back to an ATP synthase. In contrast to the first two reversals which required a partial loss in function, the change in coupling ratio required for the third reversal is explained by a gain in function. PMID:15473999

Cross, Richard L; Müller, Volker

2004-10-01

371

Modification of the ATP inhibitory site of the Ascaris suum phosphofructokinase results in the stabilization of an inactive T state  

SciTech Connect

Treatment of the Ascaris suum phosphofructokinase (PFK) with 2{prime},3{prime}-dialdehyde ATP (oATP) results in an enzyme form that is inactive. The conformational integrity of the active site, however, is preserved, suggesting that oATP modification locks the PFK into an inactive T state that cannot be activated. A rapid, irreversible first-order inactivation of the PFK is observed in the presence of oATP. The rate of inactivation is saturable and gives a K{sub oATP} of 1.07 {plus minus} 0.27 mM. Complete protection against inactivation is afforded by high concentrations of ATP. This desensitized enzyme incorporates only 0.2-0.3 mol of ({sup 3}H)oATP/subunit, suggesting that in te native enzyme inactivation perhaps results from the modification of the ATP inhibitory site rather than the catalytic site. Modification of an active-site thiol by 4,4{prime}-dithiodipyridine is prevented yb ATP before and after oATP treatment. Finally, gel filtration HPLC studies show that the oATP-modified enzyme retains its tetrameric state and neither the tryptophan fluorescence nor the circular dichroic spectra of the modified enzyme are affected by fructose 2,6-bisphosphate, suggesting that the enzyme is locked into a tetrameric inactive T state.

Rao, G.S.J.; Cook, P.F.; Harris, B.G. (Univ. of North Texas, Fort Worth (United States))

1991-10-15

372

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

EPA Science Inventory

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

373

The Chloroplast atpA Gene Cluster in Chlamydomonas reinhardtii1  

E-print Network

The Chloroplast atpA Gene Cluster in Chlamydomonas reinhardtii1 Functional Analysis University, Ithaca, New York 14853 Most chloroplast genes in vascular plants are organized into polycistronic, most Chlamydomonas reinhardtii chloroplast transcripts characterized to date have been monocistronic

374

75 FR 8467 - Airworthiness Directives; BAE SYSTEMS (Operations) Limited Model ATP Airplanes  

Federal Register 2010, 2011, 2012, 2013

...tasks were introduced by Service Bulletin (SB) ATP-51-002 * * *. As it was determined...issued * * *. Since the original Issue of the SB, three revisions have been published. Revision 1 of the SB included only editorial changes....

2010-02-25

375

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

E-print Network

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

376

Single-Molecule Protein Unfolding and Translocation by an ATP-Fueled Proteolytic Machine  

E-print Network

All cells employ ATP-powered proteases for protein-quality control and regulation. In the ClpXP protease, ClpX is a AAA+ machine that recognizes specific protein substrates, unfolds these molecules, and then translocates ...

Aubin-Tam, Marie-Eve

377

An ATP-competitive inhibitor modulates the allosteric function of the HER3 pseudokinase.  

PubMed

Human epidermal growth factor receptor 3 (HER3) is a receptor tyrosine kinase that lacks catalytic activity but is essential for cellular homeostasis due to its ability to allosterically activate EGFR and HER2. Although catalytically inactive, HER3 binds ATP tightly, hinting at a possible role of the nucleotide-binding pocket in modulating HER3 function. We report a structure of the HER3 pseudokinase bound to the ATP-competitive inhibitor bosutinib. Previously solved structures show that bosutinib can potently interact with multiple kinase domain conformations. In complex with HER3, bosutinib binds to yet another conformation, which is nearly identical to that observed in the HER3-ATP complex. Interestingly, occupation of the ATP-binding site by bosutinib improves the ability of HER3 to act as an allosteric activator of EGFR in vitro by increasing the affinity of the HER3-EGFR heterodimer in a membrane-dependent manner. PMID:24656791

Littlefield, Peter; Moasser, Mark M; Jura, Natalia

2014-04-24

378

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

379

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

E-print Network

by recrea- tional and competitive athletes as ergogenic aids to improve their physique and performance capabilities. For etal muscle phosphocreatine and free creatine concentra- tions, which may enhance the ability to sustain high adenosine triphosphate (ATP...

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

2008-01-01

380

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

381

ATP citrate lyase knockdown impacts cancer stem cells in vitro.  

PubMed

ATP citrate lyase (ACL) knockdown (KD) causes tumor suppression and induces differentiation. We have previously reported that ACL KD reverses epithelial-mesenchymal transition (EMT) in lung cancer cells. Because EMT is often associated with processes that induce stemness, we hypothesized that ACL KD impacts cancer stem cells. By assessing tumorsphere formation and expression of stem cell markers, we showed this to be the case in A549 cells, which harbor a Ras mutation, and in two other non-small-cell lung cancer cell lines, H1975 and H1650, driven by activating EGFR mutations. Inducible ACL KD had the same effect as stable ACL KD. Similar effects were noted in another well-characterized Ras-induced mammary model system (HMLER). Moreover, treatment with hydroxycitrate phenocopied the effects of ACL KD, suggesting that the enzymatic activity of ACL was critical. Indeed, acetate treatment reversed the ACL KD phenotype. Having previously established that ACL KD impacts signaling through the phosphatidylinositol 3-kinase (PI3K) pathway, not the Ras-mitogen-activated protein kinase (MAPK) pathway, and that EMT can be reversed by PI3K inhibitors, we were surprised to find that stemness in these systems was maintained through Ras-MAPK signaling, and not via PI3K signaling. Snail is a downstream transcription factor impacted by Ras-MAPK signaling and known to promote EMT and stemness. We found that snail expression was reduced by ACL KD. In tumorigenic HMLER cells, ACL overexpression increased snail expression and stemness, both of which were reduced by ACL KD. Furthermore, ACL could not initiate either tumorigenesis or stemness by itself. ACL and snail proteins interacted and ACL expression regulated the transcriptional activity of snail. Finally, ACL KD counteracted stem cell characteristics induced in diverse cell systems driven by activation of pathways outside of Ras-MAPK signaling. Our findings unveil a novel aspect of ACL function, namely its impact on cancer stemness in a broad range of genetically diverse cell types. PMID:23807225

Hanai, J-I; Doro, N; Seth, P; Sukhatme, V P

2013-01-01

382

Use of ATP to characterize biomass viability in freely suspended and immobilized cell bioreactors.  

PubMed

This work describes investigations into the viability of cells growing on 3,4-dichloroaniline (34DCA). Two bioreactors are employed for microbial growth, a continuous stirred tank (CST) bioreactor with a 2-L working volume, and a three-phase air lift (TPAL) bioreactor with a 3-L working volume. Experiments have been performed at several dilution rates between 0.027 and 0.115 h(-1) in the CST bioreactor and between 0.111 and 0.500 h(-1) in the TPAL bioreactor. The specific ATP concentration was calculated at each dilution rate in the suspended biomass in both bioreactors as well as in the immobilized biomass in the TPAL bioreactor. The ATP was extracted from the cells using boiling tris-EDTA buffer (pH 7.75), and the quantity determined using a firefly (bioluminescence) technique. The cultures were inspected under an electron microscope to monitor compositional changes. Results from the CST bioreactor showed that the biomass-specific ATP concentration increases from 0.44 to 1.86 mg ATP g(-1) dry weight (dw) as dilution rate increases from 0.027 to 0.115 h(-1). At this upper dilution rate the cells were washed out. The specific ATP concentration reached a limiting average value of 1.73 mg ATP g(-1) dw, which is assumed to be the quantity of ATP in 100% viable biomass. In the TPAL bioreactor, the ATP level increased with dilution rate in both the immobilized and suspended biomass. The specific ATP concentration in the immobilized biomass increased from approximately 0.051 mg ATP g(-1) dw at dilution rates between 0.111 and 0.200 h(-1) to approximately 0.119 mg ATP g(-1) dw at dilution rates between 0.300 and 0.500 h(-1). This indicates that the immobilized biomass contained a viable cell fraction of around 5%. Based on these results, kinetic data for freely suspended cells should not be applied to the modeling of immobilized cell systems on the assumption that immobilized biomass is 100% viable. PMID:18612962

Gikas, P; Livingston, A G

1993-12-01

383

Extracellular ATP triggers and maintains asthmatic airway inflammation by activating dendritic cells  

Microsoft Academic Search

Extracellular ATP serves as a danger signal to alert the immune system of tissue damage by acting on P2X or P2Y receptors. Here we show that allergen challenge causes acute accumulation of ATP in the airways of asthmatic subjects and mice with experimentally induced asthma. All the cardinal features of asthma, including eosinophilic airway inflammation, Th2 cytokine production and bronchial

Hamida Hammad; Menno van Nimwegen; Mirjam Kool; Monique A M Willart; Femke Muskens; Henk C Hoogsteden; Werner Luttmann; Davide Ferrari; Francesco Di Virgilio; J Christian Virchow; Marco Idzko; Bart N Lambrecht

2007-01-01

384

Cardiolipin Affects the Supramolecular Organization of ATP Synthase in Mitochondria  

Microsoft Academic Search

F1F0 ATP synthase forms dimers that tend to assemble into large supramolecular structures. We show that the presence of cardiolipin is critical for the degree of oligomerization and the degree of order in these ATP synthase assemblies. This conclusion was drawn from the statistical analysis of cryoelectron tomograms of cristae vesicles isolated from Drosophila flight-muscle mitochondria, which are very rich

Devrim Acehan; Ashim Malhotra; Yang Xu; Mindong Ren; Michael Schlame

2011-01-01

385

Structure, function, and evolution of bacterial ATP-binding cassette systems  

Microsoft Academic Search

The ATP-binding cassette (ABC) systems constitute one of the largest superfamilies of paralogous sequences. All ABC systems share a highly conserved ATP-hydrolyzing domain or protein (the ABC; also referred to as a nucleotide-binding domain [NBD]) that is unequivocally characterized by three short sequence motifs (Fig. 1): these are the Walker A and Walker B motifs, indicative of the presence of

A. L. Davidson; E. Dassa; C. Orelle; J. Chen

2010-01-01

386

Quantal ATP release in rat ?-cells by exocytosis of insulin-containing LDCVs  

Microsoft Academic Search

Quantal release of adenosine triphosphate (ATP) was monitored in rat pancreatic ?-cells expressing P2X2 receptors. Stimulation of exocytosis evoked rapidly activating and deactivating ATP-dependent transient inward currents (TICs).\\u000a The unitary charge (q) of the events recorded at 0.2 ?M [Ca2+]i averaged 4.3 pC. The distribution of the 3?q of these events could be described by a single Gaussian. The rise times averaged

Jovita Karanauskaite; Michael B. Hoppa; Matthias Braun; Juris Galvanovskis; Patrik Rorsman

2009-01-01

387

The T1048I mutation in ATP7A gene causes an unusual Menkes disease presentation  

PubMed Central

Background The ATP7A gene encodes the ATP7A protein, which is a trans-Golgi network copper transporter expressed in the brain and other organs. Mutations in this gene cause disorders of copper metabolism, such as Menkes disease. Here we describe the novel and unusual mutation (p.T1048I) in the ATP7A gene of a child with Menkes disease. The mutation affects a conserved DKTGT1048 phosphorylation motif that is involved in the catalytic activity of ATP7A. We also describe the clinical course and the response to copper treatment in this patient. Case presentation An 11-month-old male Caucasian infant was studied because of hypotonia, ataxia and global developmental delay. The patient presented low levels of serum copper and ceruloplasmin, and was shown to be hemizygous for the p.T1048I mutation in ATP7A. The diagnosis was confirmed when the patient was 18 months old, and treatment with copper-histidinate (Cu-His) was started immediately. The patient showed some neurological improvement and he is currently 8 years old. Because the p.T1048I mutation affects its catalytic site, we expected a complete loss of functional ATP7A and a classical Menkes disease presentation. However, the clinical course of the patient was mild, and he responded to Cu-His treatment, which suggests that this mutation leads to partial conservation of the activity of ATP7A. Conclusion This case emphasizes the important correlation between genotype and phenotype in patients with Menkes disease. The prognosis in Menkes disease is associated with early detection, early initiation of treatment and with the preservation of some ATP7A activity, which is necessary for Cu-His treatment response. The description of this new mutation and the response of the patient to Cu-His treatment will contribute to the growing body of knowledge about treatment response in Menkes disease. PMID:22992316

2012-01-01

388

Characterization and classification of ATP-binding cassette transporter ABCA3 mutants in fatal surfactant deficiency.  

PubMed

The ATP-binding cassette transporter ABCA3 is expressed predominantly at the limiting membrane of the lamellar bodies in lung alveolar type II cells. Recent study has shown that mutation of the ABCA3 gene causes fatal surfactant deficiency in newborns. In this study, we investigated in HEK293 cells the intracellular localization and N-glycosylation of the ABCA3 mutants so far identified in fatal surfactant deficiency patients. Green fluorescent protein-tagged L101P, L982P, L1553P, Q1591P, and Ins1518fs/ter1519 mutant proteins remained localized in the endoplasmic reticulum, and processing of oligosaccharide was impaired, whereas wild-type and N568D, G1221S, and L1580P mutant ABCA3 proteins trafficked to the LAMP3-positive intracellular vesicle, accompanied by processing of oligosaccharide from high mannose type to complex type. Vanadate-induced nucleotide trapping and ATP-binding analyses showed that ATP hydrolysis activity was dramatically decreased in the N568D, G1221S, and L1580P mutants, accompanied by a moderate decrease in ATP binding in N568D and L1580P mutants but not in the G1221S mutant, compared with the wild-type ABCA3 protein. In addition, mutational analyses of the Gly-1221 residue in the 11th transmembrane segment and the Leu-1580 residue in the cytoplasmic tail, and homology modeling of nucleotide binding domain 2 demonstrate the significance of these residues for ATP hydrolysis and suggest a mechanism for impaired ATP hydrolysis in G1221S and L1580P mutants. Thus, surfactant deficiency because of ABCA3 gene mutation may be classified into two categories as follows: abnormal intracellular localization (type I) and normal intracellular localization with decreased ATP binding and/or ATP hydrolysis of the ABCA3 protein (type II). These distinct pathophysiologies may reflect both the severity and effective therapy for surfactant deficiency. PMID:16959783

Matsumura, Yoshihiro; Ban, Nobuhiro; Ueda, Kazumitsu; Inagaki, Nobuya

2006-11-10

389

Extracellular ATP Released by Osteoblasts Is A Key Local Inhibitor of Bone Mineralisation  

PubMed Central

Previous studies have shown that exogenous ATP (>1µM) prevents bone formation in vitro by blocking mineralisation of the collagenous matrix. This effect is thought to be mediated via both P2 receptor-dependent pathways and a receptor-independent mechanism (hydrolysis of ATP to produce the mineralisation inhibitor pyrophosphate, PPi). Osteoblasts are also known to release ATP constitutively. To determine whether this endogenous ATP might exert significant biological effects, bone-forming primary rat osteoblasts were cultured with 0.5-2.5U/ml apyrase (which sequentially hydrolyses ATP to ADP to AMP + 2Pi). Addition of 0.5U/ml apyrase to osteoblast culture medium degraded extracellular ATP to <1% of control levels within 2 minutes; continuous exposure to apyrase maintained this inhibition for up to 14 days. Apyrase treatment for the first 72 hours of culture caused small decreases (?25%) in osteoblast number, suggesting a role for endogenous ATP in stimulating cell proliferation. Continuous apyrase treatment for 14 days (?0.5U/ml) increased mineralisation of bone nodules by up to 3-fold. Increases in bone mineralisation were also seen when osteoblasts were cultured with the ATP release inhibitors, NEM and brefeldin A, as well as with P2X1 and P2X7 receptor antagonists. Apyrase decreased alkaline phosphatase (TNAP) activity by up to 60%, whilst increasing the activity of the PPi-generating ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) up to 2.7-fold. Both collagen production and adipocyte formation were unaffected. These data suggest that nucleotides released by osteoblasts in bone could act locally, via multiple mechanisms, to limit mineralisation. PMID:23874866

Orriss, Isabel R.; Key, Michelle L.; Hajjawi, Mark O. R.; Arnett, Timothy R.

2013-01-01

390

Clathrin-Coated Vesicles Contain an ATP-Dependent Proton Pump  

Microsoft Academic Search

Clathrin-coated vesicles isolated from calf brain contain an ATP-dependent proton pump. Proton movement was monitored by measuring [14C]methylamine distribution. Addition of Mg2+ and ATP to coated vesicles equilibrated with [14C]methylamine resulted in the generation of a 4- to 5-fold concentration gradient, corresponding to a Delta pH of 0.6-0.7 units between the medium and the acidic inside of the coated vesicles.

Michael Forgac; Lewis Cantley; Bertram Wiedenmann; Larry Altstiel; Daniel Branton

1983-01-01

391

ATP-mediated vasodilatation occurs via activation of inwardly rectifying potassium channels in humans  

PubMed Central

Circulating ATP possesses unique vasomotor properties in humans and has been hypothesized to play a role in vascular control under a variety of physiological conditions. However, the primary downstream signalling mechanisms underlying ATP-mediated vasodilatation remain unclear. The purpose of the present experiment was to determine whether ATP-mediated vasodilatation is independent of nitric oxide (NO) and prostaglandin (PG) synthesis and occurs pr