Science.gov

Sample records for atp iii definition

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

    PubMed Central

    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

    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

  2. A Raman study of the binding of Fe(III) to ATP and AMP.

    PubMed Central

    Zhelyaskov, V; Yue, K T

    1992-01-01

    ATP-Fe and AMP-Fe complexes in water (H2O and 2H2O) at pH 7.5 were studied using Raman spectroscopy. Parallel and perpendicular polarization spectra were recorded in the spectral range 200-1650 cm-1, and the depolarization ratios for most of the bands were calculated. The changes in the frequencies, intensities and depolarization ratios of the ATP and AMP bands after the addition of FeCl3 showed that the adenine moiety, in addition to the phosphate(s), was involved in the binding of Fe to both ATP and AMP. Direct interactions of Fe(III) with the phosphate chain and the N-7 nitrogen and indirect interaction (via water molecules) with the amide group were proposed for the ATP-Fe complex. In contrast, direct interaction with the phosphate group and indirect interaction with the amide group were observed for the AMP-Fe complex. The different interactions of the two complexes suggest an 'anti' conformation for the ATP-Fe complex and a 'syn' conformation for the AMP-Fe complex. The strong binding of Fe to ATP compared with AMP and the difference in the conformation of the ATP-Fe and the AMP-Fe complexes may be significant in the pathway of Fe release in mitochondria. PMID:1445215

  3. Metabolic syndrome prevalence according to ATP III and IDF criteria and related factors in Turkish adults

    PubMed Central

    Bayram, Fahri; Gedik, Vedia; Kaya, Ahmet; Karaman, Ahmet; Demir, Ozgür; Sabuncu, Tevfik; Kocer, Derya; Coskun, Ramazan

    2013-01-01

    Introduction The aim of this study is to investigate the prevalence of metabolic syndrome (MS) and its components according to Adult Treatment Panel III (ATP III) and International Diabetes Federation (IDF) criteria and the risk factors affecting MS. Metabolic syndrome prevalence was evaluated according to certain quintet age groups, altitude, location and demographic features. Material and methods This study was a cross-sectional survey conducted in 24 provinces from the 7 regions of Turkey. A total of 4309 adults from 7 regions participated in the study (1947 males, 45.2%). Results The mean age of participants was 47 ±14 years. Metabolic syndrome prevalence was found as 36.6% according to ATP III and 44.0% according to IDF. The MS rate was found to be higher in females compared to males in both groups (p < 0.01). According to both criteria, MS prevalence was found to be higher in subjects who lived in coastal regions when evaluated according to altitude and in subjects who lived in district centers when evaluated according to location. The MS risk is 1.62-fold higher in females compared to males. Metabolic syndrome risk increases as age increases and is highest in the 61-65 age group. Metabolic syndrome risk increases 2.75-fold in the overweight compared to normal weighing subjects and 7.80-fold in the obese. Conclusions Metabolic syndrome prevalence was found to be high in Turkey according to both criteria. Metabolic syndrome prevalence increases as age and body mass index (BMI) increase. Age, female gender and obesity are independent risk factors for MS development. PMID:23671434

  4. The Association Between Subclinical Hypothyroidism and Metabolic Syndrome as Defined by the ATP III Criteria.

    PubMed

    Eftekharzadeh, Anita; Khamseh, Mohammad Ebrahim; Farshchi, Amir; Malek, Mojtaba

    2016-04-01

    The association between subclinical hypothyroidism (SCH) and metabolic syndrome (MetS) is a matter of debate. In this study, we aimed to examine the risk of MetS in SCH through a systematic review and meta-analysis. A systematic search of published literature up to September 2015 was conducted. General population studies were included if they had considered SCH as the independent variable. After data extraction, a meta-analysis was run to assess the odds ratio (OR) of MetS and its components between subclinical hypothyroid and euthyroid groups. Only studies employing Adult Treatment Panel III (ATP III) criteria for MetS were included in the meta-analysis. In the SCH group, female gender was more prevalent [OR = 1.65, 95% confidence interval (95% CI) 1.29-2.13]. Meta-analysis showed no significant difference in MetS prevalence between SCH and euthyroid individuals (OR = 1.13, 95% CI 0.95-1.34). However, the prevalence of central obesity (OR = 1.43, 95% CI 1.04-1.96) was significantly higher in the SCH group. On the other hand, hypertriglyceridemia was more prevalent in the female-only SCH subgroup. In conclusion, although we found some MetS components to be more prevalent in SCH, the prevalence of MetS as defined by ATP III criteria was not increased in SCH. Considerable heterogeneity observed may have influenced the results on MetS components. PMID:26824297

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-12-15

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

  7. A Label-Free Luminescent Switch-On Assay for ATP Using a G-Quadruplex-Selective Iridium(III) Complex

    PubMed Central

    Wang, Modi; Mak, Tsun-Yin; Chan, Daniel Shiu-Hin; Tang, Fung-Kit; Leung, Chung-Hang; Kwan, Hiu-Yee; Yu, Zhiling; Ma, Dik-Lung

    2013-01-01

    We report herein the G-quadruplex-selective property of a luminescent cyclometallated iridium(III) complex for the detection of adenosine-5′-triphosphate (ATP) in aqueous solution. The ATP-binding aptamer was employed as the ATP recognition unit, while the iridium(III) complex was used to monitor the formation of the G-quadruplex structure induced by ATP. The sensitivity and fold enhancement of the assay were higher than those of the previously reported assay using the organic dye crystal violet as a fluorescent probe. This label-free luminescent switch-on assay exhibits high sensitivity and selectivity towards ATP with a limit of detection of 2.5 µM. PMID:24204723

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

    PubMed Central

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

    1993-01-01

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

  9. ATP synthase.

    PubMed

    Junge, Wolfgang; Nelson, Nathan

    2015-01-01

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

  10. Optical ATP Biosensor for Extracellular ATP Measurement

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2016-07-01

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

  12. Functional dyspepsia--symptoms, definitions and validity of the Rome III criteria.

    PubMed

    Tack, Jan; Talley, Nicholas J

    2013-03-01

    Dyspepsia refers to a heterogeneous group of symptoms that are localized in the epigastric region. Typical dyspeptic symptoms include postprandial fullness, early satiation, epigastric pain and epigastric burning, but other upper gastrointestinal symptoms such as nausea, belching or abdominal bloating often occur. Functional dyspepsia is defined as the presence of dyspeptic symptoms in the absence of an organic cause that readily explains them. The Rome III consensus proposed the subdivision of functional dyspepsia into postprandial distress syndrome (PDS), characterized by postprandial fullness and early satiation, and epigastric pain syndrome (EPS), characterized by epigastric pain or burning. Epidemiological studies in the USA and Europe confirmed the presence of both subgroups, with good separation between EPS and PDS. By contrast, other studies have found major overlap between EPS and PDS in patients with functional dyspepsia in specialist care centres in Europe and Asia. Preliminary pathophysiological studies suggest that PDS might be characterized by a higher prevalence of impaired gastric accommodation than EPS and raised duodenal eosinophil counts. Whether different treatment approaches are needed for EPS and PDS is currently unclear; only acotiamide, a new drug for the treatment of functional dyspepsia, has been found to be efficacious in PDS but not in EPS. Further randomized controlled trials testing treatment response by subgroup are urgently needed. PMID:23399526

  13. Electron transfer precedes ATP hydrolysis during nitrogenase catalysis.

    PubMed

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

    2013-10-01

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

  14. 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

    2011-01-01

    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

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

    SciTech Connect

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

    2012-05-01

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

  16. Treatment outcomes of patients with FIGO Stage I/II uterine cervical cancer treated with definitive radiotherapy: a multi-institutional retrospective research study.

    PubMed

    Ariga, Takuro; Toita, Takafumi; Kato, Shingo; Kazumoto, Tomoko; Kubozono, Masaki; Tokumaru, Sunao; Eto, Hidehiro; Nishimura, Tetsuo; Niibe, Yuzuru; Nakata, Kensei; Kaneyasu, Yuko; Nonoshita, Takeshi; Uno, Takashi; Ohno, Tatsuya; Iwata, Hiromitsu; Harima, Yoko; Wada, Hitoshi; Yoshida, Kenji; Gomi, Hiromichi; Numasaki, Hodaka; Teshima, Teruki; Yamada, Shogo; Nakano, Takashi

    2015-09-01

    The purpose of this study was to analyze the patterns of care and outcomes of patients with FIGO Stage I/II cervical cancer who underwent definitive radiotherapy (RT) at multiple Japanese institutions. The Japanese Radiation Oncology Study Group (JROSG) performed a questionnaire-based survey of their cervical cancer patients who were treated with definitive RT between January 2000 and December 2005. A total of 667 patients were entered in this study. Although half of the patients were considered suitable for definitive RT based on the clinical features of the tumor, about one-third of the patients were prescribed RT instead of surgery because of poor medical status. The RT schedule most frequently utilized was whole-pelvic field irradiation (WP) of 30 Gy/15 fractions followed by WP with midline block of 20 Gy/10 fractions, and high-dose-rate intracavitary brachytherapy (HDR-ICBT) of 24 Gy/4 fractions prescribed at point A. Chemotherapy was administered to 306 patients (46%). The most frequent regimen contained cisplatin (CDDP). The median follow-up time for all patients was 65 months (range, 2-135 months). The 5-year overall survival (OS), pelvic control (PC) and disease-free survival (DFS) rates for all patients were 78%, 90% and 69%, respectively. Tumor diameter and nodal status were significant prognostic indicators for OS, PC and DFS. Chemotherapy has potential for improving the OS and DFS of patients with bulky tumors, but not for non-bulky tumors. This study found that definitive RT for patients with Stage I/II cervical cancer achieved good survival outcomes. PMID:26109680

  17. Treatment outcomes of patients with FIGO Stage I/II uterine cervical cancer treated with definitive radiotherapy: a multi-institutional retrospective research study

    PubMed Central

    Ariga, Takuro; Toita, Takafumi; Kato, Shingo; Kazumoto, Tomoko; Kubozono, Masaki; Tokumaru, Sunao; Eto, Hidehiro; Nishimura, Tetsuo; Niibe, Yuzuru; Nakata, Kensei; Kaneyasu, Yuko; Nonoshita, Takeshi; Uno, Takashi; Ohno, Tatsuya; Iwata, Hiromitsu; Harima, Yoko; Wada, Hitoshi; Yoshida, Kenji; Gomi, Hiromichi; Numasaki, Hodaka; Teshima, Teruki; Yamada, Shogo; Nakano, Takashi

    2015-01-01

    The purpose of this study was to analyze the patterns of care and outcomes of patients with FIGO Stage I/II cervical cancer who underwent definitive radiotherapy (RT) at multiple Japanese institutions. The Japanese Radiation Oncology Study Group (JROSG) performed a questionnaire-based survey of their cervical cancer patients who were treated with definitive RT between January 2000 and December 2005. A total of 667 patients were entered in this study. Although half of the patients were considered suitable for definitive RT based on the clinical features of the tumor, about one-third of the patients were prescribed RT instead of surgery because of poor medical status. The RT schedule most frequently utilized was whole-pelvic field irradiation (WP) of 30 Gy/15 fractions followed by WP with midline block of 20 Gy/10 fractions, and high-dose-rate intracavitary brachytherapy (HDR-ICBT) of 24 Gy/4 fractions prescribed at point A. Chemotherapy was administered to 306 patients (46%). The most frequent regimen contained cisplatin (CDDP). The median follow-up time for all patients was 65 months (range, 2135 months). The 5-year overall survival (OS), pelvic control (PC) and disease-free survival (DFS) rates for all patients were 78%, 90% and 69%, respectively. Tumor diameter and nodal status were significant prognostic indicators for OS, PC and DFS. Chemotherapy has potential for improving the OS and DFS of patients with bulky tumors, but not for non-bulky tumors. This study found that definitive RT for patients with Stage I/II cervical cancer achieved good survival outcomes. PMID:26109680

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

    PubMed Central

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

    2015-01-01

    Background Several approaches have been proposed for risk-stratification and primary prevention of coronary heart disease (CHD), but their comparative and cost-effectiveness is unknown. Methods We constructed a state-transition microsimulation model to compare multiple approaches to the primary prevention of CHD in a simulated cohort of men aged 45–75 and women 55–75. Risk-stratification strategies included the 2013 American College of Cardiology/American Heart Association (ACC/AHA) guidelines on the treatment of blood cholesterol, the Adult Treatment Panel (ATP) III guidelines, and approaches based on coronary artery calcium (CAC) scoring and C-reactive protein (CRP). Additionally we assessed a treat-all strategy in which all individuals were prescribed either moderate-dose or high-dose statins and all males received low-dose aspirin. Outcome measures included CHD events, costs, medication-related side effects, radiation-attributable cancers, and quality-adjusted-life-years (QALYs) over a 30-year timeframe. Results Treat-all with high-dose statins dominated all other strategies for both men and women, gaining 15.7 million QALYs, preventing 7.3 million myocardial infarctions, and saving over $238 billion, compared to the status quo, far outweighing its associated adverse events including bleeding, hepatitis, myopathy, and new-onset diabetes. ACC/AHA guidelines were more cost-effective than ATP III guidelines for both men and women despite placing 8.7 million more people on statins. For women at low CHD risk, treat-all with high-dose statins was more likely to cause a statin-related adverse event than to prevent a CHD event. Conclusions Despite leading to a greater proportion of the population placed on statin therapy, the ACC/AHA guidelines are more cost-effective than ATP III. Even so, at generic prices, treating all men and women with statins and all men with low-dose aspirin appears to be more cost-effective than all risk-stratification approaches for the primary prevention of CHD. Especially for low-CHD risk women, decisions on the appropriate primary prevention strategy should be based on shared decision making between patients and healthcare providers. PMID:26422204

  19. Optogenetic control of ATP release

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

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

  20. Type III and type IV endoleak: toward a complete definition of blood flow in the sac after endoluminal AAA repair.

    PubMed

    White, G H; May, J; Waugh, R C; Chaufour, X; Yu, W

    1998-11-01

    In this document the authors continue to refine their seminal categorization of endoleak, a major complication of endovascular aneurysm repair. In addition to type I (related to the graft device itself) and type II (retrograde flow from collateral branches) endoleak, they propose two new categories: endoleak due to fabric tears, graft disconnection, or disintegration would be classified type III, and flow through the graft presumed to be associated with graft wall "porosity" would be categorized as type IV endoleak. PMID:9867318

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

    SciTech Connect

    Schubert,H.; Hill, C.

    2006-01-01

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

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

    PubMed

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

    2016-06-01

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

  3. Energy transduction in ATP synthase

    NASA Astrophysics Data System (ADS)

    Elston, Timothy; Wang, Hongyun; Oster, George

    1998-01-01

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

  4. Does ATP cross the cell plasma membrane.

    PubMed Central

    Chaudry, I. H.

    1982-01-01

    Although there is an abundance of evidence which indicates that ATP is released as well as taken up by cells, the concept that ATP cannot cross the cell membrane has tended to prevail. This article reviews the evidence for the release as well as uptake of ATP by cells. The evidence presented by various investigators clearly indicates that ATP can cross the cell membrane and suggests that the release and uptake of ATP are physiological processes. PMID:7051582

  5. Extracellular ATP signaling in plants

    PubMed Central

    Tanaka, Kiwamu; Gilroy, Simon; Jones, Alan M.; Stacey, Gary

    2016-01-01

    Extracellular adenosine-5′-triphosphate (ATP) induces a number of cellular responses in plants and animals. Some of the molecular components for purinergic signaling in animal cells appear to be lacking in plant cells, although some cellular responses are similar in both systems [e.g. increased levels of cytosolic free calcium, nitric oxide (NO), and reactive oxygen species (ROS)]. The purpose of this review is to compare and contrast purinergic signaling mechanisms in animal and plant cells. This comparison will aid our overall understanding of plant physiology and also provide details of the general fundamentals of extracellular ATP signaling in eukaryotes. PMID:20817461

  6. Phase I/II study of definitive radiotherapy and chemotherapy (cisplatin and 5-fluorouracil) for advanced or recurrent gynecologic malignancies. Preliminary report.

    PubMed

    Kuske, R R; Perez, C A; Grigsby, P W; Lovett, R D; Jacobs, A J; Galakatos, A E; Camel, H M; Kao, M S

    1989-12-01

    Twenty-three patients with advanced gynecologic malignancy were treated with definitive irradiation and synchronous sensitizing chemotherapy (CT) consisting of cisplatin (CDDP), 50 mg/m2 i.v. rapid infusion, and a 5-day continuous infusion of 5-fluorouracil (5-FU), 750 mg/m2/day. A total of three cycles were administered every 3-4 weeks. Fifteen patients had primary cervical epidermoid carcinoma (three bulky stage IIB, one stage IIIA, ten stage IIIB, one stage IV), four had pelvic recurrences of carcinoma of the cervix, two had endometrial adenocarcinomas (stage IV), and two had vulvar epidermoid carcinoma (one stage III and one stage IV). Radiotherapy (RT) for implantable tumors consisted of 2,000 cGy whole pelvis, 3,000-4,000 cGy split field, and two intracavitary or interstitial insertions, resulting in a total dose of 7,500-8,000 cGy to point A. Three courses of CT were delivered simultaneously with irradiation of the central bulk of tumor: during the first week of whole pelvis RT and with each of the two brachytherapy procedures. Nonimplantable tumors were treated with protracted external beam RT (5,500 cGy tumor dose) and three courses of CT during weeks 1, 4, and 7 of RT. Twenty-one of 23 patients completed RT and 18 of 23 patients completed CT as planned, but half had delays in either RT or CT. Grade 2 or 3 late sequelae consisted of leg edema (one patient), proctosigmoiditis (one patient), bowel obstruction (one patient), vesicovaginal fistula (one patient), and pulmonary embolus (two--one fatal). The incidence of grade 2 and 3 sequelae were 18 and 22%, respectively. With 1-3 years of follow-up evaluation, 12 of 23 (52%) patients are free of disease, and 9 of 22 evaluable patients (41%) have had failure within the pelvis. We conclude that high-dose definitive RT can be delivered with synchronous CDDP and 5-FU at the doses given, with acceptable toxicity. Further study is required to evaluate the impact of radiosensitization on tumor control and late morbidity of therapy. Optimization of irradiation and drug doses as well as the best schedules that may enhance the interaction of these two modalities should be further investigated. PMID:2686392

  7. Comparison of the prevalence of metabolic syndrome and its association with diabetes and cardiovascular disease in the rural population of Bangladesh using the modified National Cholesterol Education Program Expert Panel Adult Treatment Panel III and International Diabetes Federation definitions

    PubMed Central

    Bhowmik, Bishwajit; Afsana, Faria; Siddiquee, Tasnima; Munir, Sanjida B; Sheikh, Fareeha; Wright, Erica; Bhuiyan, Farjana R; Ashrafuzzaman, Sheikh Mohammad; Mahtab, Hajera; Azad Khan, Abul Kalam; Hussain, Akhtar

    2015-01-01

    Aims/Introduction To compare the prevalence of metabolic syndrome (MS) using the modified National Cholesterol Education Program Adult Treatment Plan III (NCEP) and the International Diabetes Federation (IDF) definitions and, using both definitions, determine and compare the association of MS, prediabetes, type 2 diabetes, hypertension (HTN) and cardiovascular disease risk (CVD). Materials and Methods A total of 2,293 randomly selected participants (aged ≥20 years) in a rural community in Bangladesh were investigated in a population-based cross-sectional study. Sociodemographic and anthropometric characteristics, blood pressure, blood glucose, and lipid profiles were studied. Age-adjusted data for MS and cardiometabolic risk factors were assessed, and their relationships were examined. Results The age-adjusted prevalence of MS was 30.7% (males 30.5%; females 30.5%) using the NCEP definition, and 24.5% (males 19.2%, females 27.5%) using the IDF definition. The prevalence of MS using the NCEP definition was also higher in study participants with prediabetes, type 2 diabetes, HTN and CVD risk. The agreement rate between both definitions was 92% (k = 0.80). The NCEP definition had a stronger association with type 2 diabetes and HTN (odds ratio 12.4 vs 5.2; odds ratio 7.0 vs 4.7, respectively) than the IDF definition. However, the odds ratios for prediabetes and CVD risk were not significantly different. Conclusions The prevalence of MS was higher using the NCEP definition, and was more strongly associated with prediabetes, type 2 diabetes, HTN and CVD in this Bangladeshi population. PMID:25969712

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

    SciTech Connect

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

    1999-05-15

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

  9. 7 CFR 3300.4 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... transport equipment that meets the definitions and standards set forth in ATP, Annex 1, including, but not limited to, railcars, trucks, trailers, semitrailers, and intermodal freight containers that have...

  10. Circadian regulation of ATP release in astrocytes.

    PubMed

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

    2011-06-01

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

  11. Circadian regulation of ATP release in astrocytes

    PubMed Central

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

    2011-01-01

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

  12. ATPase Activity Measurements Using Radiolabeled ATP.

    PubMed

    Swarts, Herman G P; Koenderink, Jan B

    2016-01-01

    ATP provides the energy that is essential for all P-type ATPases to actively transport their substrates against an existing gradient. This ATP hydrolysis can be measured using different methods. Here, we describe a method that uses radiolabeled [γ-(32)P]ATP, which is hydrolyzed by P-type ATPases to ADP and (32)Pi. Activated charcoal is used to bind the excess of [γ-(32)P]ATP, which can be separated from the unbound (32)Pi by centrifugation. With this method, a wide range (0.1 μM-10 mM) of ATP can be used. In addition, we also describe in detail how ATP hydrolysis is translated into ATPase activity. PMID:26695028

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

    PubMed

    Krah, Alexander; Takada, Shoji

    2016-04-01

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

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

    PubMed

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

    2012-08-21

    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 F(1)-ATPase and actin whereas compact structures are favored in motor proteins such as RNA polymerase and DNA helicase. The incorporation of Mg(2+) 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. PMID:22920142

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

    NASA Astrophysics Data System (ADS)

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

    2012-08-01

    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.

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

    PubMed Central

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

    2008-01-01

    Reconstruction after multilevel decompression of the cervical spine, especially in the weakened osteoporotic, neoplastic or infectious spine often requires circumferential stabilization and fusion. To avoid the additional posterior surgery in these cases while increasing rigidity of anterior-only screw-plate constructs, the authors introduce the concept of anterior transpedicular screw (ATPS) fixation. We demonstrated its morphological feasibility as well as its indications in a previous study in Part I of our project. Consequently, the objectives of the current study were to assess the ex vivo accuracy of placing ATPS into the cervical vertebra as well as the biomechanical performance of ATPS in comparison to traditional vertebral body screws (VBS) in terms of pull-out strength (POS). Twenty-three ATPS were inserted alternately to two screws into the pedicles and vertebral bodies, respectively, of six cadaveric specimens from C3–T1. For insertion of ATPS, a manual fluoroscopically assisted technique was used. Pre- and post insertional CT-scans were used to assess accuracy of ATPS insertion in the axial and sagittal planes. A newly designed grading system and accuracy score were used to delineate accuracy of ATPS insertion. Following insertion of screws, 23 ATPS and 22 VBS were subjected to pull-out testing (POT). The bone mineral density (BMD) of each specimen was assessed prior to POT. Statistical analysis showed that the incidence of correctly placed screws and non-critical pedicles breaches in axial plane was 78.3%, and 95.7% in sagittal plane. Hence, according to our definition of “critical” pedicle breach that exposes neurovascular structures at risk, 21.7% (n = 5) of all ATPS inserted showed a critical pedicle breach in axial plane. Notably, no critical pedicle perforation occurred at the C6 to T1 levels. Pull-out testing of ATPS and VBS revealed that pull-out resistance of ATPS was 2.5-fold that of VBS. Mean POS of 23 ATPS with a mean BMD of 0.566 g/cm2 and a mean osseus screw purchase of 27.2 mm was 467.8 N. In comparison, POS of 22 VBS screws with a mean BMD of 0.533 g/cm2 and a mean osseus screw purchase of 16.0 mm was 181.6 N. The difference in ultimate pull-out strength between the ATPS and VBS group was significant (p < 0.000001). Also, accuracy of ATPS placement in axial plane was shown to be significantly correlated with POS. In contrast, there was no correlation between screw-length, BMD, or level of insertion and the POS of ATPS or VBS. The study demonstrated that the use of ATPS might be a new technique worthy of further investigation. The use of ATPS shows the potential to increase construct rigidity in terms of screw-plate pull-out resistance. It might diminish construct failures during anterior-only reconstructions of the highly unstable decompressed cervical spine. Electronic supplementary material The online version of this article (doi:10.1007/s00586-007-0573-x) contains supplementary material, which is available to authorized users. PMID:18224357

  17. ATP-triggered anticancer drug delivery

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  18. Light-driven production of ATP catalysed by F0F1-ATP synthase in an artificial photosynthetic membrane

    NASA Astrophysics Data System (ADS)

    Steinberg-Yfrach, Gali; Rigaud, Jean-Louis; Durantini, Edgardo N.; Moore, Ana L.; Gust, Devens; Moore, Thomas A.

    1998-04-01

    Energy-transducing membranes of living organisms couple spontaneous to non-spontaneous processes through the intermediacy of protonmotive force (p.m.f.) - an imbalance in electrochemical potential of protons across the membrane. In most organisms, p.m.f. is generated by redox reactions that are either photochemically driven, such as those in photosynthetic reaction centres, or intrinsically spontaneous, such as those of oxidative phosphorylation in mitochondria. Transmembrane proteins (such as the cytochromes and complexes I, III and IV in the electron-transport chain in the inner mitochondrial membrane) couple the redox reactions to proton translocation, thereby conserving a fraction of the redox chemical potential as p.m.f. Many transducer proteins couple p.m.f. to the performance of biochemical work, such as biochemical synthesis and mechanical and transport processes. Recently, an artificial photosynthetic membrane was reported in which a photocyclic process was used to transport protons across a liposomal membrane, resulting in acidification of the liposome's internal volume. If significant p.m.f. is generated in this system, then incorporating an appropriate transducer into the liposomal bilayer should make it possible to drive a non-spontaneous chemical process. Here we report the incorporation of FOF1-ATP synthase into liposomes containing the components of the proton-pumping photocycle. Irradiation of this artificial membrane with visible light results in the uncoupler- and inhibitor-sensitive synthesis of adenosine triphosphate (ATP) against an ATP chemical potential of ~12kcalmol-1, with a quantum yield of more than 7%. This system mimics the process by which photosynthetic bacteria convert light energy into ATP chemical potential.

  19. Thermodynamics of proton transport coupled ATP synthesis.

    PubMed

    Turina, Paola; Petersen, Jan; Gräber, Peter

    2016-06-01

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

  20. A new function for ATP: activating cardiac sympathetic afferents during myocardial ischemia

    PubMed Central

    Longhurst, John C.

    2010-01-01

    Myocardial ischemia activates cardiac sympathetic afferents leading to chest pain and reflex cardiovascular responses. Brief myocardial ischemia leads to ATP release in the interstitial space. Furthermore, exogenous ATP and α,β-methylene ATP (α,β-meATP), a P2X receptor agonist, stimulate cutaneous group III and IV sensory nerve fibers. The present study tested the hypothesis that endogenous ATP excites cardiac afferents during ischemia through activation of P2 receptors. Nerve activity of single unit cardiac sympathetic afferents was recorded from the left sympathetic chain or rami communicates (T2-T5) in anesthetized cats. Single fields of 45 afferents (conduction velocities = 0.25–4.92 m/s) were identified in the left ventricle with a stimulating electrode. Five minutes of myocardial ischemia stimulated 39 of 45 cardiac afferents (8 Aδ, 37 C fibers). Epicardial application of ATP (1–4 μmol) stimulated six ischemically sensitive cardiac afferents in a dose-dependent manner. Additionally, epicardial ATP (2 μmol), ADP (2 μmol), a P2Y agonist, and α,β-meATP (0.5 μmol) significantly activated eight other ischemically sensitive afferents. Third, pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid, a P2 receptor antagonist, abolished the responses of six afferents to epicardial ATP (2 μmol) and attenuated the ischemia-related increase in activity of seven other afferents by 37%. In the absence of P2 receptor blockade, cardiac afferents responded consistently to repeated application of ATP (n = 6) and to recurrent myocardial ischemia (n = 6). Finally, six ischemia-insensitive cardiac spinal afferents did not respond to epicardial ATP (2–4 μmol), although these afferents did respond to epicardial bradykinin. Taken together, these data indicate that, during ischemia, endogenously released ATP activates ischemia-sensitive, but not ischemia-insensitive, cardiac spinal afferents through stimulation of P2 receptors likely located on the cardiac sensory neurites. PMID:20870803

  1. Expression in mouse kidney of membrane copper transporters Atp7a and Atp7b.

    PubMed

    Moore, Steven D P; Cox, Diane W

    2002-01-01

    Copper is essential for activity of many enzymes, but is toxic in excess. Several copper proteins are required for copper homeostasis. ATP7A and ATP7B are genes encoding membrane copper transporters. ATP7A, defective in Menkes disease (MNK), is expressed in many tissues involved primarily in copper uptake from dietary sources. ATP7B, defective in Wilson disease (WND), is essential for copper excretion. Although MNK patients have a copper deficiency in most tissues, copper accumulates in proximal tubules in the kidney. WND patients also have copper accumulation in the proximal tubules. In some WND patients this copper accumulation may result in tubular dysfunction, resulting in the increased excretion of low molecular weight substances (e.g. amino acids and calcium). In mouse, we have demonstrated, by in situ hybridization, the expression pattern in the kidney of mouse orthologues, Atp7a and Atp7b, and have confirmed Atp7b expression by immunohistochemistry. Both Atp7a and Atp7b are expressed in glomeruli; however, Atp7b is also seen in the kidney medulla. This suggests that glomeruli are responsible for regulating copper levels in the filtrate. In WND patients, urinary copper levels are extremely high suggesting Atp7b in the loops of Henle may have a role in copper reabsorption. PMID:12372948

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

    PubMed

    Cavieres, J D; Glynn, I M

    1979-12-01

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

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

    PubMed Central

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

    2015-01-01

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

  4. ATP-dependent nucleosome remodeling.

    PubMed

    Becker, Peter B; Hrz, Wolfram

    2002-01-01

    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

  5. Interactions of caged-ATP and photoreleased ATP with alkaline phosphatase.

    PubMed

    Zhang, Le; Buchet, Rene; Azzar, Gerard

    2005-03-11

    Photolytic release of ATP from inactive P(3)-[1-(2-nitrophenyl)]ethyl ester of ATP (NPE-caged ATP) provides a means to reveal molecular interactions between nucleotide and enzyme by using infrared spectroscopy. Reaction-induced infrared difference spectra of bovine intestinal alkaline phosphatase (BIAP) and of NPE-caged ATP revealed small structural alterations on the peptide backbone affecting one or two amino-acid residues. After photorelease of ATP, the substrate could be hydrolyzed sequentially by the enzyme producing three Pi, adenosine, and the photoproduct nitrosoacetophenone. It was concluded that NPE-caged ATP could bind to BIAP prior to the photolytic cleavage of ATP and that Pi could interact with BIAP after photolysis of NPE-caged ATP and hydrolysis, yielding infrared spectra with distinct structure changes of BIAP. This suggests that the molecular mechanism of ATP hydrolysis by BIAP involved small structural adjustments of the peptide backbone in the vicinity of the active site during ATP hydrolysis which continued during Pi binding. PMID:15694389

  6. Cleanup MAC and MBA code ATP

    SciTech Connect

    Russell, V.K.

    1994-10-17

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

  7. The Role of ATP in Sleep Regulation

    PubMed Central

    Chikahisa, Sachiko; Séi, Hiroyoshi

    2011-01-01

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

  8. Customized ATP towpreg. [Automated Tow Placement

    NASA Technical Reports Server (NTRS)

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

    1992-01-01

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

  9. An RNA motif that binds ATP

    NASA Technical Reports Server (NTRS)

    Sassanfar, M.; Szostak, J. W.

    1993-01-01

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

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

    SciTech Connect

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

    2015-01-01

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

  11. Decreased ATP cost of isometric contractions in ATP-depleted rat fast-twitch muscle.

    PubMed

    Foley, J M; Harkema, S J; Meyer, R A

    1991-11-01

    ATP was depleted to inosine 5'-monophosphate (IMP) in gastrocnemius muscles of anesthetized rats during two consecutive bouts of intermittent tetanic stimulation (each 1 Hz at 100 Hz, 100-ms duration, for 3 min) separated by 5 min. During a subsequent 75-min recovery period, resynthesis of adenine nucleotide from IMP was inhibited in one group of muscles with hadacidin (N-formyl-N-hydroxyaminoacetate, total dose 300 mg/kg ip), an inhibitor of adenylosuccinate synthase. In vivo 31P-nuclear magnetic resonance spectra and analysis of muscle extracts confirmed that ATP was reduced by over 30% in hadacidin-treated muscles compared both with nonstimulated muscles and with identically stimulated and rested control muscles in saline-injected rats. After the recovery period, calculated [ADP] in ATP-depleted muscles was half that in controls, while phosphorylation potential [PP = ln([ATP]/[ADP] [Pi]), where Pi is inorganic phosphate] and twitch force were similar. During a final 8 min of 0.75-Hz twitch stimulation, [ADP] and the initial rate of phosphocreatine (PCr) hydrolysis were less in ATP-depleted compared with control muscles, while changes in PP and twitch force were similar in the two groups. During a final 3 min of tetanic stimulation, ATP, PCr, and pH decreased less in ATP-depleted compared with control muscles. The results indicate that the ATP cost of isometric contraction is decreased by acute ATP depletion. PMID:1951672

  12. ATP Synthesis in the Extremely Halophilic Bacteria

    NASA Technical Reports Server (NTRS)

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

    1994-01-01

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

  13. Differential patterns of peroxynitrite mediated apoptosis in proximal tubular epithelial cells following ATP depletion recovery

    PubMed Central

    Nilakantan, Vani; Liang, Huanling; Maenpaa, Cheryl J.; Johnson, Christopher P.

    2008-01-01

    Ischemia-reperfusion injury (IRI) is characterized by ATP depletion in the ischemic phase, followed by a rapid increase in reactive oxygen species, including peroxynitrite in the reperfusion phase. In this study, we examined the role of peroxynitrite on cytotoxicity and apoptosis in an in vitro model of ATP depletion-recovery. Porcine proximal tubular epithelial (LLC-PK1) cells were ATP depleted for either 2 h (2/2) or 4 h (4/2) followed by recovery in serum free medium for 2 h. A subset of cells was treated with 100 μM of the peroxynitrite scavenger, iron (III) tetrakis (N-methyl-4′pyridyl) porphyrin pentachloride (FeTMPyP) 30 min prior to and during treatment/recovery. Treatment with FeTMPyP reduced cytotoxicity and superoxide levels at both the 2/2 and 4/2 time points, however FeTMPyP decreased nitric oxide only at the 2/2 time point. FeTMPyP also partially blocked caspase-3 and caspase-8 activation at both 2/2 and 4/2 time points. At the 4/2 time point, FeTMPyP also partially inhibited the ATP depletion mediated increase in tumor necrosis factor alpha (TNF-α) and decreased Bax and FasL gene expression. These data show that peroxynitrite induces apoptosis by activation of multiple pathways depending on length and severity of insult following ATP depletion-recovery. PMID:18357533

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  15. Mechanisms for ATP-dependent chromatin remodelling.

    PubMed

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

    2000-01-01

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

  16. Assembly of the Rotor Component of Yeast Mitochondrial ATP Synthase Is Enhanced When Atp9p Is Supplied by Atp9p-Cox6p Complexes*

    PubMed Central

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

    2014-01-01

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

  17. BRET-linked ATP assay with luciferase.

    PubMed

    Borghei, Golnaz; Hall, Elizabeth A H

    2014-09-01

    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

  18. Dynein ATPase pathway: ATP analogs and regulation by phosphorylation

    SciTech Connect

    Chilcote, T.J.

    1988-01-01

    Three biochemical aspects of 22S dynein from Tetrahymena cilia have been investigated: its ATP binding polypeptides and the manner in which they bind ATP, its AMPPNP-induced dissociation from microtubules, and its phosphorylation. We have attempted to identify the polypeptides of dynein that bind ATP, i.e., the active site polypeptides, with the photoaffinity ATP analog 8-N{sub 3}ATP. The 8-N{sub 3}ATP has been shown to bind to dyneins active sites and in a manner similar to that of ATP. Upon irradiation, (2-{sup 3}H)8-N{sub 3}ATP covalently labels the three heavy chains, i.e., heads, which is detected by autoradiography of SDS PAG's. Thus, the three heads are considered to be the three active sites of dynein. AMPPNP is a nonhydrolyzable ATP analog which we have assayed for the ability to induce dynein dissociation from microtubules.

  19. Sulfide-based ATP production in Urechis unicinctus

    NASA Astrophysics Data System (ADS)

    Ma, Zhuojun; Bao, Zhenmin; Wang, Sifeng; Zhang, Zhifeng

    2010-05-01

    We measured sulfide-based ATP production by isolated mitochondria from four tissues of Urechis unicinctus and the effects of inhibitors of respiratory complexes on ATP production were evaluated. The results show that these mitochondria could oxidize sulfide to produce ATP. The yield of sulfide-stimulated ATP varied from 5 nmol ATP/min/mg to 90 nmol ATP/min/mg according to the sulfide concentration and the source of the mitochondria. The maximum ATP synthesis occurred in hindgut mitochondria using 5 μmol/L sulfide as a substrate. The effects of inhibitors (Rotenone, Antimycin A, Cyanide, and Salicylhydroxamic acid) on mitochondrial ATP production varied with the source of the mitochondria. Our results indicate that sulfide-based ATP production and the associated electron transport pathway are tissue-specific in U. unicinctus.

  20. The Structural Basis of ATP as an Allosteric Modulator

    PubMed Central

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

    2014-01-01

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

  1. ATP-sensitive potassium channel (K(ATP))-dependent regulation of cardiotropic viral infections.

    PubMed

    Eleftherianos, Ioannis; Won, Sungyong; Chtarbanova, Stanislava; Squiban, Barbara; Ocorr, Karen; Bodmer, Rolf; Beutler, Bruce; Hoffmann, Jules A; Imler, Jean-Luc

    2011-07-19

    The effects of the cellular environment on innate immunity remain poorly characterized. Here, we show that in Drosophila ATP-sensitive potassium channels (K(ATP)) mediate resistance to a cardiotropic RNA virus, Flock House virus (FHV). FHV viral load in the heart rapidly increases in K(ATP) mutant flies, leading to increased viremia and accelerated death. The effect of K(ATP) channels is dependent on the RNA interference genes Dcr-2, AGO2, and r2d2, indicating that an activity associated with this potassium channel participates in this antiviral pathway in Drosophila. Flies treated with the K(ATP) agonist drug pinacidil are protected against FHV infection, thus demonstrating the importance of this regulation of innate immunity by the cellular environment in the heart. In mice, the Coxsackievirus B3 replicates to higher titers in the hearts of mayday mutant animals, which are deficient in the Kir6.1 subunit of K(ATP) channels, than in controls. Together, our data suggest that K(ATP) channel deregulation can have a critical impact on innate antiviral immunity in the heart. PMID:21719711

  2. A reusable prepositioned ATP reaction chamber

    NASA Technical Reports Server (NTRS)

    Hoffman, D. G.

    1972-01-01

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

  3. Torque generation mechanism of ATP synthase

    NASA Astrophysics Data System (ADS)

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

    2010-03-01

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

  4. Electric Field Driven Torque in ATP Synthase

    PubMed Central

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

    2013-01-01

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

  5. Effect of ATP on actin filament stiffness.

    PubMed

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

    1990-09-01

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

  6. ATP specifically drives refolding of non-native conformations of cytochrome c.

    PubMed

    Sinibaldi, Federica; Mei, Giampiero; Polticelli, Fabio; Piro, M Cristina; Howes, Barry D; Smulevich, Giulietta; Santucci, Roberto; Ascoli, Franca; Fiorucci, Laura

    2005-04-01

    An increasing body of evidence ascribes to misfolded forms of cytochrome c (cyt c) a role in pathophysiological events such as apoptosis and disease. Here, we examine the conformational changes induced by lipid binding to horse heart cyt c at pH 7 and study the ability of ATP (and other nucleotides) to refold several forms of unfolded cyt c such as oleic acid-bound cyt c, nicked cyt c, and acid denatured cyt c. The CD and fluorescence spectra demonstrate that cyt c unfolded by oleic acid has an intact secondary structure, and a disrupted tertiary structure and heme environment. Furthermore, evidence from the Soret CD, electronic absorption, and resonance Raman spectra indicates the presence of an equilibrium of at least two low-spin species having distinct heme-iron(III) coordination. As a whole, the data indicate that binding of cyt c to oleic acid leads to a partially unfolded conformation of the protein, resembling that typical of the molten globule state. Interestingly, the native conformation is almost fully recovered in the presence of ATP or dATP, while other nucleotides, such as GTP, are ineffective. Molecular modeling of ATP binding to cyt c and mutagenesis experiments show the interactions of phosphate groups with Lys88 and Arg91, with adenosine ring interaction with Glu62 explaining the unfavorable binding of GTP. The finding that ATP and dATP are unique among the nucleotides in being able to turn non-native states of cyt c back to native conformation is discussed in the light of cyt c involvement in cell apoptosis. PMID:15741329

  7. Torque Generation and Utilization in Motor Enzyme F0F1-ATP Synthase

    PubMed Central

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

    2012-01-01

    ATP synthase (F0F1) is made of two motors, a proton-driven motor (F0) and an ATP-driven motor (F1), connected by a common rotary shaft, and catalyzes proton flow-driven ATP synthesis and ATP-driven proton pumping. In F1, the central γ subunit rotates inside the α3β3 ring. Here we report structural features of F1 responsible for torque generation and the catalytic ability of the low-torque F0F1. (i) Deletion of one or two turns in the α-helix in the C-terminal domain of catalytic β subunit at the rotor/stator contact region generates mutant F1s, termed F1(1/2)s, that rotate with about half of the normal torque. This helix would support the helix-loop-helix structure acting as a solid “pushrod” to push the rotor γ subunit, but the short helix in F1(1/2)s would fail to accomplish this task. (ii) Three different half-torque F0F1(1/2)s were purified and reconstituted into proteoliposomes. They carry out ATP-driven proton pumping and build up the same small transmembrane ΔpH, indicating that the final ΔpH is directly related to the amount of torque. (iii) The half-torque F0F1(1/2)s can catalyze ATP synthesis, although slowly. The rate of synthesis varies widely among the three F0F1(1/2)s, which suggests that the rate reflects subtle conformational variations of individual mutants. PMID:22128167

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

    SciTech Connect

    Russell, V.K.

    1994-10-17

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

  9. Inhibition of glutamate release by delaying ATP fall accounts for neuroprotective effects of antioxidants in experimental stroke.

    PubMed

    Hurtado, Olivia; De Cristóbal, Javier; Sánchez, Verónica; Lizasoain, Ignacio; Cárdenas, Antonio; Pereira, Marta P; Colado, M Isabel; Leza, Juan C; Lorenzo, Pedro; Moro, María A

    2003-11-01

    Excitotoxic neuronal injury related to excessive glutamate release is believed to play a key role in the pathogenesis of focal cerebral ischemia. Reversal of neuronal glutamate transporters caused by ATP fall and subsequent imbalance of membrane ionic gradients accounts for most glutamate release after cerebral ischemia. ATP synthesis from oxidative phosphorylation derives from the coupled functioning of the mitochondrial respiratory chain (MRC) and the ATP synthase; interestingly, the MRC is one of the main sites of cellular reactive oxygen species (ROS) generation even in physiological circumstances. Hence, we have studied the effect of the antioxidants glutathione, superoxide dismutase, and alpha-tocopherol on infarct outcome, brain ATP, and glutamate levels after permanent middle cerebral artery occlusion (MCAO) in Fischer rats; we have also characterized the actions of antioxidants on MRC complexes. Our results show that intraperitoneal administration of antioxidants 2 h before MCAO enhances ATP synthesis and causes a neuroprotective effect concomitant to inhibition of ischemia-induced increase in brain glutamate. Antioxidants also increased mitochondrial ATP and MRC complex I-III activity and respiration, suggesting that these actions are due to removal of the inhibition caused by endogenous ROS on MRC. These findings may possess important therapeutic repercussions in the management of ischemic stroke. PMID:14500556

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

    PubMed

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

    2016-04-01

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

  11. Genomic Analysis of ATP Efflux in Saccharomyces cerevisiae.

    PubMed

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

    2016-01-01

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

  12. Genomic Analysis of ATP Efflux in Saccharomyces cerevisiae

    PubMed Central

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

    2015-01-01

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

  13. Space shuttle (ATP configuration) abort staging investigation

    NASA Technical Reports Server (NTRS)

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

    1973-01-01

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

  14. ATP-binding cassette transporters in liver.

    PubMed

    Wlcek, Katrin; Stieger, Bruno

    2014-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-03-15

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

  17. ATP-mediated glucosensing by hypothalamic tanycytes

    PubMed Central

    Frayling, Cameron; Britton, Ruth; Dale, Nicholas

    2011-01-01

    Abstract The brain plays a vital role in the regulation of food intake, appetite and ultimately bodyweight. Neurons in the hypothalamic arcuate nucleus, the ventromedial hypothalamic nuclei (VMH) and the lateral hypothalamus are sensitive to a number of circulating signals such as leptin, grehlin, insulin and glucose. These neurons are part of a network that integrates this information to regulate feeding and appetite. Hypothalamic tanycytes contact the cerebral spinal fluid of the third ventricle and send processes into the parenchyma. A subset of tanycytes are located close to, and send processes towards, the hypothalamic nuclei that contain neurons that are glucosensitive and are involved in the regulation of feeding. Nevertheless the signalling properties of tanycytes remain largely unstudied. We now demonstrate that tanycytes signal via waves of intracellular Ca2+; they respond strongly to ATP, histamine and acetylcholine – transmitters associated with the drive to feed. Selective stimulation by glucose of tanycyte cell bodies evokes robust ATP-mediated Ca2+ responses. Tanycytes release ATP in response to glucose. Furthermore tanycytes also respond to non-metabolisable analogues of glucose. Although tanycytes have been proposed as glucosensors, our study provides the first direct demonstration of this hypothesis. Tanycytes must therefore now be considered as active signalling cells within the brain that can respond to a number of neuronally derived and circulating transmitters and metabolites. PMID:21486800

  18. Loss of LRPPRC causes ATP synthase deficiency.

    PubMed

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

    2014-05-15

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

  19. 7 CFR 3300.4 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 15 2010-01-01 2010-01-01 false Definitions. 3300.4 Section 3300.4 Agriculture Regulations of the Department of Agriculture (Continued) OFFICE OF TRANSPORTATION, DEPARTMENT OF AGRICULTURE AGREEMENT ON THE INTERNATIONAL CARRIAGE OF PERISHABLE FOODSTUFFS AND ON THE SPECIAL EQUIPMENT TO BE USED FOR SUCH CARRIAGE (ATP);...

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

    NASA Technical Reports Server (NTRS)

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

    1974-01-01

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

  1. 5 CFR 630.201 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 5 Administrative Personnel 1 2010-01-01 2010-01-01 false Definitions. 630.201 Section 630.201 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL SERVICE REGULATIONS ABSENCE AND LEAVE Definitions and General Provisions for Annual and Sick Leave § 630.201 Definitions. (a) In section 6301(2)(iii)...

  2. ATP synthase of yeast mitochondria. Isolation of subunit j and disruption of the ATP18 gene.

    PubMed

    Arnold, I; Pfeiffer, K; Neupert, W; Stuart, R A; Schägger, H

    1999-01-01

    The subunit composition of the mitochondrial ATP synthase from Saccharomyces cerevisiae was analyzed using blue native gel electrophoresis and high resolution SDS-polyacrylamide gel electrophoresis. We report here the identification of a novel subunit of molecular mass of 6,687 Da, termed subunit j (Su j). An open reading frame of 127 base pairs (ATP18), which encodes for Su j, was identified on chromosome XIII. Su j does not display sequence similarity to ATP synthase subunits from other organisms. Data base searches, however, identified a potential homolog from Schizosaccharomyces pombe with 51% identity to Su j of S. cerevisiae. Su j, a small protein of 59 amino acid residues, has the characteristics of an integral inner membrane protein with a single transmembrane segment. Deletion of the ATP18 gene encoding Su j led to a strain (Deltasu j) completely deficient in oligomycin-sensitive ATPase activity and unable to grow on nonfermentable carbon sources. The presence of Su j is required for the stable expression of subunits 6 and f of the F0 membrane sector. In the absence of Su j, spontaneously arising rho- cells were observed that lacked also ubiquinol-cytochrome c reductase and cytochrome c oxidase activities. We conclude that Su j is a novel and essential subunit of yeast ATP synthase. PMID:9867807

  3. Efficient ATP synthesis by thermophilic Bacillus FoF1-ATP synthase.

    PubMed

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

    2011-08-01

    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 ε-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 μM and 0.55 mM, respectively, which are an order of magnitude lower than previous estimates and are suited to efficient ATP synthesis. PMID:21605343

  4. Efficient ATP synthesis by thermophilic Bacillus FoF1-ATP synthase

    PubMed Central

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

    2011-01-01

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

  5. Real-time luminescence imaging of cellular ATP release.

    PubMed

    Furuya, Kishio; Sokabe, Masahiro; Grygorczyk, Ryszard

    2014-03-15

    Extracellular ATP and other purines are ubiquitous mediators of local intercellular signaling within the body. While the last two decades have witnessed enormous progress in uncovering and characterizing purinergic receptors and extracellular enzymes controlling purinergic signals, our understanding of the initiating step in this cascade, i.e., ATP release, is still obscure. Imaging of extracellular ATP by luciferin-luciferase bioluminescence offers the advantage of studying ATP release and distribution dynamics in real time. However, low-light signal generated by bioluminescence reactions remains the major obstacle to imaging such rapid processes, imposing substantial constraints on its spatial and temporal resolution. We have developed an improved microscopy system for real-time ATP imaging, which detects ATP-dependent luciferin-luciferase luminescence at ∼10 frames/s, sufficient to follow rapid ATP release with sensitivity of ∼10 nM and dynamic range up to 100 μM. In addition, simultaneous differential interference contrast cell images are acquired with infra-red optics. Our imaging method: (1) identifies ATP-releasing cells or sites, (2) determines absolute ATP concentration and its spreading manner at release sites, and (3) permits analysis of ATP release kinetics from single cells. We provide instrumental details of our approach and give several examples of ATP-release imaging at cellular and tissue levels, to illustrate its potential utility. PMID:23973809

  6. ATP promotes extracellular matrix biosynthesis of intervertebral disc cells.

    PubMed

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

    2015-02-01

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

  7. Prevalence of Metabolic Syndrome in Type 2 Diabetes Mellitus Using NCEP-ATPIII, IDF and WHO Definition and Its Agreement in Gwalior Chambal Region of Central India

    PubMed Central

    Yadav, Dhananjay; Mahajan, Sunil; Subramanian, Senthil K.; Bisen, Prakash Singh; Chung, Choon Hee; Prasad, GBKS

    2013-01-01

    The aim of study was to determine the prevalence of metabolic syndrome (MetS) in people with type 2 diabetes mellitus (T2DM). National Cholesterol Education Program (NCEP) ATPIII Criteria, International Diabetes Federation and the World Health Organization (WHO) definitions were used in quantifying the metabolic syndrome and also the concordance between these three criteria’s used for identifying metabolic syndrome. Methods: This cross-sectional study involved 700 type 2 diabetic subjects from the urban areas of Gwalior Chambal region (Central India). Subjects in the age group of 28-87 yrs were included in the study. Type I diabetics, pregnant ladies and those with chronic viral and bacterial infections and serious metabolic disorders were excluded from the study. Fasting blood glucose, Blood lipids (T-cholesterol, triglyceride, HDL-cholesterol) were assessed and anthropometry blood pressure were measured from all the subjects. Results: The Prevalence of metabolic syndrome was found to be 45.8%, 57.7% and 28% following NCEP-ATPIII Criteria, IDF and WHO definitions, respectively. Using all the three definitions the prevalence was higher in women in all age groups. ATP III and IDF criteria showed good agreement (κ 0.68) compared to ATP III with WHO (κ 0.54) and IDF with WHO (κ 0.34) criteria. Highest prevalence was observed following IDF definition. Conclusions: A good agreement was observed between ATPIII and IDF criteria. Maximum prevalence of Metabolic syndrome was recorded when IDF criteria was followed. NCEP-ATPIII criteria for the diagnosis of MetS and this criterion reflected equal importance to the every variable and showed a good agreement between the different criteria used. PMID:24171882

  8. Suppressors of superoxide production from mitochondrial complex III.

    PubMed

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

    2015-11-01

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

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

    SciTech Connect

    Jason Alan Gruenhagen

    2003-12-12

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

  10. Relation of rumen ATP concentration to bacterial and protozoal numbers.

    PubMed Central

    Nuzback, D E; Bartley, E E; Dennis, S M; Nagaraja, T G; Galitzer, S J; Dayton, A D

    1983-01-01

    Cultures of Streptococcus bovis and mixed populations of rumen bacteria were used to investigate the concentration of ATP and rumen bacterial numbers at various stages of growth. ATP, extracted with Tris buffer, was analyzed using the firefly luciferin-luciferase bioluminescent reaction. ATP concentrations of S. bovis and mixed cultures of rumen bacteria significantly correlated with live cell counts during the log phase of growth but not during the stationary phase. The average cellular ATP concentration of rumen bacteria was calculated to be 0.3 fg of ATP per cell. Studies done with in vivo artificial rumen apparatus revealed that the protozoal contribution to rumen fluid ATP pool size was much more substantial than was the bacterial contribution. The rumen fluid ATP concentration was greater in cattle with protozoa than in those that were defaunated. Differences in ATP concentration due to size differences of ciliate protozoa were observed. Due to the unbalanced distribution of ATP in rumen microbes, ATP appears to be an unsuitable indicator of rumen microbial biomass. PMID:6639012

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

    SciTech Connect

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

    2009-01-01

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

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

    PubMed Central

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

    2009-01-01

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

  13. Sequential Action of MalE and Maltose Allows Coupling ATP Hydrolysis to Translocation in the MalFGK2 Transporter.

    PubMed

    Bao, Huan; Dalal, Kush; Cytrynbaum, Eric; Duong, Franck

    2015-10-16

    ATP-binding cassette (ABC) transporters have evolved an ATP-dependent alternating-access mechanism to transport substrates across membranes. Despite important progress, especially in their structural analysis, it is still unknown how the substrate stimulates ATP hydrolysis, the hallmark of ABC transporters. In this study, we measure the ATP turnover cycle of MalFGK2 in steady and pre-steady state conditions. We show that (i) the basal ATPase activity of MalFGK2 is very low because the cleavage of ATP is rate-limiting, (ii) the binding of open-state MalE to the transporter induces ATP cleavage but leaves release of Pi limiting, and (iii) the additional presence of maltose stimulates release of Pi, and therefore increases the overall ATP turnover cycle. We conclude that open-state MalE stabilizes MalFGK2 in the outward-facing conformation until maltose triggers return to the inward-facing state for substrate and Pi release. This concerted action explains why ATPase activity of MalFGK2 depends on maltose, and why MalE is essential for transport. PMID:26338707

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

    PubMed

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

    2015-02-01

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

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

    PubMed Central

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

    2013-01-01

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

  16. Welding III.

    ERIC Educational Resources Information Center

    Allegheny County Community Coll., Pittsburgh, PA.

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

  17. LANDVIEW III

    EPA Science Inventory

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

  18. Welding III.

    ERIC Educational Resources Information Center

    Allegheny County Community Coll., Pittsburgh, PA.

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

  19. SAGE III

    Atmospheric Science Data Center

    2014-12-04

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

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

    SciTech Connect

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

    1994-10-17

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

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

    NASA Astrophysics Data System (ADS)

    Feofilova, Maria; Howard, Jonathon

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

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

    PubMed Central

    Sengupta, Souvik; Ganguly, Agneyo; Roy, Amit; BoseDasgupta, Somdeb; D’Annessa, Ilda; Desideri, Alessandro; Majumder, Hemanta K.

    2011-01-01

    Most type IB topoisomerases do not require ATP and Mg2+ for activity. However, as shown previously for vaccinia topoisomerase I, we demonstrate that ATP stimulates the relaxation activity of the unusual heterodimeric type IB topoisomerase from Leishmania donovani (LdTOP1L/S) in the absence of Mg2+. The stimulation is independent of ATP hydrolysis but requires salt as a co-activator. ATP binds to LdTOP1L/S and increases its rate of strand rotation. Docking studies indicate that the amino acid residues His93, Tyr95, Arg188 and Arg190 of the large subunit may be involved in ATP binding. Site directed mutagenesis of these four residues individually to alanine and subsequent relaxation assays reveal that the R190A mutant topoisomerase is unable to exhibit ATP-mediated stimulation in the absence of Mg2+. However, the ATP-independent relaxation activities of all the four mutant enzymes remain unaffected. Additionally, we provide evidence that ATP binds LdTOP1L/S and modulates the activity of the otherwise ATP-independent enzyme. This study establishes ATP as an activator of LdTOP1L/S in the absence of Mg2+. PMID:21186185

  3. Granule-specific ATP requirements for Ca2+-induced exocytosis in human neutrophils. Evidence for substantial ATP-independent release.

    PubMed

    Theander, Sten; Lew, Daniel P; Nüsse, Oliver

    2002-07-15

    Ca2+-induced exocytosis in neuronal and neuroendocrine cells involves ATP-dependent steps believed to 'prime' vesicles for exocytosis. Primed, docked vesicles are released in response to Ca2+ influx through voltage-gated Ca2+ channels. Neutrophils, however, do not possess voltage-gated Ca2+ channels and appear to have no docked vesicles. Furthermore, neutrophils have several types of granules with markedly different Ca2+ requirements for exocytosis. These differential Ca2+ dependencies were used as a tool to investigate the ATP dependence of different granule populations. Here we demonstrate distinct ATP requirements for release of neutrophil granule populations, with respect to rate as well as amplitude. Intracellular ATP was depleted to various levels, and exocytosis was stimulated with different Ca2+ concentrations and measured with the patch-clamp capacitance technique or by detecting enzyme release. Primary granule exocytosis displayed a distinct ATP dependence with an apparent K(m) of approximately 80 microM ATP and no ATP-independent exocytosis. Release of secondary and tertiary granules displayed a more shallow ATP dependence (K(m) approximately 330 microM), and more than 50% of secondary and tertiary granules appeared not to need ATP at all for their release. Individual granules in human neutrophils have distinct ATP requirements for exocytosis, suggesting that the ATP-sensitive elements are localised to the granules. Primary granule exocytosis has a very low affinity for ATP. Furthermore, substantial ATP-independent exocytosis of secondary and tertiary granule occurs despite the absence of docked granules. These characteristics should help neutrophils to fulfil their bactericidal functions at poorly irrigated sites of infection with low glucose supply. PMID:12082157

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

    PubMed Central

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

    2008-01-01

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

  5. Snapshots of the maltose transporter during ATP hydrolysis

    SciTech Connect

    Oldham, Michael L.; Chen, Jue

    2011-12-05

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

  6. ATP7B detoxifies silver in ciliated airway epithelial cells

    SciTech Connect

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

    2010-03-15

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

  7. Diversity and regulation of ATP sulfurylase in photosynthetic organisms.

    PubMed

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

    2014-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1977-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  10. Diversity and regulation of ATP sulfurylase in photosynthetic organisms

    PubMed Central

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

    2014-01-01

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

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

    SciTech Connect

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

    1990-07-03

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

  12. Mitochondrial K(ATP) channels: role in cardioprotection.

    PubMed

    Oldenburg, Olaf; Cohen, Michael V; Yellon, Derek M; Downey, James M

    2002-08-15

    The role of the mitochondrial ATP-sensitive potassium channel (mK(ATP)) in ischemic preconditioning and cardioprotection is reviewed. A great deal of accumulated evidence implicatese opening of this channel as an important step in the anti-infarct effect of ischemic preconditioning. Recent studies, however, reveal that channel opening can actually serve as a signal transduction element. Data indicate that mK(ATP) opening causes mitochondria to generate reactive oxygen species (ROS) which then activate downstream kinases. Opening of mK(ATP) prior to ischemia can serve as a trigger since the critical time for its opening is prior to the onset of the lethal ischemic insult. Most G(i)-coupled receptors trigger protection through the mK(ATP)/ROS pathway except for the adenosine receptor which uses some other, as yet unidentified, pathway. Possible coupling schemes between the receptors and the mK(ATP) are discussed. Protection from preconditioning can also be aborted when a mK(ATP) blocker is present only during the lethal ischemic insult (mediator phase), but a much higher concentration of the blocker is required. Thus the mK(ATP) probably serves a dual role as both a trigger and a mediator. Possible end-effectors of preconditioning's protection are discussed including the mK(ATP) itself. PMID:12160940

  13. Regulation of mitochondrial ATP synthase in cardiac pathophysiology

    PubMed Central

    Long, Qinqiang; Yang, Kevin; Yang, Qinglin

    2015-01-01

    Mitochondrial function is paramount to energy homeostasis, metabolism, signaling, and apoptosis in cells. Mitochondrial complex V (ATP synthase), a molecular motor, is the ultimate ATP generator and a key determinant of mitochondrial function. ATP synthase catalyzes the final coupling step of oxidative phosphorylation to supply energy in the form of ATP. Alterations at this step will crucially impact mitochondrial respiration and hence cardiac performance. It is well established that cardiac contractility is strongly dependent on the mitochondria, and that myocardial ATP depletion is a key feature of heart failure. ATP synthase dysfunction can cause and exacerbate human diseases, such as cardiomyopathy and heart failure. While ATP synthase has been extensively studied, essential questions related to how the regulation of ATP synthase determines energy metabolism in the heart linger and therapies targeting this important mechanism remain scarce. This review will visit the main findings, identify unsolved issues and provide insights into potential future perspectives related to the regulation of ATP synthase and cardiac pathophysiology. PMID:26064790

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

    PubMed Central

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

    2008-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    Boada, M; Antnez, C; Ramrez-Lorca, R; DeStefano, A L; Gonzlez-Prez, A; Gayn, J; Lpez-Arrieta, J; Ikram, M A; Hernndez, I; Marn, J; Galn, J J; Bis, J C; Maulen, A; Rosende-Roca, M; Moreno-Rey, C; Gudnasson, V; Morn, F J; Velasco, J; Carrasco, J M; Alegret, M; Espinosa, A; Vinyes, G; Lafuente, A; Vargas, L; Fitzpatrick, A L; Launer, L J; Sez, M E; Vzquez, E; Becker, J T; Lpez, O L; Serrano-Ros, M; Trraga, L; van Duijn, C M; Real, L M; Seshadri, S; Ruiz, A

    2014-06-01

    To identify loci associated with Alzheimer disease, we conducted a three-stage analysis using existing genome-wide association studies (GWAS) and genotyping in a new sample. In Stage I, all suggestive single-nucleotide polymorphisms (at P<0.001) in a previously reported GWAS of seven independent studies (8082 Alzheimer's disease (AD) cases; 12?040 controls) were selected, and in Stage II these were examined in an in silico analysis within the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium GWAS (1367 cases and 12904 controls). Six novel signals reaching P<5 10(-6) were genotyped in an independent Stage III sample (the Fundaci 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 Fundaci 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

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

    NASA Technical Reports Server (NTRS)

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

    1996-01-01

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

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

    PubMed

    Yi, Ling; Kaler, Stephen

    2014-05-01

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

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

    NASA Technical Reports Server (NTRS)

    Hochstein, L. I.

    1992-01-01

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

  20. ATP drives eosinophil effector responses through P2 purinergic receptors

    PubMed Central

    Kobayashi, Takehito; Soma, Tomoyuki; Noguchi, Toru; Nakagome, Kazuyuki; Nakamoto, Hidetomo; Kita, Hirohito; Nagata, Makoto

    2015-01-01

    Background Eosinophils recognize various stimuli, such as cytokines, chemokines, immunoglobulins, complement, and external pathogens, resulting in their accumulation in mucosal tissues and the progression of inflammation. Eosinophils are also involved in innate Th2-type immune responses mediated through endogenous danger signals, including IL-33, uric acid (UA), or ATP, in non-sensitized mice exposed to environmental allergens. However, the mechanism involved in eosinophil responses to these danger signals remains insufficiently understood. Methods We examined migration, adhesion, superoxide production and degranulation of human eosinophils. Isolated eosinophils were incubated with monosodium urate (MSU) crystals and ATPγS, a nonhydrolysable ATP analogue. To determine the involvement of P2 or P2Y2 receptors in eosinophil responses to UA and ATP, eosinophils were preincubated with a pan-P2 receptor inhibitor, oxidized ATP (oATP), or anti-P2Y2 antibody before incubation with MSU crystals or ATPγS. Results MSU crystals induced adhesion of eosinophils to recombinant human (rh)-ICAM-1 and induced production of superoxide. oATP abolished eosinophil responses to MSU crystals, suggesting involvement of endogenous ATP and its receptors. Furthermore, exogenous ATP, as ATPγS, induced migration of eosinophils through a model basement membrane, adhesion to rh-ICAM-1, superoxide generation, and degranulation of eosinophil-derived neurotoxin (EDN). oATP and anti-P2Y2 significantly reduced these eosinophil responses. Conclusions ATP serves as an essential mediator of functional responses in human eosinophils. Eosinophil responses to ATP may be implicated in airway inflammation in patients with asthma. PMID:26344078

  1. Effects of organophosphorus compounds on ATP production and mitochondrial integrity in cultured cells.

    PubMed

    Massicotte, C; Knight, K; Van der Schyf, C J; Jortner, B S; Ehrich, M

    2005-01-01

    Recent studies in vivo and in vitro suggested that mitochondrial dysfunction follows exposure to organophosphorus (OP) esters. As mitochondrial ATP production is important for cellular integrity, ATP production in the presence of OP neurotoxicants was examined in a human neuronal cell line (SH-SY5Y neuroblastoma cells) and primary dorsal root ganglia (DRG) cells isolated from chick embryos and subsequently cultured to achieve maturation with axons. These cell culture systems were chosen to evaluate toxic effects on the mitochondrial respiratory chain associated with exposure to OP compounds that do and do not cause OP-induced delayed neuropathy (OPIDN), a disorder preceded by inhibition of neurotoxic esterase (NTE). Concentration- and time-response studies were done in neuroblastoma cells exposed to phenyl saligenin phosphate (PSP) and mipafox, both compounds that readily induce delayed neuropathy in hens, or paraoxon, which does not. Phenylmethylsulfonyl fluoride (PMSF) was included as a non-neuropathic inhibitor of NTE. Purified neuronal cultures from 9 day-old chick embryo DRG were treated for 12 h with 1 microM PSP, mipafox, or paraoxon. In situ evaluation of ATP production measured by bioluminescence assay demonstrated decreased ATP concentrations both in neuroblastoma cells and chick DRG neurons treated with PSP. Mipafox decreased ATP production in DRG but not in SH-SY5Y cells. This low energy state was present at several levels of the mitochondrial respiratory chain, including Complexes I, II, III, and IV, although Complex I was the most severely affected. Paraoxon and PMSF were not effective at all complexes, and, when effective, required higher concentrations than needed for PSP. Results suggest that mitochondria are an important early target for OP compounds, with exposure resulting in depletion of ATP production. The targeting of neuronal, rather than Schwann cell mitochondria in DRG following exposure to PSP and mipafox was verified by loss of the mitochondrial-specific dye, tetramethylrhodamine, in these cells. No such loss was seen in paraoxon exposed neurons isolated from DRG or in Schwann cells treated with any of the test compounds. PMID:15897155

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

    PubMed

    Yoshida, M; Muneyuki, E; Hisabori, T

    2001-09-01

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

  3. Renal epithelial cells can release ATP by vesicular fusion

    PubMed Central

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

    2013-01-01

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

  4. ATP hydrolysis is required to reset the ATP-binding cassette dimer into the resting-state conformation.

    PubMed

    Lu, Gang; Westbrooks, James M; Davidson, Amy L; Chen, Jue

    2005-12-13

    ATP-binding cassette (ABC) transporters couple ATP binding and hydrolysis to the movement of substances across the membrane; conformational changes clearly play an important role in the transporter mechanism. Previously, we have shown that a dimer of MalK, the ATPase subunit of the maltose transporter from Escherichia coli, undergoes a tweezers-like motion in a transport cycle. The MalK monomer consists of an N-terminal nucleotide binding domain and a C-terminal regulatory domain. The two nucleotide-binding domains in a dimer are either open or closed, depending on whether ATP is present, while the regulatory domains maintain contacts to hold the dimer together. In this work, the structure of MalK in a posthydrolysis state is presented, obtained by cocrystallizing MalK with ATP-Mg(2+). ATP was hydrolyzed in the crystallization drop, and ADP-Mg(2+) was found in the resulting crystal structure. In contrast to the ATP-bound form where two ATP molecules are buried in a closed interface between the nucleotide-binding domains, the two nucleotide-binding domains of the ADP-bound form are open, indicating that ADP, unlike ATP, cannot stabilize the closed form. This conclusion is further supported by oligomerization studies of MalK in solution. At low protein concentrations, ATP promotes dimerization of MalK, whereas ADP does not. The structures of dimeric MalK in the nucleotide-free, ATP-bound, and ADP-bound forms provide a framework for understanding the nature of the conformational changes that occur in an ATP-binding cassette transporter hydrolysis cycle, as well as how conformational changes in MalK are coupled to solute transport. PMID:16326809

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

    PubMed Central

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

    2014-01-01

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

  6. The mitochondrial ATP synthase of Trypanosoma brucei: structure and regulation.

    PubMed

    Williams, N

    1994-04-01

    The structure and regulation of the Trypanosoma brucei mitochondrial ATP synthase is reviewed. This enzyme complex which catalyzes the synthesis and hydrolysis of ATP within the mitochondrion is a multisubunit complex which is regulated in several ways. Several lines of evidence have shown that the ATP synthase is regulated through the life cycle of Trypanosoma brucei. The enzyme complex is present at maximal levels in the procyclic form where mitochondrial activity is the highest and cytochromes and Kreb's cycle components are present. The levels of the ATP synthase are decreased in the bloodstream forms where the levels of the mitochondrial cytochromes are absent or substantially decreased. In recent preliminary work we have shown the presence of an ATP synthase inhibitor peptide which may indicate an additional level of complexity to the regulation. PMID:8056784

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

    PubMed Central

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

    2015-01-01

    Sulfur (S) stands fourth in the list of major plant nutrients after N, P, and K. Sulfate (SO42-), a form of soil-S taken up by plant roots is metabolically inert. As the first committed step of S-assimilation, ATP-sulfurylase (ATP-S) catalyzes SO42--activation and yields activated high-energy compound adenosine-5′-phosphosulfate that is reduced to sulfide (S2-) and incorporated into cysteine (Cys). In turn, Cys acts as a precursor or donor of reduced S for a range of S-compounds such as methionine (Met), glutathione (GSH), homo-GSH (h-GSH), and phytochelatins (PCs). Among S-compounds, GSH, h-GSH, and PCs are known for their involvement in plant tolerance to varied abiotic stresses, Cys is a major component of GSH, h-GSH, and PCs; whereas, several key stress-metabolites such as ethylene, are controlled by Met through its first metabolite S-adenosylmethionine. With the major aim of briefly highlighting S-compound-mediated role of ATP-S in plant stress tolerance, this paper: (a) overviews ATP-S structure/chemistry and occurrence, (b) appraises recent literature available on ATP-S roles and regulations, and underlying mechanisms in plant abiotic and biotic stress tolerance, (c) summarizes ATP-S-intrinsic regulation by major S-compounds, and (d) highlights major open-questions in the present context. Future research in the current direction can be devised based on the discussion outcomes. PMID:25904923

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

    PubMed

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

    2015-01-01

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

  9. Intracellular ATP can regulate afferent arteriolar tone via ATP-sensitive K+ channels in the rabbit.

    PubMed Central

    Lorenz, J N; Schnermann, J; Brosius, F C; Briggs, J P; Furspan, P B

    1992-01-01

    Studies were performed to assess whether ATP-sensitive K+ (KATP) channels on rabbit preglomerular vessels can influence afferent arteriolar (AA) tone. K+ channels with a slope conductance of 258 +/- 13 (n = 7) pS and pronounced voltage dependence were demonstrated in excised patches from vascular smooth muscle cells of microdissected preglomerular segments. Channel activity was markedly reduced by 1 mM ATP and in a dose-dependent fashion by glibenclamide (10(-9) M to 10(-6) M), a specific antagonist of KATP channels. 10(-5) M diazoxide, a K+ channel opener, activated these channels in the presence of ATP, and this effect was also blocked by glibenclamide. To determine the role of these KATP channels in the control of vascular tone, diazoxide was tested on isolated perfused AA. After preconstriction from a control diameter of 13.1 +/- 1.1 to 3.5 +/- 2.1 microns with phenylephrine (PE), addition of 10(-5) M diazoxide dilated vessels to 11.2 +/- 0.7 microns, which was not different from control. Further addition of 10(-5) M glibenclamide reconstricted the vessels to 5.8 +/- 1.5 microns (n = 5; P less than 0.03). In support of its specificity for KATP channels, glibenclamide did not reverse verapamil induced dilation in a separate series of experiments. To determine whether intracellular ATP levels can effect AA tone, studies were conducted to test the effect of the glycolytic inhibitor 2-deoxy-D-glucose. After preconstriction from 13.4 +/- 3.2 to 7.7 +/- 1.3 microns with PE, bath glucose was replaced with 6 mM 2-deoxy-D-glucose. Within 10 min, the arteriole dilated to a mean value of 11.8 +/- 1.4 microns (n = 6; NS compared to control). Subsequent addition of 10(-5) M glibenclamide significantly reconstricted the vessels to a diameter of 8.6 +/- 0.5 micron (P less than 0.04). These data demonstrate that KATP channels are present on the preglomerular vasculature and that changes in intracellular ATP can directly influence afferent arteriolar tone via these channels. Images PMID:1522228

  10. Solution structures of the actuator domain of ATP7A and ATP7B, the Menkes and Wilson disease proteins.

    PubMed

    Banci, Lucia; Bertini, Ivano; Cantini, Francesca; Migliardi, Manuele; Natile, Giovanni; Nushi, Fiorentin; Rosato, Antonio

    2009-08-25

    ATP7A and ATP7B are two human P(1B)-type ATPases that have a crucial role in maintaining copper(I) homeostasis. Among the various domains of these enzymes, one, called the Actuator or A-domain, has a regulatory function and is required for the phosphatase step of the catalytic cycle (dephosphorylation of the intermediate formed during ATP hydrolysis). Here we report the solution structures of the A-domain of both proteins, solved by heteronuclear NMR spectroscopy and a characterization of the dynamics of the A-domain of ATP7A. We observed that the catalytically important TGE loop protrudes from the structure ready for interaction with the phosphorylated site in the ATP-binding domain. The loop is rigid, suggesting that the catalytic step does not require substantial structural flexibility or rearrangements. The present structures were useful to rationalize the molecular effects of disease-causing mutations. In particular, it can be concluded that mutations occurring in the A-domain either destabilize the fold of the domain (such as Gly860Val in ATP7A) or affect the network of communication within the domain (such as Leu873Arg in ATP7A) or with the other domains of the enzyme (such as Gly853Arg in ATP7A). PMID:19645496

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2011-12-01

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

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

    PubMed

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

    2015-12-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-05-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-04-01

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

  17. Authentic role of ATP signaling in micturition reflex

    PubMed Central

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

    2016-01-01

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

  18. Authentic role of ATP signaling in micturition reflex.

    PubMed

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

    2016-01-01

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

  19. Simultaneous release of acetylcholine and ATP from stimulated cholinergic synaptosomes.

    PubMed

    Morel, N; Meunier, F M

    1981-05-01

    The release of acetylcholine (ACh) and ATP from pure cholinergic synaptosomes isolated from the electric organ of Torpedo was studied in the same perfused sample. A presynaptic ATP release was demonstrated either by depolarization with KCl or after the action of a venom extracted from the annelid Glycera convoluta (GV). The release of ATP exhibited similar kinetics to that of ACh release and was therefore probably closely related to the latter. The ACh/ATP ratio in perfusates after KCl depolarization was 45; this was much higher than the ACh/ATP ratio in cholinergic synaptic vesicles, which was 5. The ACh/ATP ratio released after the action of GV was also higher than that of synaptic vesicles. These differences are discussed. The stoichiometry of that of synaptic vesicles. These differences are discussed. The stoichiometry of ACh and ATP release is not consistent with the view that the whole synaptic vesicle content is released by exocytosis after KCl depolarization, as is the case for chromaffin cells in the adrenal medulla. PMID:7241136

  20. The Chloroplast ATP Synthase Features the Characteristic Redox Regulation Machinery

    PubMed Central

    Sunamura, Ei-Ichiro; Kim, Yusung; Konno, Hiroki

    2013-01-01

    Abstract Significance: Regulation of the activity of the chloroplast ATP synthase is largely accomplished by the chloroplast thioredoxin system, the main redox regulation system in chloroplasts, which is directly coupled to the photosynthetic reaction. We review the current understanding of the redox regulation system of the chloroplast ATP synthase. Recent Advances: The thioredoxin-targeted portion of the ATP synthase consists of two cysteines located on the central axis subunit γ. The redox state of these two cysteines is under the influence of chloroplast thioredoxin, which directly controls rotation during catalysis by inducing a conformational change in this subunit. The molecular mechanism of redox regulation of the chloroplast ATP synthase has recently been determined. Critical Issues: Regulation of the activity of the chloroplast ATP synthase is critical in driving efficiency into the ATP synthesis reaction in chloroplasts. Future Directions: The molecular architecture of the chloroplast ATP synthase, which confers redox regulatory properties requires further investigation, in light of the molecular structure of the enzyme complex as well as the physiological significance of the regulation system. Antioxid. Redox Signal. 19, 1846–1854. PMID:23145525

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

    PubMed Central

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

    2014-01-01

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

  2. ATP stimulates pannexin 1 internalization to endosomal compartments.

    PubMed

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

    2015-09-15

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

  3. Modeling K,ATP--dependent excitability in pancreatic islets.

    PubMed

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

    2014-11-01

    In pancreatic ?-cells, K,ATP channels respond to changes in glucose to regulate cell excitability and insulin release. Confirming a high sensitivity of electrical activity to K,ATP activity, mutations that cause gain of K,ATP function cause neonatal diabetes. Our aim was to quantitatively assess the contribution of K,ATP current to the regulation of glucose-dependent bursting by reproducing experimentally observed changes in excitability when K,ATP conductance is altered by genetic manipulation. A recent detailed computational model of single cell pancreatic ?-cell excitability reproduces the ?-cell response to varying glucose concentrations. However, initial simulations showed that the model underrepresents the significance of K,ATP activity and was unable to reproduce K,ATP conductance-dependent changes in excitability. By altering the ATP and glucose dependence of the L-type Ca(2+) channel and the Na-K ATPase to better fit experiment, appropriate dependence of excitability on K,ATP conductance was reproduced. Because experiments were conducted in islets, which contain cell-to-cell variability, we extended the model from a single cell to a three-dimensional model (10×10×10 cell) islet with 1000 cells. For each cell, the conductance of the major currents was allowed to vary as was the gap junction conductance between cells. This showed that single cell glucose-dependent behavior was then highly variable, but was uniform in coupled islets. The study highlights the importance of parameterization of detailed models of ?-cell excitability and suggests future experiments that will lead to improved characterization of ?-cell excitability and the control of insulin secretion. PMID:25418087

  4. 47 CFR 301.2 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 5 2010-10-01 2010-10-01 false Definitions. 301.2 Section 301.2 Telecommunication NATIONAL TELECOMMUNICATIONS AND INFORMATION ADMINISTRATION, DEPARTMENT OF COMMERCE DIGITAL-TO-ANALOG CONVERTER BOX COUPON PROGRAM § 301.2 Definitions. Act means Title III of the Deficit Reduction Act of 2005, Pub. L. No. 109-171, 120 Stat. 4,...

  5. 15 CFR 325.2 - Definitions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 15 Commerce and Foreign Trade 2 2011-01-01 2011-01-01 false Definitions. 325.2 Section 325.2... REVIEW § 325.2 Definitions. As used in this part: (a) Act means title III of Pub. L. 97-290, Export Trade... the outstanding voting securities of an issuer; or (2) having the contractual power presently...

  6. 7 CFR 786.102 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS DAIRY DISASTER ASSISTANCE PAYMENT PROGRAM (DDAP-III) § 786.102 Definitions. The definitions in 7 CFR part 718 apply to this part except to the extent they are inconsistent with...

  7. 7 CFR 786.102 - Definitions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS DAIRY DISASTER ASSISTANCE PAYMENT PROGRAM (DDAP-III) § 786.102 Definitions. The definitions in 7 CFR part 718 apply to this part except to the extent they are inconsistent with...

  8. 12 CFR 26.2 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... million or more; (iii) A senior executive officer as that term is defined in 12 CFR 5.51(c)(3); (iv) A... Definitions. For purposes of this part, the following definitions apply: (a) Affiliate. (1) The term affiliate... of the area median income. (j) Management official. (1) The term management official means: (i)...

  9. Alternating electric fields stimulate ATP synthesis in Escherichia coli.

    PubMed

    Zrimec, Alexis; Jerman, Igor; Lahajnar, Gojmir

    2002-01-01

    External alternating electric fields of low intensity stimulated membrane bound ATP synthesis in starving Escherichia coli cells with electric field amplitudes of 2.5-50 V/cm and a frequency optimum at 100 Hz. The model of electrocon-formational coupling was used to analyze the frequency and amplitude responses of ATP synthesis. Two relaxation frequencies of the system were obtained at 44 Hz and 220 Hz, and an estimate of roughly 12 was obtained as the effective charge displacement for the catalytic cycle of ATP synthesis. PMID:11944082

  10. Two-dimensional diffusion of F1F0-ATP synthase and ADP/ATP translocator. Testing a hypothesis for ATP synthesis in the mitochondrial inner membrane.

    PubMed

    Gupte, S S; Chazotte, B; Leesnitzer, M A; Hackenbrock, C R

    1991-11-01

    We report here the first experimentally determined lateral diffusion coefficients of the F1F0-ATP synthase and the ADP/ATP translocator in isolated inner membranes of rat liver mitochondria. Rabbit IgG developed against the F1F0-ATP synthase isolated from rat liver mitochondria was determined to be immunospecific for the synthase subunits, notably the alpha-beta doublet, gamma and delta subunits of F1 and subunits two, three and four of F0. This IgG, conjugated with lissamine-rhodamine, was used as a fluorescent probe to monitor the diffusion of the synthase in the membrane. IgG to cytochrome bc1 complex, prepared and labeled similarly, was used as a fluorescent probe for diffusion of this redox component. Eosin maleimide was determined to specifically label the ADP/ATP translocator in the isolated inner membrane and was used as a specific probe for the diffusion of the translocator. Using fluorescence recovery after photobleaching, the experimental average lateral diffusion coefficient of the F1F0-ATP synthase was determined to be 8.4 x 10(-10) cm2/s or twice that of cytochrome bc1 complex while the diffusion coefficient of the ADP/ATP translocator was 1.7 x 10(-9) cm2/s or four times that of cytochrome bc1 complex suggesting that all three components are independent two-dimensional diffusants. Using these diffusion coefficients and applying a number of basic assumptions, we calculated the theoretical two-dimensional diffusion-controlled collision frequencies and derived collision efficiencies (protons transferred per collision) between each of the three proton-transferring redox complexes and both the F1F0-ATP synthase and ADP/ATP translocator by treating the redox components as proton donors and the synthase and translocator as proton acceptors. These collision efficiencies support the physical possibility of a diffusion-based, random collision process of proton transfer and ATP synthesis in the mitochondrial inner membrane. PMID:1718429

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

    PubMed

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

    2006-09-01

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

  12. Data for proteomic analysis of ATP-binding proteins and kinase inhibitor target proteins using an ATP probe

    PubMed Central

    Adachi, Jun; Kishida, Marina; Watanabe, Shio; Hashimoto, Yuuki; Fukamizu, Kazuna; Tomonaga, Takeshi

    2015-01-01

    Interactions between ATP and ATP-binding proteins (ATPome) are common and are required for most cellular processes. Thus, it is clearly important to identify and quantify these interactions for understanding basic cellular mechanisms and the pathogenesis of various diseases. We used an ATP competition assay (competition between ATP and acyl-ATP probes) that enabled us to distinguish specific ATP-binding proteins from non-specific proteins (Adachi et al., 2014) [1]. As a result, we identified 539 proteins, including 178 novel ATP-binding protein candidates. We also established an ATPome selectivity profiling method for kinase inhibitors using our cataloged ATPome list. Normally only kinome selectivity is profiled in selectivity profiling of kinase inhibitors. In this data, we expand the profiled targets from the kinome to the ATPome through performance of ATPome selectivity profiling and obtained target profiles of staurosporine and (S)-crizotinib. The data accompanying the manuscript on this approach (Adachi et al., 2014) [1] have been deposited to the ProteomeXchange with identifier PXD001200. PMID:26693503

  13. Bringing Definitions into High Definition

    ERIC Educational Resources Information Center

    Mason, John

    2010-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-12-15

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

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

    PubMed

    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

    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

  17. Distinct neurological disorders with ATP1A3 mutations

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2015-06-01

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

  19. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth.

    PubMed

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

    2015-01-01

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

  20. Distinct neurological disorders with ATP1A3 mutations.

    PubMed

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

    2014-05-01

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

  1. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2001-03-01

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

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

    PubMed

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

    2016-08-01

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

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

    SciTech Connect

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

    1996-12-01

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

  5. Highly Divergent Mitochondrial ATP Synthase Complexes in Tetrahymena thermophila

    PubMed Central

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

    2010-01-01

    The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F1 sector catalyzes ATP synthesis, whereas the Fo sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F1 and Fo sectors are highly conserved across prokaryotes and eukaryotes. Therefore, it was a surprise that genes encoding the a and b subunits as well as other components of the Fo sector were undetectable in the sequenced genomes of a variety of apicomplexan parasites. While the parasitic existence of these organisms could explain the apparent incomplete nature of ATP synthase in Apicomplexa, genes for these essential components were absent even in Tetrahymena thermophila, a free-living ciliate belonging to a sister clade of Apicomplexa, which demonstrates robust oxidative phosphorylation. This observation raises the possibility that the entire clade of Alveolata may have invented novel means to operate ATP synthase complexes. To assess this remarkable possibility, we have carried out an investigation of the ATP synthase from T. thermophila. Blue native polyacrylamide gel electrophoresis (BN-PAGE) revealed the ATP synthase to be present as a large complex. Structural study based on single particle electron microscopy analysis suggested the complex to be a dimer with several unique structures including an unusually large domain on the intermembrane side of the ATP synthase and novel domains flanking the c subunit rings. The two monomers were in a parallel configuration rather than the angled configuration previously observed in other organisms. Proteomic analyses of well-resolved ATP synthase complexes from 2-D BN/BN-PAGE identified orthologs of seven canonical ATP synthase subunits, and at least 13 novel proteins that constitute subunits apparently limited to the ciliate lineage. A mitochondrially encoded protein, Ymf66, with predicted eight transmembrane domains could be a substitute for the subunit a of the Fo sector. The absence of genes encoding orthologs of the novel subunits even in apicomplexans suggests that the Tetrahymena ATP synthase, despite core similarities, is a unique enzyme exhibiting dramatic differences compared to the conventional complexes found in metazoan, fungal, and plant mitochondria, as well as in prokaryotes. These findings have significant implications for the origins and evolution of a central player in bioenergetics. PMID:20644710

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

    PubMed Central

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

    2014-01-01

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

  7. Effect of chemical sanitizing agents on ATP bioluminescence measurements.

    PubMed

    Green, T A; Russell, S M; Fletcher, D L

    1998-08-01

    Experiments were conducted to determine the effects of sodium hypochlorite (SH), quaternary ammonium (QA), trisodium phosphate (TSP), lactic acid (LA), hydrogen peroxide (HP), and trichlosan (TR) on ATP bioluminescence measurements. Each sanitizer was tested at three different levels and compared to a control group containing no sanitizer. ATP from three sources was analyzed, Escherichia coli, chicken blood, and a pure ATP standard. The effect of each sanitizer was reported as a percent decrease in log10 relative light units (LRLU) for the treatment groups when compared to LRLU from the control groups. SH concentrations of 30, 50, and 70 ppm and quaternary ammonium at concentrations of 10, 20, and 30 ppm had no effect on LRLU measurements, regardless of the ATP source. LA concentrations of 0.5% and higher reduced LRLU by 75%. LRLU measurements were significantly (P < or = 0.05) reduced by approximately 60% when levels of TSP exceeded 1%. HP had no effect on LRLU measurements from any of the ATP sources at 0.1%; however, at 1% HP significantly (P < or = 0.05) decreased LRLU measurements by approximately 60% for all ATP sources. TR at 0.25% had no significant effect on LRLU measurements from any of the ATP sources. TR at 0.5 and 1% reduced LRLU measurements by 30 and 50%, respectively. These results indicate that commercial sanitizers containing LA, TSP, HP, or TR may negatively affect LRLU measurements if the sanitizer is allowed to come into direct contact with the ATP bioluminescence reagents. PMID:9713763

  8. Definitely Life but not Definitively

    NASA Astrophysics Data System (ADS)

    Oliver, Joan D.; Perry, Randall S.

    2006-12-01

    Although there have been attempts at a definition of life from many disciplines, none is accepted by all as definitive. Some people believe that it is impossible to define life adequately at the moment. We agree with this point of view on linguistic grounds, examining the different types of definition, the contexts in which they are used and their relative usefulness as aids to arriving at a scientific definition of life. We look at some of the more recent definitions and analyse them in the light of our criteria for a good definition. We argue that since there are so many linguistic and philosophical difficulties with such a definition of life, what is needed is a series of working descriptions, which are suited to the audience and context in which they are used and useful for the intended purpose. We provide some ideas and examples of the forms these may take.

  9. Definitely life but not definitively.

    PubMed

    Oliver, Joan D; Perry, Randall S

    2006-12-01

    Although there have been attempts at a definition of life from many disciplines, none is accepted by all as definitive. Some people believe that it is impossible to define 'life' adequately at the moment. We agree with this point of view on linguistic grounds, examining the different types of definition, the contexts in which they are used and their relative usefulness as aids to arriving at a scientific definition of life. We look at some of the more recent definitions and analyse them in the light of our criteria for a good definition. We argue that since there are so many linguistic and philosophical difficulties with such a definition of life, what is needed is a series of working descriptions, which are suited to the audience and context in which they are used and useful for the intended purpose. We provide some ideas and examples of the forms these may take. PMID:17120126

  10. Energy transduction in the F1 motor of ATP synthase

    NASA Astrophysics Data System (ADS)

    Wang, Hongyun; Oster, George

    1998-11-01

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

  11. Piezo1 regulates mechanotransductive release of ATP from human RBCs

    PubMed Central

    Cinar, Eyup; Zhou, Sitong; DeCourcey, James; Wang, Yixuan; Waugh, Richard E.; Wan, Jiandi

    2015-01-01

    Piezo proteins (Piezo1 and Piezo2) are recently identified mechanically activated cation channels in eukaryotic cells and associated with physiological responses to touch, pressure, and stretch. In particular, human RBCs express Piezo1 on their membranes, and mutations of Piezo1 have been linked to hereditary xerocytosis. To date, however, physiological functions of Piezo1 on normal RBCs remain poorly understood. Here, we show that Piezo1 regulates mechanotransductive release of ATP from human RBCs by controlling the shear-induced calcium (Ca2+) influx. We find that, in human RBCs treated with Piezo1 inhibitors or having mutant Piezo1 channels, the amounts of shear-induced ATP release and Ca2+ influx decrease significantly. Remarkably, a critical extracellular Ca2+ concentration is required to trigger significant ATP release, but membrane-associated ATP pools in RBCs also contribute to the release of ATP. Our results show how Piezo1 channels are likely to function in normal RBCs and suggest a previously unidentified mechanotransductive pathway in ATP release. Thus, we anticipate that the study will impact broadly on the research of red cells, cellular mechanosensing, and clinical studies related to red cell disorders and vascular disease. PMID:26351678

  12. Characterization of the mitochondrial ATP synthase from yeast Saccharomyces cerevisae

    PubMed Central

    Pagadala, Vijayakanth; Vistain, Luke; Symersky, Jindrich; Mueller, David M.

    2013-01-01

    The mitochondrial ATP synthase from yeast S. cerevisiae has been genetically modified, purified in a functional form, and characterized with regard to lipid requirement, compatibility with a variety of detergents, and the steric limit with rotation of the central stalk has been assessed. The ATP synthase has been modified on the N-terminus of the β-subunit to include a His6 tag for Ni-chelate affinity purification. The enzyme is purified by a two-step procedure from submitochondrial particles and the resulting enzyme demonstrates lipid dependent oligomycin sensitive ATPase activity of 50 units/mg. The yeast ATP synthase shows a strong lipid selectivity, with cardiolipin (CL) being the most effective activating lipid and there are 30 moles CL bound per mole enzyme at saturation. Green Fluorescent Protein (GFP) has also been fused to the C-terminus of the ε-subunit to create a steric block for rotation of the central stalk. The ε-GFP fusion peptide is imported into the mitochondrion, assembled with the ATP synthase, and inhibits ATP synthetic and hydrolytic activity of the enzyme. F1Fo ATP synthase with ε-GFP was purified to homogeneity and serves as an excellent enzyme for two- and three-dimensional crystallization studies. PMID:21748405

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

    PubMed

    Wang, H; Oster, G

    1998-11-19

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2003-01-01

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

  16. BPA Definitions

    SciTech Connect

    United States. Bonneville Power Administration.

    1993-12-01

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

  17. NIF Title III engineering plan

    SciTech Connect

    Deis, G

    1998-06-01

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

  18. Molecular mechanisms of ATP secretion during immunogenic cell death

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    Pang, S S; Duggleby, R G

    2001-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Waxman, Lloyd; Goldberg, Alfred L.

    1982-08-01

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

  1. VopA inhibits ATP binding by acetylating the catalytic loop of MAPK kinases.

    PubMed

    Trosky, Jennifer E; Li, Yan; Mukherjee, Sohini; Keitany, Gladys; Ball, Haydn; Orth, Kim

    2007-11-23

    The bacterial pathogen Vibrio parahemeolyticus manipulates host signaling pathways during infections by injecting type III effectors. One of these effectors, Vibrio outer protein A (VopA), inhibits MAPK signaling via a novel mechanism, distinct from those described for other bacterial toxins, that disrupts this signaling pathway. VopA is an acetyltransferase that potently inhibits MAPK signaling pathways not only by preventing the activation of MAPK kinases (MKKs) but also by inhibiting the activity of activated MKKs. VopA acetylates a conserved lysine found in the catalytic loop of all kinases and blocks the binding of ATP, but not ADP, on the MKKs, resulting in an inactive phosphorylated kinase. Acetylation of this conserved lysine inhibits kinase activity by a new mechanism of regulation that has not been observed previously. Identifying the target of VopA reveals a way that the reversible post-translational modification of lysine acetylation can be used to regulate the activity of an enzyme. PMID:17881352

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

    NASA Astrophysics Data System (ADS)

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

    2014-06-01

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

  3. ATP and ADP hydrolysis in cell membranes from rat myometrium.

    PubMed

    Milošević, Maja; Petrović, Snježana; Veličković, Nataša; Grković, Ivana; Ignjatović, Marija; Horvat, Anica

    2012-12-01

    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 μM, 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

  4. ATP synthase: from single molecule to human bioenergetics

    PubMed Central

    KAGAWA, Yasuo

    2010-01-01

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

  5. Rotation and structure of FoF1-ATP synthase.

    PubMed

    Okuno, Daichi; Iino, Ryota; Noji, Hiroyuki

    2011-06-01

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

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

    PubMed Central

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

    2013-01-01

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

  7. ATP-dependent looping of DNA by ISWI.

    PubMed

    Lia, Giuseppe; Indrieri, Marco; Owen-Hughes, Tom; Finzi, Laura; Podesta, Alessandro; Milani, Paolo; Dunlap, David

    2008-09-01

    Snf2 related chromatin remodelling enzymes possess an ATPase subunit similar to that of the SF-II helicases which hydrolyzes ATP to track along DNA. Translocation and any resulting torque in the DNA could drive chromatin remodeling. To determine whether the ISWI protein can translocate and generate torque, tethered particle motion experiments and atomic force microscopy have been performed using recombinant ISWI expressed in E. coli. In the absence of ATP, ISWI bound to and wrapped DNA thereby shortening the overall contour length measured in atomic force micrographs. Although naked DNA only weakly stimulates ATP hydrolysis by ISWI, both atomic force microscopy and tethered particle motion data indicate that the protein generated loops in the presence of ATP. The duration of the looped state of the DNA measured using tethered particle motion was ATP-dependent. Finally, ISWI relaxed positively supercoiled plasmids visualized by atomic force microscopy. While other chromatin remodeling ATPases catalyze either DNA wrapping or looping, both are catalyzed by ISWI. PMID:19343651

  8. ATP Hydrolyzing Salivary Enzymes of Caterpillars Suppress Plant Defenses

    PubMed Central

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

    2012-01-01

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

  9. Mechanism of Feedback Allosteric Inhibition of ATP Phosphoribosyltransferase

    PubMed Central

    2012-01-01

    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 μM 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 α-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 α-amino group. These results are combined into a model that accounts for the allosteric inhibition of MtATP-phosphoribosyltransferase. PMID:22989207

  10. Structure of Dimeric F1F0-ATP Synthase*

    PubMed Central

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

    2010-01-01

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

  11. Role of divalent metal cations in ATP hydrolysis catalyzed by the hepatitis C virus NS3 helicase: magnesium provides a bridge for ATP to fuel unwinding.

    PubMed

    Frick, David N; Banik, Sukalyani; Rypma, Ryan S

    2007-01-26

    This study investigates the role of magnesium ions in coupling ATP hydrolysis to the nucleic acid unwinding catalyzed by the NS3 protein encoded by the hepatitis C virus (HCV). Analyses of steady-state ATP hydrolysis rates at various RNA and magnesium concentrations were used to determine values for the 15 dissociation constants describing the formation of a productive enzyme-metal-ATP-RNA complex and the four rate constants describing hydrolysis of ATP by the possible enzyme-ATP complexes. These values coupled with direct binding studies, specificity studies and analyses of site-directed mutants reveal only one ATP binding site on HCV helicase centered on the catalytic base Glu291. An adjacent residue, Asp290, binds a magnesium ion that forms a bridge to ATP, reorienting the nucleotide in the active site. RNA stimulates hydrolysis while decreasing the affinity of the enzyme for ATP, magnesium, and MgATP. The binding scheme described here explains the unusual regulation of the enzyme by ATP that has been reported previously. Binding of either free magnesium or free ATP to HCV helicase competes with MgATP, the true fuel for helicase movements, and leads to slower hydrolysis and nucleic acid unwinding. PMID:17084859

  12. The Detection of Micromolar Pericellular ATP Pool on Lymphocyte Surface by Using Lymphoid Ecto-Adenylate Kinase as Intrinsic ATP Sensor

    PubMed Central

    Mikhailov, Andrey; Samburski, Sergei S.; Jalkanen, Sirpa

    2006-01-01

    Current models of extracellular ATP turnover include transient release of nanomolar ATP concentrations, triggering of signaling events, and subsequent ectoenzymatic inactivation. Given the high substrate specificity for adenylate kinase for reversible reaction (ATP + AMP ↔ 2ADP), we exploited lymphoid ecto-adenylate kinase as an intrinsic probe for accurate sensing pericellular ATP. Incubation of leukemic T- and B-lymphocytes with [3H]AMP or [α-32P]AMP induces partial nucleotide conversion into high-energy phosphoryls. This “intrinsic” AMP phosphorylation occurs in time- and concentration-dependent fashions via nonlytic supply of endogenous γ-phosphate–donating ATP, remains relatively resistant to bulk extracellular ATP scavenging by apyrase, and is diminished after lymphocyte pretreatment with membrane-modifying agents. This enzyme-coupled approach, together with confocal imaging of quinacrine-labeled ATP stores, suggests that, along with predominant ATP accumulation within cytoplasmic granules, micromolar ATP concentrations are constitutively retained on lymphoid surface without convection into bulk milieu. High basal levels of inositol phosphates in the cells transfected with ATP-selective human P2Y2-receptor further demonstrate that lymphocyte-surrounding ATP is sufficient for triggering purinergic responses both in autocrine and paracrine fashions. The ability of nonstimulated lymphocytes to maintain micromolar ATP halo might represent a novel route initiating signaling cascades within immunological synapses and facilitating leukocyte trafficking between the blood and tissues. PMID:16707571

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

    PubMed

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

    1982-06-01

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

  14. Diverse Functional Properties of Wilson Disease ATP7B Variants

    PubMed Central

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

    2012-01-01

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

  15. Functional studies of ATP sulfurylase from Penicillium chrysogenum

    SciTech Connect

    Seubert, P.A.

    1985-01-01

    ATP sulfurylase from Penicillium chrysogenum has a specific activity (V/sub max/) of 6-7 units x mg protein/sup -1/ determined with the physiological substrates of MgATP and SO/sub 4//sup 2 -/ and assayed by (A) initial velocity measurements with APS kinase and inorganic pyrophosphatase present and (B) analysis of nonlinear reaction progress curves. The fact both assays give the same results show the intrinsic activity of ATP sulfurylase is much higher than previously reported. In initial velocity dead-end inhibition studies, the sulfate analog S/sub 2/O/sub 3//sup 2 -/ is a competitive inhibitor of SO/sub 42/..sqrt.. and a noncompetitive inhibitor of MgATP. Monovalent oxyanions such as NO/sub 3//sup -/, ClO/sub 3//sup -/, ClO/sub 4//sup -/, and FSO/sub 3//sup -/ behave as uncompetitive inhibitors of MgATP and thus seem not to be true sulfate analogs. The reverse reaction was assayed by the pyrophosphate dependent release of /sup 35/SO/sub 4//sup 2 -/ from AP/sup 35/S. Product inhibition by MgATP or SO/sub 4//sup 2 -/ is competitive with APS and mixed-type with PP/sub i/. Imidodiphosphate can serve as an alternative substrate for PP/sub i/. ATP sulfurylase binds (but does not hydrolyze) APS. A Scatchard plot of the APS binding is nonlinear, suggesting at least two types of sites. The cumulative results are qualitatively consistent with the random addition of MgATP and SO/sub 4//sup 2 -/ and the ordered release of first MgPP/sub i/ then APS, with APS release being partially rate limiting. Certain quantitative discrepancies suggest either an unknown variable (e.g. enzyme concentration) complicates the analysis or, in light of binding studies that the actual mechanism is more complicated (e.g. alternating sites) than any of the conventional models examined.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2013-02-15

    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

  18. Timely management of developing class III malocclusion.

    PubMed

    Yelampalli, M R; Rachala, M R

    2012-01-01

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

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

    SciTech Connect

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

    2008-07-15

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-10-01

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

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

    PubMed Central

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

    2015-01-01

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

  3. A Fluorescent, Reagentless Biosensor for ATP, Based on Malonyl-Coenzyme A Synthetase

    PubMed Central

    2015-01-01

    A fluorescent reagentless biosensor for ATP has been developed, based on malonyl-coenzyme A synthetase from Rhodopseudomonas palustris as the protein scaffold and recognition element. Two 5-iodoacetamidotetramethylrhodamines were covalently bound to this protein to provide the readout. This adduct couples ATP binding to a 3.7-fold increase in fluorescence intensity with excitation at 553 nm and emission at 575 nm. It measures ATP concentrations with micromolar sensitivity and is highly selective for ATP relative to ADP. Its ability to monitor enzymatic ATP production or depletion was demonstrated in steady-state kinetic assays in which ATP is a product or substrate, respectively. PMID:26355992

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

    ERIC Educational Resources Information Center

    Selig, Ted C.; And Others

    1984-01-01

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

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

    PubMed

    Emel'ianov, V V

    2007-01-01

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

  6. Treatment of heterotopic ossification through remote ATP hydrolysis

    PubMed Central

    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

    2015-01-01

    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

  7. ATP-induced noncooperative thermal unfolding of hen lysozyme

    SciTech Connect

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

    2010-07-02

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

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

    PubMed

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

    2015-10-15

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-06-01

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

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

    PubMed

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

    2013-09-01

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

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

    PubMed Central

    Lan, Ethan I.; Liao, James C.

    2012-01-01

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

  12. Pentachlorophenol decreases ATP levels in human natural killer cells

    PubMed Central

    Nnodu, Ugochukwu; Whalen, Margaret M.

    2008-01-01

    Pentachlorophenol (PCP) is used as a wood preservative and is found in human blood and urine. PCP causes significant decreases in the tumor-killing (lytic) function of human natural killer (NK) cells, a critical immune defense. The current study examined the association between decreased lytic function and decreased ATP levels, as well as the ability of antioxidants (vitamin E and reduced glutathione) to prevent PCP-induced decreases in either ATP levels or lytic function. Exposure of NK cells to 10?M PCP decreased ATP levels by 15% at 24 h, and exposure to 5 ?M PCP decreased ATP levels by 32% at 48 h. No effects were seen with 0.5 ?M at 48 h or with 5 ?M at 24 h. However, 10 ?M PCP decreased lytic function by 69% at 24h and 5 ?M decreased it by 90% at 48 h. Even 0.5 ?M PCP decreased lytic function by 46% at 48 h. None of these effects were prevented by pretreatment with 1 mM vitamin E or reduced glutathione. PMID:18623605

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

    NASA Technical Reports Server (NTRS)

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

    1982-01-01

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

  14. Teacher Development for ATP 2000. Project Report 1993-1994.

    ERIC Educational Resources Information Center

    Newsom-Stewart, Mhora; Sutphin, Dean

    Agri Tech Prep 2000 (ATP 2000) is a 4-year tech prep program intended to link high school and postsecondary curricula preparing New York students for careers in agriculture or acceptance into a college program in agriculture. Because teacher development was designated an integral project component for fiscal year 1992-93, a weeklong teacher…

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

    ERIC Educational Resources Information Center

    Sutphin, Dean; And Others

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

  16. ATP Interior Noise Technology and Flight Demonstration Program

    NASA Technical Reports Server (NTRS)

    Stephens, David G.; Powell, Clemans A.

    1988-01-01

    The paper provides an overview of the ATP (Advanced Turboprop Program) acoustics program with emphasis on the NASA technology program and the recent NASA/Industry demonstration programs aimed at understanding and controlling passenger cabin noise. Technology developments in propeller (source) noise, cabin noise transmission, and subjective acoustics are described. Finally, an overview of the industry demonstrator programs is presented.

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

    USGS Publications Warehouse

    Shultz, David J.; Stephens, Doyle W.

    1980-01-01

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

  18. Integration and test processes for precision ATP payload

    NASA Astrophysics Data System (ADS)

    Sebesta, Henry R.; Krainiak, Joe; Baughman, David; Dillow, Michael A.; Birenboim, Aaron C.; McKechnie, T. S.

    1998-09-01

    The High Altitude Balloon Experiment (HABE) is being developed by the U.S. Air Force Research Laboratory, Space Vehicle Directorate at Kirtland Air Force Base, to investigate technologies needed to perform acquisition, tracking, and pointing (ATP) functions against boosting missiles in near-space environments. HABE is designed to demonstrate ATP sequence steps that start with acquisition of a missile plume, transition through passive IR tracking of the plume, and handover to precision tracking, which employs an active laser illuminator and imaging camera to image and track the missile nose. The Inertial Pseudo Star Reference Unit provides inertially stabilized line-of-sights (LOSs) for the illuminator laser, active fine track camera, and the marker scoring. The latter serves to measure and score the payload's pointing performance. The payload will be operated and carried aloft under a large, scientific balloon. The engagement parameters and timelines for the HABE ATP payload are consistent with scenarios encountered in space-based missile defense applications. In HABE experiments, target missiles will pass at ranges from 50 to 200 km. The performance goals of the ATP payload's LOS stabilization and marker laser pointing are required to exceed 1 microradian RMS or better in jitter, drift, and accuracy (two-axis, one sigma metrics), a requirement which stresses testing capabilities.

  19. ATP-dependent remodeling of the spliceosome: intragenic suppressors of release-defective mutants of Saccharomyces cerevisiae Prp22.

    PubMed Central

    Campodonico, Eva; Schwer, Beate

    2002-01-01

    The essential splicing factor Prp22 is a DEAH-box helicase that catalyzes the release of mRNA from the spliceosome. ATP hydrolysis by Prp22 is necessary but not sufficient for spliceosome disassembly. Previous work showed that mutations in motif III (635SAT637) of Prp22 that uncouple ATP hydrolysis from spliceosome disassembly lead to severe cold-sensitive (cs) growth defects and to impaired RNA unwinding activity in vitro. The cs phenotype of S635A (635AAT) can be suppressed by intragenic mutations that restore RNA unwinding. We now report the isolation and characterization of new intragenic mutations that suppress the cold-sensitive growth phenotypes of the T637A motif III mutation (SAA), the H606A mutation in the DEAH-box (DEAA), and the R805A mutation in motif VI (804QAKGRAGR811). Whereas the T637A and H606A proteins are deficient in releasing mRNA from the spliceosome at nonpermissive temperature in vitro, the suppressor proteins have recovered mRNA release activity. To address the mechanisms of suppression, we tested ATPase and helicase activities of Prp22 suppressor mutant proteins and found that the ability to unwind a 25-bp RNA duplex was not restored in every case. This finding suggests that release of mRNA from the spliceosome is less demanding than unwinding of a 25-bp duplex RNA; the latter reaction presumably reflects the result of several successive cycles of ATP binding, hydrolysis, and unwinding. Increasing the reaction temperature allows H606A and T637A to effect mRNA release in vitro, but does not restore RNA unwinding by T637A. PMID:11861548

  20. Mechanism of feedback allosteric inhibition of ATP phosphoribosyltransferase.

    PubMed

    Pedreño, Sònia; Pisco, João Pedro; Larrouy-Maumus, Gérald; Kelly, Geoff; de Carvalho, Luiz Pedro Sório

    2012-10-01

    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 K(ii) value of 4 μM 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 α-amino group. The pH dependence of inhibition (K(ii)), on the contrary, indicates that l-histidine better inhibits MtATP-phosphoribosytransferase with a neutral imidazole and an ionized α-amino group. These results are combined into a model that accounts for the allosteric inhibition of MtATP-phosphoribosyltransferase. PMID:22989207

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

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-06-01

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

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

    PubMed

    de Kloet, S R

    2001-08-01

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

  5. H+/ATP ratio of proton transport-coupled ATP synthesis and hydrolysis catalysed by CF0F1–liposomes

    PubMed Central

    Turina, Paola; Samoray, Dietrich; Gräber, Peter

    2003-01-01

    The H+/ATP ratio and the standard Gibbs free energy of ATP synthesis were determined with a new method using a chemiosmotic model system. The purified H+-translocating ATP synthase from chloroplasts was reconstituted into phosphatidylcholine/phosphatidic acid liposomes. During reconstitution, the internal phase was equilibrated with the reconstitution medium, and thereby the pH of the internal liposomal phase, pHin, could be measured with a conventional glass electrode. The rates of ATP synthesis and hydrolysis were measured with the luciferin/luciferase assay after an acid–base transition at different [ATP]/([ADP][Pi]) ratios as a function of ΔpH, analysing the range from the ATP synthesis to the ATP hydrolysis direction and the ΔpH at equilibrium, ΔpH (eq) (zero net rate), was determined. The analysis of the [ATP]/([ADP][Pi]) ratio as a function of ΔpH (eq) and of the transmembrane electrochemical potential difference, Δµ̃H+ (eq), resulted in H+/ATP ratios of 3.9 ± 0.2 at pH 8.45 and 4.0 ± 0.3 at pH 8.05. The standard Gibbs free energies of ATP synthesis were determined to be 37 ± 2 kJ/mol at pH 8.45 and 36 ± 3 kJ/mol at pH 8.05. PMID:12554643

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

    PubMed Central

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

    1997-01-01

    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

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

    PubMed Central

    Ohmiya, Yoshihiro

    2013-01-01

    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

  8. Cationic channels sensitive to extracellular ATP in rat lacrimal cells.

    PubMed Central

    Vincent, P

    1992-01-01

    1. Responses of isolated rat lacrimal cells to local applications of ATP were studied using tight-seal whole-cell recording and/or Fura-2-derived calcium concentration measurements. 2. In cells where variations in Ca2+ concentration were prevented by use of a strong Ca2+ buffer, ATP was found to induce an inward current response at negative holding potentials. With 10 microM-ATP, the current amplitude ranged between 20 and 200 pA. The reversal potential of this ATP-induced current was close to 0 mV with normal external solution and shifted to -19 +/- 3 mV (mean +/- S.D.) when the concentration of external monovalent cations was halved. These results indicate that the channels have a cationic selectivity. The response amplitude decreased markedly from trial to trial, indicating a desensitization process which was irreversible on the time scale of the recordings. 3. Steady state I-V curves for the ATP-induced current in normal saline showed a marked inward rectification. This rectification appeared to be linked to a time-dependent activation of the channels, as hyperpolarizing voltage jumps elicited a time-dependent current increase. This relaxation could be fitted by a double-exponential function, with time constants (at -120 mV) of 0.9 +/- 0.3 ms and 110 +/- 6.4 ms. 4. Variance analysis of the ATP-induced current gave a single-channel current value of 0.34 pA at -60 mV. The single-channel current amplitude varied linearly with potential, with a slope close to 6 pS. The relation between noise covariance and time could be fitted by a double-exponential function, with time constants (at -60 mV) of 0.8 +/- 0.4 ms and 6.8 +/- 3.4 ms (mean +/- S.D.). 5. In an isotonic Ca2+ solution, 10 microM-ATP induced an inward current at -60 mV with a calculated single-channel current amplitude obtained from noise analysis close to 0.2 pA. In an external solution containing 10 mM-calcium and no sodium, 50 microM-ATP elicited a current with a reversal potential of -19 mV. 6. Fura-2 measurements were performed in intact cells or in cells dialysed with a low concentration of Ca2+ buffer (e.g. 0.5 mM-EGTA). Under such conditions ATP induced increases of the internal Ca2+ concentration with very variable amplitudes. In some cells Ca2+ rises of 50 nM or lower were found. Minimal activation of Ca(2+)-dependent channels was then observed. In other cells large Ca2+ rises (up to 500 nM) were observed and were then correlated with marked activation of Ca(2+)-dependent channels.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:1381749

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2014-01-01

    In human erythrocytes (h-RBCs) various stimuli induce increases in [cAMP] that trigger ATP release. The resulting pattern of extracellular ATP accumulation (ATPe kinetics) depends on both ATP release and ATPe degradation by ectoATPase activity. In this study we evaluated ATPe kinetics from primary cultures of h-RBCs infected with P. falciparum at various stages of infection (ring, trophozoite and schizont stages). A "3V" mixture containing isoproterenol (β-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 ΔATP1 (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 ΔATP1 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 ΔATP1 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, ΔATP1 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

  12. Regulation of Extracellular ATP in Human Erythrocytes Infected with Plasmodium falciparum

    PubMed Central

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

    2014-01-01

    In human erythrocytes (h-RBCs) various stimuli induce increases in [cAMP] that trigger ATP release. The resulting pattern of extracellular ATP accumulation (ATPe kinetics) depends on both ATP release and ATPe degradation by ectoATPase activity. In this study we evaluated ATPe kinetics from primary cultures of h-RBCs infected with P. falciparum at various stages of infection (ring, trophozoite and schizont stages). A “3V” mixture containing isoproterenol (β-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 ΔATP1 (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 ΔATP1 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 ΔATP1 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, ΔATP1 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

  13. Mutational analysis in patients with neuromuscular disorders: Detection of mitochondrial deletion and double mutations in the MT-ATP6 gene.

    PubMed

    Felhi, Rahma; Mkaouar-Rebai, Emna; Sfaihi-Ben Mansour, Lamia; Alila-Fersi, Olfa; Tabebi, Mouna; Ben Rhouma, Bochra; Ammar, Marwa; Keskes, Leila; Hachicha, Mongia; Fakhfakh, Faiza

    2016-04-22

    Mitochondrial diseases encompass a wide variety of pathologies characterized by a dysfunction of the mitochondrial respiratory chain resulting in an energy deficiency. The respiratory chain consists of five multi-protein complexes providing coupling between nutrient oxidation and phosphorylation of ADP to ATP. In the present report, we studied mitochondrial genes of complex I, III, IV and V in 2 Tunisian patients with mitochondrial neuromuscular disorders. In the first patient, we detected the m.8392C>T variation (P136S) in the mitochondrial ATPase6 gene and the m.8527A>G transition at the junction MT-ATP6/MT-ATP8 which change the initiation codon AUG to GUG. The presence of these two variations in such an important gene could probably affect the ATP synthesis in the studied patient. In the second patient, we detected several known variations in addition to a mitochondrial deletion in the major arc of the mtDNA eliminating tRNA and respiratory chain protein genes. This deletion could be responsible of an inefficient translation leading to an inefficient mitochondrial protein synthesis in P2. PMID:26993169

  14. ATPase-Independent Type-III Protein Secretion in Salmonella enterica

    PubMed Central

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

    2014-01-01

    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

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

    PubMed

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

    2014-10-01

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

  16. Measurement of ATP production and respiratory chain enzyme activities in mitochondria isolated from small muscle biopsy samples.

    PubMed

    Wibom, Rolf; Hagenfeldt, Lars; von Döbeln, Ulrika

    2002-12-15

    A set of methods suitable for assessment of respiratory chain function in mitochondria isolated from 25mg of muscle is described. This set of methods includes determination of the mitochondrial ATP production rate (MAPR) and the activities of the respiratory chain complexes I, I+III, II+III, and IV and citrate synthase. MAPR is determined with an optimized version of a luminometric method previously described. The optimized method measures 50-220% higher activities than the original method. The highest MAPRs are recorded using the substrate combinations glutamate+succinate and N,N,N(1),N(1)-tetramethyl-1,4-phenyldiamine+ascorbate. The respiratory chain complex activities are determined with standard spectrophotometric methods, adapted to an automated photometer. The sensitivity in the determination of complex I, I+III, and II+III activities was increased considerably by pretreating the samples with saponin. The set of methods was evaluated on double biopsy samples from five healthy volunteers and showed coefficients of variation between 7 and 14% when citrate synthase was used as reference base. All of the various measures of mitochondrial function showed high correlation coefficients to each other (r=0.84-0.98; p<0.01). It is concluded that the set of methods is suitable for diagnosis of mitochondrial disorders in adults and small children. PMID:12470673

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

  20. 7 CFR 3300.88 - Fees for U.S. ATP certificates.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

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

  1. P2Y Receptor Modulation of ATP Release in the Urothelium

    PubMed Central

    Mansfield, Kylie J.; Hughes, Jessica R.

    2014-01-01

    The release of ATP from the urothelium in response to stretch during filling demonstrates the importance of the purinergic system for the physiological functioning of the bladder. This study examined the effect of P2 receptor agonists on ATP release from two urothelial cell lines (RT4 and UROtsa cells). Hypotonic Krebs was used as a stretch stimulus. Incubation of urothelial cells with high concentrations of the P2Y agonist ADP induced ATP release to a level that was 40-fold greater than hypotonic-stimulated ATP release (P < 0.0011, ADP EC50 1.8 µM). Similarly, an increase in ATP release was also observed with the P2Y agonist, UTP, up to a maximum of 70% of the hypotonic response (EC50 0.62 µM). Selective P2 receptor agonists, αβ-methylene-ATP, ATP-γ-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

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

    PubMed Central

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

    2014-01-01

    Multiple functions of the endoplasmic reticulum (ER) essentially depend on ATP within this organelle. However, little is known about ER ATP dynamics and the regulation of ER ATP import. Here we describe real-time recordings of ER ATP fluxes in single cells using an ER-targeted, genetically encoded ATP sensor. In vitro experiments prove that the ATP sensor is both Ca2+ and redox insensitive, which makes it possible to monitor Ca2+-coupled ER ATP dynamics specifically. The approach uncovers a cell typespecific 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

    Laughlin, Thomas F; Ahmad, Zulfiqar

    2010-04-01

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

  5. Interaction of stilbene disulphonates with cloned K(ATP) channels.

    PubMed

    Proks, P; Jones, P; Ashcroft, F M

    2001-03-01

    In this study, we tested the effects of the stilbene disulphonates DIDS and SITS on three different types of cloned K(ATP) channel (Kir6.2/SUR1, Kir6.2/SUR2A and Kir6.2DeltaC) heterologously expressed in Xenopus oocytes, with the aim of identifying the part of the channel which is involved in mediating disulphonate inhibition. We found that the inhibitory site(s) for these drugs lies within the Kir6.2 subunit of the channel, although its properties are further modulated by the sulphonylurea (SUR) subunit. In particular, SUR2A reduces both the rate and extent of block, by impairing the ability of DIDS binding to produce channel closure. The disulphonate-binding site interacts with the ATP inhibitory site on Kir6.2 because ATP is able to protect against irreversible channel inhibition by disulphonates. This effect is not mimicked by tolbutamide (at a concentration that interacts with Kir6.2) and is abolished by mutations that render the channel ATP insensitive. A number of point mutations in both the N and C termini of Kir6.2 reduced the extent and reversibility of channel inhibition by SITS. The results are consistent with the idea that residue C42 of Kir6.2 is likely to be involved in covalently linking of SITS to the channel. Other types of Kir channel (Kir1.1, Kir2.1 and Kir4.1) were also irreversibly blocked by DIDS, suggesting that these channels may share common binding sites for these stilbene disulphonates. PMID:11226127

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

    PubMed Central

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

    2004-01-01

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

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

    SciTech Connect

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

    2006-12-15

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

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

    PubMed Central

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

    2009-01-01

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

  9. ATP5A1 and ATP5B are highly expressed in glioblastoma tumor cells and endothelial cells of microvascular proliferation.

    PubMed

    Xu, Guiyan; Li, Jian Yi

    2016-02-01

    Glioblastoma (GBM) is the most common primary malignant brain tumor. Microvascular proliferation is one of the characteristic pathologic features of GBM. Mitochondrial dysfunction plays an important role in the pathogenesis of GBM. In this study, microvascular proliferation from GBM and normal brain blood vessels were laser microdissected and total RNA was isolated from these microvasculatures. The difference of mRNA expression profiles among GBM microvasculature, normal brain blood vessels and GBM tumor cells was evaluated by mitochondria and metabolism PCR gene arrays. It was found that the mRNA levels of ATP5A1 and ATP5B in GBM tumor cells as well as microvascular proliferation were significantly higher compared with normal brain blood vessels. Immunohistochemical stains with anti-ATP5A1 antibody or anti-ATP5B antibody were performed on tissue microarray, which demonstrated strongly positive expression of ATP5A1 and ATP5B in GBM tumor cells and GBM microvascular proliferation while normal blood vessels were negative. By analyzing The Cancer Genome Atlas data sets for GBM and other cancers, genomic DNA alterations (mutation, amplification or deletion) were less likely the reason for the high expression of ATP5A1 and ATP5B in GBM. Our miRNA microarray data showed that miRNAs that target ATP5A1 or ATP5B were down-regulated, which might be the most likely reason for the high expression of ATP5A1 and ATP5B in GBM tumor cells and microvascular proliferation. These findings help us better understand the pathogenesis of GBM, and agents against ATP5A1 and/or ATP5B might effectively kill both tumor cells and microvascular proliferation in GBM. MiRNAs, such as Let-7f, miR-16, miR-23, miR-100 and miR-101, that target ATP5A1 or ATP5B, might be potential therapeutic agents for GBM. PMID:26526033

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

    PubMed

    Yu, Weiqun

    2015-11-15

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

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

    PubMed Central

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

    2006-01-01

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

  12. ATP-noncompetitive inhibitors of CDK-cyclin complexes.

    PubMed

    Orzáez, Mar; Gortat, Anna; Mondragón, Laura; Bachs, Oriol; Pérez-Payá, Enrique

    2009-01-01

    Progression through the cell division cycle is controlled by a family of cyclin-dependent kinases (CDKs), the activity of which depends on their binding to regulatory partners (cyclins A-H). Deregulation of the activity of CDKs has been associated with the development of infectious, neurodegenerative, and proliferative diseases such as Alzheimer's, Parkinson's, or cancer. Most cancer cells contain mutations in the pathways that control the activity of CDKs. This observation led this kinase family to become a central target for the development of new drugs for cancer therapy. A range of structurally diverse molecules has been shown to inhibit the activity of CDKs through their activity as ATP antagonists. Nevertheless, the ATP binding sites on CDKs are highly conserved, limiting the kinase specificity of these inhibitors. Various genetic and crystallographic approaches have provided essential information about the mechanism of formation and activation of CDK-cyclin complexes, providing new ways to implement novel research strategies toward the discovery of new, more effective and selective drugs. Herein we review the progress made in the development of ATP-noncompetitive CDK-cyclin inhibitors. PMID:19039815

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

    PubMed Central

    Corrigan, A P; Rider, C C

    1983-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-07-01

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

  16. 34 CFR 350.5 - What definitions apply?

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... PROGRAM General § 350.5 What definitions apply? (a) The following definitions in 34 CFR part 77 apply to... expected to require multiple vocational rehabilitation services over an extended period of time; and (iii..., hemiplegia, hemophilia, respiratory or pulmonary dysfunction, mental retardation, mental illness,...

  17. 34 CFR 350.5 - What definitions apply?

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... PROGRAM General § 350.5 What definitions apply? (a) The following definitions in 34 CFR part 77 apply to... expected to require multiple vocational rehabilitation services over an extended period of time; and (iii..., hemiplegia, hemophilia, respiratory or pulmonary dysfunction, mental retardation, mental illness,...

  18. 34 CFR 350.5 - What definitions apply?

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... PROGRAM General § 350.5 What definitions apply? (a) The following definitions in 34 CFR part 77 apply to... expected to require multiple vocational rehabilitation services over an extended period of time; and (iii..., hemiplegia, hemophilia, respiratory or pulmonary dysfunction, mental retardation, mental illness,...

  19. 34 CFR 350.5 - What definitions apply?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... PROGRAM General § 350.5 What definitions apply? (a) The following definitions in 34 CFR part 77 apply to... expected to require multiple vocational rehabilitation services over an extended period of time; and (iii..., hemiplegia, hemophilia, respiratory or pulmonary dysfunction, mental retardation, mental illness,...

  20. 34 CFR 350.5 - What definitions apply?

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... PROGRAM General § 350.5 What definitions apply? (a) The following definitions in 34 CFR part 77 apply to... expected to require multiple vocational rehabilitation services over an extended period of time; and (iii..., hemiplegia, hemophilia, respiratory or pulmonary dysfunction, mental retardation, mental illness,...

  1. 34 CFR 636.7 - What definitions apply?

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ..., DEPARTMENT OF EDUCATION URBAN COMMUNITY SERVICE PROGRAM General § 636.7 What definitions apply? (a) Definitions in EDGAR. The following terms used in this part are defined in 34 CFR 77.1: Applicant Application... of the following: (i) A community college. (ii) An urban school system. (iii) A local government....

  2. 34 CFR 636.7 - What definitions apply?

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ..., DEPARTMENT OF EDUCATION URBAN COMMUNITY SERVICE PROGRAM General § 636.7 What definitions apply? (a) Definitions in EDGAR. The following terms used in this part are defined in 34 CFR 77.1: Applicant Application... of the following: (i) A community college. (ii) An urban school system. (iii) A local government....

  3. 34 CFR 636.7 - What definitions apply?

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ..., DEPARTMENT OF EDUCATION URBAN COMMUNITY SERVICE PROGRAM General § 636.7 What definitions apply? (a) Definitions in EDGAR. The following terms used in this part are defined in 34 CFR 77.1: Applicant Application... of the following: (i) A community college. (ii) An urban school system. (iii) A local government....

  4. Mammalian copper-transporting P-Type ATPases, ATP7A and ATP7B: Emerging roles

    PubMed Central

    La Fontaine, Sharon; Ackland, M. Leigh; Mercer, Julian F.B.

    2010-01-01

    Copper (Cu) has a role in a diverse and increasing number of pathways, physiological and disease processes. These roles are testament to the fundamental importance of Cu in biology and the need to understand the mechanisms that regulate Cu homeostasis. The mammalian Cu-transporting P-type ATPases ATP7A and ATP7B are two key proteins that regulate the Cu status of the body. They transport Cu across cellular membranes for biosynthetic and protective functions, enabling Cu to fulfill its role as a structural cofactor for many essential enzymes, and to prevent a toxic build-up of Cu inside cells. A variety of regulatory mechanisms operate at transcriptional and post-translational levels to ensure adequate Cu supplies for both physiological and pathophysiological processes. This review summarizes the recent literature that is revealing the emerging roles of the Cu-ATPases in health and disease. PMID:19922814

  5. Protons, the thylakoid membrane, and the chloroplast ATP synthase.

    PubMed

    Junge, W

    1989-01-01

    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, all of which have been loosely subsumed under 'localized coupling' in the literature, 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. "Rotation catalysis" has been proposed. It is of heuristic value to visualize CFoCF1 as a mechanical coupling device. Its maximum turnover number ranges up to 400 s-1 for ATP and 1200 s-1 for protons. At about 200 mV electric driving force this implied a conductance of about 1 fS. Its channel portion (CFo), however, has revealed a very large protonic conductance of 1 pS (three orders of magnitude greater than the protonic conductance of gramicidin around neutral pH). (6) The sight and smell of food increased LH serotonin release; this effect was detectable when local fluoxetine was used to block serotonin reuptake.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2483874

  6. 10 CFR 1040.83 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... age or an age-related term (for example, “18 or over”). (e) Age-related term means a word or words... means the official in each DOE field office with responsibility for administering DOE's Civil Rights... Definitions. (a) Act means the Age Discrimination Act of 1975 as amended title III of Pub. L. 94-135, 42...

  7. 10 CFR 1040.83 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... age or an age-related term (for example, “18 or over”). (e) Age-related term means a word or words... means the official in each DOE field office with responsibility for administering DOE's Civil Rights... Definitions. (a) Act means the Age Discrimination Act of 1975 as amended title III of Pub. L. 94-135, 42...

  8. 10 CFR 1040.83 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... age or an age-related term (for example, “18 or over”). (e) Age-related term means a word or words... means the official in each DOE field office with responsibility for administering DOE's Civil Rights... Definitions. (a) Act means the Age Discrimination Act of 1975 as amended title III of Pub. L. 94-135, 42...

  9. 10 CFR 1040.83 - Definitions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... age or an age-related term (for example, “18 or over”). (e) Age-related term means a word or words... means the official in each DOE field office with responsibility for administering DOE's Civil Rights... Definitions. (a) Act means the Age Discrimination Act of 1975 as amended title III of Pub. L. 94-135, 42...

  10. 10 CFR 1040.83 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... age or an age-related term (for example, “18 or over”). (e) Age-related term means a word or words... means the official in each DOE field office with responsibility for administering DOE's Civil Rights... Definitions. (a) Act means the Age Discrimination Act of 1975 as amended title III of Pub. L. 94-135, 42...

  11. 14 CFR 1264.101 - Definitions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... with Chapter 51 of such Title and Subchapter III of Chapter 53 of such Title; (6) Is not subject to... reduced in grade or pay only for good cause established and determined by the Merit Systems Protection... 14 Aeronautics and Space 5 2011-01-01 2010-01-01 true Definitions. 1264.101 Section...

  12. 48 CFR 9904.420-30 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 7 2012-10-01 2012-10-01 false Definitions. 9904.420-30 Section 9904.420-30 Federal Acquisition Regulations System COST ACCOUNTING STANDARDS BOARD, OFFICE OF... following three areas: (i) Basic and applied research, (ii) Development, and (iii) Systems and other...

  13. 48 CFR 9904.420-30 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 7 2014-10-01 2014-10-01 false Definitions. 9904.420-30 Section 9904.420-30 Federal Acquisition Regulations System COST ACCOUNTING STANDARDS BOARD, OFFICE OF... following three areas: (i) Basic and applied research, (ii) Development, and (iii) Systems and other...

  14. 48 CFR 9904.420-30 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 7 2013-10-01 2012-10-01 true Definitions. 9904.420-30 Section 9904.420-30 Federal Acquisition Regulations System COST ACCOUNTING STANDARDS BOARD, OFFICE OF... following three areas: (i) Basic and applied research, (ii) Development, and (iii) Systems and other...

  15. 5 CFR 831.1901 - Definitions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

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

  16. 5 CFR 831.1901 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

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

  17. 5 CFR 831.1901 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

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

  18. 5 CFR 831.1901 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

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

  19. 5 CFR 831.1901 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

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

  20. 24 CFR 290.3 - Definitions.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

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

  1. 24 CFR 290.3 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

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

  2. 24 CFR 290.3 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

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

  3. 24 CFR 290.3 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

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

  4. 28 CFR 32.33 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... BENEFIT CLAIMS Educational Assistance Benefit Claims § 32.33 Definitions. Application means claim (i.e., a...— (i) Room and board (if incurred for attendance on at least a half-time basis); (ii) Books; (iii) Computer equipment; (iv) Supplies; (v) Transportation; and (3) For attendance on at least a...

  5. 45 CFR 1626.2 - Definitions.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... TO ALIENS § 1626.2 Definitions. (a) Citizen includes persons described or defined as citizens or nationals of the United States in 8 U.S.C. 1101(a)(22) and Title III of the Immigration and Nationality Act....) (citizens by naturalization) or antecedent citizen statutes. (b) Eligible alien means a person who is not...

  6. 13 CFR 117.3 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Definitions. As used in this part: (a) The term act means the Age Discrimination Act of 1975, as amended (Title III of Pub. L. 94-135). (b) The term action means any act, activity, policy, rule, standard, or... term age means how old a person is, or the number of years from the date of a person's birth. (d)...

  7. 7 CFR 3052.105 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... development (R&D), student financial aid (SFA), and other clusters. “Other clusters” are as defined by the... (SFA); and (iii) “Other clusters,” as described in the definition of cluster of programs in this... defined in this section. Student Financial Aid (SFA) includes those programs of general student...

  8. 34 CFR 5.2 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

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

  9. 34 CFR 5.2 - Definitions.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

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

  10. 34 CFR 5.2 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

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

  11. 34 CFR 5.2 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

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

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

    PubMed Central

    2013-01-01

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

  13. Hemolysis is a primary ATP-release mechanism in human erythrocytes

    PubMed Central

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

    2014-01-01

    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

  14. Hemolysis is a primary ATP-release mechanism in human erythrocytes.

    PubMed

    Sikora, Jacek; Orlov, Sergei N; Furuya, Kishio; Grygorczyk, Ryszard

    2014-09-25

    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

  15. ATP-responsive DNA-graphene hybrid nanoaggregates for anticancer drug delivery.

    PubMed

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

    2015-05-01

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

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

    PubMed

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

    2016-01-26

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

  17. Structure and conformational states of the bovine mitochondrial ATP synthase by cryo-EM.

    PubMed

    Zhou, Anna; Rohou, Alexis; Schep, Daniel G; Bason, John V; Montgomery, Martin G; Walker, John E; Grigorieff, Nikolaus; Rubinstein, John L

    2015-01-01

    Adenosine triphosphate (ATP), the chemical energy currency of biology, is synthesized in eukaryotic cells primarily by the mitochondrial ATP synthase. ATP synthases operate by a rotary catalytic mechanism where proton translocation through the membrane-inserted FO region is coupled to ATP synthesis in the catalytic F1 region via rotation of a central rotor subcomplex. We report here single particle electron cryomicroscopy (cryo-EM) analysis of the bovine mitochondrial ATP synthase. Combining cryo-EM data with bioinformatic analysis allowed us to determine the fold of the a subunit, suggesting a proton translocation path through the FO region that involves both the a and b subunits. 3D classification of images revealed seven distinct states of the enzyme that show different modes of bending and twisting in the intact ATP synthase. Rotational fluctuations of the c8-ring within the FO region support a Brownian ratchet mechanism for proton-translocation-driven rotation in ATP synthases. PMID:26439008

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

    PubMed

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

    2016-09-15

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

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

    PubMed

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

    2008-11-01

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

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

    PubMed

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

    2015-08-01

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

  1. CELL-SPECIFIC TRAFFICKING SUGGESTS A NEW ROLE FOR RENAL ATP7B IN THE INTRACELLULAR COPPER STORAGE

    PubMed Central

    Barnes, Natalie; Bartee, Mee Y.; Braiterman, Lita; Gupta, Arnab; Ustiyan, Vladimir; Zuzel, Vesna; Kaplan, Jack H.; Hubbard, Ann L.; Lutsenko, Svetlana

    2012-01-01

    Human Cu-ATPases ATP7A and ATP7B maintain copper homeostasis through regulated trafficking between intracellular compartments. Inactivation of these transporters causes Menkes disease and Wilson disease, respectively. In Menkes disease, copper accumulates in kidneys and causes tubular damage, indicating that the renal ATP7B does not compensate for the loss of ATP7A function. We show that this is likely due to a kidney-specific regulation of ATP7B. Unlike ATP7A (or hepatic ATP7B) which traffics from the TGN to export copper, renal ATP7B does not traffic and therefore is unlikely to mediate copper export. The lack of ATP7B trafficking is not due to the loss of a kinase-mediated phosphorylation or simultaneous presence of ATP7A in renal cells. Rather, the renal ATP7B appears 2-3 kDa smaller than hepatic ATP7B. Recombinant ATP7B expressed in renal cells is similar to hepatic protein in size and trafficking. The analysis of ATP7B mRNA revealed a complex behavior of exon 1 upon amplification, suggesting that it could be inefficiently translated. Recombinant ATP7B lacking exon 1 traffics differently in renal and hepatic cells, but does not fully recapitulate the endogenous phenotype. We discuss factors that may contribute to cell-specific behavior of ATP7B and propose a role for renal ATP7B in intracellular copper storage. PMID:19416479

  2. Electrical currents associated with nucleotide transport by the reconstituted mitochondrial ADP/ATP carrier.

    PubMed

    Brustovetsky, N; Becker, A; Klingenberg, M; Bamberg, E

    1996-01-23

    The electrophoretic export of ATP against the import of ADP in mitochondria bridges the intra- versus extramitochondrial ATP potential gap. Here we report that the electrical nature of the ADP/ATP exchange by the mitochondrial ADP/ATP carrier (AAC) can be directly studied by measuring the electrical currents via capacitive coupling of AAC-containing vesicles on a planar lipid membrane. The currents were induced by the rapid liberation of ATP or ADP with UV flash photolysis from caged nucleotides. Six different transport modes of the AAC were studied: heteroexchange with either ADP or ATP inside the vesicles, initiated by photolysis of caged ATP or ADP; homoexchange with ADPex/ADPin or ATPex/ATPin; and caged ADP or ATP with unloaded vesicles. The heteroexchange produced the largest currents with the longest duration in line with the electrical charge difference ATP4- versus ADP3-. Surprisingly, also in the homoexchange and with unloaded vesicles, small currents were measured with shorter duration. In all three modes with caged ATP, a negative charge moved into the vesicles and with caged ADP it moved out of the vesicles. All currents were completely inhibited by a mixture of the inhibitors of the AAC, carboxyatractyloside and hongkrekate, which proves that the currents are exclusively due to AAC function. The observed charge movements in the heteroexchange system agree with the prediction from transport studies in mitochondria and reconstituted vesicles. The unexpected charge movements in the homoexchange or unloaded systems are interpreted to reveal transmembrane rearrangements of charged sites in the AAC when occupied with ADP or ATP. The results also indicate that not only ATP4- but also ADP3- contribute, albeit in opposite direction, to the electrical nature of the ADP/ATP exchange, which is at variance with former conclusions from biochemical transport studies. These measurements open up new avenues of studying the electrical interactions of ADP and ATP with the AAC. PMID:8570612

  3. Kinetics of ATP binding to the origin recognition complex of Saccharomyces cerevisiae.

    PubMed

    Makise, Masaki; Takenaka, Hitomi; Kuwae, Wakako; Takahashi, Naoko; Tsuchiya, Tomofusa; Mizushima, Tohru

    2003-11-21

    Origin recognition complex (ORC), a candidate initiator of chromosomal DNA replication in eukaryotes, binds specifically to ATP through two of its subunits (Orc1p and Orc5p). In this study, we investigated the kinetics of ATP binding to ORC by a filter binding assay. The Kd values for the ATP of wild-type ORC and ORC-1A (mutant ORC containing Orc1p with a defective Walker A motif) were less than 10 nm, suggesting that the affinity of Orc5p for ATP is very high. On the other hand, the Kd values for the ATP of ORC-5A (mutant ORC containing Orc5p with a defective Walker A motif) was much higher (about 1.5 microm), suggesting that the affinity of Orc1p for ATP is relatively low in the absence of origin DNA. ATP dissociated more rapidly from its complex with ORC-5A than from its complex with ORC-1A, suggesting that the ATP-Orc5p complex is more stable than ATP-Orc1p complex. Origin DNA fragments decreased the Kd value of ORC-5A for ATP and stabilized the complex of ATP with ORC-5A. Wild-type ORC, ORC-1A, and ORC-5A required different concentrations of ATP for specific binding to origin DNA. All of these results imply that ATP binding to Orc5p, ATP binding to Orc1p, and origin DNA binding to ORC are co-operatively regulated, which may be important for the initiation of DNA replication. PMID:12966094

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2011-07-01

    ATP binding to the ϵ 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 ϵ 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 ϵ 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 ϵ 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 ϵ subunit, even with ATP as a substrate. Further analysis suggested that the compact conformation of the ϵ subunit induced by ATP binding was required to couple ATPase and H+ pumping activities in TFoF1 unless the ϵ subunit was in its extended-state conformation. The present study reveals a novel role of the ϵ subunit as an ATP-sensitive regulator of the coupling of ATPase and H+ pumping activities of TFoF1. PMID:21510843

  6. The a subunit of the A1AO ATP synthase of Methanosarcina mazei G1 contains two conserved arginine residues that are crucial for ATP synthesis.

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2011-01-01

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

  9. SUPERSTARS III: K-2.

    ERIC Educational Resources Information Center

    North Carolina State Dept. of Public Education, Raleigh.

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

  10. CITY III Player's Manual.

    ERIC Educational Resources Information Center

    Envirometrics, Inc., Washington, DC.

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2008-01-01

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

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

    PubMed

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

    2010-02-01

    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

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

    PubMed Central

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

    2011-01-01

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

  15. ATP antagonizes thrombin-induced signal transduction through 12(S)-HETE and cAMP.

    PubMed

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

    2013-01-01

    In this study we have investigated the role of extracellular ATP on thrombin induced-platelet aggregation (TIPA) in washed human platelets. ATP inhibited TIPA in a dose-dependent manner and this inhibition was abolished by apyrase but not by adenosine deaminase (ADA) and it was reversed by extracellular magnesium. Antagonists of P2Y1 and P2Y12 receptors had no effect on this inhibition suggesting that a P2X receptor controlled ATP-mediated TIPA inhibition. ATP also blocked inositol phosphates (IP1, IP2, IP3) generation and [Ca(2+)]i mobilization induced by thrombin. Thrombin reduced cAMP levels which were restored in the presence of ATP. SQ-22536, an adenylate cyclase (AC) inhibitor, partially reduced the inhibition exerted by ATP on TIPA. 12-lipoxygenase (12-LO) inhibitors, nordihidroguaretic acid (NDGA) and 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15(S)-HETE), strongly prevented ATP-mediated TIPA inhibition. Additionally, ATP inhibited the increase of 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE) induced by thrombin. Pretreatment with both SQ-22536 and NDGA almost completely abolished ATP-mediated TIPA inhibition. Our results describe for the first time that ATP implicates both AC and 12-LO pathways in the inhibition of human platelets aggregation in response to agonists. PMID:23826207

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

    PubMed Central

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

    2016-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1992-01-01

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

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

    PubMed

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

    1992-08-01

    Most models of metabolic control concentrate on the regulation of ATP production and largely ignore the regulation of ATP demand. We describe a model, based on the results of Hogan et al. (J. Appl. Physiol. 73: 728-736, 1992), that incorporates the effects of ATP demand. The model is developed from the premise that a unique set of intracellular conditions can be measured at each level of ATP turnover and that this relationship is best described by energetic state. Current concepts suggest that cells are capable of maintaining oxygen consumption in the face of declines in the concentration of oxygen through compensatory changes in cellular metabolites. We show that these compensatory changes can cause significant declines in ATP demand and result in a decline in oxygen consumption and ATP turnover. Furthermore we find that hypoxia does not directly affect the rate of anaerobic ATP synthesis and associated lactate production. Rather, lactate production appears to be related to energetic state, whatever the PO2. The model is used to describe the interaction between ATP demand and ATP supply in determining final ATP turnover. PMID:1400004

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

    PubMed Central

    Omidbakhsh, Navid; Ahmadpour, Faraz; Kenny, Nicole

    2014-01-01

    Background Meters based on adenosine triphosphate (ATP) bioluminescence measurements in relative light units (RLU) are often used to rapidly assess the level of cleanliness of environmental surfaces in healthcare and other settings. Can such ATP measurements be adversely affected by factors such as soil and cleaner-disinfectant chemistry? Objective This study tested a number of leading ATP meters for their sensitivity, linearity of the measurements, correlation of the readings to the actual microbial contamination, and the potential disinfectant chemicals interference in their readings. Methods First, solutions of pure ATP in various concentrations were used to construct a standard curve and determine linearity and sensitivity. Serial dilutions of a broth culture of Staphylococcus aureus, as a representative nosocomial pathogen, were then used to determine if a given meters 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

  20. Intracellular ATP binding is required to activate the slowly activating K+ channel I(Ks).

    PubMed

    Li, Yang; Gao, Junyuan; Lu, Zhongju; McFarland, Kelli; Shi, Jingyi; Bock, Kevin; Cohen, Ira S; Cui, Jianmin

    2013-11-19

    Gating of ion channels by ligands is fundamental to cellular function, and ATP serves as both an energy source and a signaling molecule that modulates ion channel and transporter functions. The slowly activating K(+) channel I(Ks) in cardiac myocytes is formed by KCNQ1 and KCNE1 subunits that conduct K(+) to repolarize the action potential. Here we show that intracellular ATP activates heterologously coexpressed KCNQ1 and KCNE1 as well as I(Ks) in cardiac myocytes by directly binding to the C terminus of KCNQ1 to allow the pore to open. The channel is most sensitive to ATP near its physiological concentration, and lowering ATP concentration in cardiac myocytes results in I(Ks) reduction and action potential prolongation. Multiple mutations that suppress I(Ks) by decreasing the ATP sensitivity of the channel are associated with the long QT (interval between the Q and T waves in electrocardiogram) syndrome that predisposes afflicted individuals to cardiac arrhythmia and sudden death. A cluster of basic and aromatic residues that may form a unique ATP binding site are identified; ATP activation of the wild-type channel and the effects of the mutations on ATP sensitivity are consistent with an allosteric mechanism. These results demonstrate the activation of an ion channel by intracellular ATP binding, and ATP-dependent gating allows I(Ks) to couple myocyte energy state to its electrophysiology in physiologic and pathologic conditions. PMID:24190995

  1. Heat shock protein 70 and ATP as partners in cell homeostasis (Review).

    PubMed

    Mallouk, Y; Vayssier-Taussat, M; Bonventre, J V; Polla, B S

    1999-11-01

    Heat shock proteins (HSP) are molecular chaperones, involved in many cellular functions such as protein folding, transport, maturation and degradation. Since they control the quality of newly synthesized proteins, HSP take part in cellular homeostasis. The Hsp70 family in particular exerts these functions in an adenosine triphosphate (ATP)-dependent manner. ATP is the main energy source used by cells to assume fundamental functions (respiration, proliferation, differentiation, apoptosis). Therefore, ATP levels have to be adapted to the requirements of the cells and ATP generation must constantly compensate ATP consumption. Nevertheless, under particular stress conditions, ATP levels decrease, threatening cell homeostasis and integrity. Cells have developed adaptive and protective mechanisms, among which Hsp70 synthesis and overexpression. In this review, we focus on the mechanisms which regulate Hsp70 expression under ATP depletion, using ischaemia as a paradigmatic model for ATP depletion in vivo, and analyze the molecular targets for Hsp70-mediated protection against ATP depletion. We also consider how these Hsp70-mediated protective effects could be applied in the therapeutical approaches of human diseases associated with cellular ATP depletion. PMID:10534567

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

    PubMed

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

    2011-10-21

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

  3. Air-stimulated ATP release from keratinocytes occurs through connexin hemichannels.

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    1993-04-15

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

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

    PubMed Central

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

    2013-01-01

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

  6. The effects of extracellular ATP and its receptor antagonists on pig oocytes during in vitro maturation.

    PubMed

    Wakizoe, Erika; Ashizawa, Koji; Sakamoto, Shinsuke H; Hemmi, Koichiro; Kobayashi, Ikuo; Tsuzuki, Yasuhiro

    2015-12-01

    We measured the ATP concentrations in the porcine follicular fluid derived from three sizes of follicles (small: 6 mm in diameter). Then, the effects of pre-treatment (100 ?M each for 30 min before maturation) with antagonists for extracellular ATP receptor P2X or P2Y on the nuclear maturation rate of cumulus-cell-enclosed (COs) or -denuded oocytes (DOs) up to the preovulatory stage in the presence or absence of 20 nM ATP (a similar concentration to that of medium-sized follicle fluid) were investigated. The antagonists used were pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) or reactive blue 2 (RB2), for extracellular ATP receptor P2X and P2Y, respectively. In addition, the embryonic development rates of COs pre-treated with RB2 were also evaluated. It was found that when the follicular sizes increased, the ATP concentrations significantly decreased (P < 0.05). No differences were observed in the nuclear maturation rates among all COs, regardless of pre-treatment with (+) or without (-) PPADS and in the presence (+) or absence (-) of ATP during maturation. In contrast, the nuclear maturation rate of the COs, but not DOs, in the ATP(-) RB2(+) group was significantly lower (P < 0.05) than that of the ATP(-) RB2(-) and ATP(+)RB2(-) groups. The pronuclear formation and blastocyst formation rates by parthenogenetic activation in the ATP(-) RB2(+) and ATP(+) RB2(+) groups were significantly lower (P < 0.05) than those in the ATP(-) RB2(-) group. In conclusion, it is suggested that the nuclear maturation of porcine oocytes may be influenced by the ATP receptor P2Y present in the cumulus cells. PMID:25438974

  7. Silencing of Atp6v1c1 Prevents Breast Cancer Growth and Bone Metastasis

    PubMed Central

    Feng, Shengmei; Zhu, Guochun; McConnell, Matthew; Deng, Lianfu; Zhao, Qiang; Wu, Mengrui; Zhou, Qi; Wang, Jinshen; Qi, Jin; Li, Yi-Ping; Chen, Wei

    2013-01-01

    Previous studies have shown that Atp6v1c1, a regulator of the assembly of the V0 and V1 domains of the V-ATPase complex, is up-regulated in metastatic oral tumors. Despite these studies, the function of Atp6v1c1 in tumor growth and metastasis is still unknown. Atp6v1c1's expression in metastatic oral squamous cell carcinoma indicates that Atp6v1c1 has an important function in cancer growth and metastasis. We hypothesized that elevated expression of Atp6v1c1 is essential to cancer growth and metastasis and that Atp6v1c1 promotes cancer growth and metastasis through activation of V-ATPase activity. To test this hypothesis, a Lentivirus-mediated RNAi knockdown approach was used to study the function of Atp6v1c1 in mouse 4T1 mammary tumor cell proliferation and migration in vitro and cancer growth and metastasis in vivo. Our data revealed that silencing of Atp6v1c1 in 4T1 cancer cells inhibited lysosomal acidification and severely impaired 4T1 cell growth, migration, and invasion through Matrigel in vitro. We also show that Atp6v1c1 knockdown with Lenti-c1s3, a lentivirus targeting Atp6v1c1 for shRNA mediated knockdown, can significantly inhibit 4T1 xenograft tumor growth, metastasis, and osteolytic lesions in vivo. Our study demonstrates that Atp6v1c1 may promote breast cancer growth and bone metastasis through regulation of lysosomal V-ATPase activity, indicating that Atp6v1c1 may be a viable target for breast cancer therapy and silencing of Atp6v1c1 may be an innovative therapeutic approach for the treatment and prevention of breast cancer growth and metastasis. PMID:24155661

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

    PubMed Central

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

    2008-01-01

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

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

    PubMed

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

    2015-05-01

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

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

    NASA Technical Reports Server (NTRS)

    Fox, S. W.; Nakashima, T.

    1981-01-01

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

  11. Nucleotide sequence of the Rhodospirillum rubrum atp operon.

    PubMed Central

    Falk, G; Hampe, A; Walker, J E

    1985-01-01

    The nucleotide sequence was determined of a 8775-base-pair region of DNA cloned from the photosynthetic non-sulphur bacterium Rhodospirillum rubrum. It contains a cluster of five genes encoding F1-ATPase subunits. The genes are arranged in the same order as F1 genes in the Escherichia coli unc operon. However, as in the related organism Rhodopseudomonas blastica, neither genes for components of F0, the membrane sector of ATP synthase, nor a homologue of the E. coli uncI gene are associated with this locus, as they are in E. coli. Images Fig. 2. PMID:2861810

  12. Detection of ATP hydrolysis through motion of nanoconfined DNA

    NASA Astrophysics Data System (ADS)

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

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

  13. Efficacy and Limitations of an ATP-Based Monitoring System

    PubMed Central

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

    2010-01-01

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

  14. Mapping ATP-dependent activation at a sigma54 promoter.

    PubMed

    Leach, Robert N; Gell, Christopher; Wigneshweraraj, Sivaramesh; Buck, Martin; Smith, Alastair; Stockley, Peter George

    2006-11-01

    The sigma(54) promoter specificity factor is distinct from other bacterial RNA polymerase (RNAP) sigma factors in that it forms a transcriptionally silent closed complex upon promoter binding. Transcriptional activation occurs through a nucleotide-dependent isomerization of sigma(54), mediated via its interactions with an enhancer-binding activator protein that utilizes the energy released in ATP hydrolysis to effect structural changes in sigma(54) and core RNA polymerase. The organization of sigma(54)-promoter and sigma(54)-RNAP-promoter complexes was investigated by fluorescence resonance energy transfer assays using sigma(54) single cysteine-mutants labeled with an acceptor fluorophore and donor fluorophore-labeled DNA sequences containing mismatches that mimic nifH early- and late-melted promoters. The results show that sigma(54) undergoes spatial rearrangements of functionally important domains upon closed complex formation. sigma(54) and sigma(54)-RNAP promoter complexes reconstituted with the different mismatched DNA constructs were assayed by the addition of the activator phage shock protein F in the presence or absence of ATP and of non-hydrolysable analogues. Nucleotide-dependent alterations in fluorescence resonance energy transfer efficiencies identify different functional states of the activator-sigma(54)-RNAP-promoter complex that exist throughout the mechano-chemical transduction pathway of transcriptional activation, i.e. from closed to open promoter complexes. The results suggest that open complex formation only occurs efficiently on replacement of a repressive fork junction with down-stream melted DNA. PMID:16926155

  15. A fast ATP method based on integral chopper

    NASA Astrophysics Data System (ADS)

    Chen, Wen-jian; Duan, Yuan-yuan; Ma, Shi-wei; Guo, Jun-chao

    2015-02-01

    Detecting angle between target and optical axis in Q-APD is an effective way of fast ATP. It is the key point in this method to acquire the ratio of some parameters. In this paper, a novel method of achieving division of analog electric signal using integral chopper is proposed. This can avoid the problem of high speed acquisition faced in the digital processing solution, that is, MHz level sampling frequency should be used in microsecond level signal. After the target is detected by using Q-APD, the four-channel signals generated by target are amplified by the emitter follower. Then using sample hold and sum difference calculation, background signal and offset signals in azimuth and elevation are generated. Because of using the integral chopper, integration time of offset signal to capacitance is controlled by the background signal, and the output value is just the ratio of the background signal and the offset signal. This value which has is independent of intensity of incident light, represents the angle between the target and optical axis. The optical axis can be driven by servo system based on the angle, therefore realizing the fast ATP.

  16. ATP citrate lyase inhibitors as novel cancer therapeutic agents.

    PubMed

    Zu, Xu-Yu; Zhang, Qing-Hai; Liu, Jiang-Hua; Cao, Ren-Xian; Zhong, Jing; Yi, Guang-Hui; Quan, Zhi-Hua; Pizzorno, Giuseppe

    2012-05-01

    ATP citrate lyase (ACL or ACLY) is an extra-mitochondrial enzyme widely distributed in various human and animal tissues. ACL links glucose and lipid metabolism by catalyzing the formation of acetyl-CoA and oxaloacetate from citrate produced by glycolysis in the presence of ATP and CoA. ACL is aberrantly expressed in many immortalized cells and tumors, such as breast, liver, colon, lung and prostate cancers, and is correlated reversely with tumor stage and differentiation, serving as a negative prognostic marker. ACL is an upstream enzyme of the long chain fatty acid synthesis, providing acetyl-CoA as an essential component of the fatty acid synthesis. Therefore, ACL is a key enzyme of cellular lipogenesis and potent target for cancer therapy. As a hypolipidemic strategy of metabolic syndrome and cancer treatment, many small chemicals targeting ACL have been designed and developed. This review article provides an update for the research and development of ACL inhibitors with a focus on their patent status, offering a new insight into their potential application. PMID:22339355

  17. RAD51 Protein ATP Cap Regulates Nucleoprotein Filament Stability*

    PubMed Central

    Amunugama, Ravindra; He, Yujiong; Willcox, Smaranda; Forties, Robert A.; Shim, Kang-Sup; Bundschuh, Ralf; Luo, Yu; Griffith, Jack; Fishel, Richard

    2012-01-01

    RAD51 mediates homologous recombination by forming an active DNA nucleoprotein filament (NPF). A conserved aspartate that forms a salt bridge with the ATP γ-phosphate is found at the nucleotide-binding interface between RAD51 subunits of the NPF known as the ATP cap. The salt bridge accounts for the nonphysiological cation(s) required to fully activate the RAD51 NPF. In contrast, RecA homologs and most RAD51 paralogs contain a conserved lysine at the analogous structural position. We demonstrate that substitution of human RAD51(Asp-316) with lysine (HsRAD51(D316K)) decreases NPF turnover and facilitates considerably improved recombinase functions. Structural analysis shows that archaebacterial Methanococcus voltae RadA(D302K) (MvRAD51(D302K)) and HsRAD51(D316K) form extended active NPFs without salt. These studies suggest that the HsRAD51(Asp-316) salt bridge may function as a conformational sensor that enhances turnover at the expense of recombinase activity. PMID:22275364

  18. Mechanism of an ATP-independent Protein Disaggregase

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  20. Copper binding triggers compaction in N-terminal tail of human copper pump ATP7B.

    PubMed

    Mondol, Tanumoy; Åden, Jörgen; Wittung-Stafshede, Pernilla

    2016-02-12

    Protein conformational changes are fundamental to biological reactions. For copper ion transport, the multi-domain protein ATP7B in the Golgi network receives copper from the cytoplasmic copper chaperone Atox1 and, with energy from ATP hydrolysis, moves the metal to the lumen for loading of copper-dependent enzymes. Although anticipated, conformational changes involved in ATP7B's functional cycle remain elusive. Using spectroscopic methods we here demonstrate that the four most N-terminal metal-binding domains in ATP7B, upon stoichiometric copper addition, adopt a more compact arrangement which has a higher thermal stability than in the absence of copper. In contrast to previous reports, no stable complex was found in solution between the metal-binding domains and the nucleotide-binding domain of ATP7B. Metal-dependent movement of the first four metal-binding domains in ATP7B may be a trigger that initiates the overall catalytic cycle. PMID:26797276

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

    PubMed Central

    Fields, R. Douglas

    2011-01-01

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

  2. A Tetrahymena Hsp90 co-chaperone promotes siRNA loading by ATP-dependent and ATP-independent mechanisms

    PubMed Central

    Woehrer, Sophie L; Aronica, Lucia; Suhren, Jan H; Busch, Clara Jana-Lui; Noto, Tomoko; Mochizuki, Kazufumi

    2015-01-01

    The loading of small interfering RNAs (siRNAs) and microRNAs into Argonaute proteins is enhanced by Hsp90 and ATP in diverse eukaryotes. However, whether this loading also occurs independently of Hsp90 and ATP remains unclear. We show that the Tetrahymena Hsp90 co-chaperone Coi12p promotes siRNA loading into the Argonaute protein Twi1p in both ATP-dependent and ATP-independent manners in vitro. The ATP-dependent activity requires Hsp90 and the tetratricopeptide repeat (TPR) domain of Coi12p, whereas these factors are dispensable for the ATP-independent activity. Both activities facilitate siRNA loading by counteracting the Twi1p-binding protein Giw1p, which is important to specifically sort the 26- to 32-nt siRNAs to Twi1p. Although Coi12p lacking its TPR domain does not bind to Hsp90, it can partially restore the siRNA loading and DNA elimination defects of COI12 knockout cells, suggesting that Hsp90- and ATP-independent loading of siRNA occurs in vivo and plays a physiological role in Tetrahymena. PMID:25588944

  3. Neuroglial ATP release through innexin channels controls microglial cell movement to a nerve injury

    PubMed Central

    Lipitz, Jeffrey B.; Dahl, Gerhard

    2010-01-01

    Microglia, the immune cells of the central nervous system, are attracted to sites of injury. The injury releases adenosine triphosphate (ATP) into the extracellular space, activating the microglia, but the full mechanism of release is not known. In glial cells, a family of physiologically regulated unpaired gap junction channels called innexons (invertebrates) or pannexons (vertebrates) located in the cell membrane is permeable to ATP. Innexons, but not pannexons, also pair to make gap junctions. Glial calcium waves, triggered by injury or mechanical stimulation, open pannexon/innexon channels and cause the release of ATP. It has been hypothesized that a glial calcium wave that triggers the release of ATP causes rapid microglial migration to distant lesions. In the present study in the leech, in which a single giant glial cell ensheathes each connective, hydrolysis of ATP with 10 U/ml apyrase or block of innexons with 10 µM carbenoxolone (CBX), which decreased injury-induced ATP release, reduced both movement of microglia and their accumulation at lesions. Directed movement and accumulation were restored in CBX by adding ATP, consistent with separate actions of ATP and nitric oxide, which is required for directed movement but does not activate glia. Injection of glia with innexin2 (Hminx2) RNAi inhibited release of carboxyfluorescein dye and microglial migration, whereas injection of innexin1 (Hminx1) RNAi did not when measured 2 days after injection, indicating that glial cells’ ATP release through innexons was required for microglial migration after nerve injury. Focal stimulation either mechanically or with ATP generated a calcium wave in the glial cell; injury caused a large, persistent intracellular calcium response. Neither the calcium wave nor the persistent response required ATP or its release. Thus, in the leech, innexin membrane channels releasing ATP from glia are required for migration and accumulation of microglia after nerve injury. PMID:20876360

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

    PubMed Central

    Guan, Siao-Syun; Sheu, Meei-Ling; Wu, Cheng-Tien; Chiang, Chih-Kang; Liu, Shing-Hwa

    2015-01-01

    In this study, we investigated the role of ATP synthase subunit-β (ATP5b) in diabetic nephropathy. Histopathological changes, fibrosis, and protein expressions of α-smooth muscle actin (α-SMA), advanced glycation end-products (AGEs), and ATP5b were obviously observed in the kidneys of db/db diabetic mice as compared with the control db/m+ mice. The increased ATP5b expression was majorly observed in diabetic renal tubules and was notably observed to locate in cytoplasm of tubule cells, but no significant increase of ATP5b in diabetic glomeruli. AGEs significantly increased protein expression of ATP5b and fibrotic factors and decreased ATP content in cultured renal tubular cells via an AGEs-receptor for AGEs (RAGE) axis pathway. Oxidative stress was also induced in diabetic kidneys and AGEs-treated renal tubular cells. The increase of ATP5b and CTGF protein expression in AGEs-treated renal tubular cells was reversed by antioxidant N-acetylcysteine. ATP5b-siRNA transfection augmented the increased protein expression of α-SMA and CTGF and CTGF promoter activity in AGEs-treated renal tubular cells. The in vivo ATP5b-siRNA delivery significantly enhanced renal fibrosis and serum creatinine in db/db mice with ATP5b down-regulation. These findings suggest that increased ATP5b plays an important adaptive or protective role in decreasing the rate of AGEs-induced renal fibrosis during diabetic condition. PMID:26449648

  5. Studies on the activation by ATP of the 26 S proteasome complex from rat skeletal muscle.

    PubMed Central

    Dahlmann, B; Kuehn, L; Reinauer, H

    1995-01-01

    The 26 S proteasome complex is thought to catalyse the breakdown of ubiquitinated proteins within eukaryotic cells. In addition it has been found that the complex also degrades short-lived proteins such as ornithine decarboxylase in a ubiquitin-independent manner. Both proteolytic processes are paralleled by the hydrolysis of ATP. Here we show that ATP also affects the hydrolytic activity towards fluorigenic peptide substrates by the 26 S proteasome complex from rat skeletal muscle tissue. Low concentrations of ATP (about 25 microM) optimally activate the so-called chymotryptic and tryptic activity by increasing the rate of peptide hydrolysis but not peptidylglutamylpeptide hydrolysis. Activation of the enzyme by ATP is transient but this effect can be enhanced and prolonged by including in the assay an ATP-regenerating system, indicating that ATP is hydrolysed by the 26 S proteasome complex. Although ATP cannot be substituted for by adenosine 5'-[beta,gamma-methylene]triphosphate or AMP, hydrolysis of the phosphoanhydride bond of ATP seems not to be necessary for the activation process of the proteasome complex, a conclusion drawn from the findings that ATP analogues such as adenosine 5'-[beta,gamma-imido]triphosphate, adenosine 5'-O-[gamma-thio]triphosphate, adenosine 5'-O-[beta-thio]-diphosphate and adenosine 5'-[alpha,beta-methylene]triphosphate give the same effect as ATP, and vanadate does not prevent ATP activation. These effects are independent of the presence of Mg2+. Thus, ATP and other nucleotides may act as allosteric activators of peptide-hydrolysing activities of the 26 S proteasome complex as has also been found with the lon protease from Escherichia coli. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:7619056

  6. 31P magnetization transfer measurements of Pi→ATP flux in exercising human muscle

    PubMed Central

    Savage, David B.; Williams, Guy B.; Porter, David; Carpenter, T. Adrian; Brindle, Kevin M.; Kemp, Graham J.

    2016-01-01

    Fundamental criticisms have been made over the use of 31P magnetic resonance spectroscopy (MRS) magnetization transfer estimates of inorganic phosphate (Pi)→ATP flux (VPi-ATP) in human resting skeletal muscle for assessing mitochondrial function. Although the discrepancy in the magnitude of VPi-ATP is now acknowledged, little is known about its metabolic determinants. Here we use a novel protocol to measure VPi-ATP in human exercising muscle for the first time. Steady-state VPi-ATP was measured at rest and over a range of exercise intensities and compared with suprabasal oxidative ATP synthesis rates estimated from the initial rates of postexercise phosphocreatine resynthesis (VATP). We define a surplus Pi→ATP flux as the difference between VPi-ATP and VATP. The coupled reactions catalyzed by the glycolytic enzymes GAPDH and phosphoglycerate kinase (PGK) have been shown to catalyze measurable exchange between ATP and Pi in some systems and have been suggested to be responsible for this surplus flux. Surplus VPi-ATP did not change between rest and exercise, even though the concentrations of Pi and ADP, which are substrates for GAPDH and PGK, respectively, increased as expected. However, involvement of these enzymes is suggested by correlations between absolute and surplus Pi→ATP flux, both at rest and during exercise, and the intensity of the phosphomonoester peak in the 31P NMR spectrum. This peak includes contributions from sugar phosphates in the glycolytic pathway, and changes in its intensity may indicate changes in downstream glycolytic intermediates, including 3-phosphoglycerate, which has been shown to influence the exchange between ATP and Pi catalyzed by GAPDH and PGK. PMID:26744504

  7. A metabolic control analysis of kinetic controls in ATP free energy metabolism in contracting skeletal muscle.

    PubMed

    Jeneson, J A; Westerhoff, H V; Kushmerick, M J

    2000-09-01

    A system analysis of ATP free energy metabolism in skeletal muscle was made using the principles of metabolic control theory. We developed a network model of ATP free energy metabolism in muscle consisting of actomyosin ATPase, sarcoplasmic reticulum (SR) Ca(2+)-ATPase, and mitochondria. These components were sufficient to capture the major aspects of the regulation of the cytosolic ATP-to-ADP concentration ratio (ATP/ADP) in muscle contraction and had inherent homeostatic properties regulating this free energy potential. As input for the analysis, we used ATP metabolic flux and the cytosolic ATP/ADP at steady state at six contraction frequencies between 0 and 2 Hz measured in human forearm flexor muscle by (31)P-NMR spectroscopy. We used the mathematical formalism of metabolic control theory to analyze the distribution of fractional kinetic control of ATPase flux and the ATP/ADP in the network at steady state among the components over this experimental range and an extrapolated range of stimulation frequencies (up to 10 Hz). The control analysis showed that the contractile actomyosin ATPase has dominant kinetic control of ATP flux in forearm flexor muscle over the 0- to 1.6-Hz range of contraction frequencies that resulted in steady states, as determined by (31)P-NMR. However, flux control begins to shift toward mitochondria at >1 Hz. This inversion of flux control from ATP demand to ATP supply control hierarchy progressed as the contraction frequency increased past 2 Hz and was nearly complete at 10 Hz. The functional significance of this result is that, at steady state, ATP free energy consumption cannot outstrip the ATP free energy supply. Therefore, this reduced, three-component muscle ATPase system is inherently homeostatic. PMID:10942732

  8. Altered ATP release and metabolism in dorsal root ganglia of neuropathic rats

    PubMed Central

    Matsuka, Yoshizo; Ono, Takeshi; Iwase, Hirotate; Mitrirattanakul, Somsak; Omoto, Kevin S; Cho, Ting; Lam, Yan Yan N; Snyder, Bradley; Spigelman, Igor

    2008-01-01

    Background Adenosine 5'-triphosphate (ATP) has a ubiquitous role in metabolism and a major role in pain responses after tissue injury. We investigated the changes in basal and KCl-evoked ATP release from rat dorsal root ganglia (DRG) after peripheral neuropathy induction by unilateral sciatic nerve entrapment (SNE). Results After SNE, rats develop long-lasting decreases in ipsilateral hindpaw withdrawal thresholds to mechanical and thermal stimulation. At 15–21 days after neuropathy induction, excised ipsilateral L4-L5 DRG display significantly elevated basal extracellular ATP levels compared to contralateral or control (naive) DRG. However, KCl-evoked ATP release is no longer observed in ipsilateral DRG. We hypothesized that the differential SNE effects on basal and evoked ATP release could result from the conversion of extracellular ATP to adenosine with subsequent activation of adenosine A1 receptors (A1Rs) on DRG neurons. Adding the selective A1R agonist, 2-chloro-N6-cyclopentyladenosine (100 nM) significantly decreased basal and evoked ATP release in DRG from naïve rats, indicating functional A1R activation. In DRG ipsilateral to SNE, adding a selective A1R antagonist, 8-cyclopentyl-1,3-dipropylxanthine (30 nM), further increased basal ATP levels and relieved the blockade of KCl-evoked ATP release suggesting that increased A1R activation attenuates evoked ATP release in neurons ipsilateral to SNE. To determine if altered ATP release was a consequence of altered DRG metabolism we compared O2 consumption between control and neuropathic DRG. DRG ipsilateral to SNE consumed O2 at a higher rate than control or contralateral DRG. Conclusion These data suggest that peripheral nerve entrapment increases DRG metabolism and ATP release, which in turn is modulated by increased A1R activation. PMID:19108746

  9. 31P magnetization transfer measurements of Pi→ATP flux in exercising human muscle.

    PubMed

    Sleigh, Alison; Savage, David B; Williams, Guy B; Porter, David; Carpenter, T Adrian; Brindle, Kevin M; Kemp, Graham J

    2016-03-15

    Fundamental criticisms have been made over the use of (31)P magnetic resonance spectroscopy (MRS) magnetization transfer estimates of inorganic phosphate (Pi)→ATP flux (VPi-ATP) in human resting skeletal muscle for assessing mitochondrial function. Although the discrepancy in the magnitude of VPi-ATP is now acknowledged, little is known about its metabolic determinants. Here we use a novel protocol to measure VPi-ATP in human exercising muscle for the first time. Steady-state VPi-ATP was measured at rest and over a range of exercise intensities and compared with suprabasal oxidative ATP synthesis rates estimated from the initial rates of postexercise phosphocreatine resynthesis (VATP). We define a surplus Pi→ATP flux as the difference between VPi-ATP and VATP. The coupled reactions catalyzed by the glycolytic enzymes GAPDH and phosphoglycerate kinase (PGK) have been shown to catalyze measurable exchange between ATP and Pi in some systems and have been suggested to be responsible for this surplus flux. Surplus VPi-ATP did not change between rest and exercise, even though the concentrations of Pi and ADP, which are substrates for GAPDH and PGK, respectively, increased as expected. However, involvement of these enzymes is suggested by correlations between absolute and surplus Pi→ATP flux, both at rest and during exercise, and the intensity of the phosphomonoester peak in the (31)P NMR spectrum. This peak includes contributions from sugar phosphates in the glycolytic pathway, and changes in its intensity may indicate changes in downstream glycolytic intermediates, including 3-phosphoglycerate, which has been shown to influence the exchange between ATP and Pi catalyzed by GAPDH and PGK. PMID:26744504

  10. Calcium release from intracellular stores evoked by extracellular ATP in a Xenopus renal epithelial cell line.

    PubMed Central

    Mori, M; Hosomi, H; Nishizaki, T; Kawahara, K; Okada, Y

    1997-01-01

    1. The signal transduction mechanism mediating extracellular adenosine 5'-triphosphate (ATP)-induced calcium release in a renal epithelial cell line (A6) was investigated using the whole-cell voltage-clamp technique and fura-2 fluorescence measurement. 2. ATP (10 microM) activated calcium-dependent non-selective cation channels in cells held under voltage clamp. 3. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S; 0.1-1.0 mM) in the pipette inhibited the ATP-activated calcium-dependent currents. With guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; 0.1-1.0 mM) in the pipette, the currents were spontaneously elicited without application of ATP. Pretreatment with pertussis toxin (PTX) affected neither the ATP-activated currents nor the increase in intracellular free calcium concentration ([Ca2+]i) evoked by ATP. 4. Intracellular application of neomycin or heparin inhibited the ATP-activated currents. Inositol 1,4,5-trisphosphate (IP3; 0.1-100 microM) in the internal solution produced currents similar to those due to ATP activation. 5. These results suggest that a PTX-insensitive guanosine 5'-triphosphate (GTP)-binding regulatory protein (G. protein) is involved in extracellular. ATP-induced phosphoinositide turnover and subsequent calcium release from IP3-sensitive stores, which subsequently activates the calcium-dependent channels in A6 cells. PMID:9263916

  11. Lipid-mediated Protein-protein Interactions Modulate Respiration-driven ATP Synthesis.

    PubMed

    Nilsson, Tobias; Lundin, Camilla Rydström; Nordlund, Gustav; Ädelroth, Pia; von Ballmoos, Christoph; Brzezinski, Peter

    2016-01-01

    Energy conversion in biological systems is underpinned by membrane-bound proton transporters that generate and maintain a proton electrochemical gradient across the membrane which used, e.g. for generation of ATP by the ATP synthase. Here, we have co-reconstituted the proton pump cytochrome bo3 (ubiquinol oxidase) together with ATP synthase in liposomes and studied the effect of changing the lipid composition on the ATP synthesis activity driven by proton pumping. We found that for 100 nm liposomes, containing 5 of each proteins, the ATP synthesis rates decreased significantly with increasing fractions of DOPA, DOPE, DOPG or cardiolipin added to liposomes made of DOPC; with e.g. 5% DOPG, we observed an almost 50% decrease in the ATP synthesis rate. However, upon increasing the average distance between the proton pumps and ATP synthases, the ATP synthesis rate dropped and the lipid dependence of this activity vanished. The data indicate that protons are transferred along the membrane, between cytochrome bo3 and the ATP synthase, but only at sufficiently high protein densities. We also argue that the local protein density may be modulated by lipid-dependent changes in interactions between the two proteins complexes, which points to a mechanism by which the cell may regulate the overall activity of the respiratory chain. PMID:27063297

  12. Structural Basis for a Unique ATP Synthase Core Complex from Nanoarcheaum equitans.

    PubMed

    Mohanty, Soumya; Jobichen, Chacko; Chichili, Vishnu Priyanka Reddy; Velázquez-Campoy, Adrián; Low, Boon Chuan; Hogue, Christopher W V; Sivaraman, J

    2015-11-01

    ATP synthesis is a critical and universal life process carried out by ATP synthases. Whereas eukaryotic and prokaryotic ATP synthases are well characterized, archaeal ATP synthases are relatively poorly understood. The hyperthermophilic archaeal parasite, Nanoarcheaum equitans, lacks several subunits of the ATP synthase and is suspected to be energetically dependent on its host, Ignicoccus hospitalis. This suggests that this ATP synthase might be a rudimentary machine. Here, we report the crystal structures and biophysical studies of the regulatory subunit, NeqB, the apo-NeqAB, and NeqAB in complex with nucleotides, ADP, and adenylyl-imidodiphosphate (non-hydrolysable analog of ATP). NeqB is ∼20 amino acids shorter at its C terminus than its homologs, but this does not impede its binding with NeqA to form the complex. The heterodimeric NeqAB complex assumes a closed, rigid conformation irrespective of nucleotide binding; this differs from its homologs, which require conformational changes for catalytic activity. Thus, although N. equitans possesses an ATP synthase core A3B3 hexameric complex, it might not function as a bona fide ATP synthase. PMID:26370083

  13. Lipid-mediated Protein-protein Interactions Modulate Respiration-driven ATP Synthesis

    PubMed Central

    Nilsson, Tobias; Lundin, Camilla Rydström; Nordlund, Gustav; Ädelroth, Pia; von Ballmoos, Christoph; Brzezinski, Peter

    2016-01-01

    Energy conversion in biological systems is underpinned by membrane-bound proton transporters that generate and maintain a proton electrochemical gradient across the membrane which used, e.g. for generation of ATP by the ATP synthase. Here, we have co-reconstituted the proton pump cytochrome bo3 (ubiquinol oxidase) together with ATP synthase in liposomes and studied the effect of changing the lipid composition on the ATP synthesis activity driven by proton pumping. We found that for 100 nm liposomes, containing 5 of each proteins, the ATP synthesis rates decreased significantly with increasing fractions of DOPA, DOPE, DOPG or cardiolipin added to liposomes made of DOPC; with e.g. 5% DOPG, we observed an almost 50% decrease in the ATP synthesis rate. However, upon increasing the average distance between the proton pumps and ATP synthases, the ATP synthesis rate dropped and the lipid dependence of this activity vanished. The data indicate that protons are transferred along the membrane, between cytochrome bo3 and the ATP synthase, but only at sufficiently high protein densities. We also argue that the local protein density may be modulated by lipid-dependent changes in interactions between the two proteins complexes, which points to a mechanism by which the cell may regulate the overall activity of the respiratory chain. PMID:27063297

  14. Mechanisms of ATP release and signalling in the blood vessel wall

    PubMed Central

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

    2012-01-01

    The nucleotide adenosine 5′-triphosphate (ATP) has classically been considered the cell's primary energy currency. Importantly, a novel role for ATP as an extracellular autocrine and/or paracrine signalling molecule has evolved over the past century and extensive work has been conducted to characterize the ATP-sensitive purinergic receptors expressed on almost all cell types in the body. Extracellular ATP elicits potent effects on vascular cells to regulate blood vessel tone but can also be involved in vascular pathologies such as atherosclerosis. While the effects of purinergic signalling in the vasculature have been well documented, the mechanism(s) mediating the regulated release of ATP from cells in the blood vessel wall and circulation are now a key target of investigation. The aim of this review is to examine the current proposed mechanisms of ATP release from vascular cells, with a special emphasis on the transporters and channels involved in ATP release from vascular smooth muscle cells, endothelial cells, circulating red blood cells, and perivascular sympathetic nerves, including vesicular exocytosis, plasma membrane F1/F0-ATP synthase, ATP-binding cassette (ABC) transporters, connexin hemichannels, and pannexin channels. PMID:22678409

  15. A Bacterial Virulence Protein Promotes Pathogenicity by Inhibiting the Bacterium's Own F1Fo ATP Synthase

    PubMed Central

    Lee, Eun-Jin; Pontes, Mauricio H.; Groisman, Eduardo A.

    2013-01-01

    SUMMARY Several intracellular pathogens including Salmonella enterica and Mycobacterium tuberculosis require the virulence protein MgtC to survive within macrophages and to cause a lethal infection in mice. We now report that, unlike secreted virulence factors that target the host vacuolar ATPase to withstand phagosomal acidity, the MgtC protein acts on Salmonella's own F1Fo ATP synthase. This complex couples proton translocation to ATP synthesis/ hydrolysis and is required for virulence. We establish that MgtC interacts with the a subunit of the F1Fo ATP synthase, hindering ATP-driven proton translocation and NADH-driven ATP synthesis in inverted vesicles. An mgtC null mutant displays heightened ATP levels and an acidic cytoplasm whereas mgtC overexpression decreases ATP levels. A single amino acid substitution in MgtC that prevents binding to the F1Fo ATP synthase abolishes control of ATP levels and attenuates pathogenicity. MgtC provides a singular example of a virulence protein that promotes pathogenicity by interfering with another virulence protein. PMID:23827679

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

    PubMed Central

    2012-01-01

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

  17. Activity-driven local ATP synthesis is required for synaptic function

    PubMed Central

    Rangaraju, Vidhya; Calloway, Nathaniel; Ryan, Timothy A.

    2014-01-01

    Summary Cognitive function is tightly related to metabolic state but the locus of this control is not well understood. Synapses are thought to present large ATP demands however it is unclear how fuel availability and electrical activity impact synaptic ATP levels, and how ATP availability controls synaptic function. We developed a quantitative genetically-encoded optical reporter of presynaptic ATP, Syn-ATP, and find that electrical activity imposes large metabolic demands that are met via activity-driven control of both glycolysis and mitochondrial function. We discovered that the primary source of activity-driven metabolic demand is the synaptic vesicle cycle. In metabolically intact synapses, activity-driven ATP synthesis is well matched to the energetic needs of synaptic function which at steady state results in ~ 106 free ATPs per nerve terminal. Despite this large reservoir of ATP we find that several key aspects of presynaptic function are severely impaired following even brief interruptions in activity-stimulated ATP synthesis. PMID:24529383

  18. ATP-containing vesicles in stria vascular marginal cell cytoplasms in neonatal rat cochlea are lysosomes

    PubMed Central

    Liu, Jun; Liu, Wenjing; Yang, Jun

    2016-01-01

    We confirmed that ATP is released from cochlear marginal cells in the stria vascular but the cell organelle in which ATP stores was not identified until now. Thus, we studied the ATP-containing cell organelles and suggest that these are lysosomes. Primary cultures of marginal cells of Sprague-Dawley rats aged 1–3 days was established. Vesicles within marginal cells stained with markers were identified under confocal laser scanning microscope and transmission electron microscope (TEM). Then ATP release from marginal cells was measured after glycyl-L-phenylalanine-ß- naphthylamide (GPN) treatment using a bioluminescent assay. Quinacrine-stained granules within marginal cells were labeled with LysoTracker, a lysosome tracer, and lysosomal-associated membrane protein 1(LAMP1), but not labeled with the mitochondrial tracer MitoTracker. Furthermore, LysoTracker-labelled puncta showed accumulation of Mant-ATP, an ATP analog. Treatment with 200 μM GPN quenched fluorescently labeled puncta after incubation with LysoTracker or quinacrine, but not MitoTracker. Quinacrine-labeled organelles observed by TEM were lysosomes, and an average 27.7 percent increase in ATP luminescence was observed in marginal cells extracellular fluid after GPN treatment. ATP-containing vesicles in cochlear marginal cells of the stria vascular from neonatal rats are likely lysosomes. ATP release from marginal cells may be via Ca2+-dependent lysosomal exocytosis. PMID:26864824

  19. Inhibition of ATP release from Erythrocytes: A role for EPACs and PKC

    PubMed Central

    Adderley, Shaquria P.; Sridharan, Meera; Bowles, Elizabeth A.; Stephenson, Alan H.; Sprague, Randy S.; Ellsworth, Mary L.

    2010-01-01

    Objective Here we demonstrate that, in human erythrocytes, increases in cAMP that are not localized to a specific receptor-mediated signaling pathway for ATP release can activate effector proteins resulting in inhibition of ATP release. Specifically we sought to establish that exchange proteins activated by cAMP (EPACs) inhibit ATP release via activation of protein kinase C (PKC). Methods ATP release stimulated by iloprost (ILO), or isoproterenol (ISO), was determined in the absence and presence of selective phosphodiesterase inhibitors and/or the EPAC activator, 8CPT2OMecAMP (8CPT). To determine whether EPACs inhibit ATP release via activation of PKC, erythrocytes were incubated with phorbol 12-myristate 13-acetate (PMA) prior to either forskolin or ILO in the absence and presence of a PKC inhibitor, calphostin C (CALC). Results Selective inhibition of PDEs in one pathway inhibited ATP release in response to activation of the other cAMP-dependent pathway. 8CPT and PMA inhibited both ILO- and ISO-induced ATP release. Inhibition of ATP release with 8CPT was rescued by CALC. Conclusion These results support the hypothesis that cAMP not localized to a specific signaling pathway can activate EPACs which inhibit ATP release via activation of PKC and suggest a novel role for EPACs in erythrocytes. PMID:21166931

  20. Inhibition of ATP-activated current by zinc in dorsal root ganglion neurones of bullfrog.

    PubMed Central

    Li, C; Peoples, R W; Weight, F F

    1997-01-01

    1. The effect of Zn2+ on ATP-activated current was studied in bullfrog dorsal root ganglion (DRG) neurones using the whole-cell patch-clamp technique. 2. Zn2+ (2-800 microM) inhibited current activated by submaximal concentrations of ATP. The Zn2+ concentration that produced 50% inhibition (IC50) of current activated by 2.5 microM ATP was 61 +/- 9.8 microM. When ATP concentrations were adjusted to account for chelation of Zn2+, the IC50 of Zn2+ was 86 +/- 18 microM. 3. The inhibitory action of Zn2+ on ATP-gated channels did not appear to be due to a decrease in the concentration of one or more species of ATP. 4. Zn2+ inhibition of ATP-activated current was independent of membrane potential between -80 and +40 mV, and did not involve a shift in the reversal potential of the current. 5. Zn2+ (100 microM) shifted the ATP concentration-response curve to the right in a parallel manner, increasing the EC50 for ATP from 2.5 +/- 0.5 microM to 5.5 +/- 0.4 microM. 6. Zn2+ decreased the time constant of deactivation of ATP-gated ion channels without affecting the time constant of activation or desensitization. 7. Dithiothreitol (DTT) reversed Zn2+ inhibition of ATP-activated current. 8. 2-Methylthio ATP, alpha,beta-methylene ATP and ADP activated current with EC50 values of 2.4 +/- 0.3. 50.1 +/- 5.8 and 303.1 +/- 53.9 microM, respectively. Adenosine, AMP or beta,gamma-methylene ATP did not evoke detectable current. 9. Reactive Blue 2 and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid inhibited ATP-activated current. 10. The results suggest that Zn2+ can inhibit P2X purinoceptor function by decreasing the affinity of the binding site for ATP. These observations provide the first evidence for this action of Zn2+ on a neurotransmitter-gated ion channel. Furthermore, the receptor-channel in these neurones appears to be a novel member of the P2X purinoceptor class. PMID:9457642

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

    PubMed Central

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

    2014-01-01

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

  2. Effect of ATP Sulfurylase Overexpression in Bright Yellow 2 Tobacco Cells1

    PubMed Central

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

    1998-01-01

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

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

    PubMed

    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

    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

  4. Antithrombin III blood test

    MedlinePlus

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

  5. Report of the Adult Treatment Panel III: the 2001 National Cholesterol Education Program guidelines on the detection, evaluation and treatment of elevated cholesterol in adults.

    PubMed

    Pasternak, Richard C

    2003-08-01

    The ATP III report represents an important advance from previous ATP reports dating back to the late 1980s. The guidelines are more tightly evidence-based than previous reports, partly because of evolution of the guideline process, requiring clearly delineated links between evidence and recommendations and also because of the robust evidence base published over the last decade. An important change in ATP III is the expansion of the high-risk category to include patients without evident vascular disease, but with a level of risk equivalent to those patients with established CHD. This group termed "coronary heart disease equivalents" now includes patients with diabetes, and those with a 10-year absolute risk of over 20 percent for CHD events. With the ATP III report, the Framingham risk score is formally introduced into the guideline process. The scoring system allows for easy calculation of the absolute risk for an individual of having a "hard" CHD event (myocardial infarction, or CHD death). The report also discusses in detail concepts of lifetime or long-term risk. ATP III has broadened recommendations for lifestyle change termed "therapeutic lifestyle change (TLC)," and eliminated the step 1 and step 2 diet approach. Finally, the report details established approaches to improve adherence and provides patients and clinicians with a set of implementation tools to enhance use of the guidelines and compliance with the guidelines' recommendations. It is hoped that by improved understanding, recognition of a firm evidence base, and education through multiple channels, that adherence with the new ATP III guidelines will improve the care of our population by more effectively targeting lipid factors that lead to the development and progression of atherosclerotic cardiovascular disease. PMID:14621453

  6. Two ATP Binding Cassette G Transporters, Rice ATP Binding Cassette G26 and ATP Binding Cassette G15, Collaboratively Regulate Rice Male Reproduction1[OPEN

    PubMed Central

    Zhao, Guochao; Shi, Jianxin; Liang, Wanqi; Xue, Feiyang; Luo, Qian; Zhu, Lu; Qu, Guorun; Chen, Mingjiao; Schreiber, Lukas; Zhang, Dabing

    2015-01-01

    Male reproduction in higher plants requires the support of various metabolites, including lipid molecules produced in the innermost anther wall layer (the tapetum), but how the molecules are allocated among different anther tissues remains largely unknown. Previously, rice (Oryza sativa) ATP binding cassette G15 (ABCG15) and its Arabidopsis (Arabidopsis thaliana) ortholog were shown to be required for pollen exine formation. Here, we report the significant role of OsABCG26 in regulating the development of anther cuticle and pollen exine together with OsABCG15 in rice. Cytological and chemical analyses indicate that osabcg26 shows reduced transport of lipidic molecules from tapetal cells for anther cuticle development. Supportively, the localization of OsABCG26 is on the plasma membrane of the anther wall layers. By contrast, OsABCG15 is polarly localized in tapetal plasma membrane facing anther locules. osabcg26 osabcg15 double mutant displays an almost complete absence of anther cuticle and pollen exine, similar to that of osabcg15 single mutant. Taken together, we propose that OsABCG26 and OsABCG15 collaboratively regulate rice male reproduction: OsABCG26 is mainly responsible for the transport of lipidic molecules from tapetal cells to anther wall layers, whereas OsABCG15 mainly is responsible for the export of lipidic molecules from the tapetal cells to anther locules for pollen exine development. PMID:26392263

  7. Vacuolar ATPases, like F1,F0-ATPases, show a strong dependence of the reaction velocity on the binding of more than one ATP per enzyme.

    PubMed Central

    Kasho, V N; Boyer, P D

    1989-01-01

    Recent studies with vacuolar ATPases have shown that multiple copies catalytic subunits are present and that these have definite sequence homology with catalytic subunits of the F1,F0-ATPases. Experiments are reported that assess whether the vacuolar ATPases may have the unusual catalytic cooperativity with sequential catalytic site participation as in the binding change mechanism for the F1,F0-ATPases. The extent of reversal of bound ATP hydrolysis to bound ADP and Pi as medium ATP concentration was lowered was determined by 18O-exchange measurements for yeast and neurospora vacuolar ATPases. The results show a pronounced increase in the extent of water oxygen incorporation into the Pi formed as ATP concentration is decreased to the micromolar range. The F1,F0-ATPase from neurospora mitochondria showed an even more pronounced modulation, similar to that of other F1-type ATPases. The vacuolar ATPases thus appear to have a catalytic mechanism quite analogous to that of the F1,F0-ATPases. PMID:2530585

  8. Evidence for adenylate nucleotide transport (ATP-ADP translocation) in vesicles of Frankia sp. strain EAN1pec.

    PubMed Central

    Tisa, L S; Ensign, J C

    1988-01-01

    Atractyloside and carboxyatractyloside partially inhibited nitrogenase activity (acetylene reduction) by isolated vesicles of Frankia strain EAN1pec. Extracts of disrupted vesicles showed nitrogenase activity that was not affected by the inhibitors. The vesicles accumulated ATP by an atractyloside-sensitive mechanism. This inhibition of ATP uptake was reversed when vesicles were permeabilized by detergent. Uptake of ATP was inhibited by excess ATP and ADP, but not AMP or adenosine, and by a calcium-dependent ATPase inhibitor. Uptake was stimulated by calcium ions. Accumulation of ATP was accompanied by release of ADP and AMP from the vesicles. The ATP taken up by vesicles and cells grown with N2 as the nitrogen source was found in the corresponding cell pools only as ATP. The data indicate activity of an ATP-ADP translocase system in vesicles of this organism. The role of ATP translocation in the symbiosis between Frankia strain EAN1pec and plant root nodules is discussed. PMID:3164310

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

    PubMed Central

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

    2015-01-01

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

  10. A model to predict the ATP equivalents of macronutrients absorbed from food.

    PubMed

    Coles, Leah; Rutherfurd, Shane; Moughan, Paul

    2013-02-26

    Calculating the physiologically available energy of food at the cellular level (ATP), based on known stoichiometric relationships and predicted nutrient uptake from the human digestive tract may be more accurate than using currently available factorial or empirical models for estimating dietary energy. The objective was to develop a model that can be used for describing the ATP costs/yields associated with the total tract uptake of the energy-yielding nutrients for an adult human in a state of weight loss (sub-maintenance energy intakes). A series of predictive equations for determining ATP yields/costs were developed and applied to the uptake of each energy-yielding nutrient, as predicted separately in the upper-digestive tract and the hindgut using a dual in vivo-in vitro digestibility assay. The costs associated with nutrient ingestion, absorption and transport and with the synthesis and excretion of urea produced from amino acid catabolism were calculated. ATP yields (not including costs associated with digestion, absorption and transport) were predicted as 28.9 mol ATP per mol glucose; 4.7-32.4 mol ATP per mol amino acid and 10.1 mol ATP per mol ethanol, while yields for fatty acids ranged from 70.8 mol ATP per mol lauric acid (C12) to 104 mol ATP per mol linolenic acid (C18?:?3). The energetic contribution of hindgut fermentation was predicted to be 101.7 mmol ATP per g organic matter fermented. The model is not proposed as a new system for describing the energy value of foods in the diet generally, but is a means to give a relative ranking of foods in terms of physiologically available energy (ATP) with particular application in the development of specialised weight-loss foods. PMID:23233079

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

    PubMed Central

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

    2013-01-01

    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

  12. Functional domains of an ATP-dependent DNA ligase.

    PubMed

    Doherty, A J; Wigley, D B

    1999-01-01

    The crystal structure of an ATP-dependent DNA ligase from bacteriophage T7 revealed that the protein comprised two structural domains. In order to investigate the biochemical activities of these domains, we have overexpressed them separately and purified them to homogeneity. The larger N-terminal domain retains adenylation and ligase activities, though both at a reduced level. The adenylation activity of the large domain is stimulated by the presence of the smaller domain, suggesting that a conformational change is required for adenylation in the full length protein. The DNA binding properties of the two fragments have also been studied. The larger domain is able to band shift both single and double-stranded DNA, while the smaller fragment is only able to bind to double-stranded DNA. These data suggest that the specificity of DNA ligases for nick sites in DNA is produced by a combination of these different DNA binding activities in the intact enzyme. PMID:9878388

  13. Regulation of ATP hydrolysis in liver mitochondria from ground squirrel.

    PubMed

    Bronnikov, G E; Vinogradova, S O; Chernyak, B V

    1990-06-18

    The uncoupler-induced inactivation of H(+)-ATPase in liver mitochondria from ground squirrel has been studied. The dependence of this process on delta mu H+, pH and ATP indicates that it is caused by the protein inhibitor. This conclusion is also supported by the protective effect of Zn2+ and Cu2+. The inactivation can be induced by Ca2+ at low concentrations in the presence of phosphate. It is shown that the protein inhibitor inactivates ATPase almost completely under optimal conditions while its effect in mice or rat liver mitochondria does not exceed 30%. The potential efficiency of the inhibitor's action does not depend on either the season or the state of animals (hibernating or active). At the same time, the sensitivity of this system to Ca2+ is significantly lower in active (summer) animals. PMID:2142105

  14. NASA ATP Force Measurement Technology Capability Strategic Plan

    NASA Technical Reports Server (NTRS)

    Rhew, Ray D.

    2008-01-01

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

  15. ATP binding cassette G transporters and plant male reproduction.

    PubMed

    Zhao, Guochao; Shi, Jianxin; Liang, Wanqi; Zhang, Dabing

    2016-03-01

    The function of ATP Binding Cassette G (ABCG) transporters in the regulation of plant vegetative organs development has been well characterized in various plant species. In contrast, their function in reproductive development particularly male reproductive development received considerably less attention till some ABCG transporters was reported to be associated with anther and pollen wall development in Arabidopsis thaliana and rice (Oryza sativa) during the past decade. This mini-review summarizes current knowledge of ABCG transporters regarding to their roles in male reproduction and underlying genetic and biochemical mechanisms, which makes it evident that ABCG transporters represent one of those conserved and divergent components closely related to male reproduction in plants. This mini-review also discusses the current challenges and future perspectives in this particular field. PMID:26906115

  16. Invited review: Architectures and mechanisms of ATP binding cassette proteins.

    PubMed

    Hopfner, Karl-Peter

    2016-08-01

    ATP binding cassette (ABC) ATPases form chemo-mechanical engines and switches that function in a broad range of biological processes. Most prominently, a very large family of integral membrane NTPases-ABC transporters-catalyzes the import or export of a diverse molecules across membranes. ABC proteins are also important components of the chromosome segregation, recombination, and DNA repair machineries and regulate or catalyze critical steps of ribosomal protein synthesis. Recent structural and mechanistic studies draw interesting architectural and mechanistic parallels between diverse ABC proteins. Here, I review this state of our understanding how NTP-dependent conformational changes of ABC proteins drive diverse biological processes. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 492-504, 2016. PMID:27037766

  17. ATP Dependent Chromatin Remodeling Enzymes in Embryonic Stem Cells

    PubMed Central

    Saladi, Srinivas Vinod

    2010-01-01

    Embryonic stem (ES) cells are pluripotent cells that can self renew or be induced to differentiate into multiple cell lineages, and thus have the potential to be utilized in regenerative medicine. Key pluripotency specific factors (Oct 4/Sox2/Nanog/Klf4) maintain the pluripotent state by activating expression of pluripotency specific genes and by inhibiting the expression of developmental regulators. Pluripotent ES cells are distinguished from differentiated cells by a specialized chromatin state that is required to epigenetically regulate the ES cell phenotype. Recent studies show that in addition to pluripotency specific factors, chromatin remodeling enzymes play an important role in regulating ES cell chromatin and the capacity to self-renew and to differentiate. Here we review recent studies that delineate the role of ATP dependent chromatin remodeling enzymes in regulating ES cell chromatin structure. PMID:20148317

  18. The rapid, simple and improved preparation of high specific activity alpha-[32P]dATP and alpha-[32P]ATP.

    PubMed

    Symons, R H

    1977-12-01

    An improved method is described for the rapid and simple preparation of alpha-[32P]dATP and alpha-[32P]ATP from 32Pi in good yields and with specific activities from 20 - 150 Ci/mmol. The two-step procedure involves the chemical synthesis of the mononucleotide followed by its enzymic conversion to the triphosphate with myokinase (EC 2.7.4.3) and pyruvate kinase (EC 2.7.1.40) in the presence of trace amounts of dATP or ATP to prime the reaction. The two steps are carried out in the same reaction flask and the only purification step required is a step-wise elution from a column of DEAE-cellulose. PMID:600797

  19. Human K(ATP) channelopathies: diseases of metabolic homeostasis.

    PubMed

    Olson, Timothy M; Terzic, Andre

    2010-07-01

    Assembly of an inward rectifier K+ channel pore (Kir6.1/Kir6.2) and an adenosine triphosphate (ATP)-binding regulatory subunit (SUR1/SUR2A/SUR2B) forms ATP-sensitive K+ (KATP) channel heteromultimers, widely distributed in metabolically active tissues throughout the body. KATP channels are metabolism-gated biosensors functioning as molecular rheostats that adjust membrane potential-dependent functions to match cellular energetic demands. Vital in the adaptive response to (patho)physiological stress, KATP channels serve a homeostatic role ranging from glucose regulation to cardioprotection. Accordingly, genetic variation in KATP channel subunits has been linked to the etiology of life-threatening human diseases. In particular, pathogenic mutations in KATP channels have been identified in insulin secretion disorders, namely, congenital hyperinsulinism and neonatal diabetes. Moreover, KATP channel defects underlie the triad of developmental delay, epilepsy, and neonatal diabetes (DEND syndrome). KATP channelopathies implicated in patients with mechanical and/or electrical heart disease include dilated cardiomyopathy (with ventricular arrhythmia; CMD1O) and adrenergic atrial fibrillation. A common Kir6.2 E23K polymorphism has been associated with late-onset diabetes and as a risk factor for maladaptive cardiac remodeling in the community-at-large and abnormal cardiopulmonary exercise stress performance in patients with heart failure. The overall mutation frequency within KATP channel genes and the spectrum of genotype-phenotype relationships remain to be established, while predicting consequences of a deficit in channel function is becoming increasingly feasible through systems biology approaches. Thus, advances in molecular medicine in the emerging field of human KATP channelopathies offer new opportunities for targeted individualized screening, early diagnosis, and tailored therapy. PMID:20033705

  20. The Phylogenetic Signature Underlying ATP Synthase c-Ring Compliance.

    PubMed

    Pandini, Alessandro; Kleinjung, Jens; Taylor, Willie R; Junge, Wolfgang; Khan, Shahid

    2015-09-01

    The proton-driven ATP synthase (FOF1) is comprised of two rotary, stepping motors (FO and F1) coupled by an elastic power transmission. The elastic compliance resides in the rotor module that includes the membrane-embedded FO c-ring. Proton transport by FO is firmly coupled to the rotation of the c-ring relative to other FO subunits (ab2). It drives ATP synthesis. We used a computational method to investigate the contribution of the c-ring to the total elastic compliance. We performed principal component analysis of conformational ensembles built using distance constraints from the bovine mitochondrial c-ring x-ray structure. Angular rotary twist, the dominant ring motion, was estimated to show that the c-ring accounted in part for the measured compliance. Ring rotation was entrained to rotation of the external helix within each hairpin-shaped c-subunit in the ring. Ensembles of monomer and dimers extracted from complete c-rings showed that the coupling between collective ring and the individual subunit motions was independent of the size of the c-ring, which varies between organisms. Molecular determinants were identified by covariance analysis of residue coevolution and structural-alphabet-based local dynamics correlations. The residue coevolution gave a readout of subunit architecture. The dynamic couplings revealed that the hinge for both ring and subunit helix rotations was constructed from the proton-binding site and the adjacent glycine motif (IB-GGGG) in the midmembrane plane. IB-GGGG motifs were linked by long-range couplings across the ring, while intrasubunit couplings connected the motif to the conserved cytoplasmic loop and adjacent segments. The correlation with principal collective motions shows that the couplings underlie both ring rotary and bending motions. Noncontact couplings between IB-GGGG motifs matched the coevolution signal as well as contact couplings. The residue coevolution reflects the physiological importance of the dynamics that may link proton transfer to ring compliance. PMID:26331255

  1. The P2Z-purinoceptor of human lymphocytes: actions of nucleotide agonists and irreversible inhibition by oxidized ATP.

    PubMed Central

    Wiley, J. S.; Chen, J. R.; Snook, M. B.; Jamieson, G. P.

    1994-01-01

    1. Extracellular adenosine triphosphate (ATP) is known to open a receptor-operated ion channel (P2Z class) in human lymphocytes which conducts a range of cationic permeants. The activity of a range of different agonists and inhibitors towards the P2Z-purinoceptor was investigated by measuring the agonist-induced influx of Ba2+ into fura-2 loaded lymphocytes. 2. The most potent agonist was 2' & 3'-0-(4-benzoylbenzoyl)-ATP (benzoylbenzoic ATP) which gave 2 fold greater maximum Ba2+ influx and had a 10 fold lower EC50 than for ATP. The rank order of agonist potency in K(+)-media was benzoylbenzoic ATP >> ATP = 2-methylthio ATP = 2-chloro ATP > ATP-gamma-S. ADP, UTP and alpha,beta-methylene ATP were unable to stimulate Ba2+ influx. 3. Extracellular Na+ inhibited the increment of Ba2+ influx induced by all concentrations of ATP, 2-methylthio ATP, 2-chloroATP and ATP-gamma-S. This inhibitory effect of extracellular Na+ is also reflected in the different EC50s for benzoylbenzoic ATP (8 microM in K(+)-media, 18 microM in Na(+)-media) but the maximal response to this agonist was the same in the presence or absence of Na+. 4. Treatment of lymphocytes with 2,3 dialdehyde ATP (oxidized ATP0 at 300 microM for 60 min gave total and irreversible inhibition of ATP-induced Ba2+ influx. 5'-p-Fluorosulphonyl benzoyladenosine (FSBA) also was an irreversible inhibitor but the maximal inhibition achieved was 90%.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7921625

  2. Dual contractile effects of ATP released by field stimulation revealed by effects of alpha,beta-methylene ATP and suramin in rat tail artery.

    PubMed Central

    Bao, J. X.; Stjärne, L.

    1993-01-01

    1. The field stimulation-induced release of endogenous ATP and noradrenaline (NA) and contractile response in rat isolated tail artery were examined. The release of ATP was studied by extracellular electrophysiological recording and that of NA by a novel voltammetrical technique. The effects of the P2-purinceptor antagonist, suramin, on these parameters were compared with those of alpha,beta-methylene ATP, a P2X-purinoceptor desensitizing agent. 2. Neither alpha,beta-methylene ATP (10 microM) nor suramin (100-500 microM) had significant effects on the extracellularly recorded nerve terminal action potential but both abolished the ATP-induced excitatory junction current caused by stimulation at 0.1 Hz. Neither agent affected significantly the voltammetrically measured release of NA induced by 10 or 100 pulses at 20 Hz. 3. Combined blockade of both postjunctional alpha 1- and alpha 2-adrenoceptors by prazosin and yohimbine (both 0.1 microM) profoundly depressed the contractile response to 10 pulses at 20 Hz. The small and fast residual contraction in the presence of these agents was abolished by alpha,beta-methylene ATP (10 microM) and inhibited by suramin in a concentration-dependent manner (10-500 microM; IC50 75 microM) and was hence probably caused by ATP or a related nucleotide. 4. When added first, alpha,beta-methylene ATP (10 microM) or suramin (100-500 microM) delayed the onset and enhanced the amplitude of the neurogenic contraction. This enhanced response was abolished by further addition of prazosin and yohimbine (both 0.1 microM). 5. The K+ channel blocker, tetraethylammonium (10 mM), dramatically enhanced the contractile response to 100 pulses at 1 Hz and caused it to become diphasic.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8306081

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

    PubMed

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

    2006-01-01

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

  4. Functional redundancy between DNA ligases I and III in DNA replication in vertebrate cells.

    PubMed

    Arakawa, Hiroshi; Bednar, Theresa; Wang, Minli; Paul, Katja; Mladenov, Emil; Bencsik-Theilen, Alena A; Iliakis, George

    2012-03-01

    In eukaryotes, the three families of ATP-dependent DNA ligases are associated with specific functions in DNA metabolism. DNA ligase I (LigI) catalyzes Okazaki-fragment ligation at the replication fork and nucleotide excision repair (NER). DNA ligase IV (LigIV) mediates repair of DNA double strand breaks (DSB) via the canonical non-homologous end-joining (NHEJ) pathway. The evolutionary younger DNA ligase III (LigIII) is restricted to higher eukaryotes and has been associated with base excision (BER) and single strand break repair (SSBR). Here, using conditional knockout strategies for LIG3 and concomitant inactivation of the LIG1 and LIG4 genes, we show that in DT40 cells LigIII efficiently supports semi-conservative DNA replication. Our observations demonstrate a high functional versatility for the evolutionary new LigIII in DNA replication and mitochondrial metabolism, and suggest the presence of an alternative pathway for Okazaki fragment ligation. PMID:22127868

  5. Determination of the ATP Affinity of the Sarcoplasmic Reticulum Ca(2+)-ATPase by Competitive Inhibition of [γ-(32)P]TNP-8N3-ATP Photolabeling.

    PubMed

    Clausen, Johannes D; McIntosh, David B; Woolley, David G; Andersen, Jens Peter

    2016-01-01

    The photoactivation of aryl azides is commonly employed as a means to covalently attach cross-linking and labeling reagents to proteins, facilitated by the high reactivity of the resultant aryl nitrenes with amino groups present in the protein side chains. We have developed a simple and reliable assay for the determination of the ATP binding affinity of native or recombinant sarcoplasmic reticulum Ca(2+)-ATPase, taking advantage of the specific photolabeling of Lys(492) in the Ca(2+)-ATPase by [γ-(32)P]2',3'-O-(2,4,6-trinitrophenyl)-8-azido-adenosine 5'-triphosphate ([γ-(32)P]TNP-8N3-ATP) and the competitive inhibition by ATP of the photolabeling reaction. The method allows determination of the ATP affinity of Ca(2+)-ATPase mutants expressed in mammalian cell culture in amounts too minute for conventional equilibrium binding studies. Here, we describe the synthesis and purification of the [γ-(32)P]TNP-8N3-ATP photolabel, as well as its application in ATP affinity measurements. PMID:26695037

  6. Direct membrane-damaging effect of ketoconazole and tioconazole on Candida albicans demonstrated by bioluminescent assay of ATP.

    PubMed

    Anséhn, S; Nilsson, L

    1984-07-01

    Bioluminescent assays of fungal ATP in cultures of Candida albicans exposed to tioconazole and ketoconazole demonstrated that intracellular ATP levels were directly related to cell viability. At high concentrations of drug, a fungicidal effect was indicated by a very rapid and heavy leakage of ATP and a simultaneous, dramatic decrease in intracellular ATP. This leakage was due to direct membrane damage, which was less pronounced after exposure to ketoconazole than to tioconazole. After exposure to lower imidazole concentrations, intracellular ATP indicated growth inhibition without significant leakage of ATP. These findings support the hypothesis that imidazole antifungal agents basically act in the same manner but at different concentrations. PMID:6089651

  7. Structure and mechanism of soybean ATP sulfurylase and the committed step in plant sulfur assimilation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enzymes of the sulfur assimilation pathway are potential targets for improving nutrient content and environmental stress responses in plants. The committed step in this pathway is catalyzed by ATP sulfurylase, which synthesizes adenosine-5'-phosphosulfate (APS) from sulfate and ATP. To better unde...

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

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

    A new firefly luciferase bioluminescence assay method that differentiates free extracellular ATP (dead cells, etc.) from intracellular ATP (viable microbes) was used to determine the viable microbial cleanliness of various clean-room facilities. For comparison, samples were taken from both clean-rooms, where the air was filtered to remove particles >0.5 microm, and ordinary rooms with unfiltered air. The intracellular ATP was determined after enzymatically degrading the sample's free ATP. Also for comparison, cultivable microbial populations were counted on nutrient-rich trypticase soy agar (TSA) plates. Both the cultivable and ATP-based determinations indicate that the microbial burden was lower in clean-room facilities than in ordinary rooms. However, there was no direct correlation between the two sets of measurements because the two assays measured very different populations. A large fraction of the samples yielded no colony formers on TSA, but were positive for intracellular ATP. Subsequently, genomic DNA was isolated directly from selected samples and 16S rDNA fragments were cloned and sequenced, identifying nearest neighbors, many of which are known to be noncultivable in the media employed. It was concluded that viable microbial contamination can be reliably monitored by measurement of intracellular ATP, and that this method may be considered superior to cultivable colony counts due to its speed and its ability to report the presence of viable but noncultivable organisms. When the detection of nonviable microbes is of interest, the ATP assay can be supplemented with DNA analysis.

  9. Overexpression of ATP1B1 predicts an adverse prognosis in cytogenetically normal acute myeloid leukemia.

    PubMed

    Shi, Jin-Long; Fu, Lin; Ang, Qing; Wang, Guo-Jing; Zhu, Jun; Wang, Wei-Dong

    2016-01-19

    ATP1B1 encodes the Na,K-ATPase β subunit, a key regulator of the Na+ and K+ electrochemical gradients across the plasma membrane and an essential regulator of cellular activity. We used several microarray datasets to test the prognostic efficacy of ATP1B1 expression in cytogenetically normal acute myeloid leukemia (CN-AML). Within the primary cohort (n = 157), high ATP1B1 expression (ATP1B1high) was associated with shorter overall survival (OS) and event-free survival (EFS) (P = 0.0068, P = 0.0039, respectively). Similar results were also obtained in the European Leukemia Net (ELN) Intermediate-I genetic category (OS: P = 0.0035, EFS: P = 0.0007). Multivariable analyses confirmed ATP1B1high is an independent predictor of shorter OS (P = 0.042) and EFS (P = 0.035). Analysis of another CN-AML cohort confirmed that ATP1B1high is associated with shorter OS (P = 0.0046, n = 162). In addition, up-regulation of oncogenes/onco-microRNAs such as MYCN, CCND2, CDK6, KIT and miR-155, among others, was associated with ATP1B1high, which may be indicative of ATP1B1's leukemogenicity. Our results may improve risk stratification and indicate new therapeutic targets for CN-AML. PMID:26506237

  10. A SPECTROPHOTOMETRIC ASSAY TO MEASURE RUBISCO ACTIVASE ACTIVATION ACTIVITY UNDER VARYING ATP:ADP RATIOS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ratio of ATP to ADP in the stroma is an important regulatory mechanism for controlling the activation state of Rubisco via Rubisco activase (activase). Understanding the response of activase to a varying ATP:ADP ratio should reveal insights into the regulation of photosynthesis. However, the cur...

  11. Phylogeny and Identification of Enterococci by atpA Gene Sequence Analysis

    PubMed Central

    Naser, S.; Thompson, F. L.; Hoste, B.; Gevers, D.; Vandemeulebroecke, K.; Cleenwerck, I.; Thompson, C. C.; Vancanneyt, M.; Swings, J.

    2005-01-01

    The relatedness among 91 Enterococcus strains representing all validly described species was investigated by comparing a 1,102-bp fragment of atpA, the gene encoding the alpha subunit of ATP synthase. The relationships observed were in agreement with the phylogeny inferred from 16S rRNA gene sequence analysis. However, atpA gene sequences were much more discriminatory than 16S rRNA for species differentiation. All species were differentiated on the basis of atpA sequences with, at a maximum, 92% similarity. Six members of the Enterococcus faecium species group (E. faecium, E. hirae, E. durans, E. villorum, E. mundtii, and E. ratti) showed >99% 16S rRNA gene sequence similarity, but the highest value of atpA gene sequence similarity was only 89.9%. The intraspecies atpA sequence similarities for all species except E. faecium strains varied from 98.6 to 100%; the E. faecium strains had a lower atpA sequence similarity of 96.3%. Our data clearly show that atpA provides an alternative tool for the phylogenetic study and identification of enterococci. PMID:15872246

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

    ERIC Educational Resources Information Center

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

    2007-01-01

    The Autism Tissue Program (ATP) was established to oversee and manage brain donations related to neurological research in autism. The ATP Informatics Portal (www.atpportal.org) is an integrated data access system based on Oracle technology, developed to provide access for researchers to information on this rare tissue resource. It also permits…

  13. Characterization of the Saccharomyces cerevisiae ATP-Interactome using the iTRAQ-SPROX Technique

    NASA Astrophysics Data System (ADS)

    Geer, M. Ariel; Fitzgerald, Michael C.

    2016-02-01

    The stability of proteins from rates of oxidation (SPROX) technique was used in combination with an isobaric mass tagging strategy to identify adenosine triphosphate (ATP) interacting proteins in the Saccharomyces cerevisiae proteome. The SPROX methodology utilized in this work enabled 373 proteins in a yeast cell lysate to be assayed for ATP interactions (both direct and indirect) using the non-hydrolyzable ATP analog, adenylyl imidodiphosphate (AMP-PNP). A total of 28 proteins were identified with AMP-PNP-induced thermodynamic stability changes. These protein hits included 14 proteins that were previously annotated as ATP-binding proteins in the Saccharomyces Genome Database (SGD). The 14 non-annotated ATP-binding proteins included nine proteins that were previously found to be ATP-sensitive in an earlier SPROX study using a stable isotope labeling with amino acids in cell culture (SILAC)-based approach. A bioinformatics analysis of the protein hits identified here and in the earlier SILAC-SPROX experiments revealed that many of the previously annotated ATP-binding protein hits were kinases, ligases, and chaperones. In contrast, many of the newly discovered ATP-sensitive proteins were not from these protein classes, but rather were hydrolases, oxidoreductases, and nucleic acid-binding proteins.

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

    PubMed Central

    Itoh, H; Tashima, Y

    1997-01-01

    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

  15. A lipid switch unlocks Parkinson’s disease-associated ATP13A2

    PubMed Central

    Holemans, Tine; Sørensen, Danny Mollerup; van Veen, Sarah; Martin, Shaun; Hermans, Diane; Kemmer, Gerdi Christine; Van den Haute, Chris; Baekelandt, Veerle; Günther Pomorski, Thomas; Agostinis, Patrizia; Wuytack, Frank; Palmgren, Michael; Eggermont, Jan; Vangheluwe, Peter

    2015-01-01

    ATP13A2 is a lysosomal P-type transport ATPase that has been implicated in Kufor–Rakeb syndrome and Parkinson’s disease (PD), providing protection against α-synuclein, Mn2+, and Zn2+ toxicity in various model systems. So far, the molecular function and regulation of ATP13A2 remains undetermined. Here, we demonstrate that ATP13A2 contains a unique N-terminal hydrophobic extension that lies on the cytosolic membrane surface of the lysosome, where it interacts with the lysosomal signaling lipids phosphatidic acid (PA) and phosphatidylinositol(3,5)bisphosphate [PI(3,5)P2]. We further demonstrate that ATP13A2 accumulates in an inactive autophosphorylated state and that PA and PI(3,5)P2 stimulate the autophosphorylation of ATP13A2. In a cellular model of PD, only catalytically active ATP13A2 offers cellular protection against rotenone-induced mitochondrial stress, which relies on the availability of PA and PI(3,5)P2. Thus, the N-terminal binding of PA and PI(3,5)P2 emerges as a key to unlock the activity of ATP13A2, which may offer a therapeutic strategy to activate ATP13A2 and thereby reduce α-synuclein toxicity or mitochondrial stress in PD or related disorders. PMID:26134396

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

    ERIC Educational Resources Information Center

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

    2010-01-01

    An Adobe[R] animation is presented for use in undergraduate Biochemistry courses, illustrating the mechanism of Na[superscript +] and K[superscript +] translocation coupled to ATP hydrolysis by the (Na, K)-ATPase, a P[subscript 2c]-type ATPase, or ATP-powered ion pump that actively translocates cations across plasma membranes. The enzyme is also…

  17. A Computational Analysis of ATP Binding of SV40 Large Tumor Antigen Helicase Motor

    PubMed Central

    Shi, Yemin; Liu, Hanbin; Gai, Dahai; Ma, Jianpeng; Chen, Xiaojiang S.

    2009-01-01

    Simian Virus 40 Large Tumor Antigen (LTag) is an efficient helicase motor that unwinds and translocates DNA. The DNA unwinding and translocation of LTag is powered by ATP binding and hydrolysis at the nucleotide pocket between two adjacent subunits of an LTag hexamer. Based on the set of high-resolution hexameric structures of LTag helicase in different nucleotide binding states, we simulated a conformational transition pathway of the ATP binding process using the targeted molecular dynamics method and calculated the corresponding energy profile using the linear response approximation (LRA) version of the semi-macroscopic Protein Dipoles Langevin Dipoles method (PDLD/S). The simulation results suggest a three-step process for the ATP binding from the initial interaction to the final tight binding at the nucleotide pocket, in which ATP is eventually “locked” by three pairs of charge-charge interactions across the pocket. Such a “cross-locking” ATP binding process is similar to the binding zipper model reported for the F1-ATPase hexameric motor. The simulation also shows a transition mechanism of Mg2+ coordination to form the Mg-ATP complex during ATP binding, which is accompanied by the large conformational changes of LTag. This simulation study of the ATP binding process to an LTag and the accompanying conformational changes in the context of a hexamer leads to a refined cooperative iris model that has been proposed previously. PMID:19779548

  18. Characterization of the Saccharomyces cerevisiae ATP-Interactome using the iTRAQ-SPROX Technique.

    PubMed

    Geer, M Ariel; Fitzgerald, Michael C

    2016-02-01

    The stability of proteins from rates of oxidation (SPROX) technique was used in combination with an isobaric mass tagging strategy to identify adenosine triphosphate (ATP) interacting proteins in the Saccharomyces cerevisiae proteome. The SPROX methodology utilized in this work enabled 373 proteins in a yeast cell lysate to be assayed for ATP interactions (both direct and indirect) using the non-hydrolyzable ATP analog, adenylyl imidodiphosphate (AMP-PNP). A total of 28 proteins were identified with AMP-PNP-induced thermodynamic stability changes. These protein hits included 14 proteins that were previously annotated as ATP-binding proteins in the Saccharomyces Genome Database (SGD). The 14 non-annotated ATP-binding proteins included nine proteins that were previously found to be ATP-sensitive in an earlier SPROX study using a stable isotope labeling with amino acids in cell culture (SILAC)-based approach. A bioinformatics analysis of the protein hits identified here and in the earlier SILAC-SPROX experiments revealed that many of the previously annotated ATP-binding protein hits were kinases, ligases, and chaperones. In contrast, many of the newly discovered ATP-sensitive proteins were not from these protein classes, but rather were hydrolases, oxidoreductases, and nucleic acid-binding proteins. Graphical Abstract ᅟ. PMID:26530046

  19. Enzyme-based field-effect transistor for adenosine triphosphate (ATP) sensing.

    PubMed

    Migita, Satoshi; Ozasa, Kazunari; Tanaka, Tomoya; Haruyama, Tetsuya

    2007-01-01

    Adenosine triphosphate (ATP) not only functions as an energy-carrier substance and an informative molecule, but also acts as a marker substance in studies of both bio-traces and cellular/tissular viability. Due to the importance of the ATP function for living organisms, in situ assays of ATP are in demand in various fields, e.g., hygiene. In the present study, we developed an ATP sensor that combines the selective catalytic activity of enzyme and the properties of an ion selective field effect transistor (ISFET). In this system, the ATP hydrolyrase, "apyrase (EC 3.6.1.5.)" is encased in a gel and mounted on a Ta(2)O(5) ISFET gate surface. When the enzyme layer selectively catalyzes the dephosphorylation of ATP, protons are accumulated at the gate because the enzymatic reaction produces H(+) as a byproduct. Based on the interfacial enzymatic reaction, the response from the ISFET is completely dependent upon the ATP concentration in the bulk solution. This device is readily applicable to practical in situ ATP measurement, e.g. hygienic usage. PMID:17213622

  20. Different effects of gradual vs. acute adenine nucleotide depletion on ATP cost of muscle contraction.

    PubMed

    Foley, J M; Adams, G R; Meyer, R A

    1994-11-01

    ATP and phosphocreatine (PCr) levels were chronically reduced in rat fast-twitch muscle by 40 and 87%, respectively, after rats were fed a diet containing 1% beta-guanidinopropionate (beta-GPA) for 9 wk. Myosin heavy chain distribution of superficial gastrocnemius muscle changed from 22:78% type IIA/type IIB in control to 39:61% in GPA-treated muscles. ATP cost of contractions was estimated from the PCr changes measured by gated 31P-nuclear magnetic resonance after brief (< 6 s) bursts of contractions. There was no significant change in the ATP cost of either twitch (0.3 mumol.g-1.twitch-1) or tetanic (2.5 mumol.g-1.100 ms tetanus-1) contractions in gastrocnemius muscle after chronic phosphagen depletion due to beta-GPA feeding. In two other groups of rats, ATP of gastrocnemius muscles was acutely depleted to inosine 5'-monophosphate (IMP) by tetanic stimulation. IMP reanimation was inhibited during a subsequent 75-min recovery period in one group by hadacidin treatment (100 mg/kg), resulting in 46% ATP depletion compared with the similarly stimulated and recovered control group. In contrast to gradual ATP depletion due to beta-GPA feeding, this acute ATP depletion treatment was associated with a 39% decrease in ATP cost of twitch contractions. PMID:7977681

  1. Overexpression of ATP1B1 predicts an adverse prognosis in cytogenetically normal acute myeloid leukemia

    PubMed Central

    Ang, Qing; Wang, Guo-jing; Zhu, Jun; Wang, Wei-dong

    2016-01-01

    ATP1B1 encodes the Na,K-ATPase β subunit, a key regulator of the Na+ and K+ electrochemical gradients across the plasma membrane and an essential regulator of cellular activity. We used several microarray datasets to test the prognostic efficacy of ATP1B1 expression in cytogenetically normal acute myeloid leukemia (CN-AML). Within the primary cohort (n = 157), high ATP1B1 expression (ATP1B1high) was associated with shorter overall survival (OS) and event-free survival (EFS) (P = 0.0068, P = 0.0039, respectively). Similar results were also obtained in the European Leukemia Net (ELN) Intermediate-I genetic category (OS: P = 0.0035, EFS: P = 0.0007). Multivariable analyses confirmed ATP1B1high is an independent predictor of shorter OS (P = 0.042) and EFS (P = 0.035). Analysis of another CN-AML cohort confirmed that ATP1B1high is associated with shorter OS (P = 0.0046, n = 162). In addition, up-regulation of oncogenes/onco-microRNAs such as MYCN, CCND2, CDK6, KIT and miR-155, among others, was associated with ATP1B1high, which may be indicative of ATP1B1's leukemogenicity. Our results may improve risk stratification and indicate new therapeutic targets for CN-AML. PMID:26506237

  2. Development of an ATP measurement method suitable for xenobiotic treatment activated sludge biomass.

    PubMed

    Nguyen, Lan Huong; Chong, Nyuk-Min

    2015-09-01

    Activated sludge consumes a large amount of energy to degrade a xenobiotic organic compound. By tracking the energy inventory of activated sludge biomass during the sludge's degradation of a xenobiotic, any disadvantageous effect on the sludge's performance caused by energy deficiency can be observed. The purpose of this study was to develop a reliable and accurate method for measuring the ATP contents of activated sludge cells that were to degrade a xenobiotic organic. Cell disruption and cellular ATP extraction were performed by a protocol with which xenobiotic degrading activated sludge biomass was washed with SDS, treated by Tris and TCA, and followed by bead blasting. The suspension of disrupted cells was filtered before the filtrate was injected into HPLC that was set at optimal conditions to measure the ATP concentration therein. This extraction protocol and HPLC measurement of ATP was evaluated for its linearity, limits of detection, and reproducibility. Evaluation test results reported a R(2) of 0.999 of linear fit of ATP concentration versus activated sludge concentration, a LOD=0.00045mg/L, a LOQ=0.0015mg/L for HPLC measurement of ATP, a MDL=0.46mg/g SS for ATP extraction protocol, and a recovery efficiency of 96.4±2%. This method of ATP measurement was simple, rapid, reliable, and was unburdened of some limitations other methods may have. PMID:26210584

  3. ATP Synthase: A Molecular Therapeutic Drug Target for Antimicrobial and Antitumor Peptides

    PubMed Central

    Ahmad, Zulfiqar; Okafor, Florence; Azim, Sofiya; Laughlin, Thomas F.

    2015-01-01

    In this review we discuss the role of ATP synthase as a molecular drug target for natural and synthetic antimi-crobial/antitumor peptides. We start with an introduction of the universal nature of the ATP synthase enzyme and its role as a biological nanomotor. Significant structural features required for catalytic activity and motor functions of ATP synthase are described. Relevant details regarding the presence of ATP synthase on the surface of several animal cell types, where it is associated with multiple cellular processes making it a potential drug target with respect to antimicrobial peptides and other inhibitors such as dietary polyphenols, is also reviewed. ATP synthase is known to have about twelve discrete inhibitor binding sites including peptides and other inhibitors located at the interface of α/β subunits on the F1 sector of the enzyme. Molecular interaction of peptides at the β DEELSEED site on ATP synthase is discussed with specific examples. An inhibitory effect of other natural/synthetic inhibitors on ATP is highlighted to explore the therapeutic roles played by peptides and other inhibitors. Lastly, the effect of peptides on the inhibition of the Escherichia coli model system through their action on ATP synthase is presented. PMID:23432591

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

    ERIC Educational Resources Information Center

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

    2010-01-01

    An Adobe[R] animation is presented for use in undergraduate Biochemistry courses, illustrating the mechanism of Na[superscript +] and K[superscript +] translocation coupled to ATP hydrolysis by the (Na, K)-ATPase, a P[subscript 2c]-type ATPase, or ATP-powered ion pump that actively translocates cations across plasma membranes. The enzyme is also

  5. ATP: A Coherent View for School Advanced Level Studies in Biology.

    ERIC Educational Resources Information Center

    Gayford, Chris

    1986-01-01

    Discusses how instruction of biological concepts as ATP cellular energetics is related to fundamental physical science understandings. Reviews areas of common misconceptions and confusions. Summarizes results of a study which investigated students' knowledge and perception of difficulty associated with the topic of energy and ATP. (ML)

  6. Submembrane ATP and Ca2+ kinetics in α-cells: unexpected signaling for glucagon secretion.

    PubMed

    Li, Jia; Yu, Qian; Ahooghalandari, Parvin; Gribble, Fiona M; Reimann, Frank; Tengholm, Anders; Gylfe, Erik

    2015-08-01

    Cytoplasmic ATP and Ca(2+) are implicated in current models of glucose's control of glucagon and insulin secretion from pancreatic α- and β-cells, respectively, but little is known about ATP and its relation to Ca(2+) in α-cells. We therefore expressed the fluorescent ATP biosensor Perceval in mouse pancreatic islets and loaded them with a Ca(2+) indicator. With total internal reflection fluorescence microscopy, we recorded subplasma membrane concentrations of Ca(2+) and ATP ([Ca(2+)]pm; [ATP]pm) in superficial α- and β-cells of intact islets and related signaling to glucagon and insulin secretion by immunoassay. Consistent with ATP's controlling glucagon and insulin secretion during hypo- and hyperglycemia, respectively, the dose-response relationship for glucose-induced [ATP]pm generation was left shifted in α-cells compared to β-cells. Both cell types showed [Ca(2+)]pm and [ATP]pm oscillations in opposite phase, probably reflecting energy-consuming Ca(2+) transport. Although pulsatile insulin and glucagon release are in opposite phase, [Ca(2+)]pm synchronized in the same phase between α- and β-cells. This paradox can be explained by the overriding of Ca(2+) stimulation by paracrine inhibition, because somatostatin receptor blockade potently stimulated glucagon release with little effect on Ca(2+). The data indicate that an α-cell-intrinsic mechanism controls glucagon in hypoglycemia and that paracrine factors shape pulsatile secretion in hyperglycemia. PMID:25911612

  7. Three-Dimensional Structures Reveal Multiple ADP/ATP Binding Modes

    SciTech Connect

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

    2011-12-31

    The creation of synthetic enzymes with predefined functions represents a major challenge in future synthetic biology applications. Here, we describe six structures of de novo proteins that have been determined using protein crystallography to address how simple enzymes perform catalysis. Three structures are of a protein, DX, selected for its stability and ability to tightly bind ATP. Despite the addition of ATP to the crystallization conditions, the presence of a bound but distorted ATP was found only under excess ATP conditions, with ADP being present under equimolar conditions or when crystallized for a prolonged period of time. A bound ADP cofactor was evident when Asp was substituted for Val at residue 65, but ATP in a linear configuration is present when Phe was substituted for Tyr at residue 43. These new structures complement previously determined structures of DX and the protein with the Phe 43 to Tyr substitution [Simmons, C. R., et al. (2009) ACS Chem. Biol. 4, 649-658] and together demonstrate the multiple ADP/ATP binding modes from which a model emerges in which the DX protein binds ATP in a configuration that represents a transitional state for the catalysis of ATP to ADP through a slow, metal-free reaction capable of multiple turnovers. This unusual observation suggests that design-free methods can be used to generate novel protein scaffolds that are tailor-made for catalysis.

  8. Mechanism of action of ATP on canine pulmonary vagal C fibre nerve terminals.

    PubMed Central

    Pelleg, A; Hurt, C M

    1996-01-01

    1. The effects of extracellular adenosine 5'-triphosphate (ATP) on pulmonary vagal afferent fibres (n = 46) was studied in a canine model in vivo (n = 38). 2. ATP (3-6 mumol kg-1), administered as a rapid bolus into the right atrium, elicited a transient burst of action potentials in cervical vagal fibres, which was not affected by either blockade of ganglionic transmission (hexamethonium) or a drop in arterial blood pressure (nitroglycerine). 3. The fibres with ATP-sensitive terminals were otherwise quiescent with no activity related to either cardiac or respiratory cycles and their conduction velocity was 0.85 +/- 0.13 m s-1 (n = 7). 4. Inflation of the lungs to 2-3 times the tidal volume triggered brief bursts of action potentials in these fibres. 5. Capsaicin (10 micrograms kg-1), given as a rapid bolus into the right atrium, elicited a burst of action potentials in these ATP-sensitive fibres. 6. Smaller amounts of ATP and capsaicin (0.5-3 mumol kg-1 and 1-5 micrograms kg-1, respectively) had similar effects when the two compounds were given into the right pulmonary artery. 7. Adenosine, adenosine 5'-monophosphate, or adenosine 5'-diphosphate did not excite these fibres (n = 30). 8. The non-degradable analogue of ATP alpha,beta-methylene ATP (alpha,beta-mATP) was tenfold more potent than ATP while beta,gamma-methylene ATP (beta,gamma-mATP) was in active. 9. The selective P2x-purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid markedly attenuated the effect of ATP but not of capsaicin. The P2Y-purinoceptor antagonist Reactive Blue 2 was without effect. 10. Pretreatment with pertussis toxin (PTX) did not affect this action of ATP. 11. In the canine lungs ATP activates vagal C fibre nerve terminals. This action is mediated by P2X-purinoceptors and is independent of a PTX-sensitive guanine nucleotide binding protein (G protein). PMID:8745294

  9. Fusion Power Demonstration III

    SciTech Connect

    Lee, J.D.

    1985-07-01

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

  10. Structural basis of asymmetric DNA methylation and ATP-triggered long-range diffusion by EcoP15I

    NASA Astrophysics Data System (ADS)

    Gupta, Yogesh K.; Chan, Siu-Hong; Xu, Shuang-Yong; Aggarwal, Aneel K.

    2015-06-01

    Type III R-M enzymes were identified >40 years ago and yet there is no structural information on these multisubunit enzymes. Here we report the structure of a Type III R-M system, consisting of the entire EcoP15I complex (Mod2Res1) bound to DNA. The structure suggests how ATP hydrolysis is coupled to long-range diffusion of a helicase on DNA, and how a dimeric methyltransferase functions to methylate only one of the two DNA strands. We show that the EcoP15I motor domains are specifically adapted to bind double-stranded DNA and to facilitate DNA sliding via a novel `Pin' domain. We also uncover unexpected `division of labour', where one Mod subunit recognizes DNA, while the other Mod subunit methylates the target adenine--a mechanism that may extend to adenine N6 RNA methylation in mammalian cells. Together the structure sheds new light on the mechanisms of both helicases and methyltransferases in DNA and RNA metabolism.

  11. Structural basis of asymmetric DNA methylation and ATP-triggered long-range diffusion by EcoP15I

    PubMed Central

    Gupta, Yogesh K.; Chan, Siu-Hong; Xu, Shuang-yong; Aggarwal, Aneel K.

    2015-01-01

    Type III R–M enzymes were identified >40 years ago and yet there is no structural information on these multisubunit enzymes. Here we report the structure of a Type III R–M system, consisting of the entire EcoP15I complex (Mod2Res1) bound to DNA. The structure suggests how ATP hydrolysis is coupled to long-range diffusion of a helicase on DNA, and how a dimeric methyltransferase functions to methylate only one of the two DNA strands. We show that the EcoP15I motor domains are specifically adapted to bind double-stranded DNA and to facilitate DNA sliding via a novel ‘Pin' domain. We also uncover unexpected ‘division of labour', where one Mod subunit recognizes DNA, while the other Mod subunit methylates the target adenine—a mechanism that may extend to adenine N6 RNA methylation in mammalian cells. Together the structure sheds new light on the mechanisms of both helicases and methyltransferases in DNA and RNA metabolism. PMID:26067164

  12. Type III restriction-modification enzymes: a historical perspective

    PubMed Central

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

    2014-01-01

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

  13. Light-harvesting chlorophyll pigments enable mammalian mitochondria to capture photonic energy and produce ATP.

    PubMed

    Xu, Chen; Zhang, Junhua; Mihai, Doina M; Washington, Ilyas

    2014-01-15

    Sunlight is the most abundant energy source on this planet. However, the ability to convert sunlight into biological energy in the form of adenosine-5'-triphosphate (ATP) is thought to be limited to chlorophyll-containing chloroplasts in photosynthetic organisms. Here we show that mammalian mitochondria can also capture light and synthesize ATP when mixed with a light-capturing metabolite of chlorophyll. The same metabolite fed to the worm Caenorhabditis elegans leads to increase in ATP synthesis upon light exposure, along with an increase in life span. We further demonstrate the same potential to convert light into energy exists in mammals, as chlorophyll metabolites accumulate in mice, rats and swine when fed a chlorophyll-rich diet. Results suggest chlorophyll type molecules modulate mitochondrial ATP by catalyzing the reduction of coenzyme Q, a slow step in mitochondrial ATP synthesis. We propose that through consumption of plant chlorophyll pigments, animals, too, are able to derive energy directly from sunlight. PMID:24198392

  14. ROLE OF ATP IN REGULATING RENAL MICROVASCULAR FUNCTION AND IN HYPERTENSION

    PubMed Central

    Guan, Zhengrong; Inscho, Edward W.

    2011-01-01

    Adenosine triphosphate (ATP) is an essential energy substrate for cellular metabolism but it can also influence many biological processes when released into the extracellular milieu. Research has established that extracellular ATP acts as an autocrine/paracrine factor that regulates many physiological functions. Alternatively, excessive extracellular ATP levels contribute to pathophysiological processes such as inflammation, cell proliferation and apoptosis, and atherosclerosis. Renal P2 receptors are widely distributed throughout glomeruli, vasculature and tubular segments, and participate in controlling renal vascular resistance, mediating renal autoregulation, and regulating tubular transport function. This review will focus on the role of ATP-P2 receptor signaling in regulating renal microvascular function and autoregulation, recent advances on the role of ATP-P2 signaling in hypertension-associated renal vascular injury, and emerging new directions. PMID:21768526

  15. ATP hydrolysis assists phosphate release and promotes reaction ordering in F1-ATPase

    NASA Astrophysics Data System (ADS)

    Li, Chun-Biu; Ueno, Hiroshi; Watanabe, Rikiya; Noji, Hiroyuki; Komatsuzaki, Tamiki

    2015-12-01

    F1-ATPase (F1) is a rotary motor protein that can efficiently convert chemical energy to mechanical work of rotation via fine coordination of its conformational motions and reaction sequences. Compared with reactant binding and product release, the ATP hydrolysis has relatively little contributions to the torque and chemical energy generation. To scrutinize possible roles of ATP hydrolysis, we investigate the detailed statistics of the catalytic dwells from high-speed single wild-type F1 observations. Here we report a small rotation during the catalytic dwell triggered by the ATP hydrolysis that is indiscernible in previous studies. Moreover, we find in freely rotating F1 that ATP hydrolysis is followed by the release of inorganic phosphate with low synthesis rates. Finally, we propose functional roles of the ATP hydrolysis as a key to kinetically unlock the subsequent phosphate release and promote the correct reaction ordering.

  16. Limited proteolysis of Salmonella typhimurium nicotinic acid phosphoribosyltransferase reveals ATP-linked conformational change.

    PubMed

    Rajavel, M; Gross, J; Segura, E; Moore, W T; Grubmeyer, C

    1996-04-01

    Nicotinic acid phosphoribosyltransferase (NAPRTase;EC 2.4.2.11) couples stoichiometric ATP hydrolysis with formation of nicotinate mononucleotide (NAMN) from nicotinic acid and alpha-D-5-phosphoribosyl 1-pyrophosphate (PRPP). Trypsin rapidly inactivated the ATPase and NAMN synthesis activities of NAPRTase in parallel, with cleavages at Arg-384 and Lys-374 of the 399-residue protein. ATP and PRPP each provided protection against tryptic cleavage. Limited chymotryptic proteolysis of NAPRTase exhibited very similar behavior, with specific cleavage at Phe-382 and protection by substrates. Results suggest that a solvent-exposed loop encompassing Lys-374, Phe-382, and Arg-384 is protected by ATP- or PRPP-induced conformational changes. The ability of ATP to protect even under conditions in which enzyme phosphorylation was prevented by EDTA provides evidence for a distinct ATP-induced protein conformation that acts as an intermediate in energy coupling. PMID:8672421

  17. Physiological heart activation by adrenaline involves parallel activation of ATP usage and supply.

    PubMed

    Korzeniewski, Bernard; Deschodt-Arsac, Véronique; Calmettes, Guillaume; Franconi, Jean-Michel; Diolez, Philippe

    2008-07-15

    During low-to-high work transition in adult mammalian heart in vivo the concentrations of free ADP, ATP, PCr (phosphocreatine), P(i) and NADH are essentially constant, in striking contrast with skeletal muscle. The direct activation by calcium ions of ATP usage and feedback activation of ATP production by ADP (and P(i)) alone cannot explain this perfect homoeostasis. A comparison of the response to adrenaline (increase in rate-pressure product and [PCr]) of the intact beating perfused rat heart with the elasticities of the PCr producer and consumer to PCr concentration demonstrated that both the ATP/PCr-producing block and ATP/PCr-consuming block are directly activated to a similar extent during physiological heart activation. Our finding constitutes a direct evidence for the parallel-activation mechanism of the regulation of oxidative phosphorylation in heart postulated previously in a theoretical way. PMID:18377364

  18. ATP hydrolysis assists phosphate release and promotes reaction ordering in F1-ATPase

    PubMed Central

    Li, Chun-Biu; Ueno, Hiroshi; Watanabe, Rikiya; Noji, Hiroyuki; Komatsuzaki, Tamiki

    2015-01-01

    F1-ATPase (F1) is a rotary motor protein that can efficiently convert chemical energy to mechanical work of rotation via fine coordination of its conformational motions and reaction sequences. Compared with reactant binding and product release, the ATP hydrolysis has relatively little contributions to the torque and chemical energy generation. To scrutinize possible roles of ATP hydrolysis, we investigate the detailed statistics of the catalytic dwells from high-speed single wild-type F1 observations. Here we report a small rotation during the catalytic dwell triggered by the ATP hydrolysis that is indiscernible in previous studies. Moreover, we find in freely rotating F1 that ATP hydrolysis is followed by the release of inorganic phosphate with low synthesis rates. Finally, we propose functional roles of the ATP hydrolysis as a key to kinetically unlock the subsequent phosphate release and promote the correct reaction ordering. PMID:26678797

  19. Interdependence of ATP signalling and pannexin channels; the servant was really the master all along?

    PubMed

    Jackson, Michael F

    2015-12-15

    Pannexin channels are recognized as important conduits for the release of ATP, which contributes to purinergic signalling. Pathologically, ATP release via these channels acts as a find-me signal for apoptotic cell clearance. Accordingly, there is considerable and growing interest in understanding the function and regulation of pannexin channels. In a recent issue of the Biochemical Journal, Boyce et al. provide evidence that the surface expression of pannexin channels is regulated by extracellular ATP. They propose a model in which ATP triggers pannexin channel internalization through a pathway involving clathrin- and caveolin-independent entry into early endosomes. Intriguingly, their evidence suggests that internalization is initiated through the association of ATP with pannexin channels themselves as well as ionotropic purinergic receptor 7 (P2X7) receptors. PMID:26613946

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

    PubMed Central

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

    1994-01-01

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

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

    PubMed Central

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

    2015-01-01

    Plants assimilate inorganic sulfate into sulfur-containing vital metabolites. ATP sulfurylase (ATPS) is the enzyme catalyzing the key entry step of the sulfate assimilation pathway in both plastids and cytosol in plants. Arabidopsis thaliana has four ATPS genes (ATPS1, –2, –3, and –4) encoding ATPS pre-proteins containing N-terminal transit peptide sequences for plastid targeting, however, the genetic identity of the cytosolic ATPS has remained unverified. Here we show that Arabidopsis ATPS2 dually encodes plastidic and cytosolic ATPS isoforms, differentiating their subcellular localizations by initiating translation at AUGMet1 to produce plastid-targeted ATPS2 pre-proteins or at AUGMet52 or AUGMet58 within the transit peptide to have ATPS2 stay in cytosol. Translational initiation of ATPS2 at AUGMet52 or AUGMet58 was verified by expressing a tandem-fused synthetic gene, ATPS2(5′UTR-His12):Renilla luciferase:ATPS2(Ile13−Val77):firefly luciferase, under a single constitutively active CaMV 35S promoter in Arabidopsis protoplasts and examining the activities of two different luciferases translated in-frame with split N-terminal portions of ATPS2. Introducing missense mutations at AUGMet52 and AUGMet58 significantly reduced the firefly luciferase activity, while AUGMet52 was a relatively preferred site for the alternative translational initiation. The activity of luciferase fusion protein starting at AUGMet52 or AUGMet58 was not modulated by changes in sulfate conditions. The dual localizations of ATPS2 in plastids and cytosol were further evidenced by expression of ATPS2-GFP fusion proteins in Arabidopsis protoplasts and transgenic lines, while they were also under control of tissue-specific ATPS2 promoter activity found predominantly in leaf epidermal cells, guard cells, vascular tissues and roots. PMID:25601874

  2. Modeling regulation of cardiac KATP and L-type Ca2+ currents by ATP, ADP, and Mg2+

    NASA Technical Reports Server (NTRS)

    Michailova, Anushka; Saucerman, Jeffrey; Belik, Mary Ellen; McCulloch, Andrew D.

    2005-01-01

    Changes in cytosolic free Mg(2+) and adenosine nucleotide phosphates affect cardiac excitability and contractility. To investigate how modulation by Mg(2+), ATP, and ADP of K(ATP) and L-type Ca(2+) channels influences excitation-contraction coupling, we incorporated equations for intracellular ATP and MgADP regulation of the K(ATP) current and MgATP regulation of the L-type Ca(2+) current in an ionic-metabolic model of the canine ventricular myocyte. The new model: 1), quantitatively reproduces a dose-response relationship for the effects of changes in ATP on K(ATP) current, 2), simulates effects of ADP in modulating ATP sensitivity of K(ATP) channel, 3), predicts activation of Ca(2+) current during rapid increase in MgATP, and 4), demonstrates that decreased ATP/ADP ratio with normal total Mg(2+) or increased free Mg(2+) with normal ATP and ADP activate K(ATP) current, shorten action potential, and alter ionic currents and intracellular Ca(2+) signals. The model predictions are in agreement with experimental data measured under normal and a variety of pathological conditions.

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

    PubMed Central

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

    2014-01-01

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

  4. Contribution of two ionotropic purinergic receptors to ATP responses in submandibular gland ductal cells.

    PubMed

    Pochet, Stéphanie; Garcia-Marcos, Mikel; Seil, Michèle; Otto, Anne; Marino, Aida; Dehaye, Jean-Paul

    2007-10-01

    The effect of extracellular ATP on salivary gland function was compared in wild-type (WT) and P2X(7) knockout (KO) mice. The increase in the intracellular concentration of calcium ([Ca(2+)](i)) in response to carbachol was similar in submandibular ductal cells of WT and KO mice. ATP and its analog, benzoyl-ATP, induced a sustained increase in the [Ca(2+)](i) in WT animals. In KO mice, ATP slightly and transiently increased the [Ca(2+)](i) and benzoyl-ATP had no effect. The response to ATP of WT but not KO mice was blocked by KN-62, Coomassie blue and magnesium. The small response of ATP observed in KO mice was completely blocked in the absence of extracellular calcium, unchanged by U73122 and potentiated by ivermectin indicating the probable involvement of a P2X(4) receptor. A RT-PCR and a Western blot confirmed the presence of these receptors in ducts of both WT and KO mice. ATP increased the permeability of the cells to ethidium bromide and stimulated a phospholipase A(2) activity in WT but not KO mice. Mice submandibular gland cells secreted IL-1beta but this secretion was not modified by ATP and was similar in both groups of animals. The volume of saliva provoked by pilocarpine and the concentration of proteins, sodium and chloride in this saliva was similar in both groups of animals. The concentration of potassium was higher in KO mice. We can conclude that the major purinergic receptors expressed in mice submandibular ductal cells are P2X(7) receptors but that P2X(4) receptors are also involved in some ATP effects. PMID:17651941

  5. The molecular motor F-ATP synthase is targeted by the tumoricidal protein HAMLET.

    PubMed

    Ho, James; Sielaff, Hendrik; Nadeem, Aftab; Svanborg, Catharina; Grüber, Gerhard

    2015-05-22

    HAMLET (human alpha-lactalbumin made lethal to tumor cells) interacts with multiple tumor cell compartments, affecting cell morphology, metabolism, proteasome function, chromatin structure and viability. This study investigated if these diverse effects of HAMLET might be caused, in part, by a direct effect on the ATP synthase and a resulting reduction in cellular ATP levels. A dose-dependent reduction in cellular ATP levels was detected in A549 lung carcinoma cells, and by confocal microscopy, co-localization of HAMLET with the nucleotide-binding subunits α (non-catalytic) and β (catalytic) of the energy converting F1F0 ATP synthase was detected. As shown by fluorescence correlation spectroscopy, HAMLET binds to the F1 domain of the F1F0 ATP synthase with a dissociation constant (KD) of 20.5μM. Increasing concentrations of the tumoricidal protein HAMLET added to the enzymatically active α3β3γ complex of the F-ATP synthase lowered its ATPase activity, demonstrating that HAMLET binding to the F-ATP synthase effects the catalysis of this molecular motor. Single-molecule analysis was applied to study HAMLET-α3β3γ complex interaction. Whereas the α3β3γ complex of the F-ATP synthase rotated in a counterclockwise direction with a mean rotational rate of 3.8±0.7s(-1), no rotation could be observed in the presence of bound HAMLET. Our findings suggest that direct effects of HAMLET on the F-ATP synthase may inhibit ATP-dependent cellular processes. PMID:25681694

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

    SciTech Connect

    Gikas, P.; Livingston, A.G. . Dept. of Chemical Engineering)

    1993-12-01

    This work describes investigations into the viability of cells growing on 3,4-dichloroaniline (34DCA). Two bio-reactors are employed for microbial growth, a continuous stirred tank (CST) bioreactor with a 2-L working volume, and a three-phase air lift (TPAL) bioreactor with a 3-L working volume. Experiments have been performed at several dilution rates between 0.027 and 0.115 h[sup [minus]1] in the CST bioreactor and between 0.111 and 0.500h[sup [minus]1] in the TPAL bioreactor. The specific ATP concentration was calculated at each dilution rate in the suspended biomass in both bioreactors as well as in the immobilized biomass in the TPAL bioreactor. The cultures were inspected under an electron microscope to monitor compositional changes. Results from the CST bioreactor showed that the biomass-specific ATP concentration increases from 0.44 to 1.86 mg ATP g[sup [minus]1] dry weight (dw) as dilution rate increases from 0.027 to 0.115 h[sup [minus]1]. At this upper dilution rate the cells were washed out. The specific ATP concentration reached a limiting average value of 1.73 mg ATP g[sup [minus]1] dw, which is assumed to be the quantity of ATP in 100% viable biomass, In the TPAL bioreactor, the ATP level increased with dilution rat in both the immobilized and suspended biomass. The specific ATP concentration in the immobilized biomass increased from approximately 0.051 mg ATP g[sup [minus]1] dw at dilution rates between 0.111 and 0.200 h[sup [minus]1] to approximately 0.119 mg ATP g[sup [minus]1] dw at dilution rates between 0.300 and 0.500 h[sup [minus]1].

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

    PubMed

    Garg, Priyanka; Sanguinetti, Michael C

    2014-09-01

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

  8. F1F0-ATP synthases of alkaliphilic bacteria: lessons from their adaptations

    PubMed Central

    Hicks, David B.; Liu, Jun; Fujisawa, Makoto; Krulwich, Terry A.

    2010-01-01

    This review focuses on the ATP synthases of alkaliphilic bacteria and, in particular, those that successfully overcome the bioenergetic challenges of achieving robust H+-coupled ATP synthesis at external pH values > 10. At such pH values the protonmotive force, which is posited to provide the energetic driving force for ATP synthesis, is too low to account for the ATP synthesis observed. The protonmotive force is lowered at very high pH by the need to maintain a cytoplasmic pH well below the pH outside, which results in an energetically adverse pH gradient. Several anticipated solutions to this bioenergetic conundrum have been ruled out. Although the transmembrane sodium motive force is high under alkaline conditions, respiratory alkaliphilic bacteria do not use Na+-instead of H+-coupled ATP synthases. Nor do they offset the adverse pH gradient with a compensatory increase in the transmembrane electrical potential component of the protonmotive force. Moreover, studies of ATP synthase rotors indicate that alkaliphiles cannot fully resolve the energetic problem by using an ATP synthase with a large number of c-subunits in the synthase rotor ring. Increased attention now focuses on delocalized gradients near the membrane surface and H+ transfers to ATP synthases via membrane-associated microcircuits between the H+ pumping complexes and synthases. Microcircuits likely depend upon proximity of pumps and synthases, specific membrane properties and specific adaptations of the participating enzyme complexes. ATP synthesis in alkaliphiles depends upon alkaliphile-specific adaptations of the ATP synthase and there is also evidence for alkaliphile-specific adaptations of respiratory chain components. PMID:20193659

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

    PubMed

    Tarling, Elizabeth J; Edwards, Peter A

    2011-12-01

    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

  10. 12 CFR 211.32 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 12 Banks and Banking 2 2010-01-01 2010-01-01 false Definitions. 211.32 Section 211.32 Banks and... BHC Act (12 U.S.C. 1841(a)); (2) Edge and agreement corporations that are subsidiaries of bank holding... the BHC Act (12 U.S.C. 1843(c)(14)(F)(iii)); and (4) Foreign banking organizations, as defined...

  11. 12 CFR 211.32 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 12 Banks and Banking 2 2014-01-01 2014-01-01 false Definitions. 211.32 Section 211.32 Banks and... BHC Act (12 U.S.C. 1841(a)); (2) Edge and agreement corporations that are subsidiaries of bank holding... the BHC Act (12 U.S.C. 1843(c)(14)(F)(iii)); and (4) Foreign banking organizations, as defined...

  12. 12 CFR 211.32 - Definitions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 12 Banks and Banking 2 2011-01-01 2011-01-01 false Definitions. 211.32 Section 211.32 Banks and... BHC Act (12 U.S.C. 1841(a)); (2) Edge and agreement corporations that are subsidiaries of bank holding... the BHC Act (12 U.S.C. 1843(c)(14)(F)(iii)); and (4) Foreign banking organizations, as defined...

  13. 12 CFR 211.32 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 12 Banks and Banking 2 2013-01-01 2013-01-01 false Definitions. 211.32 Section 211.32 Banks and... BHC Act (12 U.S.C. 1841(a)); (2) Edge and agreement corporations that are subsidiaries of bank holding... the BHC Act (12 U.S.C. 1843(c)(14)(F)(iii)); and (4) Foreign banking organizations, as defined...

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

    PubMed Central

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

    1990-01-01

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

  15. The Phylogenetic Signature Underlying ATP Synthase c-Ring Compliance

    DOE PAGESBeta

    Pandini, Alessandro; Kleinjung, Jens; Taylor, Willie R.; Junge, Wolfgang; Khan, Shahid

    2015-09-01

    The proton-driven ATP synthase (FOF1) is comprised of two rotary, stepping motors (FO and F1) coupled by an elastic power transmission. The elastic compliance resides in the rotor module that includes the membrane-embedded FO c-ring. Proton transport by FO is firmly coupled to the rotation of the c-ring relative to other FO subunits (ab2). It drives ATP synthesis. We used a computational method to investigate the contribution of the c-ring to the total elastic compliance. We performed principal component analysis of conformational ensembles built using distance constraints from the bovine mitochondrial c-ring x-ray structure. Angular rotary twist, the dominant ringmore » motion, was estimated to show that the c-ring accounted in part for the measured compliance. Ring rotation was entrained to rotation of the external helix within each hairpin-shaped c-subunit in the ring. Ensembles of monomer and dimers extracted from complete c-rings showed that the coupling between collective ring and the individual subunit motions was independent of the size of the c-ring, which varies between organisms. Molecular determinants were identified by covariance analysis of residue coevolution and structural-alphabet-based local dynamics correlations. The residue coevolution gave a readout of subunit architecture. The dynamic couplings revealed that the hinge for both ring and subunit helix rotations was constructed from the proton-binding site and the adjacent glycine motif (IB-GGGG) in the midmembrane plane. IB-GGGG motifs were linked by long-range couplings across the ring, while intrasubunit couplings connected the motif to the conserved cytoplasmic loop and adjacent segments. The correlation with principal collective motions shows that the couplings underlie both ring rotary and bending motions. Noncontact couplings between IB-GGGG motifs matched the coevolution signal as well as contact couplings. The residue coevolution reflects the physiological importance of the dynamics that may link proton transfer to ring compliance.« less

  16. The Phylogenetic Signature Underlying ATP Synthase c-Ring Compliance

    SciTech Connect

    Pandini, Alessandro; Kleinjung, Jens; Taylor, Willie R.; Junge, Wolfgang; Khan, Shahid

    2015-09-01

    The proton-driven ATP synthase (FOF1) is comprised of two rotary, stepping motors (FO and F1) coupled by an elastic power transmission. The elastic compliance resides in the rotor module that includes the membrane-embedded FO c-ring. Proton transport by FO is firmly coupled to the rotation of the c-ring relative to other FO subunits (ab2). It drives ATP synthesis. We used a computational method to investigate the contribution of the c-ring to the total elastic compliance. We performed principal component analysis of conformational ensembles built using distance constraints from the bovine mitochondrial c-ring x-ray structure. Angular rotary twist, the dominant ring motion, was estimated to show that the c-ring accounted in part for the measured compliance. Ring rotation was entrained to rotation of the external helix within each hairpin-shaped c-subunit in the ring. Ensembles of monomer and dimers extracted from complete c-rings showed that the coupling between collective ring and the individual subunit motions was independent of the size of the c-ring, which varies between organisms. Molecular determinants were identified by covariance analysis of residue coevolution and structural-alphabet-based local dynamics correlations. The residue coevolution gave a readout of subunit architecture. The dynamic couplings revealed that the hinge for both ring and subunit helix rotations was constructed from the proton-binding site and the adjacent glycine motif (IB-GGGG) in the midmembrane plane. IB-GGGG motifs were linked by long-range couplings across the ring, while intrasubunit couplings connected the motif to the conserved cytoplasmic loop and adjacent segments. The correlation with principal collective motions shows that the couplings underlie both ring rotary and bending motions. Noncontact couplings between IB-GGGG motifs matched the coevolution signal as well as contact couplings. The residue coevolution reflects the physiological importance of the dynamics that may link proton transfer to ring compliance.

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

    PubMed

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

    2010-09-01

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

  18. The Apple III.

    ERIC Educational Resources Information Center

    Ditlea, Steve

    1982-01-01

    Describes and evaluates the features, performance, peripheral devices, available software, and capabilities of the Apple III microcomputer. The computer's operating system, its hardware, and the commercially produced software it accepts are discussed. Specific applications programs for financial planning, accounting, and word processing are…

  19. Summary of Session III

    SciTech Connect

    Furman, M.A.

    2002-06-19

    This is a summary of the talks presented in Session III ''Simulations of Electron-Cloud Build Up'' of the Mini-Workshop on Electron-Cloud Simulations for Proton and Positron Beams ECLOUD-02, held at CERN, 15-18 April 2002.

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

    PubMed Central

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

    2013-01-01

    ATP in neurons is commonly believed to be synthesized mostly by mitochondria via oxidative phosphorylation. Neuronal mitochondria have been studied primarily in culture, i.e., in neurons isolated either from embryos or from neonatal pups. Although it is generally assumed that both embryonic and postnatal cultured neurons derive their ATP from mitochondrial oxidative phosphorylation, this has never been tested experimentally. We expressed the FRET-based ATP sensor AT1.03 in cultured hippocampal neurons isolated either from E17 to E18 rat embryos or from P1 to P2 rat pups and monitored [ATP]c simultaneously with mitochondrial membrane potential (ΔΨm; 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, ΔΨm 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 ΔΨm. These signs of oxidative phosphorylation were observed in all tested P1–P2 neurons. Measurement of ΔΨm 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

  1. Use of ATP analogs to inhibit HIV-1 transcription.

    PubMed

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

    2012-10-10

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

  2. ATP-Dependent Chromatin Remodeling Factors and DNA Damage Repair

    PubMed Central

    Osley, Mary Ann; Tsukuda, Toyoko; Nickoloff, Jac A.

    2007-01-01

    The organization of eukaryotic DNA into chromatin poses a barrier to all processes that require access of enzymes and regulatory factors to their sites of action. While the majority of studies in this area have concentrated on the role of chromatin in the regulation of transcription, there has been a recent emphasis on the relationship of chromatin to DNA damage repair. In this review, we focus on the role of chromatin in nucleotide excision repair (NER) and double-strand break (DSB) repair. NER and DSB repair use very different enzymatic machineries, and these two modes of DNA damage repair are also differentially affected by chromatin. Only a small number of nucleosomes are likely to be involved in NER, while a more extensive region of chromatin is involved in DSB repair. However, a key feature of both NER and DSB repair pathways is the participation of ATP-dependent chromatin remodeling factors at various points in the repair process. We discuss recent data that have identified roles for SWI/SNF-related chromatin remodeling factors in the two repair pathways. PMID:17291544

  3. The bizarre pharmacology of the ATP release channel pannexin1.

    PubMed

    Dahl, Gerhard; Qiu, Feng; Wang, Junjie

    2013-12-01

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

  4. Mitochondrial Configurations in Peripheral Nerve Suggest Differential ATP Production

    PubMed Central

    Perkins, Guy A.; Ellisman, Mark H.

    2010-01-01

    Physiological states of mitochondria often correlate with distinctive morphology. Electron microscopy and tomographic reconstruction were used to investigate the three-dimensional structure of axonal mitochondria and mitochondria in the surrounding Schwann cells of the peripheral nervous system (PNS), both in the vicinity of nodes of Ranvier and far from these nodes. Condensed mitochondria were found to be abundant in the axoplasm, but not in the Schwann cell. Uncharacteristic of the classical morphology of condensed mitochondria, the outer and inner boundary membranes are in close apposition and the crista junctions are narrow, consistent with their function as gates for the diffusion of macromolecules. There is also less cristae surface area and lower density of crista junctions in these mitochondria. The density of mitochondria was greater at the paranode-node-paranode (PNP) as was the crista junction opening, yet there were fewer cristae in these organelles compared to those in the internodal region. The greater density of condensed mitochondria in the PNS axoplasm and in particular at the PNP suggests a need for these organelles to operate at a high workload of ATP production. PMID:20600951

  5. ATP-dependent potassium channels and type 2 diabetes mellitus.

    PubMed

    Bonfanti, Dianne Heloisa; Alcazar, Larissa Pontes; Arakaki, Priscila Akemi; Martins, Laysa Toschi; Agustini, Bruna Carla; de Moraes Rego, Fabiane Gomes; Frigeri, Henrique Ravanhol

    2015-05-01

    Diabetes mellitus is a public health problem, which affects a millions worldwide. Most diabetes cases are classified as type 2 diabetes mellitus, which is highly associated with obesity. Type 2 diabetes is considered a multifactorial disorder, with both environmental and genetic factors contributing to its development. An important issue linked with diabetes development is the failure of the insulin releasing mechanism involving abnormal activity of the ATP-dependent potassium channel, KATP. This channel is a transmembrane protein encoded by the KCNJ11 and ABCC8 genes. Furthermore, polymorphisms in these genes have been linked to type 2 diabetes because of the role of KATP in insulin release. While several genetic variations have been reported to be associated with this disease, the E23K polymorphism is most commonly associated with this pathology, as well as to obesity. Here, we review the molecular genetics of the potassium channel and discusses its most described polymorphisms and their associations with type 2 diabetes mellitus. PMID:25583094

  6. RBC filterability, oxygen saturation, ATP intracellular stock, and cerebral microcirculation.

    PubMed

    Hermann, T; Vasselon, C; Geyssant, A; Healy, J C

    1981-01-01

    The filterability of erythrocytes through Nucleopore filters of 5 micrometer has been studied with a new device (Hemorheometer SPO1). A reliable and reproducible index of filterability (IF) can be obtained which depends only on the red blood cell deformability. The results showed an alteration of red blood cell deformability in some pathological circumstances like smoking and that deformability depends on the nature of haemoglobin, the ATP intracellar stock and the saturation of haemoglobin by oxygen. The cerebral microcirculatory consequences of alterations of red blood cell deformability has been studied in vitro by measuring whole blood filterability on 5 micrometer Nucleopore filters with the classical technic of Reid & Dormandy and in vivo by 99mTc cineangioscintigraphy and by determination of the regional cerebral blood flow by the 133Xenon inhalation technic. The results showed an increase of cerebral ascensional time and mean transit time of the cineangioscintigraphy and a decrease of regional cerebral blood flow in patients presenting with a decrease of whole blood filterability flow. PMID:6948389

  7. An ATP System for Deep-Space Optical Communication

    NASA Technical Reports Server (NTRS)

    Lee, Shinhak; Irtuzm Gerardi; Alexander, James

    2008-01-01

    An acquisition, tracking, and pointing (ATP) system is proposed for aiming an optical-communications downlink laser beam from deep space. In providing for a direction reference, the concept exploits the mature technology of star trackers to eliminate the need for a costly and potentially hazardous laser beacon. The system would include one optical and two inertial sensors, each contributing primarily to a different portion of the frequency spectrum of the pointing signal: a star tracker (<10 Hz), a gyroscope (<50 Hz), and a precise fluid-rotor inertial angular-displacement sensor (sometimes called, simply, "angle sensor") for the frequency range >50 Hz. The outputs of these sensors would be combined in an iterative averaging process to obtain high-bandwidth, high-accuracy pointing knowledge. The accuracy of pointing knowledge obtainable by use of the system was estimated on the basis of an 8-cm-diameter telescope and known parameters of commercially available star trackers and inertial sensors: The single-axis pointing-knowledge error was found to be characterized by a standard deviation of 150 nanoradians - below the maximum value (between 200 and 300 nanoradians) likely to be tolerable in deep-space optical communications.

  8. ATP-Binding Cassette Efflux Transporters in Human Placenta

    PubMed Central

    Ni, Zhanglin; Mao, Qingcheng

    2010-01-01

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

  9. Mechanism of an ATP-independent Protein Disaggregase

    PubMed Central

    Nguyen, Thang X.; Jaru-Ampornpan, Peera; Lam, Vinh Q.; Cao, Peigen; Piszkiewicz, Samantha; Hess, Sonja; Shan, Shu-ou

    2013-01-01

    Protein aggregation is detrimental to the maintenance of proper protein homeostasis in all cells. To overcome this problem, cells have evolved a network of molecular chaperones to prevent protein aggregation and even reverse existing protein aggregates. The most extensively studied disaggregase systems are ATP-driven macromolecular machines. Recently, we reported an alternative disaggregase system in which the 38-kDa subunit of chloroplast signal recognition particle (cpSRP43) efficiently reverses the aggregation of its substrates, the light-harvesting chlorophyll a/b-binding (LHC) proteins, in the absence of external energy input. To understand the molecular mechanism of this novel activity, here we used biophysical and biochemical methods to characterize the structure and nature of LHC protein aggregates. We show that LHC proteins form micellar, disc-shaped aggregates that are kinetically stable and detergent-resistant. Despite the nonamyloidal nature, the LHC aggregates have a defined global organization, displaying the chaperone recognition motif on its solvent-accessible surface. These findings suggest an attractive mechanism for recognition of the LHC aggregate by cpSRP43 and provide important constraints to define the capability of this chaperone. PMID:23525109

  10. ATP changes the fluorescence lifetime of cyan fluorescent protein via an interaction with His148.

    PubMed

    Borst, Jan Willem; Willemse, Marieke; Slijkhuis, Rik; van der Krogt, Gerard; Laptenok, Sergey P; Jalink, Kees; Wieringa, Be; Fransen, Jack A M

    2010-01-01

    Recently, we described that ATP induces changes in YFP/CFP fluorescence intensities of Fluorescence Resonance Energy Transfer (FRET) sensors based on CFP-YFP. To get insight into this phenomenon, we employed fluorescence lifetime spectroscopy to analyze the influence of ATP on these fluorescent proteins in more detail. Using different donor and acceptor pairs we found that ATP only affected the CFP-YFP based versions. Subsequent analysis of purified monomers of the used proteins showed that ATP has a direct effect on the fluorescence lifetime properties of CFP. Since the fluorescence lifetime analysis of CFP is rather complicated by the existence of different lifetimes, we tested a variant of CFP, i.e. Cerulean, as a monomer and in our FRET constructs. Surprisingly, this CFP variant shows no ATP concentration dependent changes in the fluorescence lifetime. The most important difference between CFP and Cerulean is a histidine residue at position 148. Indeed, changing this histidine in CFP into an aspartic acid results in identical fluorescence properties as observed for the Cerulean fluorescent based FRET sensor. We therefore conclude that the changes in fluorescence lifetime of CFP are affected specifically by possible electrostatic interactions of the negative charge of ATP with the positively charged histidine at position 148. Clearly, further physicochemical characterization is needed to explain the sensitivity of CFP fluorescence properties to changes in environmental (i.e. ATP concentrations) conditions. PMID:21079777

  11. Control of a Salmonella virulence locus by an ATP-sensing leader messenger RNA.

    PubMed

    Lee, Eun-Jin; Groisman, Eduardo A

    2012-06-14

    The facultative intracellular pathogen Salmonella enterica resides within a membrane-bound compartment inside macrophages. This compartment must be acidified for Salmonella to survive within macrophages, possibly because acidic pH promotes expression of Salmonella virulence proteins. We reasoned that Salmonella might sense its surroundings have turned acidic not only upon protonation of the extracytoplasmic domain of a protein sensor but also by an increase in cytosolic ATP levels, because conditions that enhance the proton gradient across the bacterial inner membrane stimulate ATP synthesis. Here we report that an increase in cytosolic ATP promotes transcription of the coding region for the virulence gene mgtC, which is the most highly induced horizontally acquired gene when Salmonella is inside macrophages. This transcript is induced both upon media acidification and by physiological conditions that increase ATP levels independently of acidification. ATP is sensed by the coupling/uncoupling of transcription of the unusually long mgtC leader messenger RNA and translation of a short open reading frame located in this region. A mutation in the mgtC leader messenger RNA that eliminates the response to ATP hinders mgtC expression inside macrophages and attenuates Salmonella virulence in mice. Our results define a singular example of an ATP-sensing leader messenger RNA. Moreover, they indicate that pathogens can interpret extracellular cues by the impact they have on cellular metabolites. PMID:22699622

  12. [Periodic and fractal precipitation in ATP-Co(2+)-decoxycholate gel system].

    PubMed

    Yin, Wen-Xuan; Liu, Yu-Feng; Zhao, Ying; Bai, Song-Yan; Zhu, Wei-Wei; Weng, Shi-Fu; Gao, Qing-Yu; Wu, Jin-Guang

    2009-11-01

    In the simulation experiments in vitro of the formation of gallstone, adenosine-triphosphate(ATP)-Co(2+)-deoxycholic acid(DC) gel system was chosen to study the periodic precipitation progress. The effect of ATP on the Co(2+)-DC gel system was also determined, and the structure of the periodic precipitation formed was characterized by FTIR. The results show that the patterns formed in the systems with ATP are different, ATP affected the rate and structure of precipitation through its variable participation in the metal coordination complexes as judged by the phosphate P=O bands and the deoxycholate COO- symmetric and asymmetric vibration bands as measured by FTIR Theses spectroscopic differences were correlated with color and pattern differences in the precipitates. ATP has a more remarkable function than AMP to the modes of patterns, meanwhile the system patterns transform from fractal to periodic precipitation. There is a complex interaction among ATP, sodium deoxycholic and Co2+ with a transparent crystal produced. The crystal is deoxycholic acid and the periodic precipitation is composed of ATP and DC covalent to Co2+. These results indicate that stone formation and remodeling is a dynamic, nonlinear progress. Much of the precipitate, as judged by local differences in composition, is not in equilibrium with the general gel environment. The authors conclude that the formation of gallstone features complex and nonlinear chemical character, in which nucleotides as living material play a very important role. PMID:20101995

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

    PubMed Central

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

    2013-01-01

    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

  14. Measurement of ADP-ATP exchange in relation to mitochondrial transmembrane potential and oxygen consumption.

    PubMed

    Chinopoulos, Christos; Kiss, Gergely; Kawamata, Hibiki; Starkov, Anatoly A

    2014-01-01

    We have previously described a fluorometric method to measure ADP-ATP exchange rates in mitochondria of permeabilized cells, in which several enzymes that consume substantial amounts of ATP and other competing reactions interconverting adenine nucleotides are present. This method relies on recording changes in free extramitochondrial Mg(2+) with the Mg(2+)-sensitive fluorescent indicator Magnesium Green (MgGr)™, exploiting the differential affinity of ADP and ATP for Mg(2+). In particular, cells are permeabilized with digitonin in the presence of BeF3(-) and Na3VO4, inhibiting all ATP- and ADP-utilizing reactions but mitochondrial exchange of ATP with ADP catalyzed by the adenine nucleotide translocase. The rate of ATP appearing in the medium upon the addition of ADP to energized mitochondria is then calculated from the rate of change in free extramitochondrial Mg(2+) using standard binding equations. Here, we describe a variant of this method involving an improved calibration step. This step minimizes errors that may be introduced during the conversion of the MgGr™ signal into free extramitochondrial [Mg(2+)] and ATP. Furthermore, we describe an approach for combining this methodology with the measurement of mitochondrial membrane potential and oxygen consumption in the same sample. The method described herein is useful for the study of malignant cells, which are known to thrive in hypoxic environments and to harbor mitochondria with profound functional alterations. PMID:24862274

  15. Exploring the ATP-binding site of P2X receptors

    PubMed Central

    Chataigneau, Thierry; Lemoine, Damien; Grutter, Thomas

    2013-01-01

    P2X receptors are ATP-gated non-selective cation channels involved in many different physiological processes, such as synaptic transmission, inflammation, and neuropathic pain. They form homo- or heterotrimeric complexes and contain three ATP-binding sites in their extracellular domain. The recent determination of X-ray structures of a P2X receptor solved in two states, a resting closed state and an ATP-bound, open-channel state, has provided unprecedented information not only regarding the three-dimensional shape of the receptor, but also on putative conformational changes that couple ATP binding to channel opening. These data provide a structural template for interpreting the huge amount of functional, mutagenesis, and biochemical data collected during more than fifteen years. In particular, the interfacial location of the ATP binding site and ATP orientation have been successfully confirmed by these structural studies. It appears that ATP binds to inter-subunit cavities shaped like open jaws, whose tightening induces the opening of the ion channel. These structural data thus represent a firm basis for understanding the activation mechanism of P2X receptors. PMID:24415999

  16. Homocysteine induces cardiac hypertrophy by up-regulating ATP7a expression

    PubMed Central

    Cao, Zhanwei; Zhang, Yanzhou; Sun, Tongwen; Zhang, Shuguang; Yu, Weiya; Zhu, Jie

    2015-01-01

    Aims: The aim of the study is to investigate the molecular mechanism by which homocysteine (Hcy) induces cardiac hypertrophy. Methods: Primary cardiomyocytes were obtained from baby Sprague-Dawley rats within 3 days after birth. Flow cytometry was used to measure cell sizes. Quantitative real-time polymerase chain reaction was performed to measure the expression of β-myosin heavy chain and atrial natriuretic peptide genes. Western blotting assay was employed to determine ATP7a protein expression. Cytochrome C oxidase (COX) activity test was used to evaluate the activity of COX. Atomic absorption spectroscopy was performed to determine copper content. siRNAs were used to target-silence the expression of ATP7a. Results: Hcy induced cardiac hypertrophy and increased the expression of cardiac hypertrophy-related genes. ATP7a was a key factor in cardiac hypertrophy induced by Hcy. Reduced ATP7a expression inhibited cardiac hypertrophy induced by Hcy. Elevated ATP7a expression induced by Hcy inhibited COX activity. Enhanced ATP7a expression inhibited COX activity by lowering intracellular copper content. Conclusions: Hcy elevates ATP7a protein expression, reduces copper content, and lowers COX activity, finally leading to cardiac hypertrophy. PMID:26722473

  17. Prokineticin 2 facilitates mechanical allodynia induced by α,β-methylene ATP in rats.

    PubMed

    Ren, Cuixia; Qiu, Chun-Yu; Gan, Xiong; Liu, Ting-Ting; Qu, Zu-Wei; Rao, Zhiguo; Hu, Wang-Ping

    2015-11-15

    Prokineticin 2 (PK2), a new chemokine, causes mechanical hypersensitivity in the rat hind paw, but little is known about the molecular mechanism. Here, we have found that ionotropic P2X receptor is essential to mechanical allodynia induced by PK2. First, intraplantar injection of high dose (3 or 10 pmol) of PK2 significantly increased paw withdrawal response frequency (%) to innocuous mechanical stimuli (mechanical allodynia). And the mechanical allodynia induced by PK2 was prevented by co-administration of TNP-ATP, a selective P2X receptor antagonist. Second, although low dose (0.3 or 1 pmol) of PK2 itself did not produce an allodynic response, it significantly facilitated the mechanical allodynia evoked by intraplantar injection of α,β-methylene ATP (α,β-meATP). Third, PK2 concentration-dependently potentiated α,β-meATP-activated currents in rat dorsal root ganglion (DRG) neurons. Finally, PK2 receptors and intracellular signal transduction were involved in PK2 potentiation of α,β-meATP-induced mechanical allodynia and α,β-meATP-activated currents, since the potentiation were blocked by PK2 receptor antagonist PKRA and selective PKC inhibitor GF 109203X. These results suggested that PK2 facilitated mechanical allodynia induced by α,β-meATP through a mechanism involved in sensitization of cutaneous P2X receptors expressed by nociceptive nerve endings. PMID:26435025

  18. Inhibition of neutrophil apoptosis by ATP is mediated by the P2Y11 receptor.

    PubMed

    Vaughan, Kathryn R; Stokes, Leanne; Prince, Lynne R; Marriott, Helen M; Meis, Sabine; Kassack, Matthias U; Bingle, Colin D; Sabroe, Ian; Surprenant, Annmarie; Whyte, Moira K B

    2007-12-15

    Neutrophils undergo rapid constitutive apoptosis that is delayed by a range of pathogen- and host-derived inflammatory mediators. We have investigated the ability of the nucleotide ATP, to which neutrophils are exposed both in the circulation and at sites of inflammation, to modulate the lifespan of human neutrophils. We found that physiologically relevant concentrations of ATP cause a concentration-dependent delay of neutrophil apoptosis (assessed by morphology, annexin V/To-Pro3 staining, and mitochondrial membrane permeabilization). We found that even brief exposure to ATP (10 min) was sufficient to cause a long-lasting delay of apoptosis and showed that the effects were not mediated by ATP breakdown to adenosine. The P2 receptor mediating the antiapoptotic actions of ATP was identified using a combination of more selective ATP analogs, receptor expression studies, and study of downstream signaling pathways. Neutrophils were shown to express the P2Y11 receptor and inhibition of P2Y11 signaling using the antagonist NF157 abrogated the ATP-mediated delay of neutrophil apoptosis, as did inhibition of type I cAMP-dependent protein kinases activated downstream of P2Y11, without effects on constitutive apoptosis. Specific targeting of P2Y11 could retain key immune functions of neutrophils but reduce the injurious effects of increased neutrophil longevity during inflammation. PMID:18056402

  19. Inhibition of neutrophil apoptosis by ATP is mediated by the P2Y11 receptor1

    PubMed Central

    Vaughan, Kathryn R.; Stokes, Leanne; Prince, Lynne R.; Meis, Sabine; Kassack, Matthias U.; Bingle, Colin D.; Sabroe, Ian; Surprenant, Annmarie; Whyte, Moira K.B.

    2008-01-01

    Neutrophils undergo rapid constitutive apoptosis that is delayed by a range of pathogen and host derived inflammatory mediators. We have investigated the ability of the nucleotide ATP, to which neutrophils are exposed both in the circulation and at sites of inflammation, to modulate the lifespan of human neutrophils. We found physiologically relevant concentrations of ATP cause a concentration-dependent delay of neutrophil apoptosis (assessed by morphology, Annexin V/To-Pro3 staining and mitochondrial membrane permeabilisation). We found even brief exposure to ATP (10 mins) was sufficient to cause a long-lasting delay of apoptosis and showed the effects were not mediated by ATP breakdown to adenosine. The P2 receptor mediating the anti-apoptotic actions of ATP was identified using a combination of more selective ATP analogues, receptor expression studies and study of downstream signalling pathways. Neutrophils were shown to express the P2Y11 receptor and inhibition of P2Y11 signalling using the antagonist NF157 abrogated the ATP-mediated delay of neutrophil apoptosis, as did inhibition of type-I cAMP-dependent protein kinases activated downstream of P2Y11, without effects upon constitutive apoptosis. Specific targeting of P2Y11 could retain key immune functions of neutrophils but reduce the injurious effects of increased neutrophil longevity during inflammation. PMID:18056402

  20. Fine-tuned ATP signals are acute mediators in osteocyte mechanotransduction.

    PubMed

    Kringelbach, Tina M; Aslan, Derya; Novak, Ivana; Ellegaard, Maria; Syberg, Susanne; Andersen, Christina K B; Kristiansen, Kim A; Vang, Ole; Schwarz, Peter; Jørgensen, Niklas R

    2015-12-01

    Osteocytes are considered the primary mechanosensors of bone, but the signaling pathways they apply in mechanotransduction are still incompletely investigated and characterized. A growing body of data strongly indicates that P2 receptor signaling among osteoblasts and osteoclasts has regulatory effects on bone remodeling. Therefore, we hypothesized that ATP signaling is also applied by osteocytes in mechanotransduction. We applied a short fluid pulse on MLO-Y4 osteocyte-like cells during real-time detection of ATP and demonstrated that mechanical stimulation activates the acute release of ATP and that these acute ATP signals are fine-tuned according to the magnitude of loading. ATP release was then challenged by pharmacological inhibitors, which indicated a vesicular release pathway for acute ATP signals. Finally, we showed that osteocytes express functional P2X2 and P2X7 receptors and respond to even low concentrations of nucleotides by increasing intracellular calcium concentration. These results indicate that in osteocytes, vesicular ATP release is an acute mediator of mechanical signals and the magnitude of loading. These and previous results, therefore, implicate purinergic signaling as an early signaling pathway in osteocyte mechanotransduction. PMID:26327582

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

    PubMed Central

    Linkerhägner, K; Oelze, J

    1995-01-01

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

  2. Selective Migration of Subpopulations of Bone Marrow Cells along an SDF-1α and ATP Gradient

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2015-08-01

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

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

    SciTech Connect

    Hattori, Motoyuki; Gouaux, Eric

    2012-10-24

    P2X receptors are trimeric ATP-activated ion channels permeable to Na{sup +}, K{sup +} and Ca{sup 2+}. The seven P2X receptor subtypes are implicated in physiological processes that include modulation of synaptic transmission, contraction of smooth muscle, secretion of chemical transmitters and regulation of immune responses. Despite the importance of P2X receptors in cellular physiology, the three-dimensional composition of the ATP-binding site, the structural mechanism of ATP-dependent ion channel gating and the architecture of the open ion channel pore are unknown. Here we report the crystal structure of the zebrafish P2X4 receptor in complex with ATP and a new structure of the apo receptor. The agonist-bound structure reveals a previously unseen ATP-binding motif and an open ion channel pore. ATP binding induces cleft closure of the nucleotide-binding pocket, flexing of the lower body {beta}-sheet and a radial expansion of the extracellular vestibule. The structural widening of the extracellular vestibule is directly coupled to the opening of the ion channel pore by way of an iris-like expansion of the transmembrane helices. The structural delineation of the ATP-binding site and the ion channel pore, together with the conformational changes associated with ion channel gating, will stimulate development of new pharmacological agents.

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

    PubMed Central

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

    2014-01-01

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

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

    SciTech Connect

    Shin, Youn Ho; Lee, Seo Jin; Jung, Junyang

    2013-01-11

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

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

    PubMed Central

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

    2010-01-01

    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

  8. Promiscuous archaeal ATP synthase concurrently coupled to Na+ and H+ translocation

    PubMed Central

    Schlegel, Katharina; Leone, Vanessa; Faraldo-Gómez, José D.; Müller, Volker

    2012-01-01

    ATP synthases are the primary source of ATP in all living cells. To catalyze ATP synthesis, these membrane-associated complexes use a rotary mechanism powered by the transmembrane diffusion of ions down a concentration gradient. ATP synthases are assumed to be driven either by H+ or Na+, reflecting distinct structural motifs in their membrane domains, and distinct metabolisms of the host organisms. Here, we study the methanogenic archaeon Methanosarcina acetivorans using assays of ATP hydrolysis and ion transport in inverted membrane vesicles, and experimentally demonstrate that the rotary mechanism of its ATP synthase is coupled to the concurrent translocation of both H+ and Na+ across the membrane under physiological conditions. Using free-energy molecular simulations, we explain this unprecedented observation in terms of the ion selectivity of the binding sites in the membrane rotor, which appears to have been tuned via amino acid substitutions so that ATP synthesis in M. acetivorans can be driven by the H+ and Na+ gradients resulting from methanogenesis. We propose that this promiscuity is a molecular mechanism of adaptation to life at the thermodynamic limit. PMID:22219361

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

    PubMed

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

    2014-01-01

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

  10. A nuclear gene encoding the beta subunit of the mitochondrial ATP synthase in Nicotiana plumbaginifolia.

    PubMed Central

    Boutry, M; Chua, N H

    1985-01-01

    The beta subunit of the mitochondrial ATP synthase in Nicotiana plumbaginifolia is encoded by two nuclear genes, atp2-1 and atp2-2, which are both expressed. The complete nucleotide sequence of atp2-1 has been determined. It contains eight introns ranging from 88 to 1453 bp. The last intron contains a putative insertion element (Inp), of 812 bp bordered by 35-bp inverted repeats which share an 11-bp homology with Agrobacterium tumefaciens T-DNA borders. Sequences homologous to Inp are present in multiple copies in the N. plumbaginifolia and the N. tabacum genome but not in more distant species. The atp2-1 encoded polypeptide is highly homologous to beta subunits from other ATP synthases but it contains an extension at the N terminus which is probably involved in mitochondrial targeting. A sequence homology between exon 4 of atp2-1 and exon 1 of the human ras genes suggests a common ancestral origin for these exons. Images Fig. 3. Fig. 4. Fig. 5. PMID:2866954

  11. Wilson Disease Protein ATP7B Utilizes Lysosomal Exocytosis to Maintain Copper Homeostasis

    PubMed Central

    Polishchuk, ElenaV.; Concilli, Mafalda; Iacobacci, Simona; Chesi, Giancarlo; Pastore, Nunzia; Piccolo, Pasquale; Paladino, Simona; Baldantoni, Daniela; vanIJzendoorn, SvenC.D.; Chan, Jefferson; Chang, ChristopherJ.; Amoresano, Angela; Pane, Francesca; Pucci, Piero; Tarallo, Antonietta; Parenti, Giancarlo; Brunetti-Pierri, Nicola; Settembre, Carmine; Ballabio, Andrea; Polishchuk, RomanS.

    2014-01-01

    Summary Copper is an essential yet toxic metal and its overload causes Wilson disease, a disorder due to mutations in copper transporter ATP7B. To remove excess copper into the bile, ATP7B traffics toward canalicular area of hepatocytes. However, the trafficking mechanisms of ATP7B remain elusive. Here, we show that, in response to elevated copper, ATP7B moves from the Golgi to lysosomes and imports metal into their lumen. ATP7B enables lysosomes to undergo exocytosis through the interaction with p62 subunit of dynactin that allows lysosome translocation toward the canalicular pole of hepatocytes. Activation of lysosomal exocytosis stimulates copper clearance from the hepatocytes and rescues the most frequent Wilson-disease-causing ATP7B mutant to the appropriate functional site. Our findings indicate that lysosomes serve as an important intermediate in ATP7B trafficking, whereas lysosomal exocytosis operates as an integral process in copper excretion and hence can be targeted for therapeutic approaches to combat Wilson disease. PMID:24909901

  12. Mutation in cysteine bridge domain of the gamma-subunit affects light regulation of the ATP synthase in Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The chloroplast ATP synthase functions to synthesize ATP from ADP and free phosphate coupled by the electrochemical potential across the thylakoid membrane in the light. The light-dependent regulation of ATP synthase activity is carried out in part through redox modulation of a cysteine bridge in CF...

  13. Regulation of Purinergic Signaling in Biliary Epithelial Cells by Exocytosis of SLC17A9-dependent ATP-enriched Vesicles*

    PubMed Central

    Sathe, Meghana N.; Woo, Kangmee; Kresge, Charles; Bugde, Abhijit; Luby-Phelps, Kate; Lewis, Matthew A.; Feranchak, Andrew P.

    2011-01-01

    ATP in bile is a potent secretogogue, stimulating biliary epithelial cell (BEC) secretion through binding apical purinergic receptors. In response to mechanosensitive stimuli, BECs release ATP into bile, although the cellular basis of ATP release is unknown. The aims of this study in human and mouse BECs were to determine whether ATP release occurs via exocytosis of ATP-enriched vesicles and to elucidate the potential role of the vesicular nucleotide transporter SLC17A9 in purinergic signaling. Dynamic, multiscale, live cell imaging (confocal and total internal reflection fluorescence microscopy and a luminescence detection system with a high sensitivity charge-coupled device camera) was utilized to detect vesicular ATP release from cell populations, single cells, and the submembrane space of a single cell. In response to increases in cell volume, BECs release ATP, which was dependent on intact microtubules and vesicular trafficking pathways. ATP release occurred as stochastic point source bursts of luminescence consistent with exocytic events. Parallel studies identified ATP-enriched vesicles ranging in size from 0.4 to 1 ?m that underwent fusion and release in response to increases in cell volume in a protein kinase C-dependent manner. Present in all models, SLC17A9 contributed to ATP vesicle formation and regulated ATP release. The findings are consistent with the existence of an SLC17A9-dependent ATP-enriched vesicular pool in biliary epithelium that undergoes regulated exocytosis to initiate purinergic signaling. PMID:21613220

  14. Adsorption of nucleotides on biomimetic apatite: The case of adenosine 5‧ triphosphate (ATP)

    NASA Astrophysics Data System (ADS)

    Hammami, Khaled; El-Feki, Hafed; Marsan, Olivier; Drouet, Christophe

    2016-01-01

    ATP is a well-known energy supplier in cells. The idea to associate ATP to pharmaceutical formulations/biotechnological devices to promote cells activity by potentially modulating their microenvironment thus appears as an appealing novel approach. Since biomimetic nanocrystalline apatites have shown great promise for biomedical applications (bone regeneration, cells diagnostics/therapeutics, …), thanks to a high surface reactivity and an intrinsically high biocompatibility, the present contribution was aimed at exploring ATP/apatite interactions. ATP adsorption on a synthetic carbonated nanocrystalline apatite preliminarily characterized (by XRD, FTIR, Raman, TG-DTA and SEM-EDX) was investigated in detail, pointing out a good agreement with Sips isothermal features. Adsorption characteristics were compared to those previously obtained on monophosphate nucleotides (AMP, CMP), unveiling some specificities. ATP was found to adsorb effectively onto biomimetic apatite: despite smaller values of the affinity constant KS and the exponential factor m, larger adsorbed amounts were reached for ATP as compared to AMP for any given concentration in solution. m < 1 suggests that the ATP/apatite adsorption process is mostly guided by direct surface bonding rather than through stabilizing intermolecular interactions. Although standard ΔGads ° was estimated to only -4 kJ/mol, the large value of Nmax led to significantly negative effective ΔGads values down to -33 kJ/mol, reflecting the spontaneous character of adsorption process. Vibrational spectroscopy data (FTIR and Raman) pointed out spectral modifications upon adsorption, confirming chemical-like interactions where both the triphosphate group of ATP and its nucleic base were involved. The present study is intended to serve as a basis for future research works involving ATP and apatite nanocrystals/nanoparticles in view of biomedical applications (e.g. bone tissue engineering, intracellular drug delivery, …).

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

    PubMed Central

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

    2012-01-01

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

  16. In vivo ATP production during free-flow and ischaemic muscle contractions in humans.

    PubMed

    Lanza, Ian R; Wigmore, Danielle M; Befroy, Douglas E; Kent-Braun, Jane A

    2006-11-15

    The aim of this study was to determine how ATP synthesis and contractility in vivo are altered by ischaemia in working human skeletal muscle. The hypotheses were: (1) glycolytic flux would be higher during ischaemic (ISC) compared to free-flow (FF) muscle contractions, in compensation for reduced oxidative ATP synthesis, and (2) ischaemic muscle fatigue would be related to the accumulation of inhibitory metabolic by-products rather than to the phosphorylation potential ([ATP]/[ADP][P(i)]) of the muscle. Twelve healthy adults (6 men, 6 women) performed six intermittent maximal isometric contractions of the ankle dorsiflexors (12 s contract, 12 s relax), once with intact blood flow and once with local ischaemia by thigh cuff inflation to 220 Torr. Intracellular phosphorous metabolites and pH were measured non-invasively with magnetic resonance spectroscopy, and rates of ATP synthesis through oxidative phosphorylation, anaerobic glycolysis, and the creatine kinase reaction were determined. The force-time integral declined more during ISC (66 +/- 3% initial) than FF (75 +/- 2% initial, P = 0.002), indicating greater fatigue in ISC. [ATP] was preserved in both protocols, indicating matching of ATP production and use under both conditions. Glycolytic flux (mm s(-1)) was similar during FF and ISC (P = 0.16). Total ATP synthesis rate was lower during ISC, despite adjustment for the greater muscle fatigue in this condition (P < 0.001). Fatigue was linearly associated with diprotonated inorganic phosphate (FF r = 0.94 +/- 0.01, ISC r = 0.92 +/- 0.02), but not phosphorylation potential. These data provide novel evidence that ATP supply and demand in vivo are balanced in human skeletal muscle during ischaemic work, not through higher glycolytic flux, but rather through increased metabolic economy and decreased rates of ATP consumption as fatigue ensues. PMID:16945975

  17. Decline of Phosphotransfer and Substrate Supply Metabolic Circuits Hinders ATP Cycling in Aging Myocardium.

    PubMed

    Nemutlu, Emirhan; Gupta, Anu; Zhang, Song; Viqar, Maria; Holmuhamedov, Ekhson; Terzic, Andre; Jahangir, Arshad; Dzeja, Petras

    2015-01-01

    Integration of mitochondria with cytosolic ATP-consuming/ATP-sensing and substrate supply processes is critical for muscle bioenergetics and electrical activity. Whether age-dependent muscle weakness and increased electrical instability depends on perturbations in cellular energetic circuits is unknown. To define energetic remodeling of aged atrial myocardium we tracked dynamics of ATP synthesis-utilization, substrate supply, and phosphotransfer circuits through adenylate kinase (AK), creatine kinase (CK), and glycolytic/glycogenolytic pathways using 18O stable isotope-based phosphometabolomic technology. Samples of intact atrial myocardium from adult and aged rats were subjected to 18O-labeling procedure at resting basal state, and analyzed using the 18O-assisted HPLC-GC/MS technique. Characteristics for aging atria were lower inorganic phosphate Pi[18O], γ-ATP[18O], β-ADP[18O], and creatine phosphate CrP[18O] 18O-labeling rates indicating diminished ATP utilization-synthesis and AK and CK phosphotransfer fluxes. Shift in dynamics of glycolytic phosphotransfer was reflected in the diminished G6P[18O] turnover with relatively constant glycogenolytic flux or G1P[18O] 18O-labeling. Labeling of G3P[18O], an indicator of G3P-shuttle activity and substrate supply to mitochondria, was depressed in aged myocardium. Aged atrial myocardium displayed reduced incorporation of 18O into second (18O2), third (18O3), and fourth (18O4) positions of Pi[18O] and a lower Pi[18O]/γ-ATP[18 O]-labeling ratio, indicating delayed energetic communication and ATP cycling between mitochondria and cellular ATPases. Adrenergic stress alleviated diminished CK flux, AK catalyzed β-ATP turnover and energetic communication in aging atria. Thus, 18O-assisted phosphometabolomics uncovered simultaneous phosphotransfer through AK, CK, and glycolytic pathways and G3P substrate shuttle deficits hindering energetic communication and ATP cycling, which may underlie energetic vulnerability of aging atrial myocardium. PMID:26378442

  18. Decline of Phosphotransfer and Substrate Supply Metabolic Circuits Hinders ATP Cycling in Aging Myocardium

    PubMed Central

    Nemutlu, Emirhan; Gupta, Anu; Zhang, Song; Viqar, Maria; Holmuhamedov, Ekhson; Terzic, Andre; Jahangir, Arshad; Dzeja, Petras

    2015-01-01

    Integration of mitochondria with cytosolic ATP-consuming/ATP-sensing and substrate supply processes is critical for muscle bioenergetics and electrical activity. Whether age-dependent muscle weakness and increased electrical instability depends on perturbations in cellular energetic circuits is unknown. To define energetic remodeling of aged atrial myocardium we tracked dynamics of ATP synthesis-utilization, substrate supply, and phosphotransfer circuits through adenylate kinase (AK), creatine kinase (CK), and glycolytic/glycogenolytic pathways using 18O stable isotope-based phosphometabolomic technology. Samples of intact atrial myocardium from adult and aged rats were subjected to 18O-labeling procedure at resting basal state, and analyzed using the 18O-assisted HPLC-GC/MS technique. Characteristics for aging atria were lower inorganic phosphate Pi[18O], γ-ATP[18O], β-ADP[18O], and creatine phosphate CrP[18O] 18O-labeling rates indicating diminished ATP utilization-synthesis and AK and CK phosphotransfer fluxes. Shift in dynamics of glycolytic phosphotransfer was reflected in the diminished G6P[18O] turnover with relatively constant glycogenolytic flux or G1P[18O] 18O-labeling. Labeling of G3P[18O], an indicator of G3P-shuttle activity and substrate supply to mitochondria, was depressed in aged myocardium. Aged atrial myocardium displayed reduced incorporation of 18O into second (18O2), third (18O3), and fourth (18O4) positions of Pi[18O] and a lower Pi[18O]/γ-ATP[18 O]-labeling ratio, indicating delayed energetic communication and ATP cycling between mitochondria and cellular ATPases. Adrenergic stress alleviated diminished CK flux, AK catalyzed β-ATP turnover and energetic communication in aging atria. Thus, 18O-assisted phosphometabolomics uncovered simultaneous phosphotransfer through AK, CK, and glycolytic pathways and G3P substrate shuttle deficits hindering energetic communication and ATP cycling, which may underlie energetic vulnerability of aging atrial myocardium. PMID:26378442

  19. [ATP-sensitive K(+)-channels in muscle cells: features and physiological role].

    PubMed

    Vadziuk, O B

    2014-01-01

    ATP-sensitive K(+)-channels of plasma membranes belong to the inward rectifier potassium channels type. They are involved in coupling of electrical activity of muscle cell with its metabolic state. These channels are heterooctameric and consist of two types of subunits: four poreforming (Kir 6.x) and four regulatory (SUR, sulfonylurea receptor). The Kir subunits contain highly selective K+ filter and provide for high-velocity K+ currents. The SUR subunits contain binding sites for activators and blockers and have metabolic sensor, which enables channel activation under conditions of metabolic stress. ATP blocks K+ currents through the ATP-sensitive K(+)-channels in the most types of muscle cells. However, functional activity of these channels does not depend on absolute concentration of ATP but on the ATP/ADP ratio and presence of Mg2+. Physiologically active substances, such as phosphatidylinositol bisphosphate and fatty acid esters can regulate the activity of these structures in muscle cells. Activation of these channels under ischemic conditions underlies their cytoprotective action, which results in prevention of Ca2+ overload in cytosol. In contrast to ATP-sensitive K(+)-channels of plasma membranes, the data regarding the structure and function of ATP-sensitive K(+)-channels of mitochondrial membrane are contradictory. Pore-forming subunits of this channel have not been firmly identified yet. ATP-sensitive K+ transport through the mitochondrial membrane is easily tested by different methods, which are briefly reviewed in this paper. Interaction of mitoK(ATP) with physiological and pharmacological ligands is discussed as well. PMID:25033550

  20. ATP4A autoimmunity and Helicobacter pylori infection in children with type 1 diabetes

    PubMed Central

    Chobot, A; Wenzlau, J; Bak-Drabik, K; Kwiecien, J; Polanska, J; Rewers, M

    2014-01-01

    Persistent presence of ATP4A autoantibodies (ATP4AA) directed towards parietal cells is typical for atrophic body gastritis (ABG), an autoimmune disease associated with type 1 diabetes. We assessed whether Helicobacter pylori (Hp) infection might be associated with positivity for ATP4AA in children with type 1 diabetes. Sera were collected from 70 (38♀) type 1 diabetes children [aged 13·2 ± 4·5 years, age at diagnosis 8·8 ± 4·3 years, diabetes duration 4·5 ± 3·8 years, mean HbA1c 7·8 ± 1·6% (62 ± 17·5 mmol/mol)] seen at the regional diabetes clinic in Katowice, Poland. Patients were tested concurrently for Hp infection by means of a 13C urea breath test. ATP4AA were measured using a novel radioimmunoprecipitation assay developed at the Barbara Davies Center for Childhood Diabetes, University of Colorado. ATP4AA were present in 21 [30%, 95% confidence interval (CI) = 19–41%] and Hp infection was detected in 23 (33%, 95% CI = 22–44%) children. There was no statistically significant association between ATP4AA presence and Hp status. ATP4AA presence was not associated with current age, age at type 1 diabetes diagnosis, diabetes duration or current HbA1c. ATP4AA were more prevalent in females [42% (26–58%)] than males [16% (3–28%)], P = 0·016. ATP4A are found in nearly one-third of children with type 1 diabetes and more common among females. In this cross-sectional analysis, Hp infection was not associated with autoimmunity against parietal cells. PMID:24773566

  1. ATP and Mg2+ Promote the Reversible Oligomerization and Aggregation of Chloroplast 2-Cys Peroxiredoxin*

    PubMed Central

    Aran, Martín; Ferrero, Diego; Wolosiuk, Alejandro; Mora-García, Santiago; Wolosiuk, Ricardo A.

    2011-01-01

    2-Cys peroxiredoxins (2-Cys Prxs) are ubiquitous peroxidases with important roles in cellular antioxidant defense and hydrogen peroxide-mediated signaling. Post-translational modifications of conserved cysteines cause the transition from low to high molecular weight oligomers, triggering the functional change from peroxidase to molecular chaperone. However, it remains unclear how non-covalent interactions of 2-Cys Prx with metabolites modulate the quaternary structure. Here, we disclose that ATP and Mg2+ (ATP/Mg) promote the self-polymerization of chloroplast 2-Cys Prx (polypeptide 23.5 kDa) into soluble higher order assemblies (>2 MDa) that proceed to insoluble aggregates beyond 5 mm ATP. Remarkably, the withdrawal of ATP or Mg2+ brings soluble oligomers and insoluble aggregates back to the native conformation without compromising the associated functions. As confirmed by transmission electron microscopy, ATP/Mg drive the toroid-like decamers (diameter 13 nm) to the formation of large sphere-like particles (diameter ∼30 nm). Circular dichroism studies on ATP-labeled 2-Cys Prx reveal that ATP/Mg enhance the proportion of β-sheets with the concurrent decrease in the content of α-helices. In line with this observation, the formation of insoluble aggregates is strongly prevented by 2,2,2-trifluoroethanol, a cosolvent employed to induce α-helical conformations. We further find that the response of self-polymerization to ATP/Mg departs abruptly from that of the associated peroxidase and chaperone activities when two highly conserved residues, Arg129 and Arg152, are mutated. Collectively, our data uncover that non-covalent interactions of ATP/Mg with 2-Cys Prx modulate dynamically the quaternary structure, thereby coupling the non-redox chemistry of cell energy with redox transformations at cysteine residues. PMID:21525006

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

    PubMed

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

    2014-09-15

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

  3. Unexpected role of the copper transporter ATP7A in PDGF-induced vascular smooth

    SciTech Connect

    Ashino, T.; Varadarajan, S.; Urao, N.; Oshikawa, J.; Chen, G. -F.; Wang, H.; Huo, Y.; Finney, L.; Vogt, S.; McKinney, R. D.; Maryon, E. B.; Kaplan, J. H.; Ushio-Fukai, M.; Fukai, T.

    2010-09-09

    Copper, an essential nutrient, has been implicated in vascular remodeling and atherosclerosis with unknown mechanism. Bioavailability of intracellular copper is regulated not only by the copper importer CTR1 (copper transporter 1) but also by the copper exporter ATP7A (Menkes ATPase), whose function is achieved through copper-dependent translocation from trans-Golgi network (TGN). Platelet-derived growth factor (PDGF) promotes vascular smooth muscle cell (VSMC) migration, a key component of neointimal formation. To determine the role of copper transporter ATP7A in PDGF-induced VSMC migration. Depletion of ATP7A inhibited VSMC migration in response to PDGF or wound scratch in a CTR1/copper-dependent manner. PDGF stimulation promoted ATP7A translocation from the TGN to lipid rafts, which localized at the leading edge, where it colocalized with PDGF receptor and Rac1, in migrating VSMCs. Mechanistically, ATP7A small interfering RNA or CTR small interfering RNA prevented PDGF-induced Rac1 translocation to the leading edge, thereby inhibiting lamellipodia formation. In addition, ATP7A depletion prevented a PDGF-induced decrease in copper level and secretory copper enzyme precursor prolysyl oxidase (Pro-LOX) in lipid raft fraction, as well as PDGF-induced increase in LOX activity. In vivo, ATP7A expression was markedly increased and copper accumulation was observed by synchrotron-based x-ray fluorescence microscopy at neointimal VSMCs in wire injury model. These findings suggest that ATP7A plays an important role in copper-dependent PDGF-stimulated VSMC migration via recruiting Rac1 to lipid rafts at the leading edge, as well as regulating LOX activity. This may contribute to neointimal formation after vascular injury. Our findings provide insight into ATP7A as a novel therapeutic target for vascular remodeling and atherosclerosis.

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

    PubMed

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

    2014-10-17

    ATP has dual roles in the reaction cycle of sarcoplasmic reticulum Ca(2+)-ATPase. Upon binding to the Ca2E1 state, ATP phosphorylates the enzyme, and by binding to other conformational states in a non-phosphorylating modulatory mode ATP stimulates the dephosphorylation and other partial reaction steps of the cycle, thereby ensuring a high rate of Ca(2+) transport under physiological conditions. The present study elucidates the mechanism underlying the modulatory effect on dephosphorylation. In the intermediate states of dephosphorylation the A-domain residues Ser(186) and Asp(203) interact with Glu(439) (N-domain) and Arg(678) (P-domain), respectively. Single mutations to these residues abolish the stimulation of dephosphorylation by ATP. The double mutation swapping Asp(203) and Arg(678) rescues ATP stimulation, whereas this is not the case for the double mutation swapping Ser(186) and Glu(439). By taking advantage of the ability of wild type and mutant Ca(2+)-ATPases to form stable complexes with aluminum fluoride (E2·AlF) and beryllium fluoride (E2·BeF) as analogs of the E2·P phosphoryl transition state and E2P ground state, respectively, of the dephosphorylation reaction, the mutational effects on ATP binding to these intermediates are demonstrated. In the wild type Ca(2+)-ATPase, the ATP affinity of the E2·P phosphoryl transition state is higher than that of the E2P ground state, thus explaining the stimulation of dephosphorylation by nucleotide-induced transition state stabilization. We find that the Asp(203)-Arg(678) and Ser(186)-Glu(439) interdomain bonds are critical, because they tighten the interaction with ATP in the E2·P phosphoryl transition state. Moreover, ATP binding and the Ser(186)-Glu(439) bond are mutually exclusive in the E2P ground state. PMID:25193668

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

    PubMed Central

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

    2014-01-01

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

  6. Medical tele-education system with superhigh-definition (SHD) image viewer

    NASA Astrophysics Data System (ADS)

    Tsumura, Hiroshi; Ashihara, Tsukasa; Urata, Yoji; Hata, Jun-ichi; Fukuhara, Yoshimi; Ono, Sadayasu

    1996-02-01

    We have been studying a medical tele-education support system by an individual tutoring system, called CALAT, and a super high definition (SHD) image processing system, called SuperFM-III. Now, we are in a trial operation to use the SuperFM-III for a super high definition image control viewer on the CALAT client side, and have created the courseware of the pathological images. In this paper, we show the concept and the implementation of this system.

  7. An investigation into the role of ATP in the mammalian pre-mRNA 3' cleavage reaction.

    PubMed

    Khleborodova, Asya; Pan, Xiaozhou; Nagre, Nagaraja N; Ryan, Kevin

    2016-06-01

    RNA Polymerase II transcribes beyond what later becomes the 3' end of a mature messenger RNA (mRNA). The formation of most mRNA 3' ends results from pre-mRNA cleavage followed by polyadenylation. In vitro studies have shown that low concentrations of ATP stimulate the 3' cleavage reaction while high concentrations inhibit it, but the origin of these ATP effects is unknown. ATP might enable a cleavage factor kinase or activate a cleavage factor directly. To distinguish between these possibilities, we tested several ATP structural analogs in a pre-mRNA 3' cleavage reaction reconstituted from DEAE-fractionated cleavage factors. We found that adenosine 5'-(β,γ-methylene)triphosphate (AMP-PCP) is an effective in vitro 3' cleavage inhibitor with an IC50 of ∼300 μM, but that most other ATP analogs, including adenosine 5'-(β,γ-imido)triphosphate, which cannot serve as a protein kinase substrate, promoted 3' cleavage but less efficiently than ATP. In combination with previous literature data, our results do not support ATP stimulation of 3' cleavage through cleavage factor phosphorylation in vitro. Instead, the more likely mechanism is that ATP stimulates cleavage factor activity through direct cleavage factor binding. The mammalian 3' cleavage factors known to bind ATP include the cleavage factor II (CF IIm) Clp1 subunit, the CF Im25 subunit and poly(A) polymerase alpha (PAP). The yeast homolog of the CF IIm complex also binds ATP through yClp1. To investigate the mammalian complex, we used a cell-line expressing FLAG-tagged Clp1 to co-immunoprecipitate Pcf11 as a function of ATP concentration. FLAG-Clp1 co-precipitated Pcf11 with or without ATP and the complex was not affected by AMP-PCP. Diadenosine tetraphosphate (Ap4A), an ATP analog that binds the Nudix domain of the CF Im25 subunit with higher affinity than ATP, neither stimulated 3' cleavage in place of ATP nor antagonized ATP-stimulated 3' cleavage. The ATP-binding site of PAP was disrupted by site directed mutagenesis but a reconstituted 3' cleavage reaction containing a mutant PAP unable to bind ATP nevertheless underwent ATP-stimulated 3' cleavage. Fluctuating ATP levels might contribute to the regulation of pre-mRNA 3' cleavage, but the three subunits investigated here do not appear to be responsible for the ATP-stimulation of pre-mRNA cleavage. PMID:27060432

  8. A silent nucleotide substitution in the ATP7A gene in a child with Menkes disease.

    PubMed

    Møller, Lisbeth Birk; Rea, Gillian; Yasmeen, Saiqa; Skjørringe, Tina; Thorborg, Sidsel Salling; Morrison, Patrick J; Donnelly, Deirdre E

    2013-12-01

    We present a case of classical Menkes disease (MD) due to a novel "silent" substitution in the ATP7A gene; c.2781G>A (p.K927K). The affected nucleotide is the last nucleotide in exon 13, and affects mRNA splicing. Transcripts missing exon 13; and transcripts missing exons 11, 12 and 13 in addition to a very small amount of normal spliced ATP7A transcripts were expressed. This is the first report of a synonymous ATP7A substitution being responsible for MD. PMID:24100245

  9. Small amounts of functional ATP7A protein permit mild phenotype.

    PubMed

    Møller, Lisbeth Birk

    2015-01-01

    Mutations in ATP7A lead to at least three allelic disorders: Menkes disease (MD), Occipital horn syndrome and X-linked distal motor neuropathy. These disorders are mainly seen in male individuals, but a few affected females have been described. More than 400 different mutations have been identified in the ATP7A gene. We have conducted several studies in the hope of uncovering the relationship between genotype and phenotype. We have examined the X-inactivation pattern in affected females, the effect of exon-deletions and--duplications, and splice-site mutations on the composition and amount of ATP7A transcript, and we have examined the structural location of missense mutations. The X-inactivation pattern did not fully explain the manifestation of MD in a small fraction of carriers. Most of the affected females had preferential inactivation of the X-chromosome with the normal ATP7A gene, but a few individuals exhibited preferential inactivation of the X-chromosome with the mutated ATP7A gene. The observed mild phenotype in some patients with mutations that effect the composition of the ATP7A transcript, seems to be explained by the presence of a small amount of normal ATP7A transcript. The location of missense mutations on structural models of the ATP7A protein suggests that affected conserved residues generally lead to a severe phenotype. The ATP7A protein traffics within the cells. At low copper levels, ATP7A locates to the Trans-Golgi Network (TGN) to load cuproenzymes with copper, whereas at higher concentrations, ATP7A shifts to the post-Golgi compartments or to the plasma membrane to export copper out of the cell. Impaired copper-regulation trafficking has been observed for ATP7A mutants, but its impact on the clinical outcome is not clear. The major problem in patients with MD seems to be insufficient amounts of copper in the brain. In fact, prenatal treatment of mottled mice as a model for human MD with a combination of chelator and copper, produces a slight increase in copper levels in the brain which perhaps leads to longer survival and more active behavior. In conclusion, small amounts of copper at the right location seem to relieve the symptoms. PMID:25172213

  10. H2 -Fueled ATP Synthesis on an Electrode: Mimicking Cellular Respiration.

    PubMed

    Gutiérrez-Sanz, Óscar; Natale, Paolo; Márquez, Ileana; Marques, Marta C; Zacarias, Sonia; Pita, Marcos; Pereira, Inês A C; López-Montero, Iván; De Lacey, Antonio L; Vélez, Marisela

    2016-05-17

    ATP, the molecule used by living organisms to supply energy to many different metabolic processes, is synthesized mostly by the ATPase synthase using a proton or sodium gradient generated across a lipid membrane. We present evidence that a modified electrode surface integrating a NiFeSe hydrogenase and a F1 F0 -ATPase in a lipid membrane can couple the electrochemical oxidation of H2 to the synthesis of ATP. This electrode-assisted conversion of H2 gas into ATP could serve to generate this biochemical fuel locally when required in biomedical devices or enzymatic synthesis of valuable products. PMID:26991333

  11. Probing the ATP Site of GRP78 with Nucleotide Triphosphate Analogs

    PubMed Central

    Chen, Yun; Lu, Hua; Pizarro, Juan C.; Park, Hee-Won

    2016-01-01

    GRP78, a member of the ER stress protein family, can relocate to the surface of cancer cells, playing key roles in promoting cell proliferation and metastasis. GRP78 consists of two major functional domains: the ATPase and protein/peptide-binding domains. The protein/peptide-binding domain of cell-surface GRP78 has served as a novel functional receptor for delivering cytotoxic agents (e.g., a apoptosis-inducing peptide or taxol) across the cell membrane. Here, we report our study on the ATPase domain of GRP78 (GRP78ATPase), whose potential as a transmembrane delivery system of cytotoxic agents (e.g., ATP-based nucleotide triphosphate analogs) remains unexploited. As the binding of ligands (ATP analogs) to a receptor (GRP78ATPase) is a pre-requisite for internalization, we determined the binding affinities and modes of GRP78ATPase for ADP, ATP and several ATP analogs using surface plasmon resonance and x-ray crystallography. The tested ATP analogs contain one of the following modifications: the nitrogen at the adenine ring 7-position to a carbon atom (7-deazaATP), the oxygen at the β-γ bridge position to a carbon atom (AMPPCP), or the removal of the 2’-OH group (2’-deoxyATP). We found that 7-deazaATP displays an affinity and a binding mode that resemble those of ATP regardless of magnesium ion (Mg++) concentration, suggesting that GRP78 is tolerant to modifications at the 7-position. By comparison, AMPPCP’s binding affinity was lower than ATP and Mg++-dependent, as the removal of Mg++ nearly abolished binding to GRP78ATPase. The AMPPCP-Mg++ structure showed evidence for the critical role of Mg++ in AMPPCP binding affinity, suggesting that while GRP78 is sensitive to modifications at the β-γ bridge position, these can be tolerated in the presence of Mg++. Furthermore, 2’-deoxyATP’s binding affinity was significantly lower than those for all other nucleotides tested, even in the presence of Mg++. The 2’-deoxyATP structure showed the conformation of the bound nucleotide flipped out of the active site, explaining the low affinity binding to GRP78 and suggesting that the 2’-OH group is essential for the high affinity binding to GRP78. Together, our results demonstrate that GRP78ATPase possesses nucleotide specificity more relaxed than previously anticipated and can tolerate certain modifications to the nucleobase 7-position and, to a lesser extent, the β-γ bridging atom, thereby providing a possible atomic mechanism underlying the transmembrane transport of the ATP analogs. PMID:27144892

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

    SciTech Connect

    Aulio, K.

    1984-01-01

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

  13. Near-infrared light increases ATP, extends lifespan and improves mobility in aged Drosophila melanogaster

    PubMed Central

    Begum, Rana; Calaza, Karin; Kam, Jaimie Hoh; Salt, Thomas E.; Hogg, Chris; Jeffery, Glen

    2015-01-01

    Ageing is an irreversible cellular decline partly driven by failing mitochondrial integrity. Mitochondria accumulate DNA mutations and reduce ATP production necessary for cellular metabolism. This is associated with inflammation. Near-infrared exposure increases retinal ATP in old mice via cytochrome c oxidase absorption and reduces inflammation. Here, we expose fruitflies daily to 670 nm radiation, revealing elevated ATP and reduced inflammation with age. Critically, there was a significant increase in average lifespan: 100–175% more flies survived into old age following 670 nm exposure and these had significantly improved mobility. This may be a simple route to extending lifespan and improving function in old age. PMID:25788488

  14. Near-infrared light increases ATP, extends lifespan and improves mobility in aged Drosophila melanogaster.

    PubMed

    Begum, Rana; Calaza, Karin; Kam, Jaimie Hoh; Salt, Thomas E; Hogg, Chris; Jeffery, Glen

    2015-03-01

    Ageing is an irreversible cellular decline partly driven by failing mitochondrial integrity. Mitochondria accumulate DNA mutations and reduce ATP production necessary for cellular metabolism. This is associated with inflammation. Near-infrared exposure increases retinal ATP in old mice via cytochrome c oxidase absorption and reduces inflammation. Here, we expose fruitflies daily to 670 nm radiation, revealing elevated ATP and reduced inflammation with age. Critically, there was a significant increase in average lifespan: 100-175% more flies survived into old age following 670 nm exposure and these had significantly improved mobility. This may be a simple route to extending lifespan and improving function in old age. PMID:25788488

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

    PubMed Central

    Galimov, Erik M.

    2009-01-01

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

  16. Fueling type III secretion

    PubMed Central

    Lee, Pei-Chung

    2015-01-01

    Type III secretion systems are complex nanomachines that export proteins from the bacterial cytoplasm across the cell envelope in a single step. They are at the core of the machinery used to assemble the bacterial flagellum, and the needle complex many Gram-negative pathogens use to inject effector proteins into host cells and cause disease. Several models have been put forward to explain how this export is energized, and the mechanism has been the subject of considerable debate. Here we present an overview of these models and discuss their relative merits. Recent evidence suggests that the proton motive force is the primary energy source for type III secretion, although contribution from refolding of secreted proteins has not been ruled out. The mechanism, by which the proton motive force is converted to protein export, remains enigmatic. PMID:25701111

  17. Fueling type III secretion.

    PubMed

    Lee, Pei-Chung; Rietsch, Arne

    2015-05-01

    Type III secretion systems (T3SSs) are complex nanomachines that export proteins from the bacterial cytoplasm across the cell envelope in a single step. They are at the core of the machinery used to assemble the bacterial flagellum, and the needle complex many Gram-negative pathogens use to inject effector proteins into host cells and cause disease. Several models have been put forward to explain how this export is energized, and the mechanism has been the subject of considerable debate. Here we present an overview of these models and discuss their relative merits. Recent evidence suggests that the proton motive force (pmf) is the primary energy source for type III secretion, although contribution from refolding of secreted proteins has not been ruled out. The mechanism by which the pmf is converted to protein export remains enigmatic. PMID:25701111

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

    SciTech Connect

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

    2005-07-21

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

  19. Phase III design: principles.

    PubMed

    Buyse, Marc

    2016-02-01

    Phase III clinical trials are the gold standard to demonstrate the effects of an experimental therapy compared to standard therapy for a disease of interest. The first step when planning a phase III trial is to specify the statistical hypothesis that the trial purports to test, which is usually that the experimental therapy provides some efficacy benefit over standard therapy, without adding significant harm. In a phase III trial, a pre-specified number of patients from the target population are randomized to receive experimental or standard therapy. The patients are treated and followed up according to a protocol that also defines the endpoints of interest, in particular the primary endpoint which is chosen to reflect a clinical benefit of experimental therapy over standard therapy. The trial data are typically monitored by an independent committee who may recommend stopping the trial early, if appropriate. The benefit of experimental therapy over standard therapy, if any, may be observed across all patients, or may be confined to a subset of patients. PMID:26932434

  20. Definition of "experimental procedures".

    PubMed

    2009-11-01

    This Practice Committee Opinion provides a revised definition of "experimental procedures." This version replaces the document "Definition of Experimental" that was published most recently in November 2008. PMID:19836733