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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 20072011 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. Inactivation and phosphorylation of sarcoplasmic reticulum Ca(2+)-ATPase by Mg.ATP analogues Rh(III)-ATP and Co(III)-ATP.

    PubMed

    Kuntzweiler, T A; Grisham, C M

    1992-05-15

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

  3. 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

  4. 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

  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. The modified NCEP ATP III criteria maybe better than the IDF criteria in diagnosing Metabolic Syndrome among Malays in Kuala Lumpur

    PubMed Central

    2010-01-01

    Background Metabolic Syndrome is associated with increased risk for type 2 diabetes and cardiovascular diseases. However, different diagnostic criteria have been recommended by different expert groups. In Malaysia, there is a lack of research comparing these different diagnostic criteria. Therefore, it is our aim to study the concordance between the IDF and the modified NCEP ATP III definitions of Metabolic Syndrome among a Malay cohort in Kuala Lumpur; and to demonstrate if all participants have the same cardiometabolic risks. Methods This was an analytical cross sectional study. Ethics approval was obtained and informed consent was given by all participants. Anthropometric measurements, blood pressure, fasting blood glucose and lipid profile were taken following standard protocols. Results Metabolic Syndrome was diagnosed in 41.4% and 38.2% participants using the modified NCEP and IDF criteria respectively. Among those diagnosed with Metabolic Syndrome by modified NCEP, 7.6% were missed by the IDF criteria. Participants diagnosed by the modified NCEP criteria had lower BMI and waist circumference but had higher cardiometabolic risks than those diagnosed with both criteria. Their blood pressure, glucose, total cholesterol and triglyceride were more adverse than the IDF group. This demonstrated that central obesity may not be a prerequisite for the development of increased cardiometabolic risks within this Malay cohort. Conclusion Metabolic syndrome is common in this Malay cohort regardless of the criterion used. The modified NCEP ATP III criteria may be more suitable in diagnosis of metabolic syndrome for this Malay cohort. PMID:21054885

  7. Inhibition of the Fe(III)-Catalyzed Dopamine Oxidation by ATP and Its Relevance to Oxidative Stress in Parkinsons Disease

    PubMed Central

    2013-01-01

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

  8. 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

  9. 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

  10. ATP synthase.

    PubMed

    Junge, Wolfgang; Nelson, Nathan

    2015-01-01

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

  11. Making ATP

    PubMed Central

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

    2005-01-01

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

  12. 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

  13. ATP regulation in bioproduction.

    PubMed

    Hara, Kiyotaka Y; Kondo, Akihiko

    2015-01-01

    Adenosine-5'-triphosphate (ATP) is consumed as a biological energy source by many intracellular reactions. Thus, the intracellular ATP supply is required to maintain cellular homeostasis. The dependence on the intracellular ATP supply is a critical factor in bioproduction by cell factories. Recent studies have shown that changing the ATP supply is critical for improving product yields. In this review, we summarize the recent challenges faced by researchers engaged in the development of engineered cell factories, including the maintenance of a large ATP supply and the production of cell factories. The strategies used to enhance ATP supply are categorized as follows: addition of energy substrates, controlling pH, metabolic engineering of ATP-generating or ATP-consuming pathways, and controlling reactions of the respiratory chain. An enhanced ATP supply generated using these strategies improves target production through increases in resource uptake, cell growth, biosynthesis, export of products, and tolerance to toxic compounds. PMID:26655598

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

    PubMed Central

    Moebus, Susanne; Hanisch, Jens Ulrich; Neuhuser, Markus; Aidelsburger, Pamela; Wasem, Jrgen; Jckel, Karl-Heinz

    2006-01-01

    Objective: Metabolic Syndrome (MetSyn) describes a cluster of metabolic disorders and is considered a risk factor for development of cardiovascular disease. Although a high prevalence is commonly assumed in Germany data about the degree of its occurrence in the population and in subgroups are still missing. The aim of this study was to assess the prevalence of the MetSyn according to the NCEP ATP-III (National Cholesterol Education Program Adult Treatment Panel III) criteria in persons aged ?18 years attending a general practitioner in Germany. Here we describe in detail the methods used and the feasibility of determining the MetSyn in a primary health care setting. Research design and methods: The German-wide cross-sectional study was performed during two weeks in October 2005. Blood samples were analyzed in a central laboratory. Waist circumference and blood pressure were assessed, data on smoking, life style, fasting status, socio-demographic characteristics and core information from non-participants collected. Quality control procedures included telephone-monitoring and random on-site visits. In order to achieve a maximal number of fasting blood samples with a minimal need for follow-up appointments a stepwise approach was developed. Basic descriptive statistics were calculated, the Taylor expansion method used to estimate standard errors needed for calculation of confidence intervals for clustered observations. Results: In total, 1511 randomly selected general practices from 397 out of 438 German cities and administrative districts enrolled 35,869 patients (age range: 18-99, women 61.1%). More than 50,000 blood samples were taken. Fasting blood samples were available for 49% of the participants. Of the participating patients 99.3% returned questionnaires to the GP, only 12% were not filled out completely. The overall prevalence of the MetSyn (NCEP/ATP III 2001) was found to be 19.8%, with men showing higher prevalence rates than women (22.7% respective 18.0%). Conclusions: This study was designed to provide data as robust as possible within the confines of an epidemiological study. Judging by the low degree of missing data and the high data quality, the feasibility for this kind of a research setting (short evaluation period, practitioners as data assessment sites) was found to be very good. The results will help to gain a more comprehensive insight into the prevalence of MetSyn for patients in primary health care in Germany. PMID:19675698

  15. 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

  16. Definitive Chemoradiotherapy Versus Surgery Followed by Adjuvant Radiotherapy in Resectable Stage III/IV Hypopharyngeal Cancer

    PubMed Central

    Kim, Jun Won; Kim, Mi Sun; Kim, Se-Heon; Kim, Joo Hang; Lee, Chang Geol; Kim, Gwi Eon; Keum, Ki Chang

    2016-01-01

    Purpose The purpose of this study is to compare the treatment outcomes for locally advanced resectable hypopharyngeal cancer between organ-preserving chemoradiotherapy (CRT) and surgery followed by radiotherapy (SRT). Materials and Methods We reviewed 91 patients with stage III/IV hypopharyngeal squamous cell carcinoma treated with radiotherapy (RT). In the CRT group (n=34), 18 patients were treated with concurrent CRT and 16 patients with induction chemotherapy plus concurrent CRT. In the SRT group (n=57), six patients were treated with total laryngopharyngectomy, 34 patients with total laryngectomy (TL) and partial pharyngectomy (PP), and 17 patients with PP, which were followed by adjuvant radiotherapy (n=41) or CRT (n=16). The median RT dose was 70 Gy for CRT and 59.4 Gy for SRT. Results Five-year local control (84.1% vs. 90.9%), and disease-free survival (DFS, 51.0% vs. 52.7%) and overall survival (OS, 58.6% vs. 56.6%) showed no significant difference between the CRT and SRT groups. The functional larynx-preservation rate was higher in the CRT group (88.2% vs. 29.8%). Treatment-related toxicity, requiring surgical intervention, occurred more frequently in the SRT group (37% vs. 12%). In the SRT group, TL resulted in a significantly higher DFS than larynx-sparing surgery (63.9% vs. 26.5%, p=0.027). Treatment outcome of the SRT group improved when only patients with TL were considered (n=40); however, 5-year OS (67.1% vs. 58.6%, p=0.830) and DFS (63.9% vs. 51.0%, p=0.490) did not improve significantly when compared to the CRT group. Conclusion Organ preserving CRT provided a treatment outcome that is comparable to SRT for locally advanced hypopharyngeal cancer, while offering an opportunity for functional larynx-preservation and reduced treatment-related toxicity. PMID:25779363

  17. Customized ATP towpreg

    NASA Astrophysics Data System (ADS)

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

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

  18. 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

  19. Feasibility of endoscopic mucosal resection as salvage treatment for patients with local failure after definitive chemoradiotherapy for stage IB, II, and III esophageal squamous cell cancer.

    PubMed

    Makazu, M; Kato, K; Takisawa, H; Yoshinaga, S; Oda, I; Saito, Y; Mayahara, H; Ito, Y; Itami, J; Hamaguchi, T; Yamada, Y; Shimada, Y

    2014-01-01

    Local failure after definitive chemoradiotherapy (CRT) for stage IB, II, and III esophageal cancer is one of the causes of poor outcome. Endoscopic mucosal resection (EMR) is an effective treatment for superficial esophageal cancer. However, its feasibility as a salvage treatment for local recurrent or residual tumors after definitive CRT for stage IB, II, and III esophageal cancer remains unclear. Between January 2000 and February 2008, 274 patients with stage IB, II, and III esophageal squamous cell cancer excluding T4 received definitive CRT at the National Cancer Center Hospital, Japan. Of these patients, nine patients with local recurrence after achieving complete response and two patients with residual tumor underwent salvage EMR. The technique of salvage EMR involved a strip biopsy method. We retrospectively reviewed the 11 patients (13 lesions). Characteristics of all 11 patients were as follows: median age of 69 (range: 45-78); male/female: 10/1; baseline clinical stage (Union for International Cancer Control 7th) IB/IIA/IIB/III: 1/3/7/0. The depth of resected tumor was limited to the mucosal layer in seven lesions and submucosal in six lesions. En bloc resection was performed on six lesions (46%). The vertical margin was free of cancer cells in 11 lesions (84.6%). No major complications, such as hemorrhage requiring blood transfusion and perforation, were experienced. At a median follow-up period of 38.9 months (range: 5.3-94 months) after salvage EMR, no recurrence was detected in six patients (54%). Local recurrence was detected in five patients (27%). Of these patients, two had lung metastasis simultaneously, and one was also detected lung metastasis 2 months after the detection of local recurrence. The 5-year survival rate after salvage EMR was 41.6%. Salvage EMR is a feasible treatment option for local recurrent or residual lesions after definitive chemotherapy and/or radiotherapy for stage IB, II, and III esophageal squamous cell cancer. PMID:23442160

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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.

  5. ATP release via anion channels.

    PubMed

    Sabirov, Ravshan Z; Okada, Yasunobu

    2005-12-01

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

  6. Microglia release ATP by exocytosis.

    PubMed

    Imura, Yoshio; Morizawa, Yosuke; Komatsu, Ryohei; Shibata, Keisuke; Shinozaki, Youichi; Kasai, Hirotake; Moriishi, Kohji; Moriyama, Yoshinori; Koizumi, Schuichi

    2013-08-01

    Microglia survey the brain environment by sensing several types of diffusible molecules, among which extracellular nucleotides released/leaked from damaged cells have central roles. Microglia sense ATP or other nucleotides by multiple P2 receptors, after which they change into several different phenotypes. However, so far, it is largely unknown whether microglia themselves release ATP and, if so, by what mechanism. Here we show that exocytosis is the mechanism by which microglia release ATP. When we stimulated microglia with ionomycin, they released ATP and the release was dependent on Ca?, vesicular H?-ATPase, or SNAREs but independent of connexin/pannexin hemichannels. VNUT was found to be expressed in microglia and exhibited no colocalization with lysosome. We also visualized the exocytosis of ATP by a quinacrine-based fluorescent time-lapse imaging. Moreover, we found that lipopolysaccharide increased the ionomycin-induced release of ATP, which was dependent on the increase in VNUT. Taken together, our data suggested that exocytosis is the mechanism of ATP release from microglia. When activated, they would release ATP by increasing VNUT-dependent exocytotic mechanisms. PMID:23832620

  7. 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

  8. 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.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

    Vang, luva K; Corfitzen, Charlotte B; Smith, Christian; Albrechtsen, Hans-Jrgen

    2014-11-01

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

  11. Genetics Home Reference: Mucolipidosis III gamma

    MedlinePLUS

    ... literature OMIM Genetic disorder catalog Conditions > Mucolipidosis III gamma On this page: Description Genetic changes Inheritance Diagnosis ... definitions Reviewed May 2015 What is mucolipidosis III gamma? Mucolipidosis III gamma is a slowly progressive disorder ...

  12. 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 ?20years) 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

  13. 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

  14. 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.

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

    PubMed Central

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

    2012-01-01

    Purpose The purpose of this study was to estimate the presence of metabolic syndrome (MS) in a group of children and adolescents with a body mass index (BMI) above the 85th percentile for their age and sex in Qazvin Province, Iran; to evaluate the relationship between obesity and metabolic abnormalities; and to compare two proposed definitions of MS. Patients and methods The study was conducted on 100 healthy subjects aged between 6 and 16 years (average age, 10.52 2.51 years) with a high BMI for their age and sex. Fifty- eight percent of subjects were female. Physical examination including evaluation of weight, height, BMI, and blood pressure measurement was performed (overweight was defined as a BMI between the 85th and 95th percentiles for children of the same age and sex; obese was defined as a BMI over the 95th percentile for children of the same age and sex). Blood levels of glucose, insulin, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, and uric acid were measured after a 12-hour overnight fast. The authors used and compared two definitions of MS: the National Cholesterol Education Programs Adult Treatment Panel III (NCEP ATP III) criteria and a modified definition by Weiss et al. Variables were compared using the Students t-test and chi-square and Mann-Whitney U tests, and agreement between the two definitions was analyzed using kappa values. Results The subjects had a mean BMI of 26.02 4.38 and 80% had obesity. Insulin resistance was found in 81% of the study population. MS was present in ten (50%) of the overweight and 53 (66.2%) of the obese subjects using the NCEP ATP III criteria. MS was present in five (25%) of the overweight and 34 (42.5%) of the obese subjects using the definition by Weiss et al. The overall kappa value for the two definitions of MS was 0.533. There were no statistically significant differences between the two definitions of MS in participants. Conclusion The prevalence of MS in children and adolescents depends on the criteria chosen and their respective cutoff points. The NCEP ATP III criteria, the parameters of which include higher cutoff values for high-density lipoprotein cholesterol and triglycerides, detected the higher prevalence and therefore the NCEP ATP III criteria are able to diagnose a larger number of children and adolescents at metabolic risk. PMID:22346358

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

    PubMed

    Igamberdiev, Abir U; Kleczkowski, Leszek A

    2015-01-01

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

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

    PubMed Central

    Igamberdiev, Abir U.; Kleczkowski, Leszek A.

    2015-01-01

    The bulk of ATP synthesis in plants is performed by ATP synthase, the main bioenergetics engine of cells, operating both in mitochondria and in chloroplasts. The reaction mechanism of ATP synthase has been studied in detail for over half a century; however, its optimal performance depends also on the steady delivery of ATP synthase substrates and the removal of its products. For mitochondrial ATP synthase, we analyze here the provision of stable conditions for (i) the supply of ADP and Mg2+, supported by adenylate kinase (AK) equilibrium in the intermembrane space, (ii) the supply of phosphate via membrane transporter in symport with H+, and (iii) the conditions of outflow of ATP by adenylate transporter carrying out the exchange of free adenylates. We also show that, in chloroplasts, AK equilibrates adenylates and governs Mg2+ contents in the stroma, optimizing ATP synthase and Calvin cycle operation, and affecting the import of inorganic phosphate in exchange with triose phosphates. It is argued that chemiosmosis is not the sole component of ATP synthase performance, which also depends on AK-mediated equilibrium of adenylates and Mg2+, adenylate transport, and phosphate release and supply. PMID:25674099

  18. Assaying ATP Synthase Rotor Activity

    NASA Astrophysics Data System (ADS)

    Maguire, D.; Shah, J.; McCabe, M.

    With the development of laser excitation and detection systems, it has become possible to consider analyses that have otherwise been beyond the capacity of experimentalists. In this investigation, a case is made for the development of a device to analyse ATP synthase activity in-vivo. Such analyses ultimately have applications in clinical practice, particularly in the field of mitochondrial disorders, of both nuclear and mitochondrial DNA origin.

  19. 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

  20. 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

  1. 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

  2. Assembly of F1F0-ATP synthases.

    PubMed

    Rhle, Thilo; Leister, Dario

    2015-09-01

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

  3. 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??M in MDA-MB-231 cells, a 3.6-fold increase in the cytotoxicity compared with that of non-ATP-responsive nanovehicles. Equipped with an outer shell crosslinked by hyaluronic acid, a specific tumour-targeting ligand, the ATP-responsive nanocarriers present an improvement in the chemotherapeutic inhibition of tumour growth using xenograft MDA-MB-231 tumour-bearing mice. This ATP-triggered drug release system provides a more sophisticated drug delivery system, which can differentiate ATP levels to facilitate the selective release of drugs.

  4. 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.

  5. ATP-induced protein-Hsp70 complex dissociation requires K+ but not ATP hydrolysis.

    PubMed

    Palleros, D R; Reid, K L; Shi, L; Welch, W J; Fink, A L

    1993-10-14

    The molecular chaperone proteins, particularly Hsp60 and Hsp70, have been implicated in essential cell functions under both normal and stress conditions (reviewed in refs 1-5). Members of the family of heat-shock proteins of M(r) 70K, Hsp70, bind to unfolded proteins and short peptides. Addition of Mg-ATP results in the dissociation of the substrate polypeptides from the chaperone, but as ATP-gamma S (an ATP analogue that is only slowly hydrolysable) cannot substitute for ATP in this reaction, it has been concluded that ATP hydrolysis is necessary to dissociate Hsp70-substrate protein complexes. By independently measuring the rates of ATP hydrolysis and substrate protein dissociation, we show here that Mg-ATP binding but not Mg-ATP hydrolysis is essential for substrate dissociation. We also show that there is an absolute requirement for K+ for the effect of Mg-ATP: only the combination of K+ and Mg-ATP will cause the conformational change in Hsp70 that is necessary for substrate dissociation. Moreover, in the absence of K+, Mg-ATP favours complex formation. We consider these results in terms of a G-protein-like model. PMID:8413631

  6. 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

  7. 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

  8. 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

  9. 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

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

    PubMed

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

    2011-12-16

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

  11. 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.

  12. 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.

  13. 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.

  14. Mussel and mammalian ATP synthase share the same bioenergetic cost of ATP.

    PubMed

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

    2013-06-01

    The molecular mechanism by which the membrane-embedded FO sector of the mitochondrial ATP synthase translocates protons, thus dissipating the transmembrane protonmotive force and leading to ATP synthesis, involves the neutralization of the carboxylate residues of the c-ring. Carboxylates are thought to constitute the binding sites for ion translocation. In order to cast light on this mechanism, we exploited N,N'-dicyclohexylcarbodiimide, which covalently binds to FO c-ring carboxylates, and ionophores which selectively modulate the transmembrane electric (??) and chemical (?pH) gradients such as valinomycin, nigericin and dinitrophenol. ATP hydrolysis was evaluated in mitochondrial preparations and/or inside-out submitochondrial particles from mussel and mammalian tissues under different experimental conditions. The experiments pointed out striking similarities between mussel and mammalian mitochondrial ATP synthase. Our results support the hypothesis that the ATP synthase of Mytilus galloprovincialis induces intersubunit torque generation and translocates H(+) by coordinating the hydronium ion (H3O(+)) in the ion binding site of FO. Our results are consistent with the hypothesis that in mussel mitochondria the main component of the electrochemical gradient driving proton flux and ATP synthesis is ??. Therefore, mussel FO probably contains a small c-ring, which implies a low bioenergetic cost of making ATP as in mammals. These features which make mussel mitochondria as efficient in ATP production as mammalian ones may be especially advantageous in facultative aerobic species which intermittently exploit mitochondrial respiration to generate ATP. PMID:23456170

  15. 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

  16. Cell-free expression and assembly of ATP synthase.

    PubMed

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

    2011-10-28

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

  17. 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.

  18. 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

  19. 28 CFR 35.104 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... such impairment; or (iii) Has none of the impairments defined in paragraph (1) of this definition but...— (i) Transvestism, transsexualism, pedophilia, exhibitionism, voyeurism, gender identity disorders...

  20. 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 hosts 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...

  1. SAGE III

    Atmospheric Science Data Center

    2014-12-04

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

  2. 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

  3. Electron Spin Resonance in ATP and RNA

    PubMed Central

    Maling, J. E.; Taskovich, L. T.; Blois, M. S.

    1963-01-01

    Cu++, Mn++, and Fe+++ account for the electron spin resonances observed in certain samples of ATP and RNA. The copper ion seems more loosely bound to these substances than either iron or manganese. A striking similarity is observed between the manganese spectra in manganese RNA, ATP, and ADP suggesting that the binding sites are similar in the three compounds. The similarity of the e.s.r. spectra of iron ATP and of iron and manganese RNA, except for hyperfine spectrum (hfs) in the latter, suggests that the two ions bind similarly in the two compounds. A detailed interpretation of the spectra is lacking however and these conclusions can only be tentative. When manganese TPP and ATP are heated or pH changed the e.s.r. alters indicating a change in the environment of the ion. The sharp 6 line manganese spectrum in both TPP and ATP at pH 1 suggests an almost free ion at this pH in the sense of an almost isotropic average environment. PMID:13932528

  4. 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

  5. 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.

  6. 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 ?mol/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.

  7. 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

  8. 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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-06-01

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

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

    SciTech Connect

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

    1988-07-12

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

  12. 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.

  13. 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

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

  15. The molecular mechanism of ATP synthesis by F1F0-ATP synthase.

    PubMed

    Senior, Alan E; Nadanaciva, Sashi; Weber, Joachim

    2002-02-15

    ATP synthesis by oxidative phosphorylation and photophosphorylation, catalyzed by F1F0-ATP synthase, is the fundamental means of cell energy production. Earlier mutagenesis studies had gone some way to describing the mechanism. More recently, several X-ray structures at atomic resolution have pictured the catalytic sites, and real-time video recordings of subunit rotation have left no doubt of the nature of energy coupling between the transmembrane proton gradient and the catalytic sites in this extraordinary molecular motor. Nonetheless, the molecular events that are required to accomplish the chemical synthesis of ATP remain undefined. In this review we summarize current state of knowledge and present a hypothesis for the molecular mechanism of ATP synthesis. PMID:11997128

  16. ACRIM III

    Atmospheric Science Data Center

    2015-12-30

    ACRIM III Data and Information Active Cavity Radiometer Irradiance ... the ACRIMSAT spacecraft on December 20, 1999. ACRIM III data are reprocessed every 90 days to utilize instrument recalibration. ... stopped communicating in December 2013. A reprocessed data set spanning April 5 2000 to March 5 2013 was uploaded in May 2013. Data ...

  17. Dynamic imaging of free cytosolic ATP concentration during fuel sensing by rat hypothalamic neurones: evidence for ATP-independent control of ATP-sensitive K+ channels

    PubMed Central

    Ainscow, Edward K; Mirshamsi, Shirin; Tang, Teresa; Ashford, Michael L J; Rutter, Guy A

    2002-01-01

    Glucose-responsive (GR) neurons from hypothalamic nuclei are implicated in the regulation of feeding and satiety. To determine the role of intracellular ATP in the closure of ATP-sensitive K+ (KATP) channels in these cells and associated glia, the cytosolic ATP concentration ([ATP]c) was monitored in vivo using adenoviral-driven expression of recombinant targeted luciferases and bioluminescence imaging. Arguing against a role for ATP in the closure of KATP channels in GR neurons, glucose (3 or 15 mm) caused no detectable increase in [ATP]c, monitored with cytosolic luciferase, and only a small decrease in the concentration of ATP immediately beneath the plasma membrane, monitored with a SNAP25luciferase fusion protein. In contrast to hypothalamic neurons, hypothalamic glia responded to glucose (3 and 15 mm) with a significant increase in [ATP]c. Both neurons and glia from the cerebellum, a glucose-unresponsive region of the brain, responded robustly to 3 or 15 mm glucose with increases in [ATP]c. Further implicating an ATP-independent mechanism of KATP channel closure in hypothalamic neurons, removal of extracellular glucose (10 mm) suppressed the electrical activity of GR neurons in the presence of a fixed, high concentration (3 mm) of intracellular ATP. Neurons from both brain regions responded to 5 mm lactate (but not pyruvate) with an oligomycin-sensitive increase in [ATP]c. High levels of the plasma membrane lactate-monocarboxylate transporter, MCT1, were found in both cell types, and exogenous lactate efficiently closed KATP channels in GR neurons. These data suggest that (1) ATP-independent intracellular signalling mechanisms lead to the stimulation of hypothalamic neurons by glucose, and (2) these effects may be potentiated in vivo by the release of lactate from neighbouring glial cells. PMID:12381816

  18. Genomic Analysis of ATP Efflux in Saccharomyces cerevisiae.

    PubMed

    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

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

    PubMed

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

    2005-04-01

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

  20. Genomic Analysis of ATP Efflux in Saccharomyces cerevisiae

    PubMed Central

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

    2015-01-01

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

  1. Polynucleotide phosphorylase and mitochondrial ATP synthase mediate reduction of arsenate to the more toxic arsenite by forming arsenylated analogues of ADP and ATP.

    PubMed

    Nmeti, Balzs; Regonesi, Maria Elena; Tortora, Paolo; Gregus, Zoltn

    2010-10-01

    We have demonstrated that phosphorolytic-arsenolytic enzymes can promote reduction of arsenate (AsV) into the more toxic arsenite (AsIII) because they convert AsV into an arsenylated product in which the arsenic is more reducible by glutathione (GSH) or other thiols to AsIII than in inorganic AsV. We have also shown that mitochondria can rapidly reduce AsV in a process requiring intact oxidative phosphorylation and intramitochondrial GSH. Thus, these organelles might reduce AsV because mitochondrial ATP synthase, using AsV instead of phosphate, arsenylates ADP to ADP-AsV, which in turn is readily reduced by GSH. To test this hypothesis, we first examined whether the RNA-cleaving enzyme polynucleotide phosphorylase (PNPase), which can split poly-adenylate (poly-A) by arsenolysis into units of AMP-AsV (a homologue of ADP-AsV), could also promote reduction of AsV to AsIII in presence of thiols. Indeed, bacterial PNPase markedly facilitated formation of AsIII when incubated with poly-A, AsV, and GSH. PNPase-mediated AsV reduction depended on arsenolysis of poly-A and presence of a thiol. PNPase can also form AMP-AsV from ADP and AsV (termed arsenolysis of ADP). In presence of GSH, this reaction also facilitated AsV reduction in proportion to AMP-AsV production. Although various thiols did not influence the arsenolytic yield of AMP-AsV, they differentially promoted the PNPase-mediated reduction of AsV, with GSH being the most effective. Circumstantial evidence indicated that AMP-AsV formed by PNPase is more reducible to AsIII by GSH than inorganic AsV. Then, we demonstrated that AsV reduction by isolated mitochondria was markedly inhibited by an ADP analogue that enters mitochondria but is not phosphorylated or arsenylated. Furthermore, inhibitors of the export of ATP or ADP-AsV from the mitochondria diminished the increment in AsV reduction caused by adding GSH externally to these organelles whose intramitochondrial GSH had been depleted. Thus, whereas PNPase promotes reduction of AsV by incorporating it into AMP-AsV, the mitochondrial ATP synthase facilitates AsV reduction by forming ADP-AsV; then GSH can easily reduce these arsenylated nucleotides to AsIII. PMID:20457661

  2. 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

  3. Space shuttle (ATP configuration) abort staging investigation

    NASA Technical Reports Server (NTRS)

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

    1973-01-01

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

  4. The catalytic transition state in ATP synthase.

    PubMed

    Senior, A E; Weber, J; Nadanaciva, S

    2000-10-01

    The catalytic transition state of ATP synthase has been characterized and modeled by combined use of (1) Mg-ADP-fluoroaluminate, Mg-ADP-fluoroscandium, and corresponding Mg-IDP-fluorometals as transition-state analogs; (2) fluorescence signals of beta-Trp331 and beta-Trp148 as optical probes to assess formation of the transition state; (3) mutations of critical catalytic residues to determine side-chain ligands required to stabilize the transition state. Rate acceleration by positive catalytic site cooperativity is explained as due to mobility of alpha-Arg376, acting as an "arginine finger" residue, which interacts with nucleotide specifically at the transition state step of catalysis, not with Mg-ATP- or Mg-ADP-bound ground states. We speculate that formation and collapse of the transition state may engender catalytic site alpha/beta subunit-interface conformational movement, which is linked to gamma-subunit rotation. PMID:15254388

  5. Loss of LRPPRC causes ATP synthase deficiency.

    PubMed

    Mourier, Arnaud; Ruzzenente, Benedetta; Brandt, Tobias; Khlbrandt, Werner; Larsson, Nils-Gran

    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

  6. 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

  7. 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.

  8. 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 ?-subunit, we have developed highly reproducible, simple procedures for the preparation of active proteoliposomes and for kinetic analysis of ATP synthesis, which was driven by acid-base transition and K(+)-diffusion potential. The synthesis activity reached ? 16 s(-1) at 30 C with a Q(10) temperature coefficient of 3-4 between 10 and 30 C, suggesting a high level of activity at the physiological temperature of ? 60 C. The Michaelis-Menten constants for the substrates ADP and inorganic phosphate were 13 ?M and 0.55 mM, respectively, which are an order of magnitude lower than previous estimates and are suited to efficient ATP synthesis. PMID:21605343

  9. 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

  10. Rate acceleration of ATP hydrolysis by F(1)F(o)-ATP synthase.

    PubMed

    Senior, A E; Nadanaciva, S; Weber, J

    2000-01-01

    The rate acceleration of ATP hydrolysis by F(1)F(o)-ATP synthase is of the order of 10(11)-fold. We present a cyclic enzyme mechanism for the reaction, relate it to known F(1) X-ray structure and speculate on the linkage between enzyme reaction intermediates and subunit rotation. Next, we describe five factors known to be important in the Escherichia coli enzyme for the rate acceleration. First, the provision of substrate binding energy by residues lining the catalytic site is substantial; beta-Lys155 and beta-Arg182 are specific examples, both of which differentially support substrate MgATP versus product MgADP binding. Second, octahedral coordination of the Mg(2+) in MgATP is crucial for both catalysis and catalytic site asymmetry. The residues involved are beta-Thr156, beta-Glu185 and beta-Asp242. Third, there is stabilization of a pentacoordinate phosphorus catalytic transition state by residues beta-Lys155, beta-Arg182 and alpha-Arg376. Fourth, residue beta-Glu181 binds the substrate water and stabilizes the catalytic transition state. Fifth, there is strong positive catalytic cooperativity, with binding of MgATP at all three sites yielding the maximum rate (V(max)); the molecular basis of this factor remains to be elucidated. PMID:10600671

  11. 12 CFR 212.2 - 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 225.71(c); (iv) A... 12 Banks and Banking 2 2010-01-01 2010-01-01 false Definitions. 212.2 Section 212.2 Banks and... INTERLOCKS § 212.2 Definitions. For purposes of this part, the following definitions apply: (a) Affiliate....

  12. 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

  13. ATP Promotes the Extracellular Matrix Biosynthesis of Intervertebral Disc Cells

    PubMed Central

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

    2014-01-01

    A recent study by our lab found 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 may regulate biological activities in the IVD. Therefore, the objective of this study was to investigate the effects of extracellular ATP on the extracellular matrix (ECM) biosynthesis of porcine IVD cells isolated from two distinct anatomical regions: annulus fibrosus (AF) and nucleus pulposus (NP). The ATP treatment significantly promoted the ECM deposition and corresponding gene expression (aggrecan and type II collagen) by both cell types in 3-dimensional agarose culture. A significant increase in ECM accumulation was found in AF cells at a lower ATP treatment level (20 M) compared to NP cells (100 M), indicating that AF cells may be more sensitive to extracellular ATP than NP cells. NP cells also exhibited higher ECM accumulation and intracellular ATP than AF cells under Control and treatment conditions, suggesting that NP cells are intrinsically more metabolically active. Moreover, the ATP treatment also augmented the intracellular ATP level in NP and AF cells. Our findings suggest that extracellular ATP not only promotes ECM biosynthesis via molecular pathway but also increases energy supply to fuel that process. PMID:25407524

  14. 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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed Central

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

    1998-01-01

    Heat shock protein 90 (Hsp90), an abundant molecular chaperone in the eukaryotic cytosol, is involved in the folding of a set of cell regulatory proteins and in the re-folding of stress-denatured polypeptides. The basic mechanism of action of Hsp90 is not yet understood. In particular, it has been debated whether Hsp90 function is ATP dependent. A recent crystal structure of the NH2-terminal domain of yeast Hsp90 established the presence of a conserved nucleotide binding site that is identical with the binding site of geldanamycin, a specific inhibitor of Hsp90. The functional significance of nucleotide binding by Hsp90 has remained unclear. Here we present evidence for a slow but clearly detectable ATPase activity in purified Hsp90. Based on a new crystal structure of the NH2-terminal domain of human Hsp90 with bound ADP-Mg and on the structural homology of this domain with the ATPase domain of Escherichia coli DNA gyrase, the residues of Hsp90 critical in ATP binding (D93) and ATP hydrolysis (E47) were identified. The corresponding mutations were made in the yeast Hsp90 homologue, Hsp82, and tested for their ability to functionally replace wild-type Hsp82. Our results show that both ATP binding and hydrolysis are required for Hsp82 function in vivo. The mutant Hsp90 proteins tested are defective in the binding and ATP hydrolysisdependent cycling of the co-chaperone p23, which is thought to regulate the binding and release of substrate polypeptide from Hsp90. Remarkably, the complete Hsp90 protein is required for ATPase activity and for the interaction with p23, suggesting an intricate allosteric communication between the domains of the Hsp90 dimer. Our results establish Hsp90 as an ATP-dependent chaperone. PMID:9817749

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

    PubMed Central

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

    2012-01-01

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

  18. 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.

  19. Coupled ATP and potassium efflux from intercalated cells

    PubMed Central

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

    2011-01-01

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

  20. The role of mitochondrially derived ATP in synaptic vesicle recycling.

    PubMed

    Pathak, Divya; Shields, Lauren Y; Mendelsohn, Bryce A; Haddad, Dominik; Lin, Wei; Gerencser, Akos A; Kim, Hwajin; Brand, Martin D; Edwards, Robert H; Nakamura, Ken

    2015-09-11

    Synaptic mitochondria are thought to be critical in supporting neuronal energy requirements at the synapse, and bioenergetic failure at the synapse may impair neural transmission and contribute to neurodegeneration. However, little is known about the energy requirements of synaptic vesicle release or whether these energy requirements go unmet in disease, primarily due to a lack of appropriate tools and sensitive assays. To determine the dependence of synaptic vesicle cycling on mitochondrially derived ATP levels, we developed two complementary assays sensitive to mitochondrially derived ATP in individual, living hippocampal boutons. The first is a functional assay for mitochondrially derived ATP that uses the extent of synaptic vesicle cycling as a surrogate for ATP level. The second uses ATP FRET sensors to directly measure ATP at the synapse. Using these assays, we show that endocytosis has high ATP requirements and that vesicle reacidification and exocytosis require comparatively little energy. We then show that to meet these energy needs, mitochondrially derived ATP is rapidly dispersed in axons, thereby maintaining near normal levels of ATP even in boutons lacking mitochondria. As a result, the capacity for synaptic vesicle cycling is similar in boutons without mitochondria as in those with mitochondria. Finally, we show that loss of a key respiratory subunit implicated in Leigh disease markedly decreases mitochondrially derived ATP levels in axons, thus inhibiting synaptic vesicle cycling. This proves that mitochondria-based energy failure can occur and be detected in individual neurons that have a genetic mitochondrial defect. PMID:26126824

  1. Bioluminescence detection of ATP release mechanisms in epithelia.

    PubMed

    Taylor, A L; Kudlow, B A; Marrs, K L; Gruenert, D C; Guggino, W B; Schwiebert, E M

    1998-11-01

    Autocrine and paracrine release of and extracellular signaling by ATP is a ubiquitous cell biological and physiological process. Despite this knowledge, the mechanisms and physiological roles of cellular ATP release are unknown. We tested the hypothesis that epithelia release ATP under basal and stimulated conditions by using a newly designed and highly sensitive assay for bioluminescence detection of ATP released from polarized epithelial monolayers. This bioluminescence assay measures ATP released from cystic fibrosis (CF) and non-CF human epithelial monolayers in a reduced serum medium through catalysis of the luciferase-luciferin reaction, yielding a photon of light collected by a luminometer. This novel assay measures ATP released into the apical or basolateral medium surrounding epithelia. Of relevance to CF, CF epithelia fail to release ATP across the apical membrane under basal conditions. Moreover, hypotonicity is an extracellular signal that stimulates ATP release into both compartments of non-CF epithelia in a reversible manner; the response to hypotonicity is also lost in CF epithelia. The bioluminescence detection assay for ATP released from epithelia and other cells will be useful in the study of extracellular nucleotide signaling in physiological and pathophysiological paradigms. Taken together, these results suggest that extracellular ATP may be a constant regulator of epithelial cell function under basal conditions and an autocrine regulator of cell volume under hypotonic conditions, two functions that may be lost in CF and contribute to CF pathophysiology. PMID:9814989

  2. Kinetic mechanism of the dimeric ATP sulfurylase from plants

    PubMed Central

    Ravilious, GeoffreyE.; Herrmann, Jonathan; Goo Lee, Soon; Westfall, CoreyS.; Jez, JosephM.

    2013-01-01

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

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

    PubMed

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

    2013-01-01

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

  4. 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

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

    PubMed

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

    1983-09-01

    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

  6. ATP-induced reverse temperature effect in isohemoglobins from the endothermic porbeagle shark (Lamna nasus).

    PubMed

    Larsen, Christina; Malte, Hans; Weber, Roy E

    2003-08-15

    The evolutionary convergence of endothermic tunas and lamnid sharks is unique. Their heat exchanger-mediated endothermy represents an interesting example of the evolutionary pressure associated with this specific characteristic. To assess the implications of endothermy for gas transport and the possible contribution of hemoglobin (Hb), we investigated the effect of temperature on the oxygen equilibria of purified isohemoglobin components V and III from the porbeagle shark (Lamna nasus). In the absence of ATP the effect of temperature on oxygen affinity is normal in both Hb III (P50 = 0.9 and 2.2 torr at 10 and 26 degrees C, respectively) and Hb V (P50 = 1.5 and 2.5 torr at 10 and 26 degrees C, respectively). In the presence of this effector P50 decreases with increasing temperature in both components (P50 at 10 and 26 degrees C = 9.9 and 8.4 torr (Hb III), respectively, and 9.6 and 7.4 torr (Hb V), respectively. The reverse temperature effect in the presence of ATP will reduce the risk of oxygen loss from the arterial to the venous blood by lowering the oxygen tension gradient between the blood vessels. The mechanism behind the reverse temperature effect resembles that found in the bluefin tuna (Thunnus thynnus), an endothermic teleost, thus evidencing further convergent evolution. PMID:12773532

  7. 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

  8. 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

  9. 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.

  10. 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, zkan; 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 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

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

    PubMed

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

    2015-02-01

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

  12. A New Metal-Binding Site for Yeast Phosphoglycerate Kinase as Determined by the Use of a Metal-ATP Analog

    PubMed Central

    Pappu, Kameshwari M.; Kunnumal, Baburaj; Serpersu, Engin H.

    1997-01-01

    Suicide substrate ?, ?-bidentate Rh(III)ATP (RhATP) was used to map the metal ion-binding site in yeast phosphoglycerate kinase (PGK). Cleavage of the RhATP-inactivated enzyme with pepsin and subsequent separation of peptides by reverse-phase high-performance liquid chromatography gave two Rh-nucleotide bound peptides. One of the peptides corresponded to the C-terminal residues of PGK, and the other to a part of helix V. Of the four glutamates present in the C-terminal peptide, Glu 398 may be a likely metal coordination site. Therefore, importance of the C-terminal residues in PGK catalysis may be attributed, in part, to the coordination of metal ion of the metal-ATP substrate. Metal coordination may then align the C-terminal peptide to extend toward the N-terminal domain and form the closed active site. Results presented in this paper suggest that one or more side chains of the enzyme may be coordinated to the metal ion in the PGK3-phospho-D-glycerateRhATP complex, and that exchange-inert metal-ATP analogs could be used to determine metal coordination sites on kinases and other metal-ATP-utilizing enzymes. ImagesFIGURE 6 PMID:9017217

  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. 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

  15. 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.

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

    NASA Astrophysics Data System (ADS)

    Toyabe, Shoichi; Muneyuki, Eiro

    2015-01-01

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

  17. 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

  18. 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...

  19. 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…

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

    PubMed

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

    2015-12-01

    Extracellular adenosine triphosphate (ATP) regulates pancreatic duct function via P2Y and P2X receptors. It is well known that ATP is released from upstream pancreatic acinar cells. The ATP homeostasis in pancreatic ducts, which secrete bicarbonate-rich fluid, has not yet been examined. First, our aim was to reveal whether pancreatic duct cells release ATP locally and whether they enzymatically modify extracellular nucleotides/sides. Second, we wished to explore which physiological and pathophysiological factors may be important in these processes. Using a human pancreatic duct cell line, Capan-1, and online luminescence measurement, we detected fast ATP release in response to pH changes, bile acid, mechanical stress and hypo-osmotic stress. ATP release following hypo-osmotic stress was sensitive to drugs affecting exocytosis, pannexin-1, connexins, maxi-anion channels and transient receptor potential cation channel subfamily V member 4 (TRPV4) channels, and corresponding transcripts were expressed in duct cells. Direct stimulation of intracellular Ca(2+) and cAMP signalling and ethanol application had negligible effects on ATP release. The released ATP was sequentially dephosphorylated through ecto-nucleoside triphosphate diphosphohydrolase (NTPDase2) and ecto-5'-nucleotidase/CD73 reactions, with respective generation of adenosine diphosphate (ADP) and adenosine and their maintenance in the extracellular medium at basal levels. In addition, Capan-1 cells express counteracting adenylate kinase (AK1) and nucleoside diphosphate kinase (NDPK) enzymes (NME1, 2), which contribute to metabolism and regeneration of extracellular ATP and other nucleotides (ADP, uridine diphosphate (UDP) and uridine triphosphate (UTP)). In conclusion, we illustrate a complex regulation of extracellular purine homeostasis in a pancreatic duct cell model involving: ATP release by several mechanisms and subsequent nucleotide breakdown and ATP regeneration via counteracting nucleotide-inactivating and nucleotide-phosphorylating ecto-enzymes. We suggest that extracellular ATP homeostasis in pancreatic ducts may be important in pancreas physiology and potentially in pancreas pathophysiology. PMID:26431833

  1. ATP7B detoxifies silver in ciliated airway epithelial cells.

    PubMed

    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(-/-) 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+/Cu+ transporters ATP7A, ATP7B, and CTR1 in airway epithelial cells and a role for ATP7B in detoxification of these metals in the lung. PMID:20005242

  2. 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.

  3. Role of iron in lymphoma-induction by ATP.

    PubMed

    Anghileri, L J; Plenat, F; Thouvenot, P

    2001-01-01

    Iron complexed by ATP induces lymphomas in mouse organs other than the specific targets of the lympho-adenitis provoked by sodium ATP: lymph nodes, spleen and liver. The reduction of life spans and the production of substantial volumes of ascites, that are lacking in the case of sodium ATP, are an index of the degree of malignancy of the induced lymphomas. On the basis of the known characteristics of iron-ATP complex of cellular calcium homeostasis alteration, the mechanism of these phenomena is discussed. PMID:11496334

  4. Regulation of mitochondrial ATP synthase in cardiac pathophysiology.

    PubMed

    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

  5. 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

  6. 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

  7. 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.

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

    PubMed Central

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

    2013-01-01

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

  10. 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

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

    PubMed Central

    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

    2014-01-01

    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 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

  12. 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.

  13. 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.

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

    PubMed Central

    Yi, Ling; Kaler, Stephen

    2014-01-01

    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 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 ATP7AT994I and p97/VCP, a protein mutated in two autosomal dominant forms of motor neuron disease. Small-interfering RNA knockdown of p97/VCP corrected ATP7AT994I mislocalization. For ATP7AP1386S, flow cytometry documented that non-permeabilized fibroblasts bound a C-terminal ATP7A antibody, suggesting unstable insertion of the 8th transmembrane segment due to a helix-breaker effect of the amino acid substitution. This could sabotage interaction of ATP7AP1386S 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

  15. 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

  16. 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

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

    SciTech Connect

    Czechowski, M.H.

    1996-11-01

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

  18. 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

  19. Constitutive and agonist stimulated ATP secretion in leukocytes

    PubMed Central

    Campwala, Hinnah; Fountain, Samuel J.

    2013-01-01

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

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

    PubMed

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

    2004-09-15

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

  1. 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

  2. Radioprotective effects of ATP in human blood ex vivo

    SciTech Connect

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

    2008-03-07

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

  3. 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

  4. Pancreatic beta-cells communicate via intermittent release of ATP.

    PubMed

    Hellman, Bo; Dansk, Helne; Grapengiesser, Eva

    2004-05-01

    The role of external ATP for intercellular communication was studied in glucose-stimulated pancreatic beta-cells isolated from ob/ob mice. Digital image analyses with fura-2 revealed spontaneous transients of cytoplasmic Ca2+ appearing in synchrony in the absence of cell contacts. After removal of slow oscillations with methoxyverapamil, addition of ATP (0.1-100 microM) resulted in prompt firing of a transient, followed by suppression of the generation and synchronization of spontaneously occurring transients. It was possible to trigger transients during the suppressive phase by raising the concentration of ATP. The dual action of ATP was mimicked by ADP or 2-methylthio-ATP but not by AMP or UTP. The number of spontaneous transients and their synchronization were reduced in the presence of the dephosphorylating agent apyrase. Additional evidence that intermittent release of ATP participates in the generation of spontaneous Ca2+ transients was obtained from the suppression observed from use of antagonists of the purinoceptors [suramin (0.3-30 microM), pyridoxalphosphate-6-azophenyl-2,4-disulfonic acid (PPADS; 10-30 microM) and 2-deoxy-N-methyladenosine (MRS 2179; 0.3-30 microM)] or from counteracting beta-cell release of ATP by inhibiting exocytosis with 100 nM epinephrine, 100 nM somatostatin, or lowering the temperature below 30 degrees C. The data indicate that ATP has time-dependent actions (prompt stimulation followed by inhibition) on the generation of Ca2+ transients mediated by P2Y receptors. It is proposed that beta-cells both receive a neural ATP signal with coordinating effects on their Ca2+ oscillations and propagate this message to adjacent cells via intermittent release of ATP combined with gap junction coupling. PMID:14722025

  5. 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

  6. Adenosine triphosphate (ATP) reduces amyloid-? protein misfolding in vitro.

    PubMed

    Coskuner, Orkid; Murray, Ian V J

    2014-01-01

    Alzheimer's disease (AD) is a devastating disease of aging that initiates decades prior to clinical manifestation and represents an impending epidemic. Two early features of AD are metabolic dysfunction and changes in amyloid-? protein (A?) levels. Since levels of ATP decrease over the course of the disease and A? is an early biomarker of AD, we sought to uncover novel linkages between the two. First and remarkably, a GxxxG motif is common between both A? (oligomerization motif) and nucleotide binding proteins (Rossmann fold). Second, ATP was demonstrated to protect against A? mediated cytotoxicity. Last, there is structural similarity between ATP and amyloid binding/inhibitory compounds such as ThioT, melatonin, and indoles. Thus, we investigated whether ATP alters misfolding of the pathologically relevant A?42. To test this hypothesis, we performed computational and biochemical studies. Our computational studies demonstrate that ATP interacts strongly with Tyr10 and Ser26 of A? fibrils in solution. Experimentally, both ATP and ADP reduced A? misfolding at physiological intracellular concentrations, with thresholds at ~500 ?M and 1 mM respectively. This inhibition of A? misfolding is specific; requiring Tyr10 of A? and is enhanced by magnesium. Last, cerebrospinal fluid ATP levels are in the nanomolar range and decreased with AD pathology. This initial and novel finding regarding the ATP interaction with A? and reduction of A? misfolding has potential significance to the AD field. It provides an underlying mechanism for published links between metabolic dysfunction and AD. It also suggests a potential role of ATP in AD pathology, as the occurrence of misfolded extracellular A? mirrors lowered extracellular ATP levels. Last, the findings suggest that A? conformation change may be a sensor of metabolic dysfunction. PMID:24625803

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

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

    PubMed Central

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

    2011-01-01

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

  9. Expression of the atpE Gene Coding for the epsilon Subunit of Maize Chloroplast Coupling Factor.

    PubMed

    Shi, Jin; Wei, Jian-Mian; Shen, Yun-Gang

    1996-01-01

    The entire atpE gene of the maize chloroplast coupling factor was inserted into the polylinker region of vectors pJLA505 and pWA to form recombinant plasmids pJLA505-atpE and pWA-atpE respectively. These expression plasmids were transformed into E. coli NM522 which induced at 42 degrees. By the analysis of SDS-PAGE, the expressed product of interest was observed to account fore more than 3o% of total E. coliproteins. The identification of the expressed product demonstrated that its immunological specificity was well retained. The antiserum cross-reacted with the expressed epsilon protein and CF(1)-epsilon protein of spinach and produced precipitin lines on Ouchterlony immunodiffusion test. The expressed product aggregated insolubly as the inclusion body and was purified to over 80% purity. The purified product had the same function as that of the native epsilon subunit. PMID:12237708

  10. 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

  11. Dimers of mitochondrial ATP synthase form the permeability transition pore.

    PubMed

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

    2013-04-01

    Here we define the molecular nature of the mitochondrial permeability transition pore (PTP), a key effector of cell death. The PTP is regulated by matrix cyclophilin D (CyPD), which also binds the lateral stalk of the FOF1 ATP synthase. We show that CyPD binds the oligomycin sensitivity-conferring protein subunit of the enzyme at the same site as the ATP synthase inhibitor benzodiazepine 423 (Bz-423), that Bz-423 sensitizes the PTP to Ca(2+) like CyPD itself, and that decreasing oligomycin sensitivity-conferring protein expression by RNAi increases the sensitivity of the PTP to Ca(2+). Purified dimers of the ATP synthase, which did not contain voltage-dependent anion channel or adenine nucleotide translocator, were reconstituted into lipid bilayers. In the presence of Ca(2+), addition of Bz-423 triggered opening of a channel with currents that were typical of the mitochondrial megachannel, which is the PTP electrophysiological equivalent. Channel openings were inhibited by the ATP synthase inhibitor AMP-PNP (?-imino ATP, a nonhydrolyzable ATP analog) and Mg(2+)/ADP. These results indicate that the PTP forms from dimers of the ATP synthase. PMID:23530243

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

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

    PubMed Central

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

    2013-01-01

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

  19. 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

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

    PubMed Central

    Sielaff, Hendrik; Brsch, Michael

    2013-01-01

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

  1. Regulation of ATP production by mitochondrial Ca2+

    PubMed Central

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

    2012-01-01

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

  2. 40 CFR 350.1 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... INFORMATION: AND TRADE SECRET DISCLOSURES TO HEALTH PROFESSIONALS Trade Secrecy Claims § 350.1 Definitions... secret. Sanitized means a version of a document from which information claimed as trade secret or... support of a claim that chemical identity is a trade secret. Title III means Title III of the...

  3. 40 CFR 350.1 - Definitions.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... INFORMATION: AND TRADE SECRET DISCLOSURES TO HEALTH PROFESSIONALS Trade Secrecy Claims § 350.1 Definitions... secret. Sanitized means a version of a document from which information claimed as trade secret or... support of a claim that chemical identity is a trade secret. Title III means Title III of the...

  4. 40 CFR 350.1 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... INFORMATION: AND TRADE SECRET DISCLOSURES TO HEALTH PROFESSIONALS Trade Secrecy Claims § 350.1 Definitions... secret. Sanitized means a version of a document from which information claimed as trade secret or... support of a claim that chemical identity is a trade secret. Title III means Title III of the...

  5. 40 CFR 350.1 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... INFORMATION: AND TRADE SECRET DISCLOSURES TO HEALTH PROFESSIONALS Trade Secrecy Claims § 350.1 Definitions... secret. Sanitized means a version of a document from which information claimed as trade secret or... support of a claim that chemical identity is a trade secret. Title III means Title III of the...

  6. 40 CFR 350.1 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... INFORMATION: AND TRADE SECRET DISCLOSURES TO HEALTH PROFESSIONALS Trade Secrecy Claims § 350.1 Definitions... secret. Sanitized means a version of a document from which information claimed as trade secret or... support of a claim that chemical identity is a trade secret. Title III means Title III of the...

  7. 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,...

  8. 5 CFR 630.201 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... project and paid on an hourly rate. (b) In subparts B through G of this part: Accrued leave means the... 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)...

  9. 7 CFR 786.102 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... risk of producing milk, and make contributions (including land, labor, management, equipment, or... 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...

  10. 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...

  11. 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

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

    PubMed

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

    2015-01-20

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

  13. 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

  14. The evidence for two opposite, ATP-generating and ATP-consuming, extracellular pathways on endothelial and lymphoid cells.

    PubMed Central

    Yegutkin, Gennady G; Henttinen, Tiina; Samburski, Sergei S; Spychala, Jozef; Jalkanen, Sirpa

    2002-01-01

    Extracellular purines are important signalling molecules in the vasculature that are regulated by a network of cell surface ectoenzymes. By using human endothelial cells and normal and leukaemic lymphocytes as enzyme sources, we identified the following purine-converting ectoenzymes: (1) ecto-nucleotidases, NTP diphosphohydrolase/CD39 (EC 3.6.1.5) and ecto-5'-nucleotidase/CD73 (EC 3.1.3.5); (2) ecto-nucleotide kinases, adenylate kinase (EC 2.7.4.3) and nucleoside diphosphate kinase (EC 2.7.4.6); (3) ecto-adenosine deaminase (EC 3.5.4.4). Evidence for this was obtained by using enzyme assays with (3)H-labelled nucleotides and adenosine as substrates, direct evaluation of gamma-phosphate transfer from [gamma-(32)P]ATP to AMP/NDP, and bioluminescent measurement of extracellular ATP synthesis. In addition, incorporation of radioactivity into an approx. 20 kDa surface protein was observed following incubation of Namalwa B cells with [gamma-(32)P]ATP. Thus two opposite, ATP-generating and ATP-consuming, pathways coexist on the cell surface, where basal ATP release, re-synthesis of high-energy phosphoryls, and selective ecto-protein phosphorylation are counteracted by stepwise nucleotide breakdown with subsequent adenosine inactivation. The comparative measurements of enzymic activities indicated the predominance of the nucleotide-inactivating pathway via ecto-nucleotidase reactions on the endothelial cells. The lymphocytes are characterized by counteracting ATP-regenerating/adenosine-eliminating phenotypes, thus allowing them to avoid the lymphotoxic effects of adenosine and maintain surrounding ATP at a steady-state level. These results are in agreement with divergent effects of ATP and adenosine on endothelial function and haemostasis, and provide a novel regulatory mechanism of local agonist availability for nucleotide- or nucleoside-selective receptors within the vasculature. PMID:12099890

  15. Distinct neurological disorders with ATP1A3 mutations.

    PubMed

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

    2014-05-01

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

  16. 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

  17. Affinity labeling of Avena phytochrome with ATP analogs

    PubMed Central

    Wong, Yum-Shing; Lagarias, J. Clark

    1989-01-01

    The presence of ATP-dependent, polycation-stimulated protein kinase activity in highly purified phytochrome preparations [Wong, Y.-S., Cheng, H.-C., Walsh, D. A. & Lagarias, J. C. (1986) J. Biol. Chem. 261, 12089-12097] has renewed the hypothesis that the phytochrome photoreceptor possesses enzymatic activity. A prerequisite for protein kinase function is the presence of an ATP binding site. Here we present evidence for a nucleoside triphosphate binding site(s) in the phytochrome molecule. Two ATP analogs, 5?-p-fluorosulfonylbenzoyladenosine and 8-azidoadenosine 5?-triphosphate, were used to affinity label purified Avena phytochrome. Labeling with both reagents is stimulated by the polycations poly(Lys75,Ala25) and histone H1. Coincubation with ATP inhibits the polycation-stimulated labeling of phytochrome. In similar experiments GTP, CTP, UTP, ADP, and pyrophosphate, but not adenosine or AMP, also prevent photoaffinity labeling of phytochrome. Images PMID:16594043

  18. Distinct neurological disorders with ATP1A3 mutations

    PubMed Central

    Heinzen, Erin L.; Arzimanoglou, Alexis; Brashear, Allison; Clapcote, Steven J.; Gurrieri, Fiorella; Goldstein, David B.; Jhannesson, Sigurur 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 ?3 subunit of Na+/K+-ATPase, cause both rapid-onset dystonia parkinsonism and alternating hemiplegia of childhood. These discoveries link two clinically distinct neurological diseases to the same gene, however, ATP1A3 mutations are, with one exception, disease-specific. Although the exact mechanism of how these mutations lead to disease is still unknown, much knowledge has been gained about functional consequences of ATP1A3 mutations using a range of in vitro and animal model systems, and the role of Na+/K+-ATPases in the brain. Researchers and clinicians are attempting to further characterise neurological manifestations associated with mutations in ATP1A3, and to build on the existing molecular knowledge to understand how specific mutations can lead to different diseases. PMID:24739246

  19. 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

  20. Highly Divergent Mitochondrial ATP Synthase Complexes in Tetrahymena thermophila

    PubMed Central

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

    2010-01-01

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

  1. Sleep and Brain Energy Levels: ATP changes during sleep

    PubMed Central

    Dworak, Markus; McCarley, Robert W.; Kim, Tae; Kalinchuk, Anna V.; Basheer, Radhika

    2010-01-01

    Sleep is one of the most pervasive biological phenomena, but one whose function remains elusive. Although many theories of function, indirect evidence, and even common sense suggest sleep is needed for an increase in brain energy, brain energy levels have not been directly measured with modern technology. We here report that ATP levels, the energy currency of brain cells, show a surge in the initial hours of spontaneous sleep in wake-active but not in sleep-active brain regions of rat. The surge is dependent on sleep but not time of day, since preventing sleep by gentle handling of rats for 3 h or 6 h also prevents the surge in ATP. A significant positive correlation was observed between the surge in ATP and EEG NREM delta activity (0.54.5 Hz) during spontaneous sleep. Inducing sleep and delta activity by adenosine infusion into basal forebrain during the normally active dark period also increases ATP. Taken together, these observations suggest that the surge in ATP occurs when the neuronal activity is reduced, as occurs during sleep. The levels of phosphorylated AMP-activated protein kinase (P-AMPK), well known for its role in cellular energy sensing and regulation, and ATP show reciprocal changes. P-AMPK levels are lower during the sleep-induced ATP surge than during wake or sleep deprivation. Taken together, these results suggest that sleep-induced surge in ATP and the decrease in P-AMPK levels set the stage for increased anabolic processes during sleep and provides insight into the molecular events leading to the restorative biosynthetic processes occurring during sleep. PMID:20592221

  2. [Effect of adenosine triphosphoric acid (ATP) on accessory conduction bundles].

    PubMed

    Perrot, B; Faivre, G

    1982-05-01

    The effects of an injection of 40 mg of ATP were studied in 48 subjects with an overt or latent preexcitation syndrome. The results showed that the slowing of anterograde or retrograde conduction by ATP was not specific for nodal conduction. This phenomenon was observed in conduction through Kent bundles with long refractory periods and the possibility of preexcitation due to the association of James and Mahaim fibres should also be considered. PMID:6810790

  3. Catalytic Mechanism of the Maltose Transporter Hydrolyzing ATP.

    PubMed

    Huang, Wenting; Liao, Jie-Lou

    2016-01-12

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

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

    PubMed

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

    1990-01-01

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

  5. 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

  6. Piezo1 regulates mechanotransductive release of ATP from human RBCs.

    PubMed

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

    2015-09-22

    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 (Ca(2+)) influx. We find that, in human RBCs treated with Piezo1 inhibitors or having mutant Piezo1 channels, the amounts of shear-induced ATP release and Ca(2+) influx decrease significantly. Remarkably, a critical extracellular Ca(2+) 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

  7. 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

  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. 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

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

    PubMed

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

    2012-01-01

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

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

    PubMed Central

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

    2002-01-01

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

  13. Modification of the ATP-Induced Increase in

    PubMed

    Musat; Wang; Dhalla

    1998-10-01

    BACKGROUND: It is generally accepted that the plasma membrane of mammalian ventricular myocytes regulates the cytosolic concentration of Ca(2+). In this study we investigated the effects of some P2-purinoceptor antagonists and metals such as copper and zinc on the adenosine triphosphate (ATP)-induced increase in intracellular concentration of free Ca(2+) ([Ca(2+)](i)). METHODS AND RESULTS: Cardiomyocytes were isolated from adult male Sprague-Dawley rats loaded with Fura-2, and fluorescence measurements were performed by employing stirred cell suspensions at room temperature. ATP (50 M) increased [Ca(2+)](i) over the basal value, and 10 M cibacron blue or verapamil virtually abolished it. The ATP-induced increase in [Ca(2+)](i) was not observed in Ca(2+)- or Mg(2+)-free buffers. Incubation of cells with ZnCl(2) produced a significant depression of the ATP-induced increase in [Ca(2+)](i); 25 M Zn(2+) decreased the peak response to approximately 50% of the control value. The ATP-induced increase in [Ca(2+)](i), was inhibited by low concentrations (1-5 M) of Cu(2+) but was markedly augmented by high concentrations (25 M) of Cu(2+). The increase in the [Ca(2+)](i) response to cron blue, and Zn(2+), but not by ryanodine or caffeine pretreatment. CONCLUSIONS: The ATP-induced increase in [Ca(2+)](i) is dependent on the extracellular concentrations of Ca(2+) as well as Mg(2+) and is antagonized by cibacron blue and Zn(2+). On the other hand, Cu(2+) produced a biphasic response to the ATP-induced increase in [Ca(2+)](i) in cardiomyocytes. PMID:10684511

  14. 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.

  15. A Shigella effector dampens inflammation by regulating epithelial release of danger signal ATP through production of the lipid mediator PtdIns5P.

    PubMed

    Puhar, Andrea; Tronchre, Hlne; Payrastre, Bernard; Nhieu, Guy Tran Van; Sansonetti, Philippe J

    2013-12-12

    Upon infection with Shigella flexneri, epithelial cells release ATP through connexin hemichannels. However, the pathophysiological consequence and the regulation of this process are unclear. Here we showed that in intestinal epithelial cell ATP release was an early alert response to infection with enteric pathogens that eventually promoted inflammation of the gut. Shigella evolved to escape this inflammatory reaction by its type III secretion effector IpgD, which blocked hemichannels via the production of the lipid PtdIns5P. Infection with an ipgD mutant resulted in rapid hemichannel-dependent accumulation of extracellular ATP in vitro and in vivo, which preceded the onset of inflammation. At later stages of infection, ipgD-deficient Shigella caused strong intestinal inflammation owing to extracellular ATP. We therefore describe a new paradigm of host-pathogen interaction based on endogenous danger signaling and identify extracellular ATP as key regulator of mucosal inflammation during infection. Our data provide new angles of attack for the development of anti-inflammatory molecules. PMID:24332032

  16. 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.

  17. A1Ao-ATP synthase of Methanobrevibacter ruminantium couples sodium ions for ATP synthesis under physiological conditions.

    PubMed

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

    2011-11-18

    An unresolved question in the bioenergetics of methanogenic archaea is how the generation of proton-motive and sodium-motive forces during methane production is used to synthesize ATP by the membrane-bound A(1)A(o)-ATP synthase, with both proton- and sodium-coupled enzymes being reported in methanogens. To address this question, we investigated the biochemical characteristics of the A(1)A(o)-ATP synthase (MbbrA(1)A(o)) of Methanobrevibacter ruminantium M1, a predominant methanogen in the rumen. Growth of M. ruminantium M1 was inhibited by protonophores and sodium ionophores, demonstrating that both ion gradients were essential for growth. To study the role of these ions in ATP synthesis, the ahaHIKECFABD operon encoding the MbbrA(1)A(o) was expressed in Escherichia coli strain DK8 (?atp) and purified yielding a 9-subunit protein with an SDS-stable c oligomer. Analysis of the c subunit amino acid sequence revealed that it consisted of four transmembrane helices, and each hairpin displayed a complete Na(+)-binding signature made up of identical amino acid residues. The purified MbbrA(1)A(o) was stimulated by sodium ions, and Na(+) provided pH-dependent protection against inhibition by dicyclohexylcarbodiimide but not tributyltin chloride. ATP synthesis in inverted membrane vesicles lacking sodium ions was driven by a membrane potential that was sensitive to cyanide m-chlorophenylhydrazone but not to monensin. ATP synthesis could not be driven by a chemical gradient of sodium ions unless a membrane potential was imposed. ATP synthesis under these conditions was sensitive to monensin but not cyanide m-chlorophenylhydrazone. These data suggest that the M. ruminantium M1 A(1)A(o)-ATP synthase exhibits all the properties of a sodium-coupled enzyme, but it is also able to use protons to drive ATP synthesis under conditions that favor proton coupling, such as low pH and low levels of sodium ions. PMID:21953465

  18. 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

  19. 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 (?3?3???) and Fo (ab2c10) are ubiquitous, stable thermophilic FoF1 (TFoF1) has been used to elucidate molecular mechanisms, while human F1Fo (HF1Fo) has been used to study biomedical significance. Among F1s, only thermophilic F1 (TF1) can be analyzed simultaneously by reconstitution, crystallography, mutagenesis and nanotechnology for torque-driven ATP synthesis using elastic coupling mechanisms. In contrast to the single operon of TFoF1, HFoF1 is encoded by both nuclear DNA with introns and mitochondrial DNA. The regulatory mechanism, tissue specificity and physiopathology of HFoF1 were elucidated by proteomics, RNA interference, cytoplasts and transgenic mice. The ATP synthesized daily by HFoF1 is in the order of tens of kilograms, and is primarily controlled by the brain in response to fluctuations in activity. PMID:20689227

  20. 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

  1. ATP synthase promotes germ cell differentiation independent of oxidative phosphorylation

    PubMed Central

    Teixeira, Felipe K.; Sanchez, Carlos G.; Hurd, Thomas R.; Seifert, Jessica R. K.; Czech, Benjamin; Preall, Jonathan B.; Hannon, Gregory J.; Lehmann, Ruth

    2015-01-01

    The differentiation of stem cells is a tightly regulated process essential for animal development and tissue homeostasis. Through this process, attainment of new identity and function is achieved by marked changes in cellular properties. Intrinsic cellular mechanisms governing stem cell differentiation remain largely unknown, in part because systematic forward genetic approaches to the problem have not been widely used1,2. Analysing genes required for germline stem cell differentiation in the Drosophila ovary, we find that the mitochondrial ATP synthase plays a critical role in this process. Unexpectedly, the ATP synthesizing function of this complex was not necessary for differentiation, as knockdown of other members of the oxidative phosphorylation system did not disrupt the process. Instead, the ATP synthase acted to promote the maturation of mitochondrial cristae during differentiation through dimerization and specific upregulation of the ATP synthase complex. Taken together, our results suggest that ATP synthase-dependent crista maturation is a key developmental process required for differentiation independent of oxidative phosphorylation. PMID:25915123

  2. 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

  3. ATP modulates PTEN subcellular localization in multiple cancer cell lines

    PubMed Central

    Lobo, Glenn P.; Waite, Kristin A.; Planchon, Sarah M.; Romigh, Todd; Houghton, Janet A.; Eng, Charis

    2008-01-01

    The tumour suppressor gene PTEN plays an important somatic role in both hereditary and sporadic breast carcinogenesis. While the role of PTEN's lipid phosphatase activity, as a negative regulator of the cytoplasmic phosphatidylinositol-3-kinase/Akt pathway is well known, it is now well established that PTEN exists and functions in the nucleus. Multiple mechanisms of regulating PTEN's subcellular localization have been reported. However none are ubiquitous across multiple cancer cell lines and tissue types. We show here that adenosine triphosphate (ATP) regulates PTEN subcellular localization in a variety of different cancer cell lines, including those derived from breast, colon and thyroid carcinomas. Cells deficient in ATP show an increased level of nuclear PTEN protein. This increase in PTEN is reversed when cells are supplemented with ATP, ADP or AMP. In contrast, the addition of the non-hydrolyzable analogue ATP?S, did not reverse nuclear PTEN protein levels in all the cell types tested. To our knowledge, this is the first report that describes a regulation of PTEN subcellular localization that is not specific to one cell line or tissue type, but appears to be common across a variety of cell lineages. PMID:18579579

  4. 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

  5. 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

  6. QM/MM investigation of ATP hydrolysis in aqueous solution.

    PubMed

    Wang, Cui; Huang, Wenting; Liao, Jie-Lou

    2015-03-01

    Adenosine-5'-triphosphate (ATP) hydrolysis represents a most important reaction in biology. Despite extensive research efforts, the mechanism for ATP hydrolysis in aqueous solution still remains under debate. Previous theoretical studies often predefined reaction coordinates to characterize the mechanism for ATP hydrolysis in water with Mg(2+) by evaluating free energy profiles through these preassumed reaction paths. In the present work, a nudged elastic band method is applied to identify the minimum energy path calculated with a hybrid quantum mechanics and molecular mechanics approach. Along the reaction path, the free energy profile was obtained to have a single transition state and the activation energy of 32.5 kcal/mol. This transition state bears a four-centered structure that describes a concerted nature of the reaction. In the More-O'Ferrall-Jencks diagram, the results show that the reaction proceeds through a concerted path before the system reaches the transition state and along an associative path after the transition state. In addition, the calculated reaction free energy is -7.0 kcal/mol, in good agreement with experiment, capturing the exothermic feature of MgATP(2-) hydrolysis in aqueous solution, whereas the reaction was often shown to be endothermic in the previous theoretical studies. As Mg(2+) is required for ATP hydrolysis in cells, its role in the reaction is also elucidated. PMID:25658024

  7. 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.

  8. 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

  9. 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

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

    PubMed

    Lpez-Sez, J F; Mingo, R; Gonzlez-Fernndez, 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

  11. ATP in cellular calcium-overload by trivalent metal ions.

    PubMed

    Anghileri, L J; Thouvenot, P

    1998-01-01

    Extracellular Ca2+-influx induced by trivalent metal ions (Fe3+, Al3+, Cr3+, In3+, Ga3+, and La3+) in Ehrlich carcinoma cells is enhanced by ATP. This action seems to be related to the high coordination capacity of the ATP ligand that inhibits the polymerization of the solvated cations taking place at physiological pH, and consequently permits their biological activity. A general relationship between induced lipid peroxidation and increased calcium uptake was not found. These results emphasize the ATP role in the toxicity of trivalent metals, and its possible involvement, via cellular calcium overload, in a neurodegenerative process, such as Alzheimer's and Parkinson's diseases, in whose etiology the implication of aluminum and iron has been suggested. PMID:9630430

  12. Yeast mitochondrial ADP/ATP carriers are monomeric in detergents

    PubMed Central

    Bamber, Lisa; Harding, Marilyn; Butler, P. Jonathan G.; Kunji, Edmund R. S.

    2006-01-01

    Mitochondrial carriers are believed widely to be homodimers both in the inner membrane of the organelle and in detergents. The dimensions and molecular masses of the detergent and proteindetergent micelles were measured for yeast ADP/ATP carriers in a range of different detergents. The radius of the carrier at the midpoint of the membrane, its average radius, its Stokes' radius, its molecular mass, and its excluded volume were determined. These parameters are consistent with the known structural model of the bovine ADP/ATP carrier and they demonstrate that the yeast mitochondrial ADP/ATP carriers are monomeric in detergents. Therefore, models of substrate transport have to be considered in which the carrier operates as a monomer rather than as a dimer. PMID:17056710

  13. Kinetic mechanism of ATP-sulphurylase from rat chondrosarcoma.

    PubMed Central

    Lyle, S; Geller, D H; Ng, K; Westley, J; Schwartz, N B

    1994-01-01

    ATP-sulphurylase catalyses the production of adenosine 5'-phosphosulphate (APS) from ATP and free sulphate with the release of PPi. APS kinase phosphorylates the APS intermediate to produce adenosine 3'-phosphate 5'-phosphosulphate (PAPS). The kinetic mechanism of rat chondrosarcoma ATP-sulphurylase was investigated by steady-state methods in the physiologically forward direction as well as the reverse direction. The sulphurylase activity was coupled to APS kinase activity in order to overcome the thermodynamic constraints of the sulphurylase reaction in the forward direction. Double-reciprocal initial-velocity plots for the forward sulphurylase intersect to the left of the ordinate for this reaction. KmATP and Kmsulphate were found to be 200 and 97 microM respectively. Chlorate, a competitive inhibitor with respect to sulphate, showed uncompetitive inhibition with respect to ATP with an apparent Ki of 1.97 mM. Steady-state data from experiments in the physiologically reverse direction also yielded double-reciprocal initial-velocity patterns that intersect to the left of the ordinate axis, with a KmAPS of 39 microM and a Kmpyrophosphate of 18 microM. The results of steady-state experiments in which Mg2+ was varied indicated that the true substrate is the MgPPi complex. An analogue of APS, adenosine 5'-[beta-methylene]phosphosulphate, was a linear inhibitor competitive with APS and non-competitive with respect to MgPPi. The simplest formal mechanism that agrees with all the data is an ordered steady-state single displacement with MgATP as the leading substrate in the forward direction and APS as the leading substrate in the reverse direction. PMID:8042976

  14. In vivo distribution of ferric-ATP complex.

    PubMed

    Anghileri, L J; Robert, J

    1994-01-01

    At physiological pH, ferric-ATP complex presents ionic characteristics unlike those of ferric lactate. Its rapid diffusion into the tissues, and its reactivity which is not impaired by polymerization seem responsible for its toxicity. In comparison with ferric lactate, after parenteral administration there is a much higher spleen, lung, kidney and bone accumulation. After oral administration, on the other hand, the values are lower. ATP hydrolysis by the stomach's pH, and the subsequent iron insolubilization as phosphate could be the reason for this behaviour. PMID:7957445

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

    PubMed Central

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

    2015-01-01

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

  17. 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

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

    PubMed

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

    2013-11-01

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

  19. 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

  20. 48 CFR 52.234-1 - Industrial Resources Developed Under Defense Production Act Title III.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Developed Under Defense Production Act Title III. 52.234-1 Section 52.234-1 Federal Acquisition Regulations... CLAUSES Text of Provisions and Clauses 52.234-1 Industrial Resources Developed Under Defense Production... Under Defense Production Act Title III (DEC 1994) (a) Definitions. Title III industrial resource...

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

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... Developed Under Defense Production Act Title III. 52.234-1 Section 52.234-1 Federal Acquisition Regulations... CLAUSES Text of Provisions and Clauses 52.234-1 Industrial Resources Developed Under Defense Production... Under Defense Production Act Title III (DEC 1994) (a) Definitions. Title III industrial resource...

  2. 48 CFR 52.234-1 - Industrial Resources Developed Under Defense Production Act Title III.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... Developed Under Defense Production Act Title III. 52.234-1 Section 52.234-1 Federal Acquisition Regulations... CLAUSES Text of Provisions and Clauses 52.234-1 Industrial Resources Developed Under Defense Production... Under Defense Production Act Title III (DEC 1994) (a) Definitions. Title III industrial resource...

  3. 48 CFR 52.234-1 - Industrial Resources Developed Under Defense Production Act Title III.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... Developed Under Defense Production Act Title III. 52.234-1 Section 52.234-1 Federal Acquisition Regulations... CLAUSES Text of Provisions and Clauses 52.234-1 Industrial Resources Developed Under Defense Production... Under Defense Production Act Title III (DEC 1994) (a) Definitions. Title III industrial resource...

  4. 48 CFR 52.234-1 - Industrial Resources Developed Under Defense Production Act Title III.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... Developed Under Defense Production Act Title III. 52.234-1 Section 52.234-1 Federal Acquisition Regulations... CLAUSES Text of Provisions and Clauses 52.234-1 Industrial Resources Developed Under Defense Production... Under Defense Production Act Title III (DEC 1994) (a) Definitions. Title III industrial resource...

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

    NASA Astrophysics Data System (ADS)

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

    2003-07-01

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

  6. 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

  7. 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 ?-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 ?-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 ?-helix. We propose a mechanism for ATP-Mg/Pi carriers in which the amphipathic ?-helix becomes mobile upon release of calcium and could block the transport of substrates across the mitochondrial inner membrane. PMID:26164100

  8. 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 ?-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 ?-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 ?-helix. We propose a mechanism for ATP-Mg/Pi carriers in which the amphipathic ?-helix becomes mobile upon release of calcium and could block the transport of substrates across the mitochondrial inner membrane. PMID:26164100

  9. Characterization of ATP Alternations in an Alzheimers Transgenic Mouse Model

    PubMed Central

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

    2014-01-01

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

  10. 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

  11. Timely management of developing class III malocclusion.

    PubMed

    Yelampalli, M R; Rachala, M R

    2012-01-01

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

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

    PubMed

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

    2014-01-01

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

  13. 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 ?-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

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

    PubMed Central

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

    2014-01-01

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

  15. 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

  16. 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.

  17. Characterization of ATP citrate lyase from Chlorobium limicola.

    PubMed Central

    Antranikian, G; Herzberg, C; Gottschalk, G

    1982-01-01

    ATP citrate lyase (EC 4.1.3.8) from Chlorobium limicola was partially purified. It was established that the consumption of substrates and the formation of products proceeded stoichiometrically and that citrate cleavage was of the si-type. ADP and oxaloacetate inhibited enzyme activity. Oxaloacetate also inhibited the growth of C. limicola. PMID:7142107

  18. Abiogenic Photophosphorylation of ADP to ATP Sensitized by Flavoproteinoid Microspheres

    NASA Astrophysics Data System (ADS)

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

    2008-06-01

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

  19. Hair bundles are specialized for ATP delivery via creatine kinase.

    PubMed

    Shin, Jung-Bum; Streijger, Femke; Beynon, Andy; Peters, Theo; Gadzala, Laura; McMillen, Debra; Bystrom, Cory; Van der Zee, Catharina E E M; Wallimann, Theo; Gillespie, Peter G

    2007-02-01

    When stimulated strongly, a hair cell's mechanically sensitive hair bundle may consume ATP too rapidly for replenishment by diffusion. To provide a broad view of the bundle's protein complement, including those proteins participating in energy metabolism, we used shotgun mass spectrometry methods to identify proteins of purified chicken vestibular bundles. In addition to cytoskeletal proteins, proteins involved in Ca(2+) regulation, and stress-response proteins, many of the most abundant bundle proteins that were identified by mass spectrometry were involved in ATP synthesis. After beta-actin, the cytosolic brain isoform of creatine kinase was the next most abundant bundle protein; at approximately 0.5 mM, creatine kinase is capable of maintaining high ATP levels despite 1 mM/s ATP consumption by the plasma-membrane Ca(2+)-ATPase. Consistent with this critical role in hair bundle function, the creatine kinase circuit is essential for high-sensitivity hearing as demonstrated by hearing loss in creatine kinase knockout mice. PMID:17270734

  20. 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)

  1. 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…

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

    NASA Technical Reports Server (NTRS)

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

    1982-01-01

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

  3. ATP 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.

  4. 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…

  5. 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.

  6. 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,

  7. 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,…

  8. [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

  9. 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

  10. 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.

  11. 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.

  12. 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

  13. 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

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

    PubMed Central

    Sivaramakrishnan, Venketesh

    2015-01-01

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

  15. 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.

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

    PubMed

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

    2015-11-15

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

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

    PubMed

    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. PMID:12529523

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

    PubMed

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

    2015-01-01

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

  19. 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

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

    PubMed

    Villavicencio-Queijeiro, Alexa; Pardo, Juan Pablo; 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

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

    PubMed

    Hamer, E; Schoner, W

    1993-04-15

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

  2. Mechanisms by which reactions catalyzed by chloroplast coupling factor 1 are inhibited: ATP synthesis and ATP-H2O oxygen exchange

    SciTech Connect

    Spencer, J.G.; Wimmer, M.J.

    1985-07-16

    The ATP-H2O back-exchange reaction catalyzed by membrane-bound chloroplast coupling factor 1 (CF1) in the light is known to be extensive; each reacting ATP molecule nearly equilibrates its gamma-PO2 oxygens with H2O before it dissociates from the enzyme. Pi, ASi, ADP, and GDP, alternate substrates of photophosphorylation, each inhibit the exchange reaction. At all concentrations of these substrate/inhibitor molecules tested, the high extent of exchange per molecule of ATP that reacts remains the same, while the number of ATP molecules experiencing exchange decreases. Thus, these inhibitors appear to act in a competitive-type manner, decreasing ATP turnover, as opposed to modulating the rate constants responsible for the partitioning of E X ATP during the exchange reaction. This is consistent with the identity of CF1 catalytic sites for ATP-H2O back-exchange and ATP synthesis. The extent of ATP-H2O forward oxygen exchange, which occurs during net ATP synthesis prior to product dissociation, is unaffected by uncouplers, whether catalyzed by native CF1 (ATPase latent) or the dithiothreitol/light-activated ATPase form.

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

    PubMed Central

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

    2002-01-01

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

  4. 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

  5. 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

  6. 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

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

    SciTech Connect

    Inagaki, Nobuya; Seino, Susumu; Inazawa, Johji

    1995-11-01

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

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

    PubMed Central

    Vishnu, Neelanjan; Jadoon Khan, Muhammad; Karsten, Felix; Groschner, Lukas N.; Waldeck-Weiermair, Markus; Rost, Rene; 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

  9. 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....

  10. 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...

  11. 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; Gttlinger, 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- 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

  12. 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

  13. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2011-03-01

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

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

    NASA Astrophysics Data System (ADS)

    Zhang, Yun-Xin

    2014-10-01

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

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

    PubMed Central

    Bartolome, Fernando; Wu, Hsiu-Chuan; Burchell, VictoriaS.; Preza, Elisavet; Wray, Selina; Mahoney, ColinJ.; Fox, NickC.; Calvo, Andrea; Canosa, Antonio; Moglia, Cristina; Mandrioli, Jessica; Chi, Adriano; Orrell, RichardW.; Houlden, Henry; Hardy, John; Abramov, AndreyY.; Plun-Favreau, Helene

    2013-01-01

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

  17. Structure and mechanism of ATP-binding cassette transporters

    PubMed Central

    Locher, Kaspar P.

    2008-01-01

    ATP-binding cassette (ABC) transporters constitute a large superfamily of integral membrane proteins that includes both importers and exporters. In recent years, several structures of complete ABC transporters have been determined by X-ray crystallography. These structures suggest a mechanism by which binding and hydrolysis of ATP by the cytoplasmic, nucleotide-binding domains control the conformation of the transmembrane domains and therefore which side of the membrane the translocation pathway is exposed to. A basic, conserved two-state mechanism can explain active transport of both ABC importers and ABC exporters, but various questions remain unresolved. In this article, I will review some of the crystal structures and the mechanistic insight gained from them. Future challenges for a better understanding of the mechanism of ABC transporters will be outlined. PMID:18957379

  18. Pyrithione biocides as inhibitors of bacterial ATP synthesis.

    PubMed

    Dinning, A J; Al-Adham, I S; Eastwood, I M; Austin, P; Collier, P J

    1998-07-01

    Sodium pyrithione and zinc pyrithione (NaPT and ZnPT, respectively) are widely used as cosmetic preservatives and general antimicrobial agents. They have been shown to be active against fungal cell walls, associated membranes and bacterial transport processes. Investigations were undertaken into the effect of these antimicrobial agents on substrate catabolism and intracellular ATP levels using an oxygen electrode and luciferin-laciferase technology, respectively. Results indicate that, while both compounds are poor inhibitors of substrate catabolism, sub-inhibitory concentrations of biocide greatly reduces intracellular ATP levels in both Escherichia coli NCIMB 10000 and Pseudomonas aeruginosa NCIMB 10548. This is thought to be due to the action of NaPT and ZnPT on the Gram-negative bacterial membrane. PMID:9721664

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

    PubMed

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

    2009-12-01

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

  20. Adenosine and ATP: traffic regulators in the kidney

    PubMed Central

    Kriz, Wilhelm

    2004-01-01

    Glomerular filtration in the kidney is a continuous process that acts in concert with tubular reabsorption to prevent derangements of body fluid composition. Filtration is regulated by systemic factors, but it is also controlled by an intrinsic mechanism based on the anatomical connection between the distal nephron and the glomerular arterioles. Facing the threat of urinary salt loss, this mechanism causes vasoconstriction and reduces filtration by generating adenosine through the hydrolysis of nucleotide precursors such as 5?-AMP and possibly ATP . PMID:15343376

  1. 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

  2. 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

  3. Exogenous adenosine triphosphate (ATP) preserves proximal tubule microfilament structure and function in vivo in a maleic acid model of ATP depletion.

    PubMed Central

    Kellerman, P S

    1993-01-01

    The hallmark of ischemic acute renal failure is a rapid and early decline in proximal tubule ATP. Since we have previously shown that over half of apical microfilament losses occur within the first 5 min of experimental ischemic injury, we postulated that microfilament (F-actin) structure and cellular location are dependent on cellular ATP levels. To test this hypothesis, we used maleic acid to selectively inhibit renal cortical ATP production in vivo. Maleic acid significantly decreased tissue ATP and apical F-actin in a dose-dependent manner relative to equimolar sodium chloride controls, yet higher doses of maleic acid quantitatively resulted in net actin polymerization, primarily in the cytoplasm. Functionally, maleic acid decreased glomerular filtration rate (GFR) and tubular reabsorption of sodium (TRNa) in a dose-dependent manner relative to sodium chloride controls. Administration of exogenous ATP resulted in significant increases in tissue ATP, net actin depolymerization, and relocation of F-actin from the cytoplasm back to the apical surface coinciding with increases in GFR and TRNa. Thus, ATP depletion induced by maleic acid resulted in significant cytoskeletal and functional alterations that were ameliorated by exogenous ATP. We therefore conclude that the structure and cellular location of F-actin necessary for normal functioning of proximal tubule cells in vivo is dependent on tissue ATP levels. Images PMID:8408646

  4. 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.

  5. 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

  6. Association and stoichiometry of K(ATP) channel subunits.

    PubMed

    Clement, J P; Kunjilwar, K; Gonzalez, G; Schwanstecher, M; Panten, U; Aguilar-Bryan, L; Bryan, J

    1997-05-01

    ATP-sensitive potassium channels (K(ATP) channels) are heteromultimers of sulfonylurea receptors (SUR) and inwardly rectifying potassium channel subunits (K(IR)6.x) with a (SUR-K(IR)6.x)4 stoichiometry. Association is specific for K(IR)6.x and affects receptor glycosylation and cophotolabeling of K(IR)6.x by 125I-azidoglibenclamide. Association produces digitonin stable complexes with an estimated mass of 950 kDa. These complexes can be purified by lectin chromatography or by using Ni2(+)-agarose and a his-tagged SUR1. Expression of SUR1 approximately (K(IR)6.2)i fusion constructs shows that a 1:1 SUR1:K(IR)6.2 stoichiometry is both necessary and sufficient for assembly of active K(ATP) channels. Coexpression of a mixture of strongly and weakly rectifying triple fusion proteins, rescued by SUR1, produced the three channel types expected of a tetrameric pore. PMID:9182806

  7. Overview of photo-induced therapy for ATP production

    NASA Astrophysics Data System (ADS)

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

    2012-10-01

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

  8. Mg? coordinating dynamics in Mg:ATP fueled motor proteins.

    PubMed

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

    2014-03-21

    The coordination of Mg(2+) with the triphosphate group of adenosine triphosphate (ATP) in motor proteins is investigated using data mining and molecular dynamics. The possible coordination structures available from crystal data for actin, myosin, RNA polymerase, DNA polymerase, DNA helicase, and F1-ATPase are verified and investigated further by molecular dynamics. Coordination states are evaluated using structural analysis and quantified by radial distribution functions, coordination numbers, and pair interaction energy calculations. The results reveal a diverse range of both transitory and stable coordination arrangements between Mg(2+) and ATP. The two most stable coordinating states occur when Mg(2+) coordinates two or three oxygens from the triphosphate group of ATP. Evidence for five-site coordination is also reported involving water in addition to the triphosphate group. The stable states correspond to a pair interaction energy of either ?-2750 kJ/mol or -3500 kJ/mol. The role of water molecules in the hydration shell surrounding Mg(2+) is also reported. PMID:24655204

  9. 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.

  10. 2-Hydroxyglutarate Inhibits ATP Synthase and mTOR Signaling.

    PubMed

    Fu, Xudong; Chin, Randall M; Vergnes, Laurent; Hwang, Heejun; Deng, Gang; Xing, Yanpeng; Pai, Melody Y; Li, Sichen; Ta, Lisa; Fazlollahi, Farbod; Chen, Chuo; Prins, Robert M; Teitell, Michael A; Nathanson, David A; Lai, Albert; Faull, Kym F; Jiang, Meisheng; Clarke, Steven G; Cloughesy, Timothy F; Graeber, Thomas G; Braas, Daniel; Christofk, Heather R; Jung, Michael E; Reue, Karen; Huang, Jing

    2015-09-01

    We discovered recently that the central metabolite ?-ketoglutarate (?-KG) extends the lifespan of C. elegans through inhibition of ATP synthase and TOR signaling. Here we find, unexpectedly, that (R)-2-hydroxyglutarate ((R)-2HG), an oncometabolite that interferes with various ?-KG-mediated processes, similarly extends worm lifespan. (R)-2HG accumulates in human cancers carrying neomorphic mutations in the isocitrate dehydrogenase (IDH) 1 and 2 genes. We show that, like ?-KG, both (R)-2HG and (S)-2HG bind and inhibit ATP synthase and inhibit mTOR signaling. These effects are mirrored in IDH1 mutant cells, suggesting a growth-suppressive function of (R)-2HG. Consistently, inhibition of ATP synthase by 2-HG or ?-KG in glioblastoma cells is sufficient for growth arrest and tumor cell killing under conditions of glucose limitation, e.g., when ketone bodies (instead of glucose) are supplied for energy. These findings inform therapeutic strategies and open avenues for investigating the roles of 2-HG and metabolites in biology and disease. PMID:26190651

  11. 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

  12. 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 (4C). 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

  13. Extracellular ATP induces the accumulation of superoxide via NADPH oxidases in Arabidopsis.

    PubMed

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

    2006-04-01

    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 ATPgammaS. 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 microm, 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

  14. 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

  15. Visualization and Measurement of ATP Levels in Living Cells Replicating Hepatitis C Virus Genome RNA

    PubMed Central

    Ando, Tomomi; Imamura, Hiromi; Suzuki, Ryosuke; Aizaki, Hideki; Watanabe, Toshiki; Wakita, Takaji; Suzuki, Tetsuro

    2012-01-01

    Adenosine 5?-triphosphate (ATP) is the primary energy currency of all living organisms and participates in a variety of cellular processes. Although ATP requirements during viral lifecycles have been examined in a number of studies, a method by which ATP production can be monitored in real-time, and by which ATP can be quantified in individual cells and subcellular compartments, is lacking, thereby hindering studies aimed at elucidating the precise mechanisms by which viral replication energized by ATP is controlled. In this study, we investigated the fluctuation and distribution of ATP in cells during RNA replication of the hepatitis C virus (HCV), a member of the Flaviviridae family. We demonstrated that cells involved in viral RNA replication actively consumed ATP, thereby reducing cytoplasmic ATP levels. Subsequently, a method to measure ATP levels at putative subcellular sites of HCV RNA replication in living cells was developed by introducing a recently-established Frster resonance energy transfer (FRET)-based ATP indicator, called ATeam, into the NS5A coding region of the HCV replicon. Using this method, we were able to observe the formation of ATP-enriched dot-like structures, which co-localize with non-structural viral proteins, within the cytoplasm of HCV-replicating cells but not in non-replicating cells. The obtained FRET signals allowed us to estimate ATP concentrations within HCV replicating cells as ?5 mM at possible replicating sites and ?1 mM at peripheral sites that did not appear to be involved in HCV replication. In contrast, cytoplasmic ATP levels in non-replicating Huh-7 cells were estimated as ?2 mM. To our knowledge, this is the first study to demonstrate changes in ATP concentration within cells during replication of the HCV genome and increased ATP levels at distinct sites within replicating cells. ATeam may be a powerful tool for the study of energy metabolism during replication of the viral genome. PMID:22396648

  16. 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

  17. 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

  18. 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

  19. Photoaffinity labeling of rat liver carbamoyl phosphate synthetase I by azido-ATP

    SciTech Connect

    Corina, K.; Powers-Lee, S.G.

    1987-05-01

    8-Azido-ATP has been found to serve as a photoaffinity label for two distinct ATP sites on rat liver carbamoyl phosphate synthetase I and to allow preliminary localization of these sites. In the dark, azido-ATP acted as a competitive inhibitor with respect to ATP. Ultraviolet irradiation of carbamoyl phosphate synthetase I in the presence of azido-ATP led to an irreversible loss of activity. ATP specifically protected against this inactivation. The incorporation of 2 moles of azido-ATP per mole of enzyme was required for complete inactivation. In order to localize the azido-ATP binding sites to structural domains of carbamoyl phosphate synthetase I, the enzyme was photolabeled with /sup 32/P-azido-ATP and subjected to limited proteolytic digestion. The resulting model for the functional roles of the domains is that there is one ATP site on each of the two large internal structural domains of the enzyme. Each of these domains was found to contain the consensus sequences A and B common to many other nucleotide-binding proteins. In addition, there is extensive structural and possibly functional interaction of the smaller N-terminal domain with one of the internal ATP-binding domains.

  20. Photoaffinity labeling of rat liver carbamoyl phosphate synthetase I by 8-azido-ATP

    SciTech Connect

    Powers-Lee, S.G.; Corina, K.

    1987-07-05

    8-Azido-ATP has been found to serve as a photoaffinity label for two distinct ATP sites on rat liver carbamoyl phosphate synthetase I and to allow preliminary localization of these sites. In the dark, 8-azido-ATP acted as a competitive inhibitor with respect to ATP. Ultraviolet irradiation of carbamoyl phosphate synthetase I in the presence of 8-azido-ATP led to an irreversible loss of activity. ATP specifically protected against this inactivation. The incorporation of 2 mol of 8-azido-ATP per mol of enzyme was required for complete inactivation. To localize the 8-azido-ATP-binding sites to discrete regions of carbamoyl phosphate synthetase I which appear to be structural domains, the enzyme was photolabeled with (gamma-/sup 32/P)8-azido-ATP and subjected to limited proteolytic digestion. The resulting model for the functional roles of the domains is that there is one ATP site on each of the two large internal structural domains of the enzyme. Each of these domains was found to contain the consensus sequences A and B common to many other nucleotide-binding proteins. In addition, there is extensive structural and possibly functional interaction of the smaller N-terminal domain with one of the internal ATP-binding domains, analogous to a subunit interaction observed with the evolutionarily related Escherichia coli carbamoyl phosphate synthetase.

  1. ATP protects against FITC labeling of Solanum lycopersicon and Arabidopsis thaliana Ca2+-ATPase ATP binding domains.

    PubMed

    Galva, Charitha; Virgin, Gail K; Helms, Jeff B; Gatto, Craig

    2013-10-01

    Ca(2+)-ATPases are integral membrane proteins that actively transport Ca(2+) against substantial concentration gradients in eukaryotic cells. This active transport is energized by coupling ion translocation with ATP hydrolysis. In order to better understand this coupling mechanism, we studied the nucleotide specificities of isolated ATP binding domains (ABDs) of Solanum lycopersicon Ca(2+)-ATPase (LCA), a type IIA non-calmodulin regulated P-type pump found in tomato plants that is very similar to mammalian sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA), and Arabidopsis Ca(2+)-ATPase, isoform 2 (ACA2), a type IIB calmodulin regulated P-type ATPase found in the endoplasmic reticulum of Arabidopsis cells. We used nucleotide protection against FITC labeling as a measure of binding since both LCA and ACA contained the KGAP(S,V,F)E motif, which has been shown to be modified by fluorescein isothiocyanate (FITC) in P-type pumps from animal cells. We demonstrated that the heterologously expressed GST-tagged ABDs from both LCA and ACA2 were modified by FITC and that ATP protects against this modification. Moreover, GTP was able to reduce, but not eliminate, the level of FITC labeling in both ABD constructs, suggesting that these plant pumps may also bind GTP with low affinity, which is in contrast to mammalian SERCA and PMCA type pumps which do not bind GTP. PMID:23974359

  2. ATP protects against FITC labeling of Solanum lycopersicon and Arabidopsis thaliana Ca2+ -ATPase ATP binding domains

    PubMed Central

    Galva, Charitha; Virgin, Gail K.; Helms, Jeff B.; Gatto, Craig

    2013-01-01

    Ca2+-ATPases are integral membrane proteins that actively transport Ca2+ against substantial concentration gradients in eukaryotic cells. This active transport is energized by coupling ion translocation with ATP hydrolysis. In order to better understand this coupling mechanism, we studied the nucleotide specificities of isolated ATP binding domains (ABDs) of Solanum lycopersicon Ca2+-ATPase (LCA), a type IIA non-calmodulin regulated P-type pump found in tomato plants that is very similar to mammalian sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), and Arabidopsis Ca2+-ATPase, isoform 2 (ACA2), a type IIB calmodulin regulated P-type ATPase found in the endoplasmic reticulum of Arabidopsis cells. We used nucleotide protection against FITC labeling as a measure of binding since both LCA and ACA contained the KGAP(S,V,F)E motif, which has been shown to be modified by fluorescein isothiocyanate (FITC) in P-type pumps from animal cells. We demonstrated that the heterologously expressed GST-tagged ABDs from both LCA and ACA2 were modified by FITC and that ATP protects against this modification. Moreover, GTP was able to reduce, but not eliminate, the level of FITC labeling in both ABD constructs, suggesting that these plant pumps may also bind GTP with low affinity, which is in contrast to mammalian SERCA and PMCA type pumps which do not bind GTP. PMID:23974359

  3. 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

  4. 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

  5. Characterization of an ATP-dependent DNA ligase encoded by Haemophilus influenzae.

    PubMed Central

    Cheng, C; Shuman, S

    1997-01-01

    We report that Haemophilus influenzae encodes a 268 amino acid ATP-dependent DNA ligase. The specificity of Haemophilus DNA ligase was investigated using recombinant protein produced in Escherichia coli. The enzyme catalyzed efficient strand joining on a singly nicked DNA in the presence of magnesium and ATP (Km = 0.2 microM). Other nucleoside triphosphates or deoxynucleoside triphosphates could not substitute for ATP. Haemophilus ligase reacted with ATP in the absence of DNA substrate to form a covalent ligase-adenylate intermediate. This nucleotidyl transferase reaction required a divalent cation and was specific for ATP. The Haemophilus enzyme is the first example of an ATP-dependent DNA ligase encoded by a eubacterial genome. It is also the smallest member of the covalent nucleotidyl transferase superfamily, which includes the bacteriophage and eukaryotic ATP-dependent polynucleotide ligases and the GTP-dependent RNA capping enzymes. PMID:9060431

  6. 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...

  7. 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...

  8. 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...

  9. 48 CFR 9904.420-30 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 7 2010-10-01 2010-10-01 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...

  10. 10 CFR 35.2 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... COMMISSION MEDICAL USE OF BYPRODUCT MATERIAL General Information § 35.2 Definitions. Address of use means the... Commission master material licensee that authorizes medical use or the practice of nuclear pharmacy; (iii) A... use or the practice of nuclear pharmacy; or (iv) A permit issued by a Commission master...

  11. 32 CFR 562.3 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... (MS)-III and MS-IV. (The Ranger camp is an acceptable alternate). (c) Advanced course. The Senior ROTC.... (4) US Army Fourth ROTC Region, Fort Lewis, WA 98433. (m) ROTC ranger camp. The ranger course...' TRAINING CORPS § 562.3 Definitions. The following terms apply to the Army's Senior Reserve...

  12. 32 CFR 562.3 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... (MS)-III and MS-IV. (The Ranger camp is an acceptable alternate). (c) Advanced course. The Senior ROTC.... (4) US Army Fourth ROTC Region, Fort Lewis, WA 98433. (m) ROTC ranger camp. The ranger course...' TRAINING CORPS § 562.3 Definitions. The following terms apply to the Army's Senior Reserve...

  13. 28 CFR 905.1 - Definition.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... Administration NATIONAL CRIME PREVENTION AND PRIVACY COMPACT COUNCIL NATIONAL FINGERPRINT FILE (NFF) PROGRAM QUALIFICATION REQUIREMENTS § 905.1 Definition. “National Fingerprint File” means a database of fingerprints, or... maintained by the FBI to provide positive identification of record subjects indexed in the III System....

  14. 28 CFR 905.1 - Definition.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Administration NATIONAL CRIME PREVENTION AND PRIVACY COMPACT COUNCIL NATIONAL FINGERPRINT FILE (NFF) PROGRAM QUALIFICATION REQUIREMENTS § 905.1 Definition. “National Fingerprint File” means a database of fingerprints, or... maintained by the FBI to provide positive identification of record subjects indexed in the III System....

  15. 28 CFR 905.1 - Definition.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Administration NATIONAL CRIME PREVENTION AND PRIVACY COMPACT COUNCIL NATIONAL FINGERPRINT FILE (NFF) PROGRAM QUALIFICATION REQUIREMENTS § 905.1 Definition. “National Fingerprint File” means a database of fingerprints, or... maintained by the FBI to provide positive identification of record subjects indexed in the III System....

  16. 28 CFR 905.1 - Definition.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Administration NATIONAL CRIME PREVENTION AND PRIVACY COMPACT COUNCIL NATIONAL FINGERPRINT FILE (NFF) PROGRAM QUALIFICATION REQUIREMENTS § 905.1 Definition. “National Fingerprint File” means a database of fingerprints, or... maintained by the FBI to provide positive identification of record subjects indexed in the III System....

  17. 28 CFR 905.1 - Definition.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Administration NATIONAL CRIME PREVENTION AND PRIVACY COMPACT COUNCIL NATIONAL FINGERPRINT FILE (NFF) PROGRAM QUALIFICATION REQUIREMENTS § 905.1 Definition. “National Fingerprint File” means a database of fingerprints, or... maintained by the FBI to provide positive identification of record subjects indexed in the III System....

  18. 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...

  19. 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...

  20. 12 CFR 212.2 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 12 Banks and Banking 2 2012-01-01 2012-01-01 false Definitions. 212.2 Section 212.2 Banks and Banking FEDERAL RESERVE SYSTEM BOARD OF GOVERNORS OF THE FEDERAL RESERVE SYSTEM MANAGEMENT OFFICIAL... million or more; (iii) A senior executive officer as that term is defined in 12 CFR 225.71(c); (iv)...

  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. [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

  3. 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

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

    PubMed Central

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

    2010-01-01

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

  5. 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

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

    PubMed Central

    Morn, Mara; Lpez-Bernardo, Elia; Cadenas, Susana; Hidalgo, Beatriz; Snchez, Ricardo; Seneca, Sara; Arenas, Joaqun; Martn, 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

  7. Orientational Changes of Crossbridges During Single Turnover of ATP

    PubMed Central

    Borejdo, J.; Akopova, I.

    2003-01-01

    Muscle contraction results from rotation of actin-bound myosin crossbridges. Crossbridges consist of the globular N-terminal catalytic domain and the ?-helical C-terminal regulatory domain containing the essential and regulatory light chains. The essential light chain exists in two isoforms, of which the larger one has a 41-amino acid extension piece added at the N-terminus. The catalytic domain is responsible for binding to actin and for setting the stage for the main force-generating event, which is a swing of the regulatory domain. We measured the kinetics of the swing associated with the turnover of a single molecule of ATP. Muscle was labeled at the regulatory domain by replacing native essential or regulatory light chain with fluorescent adducts. The rotations were measured by the anisotropy of fluorescence originating from ?400 crossbridges residing in a small volume defined by a confocal aperture of a microscope. The crossbridges were synchronized by rapid photogeneration of a stoichiometric amount of ATP. The rotations reflected dissociation from thin filaments followed by a slow reattachment. The dissociation was the same for each light chain (halftime ?120 ms) but the rate of reattachment depended on the type of light chain. The halftimes were 920 50 ms and 660 100 ms for isoforms 1 and 3 of the essential light chain, respectively. The reason that the lifetimes were so long was creation of a small amount of ATP, enough only for a single turnover of crossbridges. A model was constructed that quantitized this effect. After accounting for the slowdown, the halftimes of dissociation and attachment were 34 and 200 ms, respectively. PMID:12668452

  8. 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

  9. Rho signaling regulates pannexin 1-mediated ATP release from airway epithelia.

    PubMed

    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

    2011-07-29

    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

  10. 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

  11. 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

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

    PubMed Central

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

    2013-01-01

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

  13. Glucose Triggers ATP Secretion from Bacteria in a Growth-Phase-Dependent Manner

    PubMed Central

    Hironaka, Ippei; Iwase, Tadayuki; Sugimoto, Shinya; Okuda, Ken-ichi; Tajima, Akiko; Yanaga, Katsuhiko

    2013-01-01

    ATP modulates immune cell functions, and ATP derived from gut commensal bacteria promotes the differentiation of T helper 17 (Th17) cells in the intestinal lamina propria. We recently reported that Enterococcus gallinarum, isolated from mice and humans, secretes ATP. We have since found and characterized several ATP-secreting bacteria. Of the tested enterococci, Enterococcus mundtii secreted the greatest amount of ATP (>2 ?M/108 cells) after overnight culture. Glucose, not amino acids and vitamins, was essential for ATP secretion from E. mundtii. Analyses of energy-deprived cells demonstrated that glycolysis is the most important pathway for bacterial ATP secretion. Furthermore, exponential-phase E. mundtii and Enterococcus faecalis cells secrete ATP more efficiently than stationary-phase cells. Other bacteria, including Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus, also secrete ATP in exponential but not stationary phase. These results suggest that various gut bacteria, including commensals and pathogens, might secrete ATP at any growth phase and modulate immune cell function. PMID:23354720

  14. 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

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

    PubMed

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

    2012-01-01

    F(1)-ATPase is an ATP-driven rotary molecular motor that synthesizes ATP when rotated in reverse. To elucidate the mechanism of ATP synthesis, we imaged binding and release of fluorescently labelled ADP and ATP while rotating the motor in either direction by magnets. Here we report the binding and release rates for each of the three catalytic sites for 360 of the rotary angle. We show that the rates do not significantly depend on the rotary direction, indicating ATP synthesis by direct reversal of the hydrolysis-driven rotation. ADP and ATP are discriminated in angle-dependent binding, but not in release. Phosphate blocks ATP binding at angles where ADP binding is essential for ATP synthesis. In synthesis rotation, the affinity for ADP increases by >10(4), followed by a shift to high ATP affinity, and finally the affinity for ATP decreases by >10(4). All these angular changes are gradual, implicating tight coupling between the rotor angle and site affinities. PMID:22929779

  16. 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.

  17. 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

  18. ATP-dependent taurocholate transport by rat liver canalicular membrane vesicles.

    PubMed

    Adachi, Y; Kobayashi, H; Kurumi, Y; Shouji, M; Kitano, M; Yamamoto, T

    1991-10-01

    We conducted an experimental study to examine the possibility that ATP is involved in the mechanism by which bile acids are excreted through the liver canalicular membrane in opposing the concentration gradient. Canalicular membrane vesicles were purified from the livers of Sprague-Dawley rats, and the uptake of tritiated sodium taurocholate into canalicular membrane vesicles was determined by rapid filtration technique. Vesicle-associated sodium taurocholate was increased in the presence of ATP and ATP-regenerating system. This was also observed at a voltage-clamped condition. ATP-dependent uptake into the osmotically reactive intravesicular space was saturated with increasing concentrations of sodium taurocholate (Km = 47 mumol/L, Vmax = 270 pmoles/mg protein.20s). ATP-dependent uptake increased to the point of saturation when the sodium taurocholate concentration was 50 mumol/L and the ATP concentration was increased from 0 to 1 mmol/L (Km = 64 mumol/L). Among the several nucleotides used, ATP was a potent stimulator of transport, whereas a nonhydrolyzable analogue (i.e., adenosine 5'-[beta,gamma-imino]triphosphate) showed no effect. In addition, ATP-dependent transport was inhibited by vanadate in a dose-dependent manner. From these results it was concluded that the primary active transport of sodium taurocholate is present in hepatocellular canalicular membranes. This transport is directly dependent on ATP, and hydrolysis of gamma-phosphate of ATP is required. PMID:1916666

  19. 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

  20. 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

  1. Extracellular ATP mediates inflammatory responses in colitis via P2??7 receptor signaling

    PubMed Central

    Wan, Ping; Liu, Xiaopeng; Xiong, Yan; Ren, Yuping; Chen, Jiang; Lu, Nonghua; Guo, Yuan; Bai, Aiping

    2016-01-01

    Extracellular purinergic products, particularly ATP, have recently been implicated to regulate immune cell functions and contribute to aberrant inflammatory responses of immune diseases. However, regulation of immune responses of colitis by extracellular ATP and its main receptor, P2??7, remains to be elucidated. In the study, we induced murine colitis by feeding mice with 4% dextran sulfate sodium (DSS), and noted dramatically heightened extracellular ATP levels in colon tissues during the progression of experimental colitis. Blockade of ATP release by carbenoxolone (CBX) treatment, or promoting ATP degradation by ATP diphosphohydrolase (apyrase), decreased extracellular ATP levels in colon tissues, attenuated DSS-induced colitis, whereas inhibition of extracellular ATP degradation by sodium metatungstate (POM-1) exacerbated tissue damage in the mice with colitis. Moreover, treatment with inhibitor of P2??7 receptor, A438079, decreased NF?B activation and active caspase-1 expression in lamina propria immune cells, downregulated proinflammatory cytokine production in colon tissues, and attenuated murine colitis. Collectively, these data suggest extracellular ATP participates in regulation of inflammatory responses of experimental colitis, through P2??7 receptor and inflammasome and NF?B signaling, which provides potential alternatives to the current clinical approaches to suppress extracellular ATP-mediated immune responsiveness. PMID:26739809

  2. 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

  3. 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

  4. 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.

  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. Bacterial RTX Toxins Allow Acute ATP Release from Human Erythrocytes Directly through the Toxin Pore*

    PubMed Central

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

    2014-01-01

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

  7. 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

  8. 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

  9. Congenital antithrombin III deficiency

    MedlinePLUS

    ... from a parent with the disease. The abnormal gene leads to a low level of the antithrombin III protein. This low level of antithrombin III can cause abnormal blood clots (thrombi) that can block blood flow and damage organs. People with this condition will ...

  10. 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…

  11. Molecular mechanism for ATP-dependent closure of the K+ channel Kir6.2.

    PubMed

    John, Scott A; Weiss, James N; Xie, Lai-Hua; Ribalet, Bernard

    2003-10-01

    In the ATP-dependent K+ (KATP) channel pore-forming protein Kir6.2, mutation of three positively charged residues, R50, K185 and R201, impairs the ability of ATP to close the channel. The mutations do not change the channel open probability (Po) in the absence of ATP, supporting the involvement of these residues in ATP binding. We recently proposed that at least two of these positively charged residues, K185 and R201, interact with ATP phosphate groups to cause channel closure: the beta phosphate group of ATP interacts with K185 to initiate closure, while the alpha phosphate interacts with R201 to stabilize the channel's closed state. In the present study we replaced these three positive residues with residues of different charge, size and hydropathy. For K185 and R201, we found that charge, more than any other property, controls the interaction of ATP with Kir6.2. At these positions, replacement with another positive residue had minor effects on ATP sensitivity. In contrast, replacement of K185 with a negative residue (K185D/E) decreased ATP sensitivity much more than neutral substitutions, suggesting that an electrostatic interaction between the beta phosphate group of ATP and K185 destabilizes the open state of the channel. At R201, replacement with a negative charge (R201E) had multiple effects, decreasing ATP sensitivity and preventing full channel closure at high concentrations. In contrast, the R50E mutation had a modest effect on ATP sensitivity, and only residues such as proline and glycine that affect protein structure caused major decreases in ATP sensitivity at the R50 position. Based on these results and the recently published structure of Kir3.1 cytoplasmic domain, we propose a scheme where binding of the beta phosphate group of ATP to K185 induces a motion of the surrounding region, which destabilizes the open state, favouring closure of the M2 gate. Binding of the alpha phosphate group of ATP to R201 then stabilizes the closed state. R50 on the N-terminus controls ATP binding by facilitating the interaction of the beta phosphate group of ATP with K185 to destabilize the open state. PMID:12860923

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

    PubMed Central

    2013-01-01

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

  13. Loss of ATP Diphosphohydrolase Activity with Endothelial Cell Activation

    PubMed Central

    Robson, Simon C.; Kaczmarek, Elzbieta; Siegel, Jonathan B.; Candinas, Daniel; Koziak, Katarzyna; Millan, Maria; Hancock, Wayne W.; Bach, Fritz H.

    1997-01-01

    Quiescent endothelial cells (EC) regulate blood flow and prevent intravascular thrombosis. This latter effect is mediated in a number of ways, including expression by EC of thrombomodulin and heparan sulfate, both of which are lost from the EC surface as part of the activation response to proinflammatory cytokines. Loss of these anticoagulant molecules potentiates the procoagulant properties of the injured vasculature. An additional thromboregulatory factor, ATP diphosphohydrolase (ATPDase; designated as EC 3.6.1.5) is also expressed by quiescent EC, and has the capacity to degrade the extracellular inflammatory mediators ATP and ADP to AMP, thereby inhibiting platelet activation and modulating vascular thrombosis. We describe here that the antithrombotic effects of the ATPDase, like heparan sulfate and thrombomodulin, are lost after EC activation, both in vitro and in vivo. Because platelet activation and aggregation are important components of the hemostatic changes that accompany inflammatory diseases, we suggest that the loss of vascular ATPDase may be crucial for the progression of vascular injury. PMID:8996251

  14. ATP in iron overload-induced intracellular calcium changes.

    PubMed

    Anghileri, L J; Thouvenot, P

    1998-05-01

    The cellular iron uptake from low molecular weight iron complexes (ferric citrate, ferric lactate and ferric ATP complex) is concentration-dependent, and only a small part of the iron penetrates the cell as shown by deferoxamine treatment. A threshold of iron concentration in the cell must be reached for the iron complex-induced increase of cellular Ca2+-uptake. ATP seems to play a key role in an iron translocation that enhances the effects of the iron complexes. A non-specific and competitive iron-binding by proteins seems to act as a buffer system that reduces the iron overload effects. Calcium channel blockers have no effects on the iron complex-cell interaction or iron-induced Ca2+-uptake modification. An iron complex concentration-dependent inhibition of the CaATPase activity, and its consequent Ca2+-extrusion impairment appear as the likely cause of calcium overload. The relevance of these findings in iron overload-induced pathologies is discussed. PMID:9852309

  15. The human ATP-binding cassette (ABC) transporter superfamily.

    PubMed

    Dean, M; Rzhetsky, A; Allikmets, R

    2001-07-01

    The ATP-binding cassette (ABC) transporter superfamily contains membrane proteins that translocate a variety of substrates across extra- and intra-cellular membranes. Genetic variation in these genes is the cause of or contributor to a wide variety of human disorders with Mendelian and complex inheritance, including cystic fibrosis, neurological disease, retinal degeneration, cholesterol and bile transport defects, anemia, and drug response. Conservation of the ATP-binding domains of these genes has allowed the identification of new members of the superfamily based on nucleotide and protein sequence homology. Phylogenetic analysis is used to divide all 48 known ABC transporters into seven distinct subfamilies of proteins. For each gene, the precise map location on human chromosomes, expression data, and localization within the superfamily has been determined. These data allow predictions to be made as to potential functions or disease phenotypes associated with each protein. In this paper, we review the current state of knowledge on all human ABC genes in inherited disease and drug resistance. In addition, the availability of the complete Drosophila genome sequence allows the comparison of the known human ABC genes with those in the fly genome. The combined data enable an evolutionary analysis of the superfamily. Complete characterization of all ABC from the human genome and from model organisms will lead to important insights into the physiology and the molecular basis of many human disorders. PMID:11435397

  16. 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

  17. ATP-induced helicase slippage reveals highly coordinated subunits

    NASA Astrophysics Data System (ADS)

    Wang, Michelle D.

    2012-02-01

    Helicases are vital enzymes that carry out strand separation of duplex nucleic acids during replication, repair and recombination. T7 helicase, a model hexameric motor, has been observed to use dTTP, but not ATP, to unwind dsDNA as it translocates along ssDNA. Whether and how different subunits of the helicase coordinate their chemo-mechanical activities and DNA binding during translocation is still under debate. Here we address this question using a single-molecule approach to monitor helicase unwinding. We found that T7 helicase does in fact unwind dsDNA in the presence of ATP and that the unwinding rate is even faster than that with dTTP. However, unwinding was repeatedly interrupted by sudden slippage events, ultimately preventing unwinding over a substantial distance. This behaviour was greatly reduced with the supplement of a small amount of dTTP. These findings presented an opportunity to use nucleotide mixtures to investigate helicase subunit coordination. Our results support a model where nearly all subunits coordinate their chemo-mechanical activities and DNA binding. Such subunit coordination may be general to many ring-shaped helicases and reveals a potential mechanism for regulation of DNA unwinding during replication.

  18. Mechanosensitive ATP Release Maintains Proper Mucus Hydration of Airways

    PubMed Central

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

    2013-01-01

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

  19. Methodological problems of direct bioluminescent ATP assay in platelets and erythrocytes.

    PubMed

    Girotti, S; Ferri, E; Cascione, M L; Comuzio, S; Mazzuca, A; Orlandini, A; Breccia, A

    1989-07-01

    Direct bioluminescent ATP determination in platelets and erythrocytes involves the study of different parameters which are discussed here. Some parameters are linked to the bioluminescent reaction and to the analyte (ATP); others have regard to the biological matrix. The composition of bioluminescent reagents and the preparation and conservation of the ATP standard, also in the presence of excipients, are among the first given. Matrix problems involve cell characteristics related to age and form, lysis resistance and the possible formation of aggregates (platelets) that may inhibit the complete release of ATP. For these reasons we used the most efficient ATP release agent with the lowest inhibitory effect on luciferase. The data obtained correlate well with a bioluminescent method requiring extraction with ethanol/EDTA, and therefore more time, for ATP determination in platelets and erythrocytes. PMID:2801244

  20. Toxic and carcinogenic effects of parenteral and percutaneous ATP and its iron complex.

    PubMed

    Anghileri, Leopold J; Mayayo, Emilio; Domingo, Jos L; Thouvenot, Pierre

    2002-08-01

    The long term effects of percutaneous, subcutaneous and intraperitoneal administration of sodium-ATP (NaATP) and ferric iron-ATP (FeATP) were studied on an animal model. Both compounds induce a generalized lymphoadenitis which in the case of FeATP led to lymphomas. The analytical study of the involved target tissues showed intracellular composition changes that result from the impairment of the cell membrane permeability. The morbidity and mortality rate were higher with FeATP which seems to be the result of two different, in intensity and duration, interactions with the cell plasma membrane. The influence of the changes in cellular calcium homeostasis, and its relationship with carcinogenesis and immuno response are discussed. PMID:12173248

  1. Role of iron-ATP complex in lymphocyte proliferation and infiltration.

    PubMed

    Anghileri, Leopold J; Ortal, Emilio Mayayo; Thouvenot, Pierre

    2005-01-01

    The roles of sodium-ATP (NaATP) and ferric-ATP complex (FeATP) on the proliferation and infiltration of lymphocytes have been studied by evaluation of the hypertrophy and histopathologic examination of spleen and liver, as well of the modifications in the elemental balance of iron, calcium, magnesium, and phosphorus. The results showed that in the implicated biochemical processes, calcium and magnesium have a principal role. An in vitro study on a cell model has permited one to evaluate the effects of deferoxamine, a known iron chelator and inhibitor of human lymphocyte proliferation, on FeATP-modified cellular calcium fluxes. These findings showing a reduced 45Ca2+ uptake in the presence of deferoxamine appear to indicate that in vivo the chelation of blood-borne iron by ATP might play a key role in proliferation and infiltration of lymphocytes, leading to lymphoma development. PMID:16327061

  2. Nonsynaptic and nonvesicular ATP release from neurons and relevance to neuronglia 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

  3. SLC17A9 Protein Functions as a Lysosomal ATP Transporter and Regulates Cell Viability*

    PubMed Central

    Cao, Qi; Zhao, Kexin; Zhong, Xi Zo; Zou, Yuanjie; Yu, Haichuan; Huang, Peng; Xu, Tian-Le; Dong, Xian-Ping

    2014-01-01

    Lysosomes contain abundant ATP, which is released through lysosomal exocytosis following exposure to various stimuli. However, the molecular mechanisms underlying lysosomal ATP accumulation remain unknown. The vesicular nucleotide transporter, also known as solute carrier family 17 member 9 (SLC17A9), has been shown to function in ATP transport across secretory vesicles/granules membrane in adrenal chromaffin cells, T cells, and pancreatic cells. Here, using mammalian cell lines, we report that SLC17A9 is highly enriched in lysosomes and functions as an ATP transporter in those organelles. SLC17A9 deficiency reduced lysosome ATP accumulation and compromised lysosome function, resulting in cell death. Our data suggest that SLC17A9 activity mediates lysosomal ATP accumulation and plays an important role in lysosomal physiology and cell viability. PMID:24962569

  4. 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

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

    SciTech Connect

    Jones, P.M.

    1981-06-01

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

  6. 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 ?-barrel with an ?-helix partially occupying the central pore. To understand ATP permeation through VDAC, we solved the crystal structure of mouse VDAC1 (mVDAC1) in the presence of ATP, revealing a low-affinity binding site. Guided by these coordinates, we initiated hundreds of molecular dynamics (MD) simulations to construct a Markov State Model (MSM) of ATP permeation. These simulations indicate that ATP flows through VDAC using multiple pathways, consistent with our structural data and experimentally determined physiological rates. PMID:24908397

  7. 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

  8. Enzymatic conversion of ATP to adenosine contributes to ATP-induced inhibition of glutamate release in rat medullary dorsal horn neurons.

    PubMed

    Choi, In-Sun; Cho, Jin-Hwa; Lee, Maan-Gee; Jang, Il-Sung

    2015-06-01

    Purine nucleotides, such as ATP and ADP, activate ionotropic P2X and metabotropic P2Y receptors to regulate neurotransmitter release in the peripheral as well as central nervous system. Here we report another type of ATP-induced presynaptic modulation of glutamate release in rat medullary dorsal horn neurons. Glutamatergic excitatory postsynaptic currents (EPSCs) induced by electrical stimulation of trigeminal tract were recorded from horizontal brain stem slices using a whole-cell patch clamp technique. ATP decreased the amplitude of glutamatergic EPSCs in a reversible and concentration dependent manner and increased the paired-pulse ratio. In addition, ATP reduced the frequency of miniature EPSCs without affecting the current amplitude, suggesting that ATP acts presynaptically to reduce the probability of glutamate release. The ATP-induced decrease in glutamatergic EPSCs was not affected by P2X and P2Y receptor antagonists, but was completely blocked by DPCPX, a selective adenosine A1 receptor antagonist. The ATP-induced decrease in glutamatergic EPSCs was also inhibited by an inhibitor of tissue nonspecific alkaline phosphatase but not by inhibitors of other enzymes such as ecto-nucleoside triphosphate diphosphohydrolases and ecto-5'-nucleotidases. The results suggest that exogenously applied purine nucleotides are rapidly converted to adenosine by specific enzymes, and subsequently act on presynaptic A1 receptors to inhibit glutamate release from primary afferent terminals. This type of modulation mediated by purine nucleotides may play an important role in regulating nociceptive transmission from orofacial tissues. PMID:25656480

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

    SciTech Connect

    Masker, W.E.

    1982-07-01

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

  10. 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

  11. 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 M 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

  12. ATP testing: an anecdotal look at its use in an office-based plastic surgery setting.

    PubMed

    Kurley, Barbara

    2014-01-01

    Proper cleaning of surgical instruments prior to sterilization is an essential objective in the surgical setting. Testing for the presence of adenosine triphosphate (ATP) on a cleaned instrument can help determine if it meets cleanliness requirements for sterilization. A program was piloted in our facility by using a commercial ATP testing system. In this article, our experience with the evaluation of available ATP testing systems, the implementation processes we used, and conclusions drawn from our procedures and results are described. PMID:25461633

  13. 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

  14. 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

  15. 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

  16. 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

  17. ATP synthase in mycobacteria: special features and implications for a function as drug target.

    PubMed

    Lu, Ping; Lill, Holger; Bald, Dirk

    2014-07-01

    ATP synthase is a ubiquitous enzyme that is largely conserved across the kingdoms of life. This conservation is in accordance with its central role in chemiosmotic energy conversion, a pathway utilized by far by most living cells. On the other hand, in particular pathogenic bacteria whilst employing ATP synthase have to deal with energetically unfavorable conditions such as low oxygen tensions in the human host, e.g. Mycobacterium tuberculosis can survive in human macrophages for an extended time. It is well conceivable that such ATP synthases may carry idiosyncratic features that contribute to efficient ATP production. In this review genetic and biochemical data on mycobacterial ATP synthase are discussed in terms of rotary catalysis, stator composition, and regulation of activity. ATP synthase in mycobacteria is of particular interest as this enzyme has been validated as a target for promising new antibacterial drugs. A deeper understanding of the working of mycobacterial ATP synthase and its atypical features can provide insight in adaptations of bacterial energy metabolism. Moreover, pinpointing and understanding critical differences as compared with human ATP synthase may provide input for the design and development of selective ATP synthase inhibitors as antibacterials. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. PMID:24513197

  18. 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

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

    PubMed

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

    2008-06-01

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

  20. 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

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

    PubMed Central

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

    2014-01-01

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

  2. 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

  3. 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

  4. ATP-containing vesicles in stria vascular marginal cell cytoplasms in neonatal rat cochlea are lysosomes.

    PubMed

    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-- 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??M 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 Ca(2+)-dependent lysosomal exocytosis. PMID:26864824

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

    PubMed Central

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

    2014-01-01

    In cervical cancer, HPV infection and disruption of mechanisms involving cell growth, differentiation, and apoptosis are strictly linked with tumor progression and invasion. Tumor microenvironment is ATP and adenosine rich, suggesting a role for purinergic signaling in cancer cell growth and death. Here we investigate the effect of extracellular ATP on human cervical cancer cells. We find that extracellular ATP itself has a small cytotoxic effect, whereas adenosine formed from ATP degradation by ectonucleotidases is the main factor responsible for apoptosis induction. The level of P27 receptor seemed to define the main cytotoxic mechanism triggered by ATP, since ATP itself eliminated a small subpopulation of cells that express high P27 levels, probably through its activation. Corroborating these data, blockage or knockdown of P27 only slightly reduced ATP cytotoxicity. On the other hand, cell viability was almost totally recovered with dipyridamole, an adenosine transporter inhibitor. Moreover, ATP-induced apoptosis and signalingp53 increase, AMPK activation, and PARP cleavageas well as autophagy induction were also inhibited by dipyridamole. In addition, inhibition of adenosine conversion into AMP also blocked cell death, indicating that metabolization of intracellular adenosine originating from extracellular ATP is responsible for the main effects of the latter in human cervical cancer cells. PMID:25103241

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

    PubMed Central

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

    2013-01-01

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

  7. The functional design of the rotary enzyme ATP synthase is consistent with maximum entropy production

    NASA Astrophysics Data System (ADS)

    Dewar, R. C.; Jureti?, D.; upanovi?, P.

    2006-10-01

    We show that the molecular motor ATP synthase has evolved in accordance with the statistical selection principle of Maximum Shannon Entropy and one of its corollaries, Maximum Entropy Production. These principles predict an optimal angular position for the ATP-binding transition close to the experimental value; an inverse relation between the optimal gearing ratio and the proton motive force ( pmf); optimal operation at an inflection point in the curve of ATP synthesis rate versus pmf, enabling rapid metabolic control; and a high optimal free energy conversion efficiency. Our results suggest a statistical interpretation for the evolutionary optimization of ATP synthase function.

  8. A possible subcellular mechanism underlying the "French paradox": the opening of mitochondrial K(ATP) channels.

    PubMed

    Aiello, Ernesto A; Cingolani, Horacio E

    2011-10-01

    A reduction in the risk of coronary heart disease has been associated to moderate red wine consumption. We tested whether a nonalcoholic red wine extract would open mitochondrial K(ATP) channels in guinea pig myocytes. The opening of mitochondrial K(ATP) channels was assessed by endogenous flavoprotein fluorescence. Red wine extract (100 ?gmL(-1)) increased flavoprotein oxidation (10.9% 1.2%, n = 20). This effect was prevented by the mitochondrial K(ATP) channel blocker, 5-hydroxydecanoate (500 molL(-1); 0.3% 1.1%, n = 13), confirming the hypothesis that red wine extract opens mitochondrial K(ATP) channels. PMID:21999300

  9. Glycine Uptake into Barley Mesophyll Vacuoles Is Regulated but Not Energized by ATP 1

    PubMed Central

    Goerlach, Joern; Willms-Hoff, Indra

    1992-01-01

    [U-14C]glycine uptake into barley (Hordeum vulgare cv Hasso) vacuoles was investigated. Glycine (2 millimolar) transport was stimulated two- to fourfold by NaATP. Stimulation was saturable with respect to ATP (1 millimolar) and linear up to 20 millimolar glycine. Stimulation by NaATP was suppressed by Mg2+ in equimolar amounts. Neither MgATP nor Mg-inorganic pyrophosphate had any effect on basal transport rate. Thus, the proton motive force can be excluded as the driving force. Uncouplers (valinomycine/carbonylcyanide-m-chlorophenylhydrazone) inhibited the basal rate up to 30% but had no influence on NaATP-stimulated uptake. Vanadate had no effect on either basal or NaATP-stimulated uptake. Nonhydrolyzable ATP analogs (adenylyl(?, ?-methylen)-diphosphate or adenylyl-imidodiphosphate) stimulated comparable to NaATP. Other nucleotides (UTP, ADP) had no effect. Some evidence exists that other amino acids (arginine, alanine, isoleucine, phenylalanine) are transported to a certain extent by a similar mechanism. The results indicate a high capacity channel-like translocator that is regulated but not energized by ATP. PMID:16668840

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

    PubMed

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

    2013-12-01

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

  11. 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

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

    PubMed

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

    2014-08-01

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

  13. 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

  14. 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

  15. Two ATP Binding Cassette G Transporters, Rice ATP Binding Cassette G26 and ATP Binding Cassette G15, Collaboratively Regulate Rice Male Reproduction.

    PubMed

    Zhao, Guochao; Shi, Jianxin; Liang, Wanqi; Xue, Feiyang; Luo, Qian; Zhu, Lu; Qu, Guorun; Chen, Mingjiao; Schreiber, Lukas; Zhang, Dabing

    2015-11-01

    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

  16. Antithrombin III blood test

    MedlinePLUS

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

  17. 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

  18. Cdc123, a Cell Cycle Regulator Needed for eIF2 Assembly, Is an ATP-Grasp Protein with Unique Features.

    PubMed

    Panvert, Michel; Dubiez, Etienne; Arnold, Lea; Perez, Javier; Mechulam, Yves; Seufert, Wolfgang; Schmitt, Emmanuelle

    2015-09-01

    Eukaryotic initiation factor 2 (eIF2), a heterotrimeric guanosine triphosphatase, has a central role in protein biosynthesis by supplying methionylated initiator tRNA to the ribosomal translation initiation complex and by serving as a target for translational control in response to stress. Recent work identified a novel step indispensable for eIF2 function: assembly of eIF2 from its three subunits by the cell proliferation protein Cdc123. We report the first crystal structure of a Cdc123 representative, that from Schizosaccharomyces pombe, both isolated and bound to domain III of Saccharomyces cerevisiae eIF2?. The structures show that Cdc123 resembles enzymes of the ATP-grasp family. Indeed, Cdc123 binds ATP-Mg(2+), and conserved residues contacting ATP-Mg(2+) are essential for Cdc123 to support eIF2 assembly and cell viability. A docking of eIF2?? onto Cdc123, combined with genetic and biochemical experiments, allows us to propose a model explaining how Cdc123 participates in the biogenesis of eIF2 through facilitating assembly of eIF2? to eIF2?. PMID:26211610

  19. Vacuolar ATPases, like F sup 1 ,F sup 0 -ATPases, show a strong dependence of the reaction velocity on the binding of more than one ATP per enzyme

    SciTech Connect

    Kasho, V.N.; Boyer, P.D. )

    1989-11-01

    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 F{sub 1}, F{sub 0}-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 F{sub 1},F{sub 0}-ATPases. The extent of reversal of bound ATP hydrolysis to bound ADP and P{sub i} as medium ATP concentration was lowered was determined by {sup 18}O-exchange measurements for yeast and neurospora vacuolar ATPases. The results show a pronounced increase in the extent of water oxygen incorporation into the P{sub i} formed as ATP concentration is decreased to the micromolar range. The F{sub 1},F{sub 0}-ATPase from neurospora mitochondria showed an event more pronounced modulation, similar to that of other F{sub 1}-type ATPases. The vacuolar ATPases thus appear to have a catalytic mechanism quite analogous to that of the F{sub 1},F{sub 0}-ATPases.

  20. Temporal Phosphoproteome Dynamics Induced by an ATP Synthase Inhibitor Citreoviridin.

    PubMed

    Hu, Chia-Wei; Hsu, Chia-Lang; Wang, Yu-Chao; Ishihama, Yasushi; Ku, Wei-Chi; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

    2015-12-01

    Citreoviridin, one of toxic mycotoxins derived from fungal species, can suppress lung cancer cell growth by inhibiting the activity of ectopic ATP synthase, but has limited effect on normal cells. However, the mechanism of citreoviridin triggering dynamic molecular responses in cancer cells remains unclear. Here, we performed temporal phosphoproteomics to elucidate the dynamic changes after citreoviridin treatment in cells and xenograft model. We identified a total of 829 phosphoproteins and demonstrated that citreoviridin treatment affects protein folding, cell cycle, and cytoskeleton function. Furthermore, response network constructed by mathematical modeling shows the relationship between the phosphorylated heat shock protein 90 ? and mitogen-activated protein kinase signaling pathway. This work describes that citreoviridin suppresses cancer cell growth and mitogen-activated protein kinase/extracellular signal-regulated kinase signaling by site-specific dephosphorylation of HSP90AB1 on Serine 255 and provides perspectives in cancer therapeutic strategies. PMID:26503892

  1. 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.

  2. ATP-dependent chromatin remodeling in T cells

    PubMed Central

    Wurster, Andrea L.; Pazin, Michael J.

    2012-01-01

    One of the best studied systems for mammalian chromatin remodeling is transcriptional regulation during T cell development. The variety of these studies have led to important findings in T cell gene regulation and cell fate determination. Importantly, these findings have also advanced our knowledge of the function of remodeling enzymes in mammalian gene regulation. In this review, first we briefly present biochemical/cell-free analysis of 3 types of ATP dependent remodeling enzymes (SWI/SNF, Mi2, and ISWI), to construct an intellectual framework to understand how these enzymes might be working. Second, we compare and contrast the function of these enzymes, during early (thymic) and late (peripheral) T cell development. Finally, we examine some of the gaps in our present understanding. PMID:21999456

  3. 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

  4. 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

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

    PubMed

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

    2004-06-01

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

  6. Ca2+ responses to ATP via purinoceptors in the early embryonic chick retina.

    PubMed Central

    Sugioka, M; Fukuda, Y; Yamashita, M

    1996-01-01

    1. The action of adenosine triphosphate on cytoplasmic Ca2+ concentration ([Ca2+]i) was studied in the retinal cell of early embryonic chicks with fura-2 fluorescence measurements. The fluorescence was measured from the whole neural retina dissected from chick embryos at embryonic day three (E3). 2. Bath application of ATP (> or = 30 microM; EC50, 128 microM) raised [Ca2+]i by the release of Ca2+ from intracellular Ca2+ stores, since the Ca2+ response to ATP occurred even in a Ca(2+)-free medium. 3. The Ca2+ response to ATP was mediated by P2U purinoceptors. An agonist for P2U purinoceptors, uridine triphosphate (UTP), evoked Ca2+ rises more potently (> or = 3 microM; EC50, 24 microM) than ATP. Agonists for P2X purinoceptors, alpha, beta-methylene ATP and beta, gamma-methylene ATP, or an agonist for P2Y purinoceptors, 2-methylthio ATP (500 microM each), caused no Ca2+ response. Suramin (100 microM) and Reactive Blue 2 (50 microM) almost completely blocked the Ca2+, responses to 500 microM ATP and 200 microM UTP. 4. The developmental profile of the Ca2+ response to ATP was studied from E3 to E13. The Ca2+ response to ATP was largest at E3, drastically declined towards E8 and decreased further until E11-13. 5. These results suggest that the Ca2+ mobilization by ATP via P2U purinoceptors is characteristic of early embryonic retinal cells. PMID:8799905

  7. 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

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

    PubMed Central

    Oliveira, Olga; Ferreira, Snia; Reis, Maria Jlia; Oliveira, Jos Carlos; Correia-de-S, Paulo

    2013-01-01

    Background Nowadays, there is a considerable bulk of evidence showing that ATP has a prominent role in the regulation of human urinary bladder function and in the pathophysiology of detrusor overactivity. ATP mediates nonadrenergic-noncholinergic detrusor contractions in overactive bladders. In vitro studies have demonstrated that uroepithelial cells and cholinergic nerves from overactive human bladder samples (OAB) release more ATP than controls. Here, we compared the urinary ATP concentration in samples collected non-invasively from OAB women with detrusor overactivity and age-matched controls. Methods Patients with neurologic diseases, history of malignancy, urinary tract infections or renal impairment (creatinine clearance <70 ml/min) were excluded. All patients completed a 3-day voiding diary, a 24 h urine collection and blood sampling to evaluate creatinine clearance. Urine samples collected during voluntary voids were immediately freeze-preserved for ATP determination by the luciferin-luciferase bioluminescence assay; for comparison purposes, samples were also tested for urinary nerve growth factor (NGF) by ELISA. Results The urinary content of ATP, but not of NGF, normalized to patients urine creatinine levels (ATP/Cr) or urinary volume (ATP.Vol) were significantly (P<0.05) higher in OAB women with detrusor overactivity (n?=?34) than in healthy controls (n?=?30). Significant differences between the two groups were still observed by boosting urinary ATP/Cr content after water intake, but these were not detected for NGF/Cr. In OAB patients, urinary ATP/Cr levels correlated inversely with mean voided volumes determined in a 3-day voiding diary. Conclusion A high area under the receiver operator characteristics (ROC) curve (0.741; 95% CI 0.620.86; P<0.001) is consistent with urinary ATP/Cr being a highly sensitive dynamic biomarker for assessing detrusor overactivity in women with OAB syndrome. PMID:23741373

  9. 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

  10. 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

  11. Determination of the ATP Affinity of the Sarcoplasmic Reticulum Ca(2+)-ATPase by Competitive Inhibition of [?-(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 [?-(32)P]2',3'-O-(2,4,6-trinitrophenyl)-8-azido-adenosine 5'-triphosphate ([?-(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 [?-(32)P]TNP-8N3-ATP photolabel, as well as its application in ATP affinity measurements. PMID:26695037

  12. 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

  13. [Effect of adenosine-5'-O-(beta, gamma-dichloromethane)triphosphate on ATP-activated currents in sensory neurons in rats].

    PubMed

    Marchenko, S M; Obukhov, A G; Volkova, T M

    1988-07-01

    The action of beta,gamma-dihalogenmethane derivatives of ATP on ATP receptors has been studied in the sensory neurons of rats. beta,gamma-Dichloromethane-ATP and beta,gamma-dibromomethane-ATP are competitive blockers and beta,gamma-difluoromethane-ATP is a partial agonist of ATP receptors. At present, beta,gamma-dichloromethane-ATP is the most efficient of the known competitive blockers of these receptors (Ki = 2.10(-5) M). The data obtained confirm that beta- and gamma-phosphates of ATP play an important role in the activation of ATP receptors. PMID:3255302

  14. 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 96.42%. This method of ATP measurement was simple, rapid, reliable, and was unburdened of some limitations other methods may have. PMID:26210584

  15. Action PotentialEnhanced ATP Release From Taste Cells Through Hemichannels

    PubMed Central

    Murata, Yoshihiro; Yasuo, Toshiaki; Yoshida, Ryusuke; Obata, Kunihiko; Yanagawa, Yuchio; Margolskee, Robert F.

    2010-01-01

    Only some taste cells fire action potentials in response to sapid stimuli. Type II taste cells express many taste transduction molecules but lack well-elaborated synapses, bringing into question the functional significance of action potentials in these cells. We examined the dependence of adenosine triphosphate (ATP) transmitter release from taste cells on action potentials. To identify type II taste cells we used mice expressing a green fluorescence protein (GFP) transgene from the ?-gustducin promoter. Action potentials were recorded by an electrode basolaterally attached to a single GFP-positive taste cell. We monitored ATP release from gustducin-expressing taste cells by collecting the electrode solution immediately after tastant-stimulated action potentials and using a luciferase assay to quantify ATP. Stimulation of gustducin-expressing taste cells with saccharin, quinine, or glutamate on the apical membrane increased ATP levels in the electrode solution; the amount of ATP depended on the firing rate. Increased spontaneous firing rates also induced ATP release from gustducin-expressing taste cells. ATP release from gustducin-expressing taste cells was depressed by tetrodotoxin and inhibited below the detection limit by carbenoxolone. Our data support the hypothesis that action potentials in taste cells responsive to sweet, bitter, or umami tastants enhance ATP release through pannexin 1, not connexin-based hemichannels. PMID:20519578

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

    PubMed

    Holemans, Tine; Srensen, Danny Mollerup; van Veen, Sarah; Martin, Shaun; Hermans, Diane; Kemmer, Gerdi Christine; Van den Haute, Chris; Baekelandt, Veerle; Gnther Pomorski, Thomas; Agostinis, Patrizia; Wuytack, Frank; Palmgren, Michael; Eggermont, Jan; Vangheluwe, Peter

    2015-07-21

    ATP13A2 is a lysosomal P-type transport ATPase that has been implicated in Kufor-Rakeb syndrome and Parkinson's disease (PD), providing protection against ?-synuclein, Mn(2+), and Zn(2+) toxicity in various model systems. So far, the molecular function and regulation of ATP13A2 remains undetermined. Here, we demonstrate that ATP13A2 contains a unique N-terminal hydrophobic extension that lies on the cytosolic membrane surface of the lysosome, where it interacts with the lysosomal signaling lipids phosphatidic acid (PA) and phosphatidylinositol(3,5)bisphosphate [PI(3,5)P2]. We further demonstrate that ATP13A2 accumulates in an inactive autophosphorylated state and that PA and PI(3,5)P2 stimulate the autophosphorylation of ATP13A2. In a cellular model of PD, only catalytically active ATP13A2 offers cellular protection against rotenone-induced mitochondrial stress, which relies on the availability of PA and PI(3,5)P2. Thus, the N-terminal binding of PA and PI(3,5)P2 emerges as a key to unlock the activity of ATP13A2, which may offer a therapeutic strategy to activate ATP13A2 and thereby reduce ?-synuclein toxicity or mitochondrial stress in PD or related disorders. PMID:26134396

  17. Synergistic binding of glucose and aluminium ATP to hexokinase from Saccharomyces cerevisiae.

    PubMed

    Woolfitt, A R; Kellett, G L; Hoggett, J G

    1988-08-10

    The binding of glucose, AlATP and AlADP to the monomeric and dimeric forms of the native yeast hexokinase PII isoenzyme and to the proteolytically modified SII monomeric form was monitored at pH 6.7 by the concomitant quenching of intrinsic protein fluorescence. No fluorescence changes were observed when free enzyme was mixed with AlATP at concentrations up to 7500 microM. In the presence of saturating concentrations of glucose, the maximal quenching of fluorescence induced by AlATP was between 1.5 and 3.5% depending on species, and the average value of [L]0.5, the concentration of ligand at half-saturation, over all monomeric species was 0.9 +/- 0.4 microM. The presence of saturating concentrations of AlATP diminished [L]0.5 for glucose binding by between 260- and 670-fold for hexokinase PII and SII monomers, respectively (dependent on the ionic strength), and by almost 4000-fold for PII dimer. The data demonstrate extremely strong synergistic interactions in the binding of glucose and AlATP to yeast hexokinase, arising as a consequence of conformational changes in the free enzyme induced by glucose and in enzyme-glucose complex induced by AlATP. The synergistic interactions of glucose and AlATP are related to their kinetic synergism and to the ability of AlATP to act as a powerful inhibitor of the hexokinase reaction. PMID:3042027

  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. [Activity of ATP-dependent protease and steroid metabolism in the human placenta].

    PubMed

    Ishii, A; Hamada, H; Hayashi, K; Honda, H; Maehara, T; Watabe, S; Yago, N

    1992-01-01

    Placenta play the important role of nourishment of fetus and for the regulation of fetal and maternal steroid metabolism. Steroid metabolism is synthesized in mitochondria and levels of enzymes in steroid metabolism are regulated by the rate of synthesis and degradation of these enzymes. Therefore, we studied protease in human placental mitochondria to clarify regulation mechanism of steroid-synthesizing enzymes. 50 micrograms of human early and term placenta homogenate, proteins were analyzed by immunoblotting technique after SDS-polyacrylamid gel electrophoresis. ATP-dependent protease was detected using antiserum raised against purified bovine adrenocortical ATP-dependent protease and avidin-biotin complex method. ATP-dependent protease activity was assayed using 14C-Caseins a substrate. Five cell fractions of homogenate placenta were electrophorated and antibody-stained using ATP-dependent protease, cytochrome P-450 scc and adrenodoxin antibody after the blotting, and the quantity was analyzed by the densitometry and the method of De Douve. We had the following results: 1) ATP dependent protease is present in human placenta and localized in mitochondria. 2) ATP dependent protease decomposes adrenodoxin reductase in human placenta. 3) ATP dependent protease present in almost same consistent in early and term placenta and there is no significant difference. It is suggested as follows that the ATP dependent protease in human placenta participates in the regulation of steroid hormone through the metabolism of steroid synthetic enzyme. PMID:1299082

  20. 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.

  1. Specific requirement for ATP at an early step of in vitro transcription of human mitochondrial DNA

    SciTech Connect

    Narasimhan, N.; Attardi, G.

    1987-06-01

    The ATP concentrations allowing transcription of heavy- and light-strand of human mtDNA in a HeLa cell mitochrondrial lysate were found to cover a broad range, with a maximum around 2.5 mM, and with reproducible differences in the ATP response curves for the two transcription events. Direct measurements showed that nonspecific ATP degradation during the assay did not account for the high ATP requirement. 5'-Adenylyl imidodiphosphate (p(NH)ppA), an ATP analog with a nonhydrolyzable ..beta..-..gamma.. bond, was unable to substitute for ATP in supporting mtDNA transcription but greatly stimulated this transcription in the presence of a low concentration of exogenous APT, measured with (/sup 32/P)-labeled nucleotides. Evidence was obtained indicating that p(NH)ppA did not support an early event in mtDNA transcription (formation of preinitiation complex or initiation), whereas this analog could substitute effectively for ATP in the subsequent elongation steps. These results pointed to a specific requirement for ATP at an early step of the transcription process.

  2. 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

  3. 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

  4. 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 ?/? subunits on the F1 sector of the enzyme. Molecular interaction of peptides at the ? 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

  5. 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

  6. 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...

  7. 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)

  8. A SPECTROPHOTOMETRIC ASSAY TO MEASURE RUBISCO ACTIVASE ACTIVATION ACTIVITY UNDER VARYING ATP:ADP RATIOS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ratio of ATP to ADP in the stroma is an important regulatory mechanism for controlling the activation state of Rubisco via Rubisco activase (activase). Understanding the response of activase to a varying ATP:ADP ratio should reveal insights into the regulation of photosynthesis. However, the cur...

  9. On the Mg(2+) binding site of the ? subunit from bacterial F-type ATP synthases.

    PubMed

    Krah, Alexander; Takada, Shoji

    2015-10-01

    F-type ATP synthases, central energy conversion machines of the cell synthesize adenosine triphosphate (ATP) using an electrochemical gradient across the membrane and, reversely, can also hydrolyze ATP to pump ions across the membrane, depending on cellular conditions such as ATP concentration. To prevent wasteful ATP hydrolysis, mammalian and bacterial ATP synthases possess different regulatory mechanisms. In bacteria, a low ATP concentration induces a conformational change in the ? subunit from the down- to up-states, which inhibits ATP hydrolysis. Moreover, the conformational change of the ? subunit depends on Mg(2+) concentration in some bacteria such as Bacillus subtilis, but not in others. This diversity makes the ? subunit a potential target for antibiotics. Here, performing molecular dynamics simulations, we identify the Mg(2+) binding site in the ? subunit from B. subtilis as E59 and E86. The free energy analysis shows that the first-sphere bi-dentate coordination of the Mg(2+) ion by the two glutamates is the most stable state. In comparison, we also clarify the reason for the absence of Mg(2+) dependency in the ? subunit from thermophilic Bacillus PS3, despite the high homology to that from B. subtilis. Sequence alignment suggests that this Mg(2+) binding motif is present in the ? subunits of some pathogenic bacteria. In addition we discuss strategies to stabilize an isolated ? subunit carrying the Mg(2+) binding motif by site directed mutagenesis, which also can be used to crystallize Mg(2+) dependent ? subunits in future. PMID:26028301

  10. 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…

  11. 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.

  12. 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

  13. 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…

  14. 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.

  15. Regulation of calreticulin-major histocompatibility complex (MHC) class I interactions by ATP.

    PubMed

    Wijeyesakere, Sanjeeva Joseph; Gagnon, Jessica K; Arora, Karunesh; Brooks, Charles L; Raghavan, Malini

    2015-10-13

    The MHC class I peptide loading complex (PLC) facilitates the assembly of MHC class I molecules with peptides, but factors that regulate the stability and dynamics of the assembly complex are largely uncharacterized. Based on initial findings that ATP, in addition to MHC class I-specific peptide, is able to induce MHC class I dissociation from the PLC, we investigated the interaction of ATP with the chaperone calreticulin, an endoplasmic reticulum (ER) luminal, calcium-binding component of the PLC that is known to bind ATP. We combined computational and experimental measurements to identify residues within the globular domain of calreticulin, in proximity to the high-affinity calcium-binding site, that are important for high-affinity ATP binding and for ATPase activity. High-affinity calcium binding by calreticulin is required for optimal nucleotide binding, but both ATP and ADP destabilize enthalpy-driven high-affinity calcium binding to calreticulin. ATP also selectively destabilizes the interaction of calreticulin with cellular substrates, including MHC class I molecules. Calreticulin mutants that affect ATP or high-affinity calcium binding display prolonged associations with monoglucosylated forms of cellular MHC class I, delaying MHC class I dissociation from the PLC and their transit through the secretory pathway. These studies reveal central roles for ATP and calcium binding as regulators of calreticulin-substrate interactions and as key determinants of PLC dynamics. PMID:26420867

  16. 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

  17. Intracellular ATP Decrease Mediates NLRP3 Inflammasome Activation upon Nigericin and Crystal Stimulation.

    PubMed

    Nomura, Johji; So, Alexander; Tamura, Mizuho; Busso, Nathalie

    2015-12-15

    Activation of the nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome initiates an inflammatory response, which is associated with host defense against pathogens and the progression of chronic inflammatory diseases such as gout and atherosclerosis. The NLRP3 inflammasome mediates caspase-1 activation and subsequent IL-1? processing in response to various stimuli, including extracellular ATP, although the roles of intracellular ATP (iATP) in NLRP3 activation remain unclear. In this study, we found that in activated macrophages artificial reduction of iATP by 2-deoxyglucose, a glycolysis inhibitor, caused mitochondrial membrane depolarization, leading to IL-1? secretion via NLRP3 and caspase-1 activation. Additionally, the NLRP3 activators nigericin and monosodium urate crystals lowered iATP through K(+)- and Ca(2+)-mediated mitochondrial dysfunction, suggesting a feedback loop between iATP loss and lowering of mitochondrial membrane potential. These results demonstrate the fundamental roles of iATP in the maintenance of mitochondrial function and regulation of IL-1? secretion, and they suggest that maintenance of the intracellular ATP pools could be a strategy for countering NLRP3-mediated inflammation. PMID:26546608

  18. Synchronous release of ATP and neurotransmitter within milliseconds of a motor nerve impulse in the frog.

    PubMed

    Silinsky, E M; Redman, R S

    1996-05-01

    1. It has been suggested that ATP is released together with the neurotransmitter acetylcholine (ACh) and, after hydrolysis to adenosine, is the primary physiological mediator of prejunctional neuromuscular depression. To evaluate whether ATP is released with sufficient rapidity to mediate prejunctional depression, outside-out patches containing both ATP-gated and ACh-gated ion channels were made from acutely dissociated guinea-pig sympathetic neurons and used to detect the co-release of nucleotide and neurotransmitter in frog cutaneous pectoris nerve-muscle preparations. 2. In a normal bathing solution in which muscle nicotinic receptors were blocked, a single stimulus to the motor nerve produced channel openings in the detector patch characteristic of both ATP and ACh. 3. In the remaining experiments, preparations were treated with sufficient hexamethonium (200 microM) to block nicotinic responses in the detector patch. In these experiments, a single temporally isolated nerve impulse caused the synchronous opening of ATP-gated channels in the detector patch with a latency of < 5 ms when patches were placed within 10 microns of the motor nerve ending. This multichannel phasic response was followed by trail of discrete channel openings characteristic of ATP-gated channels. 4. The selective ATP antagonist suramin (50 microM) reversibly eliminated the response to nerve stimulation. 5. The results suggest that ATP is released synchronously together with the neurotransmitter ACh in response to an individual nerve impulse and with a brief latency characteristic of quantal release from synaptic vesicles. PMID:8734992

  19. Structure and Mechanism of Soybean ATP Sulfurylase and the Committed Step in Plant Sulfur Assimilation*

    PubMed Central

    Herrmann, Jonathan; Ravilious, Geoffrey E.; McKinney, Samuel E.; Westfall, Corey S.; Lee, Soon Goo; Baraniecka, Patrycja; Giovannetti, Marco; Kopriva, Stanislav; Krishnan, Hari B.; Jez, Joseph M.

    2014-01-01

    Enzymes of the sulfur assimilation pathway are potential targets for improving nutrient content and environmental stress responses in plants. The committed step in this pathway is catalyzed by ATP sulfurylase, which synthesizes adenosine 5?-phosphosulfate (APS) from sulfate and ATP. To better understand the molecular basis of this energetically unfavorable reaction, the x-ray crystal structure of ATP sulfurylase isoform 1 from soybean (Glycine max ATP sulfurylase) in complex with APS was determined. This structure revealed several highly conserved substrate-binding motifs in the active site and a distinct dimerization interface compared with other ATP sulfurylases but was similar to mammalian 3?-phosphoadenosine 5?-phosphosulfate synthetase. Steady-state kinetic analysis of 20 G. max ATP sulfurylase point mutants suggests a reaction mechanism in which nucleophilic attack by sulfate on the ?-phosphate of ATP involves transition state stabilization by Arg-248, Asn-249, His-255, and Arg-349. The structure and kinetic analysis suggest that ATP sulfurylase overcomes the energetic barrier of APS synthesis by distorting nucleotide structure and identifies critical residues for catalysis. Mutations that alter sulfate assimilation in Arabidopsis were mapped to the structure, which provides a molecular basis for understanding their effects on the sulfur assimilation pathway. PMID:24584934

  20. Characterization of the Saccharomyces cerevisiae ATP-Interactome using the iTRAQ-SPROX Technique

    NASA Astrophysics Data System (ADS)

    Geer, M. Ariel; Fitzgerald, Michael C.

    2015-11-01

    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.

  1. 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

  2. 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

  3. ATP release mechanisms of endothelial cell-mediated stimulus-dependent hyperalgesia

    PubMed Central

    Joseph, Elizabeth K.; Green, Paul G.; Levine, Jon D.

    2014-01-01

    Endothelin-1 acts on endothelial cells to enhance mechanical stimulation-induced release of ATP, which in turn can act on sensory neurons innervating blood vessels to contribute to vascular pain, a phenomenon we have referred to as stimulus-dependent hyperalgesia (SDH). In the present study we evaluated the role of the major classes of ATP release mechanisms to SDH: vesicular exocytosis, plasma membrane associated ATP synthase, ATP-Binding Cassette (ABC) transporters, and ion channels. Inhibitors of vesicular exocytosis (i.e., monensin, brefeldin A and bafilomycin), plasma membrane associated ATPase (i.e., oligomycin and pigment epithelium-derived factor-derived peptide 34-mer) and connexin ion channels (carbenoxolone and flufenamic acid), but not ABC transporters (i.e., dipyridamole, nicardipine or CFTRinh-172) attenuated stimulus-dependent hyperalgesia. These studies support a role of ATP in SDH, and suggest novel targets for the treatment of vascular pain syndromes. PMID:24793242

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

    NASA Astrophysics Data System (ADS)

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

    1996-03-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is chloride ion channel regulated by protein kinase A and adenosine triphosphate (ATP). Loss of CFTR-mediated chloride ion conductance from the apical plasma membrane of epithelial cells is a primary physiological lesion in cystic fibrosis. CFTR has also been suggested to function as an ATP channel, although the size of the ATP anion is much larger than the estimated size of the CFTR pore. ATP was not conducted through CFTR in intact organs, polarized human lung cell lines, stably transfected mammalian cell lines, or planar lipid bilayers reconstituted with CFTR protein. These findings suggest that ATP permeation through the CFTR is unlikely to contribute to the normal function of CFTR or to the pathogenesis of cystic fibrosis.

  5. 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

  6. 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.

  7. Dynamic Structure Change due to ATP Hydrolysis in the Motor Domain of Myosin: Molecular Dynamics Simulations

    NASA Astrophysics Data System (ADS)

    Kawakubo, Tatsuyuki; Okada, Okimasa; Minami, Tomoyuki

    2007-07-01

    The muscle contraction is caused by directed movement of the myosin head along the actin filament. This movement is triggered by ATP hydrolysis, which occurs in the motor domain of myosin. The mechanism for its intra-molecular process remains unknown because of a lack of way to observe vibrational motions of each atom in a myosin molecule. We have carried out 10-ns, all-atom molecular dynamics (MD) simulations to examine what kinds of dynamic structure changes of the motor domain are produced by the energy released from an ATP hydrolysis. The result revealed that a disturbance due to the ATP hydrolysis enhances collective motions of atoms at the actin-binding site and the junction with the neck, both of which are relevant to the movement of the myosin head along the actin filament. Further, an ATP hydrolysis was found to open the gate to the ATPase pocket for the following ATP molecule to easily get into there.

  8. 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

  9. 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

  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 RM 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 RM 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 adeninea 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. 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.

  13. 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

  14. 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.

  15. 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

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

    PubMed Central

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

    2012-01-01

    Erythrocytes have been implicated as controllers of vascular caliber by virtue of their ability to release the vasodilator ATP in response to local physiological and pharmacological stimuli. The regulated release of ATP from erythrocytes requires activation of a signaling pathway involving G proteins (Gi or Gs), adenylyl cyclase, protein kinase A, and the cystic fibrosis transmembrane conductance regulator as well as a final conduit through which this highly charged anion exits the cell. Although pannexin 1 has been shown to be the final conduit for ATP release from human erythrocytes in response to reduced oxygen tension, it does not participate in transport of ATP following stimulation of the prostacyclin (IP) receptor in these cells, which suggests that an additional protein must be involved. Using antibodies directed against voltage-dependent anion channel (VDAC)1, we confirm that this protein is present in human erythrocyte membranes. To address the role of VDAC in ATP release, two structurally dissimilar VDAC inhibitors, Bcl-xL BH4423 and TRO19622, were used. In response to the IP receptor agonists, iloprost and UT-15C, ATP release was inhibited by both VDAC inhibitors although neither iloprost-induced cAMP accumulation nor total intracellular ATP concentration were altered. Together, these findings support the hypothesis that VDAC is the ATP conduit in the IP receptor-mediated signaling pathway in human erythrocytes. In addition, neither the pannexin inhibitor carbenoxolone nor Bcl-xL BH4423 attenuated ATP release in response to incubation of erythrocytes with the ?-adrenergic receptor agonist isoproterenol, suggesting the presence of yet another channel for ATP release from human erythrocytes. PMID:22159995

  17. 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].

  18. 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

  19. Lack of Direct Cytotoxicity of Extracellular ATP against Hepatocytes: Role in the Mechanism of Acetaminophen Hepatotoxicity

    PubMed Central

    Xie, Yuchao; Woolbright, Benjamin L.; Kos, Milan; McGill, Mitchell R.; Dorko, Kenneth; Kumer, Sean C.; Schmitt, Timothy M.; Jaeschke, Hartmut

    2015-01-01

    Background Acetaminophen (APAP) hepatotoxicity is a major cause of acute liver failure in many countries. Mechanistic studies in mice and humans have implicated formation of a reactive metabolite, mitochondrial dysfunction and oxidant stress as critical events in the pathophysiology of APAP-induced liver cell death. It was recently suggested that ATP released from necrotic cells can directly cause cell death in mouse hepatocytes and in a hepatoma cell line (HepG2). Aim To assess if ATP can directly cause cell toxicity in hepatocytes and evaluate their relevance in the human system. Methods Primary mouse hepatocytes, human HepG2 cells, the metabolically competent human HepaRG cell line and freshly isolated primary human hepatocytes were exposed to 10-100 ?M ATP or AT?P in the presence or absence of 5-10 mM APAP for 9-24 h. Results ATP or AT?P was unable to directly cause cell toxicity in all 4 types of hepatocytes. In addition, ATP did not enhance APAP-induced cell death observed in primary mouse or human hepatocytes, or in HepaRG cells as measured by LDH release and by propidium iodide staining in primary mouse hepatocytes. Furthermore, addition of ATP did not cause mitochondrial dysfunction or enhance APAP-induced mitochondrial dysfunction in primary murine hepatocytes, although ATP did cause cell death in murine RAW macrophages. Conclusions It is unlikely that ATP released from necrotic cells can significantly affect cell death in human or mouse liver during APAP hepatotoxicity. Relevance for Patients Understanding the mechanisms of APAP-induced cell injury is critical for identifying novel therapeutic targets to prevent liver injury and acute liver failure in APAP overdose patients. PMID:26722668

  20. The molecular motor F-ATP synthase is targeted by the tumoricidal protein HAMLET.

    PubMed

    Ho, James; Sielaff, Hendrik; Nadeem, Aftab; Svanborg, Catharina; Grber, 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 ? (non-catalytic) and ? (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 20.5?M. Increasing concentrations of the tumoricidal protein HAMLET added to the enzymatically active ?3?3? 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-?3?3? complex interaction. Whereas the ?3?3? complex of the F-ATP synthase rotated in a counterclockwise direction with a mean rotational rate of 3.80.7s(-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

  1. Clinical heterogeneity in respiratory chain complex III deficiency in childhood.

    PubMed

    Mourmans, J; Wendel, U; Bentlage, H A; Trijbels, J M; Smeitink, J A; de Coo, I F; Gabrels, F J; Sengers, R C; Ruitenbeek, W

    1997-07-01

    Six children are presented with an isolated complex III deficiency in muscle tissue. More specifically, oxidation rates and ATP+CrP production rates from both pyruvate and succinate as substrates and/or the activity of decylubiquinol:cytochrome c oxidoreductase were all markedly reduced. Complex III deficiency was also present in liver of two patients tested, but could not be demonstrated in cultured fibroblasts of four patients tested. Mitochondrial DNA, extracted from muscle, was analyzed; no deletions or common point mutations were found. Four patients presented with a multi-organ disorder. Among these patients three presented at neonatal age with neurological signs and lactate elevation in blood and CSF, of whom two had severe neonatal Fanconi syndrome. One child, aged seven years, had encephalomyopathy, ophthalmoplegia, retinopathy and Wolff-Parkinson-White syndrome. The remaining two patients exhibited myopathy only, within the first year of life. Thus, like in other respiratory chain disorders, patients with complex III deficiency may present at any age and show variable symptoms and outcome, ranging from neonatal death to failure to thrive only. Apparently there are no clinical findings which are specific for complex III deficiency. PMID:9168175

  2. 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.

  3. 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

  4. 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

  5. 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

  6. Prime type III factors

    PubMed Central

    Shlyakhtenko, Dimitri

    2000-01-01

    It is shown that for each 0 < ? < 1, the free ArakiWoods factor of type III? cannot be written as a tensor product of two diffuse von Neumann algebras (i.e., is prime) and does not contain a Cartan subalgebra. PMID:11058159

  7. 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…

  8. 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.

  9. Anionic phospholipids activate ATP-sensitive potassium channels.

    PubMed

    Fan, Z; Makielski, J C

    1997-02-28

    The ATP-sensitive potassium channel (KATP) controls insulin release in pancreatic beta-cells and also modulates important functions in other cell types. In this study we report that anionic phospholipids activated KATP in pancreatic beta-cells, cardiac myocytes, skeletal muscle cells, and a cloned KATP composed of two subunits (SUR/Kir6. 2) stably expressed in a mammalian cell line. The effectiveness was proportional to the number of negative charges on the head group of the anionic phospholipid. Screening negative charges with polyvalent cations antagonized the effect. Enzymatic treatment with phospholipases that reduced charge on the lipids also reduced or eliminated the effect. These results suggest that intact phospholipids with negative charges are the critical requirement for activation of KATP, in distinction from the usual cell signaling pathway through phospholipids that requires cleavage. Mutations of two positively charged amino acid residues at the C terminus of Kir6. 2 accelerated loss of channel activity and reduced the activating effects of phospholipids, suggesting involvement of this region in the activation. Metabolism of anionic phospholipids in plasmalemmal membrane may be a novel and general mechanism for regulation of KATP and perhaps other ion channels in the family of inward rectifiers. PMID:9038137

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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

  15. Ca2+ Entry is Required for Mechanical Stimulation-induced ATP Release from Astrocyte

    PubMed Central

    Lee, Jaekwang; Chun, Ye-Eun; Han, Kyung-Seok; Lee, Jungmoo; Woo, Dong Ho

    2015-01-01

    Astrocytes and neurons are inseparable partners in the brain. Neurotransmitters released from neurons activate corresponding G protein-coupled receptors (GPCR) expressed in astrocytes, resulting in release of gliotransmitters such as glutamate, D-serine, and ATP. These gliotransmitters in turn influence neuronal excitability and synaptic activities. Among these gliotransmitters, ATP regulates the level of network excitability and is critically involved in sleep homeostasis and astrocytic Ca2+ oscillations. ATP is known to be released from astrocytes by Ca2+-dependent manner. However, the precise source of Ca2+, whether it is Ca2+ entry from outside of cell or from the intracellular store, is still not clear yet. Here, we performed sniffer patch to detect ATP release from astrocyte by using various stimulation. We found that ATP was not released from astrocyte when Ca2+ was released from intracellular stores by activation of G?q-coupled GPCR including PAR1, P2YR, and B2R. More importantly, mechanical stimulation (MS)-induced ATP release from astrocyte was eliminated when external Ca2+ was omitted. Our results suggest that Ca2+ entry, but not release from intracellular Ca2+ store, is critical for MS-induced ATP release from astrocyte. PMID:25792866

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

    PubMed

    Hwang, Tzyh-Chang; Sheppard, David N

    2009-05-15

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

  18. ATP monitoring technology for microbial growth control in potable water systems

    NASA Astrophysics Data System (ADS)

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

    2006-05-01

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

  19. Effect of gamma radiation and oregano essential oil on murein and ATP concentration of Staphylococcus aureus.

    PubMed

    Caillet, Stphane; Ursachi, Laura; Shareck, Franois; Lacroix, Monique

    2009-01-01

    The study was carried out to evaluate the effects of gamma-irradiation alone or in combination with oregano essential oil on murein composition of Staphylococcus aureus and on the intracellular and extracellular concentration of ATP. The bacterial strain was treated with 3 irradiation doses: 1.2 kGy to induce cell damage, 2.9 kGy to obtain a viable but nonculturable state, and 3.5 kGy to cause cell death. Oregano essential oil was used at 0.010% and 0.013% (w/v), which is the minimum inhibitory concentration (MIC). All treatments had a significant effect (P < or = 0.05) on the murein composition, although some muropeptides did not seem to be affected by the treatment. Each treatment influenced differently the relative percentage and number of muropeptides. There was a significant (P < or = 0.05) correlation between the reduction of intracellular ATP and increase in extracellular ATP following treatment of the cells with oregano oil. The reduction of intracellular ATP was even more important when essential oil was combined with irradiation. Also, irradiation alone of S. aureus induced a significant decrease (P < or = 0.05) of the internal ATP and a significant increase (P < or = 0.05) of the external ATP. However, no significant difference (P > 0.05) was observed in ATP concentrations between different radiation doses. Transmission electron microscopic observation revealed that oregano oil and irradiation have an effect on cell wall structure. PMID:20492121

  20. 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.

  1. Crystallization of the c14-rotor of the chloroplast ATP synthase reveals that it contains pigments

    PubMed Central

    Varco-Merth, Benjamin; Fromme, Raimund; Wang, Meitian; Fromme, Petra

    2012-01-01

    The ATP synthase is one of the most important enzymes on earth as it couples the transmembrane electrochemical potential of protons to the synthesis of ATP from ADP and inorganic phosphate, providing the main ATP source of almost all higher life on earth. During ATP synthesis, stepwise protonation of a conserved carboxylate on each protein subunit of an oligomeric ring of 1015 c-subunits is commonly thought to drive rotation of the rotor moiety (c1014??) relative to stator moiety (?3?3?ab2). Here we report the isolation and crystallization of the c14-ring of subunit c from the spinach chloroplast enzyme diffracting as far as 2.8 . Though ATP synthase was not previously known to contain any pigments, the crystals of the c-subunit possessed a strong yellow color. The pigment analysis revealed that they contain 1 chlorophyll and 2 carotenoids, thereby showing for the first time that the chloroplast ATP synthase contains cofactors, leading to the question of the possible roles of the functions of the pigments in the chloroplast ATP synthase. PMID:18515064

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

    PubMed Central

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

    2012-01-01

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

  3. 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.

  4. 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 ?-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

  5. 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

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

    PubMed

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

    2014-01-01

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

  7. De novo mutations in ATP1A3 cause alternating hemiplegia of childhood.

    PubMed

    Heinzen, Erin L; Swoboda, Kathryn J; Hitomi, Yuki; Gurrieri, Fiorella; Nicole, Sophie; de Vries, Boukje; Tiziano, F Danilo; Fontaine, Bertrand; Walley, Nicole M; Heavin, Sinad; Panagiotakaki, Eleni; Fiori, Stefania; Abiusi, Emanuela; Di Pietro, Lorena; Sweney, Matthew T; Newcomb, Tara M; Viollet, Louis; Huff, Chad; Jorde, Lynn B; Reyna, Sandra P; Murphy, Kelley J; Shianna, Kevin V; Gumbs, Curtis E; Little, Latasha; Silver, Kenneth; Pt?ek, Louis J; Haan, Joost; Ferrari, Michel D; Bye, Ann M; Herkes, Geoffrey K; Whitelaw, Charlotte M; Webb, David; Lynch, Bryan J; Uldall, Peter; King, Mary D; Scheffer, Ingrid E; Neri, Giovanni; Arzimanoglou, Alexis; van den Maagdenberg, Arn M J M; Sisodiya, Sanjay M; Mikati, Mohamad A; Goldstein, David B

    2012-09-01

    Alternating hemiplegia of childhood (AHC) is a rare, severe neurodevelopmental syndrome characterized by recurrent hemiplegic episodes and distinct neurological manifestations. AHC is usually a sporadic disorder and has unknown etiology. We used exome sequencing of seven patients with AHC and their unaffected parents to identify de novo nonsynonymous mutations in ATP1A3 in all seven individuals. In a subsequent sequence analysis of ATP1A3 in 98 other patients with AHC, we found that ATP1A3 mutations were likely to be responsible for at least 74% of the cases; we also identified one inherited mutation in a case of familial AHC. Notably, most AHC cases are caused by one of seven recurrent ATP1A3 mutations, one of which was observed in 36 patients. Unlike ATP1A3 mutations that cause rapid-onset dystonia-parkinsonism, AHC-causing mutations in this gene caused consistent reductions in ATPase activity without affecting the level of protein expression. This work identifies de novo ATP1A3 mutations as the primary cause of AHC and offers insight into disease pathophysiology by expanding the spectrum of phenotypes associated with mutations in ATP1A3. PMID:22842232

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

    PubMed Central

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

    2015-01-01

    Adenosine triphosphate (ATP), the chemical energy currency of biology, is synthesized in eukaryotic cells primarily by the mitochondrial ATP synthase. ATP synthases operate by a rotary catalytic mechanism where proton translocation through the membrane-inserted FO region is coupled to ATP synthesis in the catalytic F1 region via rotation of a central rotor subcomplex. We report here single particle electron cryomicroscopy (cryo-EM) analysis of the bovine mitochondrial ATP synthase. Combining cryo-EM data with bioinformatic analysis allowed us to determine the fold of the a subunit, suggesting a proton translocation path through the FO region that involves both the a and b subunits. 3D classification of images revealed seven distinct states of the enzyme that show different modes of bending and twisting in the intact ATP synthase. Rotational fluctuations of the c8-ring within the FO region support a Brownian ratchet mechanism for proton-translocation-driven rotation in ATP synthases. DOI: http://dx.doi.org/10.7554/eLife.10180.001 PMID:26439008

  9. 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

  10. [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

  11. Contraction of glycerinated muscle fibers as a function of the ATP concentration.

    PubMed Central

    Cooke, R; Bialek, W

    1979-01-01

    We have measured the force-velocity curves of glycerinated rabbit psoas fibers over a range of ATP concentration from 2.5 microM to 5 mM. As the ATP concentration is increased, the isometric tension increases to a maximum around 50 microM, then decreases to a plateau at 70% of the maximum by 1 mM ATP. At low ATP concentrations the maximum velocity of contraction is low and increases with increasing ATP, reaching a plateau at approximately 2 lengths per second by 1 mM ATP. Our studies suggest that the binding of ATP dissociates the myosin head from actin in the contracting muscle, a reaction similar to that seen in solution. We have constructed models of the actin-myosin-nucleotide interactions based on a kinetic scheme derived from solution studies. The fit of these models to the data shows that the rates of some reactions in the fiber must be considerably different from the rates of the analogous reactions in solution. The data is best fit by models in which head attachment occurs rapidly at the beginning of a power stroke, head detachment occurs rapidly at the end of the power stroke, and the force produced by a myosin head in a power stroke is independent of velocity. PMID:262549

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

    PubMed

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

    2014-04-01

    Active skeletal muscle synthesizes and releases interleukin-6 (IL-6), which plays important roles in the organism's adaptation to exercise. Autocrine/paracrine ATP signaling has been shown to modulate IL-6 expression. The aim of this study was to determine whether a period of physical activity modifies the ATP-induced IL-6 expression. BalbC mice were either subject to 5 weeks voluntary wheel running (VA) or kept sedentary (SED). Flexor digitorum brevis muscles were dissected, stimulated with different ATP concentrations (0-100 ?M) and IL-6 mRNA levels were measured using qPCR. ATP evoked a concentration-dependent rise in IL-6 mRNA in both SED and VA mice. VA mice however, had significantly higher ATP sensitivity (pD2 pharmacological values: VA=5.580.02 vs. SED=4.950.04, p<0.05). Interestingly, in VA mice we observed a positive correlation between the level of physical activity and the IL-6 mRNA increase following fiber stimulation with 10??M ATP. In addition, there were lower P2Y2- and higher P2Y14-receptor mRNA levels in skeletal muscles of VA compared to SED mice, showing plasticity of nucleotide receptors with exercise. These results suggest that exercise increases skeletal muscle ATP sensitivity, a response dependent on the level of physical activity performed. This could have an important role in the mechanisms controlling skeletal muscle adaptation to exercise and training. PMID:24022572

  13. ATP-dependent bile-salt transport in canalicular rat liver plasma-membrane vesicles.

    PubMed Central

    Stieger, B; O'Neill, B; Meier, P J

    1992-01-01

    The present study identifies and characterizes a novel ATP-dependent bile-salt transport system in isolated canalicular rat liver plasma-membrane (cLPM) vesicles. ATP (1-5 mM) stimulated taurocholate uptake into cLPM vesicles between 6- and 8-fold above equilibrium uptake values (overshoot) and above values for incubations in the absence of ATP. The ATP-dependent portion of taurocholate uptake was 2-fold higher in the presence of equilibrated KNO3 as compared with potassium gluconate, indicating that the stimulatory effect of ATP was not due to the generation of an intravesicular positive membrane potential. Saturation kinetics revealed a very high affinity (Km approximately 2.1 microM) of the system for taurocholate. The system could only minimally be stimulated by nucleotides other than ATP. Furthermore, it was preferentially inhibited by conjugated univalent bile salts. Further strong inhibitory effects were observed with valinomycin, oligomycin, 4,4'-di-isothiocyano-2,2'-stilbene disulphonate, sulphobromophthalein, leukotriene C4 and N-ethylmaleimide, whereas nigericin, vanadate, GSH, GSSG and daunomycin exerted only weak inhibitory effects or none at all. These results indicate the presence of a high-affinity primary ATP-dependent bile-salt transport system in cLPM vesicles. This transport system might be regulated in vivo by the number of carriers present at the perspective transport site(s), which, in addition to the canalicular membrane, might also include pericanalicular membrane vesicles. PMID:1599411