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Sample records for adenine nucleotides atp

  1. Identification of a mitochondrial ATP synthase-adenine nucleotide translocator complex in Leishmania.

    PubMed

    Detke, Siegfried; Elsabrouty, Rania

    2008-01-01

    The ATP synthasome is a macromolecular complex consisting of ATP synthase, adenine nucleotide translocator and phosphate carrier. To determine if this complex is evolutionary old or young, we searched for its presence in Leishmania, a mitochondria containing protozoan which evolved from the main eukaryote line soon after eukaryotes split from prokaryotes. Sucrose gradient centrifugation showed that the distribution of ANT among the fractions coincided with the distribution of ATP synthase. In addition, ATP synthase co-precipitated with FLAG tagged and wild type adenine nucleotide translocator isolated with anti FLAG and anti adenine nucleotide translocator antibodies, respectively. These data indicate that the adenine nucleotide translocator interacted with the ATP synthase to form a stable structure referred to as the ATP synthasome. The presence of the ATP synthasome in Leishmania, an organism branching off the main line of eukaryotes early in the development of eukaryotes, as well as in higher eukaryotes suggests that the ATP synthasome is a phylogenetically ancient structure. PMID:17920025

  2. Changes in the adenine nucleotide content of beef-heart mitochondrial F1 ATPase during ATP synthesis in dimethyl sulfoxide.

    PubMed

    Beharry, S; Bragg, P D

    1992-01-31

    Beef-heart mitochondrial F1 ATPase can be induced to synthesize ATP from ADP and inorganic phosphate in 30% Me2SO. We have analyzed the adenine nucleotide content of the F1 ATPase during the time-course of ATP synthesis, in the absence of added medium nucleotide, and in the absence and presence of 10 mM inorganic phosphate. The enzyme used in these investigations was either pretreated or not pretreated with ATP to produce F1 with a defined nucleotide content and catalytic or noncatalytic nucleotide-binding site occupancy. We show that the mechanism of ATP synthesis in Me2SO involves (i) an initial rapid loss of bound nucleotide(s), this process being strongly influenced by inorganic phosphate; (ii) a rebinding of lost nucleotide; and (iii) synthesis of ATP from bound ADP and inorganic phosphate.

  3. The effect of dimethylsulfoxide on adenine nucleotide binding and ATP synthesis by beef-heart mitochondrial F1 ATPase.

    PubMed

    Beharry, S; Bragg, P D

    1991-04-01

    Dimethylsulfoxide (Me2SO; 30%, v/v) promotes the formation of ATP from ADP and phosphate catalyzed by soluble mitochondrial F1 ATPase. The effects of this solvent on the adenine nucleotide binding properties of beef-heart mitochondrial F1 ATPase were examined. The ATP analog adenylyl-5'-imidodiphosphate bound to F1 at 1.9 and 1.0 sites in aqueous and Me2SO systems, respectively, with a KD value of 2.2 microM. Lower affinity sites were present also. Binding of ATP or adenylyl-5'-imidodiphosphate at levels near equimolar with the enzyme occurred to a greater extent in the absence of Me2SO. Addition of ATP to the nucleotide-loaded enzyme resulted in exchange of about one-half of the bound ATP. This occurred only in an entirely aqueous medium. ATP bound in Me2SO medium was not released by exogenous ATP. Comparison of the effect of different concentrations of Me2SO on ADP binding to F1 and ATP synthesis by the enzyme showed that binding of ADP was diminished by concentrations of Me2SO lower than those required to support ATP synthesis. However, one site could still be filled by ADP at concentrations of Me2SO optimal for ATP synthesis. This site is probably a noncatalytic site, since the nucleotide bound there was not converted to ATP in 30% Me2SO. The ATP synthesized by F1 in Me2SO originated from endogenous bound ADP. We conclude that 30% Me2SO affects the adenine nucleotide binding properties of the enzyme. The role of this in the promotion of the formation of ATP from ADP and phosphate is discussed.

  4. Transport of adenine nucleotides in the mitochondria of Saccharomyces cerevisiae: interactions between the ADP/ATP carriers and the ATP-Mg/Pi carrier.

    PubMed

    Traba, Javier; Satrústegui, Jorgina; del Arco, Araceli

    2009-04-01

    The ADP/ATP and ATP-Mg/Pi carriers are widespread among eukaryotes and constitute two systems to transport adenine nucleotides in mitochondria. ADP/ATP carriers carry out an electrogenic exchange of ADP for ATP essential for oxidative phosphorylation, whereas ATP-Mg/Pi carriers perform an electroneutral exchange of ATP-Mg for phosphate and are able to modulate the net content of adenine nucleotides in mitochondria. The functional interplay between both carriers has been shown to modulate viability in Saccharomyces cerevisiae. The simultaneous absence of both carriers is lethal. In the light of the new evidence we suggest that, in addition to exchange of cytosolic ADP for mitochondrial ATP, the specific function of the ADP/ATP carriers required for respiration, both transporters have a second function, which is the import of cytosolic ATP in mitochondria. The participation of these carriers in the generation of mitochondrial membrane potential is discussed. Both are necessary for the function of the mitochondrial protein import and assembly systems, which are the only essential mitochondrial functions in S. cerevisiae.

  5. ATP/ADP Turnover and Import of Glycolytic ATP into Mitochondria in Cancer Cells Is Independent of the Adenine Nucleotide Translocator.

    PubMed

    Maldonado, Eduardo N; DeHart, David N; Patnaik, Jyoti; Klatt, Sandra C; Gooz, Monika Beck; Lemasters, John J

    2016-09-01

    Non-proliferating cells oxidize respiratory substrates in mitochondria to generate a protonmotive force (Δp) that drives ATP synthesis. The mitochondrial membrane potential (ΔΨ), a component of Δp, drives release of mitochondrial ATP(4-) in exchange for cytosolic ADP(3-) via the electrogenic adenine nucleotide translocator (ANT) located in the mitochondrial inner membrane, which leads to a high cytosolic ATP/ADP ratio up to >100-fold greater than matrix ATP/ADP. In rat hepatocytes, ANT inhibitors, bongkrekic acid (BA), and carboxyatractyloside (CAT), and the F1FO-ATP synthase inhibitor, oligomycin (OLIG), inhibited ureagenesis-induced respiration. However, in several cancer cell lines, OLIG but not BA and CAT inhibited respiration. In hepatocytes, respiratory inhibition did not collapse ΔΨ until OLIG, BA, or CAT was added. Similarly, in cancer cells OLIG and 2-deoxyglucose, a glycolytic inhibitor, depolarized mitochondria after respiratory inhibition, which showed that mitochondrial hydrolysis of glycolytic ATP maintained ΔΨ in the absence of respiration in all cell types studied. However in cancer cells, BA, CAT, and knockdown of the major ANT isoforms, ANT2 and ANT3, did not collapse ΔΨ after respiratory inhibition. These findings indicated that ANT did mediate mitochondrial ATP/ADP exchange in cancer cells. We propose that suppression of ANT contributes to low cytosolic ATP/ADP, activation of glycolysis, and a Warburg metabolic phenotype in proliferating cells.

  6. Adenine nucleotide levels and regional distribution of ATP in rabbit spinal cord after ischemia and recirculation.

    PubMed

    Danielisová, V; Chavko, M; Kehr, J

    1987-03-01

    Rabbit spinal cords were subjected to 10 to 40 minutes of ischemia with and without 4 days of recirculation and L-4 segment was analyzed for adenylates and ATP-induced bioluminiscence. ATP level and energy charge was progressively reduced by increasing durations of ischemia. Regional evaluation of ATP-induced bioluminiscence after 10 and 20 minutes of ischemia revealed ATP depletion mainly in the gray matter of spinal cord. Forty minutes of ischemia resulted in complete reduction of ATP bioluminiscence in both gray and white matter. Within 4 days of recirculation following all periods of ischemia studied, only partial metabolic recovery occurred. Restitution of ATP-induced bioluminiscence was regionally heterogeneous, reduced predominantly in the anterior horns of gray matter.

  7. Adenine nucleotide transporters in organelles: novel genes and functions.

    PubMed

    Traba, Javier; Satrústegui, Jorgina; del Arco, Araceli

    2011-04-01

    In eukaryotes, cellular energy in the form of ATP is produced in the cytosol via glycolysis or in the mitochondria via oxidative phosphorylation and, in photosynthetic organisms, in the chloroplast via photophosphorylation. Transport of adenine nucleotides among cell compartments is essential and is performed mainly by members of the mitochondrial carrier family, among which the ADP/ATP carriers are the best known. This work reviews the carriers that transport adenine nucleotides into the organelles of eukaryotic cells together with their possible functions. We focus on novel mechanisms of adenine nucleotide transport, including mitochondrial carriers found in organelles such as peroxisomes, plastids, or endoplasmic reticulum and also mitochondrial carriers found in the mitochondrial remnants of many eukaryotic parasites of interest. The extensive repertoire of adenine nucleotide carriers highlights an amazing variety of new possible functions of adenine nucleotide transport across eukaryotic organelles.

  8. Applications of adenine nucleotide measurements in oceanography

    NASA Technical Reports Server (NTRS)

    Holm-Hansen, O.; Hodson, R.; Azam, F.

    1975-01-01

    The methodology involved in nucleotide measurements is outlined, along with data to support the premise that ATP concentrations in microbial cells can be extrapolated to biomass parameters. ATP concentrations in microorganisms and nucleotide analyses are studied.

  9. Localisation of adenine nucleotide-binding sites on beef-heart mitochondrial ATPase by photolabelling with 8-azido-ADP and 8-azido-ATP.

    PubMed

    Wagenvoord, R J; van der Kraan, I; Kemp, A

    1979-10-10

    1. In addition to the previously studied 8-azido-ATP, 8-azido-ADP is a suitable photoaffinity label for beef-heart mitochondrial ATPase (F1). 2. Photolysis at 350 nm of 8-azido-ADP in the presence of isolated F1 leads to inactivation of ATPase activity. Both ATP and ADP (but not AMP) protect against the inactivation. 3. In the absence of Mg2+, 8-azido-ADP binds almost equally to the alpha and beta subunits of F1, whereas in the presence of Mg2+ the alpha subunits are predominantly labelled. 4. The ATPase activity is completely inhibited when two molecules of 8-azido-ADP are bound per molecule F1. 5. 8-Azido-ATP and ATP are competitive substrates for F1, indicating that in the presence of Mg2+ 8-azido-ATP binds to the same site as ATP. 6. The amount of tightly bound nucleotides in F1 is not significantly changed upon incubation with 8-azido-ATP either in the light or the dark. 7. 8-Azido-ATP is also a suitadrial particles, photolabelling leading to inactivation of ATPase activity. 9. Oxidative phosphorylation and the ATP-driven reduction of NAD+ by succinate are also inhibited by photolabelling Mg-ATP particles with 8-azido-ATP. 10. In contrast to the uncoupled ATPase activity, where the two ATP-binding sites do not interact, cooperation between the two sites is required for ATP hydrolysis coupled to reduction of NAD+ by succinate.

  10. The number and localisation of adenine nucleotide-binding sites in beef-heart mitochondrial ATPase (F1) determined by photolabelling with 8-azido-ATP and 8-azido-ADP.

    PubMed

    Wagenvoord, R J; Kemp, A; Slater, E C

    1980-12-01

    1. When irradiated 8-azido-ATP becomes covalently bound (as the nitreno compound) to beef-heart mitochondrial ATPase (F1) as the triphosphate, either in the absence or presence of Mg2+, label covalently bound is not hydrolysed. 2. In the presence of Mg2+ the nitreno-ATP is bound to both the alpha and beta subunits, mainly (63%) to the alpha subunits. 3. After successive photolabelling of F1 with 8-azido-ATP (no Mg2+) and 8-azido-ADP (with Mg2+) 4 mol label is bound to F1, 2 mol to the alpha and 2 mol to the beta subunits. 4. When the order of photolabelling is reversed, much less 8-nitreno-ATP is bound to F1 previously labelled with 8-nitreno-ADP. It is concluded that binding to the alpha-subunits hinders binding to the beta subunits. 5. F1 that has been photolabelled with up to 4 mol label still contains 2 mol firmly bound adenine nucleotides per mol F1. 6. It is concluded that at least 6 sites for adenine nucleotides are present in isolated F1.

  11. Adenine nucleotides as allosteric effectors of pea seed glutamine synthetase.

    PubMed

    Knight, T J; Langston-Unkefer, P J

    1988-08-15

    The effects of adenine nucleotides on pea seed glutamine synthetase (EC 6.3.1.2) activity were examined as a part of our investigation of the regulation of this octameric plant enzyme. Saturation curves for glutamine synthetase activity versus ATP with ADP as the changing fixed inhibitor were not hyperbolic; greater apparent Vmax values were observed in the presence of added ADP than the Vmax observed in the absence of ADP. Hill plots of data with ADP present curved upward and crossed the plot with no added ADP. The stoichiometry of adenine nucleotide binding to glutamine synthetase was examined. Two molecules of [gamma-32P]ATP were bound per subunit in the presence of methionine sulfoximine. These ATP molecules were bound at an allosteric site and at the active site. One molecule of either [gamma-32P]ATP or [14C]ADP bound per subunit in the absence of methionine sulfoximine; this nucleotide was bound at an allosteric site. ADP and ATP compete for binding at the allosteric site, although ADP was preferred. ADP binding to the allosteric site proceeded in two kinetic phases. A Vmax value of 1.55 units/mg was measured for glutamine synthetase with one ADP tightly bound per enzyme subunit; a Vmax value of 0.8 unit/mg was measured for enzyme with no adenine nucleotide bound at the allosteric site. The enzyme activation caused by the binding of ADP to the allosteric sites was preceded by a lag phase, the length of which was dependent on the ADP concentration. Enzyme incubated in 10 mM ADP bound approximately 4 mol of ADP/mol of native enzyme before activation was observed; the activation was complete when 7-8 mol of ADP were bound per mol of the octameric, native enzyme. The Km for ATP (2 mM) was not changed by ADP binding to the allosteric sites. ADP was a simple competitive inhibitor (Ki = 0.05 mM) of ATP for glutamine synthetase with eight molecules of ADP tightly bound to the allosteric sites of the octamer. Binding of ATP to the allosteric sites led to marked

  12. Labeling of mitochondrial adenine nucleotides of bovine sperm

    SciTech Connect

    Cheetham, J.; Lardy, H.A.

    1986-05-01

    Incorporation of /sup 32/P/sub i/ into the adenine nucleotide pool of intact bovine spermatozoa utilizing endogenous substrates results in a specific activity (S.A.) ratio ATP/ADP of 0.3 to 0.5, suggesting compartmentation of nucleotide pools or a pathway for phosphorylation of AMP in addition to the myokinase reaction. Incubation of filipin-permeabilized cells with pyruvate, acetylcarnitine, or ..cap alpha..-ketoglutarate (..cap alpha..KG) resulted in ATP-ADP S.A. ratios of 0.5, 0.8, and 1.6, respectively, for mitochondrial nucleotides. However, when malate was included with pyruvate or acetylcarnitine, the ATP/ADP S.A. ratio increased by 400% to 2.0 for pyruvate/malate and by 290% to 2.8 for acetylcarnitine/malate, while the ATP/ADP ratio increased by less than 100% in both cases. These results may indicate that under conditions of limited flux through the citric acid cycle a pathway for phosphorylation of AMP from a precursor other than ATP exists or that ATP is compartmented within the mitochondrion. In the presence of uncoupler and oligomycin with ..cap alpha..KG, pyruvate/malate, or acetylcarnitine/malate, /sup 32/P/sub i/ is incorporated primarily into ATP, resulting in an ATP/ADP S.A. ratio of 4.0 for ..cap alpha..KG, 2.7 for pyruvate/malate, and 2.8 for acetylcarnitine/malate. These data are consistent with phosphorylation of ADP during substrate level phosphorylation in the citric acid cycle.

  13. The rate of ATP export in the extramitochondrial phase via the adenine nucleotide translocator changes in aging in mitochondria isolated from heart left ventricle of either normotensive or spontaneously hypertensive rats.

    PubMed

    Atlante, Anna; Seccia, Teresa Maria; Marra, Ersilia; Passarella, Salvatore

    2011-10-01

    To find out whether and how deficit of cellular energy supply from mitochondria to cytosol occurs in aging and hypertension, we used mitochondria isolated from 5 to 72 week-old heart left ventricle of either normotensive (WKY) or spontaneous hypertensive (SH) rats as a model system. Measurements were made of the rate of ATP appearance outside mitochondria, due to externally added ADP, as an increase of NADPH absorbance which occurs when ATP is produced in the presence of glucose, hexokinase and glucose-6-phosphate dehydrogenase. Such a rate proved to mirror the function of the adenine nucleotide translocator (ANT) rather than other processes linked to the both oxidative and substrate level phosphorylation. The changes in both Ki for atractyloside and Km for ADP suggest the occurrence of modification of the ANT conformation during aging in which the ANT Vmax was found to decrease in normotensive but to increase under spontaneously hypertension in 24 week-old rats with a subsequent decrease in both cases. ANT function, as investigated in the ADP physiological range (20-60μM), is expected to decrease in normotensive, but to increase in hypertensive rats up to 48 weeks. Later a decrease in the ATP rate of export outside mitochondria should occur in both cases. PMID:21855562

  14. Phosphorus-31 NMR visibility and characterization of rat liver mitochondrial matrix adenine nucleotides

    SciTech Connect

    Hutson, S.M.; Berkich, D.; Williams, G.D.; LaNoue, K.F.; Briggs, R.W. )

    1989-05-16

    Compartmentation and NMR visibility of mitochondrial adenine nucleotides were quantitated in isolated rat liver mitochondria respiring on succinate and glutamate in vitro at 8 and 25{degree}C. Intra- and extramitochondrial nucleotides were discriminated by adding the chelator trans-1,2-diaminocyclohexane-N,N,N{prime},N{prime}-tetraacetic acid (CDTA). T{sub 1} values of about 0.2-0.3 s for magnesium-bound matrix nucleotides were determined. Adenine nucleotide T{sub 1} values were influenced by the ionic environment; only magnesium-free ATP T{sub 1}'s were affected by temperature. Intra- and extramitochondrial adenine nucleotide ratios were varied in ATP-loaded mitochondria with added ATP and phosphate using the mitochondrial inhibitors oligomycin and carboxyatractyloside, and adenine nucleotides were quantitated by using NMR and enzymatic analysis. There was good agreement between matrix ATP concentrations (magnesium-bound ATP) calculated by using NMR and standard biochemical techniques. Although matrix ADP could be detected by NMR, it was difficult to quantitate accurately by NMR. The data indicate that mitochondrial ATP is NMR-visible in isolated mitochondria in vitro.

  15. Radiation and thermal stabilities of adenine nucleotides.

    PubMed

    Demidov, V V; Potaman, V N; Solyanina, I P; Trofimov, V I

    1995-03-01

    We have investigated in detail radiation and thermal stabilities and transformations of adenosine mono- and triphosphates in liquid and frozen solid aqueous solutions within a wide range of absorbed radiation dose (up to 75 kGy) and temperature (up to 160 degrees C). Dephosphorylation is the main pathway of high temperature hydrolysis of adenine nucleotides. Basic thermodynamic and kinetic parameters of this process have been determined. Radiolysis of investigated compounds at room temperature results in scission of N-glycosidic bond with a radiation yield about of 1 mol/100 eV. Solution freezing significantly enhances radiation stability of nucleotides as well as other biomolecules. This circumstance is essential in the discussion of panspermia concepts.

  16. Measurement of liver adenine nucleotides and S-adenosyl amino acids by one-step high-performance liquid chromatography.

    PubMed

    Gourdeau, H; Lavoie, R; Grose, J H; Bélanger, L

    1986-10-01

    A reverse-phase isocratic HPLC method is described for direct simultaneous assay of ATP, ADP, AMP, S-adenosylmethionine, S-adenosylhomocysteine, S-adenosylethionine, and other adenine derivatives in liver microbiopsies. The procedure was tested in conditions which alter the hepatic content of adenine nucleotides and sulfur-adenosyl amino acids in humans, rats, and guinea pigs.

  17. Glucagon regulation of oxidative phosphorylation requires an increase in matrix adenine nucleotide content through Ca2+ activation of the mitochondrial ATP-Mg/Pi carrier SCaMC-3.

    PubMed

    Amigo, Ignacio; Traba, Javier; González-Barroso, M Mar; Rueda, Carlos B; Fernández, Margarita; Rial, Eduardo; Sánchez, Aránzazu; Satrústegui, Jorgina; Del Arco, Araceli

    2013-03-15

    It has been known for a long time that mitochondria isolated from hepatocytes treated with glucagon or Ca(2+)-mobilizing agents such as phenylephrine show an increase in their adenine nucleotide (AdN) content, respiratory activity, and calcium retention capacity (CRC). Here, we have studied the role of SCaMC-3/slc25a23, the mitochondrial ATP-Mg/Pi carrier present in adult mouse liver, in the control of mitochondrial AdN levels and respiration in response to Ca(2+) signals as a candidate target of glucagon actions. With the use of SCaMC-3 knock-out (KO) mice, we have found that the carrier is responsible for the accumulation of AdNs in liver mitochondria in a strictly Ca(2+)-dependent way with an S0.5 for Ca(2+) activation of 3.3 ± 0.9 μm. Accumulation of matrix AdNs allows a SCaMC-3-dependent increase in CRC. In addition, SCaMC-3-dependent accumulation of AdNs is required to acquire a fully active state 3 respiration in AdN-depleted liver mitochondria, although further accumulation of AdNs is not followed by increases in respiration. Moreover, glucagon addition to isolated hepatocytes increases oligomycin-sensitive oxygen consumption and maximal respiratory rates in cells derived from wild type, but not SCaMC-3-KO mice and glucagon administration in vivo results in an increase in AdN content, state 3 respiration and CRC in liver mitochondria in wild type but not in SCaMC-3-KO mice. These results show that SCaMC-3 is required for the increase in oxidative phosphorylation observed in liver mitochondria in response to glucagon and Ca(2+)-mobilizing agents, possibly by allowing a Ca(2+)-dependent accumulation of mitochondrial AdNs and matrix Ca(2+), events permissive for other glucagon actions.

  18. Similarities between UDP-Glucose and Adenine Nucleotide Release in Yeast

    PubMed Central

    Esther, Charles R.; Sesma, Juliana I.; Dohlman, Henrik G.; Ault, Addison D.; Clas, Marién L.; Lazarowski, Eduardo R.; Boucher, Richard C.

    2008-01-01

    Extracellular UDP-glucose is a natural purinergic receptor agonist, but its mechanisms of cellular release remain unclear. We studied these mechanisms in Saccharomyces cerevisiae, a simple model organism that releases ATP, another purinergic agonist. Similar to ATP, UDP-glucose was released by S. cerevisiae at a rate that was linear over time. However, unlike ATP release, UDP-glucose release was not dependent on glucose stimulation. This discrepancy was resolved by demonstrating the apparent glucose stimulation of ATP release reflected glucose-dependent changes in the intracellular pattern of adenine nucleotides, with AMP release dominating in the absence of glucose. Indeed, total adenine nucleotide release, like UDP-glucose release, did not vary with glucose concentration over the short term. The genetic basis of UDP-glucose release was explored through analysis of deletion mutants, aided by development of a novel bioassay for UDP-glucose based on signaling through heterologously expressed human P2Y14 receptors. Using this assay, an elevated rate of UDP-glucose release was demonstrated in mutants lacking the putative Golgi nucleotide sugar transporter YMD8. An increased rate of UDP-glucose release in ymd8Δ was reduced by deletion of the YEA4 UDP-N-acetylglucosamine or the HUT1 UDP-galactose transporters, and overexpression of YEA4 or HUT1 increased the rate of UDP-glucose release. These findings suggest an exocytotic release mechanism similar to that of ATP, a conclusion supported by decreased rates of ATP, AMP, and UDP-glucose release in response to the secretory inhibitor Brefeldin A. These studies demonstrate the involvement of the secretory pathway in nucleotide and nucleotide sugar efflux in yeast and offer a powerful model system for further investigation. PMID:18693752

  19. Modulation by adenine nucleotides of epileptiform activity in the CA3 region of rat hippocampal slices

    PubMed Central

    Ross, F M; Brodie, M J; Stone, T W

    1998-01-01

    Hippocampal slices (450 μm) generate epileptiform bursts of an interictal nature when perfused with a zero magnesium medium containing 4-aminopyridine (50 μM). The effect of adenine nucleotides on this activity was investigated.ATP and adenosine depressed this epileptiform activity in a concentration-dependent manner, with both purines being equipotent at concentrations above 10 μM.Adenosine deaminase 0.2 u ml−1, a concentration that annuls the effect of adenosine (50 μM), did not significantly alter the depression of activity caused by ATP (50 μM).8-Cyclopentyl-1, 3-dimethylxanthine (CPT), an A1 receptor antagonist, enhanced the discharge rate significantly and inhibited the depressant effect of both ATP and adenosine such that the net effect of ATP or adenosine plus CPT was excitatory.Several ATP analogues were also tested: α, β-methyleneATP (α, β-meATP), 2-methylthioATP (2-meSATP) and uridine triphosphate (UTP). Only α, β-meATP (10 μM) produced an increase in the frequency of spontaneous activity which suggests a lack of involvement of P2Y or P2U receptors.Suramin and pyridoxalphosphate-6-azophenyl-2′, 4′-disulphonic acid (PPADS), P2 receptor antagonists, failed to inhibit the depression produced by ATP (50 μM). The excitatory effect of α, β-meATP (10 μM) was inhibited by suramin (50 μM) and PPADS (5 μM).ATP therefore depresses epileptiform activity in this model in a manner which is not consistent with the activation of known P1 or P2 receptors, suggesting the involvement of a xanthine-sensitive nucleotide receptor. The results are also indicative of an excitatory P2X receptor existing in the hippocampal CA3 region. PMID:9484856

  20. Ethanol-induced activation of adenine nucleotide turnover. Evidence for a role of acetate

    SciTech Connect

    Puig, J.G.; Fox, I.H.

    1984-09-01

    Consumption of alcohol causes hyperuricemia by decreasing urate excretion and increasing its production. Our previous studies indicate that ethanol administration increases uric acid production by increasing ATP degradation to uric acid precursors. To test the hypothesis that ethanol-induced increased urate production results from acetate metabolism and enhanced adenosine triphosphate turnover, we gave intravenous sodium acetate, sodium chloride and ethanol (0.1 mmol/kg per min for 1 h) to five normal subjects. Acetate plasma levels increased from 0.04 +/- 0.01 mM (mean +/- SE) to peak values of 0.35 +/- 0.07 mM and to 0.08 +/- 0.01 mM during acetate and ethanol infusions, respectively. Urinary oxypurines increased to 223 +/- 13% and 316 +/- 44% of the base-line values during acetate and ethanol infusions, respectively. Urinary radioactivity from the adenine nucleotide pool labeled with (8-14C) adenine increased to 171 +/- 27% and to 128 +/- 8% of the base-line values after acetate and ethanol infusions. These data indicate that both ethanol and acetate increase purine nucleotide degradation by enhancing the turnover of the adenine nucleotide pool. They support the hypothesis that acetate metabolism contributes to the increased production of urate associated with ethanol intake.

  1. Identification and characterization of a novel plastidic adenine nucleotide uniporter from Solanum tuberosum.

    PubMed

    Leroch, Michaela; Kirchberger, Simon; Haferkamp, Ilka; Wahl, Markus; Neuhaus, H Ekkehard; Tjaden, Joachim

    2005-05-01

    Homologs of BT1 (the Brittle1 protein) are found to be phylogenetically related to the mitochondrial carrier family and appear to occur in both mono- and dicotyledonous plants. Whereas BT1 from cereals is probably involved in the transport of ADP-glucose, which is essential for starch metabolism in endosperm plastids, BT1 from a noncereal plant, Solanum tuberosum (StBT1), catalyzes an adenine nucleotide uniport when functionally integrated into the bacterial cytoplasmic membrane. Import studies into intact Escherichia coli cells harboring StBT1 revealed a narrow substrate spectrum with similar affinities for AMP, ADP, and ATP of about 300-400 mum. Transiently expressed StBT1-green fluorescent protein fusion protein in tobacco leaf protoplasts showed a plastidic localization of the StBT1. In vitro synthesized radioactively labeled StBT1 was targeted to the envelope membranes of isolated spinach chloroplasts. Furthermore, we showed by real time reverse transcription-PCR a ubiquitous expression pattern of the StBT1 in autotrophic and heterotrophic potato tissues. We therefore propose that StBT1 is a plastidic adenine nucleotide uniporter used to provide the cytosol and other compartments with adenine nucleotides exclusively synthesized inside plastids.

  2. Adenine nucleotide-dependent and redox-independent control of mitochondrial malate dehydrogenase activity in Arabidopsis thaliana.

    PubMed

    Yoshida, Keisuke; Hisabori, Toru

    2016-06-01

    Mitochondrial metabolism is important for sustaining cellular growth and maintenance; however, the regulatory mechanisms underlying individual processes in plant mitochondria remain largely uncharacterized. Previous redox-proteomics studies have suggested that mitochondrial malate dehydrogenase (mMDH), a key enzyme in the tricarboxylic acid (TCA) cycle and redox shuttling, is under thiol-based redox regulation as a target candidate of thioredoxin (Trx). In addition, the adenine nucleotide status may be another factor controlling mitochondrial metabolism, as respiratory ATP production in mitochondria is believed to be influenced by several environmental stimuli. Using biochemical and reverse-genetic approaches, we addressed the redox- and adenine nucleotide-dependent regulation of mMDH in Arabidopsis thaliana. Recombinant mMDH protein formed intramolecular disulfide bonds under oxidative conditions, but these bonds did not have a considerable effect on mMDH activity. Mitochondria-localized o-type Trx (Trx-o) did not facilitate re-reduction of oxidized mMDH. Determination of the in vivo redox state revealed that mMDH was stably present in the reduced form even in Trx-o-deficient plants. Accordingly, we concluded that mMDH is not in the class of redox-regulated enzymes. By contrast, mMDH activity was lowered by adenine nucleotides (AMP, ADP, and ATP). Each adenine nucleotide suppressed mMDH activity with different potencies and ATP exerted the largest inhibitory effect with a significantly lower K(I). Correspondingly, mMDH activity was inhibited by the increase in ATP/ADP ratio within the physiological range. These results suggest that mMDH activity is finely controlled in response to variations in mitochondrial adenine nucleotide balance. PMID:26946085

  3. Adenine nucleotide-dependent and redox-independent control of mitochondrial malate dehydrogenase activity in Arabidopsis thaliana.

    PubMed

    Yoshida, Keisuke; Hisabori, Toru

    2016-06-01

    Mitochondrial metabolism is important for sustaining cellular growth and maintenance; however, the regulatory mechanisms underlying individual processes in plant mitochondria remain largely uncharacterized. Previous redox-proteomics studies have suggested that mitochondrial malate dehydrogenase (mMDH), a key enzyme in the tricarboxylic acid (TCA) cycle and redox shuttling, is under thiol-based redox regulation as a target candidate of thioredoxin (Trx). In addition, the adenine nucleotide status may be another factor controlling mitochondrial metabolism, as respiratory ATP production in mitochondria is believed to be influenced by several environmental stimuli. Using biochemical and reverse-genetic approaches, we addressed the redox- and adenine nucleotide-dependent regulation of mMDH in Arabidopsis thaliana. Recombinant mMDH protein formed intramolecular disulfide bonds under oxidative conditions, but these bonds did not have a considerable effect on mMDH activity. Mitochondria-localized o-type Trx (Trx-o) did not facilitate re-reduction of oxidized mMDH. Determination of the in vivo redox state revealed that mMDH was stably present in the reduced form even in Trx-o-deficient plants. Accordingly, we concluded that mMDH is not in the class of redox-regulated enzymes. By contrast, mMDH activity was lowered by adenine nucleotides (AMP, ADP, and ATP). Each adenine nucleotide suppressed mMDH activity with different potencies and ATP exerted the largest inhibitory effect with a significantly lower K(I). Correspondingly, mMDH activity was inhibited by the increase in ATP/ADP ratio within the physiological range. These results suggest that mMDH activity is finely controlled in response to variations in mitochondrial adenine nucleotide balance.

  4. An alternative membrane transport pathway for phosphate and adenine nucleotides in mitochondria and its possible function.

    PubMed

    Reynafarje, B; Lehninger, A L

    1978-10-01

    This paper describes the properties and a possible biological role of a transport process across the inner membrane of rat liver mitochondria resulting in the exchange of ATP(4-) (out) for ADP(3-) (in) + 0.5 phosphate(2-) (in). This transmembrane exchange reaction, designated as the ATP-ADP-phosphate exchange, is specific for the ligands shown, electroneutral, insensitive to N-ethylmaleimide or mersalyl, inhibited by atractyloside, and appears to occur only in the direction as written. It is thus distinct from the well-known phosphate-hydroxide and phosphate-dicarboxylate exchange systems, which are inhibited by mersalyl, and from the ATP-ADP exchanger, which does not transport phosphate. During ATP hydrolysis by mitochondria, half of the phosphate formed from ATP passes from the matrix to the medium by the mersalyl-insensitive ATP-ADP-phosphate exchange and the other half by the well-known mersalyl-sensitive phosphate-hydroxide exchange. These and other considerations have led to a hypothesis for the pathway and stoichiometry of ATP-dependent reverse electron transport, characterized by a requirement of 1.33 molecules of ATP per pair of electrons reversed and by the utilization of a different membrane transport pathway for phosphate and adenine nucleotides than is taken in forward electron flow and oxidative phosphorylation. The possible occurrence of independent pathways for ATP-forming forward electron flow and ATP-consuming reverse electron flow is consonant with the fact that the opposing degradative and synthetic pathways in the central routes of cell metabolism generally have different pathways that are independently regulated.

  5. Relaxation of isolated taenia coli of guinea-pig by enantiomers of 2-azido analogues of adenosine and adenine nucleotides.

    PubMed Central

    Cusack, N. J.; Planker, M.

    1979-01-01

    1 2-Azido photoaffinity analogues of adenosine 5'triphosphate (ATP), adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP), and adenosine have been synthesized and tested on guinea-pig taenia coli. 2 2-Azido-ATP and 2-azido-ADP were approximately 20 times more potent than ATP as relaxants of taenia coli, and required prolonged washout times before recovery of the muscle. 3 2-Azido-AMP and 2-azidoadenosine were 2 to 12 times more potent than ATP, but took much longer (up to 100 s) to reach maximal relaxation. This behaviour is different from that of AMP and adenosine which were much less potent than ATP. 4 L-Enantiomers of adenosine and adenine nucleotides were also tested. L-ATP and L-ADP were 3 to 6 times less potent than ATP and ADP, and L-AMP and L-adenosine were inactive. 2-Azido-L-ATP and 2-azido-L-ADP were approximately 120 times less potent than 2-Azido-ATP and 6 times less potent than ATP as relaxants of taenia coli. 2-Azido-L-AMP and 2-azidio-L-adenosine were almost inactive. 5 2-Azido derivatives are photolysed by u.v. irradiation to reactive intermediates. 2-Azido-ATP and 2-azidoadenosine might be suitable photoaffinity ligands for labelling putative P2 and P1 purine receptors respectively. 2-Azido-L-ATP and 2-azido-L-adenosine could be useful controls for nonspecific labelling. PMID:497519

  6. Stimulation of the thiol-dependent ADP-ribosyltransferase and NAD glycohydrolase activities of Bordetella pertussis toxin by adenine nucleotides, phospholipids, and detergents.

    PubMed

    Moss, J; Stanley, S J; Watkins, P A; Burns, D L; Manclark, C R; Kaslow, H R; Hewlett, E L

    1986-05-01

    Pertussis toxin catalyzed ADP-ribosylation of the guanyl nucleotide binding protein transducin was stimulated by adenine nucleotide and either phospholipids or detergents. To determine the sites of action of these agents, their effects were examined on the transducin-independent NAD glycohydrolase activity. Toxin-catalyzed NAD hydrolysis was increased synergistically by ATP and detergents or phospholipids; the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) was more effective than the nonionic detergent Triton X-100 greater than lysophosphatidylcholine greater than phosphatidylcholine. The A0.5 for ATP in the presence of CHAPS was 2.6 microM; significantly higher concentrations of ATP were required for maximal activation in the presence of cholate or lysophosphatidylcholine. In CHAPS, NAD hydrolysis was enhanced by ATP greater than ADP greater than AMP greater than adenosine; ATP was more effective than MgATP or the nonhydrolyzable analogue adenyl-5'-yl imidodiphosphate. GTP and guanyl-5'-yl imidodiphosphate were less active than the corresponding adenine nucleotides. Activity in the presence of CHAPS and ATP was almost completely dependent on dithiothreitol; the A0.5 for dithiothreitol was significantly decreased by CHAPS alone and, to a greater extent, by CHAPS and ATP. To determine the site of action of ATP, CHAPS, and dithiothreitol, the enzymatic (S1) and binding components (B oligomer) were resolved by chromatography. The purified S1 subunit catalyzed the dithiothreitol-dependent hydrolysis of NAD; activity was enhanced by CHAPS but not ATP. The studies are consistent with the conclusion that adenine nucleotides, dithiothreitol, and CHAPS act on the toxin itself rather than on the substrate; adenine nucleotides appear to be involved in the activation of toxin but not the isolated catalytic unit.

  7. Comparison of glycogen and adenine nucleotides as indicators of metabolis stress in mummichogs

    SciTech Connect

    Vetter, R.D.; Hwang, H.M.; Hodson, R.E.

    1986-01-01

    Adenine nucleotide and glycogen concentrations were measured concurrently in white muscle of mummichogs Fundulus heteroclitus after the fish were exposed to stressors that either caused an increase in energy use (metabolic loading) or damaged metabolic function (toxic inhibition). When fish were exposed 4 h to 1% unbleached kraft mill effluent in the presence of 6 mg/L dissolved oxygen, glycogen and AMP concentrations significantly decreased below control values, whereas ATP, ADP, and total adenylate (TA) concentrations as well as the adenylate energy charge (AEC = (ATP + 1/2ADP)/TA) were unchanged. When dissolved oxygen was below 1 mg/L, the effluent caused significant decreases in glycogen, ATP, and TA, but not in ADP, AMP, or the AEC. The combined effect of effluent and hypoxia caused more significant drops in ATP or TA pool. When fish were exposed to 60..mu..g/L DDT for 4 h, none of the measured energy variables changed even though this concentration was lethal after several days. At a concentration of 100 ..mu..g/L DDT, all variables except ADP decreased significantly from control values, which may have reflected energy depletion of the muscle in response to nerve spasms rather than a direct toxic effect on the muscle itself.

  8. Dietary adenine controls adult lifespan via adenosine nucleotide biosynthesis and AMPK, and regulates the longevity benefit of caloric restriction

    PubMed Central

    Stenesen, Drew; Suh, Jae Myoung; Seo, Jin; Yu, Kweon; Lee, Kyu-Sun; Kim, Jong-Seok; Min, Kyung-Jin; Graff, Jonathan M.

    2012-01-01

    SUMMARY A common thread among conserved lifespan regulators lies within intertwined roles in metabolism and energy homeostasis. We show that heterozygous mutations of adenosine monophosphate (AMP) biosynthetic enzymes extend Drosophila lifespan. The lifespan benefit of these mutations depends upon increased AMP to adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to ATP ratios and adenosine monophosphate-activated protein kinase (AMPK). Transgenic expression of AMPK in adult fat body or adult muscle, key metabolic tissues, extended lifespan, while AMPK RNAi reduced lifespan. Supplementing adenine, a substrate for AMP biosynthesis, to the diet of long-lived AMP biosynthesis mutants reversed lifespan extension. Remarkably, this simple change in diet also blocked the pro-longevity effects of dietary restriction. These data establish AMP biosynthesis, adenosine nucleotide ratios, and AMPK as determinants of adult lifespan, provide a mechanistic link between cellular anabolism and energy sensing pathways, and indicate that dietary adenine manipulations might alter metabolism to influence animal lifespan. PMID:23312286

  9. K(ATP) channels process nucleotide signals in muscle thermogenic response.

    PubMed

    Reyes, Santiago; Park, Sungjo; Terzic, Andre; Alekseev, Alexey E

    2010-12-01

    Uniquely gated by intracellular adenine nucleotides, sarcolemmal ATP-sensitive K(+) (K(ATP)) channels have been typically assigned to protective cellular responses under severe energy insults. More recently, K(ATP) channels have been instituted in the continuous control of muscle energy expenditure under non-stressed, physiological states. These advances raised the question of how K(ATP) channels can process trends in cellular energetics within a milieu where each metabolic system is set to buffer nucleotide pools. Unveiling the mechanistic basis of the K(ATP) channel-driven thermogenic response in muscles thus invites the concepts of intracellular compartmentalization of energy and proteins, along with nucleotide signaling over diffusion barriers. Furthermore, it requires gaining insight into the properties of reversibility of intrinsic ATPase activity associated with K(ATP) channel complexes. Notwithstanding the operational paradigm, the homeostatic role of sarcolemmal K(ATP) channels can be now broadened to a wider range of environmental cues affecting metabolic well-being. In this way, under conditions of energy deficit such as ischemic insult or adrenergic stress, the operation of K(ATP) channel complexes would result in protective energy saving, safeguarding muscle performance and integrity. Under energy surplus, downregulation of K(ATP) channel function may find potential implications in conditions of energy imbalance linked to obesity, cold intolerance and associated metabolic disorders.

  10. Content of Adenine Nucleotides and Orthophosphate in Exporting and Importing Mature Maize Leaves 1

    PubMed Central

    Eschrich, Walter; Fromm, Joerg

    1985-01-01

    Events of reactivation by re-illumination were studied in predarkened detached mature maize leaves, which were arranged as distal sources and proximal sinks; the latter were kept in CO2-free atmosphere and were either illuminated or darkened. Adenine nucleotide (AdN) content and orthophosphate (Pi) concentrations were measured 10 minutes, 30 minutes, and 2, 7, and 14 hours after the onset of re-illumination. For comparison, mature leaves attached to the plant were analyzed. The sum of AdN increased up to 7 hours of re-illumination, then dark sinks and their sources showed decreasing amounts of AdN, while the increase continued up to 14 hours in sources and illuminated sinks. In leaves attached to the plant, no further increase in AdN level followed the 7-hour mark. The amount of individual AdN (ATP, ADP, AMP) differed considerably in sources and sinks of the detached leaves. Although both the source supplying the illuminated sink and the source supplying the dark sink were treated the same, they showed striking differences in AdN contents. Such relations were also observed, when ATP/ADP ratios and Pi concentrations were compared. The influence a sink can exert on its source suggests a participation of the physiological events in the sink on the regulation of AdN and Pi metabolism in the source. PMID:16664246

  11. In vitro studies of release of adenine nucleotides and adenosine from rat vascular endothelium in response to alpha 1-adrenoceptor stimulation.

    PubMed Central

    Shinozuka, K; Hashimoto, M; Masumura, S; Bjur, R A; Westfall, D P; Hattori, K

    1994-01-01

    1. Noradrenaline-induced release of endogenous adenine nucleotides (ATP, ADP, AMP) and adenosine from both rat caudal artery and thoracic aorta was characterized, using high-performance liquid chromatography with fluorescence detection. 2. Noradrenaline, in a concentration-dependent manner, increased the overflow of ATP and its metabolites from the caudal artery. The noradrenaline-induced release of adenine nucleotides and adenosine from the caudal artery was abolished by bunazosin, an alpha 1-adrenoceptor antagonist, but not by idazoxan, an alpha 2-adrenoceptor antagonist. Clonidine, an alpha 2-adrenoceptor agonist, contracted caudal artery smooth muscle but did not induce release of adenine nucleotides or adenosine. 3. Noradrenaline also significantly increased the overflow of ATP and its metabolites from the thoracic aorta in the rat; however, the amount of adenine nucleotides and adenosine released from the aorta was considerably less than that released from the caudal artery. 4. Noradrenaline significantly increased the overflow of ATP and its metabolites from cultured endothelial cells from the thoracic aorta and caudal artery. The amount released from the cultured endothelial cells from the thoracic aorta and caudal artery. The amount released from the cultured endothelial cells from the aorta was also much less than that from cultured endothelial cells from the caudal artery. In cultured smooth muscle cells from the caudal artery, a significant release of ATP or its metabolites was not observed. 5. These results suggest that there are vascular endothelial cells that are able to release ATP by an alpha 1-adrenoceptor-mediated mechanism, but that these cells are not homogeneously distributed in the vasculature. PMID:7889273

  12. PsANT, the adenine nucleotide translocase of Puccinia striiformis, promotes cell death and fungal growth

    PubMed Central

    Tang, Chunlei; Wei, Jinping; Han, Qingmei; Liu, Rui; Duan, Xiaoyuan; Fu, Yanping; Huang, Xueling; Wang, Xiaojie; Kang, Zhensheng

    2015-01-01

    Adenine nucleotide translocase (ANT) is a constitutive mitochondrial component that is involved in ADP/ATP exchange and mitochondrion-mediated apoptosis in yeast and mammals. However, little is known about the function of ANT in pathogenic fungi. In this study, we identified an ANT gene of Puccinia striiformis f. sp. tritici (Pst), designated PsANT. The PsANT protein contains three typical conserved mitochondrion-carrier-protein (mito-carr) domains and shares more than 70% identity with its orthologs from other fungi, suggesting that ANT is conserved in fungi. Immuno-cytochemical localization confirmed the mitochondrial localization of PsANT in normal Pst hyphal cells or collapsed cells. Over-expression of PsANT indicated that PsANT promotes cell death in tobacco, wheat and fission yeast cells. Further study showed that the three mito-carr domains are all needed to induce cell death. qRT-PCR analyses revealed an in-planta induced expression of PsANT during infection. Knockdown of PsANT using a host-induced gene silencing system (HIGS) attenuated the growth and development of virulent Pst at the early infection stage but not enough to alter its pathogenicity. These results provide new insight into the function of PsANT in fungal cell death and growth and might be useful in the search for and design of novel disease control strategies. PMID:26058921

  13. Enzyme activities and adenine nucleotide content in aorta, heart muscle and skeletal muscle from uraemic rats.

    PubMed Central

    Krog, M.; Ejerblad, S.; Agren, A.

    1986-01-01

    A prominent feature of arterial and myocardial lesions in uraemia is necrosis of the smooth muscle cells. In this study the possibility of detecting metabolic disturbances before necroses appear was investigated. The investigation was made on rats with moderate uraemia (mean serum creatinine 165 mumol/l) of 12 weeks duration. Enzyme activities and concentrations of adenine nucleotides were measured in aorta, heart and skeletal muscles. Histological examination disclosed no changes in these organs. Hexokinase, an important glycolytic enzyme, showed decreased activity in the skeletal muscle and aorta, whereas the hexosemonophosphate shunt enzyme glucose-6-phosphate dehydrogenase remained unchanged. The aspartate aminotransferase was increased in the skeletal muscle. Fat metabolism was not disturbed as reflected by unchanged activity of hydroxyacyl-CoA-dehydrogenase. Adenylatekinase which is important for the energy supply showed markedly increased activities in all tissues examined from the uraemic rats. Decreased ATP levels were found in the heart muscle and the aorta of the uraemic animals, whereas the total pool of adenosine phosphates remained unchanged in all tissues. The animal model described offers a useful means of detecting early changes in uraemia and should be useful for studying the effects of different treatments of uraemic complications. PMID:3718844

  14. Synthesis and enzymatic incorporation of α-L-threofuranosyl adenine triphosphate (tATP).

    PubMed

    Zhang, Su; Chaput, John C

    2013-03-01

    Threose nucleic acid (TNA) is an artificial genetic polymer in which the natural ribose sugar found in RNA has been replaced with an unnatural threose sugar. TNA can be synthesized enzymatically using Therminator DNA polymerase to copy DNA templates into TNA. Here, we expand the substrate repertoire of Therminator DNA polymerase to include threofuranosyl adenine 3'-triphsophate (tATP). We chemically synthesized tATP by two different methods from the 2'-O-acetyl derivative. Enzyme-mediated polymerization reveals that tATP functions as an efficient substrate for Therminator DNA polymerase, indicating that tATP can replace the diaminopurine analogue (tDTP) in TNA transcription reactions. PMID:23352269

  15. ATP-Releasing Nucleotides: Linking DNA Synthesis to Luciferase Signaling.

    PubMed

    Ji, Debin; Mohsen, Michael G; Harcourt, Emily M; Kool, Eric T

    2016-02-01

    A new strategy is reported for the production of luminescence signals from DNA synthesis through the use of chimeric nucleoside tetraphosphate dimers in which ATP, rather than pyrophosphate, is the leaving group. ATP-releasing nucleotides (ARNs) were synthesized as derivatives of the four canonical nucleotides. All four derivatives are good substrates for DNA polymerase, with Km values averaging 13-fold higher than those of natural dNTPs, and kcat values within 1.5-fold of those of native nucleotides. Importantly, ARNs were found to yield very little background signal with luciferase. DNA synthesis experiments show that the ATP byproduct can be harnessed to elicit a chemiluminescence signal in the presence of luciferase. When using a polymerase together with the chimeric nucleotides, target DNAs/RNAs trigger the release of stoichiometrically large quantities of ATP, thereby allowing sensitive isothermal luminescence detection of nucleic acids as diverse as phage DNAs and short miRNAs.

  16. Probing the ATP Site of GRP78 with Nucleotide Triphosphate Analogs

    PubMed Central

    Chen, Yun; Lu, Hua; Pizarro, Juan C.; Park, Hee-Won

    2016-01-01

    GRP78, a member of the ER stress protein family, can relocate to the surface of cancer cells, playing key roles in promoting cell proliferation and metastasis. GRP78 consists of two major functional domains: the ATPase and protein/peptide-binding domains. The protein/peptide-binding domain of cell-surface GRP78 has served as a novel functional receptor for delivering cytotoxic agents (e.g., a apoptosis-inducing peptide or taxol) across the cell membrane. Here, we report our study on the ATPase domain of GRP78 (GRP78ATPase), whose potential as a transmembrane delivery system of cytotoxic agents (e.g., ATP-based nucleotide triphosphate analogs) remains unexploited. As the binding of ligands (ATP analogs) to a receptor (GRP78ATPase) is a pre-requisite for internalization, we determined the binding affinities and modes of GRP78ATPase for ADP, ATP and several ATP analogs using surface plasmon resonance and x-ray crystallography. The tested ATP analogs contain one of the following modifications: the nitrogen at the adenine ring 7-position to a carbon atom (7-deazaATP), the oxygen at the β-γ bridge position to a carbon atom (AMPPCP), or the removal of the 2’-OH group (2’-deoxyATP). We found that 7-deazaATP displays an affinity and a binding mode that resemble those of ATP regardless of magnesium ion (Mg++) concentration, suggesting that GRP78 is tolerant to modifications at the 7-position. By comparison, AMPPCP’s binding affinity was lower than ATP and Mg++-dependent, as the removal of Mg++ nearly abolished binding to GRP78ATPase. The AMPPCP-Mg++ structure showed evidence for the critical role of Mg++ in AMPPCP binding affinity, suggesting that while GRP78 is sensitive to modifications at the β-γ bridge position, these can be tolerated in the presence of Mg++. Furthermore, 2’-deoxyATP’s binding affinity was significantly lower than those for all other nucleotides tested, even in the presence of Mg++. The 2’-deoxyATP structure showed the conformation of the

  17. Probing the ATP site of GRP78 with nucleotide triphosphate analogs

    DOE PAGES

    Hughes, Scott J.; Antoshchenko, Tetyana; Chen, Yun; Lu, Hua; Pizarro, Juan C.; Park, Hee -Won

    2016-05-04

    GRP78, a member of the ER stress protein family, can relocate to the surface of cancer cells, playing key roles in promoting cell proliferation and metastasis. GRP78 consists of two major functional domains: the ATPase and protein/peptide-binding domains. The protein/peptide-binding domain of cell-surface GRP78 has served as a novel functional receptor for delivering cytotoxic agents (e.g., a apoptosis-inducing peptide or taxol) across the cell membrane. Here, we report our study on the ATPase domain of GRP78 (GRP78ATPase), whose potential as a transmembrane delivery system of cytotoxic agents (e.g., ATP-based nucleotide triphosphate analogs) remains unexploited. As the binding of ligands (ATPmore » analogs) to a receptor (GRP78ATPase) is a pre-requisite for internalization, we determined the binding affinities and modes of GRP78ATPase for ADP, ATP and several ATP analogs using surface plasmon resonance and x-ray crystallography. The tested ATP analogs contain one of the following modifications: the nitrogen at the adenine ring 7-position to a carbon atom (7-deazaATP), the oxygen at the beta-gamma bridge position to a carbon atom (AMPPCP), or the removal of the 2'-OH group (2'-deoxyATP). We found that 7-deazaATP displays an affinity and a binding mode that resemble those of ATP regardless of magnesium ion (Mg++) concentration, suggesting that GRP78 is tolerant to modifications at the 7-position. By comparison, AMPPCP's binding affinity was lower than ATP and Mg++-dependent, as the removal of Mg++ nearly abolished binding to GRP78ATPase. The AMPPCP-Mg++ structure showed evidence for the critical role of Mg++ in AMPPCP binding affinity, suggesting that while GRP78 is sensitive to modifications at the β-γ bridge position, these can be tolerated in the presence of Mg++. Furthermore, 2'-deoxyATP's binding affinity was significantly lower than those for all other nucleotides tested, even in the presence of Mg++. The 2'-deoxyATP structure showed the conformation of

  18. The adenine nucleotide translocase type 1 (ANT1): a new factor in mitochondrial disease.

    PubMed

    Sharer, J Daniel

    2005-09-01

    Mitochondrial disorders of oxidative phosphorylation (OXPHOS) comprise a growing list of potentially lethal diseases caused by mutations in either mitochondrial (mtDNA) or nuclear DNA (nDNA). Two such conditions, autosomal dominant progressive external ophthalmoplegia (adPEO) and Senger's Syndrome, are associated with dysfunction of the heart and muscle-specific isoform of the adenine nucleotide translocase (ANT1), a nDNA gene product that facilitates transport of ATP and ADP across the inner mitochondrial membrane. AdPEO is a mtDNA deletion disorder broadly characterized by pathology involving the eyes, skeletal muscle, and central nervous system. In addition to ANT1, mutations in at least two other nuclear genes, twinkle and POLG, have been shown to cause mtDNA destabilization associated with adPEO. Senger's syndrome is an autosomal recessive condition characterized by congenital heart defects, abnormalities of skeletal muscle mitochondria, cataracts, and elevated circulatory levels of lactic acid. This syndrome is associated with severe depletion of ANT1, which may be the result of an as yet unidentified ANT1-specific transcriptional or translational processing error. ANT1 has also been associated with a third condition, autosomal dominant facioscapulohumeral muscular dystrophy (FSHD), an adult onset disorder characterized by variable muscle weakness in the face, feet, shoulders, and hips. FSHD patients possess specific DNA deletions on chromosome 4, which appear to cause derepression of several nearby genes, including ANT1. Early development of FSHD may involve mitochondrial dysfunction and increased oxidative stress, possibly associated with overexpression of ANT1. PMID:16203679

  19. Effects of increased heart work on glycolysis and adenine nucleotides in the perfused heart of normal and diabetic rats

    PubMed Central

    Opie, L. H.; Mansford, K. R. L.; Owen, Patricia

    1971-01-01

    1. In the isolated perfused rat heart, the contractile activity and the oxygen uptake were varied by altering the aortic perfusion pressure, or by the atrial perfusion technique (`working heart'). 2. The maximum increase in the contractile activity brought about an eightfold increase in the oxygen uptake. The rate of glycolytic flux rose, while tissue contents of hexose monophosphates, citrate, ATP and creatine phosphate decreased, and contents of ADP and AMP rose. 3. The changes in tissue contents of adenine nucleotides during increased heart work were time-dependent. The ATP content fell temporarily (30s and 2min) after the start of left-atrial perfusion; at 5 and 10min values were normal; and at 30 and 60min values were decreased. ADP and AMP values were increased in the first 15min, but were at control values 30 or 60min after the onset of increased heart work. 4. During increased heart work changes in the tissue contents of adenine nucleotide and of citrate appeared to play a role in altered regulation of glycolysis at the level of phosphofructokinase activity. 5. In recirculation experiments increased heart work for 30min was associated with increased entry of [14C]glucose (11.1mm) and glycogen into glycolysis and a comparable increase in formation of products of glycolysis (lactate, pyruvate and 14CO2). There was no major accumulation of intermediates. Glycogen was not a major fuel for respiration. 6. Increased glycolytic flux in Langendorff perfused and working hearts was obtained by the addition of insulin to the perfusion medium. The concomitant increases in the tissue values of hexose phosphates and of citrate contrasted with the decreased values of hexose monophosphates and of citrate during increased glycolytic flux obtained by increased heart work. 7. Decreased glycolytic flux in Langendorff perfused hearts was obtained by using acute alloxan-diabetic and chronic streptozotocin-diabetic rats; in the latter condition there were decreased tissue

  20. Pleiotropic effects of the yeast Sal1 and Aac2 carriers on mitochondrial function via an activity distinct from adenine nucleotide transport

    PubMed Central

    Kucejova, Blanka; Li, Li; Wang, Xiaowen; Giannattasio, Sergio; Chen, Xin Jie

    2009-01-01

    In Saccharomyces cerevisiae, SAL1 encodes a Ca2+-binding mitochondrial carrier. Disruption of SAL1 is synthetically lethal with the loss of a specific function associated with the Aac2 isoform of the ATP/ADP translocase. This novel activity of Aac2 is defined as the V function (for Viability of aac2 sal1 double mutant), which is independent of the ATP/ADP exchange activity required for respiratory growth (the R function). We found that co-inactivation of SAL1 and AAC2 leads to defects in mitochondrial translation and mitochondrial DNA (mtDNA) maintenance. Additionally, sal1Δ exacerbates the respiratory deficiency and mtDNA instability of ggc1Δ, shy1Δ and mtg1Δ mutants, which are known to reduce mitochondrial protein synthesis or protein complex assembly. The V function is complemented by the human Short Ca2+-binding Mitochondrial Carrier (SCaMC) protein, SCaMC-2, a putative ATP-Mg/Pi exchangers on the inner membrane. However, mitochondria lacking both Sal1p and Aac2p are not depleted of adenine nucleotides. The Aac2R252I and Aac2R253I variants mutated at the R252-254 triplet critical for nucleotide transport retain the V function. Likewise, Sal1p remains functionally active when the R479I and R481I mutations were introduced into the structurally equivalent R479-T480-R481 motif. Finally, we found that the naturally occurring V-R+ Aac1 isoform of adenine nucleotide translocase partially gains the V function at the expense of the R function by introducing the mutations P89L and A96V. Thus, our data support the view that the V function is independent of adenine nucleotide transport associated with Sal1p and Aac2p and this evolutionarily conserved activity affects multiple processes in mitochondria. PMID:18431598

  1. Studies on the energy metabolism of opossum (Didelphis virginiana) erythrocytes: V. Utilization of hypoxanthine for the synthesis of adenine and guanine nucleotides in vitro

    SciTech Connect

    Bethlenfalvay, N.C.; White, J.C.; Chadwick, E.; Lima, J.E. )

    1990-06-01

    High pressure liquid radiochromatography was used to test the ability of opossum erythrocytes to incorporate tracer amounts of (G-{sup 3}H) hypoxanthine (Hy) into ({sup 3}H) labelled triphosphates of adenine and guanine. In the presence of supraphysiologic (30 mM) phosphate which is optimal for PRPP synthesis, both ATP and GTP are extensively labelled. When physiologic (1 mM) medium phosphate is used, red cells incubated under an atmosphere of nitrogen accumulate ({sup 3}H) ATP in a linear fashion suggesting ongoing PRPP synthesis in red cells whose hemoglobin is deoxygenated. In contrast, a lesser increase of labelled ATP is observed in cells incubated under oxygen, suggesting that conditions for purine nucleotide formation from ambient Hy are more favorable in the venous circulation.

  2. Regulation of Ca²⁺ release through inositol 1,4,5-trisphosphate receptors by adenine nucleotides in parotid acinar cells.

    PubMed

    Park, Hyung Seo; Betzenhauser, Matthew J; Zhang, Yu; Yule, David I

    2012-01-01

    Secretagogue-stimulated intracellular Ca(2+) signals are fundamentally important for initiating the secretion of the fluid and ion component of saliva from parotid acinar cells. The Ca(2+) signals have characteristic spatial and temporal characteristics, which are defined by the specific properties of Ca(2+) release mediated by inositol 1,4,5-trisphosphate receptors (InsP(3)R). In this study we have investigated the role of adenine nucleotides in modulating Ca(2+) release in mouse parotid acinar cells. In permeabilized cells, the Ca(2+) release rate induced by submaximal [InsP(3)] was increased by 5 mM ATP. Enhanced Ca(2+) release was not observed at saturating [InsP(3)]. The EC(50) for the augmented Ca(2+) release was ∼8 μM ATP. The effect was mimicked by nonhydrolysable ATP analogs. ADP and AMP also potentiated Ca(2+) release but were less potent than ATP. In acini isolated from InsP(3)R-2-null transgenic animals, the rate of Ca(2+) release was decreased under all conditions but now enhanced by ATP at all [InsP(3)]. In addition the EC(50) for ATP potentiation increased to ∼500 μM. These characteristics are consistent with the properties of the InsP(3)R-2 dominating the overall features of InsP(3)R-induced Ca(2+) release despite the expression of all isoforms. Finally, Ca(2+) signals were measured in intact parotid lobules by multiphoton microscopy. Consistent with the release data, carbachol-stimulated Ca(2+) signals were reduced in lobules exposed to experimental hypoxia compared with control lobules only at submaximal concentrations. Adenine nucleotide modulation of InsP(3)R in parotid acinar cells likely contributes to the properties of Ca(2+) signals in physiological and pathological conditions.

  3. Role of vacuum ultraviolet (VUV) radiation in abiogenic synthesis of adenine nucleotides

    NASA Astrophysics Data System (ADS)

    Kuzicheva, E. A.; Simakov, M. B.; Mal'Ko, I. L.; Dodonova, N. Ya.; Gontareva, N. B.

    With the use of high performance liquid chromatography the products of abiogenic synthesis of adenine nucleotides in solid films were indentified and estimated quantitatively. The main products of photosynthesis appeared to be adenosine and deoxyadenosine monophosphates. Maximal yield of these products in case of adenosine has been 0.36 for 5'AMP, 0.41% for 2'(3')AMP, 0.20 for 2'3'cAMP in case of deoxyadenosine 0.13% for 5'dAMP, 0.15% for 3'dAMP, 0.24% for 3'5'cdAMP. The destruction of initial adenosine and deoxyadenosine by the end of the experiment was 10 and 15%, respectively. By the increasing of irradiation dose, 5'AMP and 5'dAMP synthesized in the cource of VUV photolysis were destructed up to adenine, its yield being 15% in both cases.

  4. Adenine Nucleotide Levels, the Redox State of the NADP System, and Assimilatory Force in Nonaqueously Purified Mesophyll Chloroplasts from Maize Leaves under Different Light Intensities 1

    PubMed Central

    Usuda, Hideaki

    1988-01-01

    Recently, a nonaqueous fractionation method of obtaining highly purified mesophyll chloroplasts from maize leaves was established. This method is now used to determine adenine nucleotide levels, the redox states of the NADP system, Pi levels and dihydroxyacetone phosphate/3-phosphoglycerate ratios in mesophyll chloroplasts of Zea mays L. leaves under different light intensities. The sum of the ATP, ADP, and AMP levels was estimated to be 1.4 millimolar and the ATP/ADP ratio was 1 in the dark and 2.5 to 4 in the light. The adenine nucleotides were equilibrated by adenylate kinase. The total concentration of NADP(H) in the chloroplasts was 0.3 millimolar in the dark and 0.48 millimolar in the light. The ratio of NADPH/NADP was 0.1 to 0.18 in the dark and 0.23 to 0.48 in the light. The Pi level was estimated to be 20 millimolar in the dark and 10 to 17 millimolar in the light. The 3-phosphoglycerate reducing system was under thermodynamic equilibrium in the light. The calculated assimilatory forces were 8 per molar and 40 to 170 per molar in the dark and the light, respectively. There was no relationship between the degree of activation of pyruvate, Pi dikinase, and adenylate energy charge, or ATP/ADP ratio or ADP level under various light intensities. Only a weak relationship was found between the degree of activation of NADP-malate dehydrogenase and the NADPH/NADP ratio or NADP(H) level with increasing light intensity. A possible regulatory mechanism which is responsible for the regulation of activation of pyruvate,Pi dikinase and NADP-malate dehydrogenase is discussed. PMID:16666481

  5. P2Y13 receptors mediate presynaptic inhibition of acetylcholine release induced by adenine nucleotides at the mouse neuromuscular junction.

    PubMed

    Guarracino, Juan F; Cinalli, Alejandro R; Fernández, Verónica; Roquel, Liliana I; Losavio, Adriana S

    2016-06-21

    It is known that adenosine 5'-triphosphate (ATP) is released along with the neurotransmitter acetylcholine (ACh) from motor nerve terminals. At mammalian neuromuscular junctions (NMJs), we have previously demonstrated that ATP is able to decrease ACh secretion by activation of P2Y receptors coupled to pertussis toxin-sensitive Gi/o protein. In this group, the receptor subtypes activated by adenine nucleotides are P2Y12 and P2Y13. Here, we investigated, by means of pharmacological and immunohistochemical assays, the P2Y receptor subtype that mediates the modulation of spontaneous and evoked ACh release in mouse phrenic nerve-diaphragm preparations. First, we confirmed that the preferential agonist for P2Y12-13 receptors, 2-methylthioadenosine 5'-diphosphate trisodium salt hydrate (2-MeSADP), reduced MEPP frequency without affecting MEPP amplitude as well as the amplitude and quantal content of end-plate potentials (EPPs). The effect on spontaneous secretion disappeared after the application of the selective P2Y12-13 antagonists AR-C69931MX or 2-methylthioadenosine 5'-monophosphate triethylammonium salt hydrate (2-MeSAMP). 2-MeSADP was more potent than ADP and ATP in reducing MEPP frequency. Then we demonstrated that the selective P2Y13 antagonist MRS-2211 completely prevented the inhibitory effect of 2-MeSADP on MEPP frequency and EPP amplitude, whereas the P2Y12 antagonist MRS-2395 failed to do this. The preferential agonist for P2Y13 receptors inosine 5'-diphosphate sodium salt (IDP) reduced spontaneous and evoked ACh secretion and MRS-2211 abolished IDP-mediated modulation. Immunohistochemical studies confirmed the presence of P2Y13 but not P2Y12 receptors at the end-plate region. Disappearance of P2Y13 receptors after denervation suggests the presynaptic localization of the receptors. We conclude that, at motor nerve terminals, the Gi/o protein-coupled P2Y receptors implicated in presynaptic inhibition of spontaneous and evoked ACh release are of the subtype P2Y

  6. P2Y13 receptors mediate presynaptic inhibition of acetylcholine release induced by adenine nucleotides at the mouse neuromuscular junction.

    PubMed

    Guarracino, Juan F; Cinalli, Alejandro R; Fernández, Verónica; Roquel, Liliana I; Losavio, Adriana S

    2016-06-21

    It is known that adenosine 5'-triphosphate (ATP) is released along with the neurotransmitter acetylcholine (ACh) from motor nerve terminals. At mammalian neuromuscular junctions (NMJs), we have previously demonstrated that ATP is able to decrease ACh secretion by activation of P2Y receptors coupled to pertussis toxin-sensitive Gi/o protein. In this group, the receptor subtypes activated by adenine nucleotides are P2Y12 and P2Y13. Here, we investigated, by means of pharmacological and immunohistochemical assays, the P2Y receptor subtype that mediates the modulation of spontaneous and evoked ACh release in mouse phrenic nerve-diaphragm preparations. First, we confirmed that the preferential agonist for P2Y12-13 receptors, 2-methylthioadenosine 5'-diphosphate trisodium salt hydrate (2-MeSADP), reduced MEPP frequency without affecting MEPP amplitude as well as the amplitude and quantal content of end-plate potentials (EPPs). The effect on spontaneous secretion disappeared after the application of the selective P2Y12-13 antagonists AR-C69931MX or 2-methylthioadenosine 5'-monophosphate triethylammonium salt hydrate (2-MeSAMP). 2-MeSADP was more potent than ADP and ATP in reducing MEPP frequency. Then we demonstrated that the selective P2Y13 antagonist MRS-2211 completely prevented the inhibitory effect of 2-MeSADP on MEPP frequency and EPP amplitude, whereas the P2Y12 antagonist MRS-2395 failed to do this. The preferential agonist for P2Y13 receptors inosine 5'-diphosphate sodium salt (IDP) reduced spontaneous and evoked ACh secretion and MRS-2211 abolished IDP-mediated modulation. Immunohistochemical studies confirmed the presence of P2Y13 but not P2Y12 receptors at the end-plate region. Disappearance of P2Y13 receptors after denervation suggests the presynaptic localization of the receptors. We conclude that, at motor nerve terminals, the Gi/o protein-coupled P2Y receptors implicated in presynaptic inhibition of spontaneous and evoked ACh release are of the subtype P2Y

  7. Changes in the expression of the human adenine nucleotide translocase isoforms condition cellular metabolic/proliferative status

    PubMed Central

    Mampel, Teresa; Viñas, Octavi

    2016-01-01

    Human cells express four mitochondrial adenine nucleotide translocase (hANT) isoforms that are tissue-specific and developmentally regulated. hANT1 is mainly expressed in terminally differentiated muscle cells; hANT2 is growth-regulated and is upregulated in highly glycolytic and proliferative cells; and hANT3 is considered to be ubiquitous and non-specifically regulated. Here, we studied how the expression of hANT isoforms is regulated by proliferation and in response to metabolic stimuli, and examined the metabolic consequences of their silencing and overexpression. In HeLa and HepG2 cells, expression of hANT3 was upregulated by shifting metabolism towards oxidation or by slowed growth associated with contact inhibition or growth-factor deprivation, indicating that hANT3 expression is highly regulated. Under these conditions, changes in hANT2 mRNA expression were not observed in either HeLa or HepG2 cells, whereas in SGBS preadipocytes (which, unlike HeLa and HepG2 cells, are growth-arrest-sensitive cells), hANT2 mRNA levels decreased. Additionally, overexpression of hANT2 promoted cell growth and glycolysis, whereas silencing of hANT3 decreased cellular ATP levels, limited cell growth and induced a stress-like response. Thus, cancer cells require both hANT2 and hANT3, depending on their proliferation status: hANT2 when proliferation rates are high, and hANT3 when proliferation slows. PMID:26842067

  8. Capillary zone electrophoresis with field enhanced sample stacking as a tool for targeted metabolome analysis of adenine nucleotides and coenzymes in Paracoccus denitrificans.

    PubMed

    Musilová, Jindra; Sedlácek, Vojtech; Kucera, Igor; Glatz, Zdenek

    2009-07-01

    The main aim of this work was to demonstrate the applicability of capillary zone electrophoresis in combination with field enhanced sample stacking in targeted metabolome analyses of adenine nucleotides--AMP, ADP, ATP, coenzymes NAD(+), NADP(+) and their reduced forms in Paracoccus denitrificans. Sodium carbonate/hydrogencarbonate buffer (100 mM, pH 9.6) with the addition of beta-CD at a concentration of 10 mM was found to be an effective BGE for their separation within 20 min. Besides this, special attention was paid to the development of the procedure for the extraction of specific metabolites from the bacterium P. denitrificans. This procedure was not only optimised to achieve the highest metabolite yields but also to obtain a sample that was fully compatible with the online preconcetration strategy used. The developed methodology was finally applied in a study of the bacterium P. denitrificans at various stages of the active respiratory chain.

  9. Effect of treated-sewage contamination upon bacterial energy charge, adenine nucleotides, and DNA content in a sandy aquifer on cape cod

    USGS Publications Warehouse

    Metge, D.W.; Brooks, M.H.; Smith, R.L.; Harvey, R.W.

    1993-01-01

    Changes in adenylate energy charge (EC(A)) and in total adenine nucleotides (A(T)) and DNA content (both normalized to the abundance of free- living, groundwater bacteria) in response to carbon loading were determined for a laboratory-grown culture and for a contaminated aquifer. The latter study involved a 3-km-long transect through a contaminant plume resulting from continued on-land discharge of secondary sewage to a shallow, sandy aquifer on Cape Cod, Mass. With the exception of the most contaminated groundwater immediately downgradient from the contaminant source, DNA and adenylate levels correlated strongly with bacterial abundance and decreased exponentially with increasing distance downgradient. EC(A)s (0.53 to 0.60) and the ratios of ATP to DNA (0.001 to 0.003) were consistently low, suggesting that the unattached bacteria in this groundwater study are metabolically stressed, despite any eutrophication that might have occurred. Elevated EC(A)s (up to 0.74) were observed in glucose-amended groundwater, confirming that the metabolic state of this microbial community could be altered. In general, per-bacterium DNA and ATP contents were approximately twofold higher in the plume than in surrounding groundwater, although EC(A) and per-bacterium levels of A(T) differed little in the plume and the surrounding uncontaminated groundwater. However, per-bacterium levels of DNA and A(T) varied six- and threefold, respectively, during a 6-h period of decreasing growth rate for an unidentified pseudomonad isolated from contaminated groundwater and grown in batch culture. These data suggest that the DNA content of groundwater bacteria may be more sensitive than their A(T) to the degree of carbon loading, which may have significant ramifications in the use of nucleic acids and adenine nucleotides for estimating the metabolic status of bacterial communities within more highly contaminated aquifers.

  10. Inhibition of the adenine nucleotide translocator by N-acetyl perfluorooctane sulfonamides in vitro

    SciTech Connect

    O'Brien, Timothy M. Oliveira, Paulo J.; Wallace, Kendall B.

    2008-03-01

    N-alkyl perfluorooctane sulfonamides have been widely used as surfactants on fabrics and papers, fire retardants, and anti-corrosion agents, among many other commercial applications. The global distribution and environmental persistence of these compounds has generated considerable interest regarding potential toxic effects. We have previously reported that perfluorooctanesulfonamidoacetate (FOSAA) and N-ethylperfluorooctanesulfonamidoacetate (N-EtFOSAA) induce the mitochondrial permeability transition (MPT) in vitro. In this study we tested the hypothesis that FOSAA and N-EtFOSAA interact with the adenine nucleotide translocator (ANT) resulting in a functional inhibition of the translocator and induction of the MPT. Respiration and membrane potential of freshly isolated liver mitochondria from Sprague-Dawley rats were measured using an oxygen electrode and a tetraphenylphosphonium-selective (TPP{sup +}) electrode, respectively. Mitochondrial swelling was measured spectrophotometrically. The ANT ligands bongkregkic acid (BKA) and carboxyatractyloside (cATR) inhibited uncoupling of mitochondrial respiration caused by 10 {mu}M N-EtFOSAA, 40 {mu}M FOSAA, and the positive control 8 {mu}M oleic acid. ADP-stimulated respiration and depolarization of mitochondrial membrane potential were inhibited by cATR, FOSAA, N-EtFOSAA, and oleic acid, but not by FCCP. BKA inhibited calcium-dependent mitochondrial swelling induced by FOSAA, N-EtFOSAA, and oleic acid. Seventy-five micromolar ADP also inhibited swelling induced by the test compounds, but cATR induced swelling was not inhibited by ADP. Results of this investigation indicate that N-acetyl perfluorooctane sulfonamides interact directly with the ANT to inhibit ADP translocation and induce the MPT, one or both of which may account for the metabolic dysfunction observed in vivo.

  11. Mitochondrial permeability transition as induced by cross-linking of the adenine nucleotide translocase.

    PubMed

    Zazueta, C; Reyes-Vivas, H; Zafra, G; Sánchez, C A; Vera, G; Chávez, E

    1998-04-01

    Mitochondrial permeability transition is caused by the opening of a transmembrane pore whose chemical nature has not been well established yet. The present work was aimed to further contribute to the knowledge of the membrane entity comprised in the formation of the non-specific channel. The increased permeability was established by analyzing the inability of rat kidney mitochondria to take up and accumulate Ca2+, as well as their failure to build up a transmembrane potential, after the cross-linking of membrane proteins by copper plus ortho-phenanthroline. To identify the cross-linked proteins, polyacrylamide gel electrophoresis was performed. The results are representative of at least three separate experiments. It is indicated that 30 microM Cu2+ induced the release of 4.3 nmol Ca2+ per mg protein. However, in the presence of 100 microM ortho-phenanthroline only 2 microM Cu2+ was required to attain the total release of the accumulated Ca2+; it should be noted that such a reaction is not inhibited by cyclosporin. The increased permeability corresponds to cross-linking of membrane proteins in which approximately 4 nmol thiol groups per mg protein appear to be involved. Such a linking process is inhibited by carboxyatractyloside. By using the fluorescent probe eosin-5-maleimide the label was found in a cross-linking 60 kDa dimer of two 30 kDa monomers. From the data presented it is concluded that copper-o-phenanthroline induces the intermolecular cross-linking of the adenine nucleotide translocase which in turn is converted to non-specific pore. PMID:9675885

  12. Adenine Nucleotide Translocase 4 Is Expressed Within Embryonic Ovaries and Dispensable During Oogenesis

    PubMed Central

    Lim, Chae Ho; Brower, Jeffrey V.; Resnick, James L.; Oh, S. Paul

    2015-01-01

    Adenine nucleotide translocase (Ant) facilitates the exchange of adenosine triphosphate across the mitochondrial inner membrane and plays a critical role for bioenergetics in eukaryotes. Mice have 3 Ant paralogs, Ant1 (Slc25a4), Ant2 (Slc25a5), and Ant4 (Slc25a31), which are expressed in a tissue-dependent manner. We previously identified that Ant4 was expressed exclusively in testicular germ cells in adult mice and essential for spermatogenesis and subsequently male fertility. Further investigation into the process of spermatogenesis revealed that Ant4 was particularly highly expressed during meiotic prophase I and indispensable for normal progression of leptotene spermatocytes to the stages thereafter. In contrast, the expression and roles of Ant4 in female germ cells have not previously been elucidated. Here, we demonstrate that the Ant4 gene is expressed during embryonic ovarian development during which meiotic prophase I occurs. We confirmed embryonic ovary-specific Ant4 expression using a bacterial artificial chromosome transgene. In contrast to male, however, Ant4 null female mice were fertile although the litter size was slightly decreased. They showed apparently normal ovarian development which was morphologically indistinguishable from the control animals. These data indicate that Ant4 is a meiosis-specific gene expressed during both male and female gametogenesis however indispensable only during spermatogenesis and not oogenesis. The differential effects of Ant4 depletion within the processes of male and female gametogenesis may be explained by meiosis-specific inactivation of the X-linked Ant2 gene in male, a somatic paralog of the Ant4 gene. PMID:25031318

  13. Activities of adenine nucleotide and nucleoside degradation enzymes in platelets of rats infected by Trypanosoma evansi.

    PubMed

    Oliveira, Camila B; Da Silva, Aleksandro S; Vargas, Lara B; Bitencourt, Paula E R; Souza, Viviane C G; Costa, Marcio M; Leal, Claudio A M; Moretto, Maria B; Leal, Daniela B R; Lopes, Sonia T A; Monteiro, Silvia G

    2011-05-31

    Nucleotide and nucleoside-degrading enzymes, such as nucleoside triphosphate diphosphohydrose (NTPDase), 5'-nucleotidase and adenosine deaminase (ADA) are present in the surface membranes of platelets, involved in clotting disturbances of Trypanosoma evansi-infected animals. Thus, this study was aimed at evaluating the activities of these enzymes in platelets of rats experimentally infected with T. evansi. Animals were divided into four groups, according to the level of parasitemia. Blood samples were collected on days 3 (group A: at the beginning of parasitemia), 5 (group B: high parasitemia) and 15 (group C: chronic infection), post-infection. Group D (control group) was composed of non-infected animals for platelet count, separation and enzymatic assays. Animals from groups A and B showed marked thrombocytopenia, but platelet count was not affected in chronically infected rats. NTPDase, 5'-nucleotidase and ADA activities decreased (p<0.05) in platelets from rats of groups A and B, when compared to the control group. In group C, only NTPDase and 5'-nucleoside activities decreased (p<0.001). The correlations between platelet count and nucleotide/nucleoside hydrolysis were positive and statistically significant (p<0.05) in groups A and B. Platelet aggregation was decreased in all infected groups, in comparison to the control group (p<0.05). It is concluded that the alterations observed in the activities of NTPDase, 5'-nucleotidase and ADA in platelets of T. evansi-infected animals might be related to thrombocytopenia, that by reducing the number of platelets, there was less release of ATP and ADP. Another possibility being suggested is that changes have occurred in the membrane of these cells, decreasing the expression of these enzymes in the cell membrane.

  14. Caffeic acid treatment alters the extracellular adenine nucleotide hydrolysis in platelets and lymphocytes of adult rats.

    PubMed

    Anwar, Javed; Spanevello, Roselia Maria; Pimentel, Victor Camera; Gutierres, Jessié; Thomé, Gustavo; Cardoso, Andreia; Zanini, Daniela; Martins, Caroline; Palma, Heloisa Einloft; Bagatini, Margarete Dulce; Baldissarelli, Jucimara; Schmatz, Roberta; Leal, Cláudio Alberto Martins; da Costa, Pauline; Morsch, Vera Maria; Schetinger, Maria Rosa Chitolina

    2013-06-01

    This study evaluated the effects of caffeic acid on ectonucleotidase activities such as NTPDase (nucleoside triphosphate diphosphohydrolase), Ecto-NPP (nucleotide pyrophosphatase/phosphodiesterase), 5'-nucleotidase and adenosine deaminase (ADA) in platelets and lymphocytes of rats, as well as in the profile of platelet aggregation. Animals were divided into five groups: I (control); II (oil); III (caffeic acid 10 mg/kg); IV (caffeic acid 50 mg/kg); and V (caffeic acid 100 mg/kg). Animals were treated with caffeic acid diluted in oil for 30 days. In platelets, caffeic acid decreased the ATP hydrolysis and increased ADP hydrolysis in groups III, IV and V when compared to control (P<0.05). The 5'-nucleotidase activity was decreased, while E-NPP and ADA activities were increased in platelets of rats of groups III, IV and V (P<0.05). Caffeic acid reduced significantly the platelet aggregation in the animals of groups III, IV and V in relation to group I (P<0.05). In lymphocytes, the NTPDase and ADA activities were increased in all groups treated with caffeic acid when compared to control (P<0.05). These findings demonstrated that the enzymes were altered in tissues by caffeic acid and this compound decreased the platelet aggregation suggesting that caffeic acid should be considered a potentially therapeutic agent in disorders related to the purinergic system.

  15. Alkaline Phosphatase, Soluble Extracellular Adenine Nucleotides, and Adenosine Production after Infant Cardiopulmonary Bypass

    PubMed Central

    Davidson, Jesse A.; Urban, Tracy; Tong, Suhong; Twite, Mark; Woodruff, Alan

    2016-01-01

    Rationale Decreased alkaline phosphatase activity after infant cardiac surgery is associated with increased post-operative cardiovascular support requirements. In adults undergoing coronary artery bypass grafting, alkaline phosphatase infusion may reduce inflammation. Mechanisms underlying these effects have not been explored but may include decreased conversion of extracellular adenine nucleotides to adenosine. Objectives 1) Evaluate the association between alkaline phosphatase activity and serum conversion of adenosine monophosphate to adenosine after infant cardiac surgery; 2) assess if inhibition/supplementation of serum alkaline phosphatase modulates this conversion. Methods and Research Pre/post-bypass serum samples were obtained from 75 infants <4 months of age. Serum conversion of 13C5-adenosine monophosphate to 13C5-adenosine was assessed with/without selective inhibition of alkaline phosphatase and CD73. Low and high concentration 13C5-adenosine monophosphate (simulating normal/stress concentrations) were used. Effects of alkaline phosphatase supplementation on adenosine monophosphate clearance were also assessed. Changes in serum alkaline phosphatase activity were strongly correlated with changes in 13C5-adenosine production with or without CD73 inhibition (r = 0.83; p<0.0001). Serum with low alkaline phosphatase activity (≤80 U/L) generated significantly less 13C5-adenosine, particularly in the presence of high concentration 13C5-adenosine monophosphate (10.4μmol/L vs 12.9μmol/L; p = 0.0004). Inhibition of alkaline phosphatase led to a marked decrease in 13C5-adenosine production (11.9μmol/L vs 2.7μmol/L; p<0.0001). Supplementation with physiologic dose human tissue non-specific alkaline phosphatase or high dose bovine intestinal alkaline phosphatase doubled 13C5-adenosine monophosphate conversion to 13C5-adenosine (p<0.0001). Conclusions Alkaline phosphatase represents the primary serum ectonucleotidase after infant cardiac surgery and low post

  16. An adenine nucleotide translocase (ANT) gene from Apostichopus japonicus; molecular cloning and expression analysis in response to lipopolysaccharide (LPS) challenge and thermal stress.

    PubMed

    Liu, Qiu-Ning; Chai, Xin-Yue; Tu, Jie; Xin, Zhao-Zhe; Li, Chao-Feng; Jiang, Sen-Hao; Zhou, Chun-Lin; Tang, Bo-Ping

    2016-02-01

    The adenine nucleotide translocases (ANTs) play a vital role in energy metabolism via ADP/ATP exchange in eukaryotic cells. Apostichopus japonicus (Echinodermata: Holothuroidea) is an important economic species in China. Here, a cDNA representing an ANT gene of A. japonicus was isolated and characterized from respiratory tree and named AjANT. The full-length AjANT cDNA is 1924 bp, including a 5'-untranslated region (UTR) of 38 bp, 3'-UTR of 980 bp and an open reading frame (ORF) of 906 bp encoding a polypeptide of 301 amino acids. The protein contains three homologous repeat Mito_carr domains (Pfam00153). The deduced AjANT protein sequence has 49-81% in comparison to ANT proteins from other individuals. The predicted tertiary structure of AjANT protein is highly similar to animal ANT proteins. Phylogenetic analysis showed that the AjANT is closely related to Holothuroidea ANT genes. Real-time quantitative reverse transcription-PCR (qPCR) analysis showed that AjANT expression is higher in the respiratory tree than in other examined tissues. After thermal stress or LPS challenge, expression of AjANT was significantly fluctuant compared to the control. These results suggested that changes in the expression of ANT gene might be involved in immune defense and in protecting A. japonicus against thermal stress. PMID:26706223

  17. Adenine Nucleotide Metabolism and a Role for AMP in Modulating Flagellar Waveforms in Mouse Sperm1

    PubMed Central

    Vadnais, Melissa L.; Cao, Wenlei; Aghajanian, Haig K.; Haig-Ladewig, Lisa; Lin, Angel M.; Al-Alao, Osama; Gerton, George L.

    2014-01-01

    ABSTRACT While most ATP, the main energy source driving sperm motility, is derived from glycolysis and oxidative phosphorylation, the metabolic demands of the cell require the efficient use of power stored in high-energy phosphate bonds. In times of high energy consumption, adenylate kinase (AK) scavenges one ATP molecule by transphosphorylation of two molecules of ADP, simultaneously yielding one molecule of AMP as a by-product. Either ATP or ADP supported motility of detergent-modeled cauda epididymal mouse sperm, indicating that flagellar AKs are functional. However, the ensuing flagellar waveforms fueled by ATP or ADP were qualitatively different. Motility driven by ATP was rapid but restricted to the distal region of the sperm tail, whereas ADP produced slower and more fluid waves that propagated down the full flagellum. Characterization of wave patterns by tracing and superimposing the images of the flagella, quantifying the differences using digital image analysis, and computer-assisted sperm analysis revealed differences in the amplitude, periodicity, and propagation of the waves between detergent-modeled sperm treated with either ATP or ADP. Surprisingly, addition of AMP to the incubation medium containing ATP recapitulated the pattern of sperm motility seen with ADP alone. In addition to AK1 and AK2, which we previously demonstrated are present in outer dense fibers and mitochondrial sheath of the mouse sperm tail, we show that another AK, AK8, is present in a third flagellar compartment, the axoneme. These results extend the known regulators of sperm motility to include AMP, which may be operating through an AMP-activated protein kinase. PMID:24740601

  18. Regulation of energy transduction and electron transfer in cytochrome c oxidase by adenine nucleotides.

    PubMed

    Kadenbach, B; Napiwotzki, J; Frank, V; Arnold, S; Exner, S; Hüttemann, M

    1998-02-01

    Cytochrome c oxidase from bovine heart contains seven high-affinity binding sites for ATP or ADP and three additional only for ADP. One binding site for ATP or ADP, located at the matrix-oriented domain of the heart-type subunit VIaH, increases the H+/e- stoichiometry of the enzyme from heart or skeletal muscle from 0.5 to 1.0 when bound ATP is exchanged by ADP. Two further binding sites for ATP or ADP, located at the cytosolic and the matrix domain of subunit IV, increases the K(M) for cytochrome c and inhibit the respiratory activity at high ATP/ADP ratios, respectively. We propose that thermogenesis in mammals is related to subunit VIaL of cytochrome c oxidase with a H+/e- stoichiometry of 0.5 compared to 1.0 in the enzyme from bacteria or ectotherm animals. This hypothesis is supported by the lack of subunit VIa isoforms in cytochrome c oxidase from fish.

  19. Activation by ATP of a P2U 'nucleotide' receptor in an exocrine cell.

    PubMed Central

    Martin, S. C.; Shuttleworth, T. J.

    1995-01-01

    1. We employed the perforated patch whole-cell technique to investigate the effects of ATP and other related nucleotides on membrane conductances in avian exocrine salt gland cells. 2. ATP (10 microM-1 mM) evoked an increase in maxi-K+ and Cl- conductances with a reversal potential of -35 mV. At lower concentrations of ATP (< or = 100 microM) responses were generally oscillatory with a sustained response observed at higher concentrations (> or = 200 microM). 3. Both oscillatory and sustained responses were abolished by the removal of bath Ca2+. In cells preincubated in extracellular saline containing reduced Ca2+, the application of ATP resulted in a transient increase in current. 4. As increasing concentrations of ATP (and related nucleotides) evoked a graded sequence of events with little run-down we were able to establish a rank order of potency in single cells. The order of potency of ATP analogues and agonists of the various P2-receptor subtypes was UTP > ATP = 2-methylthio-ATP > ADP. Adenosine (1 microM-1 mM), AMP (1 microM-1 mM), alpha,beta-methylene-ATP (1 microM-1 mM) and beta,gamma-methylene-ATP (1 microM-1 mM) were without effect. 5. In conclusion, although unable to preclude a role for a P2Y-receptor, our results suggest that ATP binds to a P2U-receptor increasing [Ca2+]i and subsequently activating Ca(2+)-sensitive K+ and Cl- currents. PMID:7670734

  20. Effect of treated-sewage contamination upon bacterial energy charge, adenine nucleotides, and DNA content in a sandy aquifer on Cape Cod.

    PubMed

    Metge, D W; Brooks, M H; Smith, R L; Harvey, R W

    1993-07-01

    Changes in adenylate energy charge (ECA) and in total adenine nucleotides (A(T) and DNA content (both normalized to the abundance of free-living, groundwater bacteria) in response to carbon loading were determined for a laboratory-grown culture and for a contaminated aquifer. The latter study involved a 3-km-long transect through a contaminant plume resulting from continued on-land discharge of secondary sewage to a shallow, sandy aquifer on Cape Cod, Mass. With the exception of the most contaminated groundwater immediately downgradient from the contaminant source, DNA and adenylate levels correlated strongly with bacterial abundance and decreased exponentially with increasing distance downgradient. ECAS (0.53 to 0.60) and the ratios of ATP to DNA (0.001 to 0.003) were consistently low, suggesting that the unattached bacteria in this groundwater study are metabolically stressed, despite any eutrophication that might have occurred. Elevated ECAS (up to 0.74) were observed in glucose-amended groundwater, confirming that the metabolic state of this microbial community could be altered. In general, per-bacterium DNA and ATP contents were approximately twofold higher in the plume than in surrounding groundwater, although ECA and per-bacterium levels of A(T) differed little in the plume and the surrounding uncontaminated groundwater. However, per-bacterium levels of DNA and A(T) varied six- and threefold, respectively, during a 6-h period of decreasing growth rate for an unidentified pseudomonad isolated from contaminated groundwater and grown in batch culture.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Studies of adenine nucleotide metabolism in bovine spermatozoa using sup 32 P sub i as tracer

    SciTech Connect

    Cheetham, J.A.

    1989-01-01

    It was previously demonstrated that incubation of bovine sperm with {sup 32}P{sub i} yields ADP of 2 to 3 times higher specific activity than that of ATP, contrary to what is seen in other types of cells. Experiments conducted to explain this phenomenon indicate that it occurs with a wide variety of substrates added to intact sperm. The incorporation of label into ATP requires the phosphorylation reactions of either mitochondrial oxidative phosphorylation or glycolysis, whereas incorporation of {sup 32}P{sub i} into ADP occurs when oxidative phosphorylation is inhibited and no glycolytic substrate is provided. The possibility that the high energy phosphate produced in the succinyl thiokinase step of the citric acid cycle accounts for this phenomenon was examined by restricting production of high energy phosphate to this reaction with an uncoupler of oxidative phosphorylation. Under these conditions addition of arsenite, an inhibitor of pyruvate oxidation, does not block incorporation of label into ADP. Furthermore, no {sup 32}P{sub i} label was incorporated into ADP when midpieces prepared from ejaculated sperm were incubated with various substrates plus uncoupler. Therefore, it appears unlikely that substrate level phosphorylation contributes to incorporation of {sup 32}P{sub i} into ADP or ATP of intact cells. Instead, it appears that predominant labeling of ADP may arise due to involvement of only a small portion of cell ATP in reactions in which {sup 32}P{sub i} is incorporated. When intact bovine ejaculated sperm are incubated with {sup 32}P{sub i} for two hrs, labeling of ADP reaches steady state but ATP does not. This is consistent with slow exchange of {sup 32}P-labeled ATP between a metabolically active pool and a larger one which is not incorporating {sup 32}P{sub i}.

  2. Direct Monitoring of Nucleotide Turnover in Human Cell Extracts and Cells by Fluorogenic ATP Analogs.

    PubMed

    Hacker, Stephan M; Buntz, Annette; Zumbusch, Andreas; Marx, Andreas

    2015-11-20

    Nucleotides containing adenosine play pivotal roles in every living cell. Adenosine triphosphate (ATP), for example, is the universal energy currency, and ATP-consuming processes also contribute to posttranslational protein modifications. Nevertheless, detecting the turnover of adenosine nucleotides in the complex setting of a cell remains challenging. Here, we demonstrate the use of fluorogenic analogs of ATP and adenosine tetraphosphate to study nucleotide hydrolysis in lysates of human cell lines and in intact human cells. We found that the adenosine triphosphate analog is completely stable in lysates of human cell lines, whereas the adenosine tetraphosphate analog is rapidly turned over. The observed activity in human cell lysates can be assigned to a single enzyme, namely, the human diadenosine tetraphosphate hydrolase NudT2. Since NudT2 has been shown to be a prognostic factor for breast cancer, the adenosine tetraphosphate analog might contribute to a better understanding of its involvement in cancerogenesis and allow the straightforward screening for inhibitors. Studying hydrolysis of the analogs in intact cells, we found that electroporation is a suitable method to deliver nucleotide analogs into the cytoplasm and show that high FRET efficiencies can be detected directly after internalization. Time-dependent experiments reveal that adenosine triphosphate and tetraphosphate analogs are both processed in the cellular environment. This study demonstrates that these nucleotide analogs indeed bear the potential to be powerful tools for the exploration of nucleotide turnover in the context of whole cells.

  3. ATP-binding site of adenylate kinase: mechanistic implications of its homology with ras-encoded p21, F1-ATPase, and other nucleotide-binding proteins.

    PubMed

    Fry, D C; Kuby, S A; Mildvan, A S

    1986-02-01

    The MgATP binding site of adenylate kinase, located by a combination of NMR and x-ray diffraction, is near three protein segments, five to seven amino acids in length, that are homologous in sequence to segments found in other nucleotide-binding phosphotransferases, such as myosin and F1-ATPase, ras p21 and transducin GTPases, and cAMP-dependent and src protein kinases, suggesting equivalent mechanistic roles of these segments in all of these proteins. Segment 1 is a glycine-rich flexible loop that, on adenylate kinase, may control access to the ATP-binding site by changing its conformation. Segment 2 is an alpha-helix containing two hydrophobic residues that interact with the adenine-ribose moiety of ATP, and a lysine that may bind to the beta- and gamma-phosphates of ATP. Segment 3 is a hydrophobic strand of parallel beta-pleated sheet, terminated by a carboxylate, that flanks the triphosphate binding site. The various reported mutations of ras p21 that convert it to a transforming agent all appear to involve segment 1, and such substitutions may alter the properties of p21 by hindering a conformational change at this segment. In F1-ATPase, the flexible loop may, by its position, control both the accessibility and the ATP/ADP equilibrium constant on the enzyme.

  4. Genetic mapping of human heart-skeletal muscle adenine nucleotide translocator and its relationship to the facioscapulohumeral muscular dystrophy locus

    SciTech Connect

    Haraguchi, Y.; Chung, A.B.; Torroni, A.; Stepien, G.; Shoffner, J.M.; Costigan, D.A.; Polak, M.; Wasmuth, J.J.; Altherr, M.R.; Winokur, S.T.

    1993-05-01

    The mitochondrial heart-skeletal muscle adenine nucleotide translocator (ANT1) was regionally mapped to 4q35-qter using somatic cell hybrids containing deleted chromosome 4. The regional location was further refined through family studies using ANT1 intron and promoter nucleotide polymorphisms recognized by the restriction endonucleases MboII, NdeI, and HaeIII. Two alleles were found, each at a frequency of 0.5. The ANT1 locus was found to be closely linked to D4S139, D4S171, and the dominant skeletal muscle disease locus facioscapulohumeral muscular dystrophy (FSHD). A crossover that separated D4S171 and ANT1 from D4S139 was found. Since previous studies have established the chromosome 4 map order as centromere-D4S171-D4S139-FSHD, it was concluded that ANT1 is located on the side of D4S139, that is opposite from FSHD. This conclusion was confirmed by sequencing the exons and analyzing the transcripts of ANT1 from several FSHD patients and finding no evidence of aberration. 35 refs., 5 figs., 1 tab.

  5. Alteration of mitochondrial oxidative phosphorylation in aged skeletal muscle involves modification of adenine nucleotide translocator.

    PubMed

    Gouspillou, Gilles; Bourdel-Marchasson, Isabelle; Rouland, Richard; Calmettes, Guillaume; Franconi, Jean-Michel; Deschodt-Arsac, Véronique; Diolez, Philippe

    2010-02-01

    The process of skeletal muscle aging is characterized by a progressive loss of muscle mass and functionality. The underlying mechanisms are highly complex and remain unclear. This study was designed to further investigate the consequences of aging on mitochondrial oxidative phosphorylation in rat gastrocnemius muscle, by comparing young (6 months) and aged (21 months) rats. Maximal oxidative phosphorylation capacity was clearly reduced in older rats, while mitochondrial efficiency was unaffected. Inner membrane properties were unaffected in aged rats since proton leak kinetics were identical to young rats. Application of top-down control analysis revealed a dysfunction of the phosphorylation module in older rats, responsible for a dysregulation of oxidative phosphorylation under low activities close to in vivo ATP turnover. This dysregulation is responsible for an impaired mitochondrial response toward changes in cellular ATP demand, leading to a decreased membrane potential which may in turn affect ROS production and ion homeostasis. Based on our data, we propose that modification of ANT properties with aging could partly explain these mitochondrial dysfunctions.

  6. Hydrolysis at One of the Two Nucleotide-binding Sites Drives the Dissociation of ATP-binding Cassette Nucleotide-binding Domain Dimers*

    PubMed Central

    Zoghbi, Maria E.; Altenberg, Guillermo A.

    2013-01-01

    The functional unit of ATP-binding cassette (ABC) transporters consists of two transmembrane domains and two nucleotide-binding domains (NBDs). ATP binding elicits association of the two NBDs, forming a dimer in a head-to-tail arrangement, with two nucleotides “sandwiched” at the dimer interface. Each of the two nucleotide-binding sites is formed by residues from the two NBDs. We recently found that the prototypical NBD MJ0796 from Methanocaldococcus jannaschii dimerizes in response to ATP binding and dissociates completely following ATP hydrolysis. However, it is still unknown whether dissociation of NBD dimers follows ATP hydrolysis at one or both nucleotide-binding sites. Here, we used luminescence resonance energy transfer to study heterodimers formed by one active (donor-labeled) and one catalytically defective (acceptor-labeled) NBD. Rapid mixing experiments in a stop-flow chamber showed that NBD heterodimers with one functional and one inactive site dissociated at a rate indistinguishable from that of dimers with two hydrolysis-competent sites. Comparison of the rates of NBD dimer dissociation and ATP hydrolysis indicated that dissociation followed hydrolysis of one ATP. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dimer dissociation. PMID:24129575

  7. A comparison of adenine and some derivatives on pig isolated tracheal muscle.

    PubMed Central

    Bach-Dieterle, Y.; Holden, W. E.; Junod, A. F.

    1983-01-01

    We studied the muscle relaxation induced by adenine and several adenine derivatives in strips of tracheal smooth muscle from pigs; in addition their metabolism by the tissue was examined. Adenine relaxed tissue which was contracted by carbachol, histamine, or KCl. Adenine's potency was similar to that of adenosine and ATP (threshold about 4 X 10(-5)M). In tissues with carbachol-induced tone, the adenine effect differed from adenosine and ATP by being slower in onset and in 'washout' time. Furthermore, neither dipyridamole nor theophylline modified the response to adenine. The relationship was examined between pharmacological effects and the metabolism of [3H]-adenosine and [3H]-adenine. Both substrates were taken up by the tissue and converted to nucleotides, but relaxation correlated with nucleotide accumulation only in the case of [3H]-adenine. We conclude that the site and mechanism of adenine-induced relaxation is different from that of adenosine and ATP in porcine tracheal muscle. PMID:6571222

  8. Adsorption of nucleotides on biomimetic apatite: The case of adenosine 5‧ triphosphate (ATP)

    NASA Astrophysics Data System (ADS)

    Hammami, Khaled; El-Feki, Hafed; Marsan, Olivier; Drouet, Christophe

    2016-01-01

    ATP is a well-known energy supplier in cells. The idea to associate ATP to pharmaceutical formulations/biotechnological devices to promote cells activity by potentially modulating their microenvironment thus appears as an appealing novel approach. Since biomimetic nanocrystalline apatites have shown great promise for biomedical applications (bone regeneration, cells diagnostics/therapeutics, …), thanks to a high surface reactivity and an intrinsically high biocompatibility, the present contribution was aimed at exploring ATP/apatite interactions. ATP adsorption on a synthetic carbonated nanocrystalline apatite preliminarily characterized (by XRD, FTIR, Raman, TG-DTA and SEM-EDX) was investigated in detail, pointing out a good agreement with Sips isothermal features. Adsorption characteristics were compared to those previously obtained on monophosphate nucleotides (AMP, CMP), unveiling some specificities. ATP was found to adsorb effectively onto biomimetic apatite: despite smaller values of the affinity constant KS and the exponential factor m, larger adsorbed amounts were reached for ATP as compared to AMP for any given concentration in solution. m < 1 suggests that the ATP/apatite adsorption process is mostly guided by direct surface bonding rather than through stabilizing intermolecular interactions. Although standard ΔGads ° was estimated to only -4 kJ/mol, the large value of Nmax led to significantly negative effective ΔGads values down to -33 kJ/mol, reflecting the spontaneous character of adsorption process. Vibrational spectroscopy data (FTIR and Raman) pointed out spectral modifications upon adsorption, confirming chemical-like interactions where both the triphosphate group of ATP and its nucleic base were involved. The present study is intended to serve as a basis for future research works involving ATP and apatite nanocrystals/nanoparticles in view of biomedical applications (e.g. bone tissue engineering, intracellular drug delivery, …).

  9. Effects of adenine nucleotide and sterol depletion on tight junction structure and function in MDCK cells

    SciTech Connect

    Ladino, C.A.

    1988-01-01

    The antitumor agent Hadacidin (H), N-formyl-hydroxyamino-acetic acid, reversibly inhibited the multiplication of clone 4 Madin-Darby canine kidney (MDCK) cells at a 4 mM concentration within 24-48 hours. Treated cells were arrested in the S phase of the cell cycle. Accompanying this action was a 16-fold increase in the area occupied b the cells and a refractoriness to trypsin treatment. To test whether this effect was due to an increase in tight junction integrity, electrical resistance (TER) was measured across H-treated monolayers. Addition of H at the onset of junction formation reversibly prevented the development of TER. ATP and cAMP levels were decreased by H, as well as the rate of ({sup 3}H)-leucine incorporation into protein. When 1 mM dibutyryl-cAMP (d.cAMP) and theophylline were added, H had no effect on cell division or protein synthesis, and TER was partially restored. The addition of 1 mM d.cAMP and 1 mM theophylline to control cultures decreased TER, indicating a biphasic effect on TER development/maintenance. In a separate study, the effect of sterol depletion on tight junctions formation/maintenance in wild-type MDCK cells was investigated.

  10. ATP binding to two sites is necessary for dimerization of nucleotide-binding domains of ABC proteins.

    PubMed

    Zoghbi, Maria E; Altenberg, Guillermo A

    2014-01-01

    ATP binding cassette (ABC) transporters have a functional unit formed by two transmembrane domains and two nucleotide binding domains (NBDs). ATP-bound NBDs dimerize in a head-to-tail arrangement, with two nucleotides sandwiched at the dimer interface. Both NBDs contribute residues to each of the two nucleotide-binding sites (NBSs) in the dimer. In previous studies, we showed that the prototypical NBD MJ0796 from Methanocaldococcus jannaschii forms ATP-bound dimers that dissociate completely following hydrolysis of one of the two bound ATP molecules. Since hydrolysis of ATP at one NBS is sufficient to drive dimer dissociation, it is unclear why all ABC proteins contain two NBSs. Here, we used luminescence resonance energy transfer (LRET) to study ATP-induced formation of NBD homodimers containing two NBSs competent for ATP binding, and NBD heterodimers with one active NBS and one binding-defective NBS. The results showed that binding of two ATP molecules is necessary for NBD dimerization. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dissociation, but two binding sites are required to form the ATP-sandwich NBD dimer necessary for hydrolysis.

  11. 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

  12. Protection from inactivation of the adenine nucleotide translocator during hypoglycaemia-induced apoptosis by mitochondrial phospholipid hydroperoxide glutathione peroxidase.

    PubMed Central

    Imai, Hirotaka; Koumura, Tomoko; Nakajima, Ryo; Nomura, Kazuhiro; Nakagawa, Yasuhito

    2003-01-01

    We demonstrated that mitochondrial phospholipid hydroperoxide glutathione peroxidase (PHGPx) first suppressed the dissociation of cytochrome c (cyt c) from cardiolipin (CL) in mitochondrial inner membranes and then apoptosis caused by the hypoglycaemia by the prevention of peroxidation of CL [Nomura, Imai, Koumura, Arai and Nakagawa (1999) J. Biol. Chem. 274, 29294-29302; Nomura, Imai, Koumura, Kobayashi and Nakagawa (2000) Biochem. J. 351, 183-193]. The present study shows the involvement of peroxidation of CL in the inactivation of adenine nucleotide translocator (ANT) and the opening of permeability transition pores by using the system of ANT-reconstituted liposome and isolated mitochondria. ANT activity appeared in dioleoyl phosphatidylcholine proteoliposome containing 10% (mol/mol) CL or phosphatidylglycerol (PG), but not other classes of phospholipids. ANT activity was competitively inhibited by the addition of cardiolipin hydroperoxide (CLOOH) in reconstituted liposomes containing CL. However, phosphatidylcholine hydroperoxide failed to inactivate the activity of ANT. The activity of ANT in reconstituted liposomes, including CLOOH, recovered when CLOOH in reconstituted liposome was reduced to hydroxycardiolipin by incubation with PHGPx. The activity of ANT was determined in rat basophil leukaemia RBL2H3 cells after their exposure to 2-deoxyglucose. ANT activity decreased to 50% of the control level by 4 h in response to apoptosis. In parallel, cyt c and apoptosis-inducing factor (AIF) were released from mitochondria. Suppression of the accumulation of CLOOH by overexpression of PHGPx in mitochondria effectively prevented the inactivation of ANT, the opening of permeability transition pores and the release of cyt c and AIF from mitochondria in hypoglycaemia-induced apoptotic cells. These findings suggest that mitochondrial PHGPx might be involved in the modulation of the activity of ANT and the opening of pores for the release of cyt c via the modulation of

  13. The Emerging Roles of ATP-Dependent Chromatin Remodeling Enzymes in Nucleotide Excision Repair

    PubMed Central

    Czaja, Wioletta; Mao, Peng; Smerdon, Michael J.

    2012-01-01

    DNA repair in eukaryotic cells takes place in the context of chromatin, where DNA, including damaged DNA, is tightly packed into nucleosomes and higher order chromatin structures. Chromatin intrinsically restricts accessibility of DNA repair proteins to the damaged DNA and impacts upon the overall rate of DNA repair. Chromatin is highly responsive to DNA damage and undergoes specific remodeling to facilitate DNA repair. How damaged DNA is accessed, repaired and restored to the original chromatin state, and how chromatin remodeling coordinates these processes in vivo, remains largely unknown. ATP-dependent chromatin remodelers (ACRs) are the master regulators of chromatin structure and dynamics. Conserved from yeast to humans, ACRs utilize the energy of ATP to reorganize packing of chromatin and control DNA accessibility by sliding, ejecting or restructuring nucleosomes. Several studies have demonstrated that ATP-dependent remodeling activity of ACRs plays important roles in coordination of spatio-temporal steps of different DNA repair pathways in chromatin. This review focuses on the role of ACRs in regulation of various aspects of nucleotide excision repair (NER) in the context of chromatin. We discuss current understanding of ATP-dependent chromatin remodeling by various subfamilies of remodelers and regulation of the NER pathway in vivo. PMID:23109894

  14. Trinitrophenyl-ATP blocks colonic Cl- channels in planar phospholipid bilayers. Evidence for two nucleotide binding sites

    PubMed Central

    1993-01-01

    Outwardly rectifying 30-50-pS Cl- channels mediate cell volume regulation and transepithelial transport. Several recent reports indicate that rectifying Cl- channels are blocked after addition of ATP to the extracellular bath (Alton, E. W. F. W., S. D. Manning, P. J. Schlatter, D. M. Geddes, and A. J. Williams. 1991. Journal of Physiology. 443:137-159; Paulmichl, M., Y. Li, K. Wickman, M. Ackerman, E. Peralta, and D. Clapham. 1992. Nature. 356:238-241). Therefore, we decided to conduct a more detailed study of the ATP binding site using a higher affinity probe. We tested the ATP derivative, 2',3',O-(2,4,6- trinitrocyclohexadienylidene) adenosine 5'-triphosphate (TNP-ATP), which has a high affinity for certain nucleotide binding sites. Here we report that TNP-ATP blocked colonic Cl- channels when added to either bath and that blockade was consistent with the closed-open-blocked kinetic model. The TNP-ATP concentration required for a 50% decrease in open probability was 0.27 microM from the extracellular (cis) side and 20 microM from the cytoplasmic (trans) side. Comparison of the off rate constants revealed that TNP-ATP remained bound 28 times longer when added to the extracellular side compared with the cytoplasmic side. We performed competition studies to determine if TNP-ATP binds to the same sites as ATP. Addition of ATP to the same bath containing TNP-ATP reduced channel amplitude and increased the time the channel spent in the open and fast-blocked states (i.e., burst duration). This is the result expected if TNP-ATP and ATP compete for block, presumably by binding to common sites. In contrast, addition of ATP to the bath opposite to the side containing TNP-ATP reduced amplitude but did not alter burst duration. This is the result expected if opposite-sided TNP- ATP and ATP bind to different sites. In summary, we have identified an ATP derivative that has a nearly 10-fold higher affinity for reconstituted rectifying colonic Cl- channels than any previously

  15. Evidence that the Severity of Depletion of Inorganic Phosphate Determines the Severity of the Disturbance of Adenine Nucleotide Metabolism in the Liver and Renal Cortex of the Fructose-Loaded Rat

    PubMed Central

    Morris, R. Curtis; Nigon, Kathleen; Reed, Elizabeth B.

    1978-01-01

    To test the hypothesis that in both the liver and renal cortex of the fructose-loaded rat, severity of depletion of inorganic phosphate (Pi), and not the magnitude of accumulation of fructose-1-phosphate (F-1-P), determines the severity of the dose-dependent reduction of ATP, we intraperitoneally injected fed rats with fructose, 20 and 40 μmol/g, alone, and at the higher load, in combination with (a) sodium phosphate, 20 μmol/g, administered shortly beforehand or subsequently or, (b) adenosine, 2 μmol/g, administered beforehand. The following observations were made: (a) With fructose loading alone, at the higher load, both Pi and total adenine nucleotides (TAN) were reduced by one third in the renal cortex and (as previously observed) by two thirds in the liver; and at either load, the reduction of ATP (and TAN) and the accumulation of F-1-P were less severe in the renal cortex than in the liver. (b) Prior phosphate loading largely prevented the reductions of ATP and TAN in the renal cortex and significantly attenuated them in the liver, yet doubled the renal cortical accumulation of F-1-P. (c) Adenosine loading substantially attenuated the reductions of ATP, TAN, and Pi only in the renal cortex. (d) ATP varied directly with Pi (P < 0.001, r = 0.98) in the domain of control and reduced values of Pi taken from both liver and renal cortex. (e) As judged from tissue and plasma concentrations of fructose and glucose, and tissue concentrations of fructose-6-phosphate and glucose-6-phosphate, the rate at which the renal cortex and liver converted fructose to glucose was much lower at the higher fructose load. (f) Prior phosphate loading prevented this decrease in rate in the renal cortex and attenuated it in the liver; adenosine loading attenuated it only in the renal cortex. We conclude that in both the renal cortex of the fructose-loaded rat: (a) Depletion of Pi is critical to the causation of the reductions in both ATP and TAN and, at the higher fructose load, of a

  16. Adenine-DNA adducts derived from the highly tumorigenic dibenzo[a,l]pyrene are resistant to nucleotide excision repair while guanine adducts are not

    PubMed Central

    Kropachev, Konstantin; Kolbanovskiy, Marina; Liu, Zhi; Cai, Yuqin; Zhang, Lu; Schwaid, Adam G.; Kolbanovskiy, Alexander; Ding, Shuang; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E.

    2013-01-01

    The structural origins of differences in susceptibilities of various DNA lesions to nucleotide excision repair (NER) are poorly understood. Here we compared, in the same sequence context, the relative NER dual incision efficiencies elicited by two stereochemically distinct pairs of guanine (N2-dG) and adenine (N6-dA) DNA lesions, derived from enantiomeric genotoxic diol epoxides of the highly tumorigenic fjord region polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P). Remarkably, in cell-free HeLa cell extracts, the guanine adduct with R absolute chemistry at the N2-dG linkage site is ~ 35 times more susceptible to NER dual incisions than the stereochemically identical N6-dA adduct. For the guanine and adenine adducts with S stereochemistry, a similar, but somewhat smaller effect (factor of ~15) is observed. The striking resistance of the bulky N6-dA in contrast to the modest to good susceptibilities of the N2-dG adducts to NER are interpreted in terms of the balance between lesion-induced DNA-distorting and DNA-stabilizing van der Waals interactions in their structures, that are partly reflected in the overall thermal stabilities of the modified duplexes. Our results are consistent with the hypothesis that the high genotoxic activity of DB[a,l]P is related to the formation of NER-resistant and persistent DB[a,l]P-derived adenine adducts in cellular DNA. PMID:23570232

  17. Structure and association of ATP-binding cassette transporter nucleotide-binding domains.

    PubMed

    Kerr, Ian D

    2002-03-19

    ATP-binding cassette transporters are responsible for the uptake and efflux of a multitude of substances across both eukaryotic and prokaryotic membranes. Members of this family of proteins are involved in diverse physiological processes including antigen presentation, drug efflux from cancer cells, bacterial nutrient uptake and cystic fibrosis. In order to understand more completely the role of these multidomain transporters an integrated approach combining structural, pharmacological and biochemical methods is being adopted. Recent structural data have been obtained on the cytoplasmic, nucleotide-binding domains of prokaryotic ABC transporters. This review evaluates both these data and the conflicting implications they have for domain communication in ABC transporters. Areas of biochemical research that attempt to resolve these conflicts will be discussed.

  18. Transmembrane gate movements in the type II ATP-binding cassette (ABC) importer BtuCD-F during nucleotide cycle.

    PubMed

    Joseph, Benesh; Jeschke, Gunnar; Goetz, Birke A; Locher, Kaspar P; Bordignon, Enrica

    2011-11-25

    ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that translocate substrates across cell membranes. The alternating access of their transmembrane domains to opposite sides of the membrane powered by the closure and reopening of the nucleotide binding domains is proposed to drive the translocation events. Despite clear structural similarities, evidence for considerable mechanistic diversity starts to accumulate within the importers subfamily. We present here a detailed study of the gating mechanism of a type II ABC importer, the BtuCD-F vitamin B(12) importer from Escherichia coli, elucidated by EPR spectroscopy. Distance changes at key positions in the translocation gates in the nucleotide-free, ATP- and ADP-bound conformations of the transporter were measured in detergent micelles and liposomes. The translocation gates of the BtuCD-F complex undergo conformational changes in line with a "two-state" alternating access model. We provide the first direct evidence that binding of ATP drives the gates to an inward-facing conformation, in contrast to type I importers specific for maltose, molybdate, or methionine. Following ATP hydrolysis, the translocation gates restore to an apo-like conformation. In the presence of ATP, an excess of vitamin B(12) promotes the reopening of the gates toward the periplasm and the dislodgment of BtuF from the transporter. The EPR data allow a productive translocation cycle of the vitamin B(12) transporter to be modeled.

  19. Cyclic Nucleotide Compartmentalization: Contributions of Phosphodiesterases and ATP-Binding Cassette Transporters

    PubMed Central

    Cheepala, Satish; Hulot, Jean-Sebastien; Morgan, Jessica A.; Sassi, Yassine; Zhang, Weiqiang; Naren, Anjaparavanda P.; Schuetz, John D.

    2015-01-01

    Cyclic nucleotides [e.g., cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP)] are ubiquitous second messengers that affect multiple cell functions from maturation of the egg to cell division, growth, differentiation, and death. The concentration of cAMP can be regulated by processes within membrane domains (local regulation) as well as throughout a cell (global regulation). The phosphodiesterases (PDEs) that degrade cAMP have well-known roles in both these processes. It has recently been discovered that ATP-binding cassette (ABC) transporters contribute to both local and global regulation of cAMP. This regulation may require the formation of macromolecular complexes. Some of these transporters are ubiquitously expressed, whereas others are more tissue restricted. Because some PDE inhibitors are also ABC transporter inhibitors, it is conceivable that the therapeutic benefits of their use result from the combined inhibition of both PDEs and ABC transporters. Deciphering the individual contributions of PDEs and ABC transporters to such drug effects may lead to improved therapeutic benefits. PMID:23072381

  20. Alterations in erythrocyte plasma membrane ATPase activity and adenine nucleotide content in a spontaneously diabetic subline of the Chinese hamster.

    PubMed

    Bettin, D; Klöting, I; Kohnert, K D

    1996-01-01

    The CHIG/Han subline of the Chinese hamster develops noninsulin-dependent diabetes mellitus characterized by hyperinsulinemia and different degrees of glucose intolerance. To study whether these abnormalities could affect transmembrane cation transport activity, we determined membrane ATPase activity and ATP concentrations in red blood cells of diabetes-resistant CHIA and diabetes-susceptible CHIG sublines of the Chinese hamster. Mg(2+)-ATPase activity was increased in red blood cell membranes of diabetic hamsters compared with that of nondiabetic CHIG and the diabetes-resistant CHIA animals and correlated with plasma triglyceride and cholesterol levels. Ca(2+)-ATPase and Na+/K+ATPase activity were not significantly different between diabetic and nondiabetic hamsters, but for the Na+/K(+)-ATPase, Km was decreased and the Vmax value increased in membrane preparations from severely diabetic hamsters. Both ATP and ADP content were lower in erythrocytes from diabetic than nondiabetic hamsters. Independently of the levels of glycemia, AMP concentrations were higher in CHIG than in CHIA hamsters. While ATP/AMP ratios were found to be decreased in erythrocytes from diabetes-susceptible CHIG hamsters compared to the diabetes-resistant CHIA animals, they were significantly correlated with the levels of glycemia. Furthermore, the relationship between blood glucose levels and kidney weight in hamsters of the diabetes-susceptible CHIG subline was such, that severely hyperglycemic animals displayed the greatest increase in kidney wet weight. These results indicate that the progressive metabolic deterioration in the development of noninsulin-dependent diabetes is associated with significant changes in the activity and kinetic parameters of cellular ATPases which could probably indicate early membrane alterations which may eventually result in the late microangiopathic complications of diabetes. PMID:8820985

  1. Beef-heart mitochondrial F1-ATPase can use endogenous bound phosphate to synthesize ATP in dimethyl sulfoxide.

    PubMed

    Beharry, S; Bragg, P D

    1991-10-21

    Beef-heart mitochondrial F1-ATPase contained 5 mol of inorganic phosphate bound per mol of F1, following pretreatment with ATP. A portion of the phosphate, bound most likely at a catalytic site, reacted in dimethylsulfoxide with endogenous adenine nucleotide to form ATP.

  2. Kinetics of the Association/Dissociation Cycle of an ATP-binding Cassette Nucleotide-binding Domain*

    PubMed Central

    Zoghbi, Maria E.; Fuson, Kerry L.; Sutton, Roger B.; Altenberg, Guillermo A.

    2012-01-01

    Most ATP binding cassette (ABC) proteins are pumps that transport substrates across biological membranes using the energy of ATP hydrolysis. Functional ABC proteins have two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP, but the molecular mechanism of nucleotide hydrolysis is unresolved. This is due in part to the limited kinetic information on NBD association and dissociation. Here, we show dimerization of a catalytically active NBD and follow in real time the association and dissociation of NBDs from the changes in fluorescence emission of a tryptophan strategically located at the center of the dimer interface. Spectroscopic and structural studies demonstrated that the tryptophan can be used as dimerization probe, and we showed that under hydrolysis conditions (millimolar MgATP), not only the dimer dissociation rate increases, but also the dimerization rate. Neither dimer formation or dissociation are clearly favored, and the end result is a dynamic equilibrium where the concentrations of monomer and dimer are very similar. We proposed that based on their variable rates of hydrolysis, the rate-limiting step of the hydrolysis cycle may differ among full-length ABC proteins. PMID:22158619

  3. Adenine nucleotide translocator isoforms 1 and 2 are differently distributed in the mitochondrial inner membrane and have distinct affinities to cyclophilin D.

    PubMed Central

    Vyssokikh, M Y; Katz, A; Rueck, A; Wuensch, C; Dörner, A; Zorov, D B; Brdiczka, D

    2001-01-01

    Different isoforms of the adenine nucleotide translocase (ANT) are expressed in a tissue-specific manner. It was assumed that ANT-1 and ANT-2 co-exist in every single mitochondrion and might be differently distributed within the membrane structures that constitute the peripheral inner membrane or the crista membrane. To discriminate between ANT originating from peripheral or from cristal inner membranes we made use of the fact that complexes between porin, the outer-membrane pore protein, and the ANT can be generated. Such complexes between porin and the ANT in the peripheral inner membrane were induced in rat heart mitochondria and isolated from rat brain and kidney. Using ANT-isotype-specific antibodies and sequence analysis of the N-terminal end, it was discovered that the peripheral inner membrane contained ANT-1 and ANT-2, whereas the cristal membrane contained exclusively ANT-2. Cyclophilin was co-purified with the porin-ANT complexes, whereas it was absent in the crista-derived ANT. This suggested that ANT-1 might have a higher affinity for cyclophilin. This specific intra-mitochondrial distribution of the two ANT isotypes and cyclophilin D suggests specific functions of the peripheral and crista-forming parts of the inner membrane and the two ANT isotypes therein. PMID:11513733

  4. Human cytomegalovirus miR-UL36-5p inhibits apoptosis via downregulation of adenine nucleotide translocator 3 in cultured cells.

    PubMed

    Guo, Xin; Huang, Yujing; Qi, Ying; Liu, Zhongyang; Ma, Yanping; Shao, Yaozhong; Jiang, Shujuan; Sun, Zhengrong; Ruan, Qiang

    2015-10-01

    Human cytomegalovirus (HCMV) encodes at least 26 microRNAs (miRNA). These miRNAs are utilized by HCMV to regulate its own genes as well as the genes of the host cell during infection. It has been reported that a cellular gene, solute carrier family 25, member 6 (SLC25A6), which is also designated adenine nucleotide translocator 3 (ANT3), was identified as a candidate target of hcmv-miR-UL36-5p by hybrid PCR. In this study, ANT3 was further demonstrated to be a direct target of hcmv-miR-UL36-5p by luciferase reporter assays. The expression level of ANT3 protein was confirmed, by western blotting, to be directly downregulated by overexpression of hcmv-miR-UL36-5p in HEK293 cells, U373 cells and HELF cells. Moreover, HCMV-infected cells showed a decrease in the ANT3 protein level. Using ANT3-specific small interfering RNA (siRNA) and an inhibitor for hcmv-miR-UL36-5p, it was shown that inhibition of apoptosis by hcmv-miR-UL36-5p in these cells specifically occurred via inhibition of ANT3 expression. These results imply that hcmv-miR-UL36-5 may play the same role during actual HCMV infection in order to establish a balance between the host cell and the virus.

  5. Tritium labelled nucleotides: Heterogeneous metal catalyzed exchange labelling of ATP with tritium gas

    SciTech Connect

    Jaiswal, D.K. ); Morimoto, H.; Williams, P.G.; Wemmer, D.E. )

    1991-09-01

    Adenosine 5{prime} triphosphate (ATP) in aqueous solution has been labeled by exchange with tritium gas in the presence of palladium oxide catalyst. Comparison with our experiments using Pd/BaSO{sub 4} as the catalyst shows that we have obtained product with higher specific activity and improved chemical purity. {sup 3}H NMR spectroscopy of the tritiated ATP shows labelling in both the C-8 and C-2 positions, and the integral ratio of these positions was found to vary from 3:1 to 1:1 under different reaction conditions. 5 refs., 1 fig., 2 tabs.

  6. Effect of genetic and physiological manipulations onthe kinetic and binding parameters of the adenine nucleotide translocator in Saccharomyces cervisiae and Candida utilis.

    PubMed

    Lauquin, G; Lunardi, J; Vignais, P V

    1976-01-01

    1. Ghe kinetic and binding parameters of adenine-nucleotide transport have been studied in mitochondria isolated from yeast cells in which the mitochondrial protein-synthetizing system had been inhibited by growth in the presence of erythromycin. These parameters have also been studied in promitochondria isolated from yeast grown in anaerobiosis aesence of ethidium bromide results in a loss of cytochromes b, alpha and alpha 3, but it does not affect the rate constant of ADP transport in isolated mitochondria, nor the number of binding sites for atractyloside, bongkrekic acid and ADP. 3. Promitochondria from S. cerevisiae grown in anaerobiosis, mitochondria from a qo mutant (qo mitochondria) and mitochondria from S. cerevisiae grown in the presence of erythromycin (ERY-mitochondria) are able to transport ADP by the same exchange-diffusion mechanism, sensitive to carboxy-atractyloside, and with the same rate constant as the wild type mitochondria. Promitochondria, qo mitochondria and ERY-mitochondria bind atractyloside, bongkrekic acid and ADP with the same high affinity as the wild type mitochondria. They only differ from the wild type mitochondria by a lower number of binding sites for ADP and for specific inhibitors of ADP transport. 4. Mitochondria isolated from the nuclear mutant p9 of S. cerevisae, called also op1, are characterized by a much lower affinity for bongkrekic acid than mitochondria from the wild type (20 times less). 5. Manipulation of the fatty acid composition of the mitochondrial membranes in the desaturase auxotroph mutant KD115 does not modify the number of sites, no their affinity of bongkrekic acid. 6. The above results are interpreted to mean that the structure and function of the mitochondrial adN translocator are not affected by any change in the mitochondrial protein synthetizing system. PMID:795470

  7. Neutrophils as sources of extracellular nucleotides: Functional consequences at the vascular interface

    PubMed Central

    Eltzschig, Holger K.; MacManus, Christopher F.; Colgan, Sean P.

    2009-01-01

    Nucleotide signaling is currently an area of intense investigation. Extracellular ATP liberated during hypoxia or inflammation can either signal directly to purinergic receptors or, following phosphohydrolytic metabolism, can activate surface adenosine (Ado) receptors. Given the association of polymorphonuclear leukocytes (PMN) with adenine nucleotide / nucleoside signaling in the inflammatory milieu, it was recently demonstrated that PMN actively release ATP via a connexin 43 (Cx43) hemichannel-dependent mechanism. Here we review the mechanisms of ATP release and subsequent functional implications of ATP metabolism at the interface between PMN and vascular endothelial cells during inflammation and in hypoxia. PMID:18436149

  8. Nucleotide-induced asymmetry within ATPase activator ring drives σ54-RNAP interaction and ATP hydrolysis

    SciTech Connect

    Sysoeva, Tatyana A.; Chowdhury, Saikat; Guo, Liang; Nixon, B. Tracy

    2013-12-10

    It is largely unknown how the typical homomeric ring geometry of ATPases associated with various cellular activities enables them to perform mechanical work. Small-angle solution X-ray scattering, crystallography, and electron microscopy (EM) reconstructions revealed that partial ATP occupancy caused the heptameric closed ring of the bacterial enhancer-binding protein (bEBP) NtrC1 to rearrange into a hexameric split ring of striking asymmetry. The highly conserved and functionally crucial GAFTGA loops responsible for interacting with σ54–RNA polymerase formed a spiral staircase. We propose that splitting of the ensemble directs ATP hydrolysis within the oligomer, and the ring's asymmetry guides interaction between ATPase and the complex of σ54 and promoter DNA. Similarity between the structure of the transcriptional activator NtrC1 and those of distantly related helicases Rho and E1 reveals a general mechanism in homomeric ATPases whereby complex allostery within the ring geometry forms asymmetric functional states that allow these biological motors to exert directional forces on their target macromolecules.

  9. Activation of the phospholipase C pathway by ATP is mediated exclusively through nucleotide type P2-purinoceptors in C2C12 myotubes.

    PubMed Central

    Henning, R. H.; Duin, M.; den Hertog, A.; Nelemans, A.

    1993-01-01

    1. The presence of a nucleotide receptor and a discrete ATP-sensitive receptor on C2C12 myotubes has been shown by electrophysiological experiments. In this study, the ATP-sensitive receptors of C2C12 myotubes were further characterized by measuring the formation of inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) and internal Ca2+. 2. The nucleotides ATP and UTP caused a concentration-dependent increase in Ins(1,4,5)P3 content with comparable time courses (EC50: ATP 33 +/- 2 microM, UTP 80 +/- 4 microM). ADP was less effective in increasing Ins(1,4,5)P3 content of the cells, while selective agonists for P1-, P2X- and P2Y-purinoceptors, adenosine, alpha,beta-methylene ATP and 2-methylthio ATP, appeared to be ineffective. 3. Under Ca(2+)-free conditions, the basal level of Ins(1,4,5)P3 was lower than in the presence of Ca2+, and the ATP- and UTP-induced formation of Ins(1,4,5)P3 was diminished. 4. The Ins(1,4,5)P3 formation induced by optimal ATP and UTP concentrations was not additive. ATP- and UTP-induced Ins(1,4,5)P3 formation showed cross-desensitization, whereas cross-desensitization was absent in responses elicited by one of the nucleotides and bradykinin. 5. The change in Ins(1,4,5)P3 content induced by effective nucleotides was inhibited by suramin. Schild plots for suramin inhibition of Ins(1,4,5)P3 formation in ATP- and UTP-stimulated myotubes showed slopes greater than unity (1.63 +/- 0.09 and 1.37 +/- 0.11, respectively). Apparent pA2 values were 4.50 +/- 0.48 and 4.41 +/- 0.63 for ATP and UTP, respectively. 6. Stimulation of the cells with ATP or UTP induced a rapid increase in intracellular Ca2+, followed by a slow decline to basal levels. Ca2+ responses reached lower maximal values and did not show the slow phase in the absence of extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8242247

  10. Decipher the mechanisms of protein conformational changes induced by nucleotide binding through free-energy landscape analysis: ATP binding to Hsp70.

    PubMed

    Nicolaï, Adrien; Delarue, Patrice; Senet, Patrick

    2013-01-01

    ATP regulates the function of many proteins in the cell by transducing its binding and hydrolysis energies into protein conformational changes by mechanisms which are challenging to identify at the atomic scale. Based on molecular dynamics (MD) simulations, a method is proposed to analyze the structural changes induced by ATP binding to a protein by computing the effective free-energy landscape (FEL) of a subset of its coordinates along its amino-acid sequence. The method is applied to characterize the mechanism by which the binding of ATP to the nucleotide-binding domain (NBD) of Hsp70 propagates a signal to its substrate-binding domain (SBD). Unbiased MD simulations were performed for Hsp70-DnaK chaperone in nucleotide-free, ADP-bound and ATP-bound states. The simulations revealed that the SBD does not interact with the NBD for DnaK in its nucleotide-free and ADP-bound states whereas the docking of the SBD was found in the ATP-bound state. The docked state induced by ATP binding found in MD is an intermediate state between the initial nucleotide-free and final ATP-bound states of Hsp70. The analysis of the FEL projected along the amino-acid sequence permitted to identify a subset of 27 protein internal coordinates corresponding to a network of 91 key residues involved in the conformational change induced by ATP binding. Among the 91 residues, 26 are identified for the first time, whereas the others were shown relevant for the allosteric communication of Hsp70 s in several experiments and bioinformatics analysis. The FEL analysis revealed also the origin of the ATP-induced structural modifications of the SBD recently measured by Electron Paramagnetic Resonance. The pathway between the nucleotide-free and the intermediate state of DnaK was extracted by applying principal component analysis to the subset of internal coordinates describing the transition. The methodology proposed is general and could be applied to analyze allosteric communication in other proteins.

  11. Decipher the Mechanisms of Protein Conformational Changes Induced by Nucleotide Binding through Free-Energy Landscape Analysis: ATP Binding to Hsp70

    PubMed Central

    Nicolaï, Adrien; Delarue, Patrice; Senet, Patrick

    2013-01-01

    ATP regulates the function of many proteins in the cell by transducing its binding and hydrolysis energies into protein conformational changes by mechanisms which are challenging to identify at the atomic scale. Based on molecular dynamics (MD) simulations, a method is proposed to analyze the structural changes induced by ATP binding to a protein by computing the effective free-energy landscape (FEL) of a subset of its coordinates along its amino-acid sequence. The method is applied to characterize the mechanism by which the binding of ATP to the nucleotide-binding domain (NBD) of Hsp70 propagates a signal to its substrate-binding domain (SBD). Unbiased MD simulations were performed for Hsp70-DnaK chaperone in nucleotide-free, ADP-bound and ATP-bound states. The simulations revealed that the SBD does not interact with the NBD for DnaK in its nucleotide-free and ADP-bound states whereas the docking of the SBD was found in the ATP-bound state. The docked state induced by ATP binding found in MD is an intermediate state between the initial nucleotide-free and final ATP-bound states of Hsp70. The analysis of the FEL projected along the amino-acid sequence permitted to identify a subset of 27 protein internal coordinates corresponding to a network of 91 key residues involved in the conformational change induced by ATP binding. Among the 91 residues, 26 are identified for the first time, whereas the others were shown relevant for the allosteric communication of Hsp70 s in several experiments and bioinformatics analysis. The FEL analysis revealed also the origin of the ATP-induced structural modifications of the SBD recently measured by Electron Paramagnetic Resonance. The pathway between the nucleotide-free and the intermediate state of DnaK was extracted by applying principal component analysis to the subset of internal coordinates describing the transition. The methodology proposed is general and could be applied to analyze allosteric communication in other proteins

  12. MgATP binding to the nucleotide-binding domains of the eukaryotic cytoplasmic chaperonin induces conformational changes in the putative substrate-binding domains.

    PubMed Central

    Szpikowska, B. K.; Swiderek, K. M.; Sherman, M. A.; Mas, M. T.

    1998-01-01

    The eukaryotic cytosolic chaperonins are large heterooligomeric complexes with a cylindrical shape, resembling that of the homooligomeric bacterial counterpart, GroEL. In analogy to GroEL, changes in shape of the cytosolic chaperonin have been detected in the presence of MgATP using electron microscopy but, in contrast to the nucleotide-induced conformational changes in GroEL, no details are available about the specific nature of these changes. The present study identifies the structural regions of the cytosolic chaperonin that undergo conformational changes when MgATP binds to the nucleotide binding domains. It is shown that limited proteolysis with trypsin in the absence of MgATP cleaves each of the eight subunits approximately in half, generating two fragments of approximately 30 kDa. Using mass spectrometry (MS) and N-terminal sequence analysis, the cleavage is found to occur in a narrow span of the amino acid sequence, corresponding to the peptide binding regions of GroEL and to the helical protrusion, recently identified in the structure of the substrate binding domain of the archeal group II chaperonin. This proteolytic cleavage is prevented by MgATP but not by ATP in the absence of magnesium, ATP analogs (MgATPyS and MgAMP-PNP) or MgADP. These results suggest that, in analogy to GroEL, binding of MgATP to the nucleotide binding domains of the cytosolic chaperonin induces long range conformational changes in the polypeptide binding domains. It is postulated that despite their different subunit composition and substrate specificity, group I and group II chaperonins may share similar, functionally-important, conformational changes. Additional conformational changes are likely to involve a flexible helix-loop-helix motif, which is characteristic for all group II chaperonins. PMID:9684884

  13. Identification of mutations in regions corresponding to the two putative nucleotide (ATP)-binding folds of the cystic fibrosis gene

    SciTech Connect

    Kerem, B.; Zielenski, J.; Markiewicz, D.; Bozon, D.; Kennedy, D.; Rommens, J.M. ); Gazit, E. ); Yahav, J. ); Riordan, J.R. ); Collins, F.S. ); Tsui, Lapchee Univ. of Toronto, Ontario )

    1990-11-01

    Additional mutations in the cystic fibrosis (CF) gene were identified in the regions corresponding to the two putative nucleotide (ATP)-binding folds (NBFs) of the predicted polypeptide. The patient cohort included 46 Canadian CF families with well-characterized DNA marker haplotypes spanning the disease locus and several other families from Israel. Eleven mutations were found in the first NBF, 2 were found in the second NBF, but none was found in the R-domain. Seven of the mutations were of the missense type affecting some of the highly conserved amino acid residues in the first NBF; 3 were nonsense mutations; 2 would probably affect mRNA splicing; 2 corresponded to small deletions, including another 3-base-pair deletion different from the major mutation ({delta}F508), which could account for 70% of the CF chromosomes in the population. Nine of these mutations accounted for 12 of the 31 non-{delta}F508 CF chromosomes in the Canadian families. The highly heterogeneous nature of the remaining CF mutations provides important insights into the structure and function of the protein, but it also suggests that DNA-based genetic screening for CF carrier status will not be straightforward.

  14. Protection of mice from endotoxic death by 2-methylthio-ATP.

    PubMed Central

    Proctor, R A; Denlinger, L C; Leventhal, P S; Daugherty, S K; van de Loo, J W; Tanke, T; Firestein, G S; Bertics, P J

    1994-01-01

    The lethal effects of endotoxin, a bacterial product shed into the blood during bacteremia, are thought to be due to macrophage release of mediators such as tumor necrosis factor alpha and interleukin 1. Although much is known about the pathophysiology of endotoxemia, relatively little is known about the cellular signaling mechanisms that are involved. The data in this study suggest that extracellular adenine nucleotides can influence the development of endotoxin shock. An adenine nucleotide analog, 2-methylthio-ATP, inhibited the endotoxin-stimulated release of toxic mediators (i.e., tumor necrosis factor alpha and interleukin 1), and it protected mice from endotoxin-induced death. These studies suggest a fundamental and unusual role for adenine nucleotides on endotoxin action, and they provide a potentially new therapeutic approach for the control of the pathophysiology of Gram-negative septicemia. Images PMID:8016108

  15. The role of ATP-sensitive potassium channels in cellular function and protection in the cardiovascular system.

    PubMed

    Tinker, Andrew; Aziz, Qadeer; Thomas, Alison

    2014-01-01

    ATP-sensitive potassium channels (K(ATP)) are widely distributed and present in a number of tissues including muscle, pancreatic beta cells and the brain. Their activity is regulated by adenine nucleotides, characteristically being activated by falling ATP and rising ADP levels. Thus, they link cellular metabolism with membrane excitability. Recent studies using genetically modified mice and genomic studies in patients have implicated K(ATP) channels in a number of physiological and pathological processes. In this review, we focus on their role in cellular function and protection particularly in the cardiovascular system.

  16. Fluorometric Determination of Adenosine Nucleotide Derivatives as Measures of the Microfouling, Detrital, and Sedimentary Microbial Biomass and Physiological Status

    PubMed Central

    Davis, William M.; White, David C.

    1980-01-01

    Adenosine, adenine, cyclic adenosine monophosphate (AMP), AMP, nicotinamide adenine dinucleotide, adenosine diphosphate, and adenosine triphosphate (ATP) were recovered quantitatively from aqueous portions of lipid extracts of microfouling, detrital, and sedimentary microbial communities. These could be detected quantitatively in the picomolar range by forming their 1-N6-etheno derivatives and analyzing by high-pressure liquid chromatography with fluorescence detection. Lipid extraction and subsequent analysis allowed the simultaneous measurement of the microbial community structure, total microbial biomass with the quantitative recovery of the adenine-containing cellular components, which were protected from enzymatic destruction. This extraction and fluorescent derivatization method showed equivalency with the luciferin-luciferase method for bacterial ATP measurements. Quick-freezing samples in the field with dry ice-acetone preserved the ATP and energy charge (a ratio of adenosine nucleotides) for analysis at remote laboratories. The metabolic lability of ATP in estuarine detrital and microfouling communities, as well as bacterial monocultures of constant biomass, showed ATP to be a precarious measure of biomass under some conditions. Combinations of adenosine and adenine nucleotides gave better correlations with microbial biomass measured as extractable lipid phosphate in the detrital and microfouling microbial communities than did ATP alone. Stresses such as anoxia or filtration are reflected in the rapid accumulation of intracellular adenosine and the excretion of adenosine and AMP into the surrounding milieu. Increases in AMP and adenosine may prove to be more sensitive indicators of metabolic status than the energy charge. PMID:16345633

  17. Specific and nonspecific metal ion-nucleotide interactions at aqueous/solid interfaces functionalized with adenine, thymine, guanine, and cytosine oligomers.

    PubMed

    Holland, Joseph G; Malin, Jessica N; Jordan, David S; Morales, Esmeralda; Geiger, Franz M

    2011-03-01

    This article reports nonlinear optical measurements that quantify, for the first time directly and without labels, how many Mg(2+) cations are bound to DNA 21-mers covalently linked to fused silica/water interfaces maintained at pH 7 and 10 mM NaCl, and what the thermodynamics are of these interactions. The overall interaction of Mg(2+) with adenine, thymine, guanine, and cytosine is found to involve -10.0 ± 0.3, -11.2 ± 0.3, -14.0 ± 0.4, and -14.9 ± 0.4 kJ/mol, and nonspecific interactions with the phosphate and sugar backbone are found to contribute -21.0 ± 0.6 kJ/mol for each Mg(2+) ion bound. The specific and nonspecific contributions to the interaction energy of Mg(2+) with oligonucleotide single strands is found to be additive, which suggests that within the uncertainty of these surface-specific experiments, the Mg(2+) ions are evenly distributed over the oligomers and not isolated to the most strongly binding nucleobase. The nucleobases adenine and thymine are found to bind only three Mg(2+) ions per 21-mer oligonucleotide, while the bases cytosine and guanine are found to bind eleven Mg(2+) ions per 21-mer oligonucleotide.

  18. Yeast mitochondria import ATP through the calcium-dependent ATP-Mg/Pi carrier Sal1p, and are ATP consumers during aerobic growth in glucose.

    PubMed

    Traba, Javier; Froschauer, Elisabeth Maria; Wiesenberger, Gerlinde; Satrústegui, Jorgina; Del Arco, Araceli

    2008-08-01

    Sal1p, a novel Ca2+-dependent ATP-Mg/Pi carrier, is essential in yeast lacking all adenine nucleotide translocases. By targeting luciferase to the mitochondrial matrix to monitor mitochondrial ATP levels, we show in isolated mitochondria that both ATP-Mg and free ADP are taken up by Sal1p with a K(m) of 0.20 +/- 0.03 mM and 0.28 +/- 0.06 mM respectively. Nucleotide transport along Sal1p is strictly Ca2+ dependent. Ca2+ increases the V(max) with a S(0.5) of 15 muM, and no changes in the K(m) for ATP-Mg. Glucose sensing in yeast generates Ca2+ transients involving Ca2+ influx from the external medium. We find that carbon-deprived cells respond to glucose with an immediate increase in mitochondrial ATP levels which is not observed in the presence of EGTA or in Sal1p-deficient cells. Moreover, we now report that during normal aerobic growth on glucose, yeast mitochondria import ATP from the cytosol and hydrolyse it through H+-ATP synthase. We identify two pathways for ATP uptake in mitochondria, the ADP/ATP carriers and Sal1p. Thus, during exponential growth on glucose, mitochondria are ATP consumers, as those from cells growing in anaerobic conditions or deprived of mitochondrial DNA which depend on cytosolic ATP and mitochondrial ATPase working in reverse to generate a mitochondrial membrane potential. In conclusion, the results show that growth on glucose requires ATP hydrolysis in mitochondria and recruits Sal1p as a Ca2+-dependent mechanism to import ATP-Mg from the cytosol. Whether this mechanism is used under similar settings in higher eukaryotes is an open question.

  19. Time-resolved Fourier transform infrared spectroscopy of the nucleotide-binding domain from the ATP-binding Cassette transporter MsbA: ATP hydrolysis is the rate-limiting step in the catalytic cycle.

    PubMed

    Syberg, Falk; Suveyzdis, Yan; Kötting, Carsten; Gerwert, Klaus; Hofmann, Eckhard

    2012-07-01

    MsbA is an essential Escherichia coli ATP-binding cassette (ABC) transporter involved in the flipping of lipid A across the cytoplasmic membrane. It is a close homologue of human P-glycoprotein involved in multidrug resistance, and it similarly accepts a variety of small hydrophobic xenobiotics as transport substrates. X-ray structures of three full-length ABC multidrug exporters (including MsbA) have been published recently and reveal large conformational changes during the transport cycle. However, how ATP hydrolysis couples to these conformational changes and finally the transport is still an open question. We employed time-resolved FTIR spectroscopy, a powerful method to elucidate molecular reaction mechanisms of soluble and membrane proteins, to address this question with high spatiotemporal resolution. Here, we monitored the hydrolysis reaction in the nucleotide-binding domain of MsbA at the atomic level. The isolated MsbA nucleotide-binding domain hydrolyzed ATP with V(max) = 45 nmol mg(-1) min(-1), similar to the full-length transporter. A Hill coefficient of 1.49 demonstrates positive cooperativity between the two catalytic sites formed upon dimerization. Global fit analysis of time-resolved FTIR data revealed two apparent rate constants of ~1 and 0.01 s(-1), which were assigned to formation of the catalytic site and hydrolysis, respectively. Using isotopically labeled ATP, we identified specific marker bands for protein-bound ATP (1245 cm(-1)), ADP (1101 and 1205 cm(-1)), and free phosphate (1078 cm(-1)). Cleavage of the β-phosphate-γ-phosphate bond was found to be the rate-limiting step; no protein-bound phosphate intermediate was resolved.

  20. Analysis of catalytic carboxylate mutants E552Q and E1197Q suggests asymmetric ATP hydrolysis by the two nucleotide-binding domains of P-glycoprotein.

    PubMed

    Carrier, Isabelle; Julien, Michel; Gros, Philippe

    2003-11-11

    In the nucleotide-binding domains (NBDs) of ABC transporters, such as mouse Mdr3 P-glycoprotein (P-gp), an invariant carboxylate residue (E552 in NBD1; E1197 in NBD2) immediately follows the Walker B motif (hyd(4)DE/D). Removal of the negative charge in mutants E552Q and E1197Q abolishes drug-stimulated ATPase activity measured by P(i) release. Surprisingly, drug-stimulated trapping of 8-azido-[alpha-(32)P]ATP is still observed in the mutants in both the presence and absence of the transition-state analogue vanadate (V(i)), and ADP can be recovered from the trapped enzymes. The E552Q and E1197Q mutants show characteristics similar to those of the wild-type (WT) enzyme with respect to 8-azido-[alpha-(32)P]ATP binding and 8-azido-[alpha-(32)P]nucleotide trapping, with the latter being both Mg(2+) and temperature dependent. Importantly, drug-stimulated nucleotide trapping in E552Q is stimulated by V(i) and resembles the WT enzyme, while it is almost completely V(i) insensitive in E1197Q. Similar nucleotide trapping properties are observed when aluminum fluoride or beryllium fluoride is used as an alternate transition-state analogue. Partial proteolytic cleavage of photolabeled enzymes indicates that, in the absence of V(i), nucleotide trapping occurs exclusively at the mutant NBD, whereas in the presence of V(i), nucleotide trapping occurs at both NBDs. Together, these results suggest that there is single-site turnover occurring in the E552Q and E1197Q mutants and that ADP release from the mutant site, or another catalytic step, is impaired in these mutants. Furthermore, our results support a model in which the two NBDs of P-gp are not functionally equivalent.

  1. Increased levels of extracellular ATP in glaucomatous retinas: Possible role of the vesicular nucleotide transporter during the development of the pathology

    PubMed Central

    Pérez de Lara, María J.; Guzmán-Aránguez, Ana; de la Villa, Pedro; Díaz-Hernández, Juan Ignacio; Miras-Portugal, María Teresa

    2015-01-01

    Purpose To study retinal extracellular ATP levels and to assess the changes in the vesicular nucleotide transporter (VNUT) expression in a murine model of glaucoma during the development of the disease. Methods Retinas were obtained from glaucomatous DBA/2J mice at 3, 9, 15, and 22 months together with C57BL/6J mice used as age-matched controls. To study retinal nucleotide release, the retinas were dissected and prepared as flattened whole mounts and stimulated in Ringer buffer with or without 59 mM KCl. To investigate VNUT expression, sections of the mouse retinas were evaluated with immunohistochemistry and western blot analysis using newly developed antibodies against VNUT. All images were examined and photographed under confocal microscopy. Electroretinogram (ERG) recordings were performed on the C57BL/6J and DBA/2J mice to analyze the changes in the electrophysiological response; a decrease in the scotopic threshold response was observed in the 15-month-old DBA/2J mice. Results In the 15-month-old control and glaucomatous mice, electrophysiological changes of 42% were observed. In addition, 50% increases in the intraocular pressure (IOP) were observed when the pathology was fully established. The responses in the retinal ATP net release as the pathology progressed varied from 0.32±0.04 pmol/retina (3 months) to 1.10±0.06 pmol/retina (15 months; threefold increase). Concomitantly, VNUT expression was significantly increased during glaucoma progression in the DBA/2J mice (58%) according to the immunohistochemical and western blot analysis. Conclusions These results may indicate a possible correlation between retinal dysfunction and increased levels of extracellular ATP and nucleotide transporter. These data support an excitotoxicity role for ATP via P2X7R in glaucoma. This modified cellular environment could contribute to explaining the functional and biochemical alterations observed during the development of the pathology. PMID:26392744

  2. A comparative electron paramagnetic resonance study of the nucleotide-binding domains' catalytic cycle in the assembled maltose ATP-binding cassette importer.

    PubMed

    Grote, Mathias; Bordignon, Enrica; Polyhach, Yevhen; Jeschke, Gunnar; Steinhoff, Heinz-Jürgen; Schneider, Erwin

    2008-09-15

    We present a quantitative analysis of conformational changes of the nucleotide-binding subunits, MalK(2), of the maltose ATP-binding cassette importer MalFGK(2) during the transport cycle. Distance changes occurring between selected residues were monitored in the full transporter by site-directed spin-labeling electron paramagnetic resonance spectroscopy and site-directed chemical cross-linking. We considered S83C and A85C from the conserved Q-loop and V117C located on the outer surface of MalK. Additionally, two native cysteines (C350, C360) were included in the study. On ATP binding, small rearrangements between the native sites, and no distance changes between positions 117 were detected. In contrast, positions 85 come closer together in the ATP-bound state and in the vanadate-trapped intermediate and move back toward the apo-state after ATP hydrolysis. The distance between positions 83 is shown to slightly decrease on ATP binding, and to further decrease after ATP hydrolysis. Results from cross-linking experiments are in agreement with these findings. The data are compared with in silico spin-labeled x-ray structures from both isolated MalK(2) and the MalFGK(2)-E complex. Our results are consistent with a slightly modified "tweezers-like" model of closure and reopening of MalK(2) during the catalytic cycle, and show an unforeseen potential interaction between MalK and the transmembrane subunit MalG.

  3. Neonatal Diabetes Caused by Mutations in Sulfonylurea Receptor 1: Interplay between Expression and Mg-Nucleotide Gating Defects of ATP-Sensitive Potassium Channels

    PubMed Central

    Zhou, Qing; Garin, Intza; Castaño, Luis; Argente, Jesús; Muñoz-Calvo, Ma. Teresa; Perez de Nanclares, Guiomar; Shyng, Show-Ling

    2010-01-01

    Context: ATP-sensitive potassium (KATP) channels regulate insulin secretion by coupling glucose metabolism to β-cell membrane potential. Gain-of-function mutations in the sulfonylurea receptor 1 (SUR1) or Kir6.2 channel subunit underlie neonatal diabetes. Objective: The objective of the study was to determine the mechanisms by which two SUR1 mutations, E208K and V324M, associated with transient neonatal diabetes affect KATP channel function. Design: E208K or V324M mutant SUR1 was coexpressed with Kir6.2 in COS cells, and expression and gating properties of the resulting channels were assessed biochemically and electrophysiologically. Results: Both E208K and V324M augment channel response to MgADP stimulation without altering sensitivity to ATP4− or sulfonylureas. Surprisingly, whereas E208K causes only a small increase in MgADP response consistent with the mild transient diabetes phenotype, V324M causes a severe activating gating defect. Unlike E208K, V324M also impairs channel expression at the cell surface, which is expected to dampen its functional impact on β-cells. When either mutation was combined with a mutation in the second nucleotide binding domain of SUR1 previously shown to abolish Mg-nucleotide response, the activating effect of E208K and V324M was also abolished. Moreover, combination of E208K and V324M results in channels with Mg-nucleotide sensitivity greater than that seen in individual mutations alone. Conclusion: The results demonstrate that E208K and V324M, located in distinct domains of SUR1, enhance transduction of Mg-nucleotide stimulation from the SUR1 nucleotide binding folds to Kir6.2. Furthermore, they suggest that diabetes severity is determined by interplay between effects of a mutation on channel expression and channel gating. PMID:20810569

  4. Quaternary structure of K[ssubscript ATP] channel SUR2A nucleotide binding domains resolved by synchrotron radiation X-ray scattering

    SciTech Connect

    Park, Sungjo; Terzic, Andre

    2010-05-25

    Heterodimeric nucleotide binding domains NBD1/NBD2 distinguish the ATP-binding cassette protein SUR2A, a recognized regulatory subunit of cardiac ATP-sensitive K{sup +} (K{sub ATP}) channels. The tandem function of these core domains ensures metabolism-dependent gating of the Kir6.2 channel pore, yet their structural arrangement has not been resolved. Here, purified monodisperse and interference-free recombinant particles were subjected to synchrotron radiation small-angle X-ray scattering (SAXS) in solution. Intensity function analysis of SAXS profiles resolved NBD1 and NBD2 as octamers. Implemented by ab initio simulated annealing, shape determination prioritized an oblong envelope wrapping NBD1 and NBD2 with respective dimensions of 168 x 80 x 37 {angstrom}{sup 3} and 175 x 81 x 37 {angstrom}{sup 3} based on symmetry constraints, validated by atomic force microscopy. Docking crystal structure homology models against SAXS data reconstructed the NBD ensemble surrounding an inner cleft suitable for Kir6.2 insertion. Human heart disease-associated mutations introduced in silico verified the criticality of the mapped protein-protein interface. The resolved quaternary structure delineates thereby a macromolecular arrangement of K{sub ATP} channel SUR2A regulatory domains.

  5. Photo-excitation of adenine cation radical [A•+] in the near UV-vis region produces sugar radicals in Adenosine and in its nucleotides

    PubMed Central

    Adhikary, Amitava; Khanduri, Deepti; Kumar, Anil; Sevilla, Michael D.

    2011-01-01

    In this study, we report the formation of ribose sugar radicals in high yields (85 – 100%) via photo-excitation of adenine cation radical (A•+) in Ado and its ribonucleotides. Photo-excitation of A•+ at low temperatures in homogenous aqueous glassy samples of Ado, 2′-AMP, 3′-AMP and 5′-AMP forms sugar radicals predominantly at C5′- and also at C3′-sites. The C5′• and C3′• sugar radicals were identified employing Ado deuterated at specific carbon sites: C1′, C2′, and at C5′. Phosphate substitution is found to deactivate sugar radical formation at the site of substitution. Thus, in 5′-AMP, C3′• is observed to be the main radical formed via photo-excitation at ca. 143 K whereas in 3′-AMP, C5′• is the only species found. These results were supported by results obtained employing 5′-AMP with specific deuteration at C5′-site (i.e., 5′,5′-D,D-5′-AMP). Moreover, contrary to the C5′• observed in 3′-dAMP, we find that C5′• in 3′-AMP shows a clear pH dependent conformational change as evidenced by a large increase in the C4′ β–hyperfine coupling on increasing the pH from 6 to 9. Calculations performed employing DFT (B3LYP/6-31G*) for C5′• in 3′-AMP show that the two conformations of C5′• result from strong hydrogen bond formation between the O5′-H and the 3′-phosphate dianion at higher pHs. Employing time-dependent density functional theory [TD-DFT, B3LYP/6-31G(d)] we show that in the excited state, the hole transfers to the sugar moiety and has significant hole localization at the C5′-site in a number of allowed transitions. This hole localization is proposed to lead to the formation of the neutral C5′-radical (C5′•) via deprotonation. PMID:19367991

  6. [Degradation of purine nucleotides in patients with chronic obstruction to airflow].

    PubMed

    Mateos Antón, F; García Puig, J; Gómez Fernández, P; Ramos Hernández, T; López Jiménez, M

    1989-03-11

    The increase in hypoxanthine (Hx), xanthine (X), uric acid (VA) and total purines (TP) that may be found in several clinical conditions associated with tissue hypoxia has been attributed to an increase in adenine nucleotides degradation by a reduced ATP synthesis caused by oxygen deprivation. To test this hypothesis we have investigated the urinary excretion of Hx, X, VA, TP and radioactivity elimination after labeling the adenine nucleotides with adenine (8-14C) in 5 patients with chronic airflow obstruction (CAFO), in the basal state and after oxygen therapy (FiO2, 24%). The results were compared with those from 4 normal individuals. Patients with COFA showed an increase of the renal elimination of Hx, X, VA, TP and radioactivity, which was significantly different from the control group (p less than 0.05). Oxygen administration was associated with a significant reduction in the excretion of purines and radioactivity (p less than 0.01), which decreased to values similar to those found in normal individuals. These findings suggest that in patients with COFA and severe hypoxemia there is a marked increase in the degradation of adenine nucleotides. The normalization of the purine and radioactivity excretion after oxygen therapy points to a basic role of oxygen in the catabolism of adenine nucleotides. PMID:2716427

  7. Small-angle X-ray scattering study of the ATP modulation of the structural features of the nucleotide binding domains of the CFTR in solution.

    PubMed

    Galeno, Lauretta; Galfrè, Elena; Moran, Oscar

    2011-07-01

    Nucleotide binding domains (NBD1 and NBD2) of the cystic fibrosis transmembrane conductance (CFTR), the defective protein in cystic fibrosis, are responsible for controlling the gating of the chloride channel and are the putative binding site for several candidate drugs in the disease treatment. We studied the structural properties of recombinant NBD1, NBD2, and an equimolar NBD1/NBD2 mixture in solution by small-angle X-ray scattering. We demonstrated that NBD1 or NBD2 alone have an overall structure similar to that observed for crystals. Application of 2 mM ATP induces a dimerization of NBD1 but does not modify the NBD2 monomeric conformation. An equimolar mixture of NBD1/NBD2 in solution shows a dimeric conformation, and the application of ATP to the solution causes a conformational change in the NBD1/NBD2 complex into a tight heterodimer. We hypothesize that a similar conformation change occurs in situ and that transition is part of the gating mechanism. To our knowledge, this is the first direct observation of a conformational change of the NBD1/NBD2 interaction by ATP. This information may be useful to understand the physiopathology of cystic fibrosis.

  8. An Adenine-DNA Adduct Derived from Nitroreduction of 6-Nitrochrysene is more Resistant to Nucleotide Excision Repair than Guanine-DNA Adducts

    PubMed Central

    Krzeminski, Jacek; Kropachev, Konstantin; Reeves, Dara; Kolbanovskiy, Aleksandr; Kolbanovskiy, Marina; Chen, Kun-Ming; Sharma, Arun K.; Geacintov, Nicholas; Amin, Shantu; El-Bayoumy, Karam

    2013-01-01

    Previous studies in rats, mice and in vitro systems showed that 6-NC can be metabolically activated by two major pathways: 1) the formation of N-hydroxy-6-aminochrysene by nitroreduction to yield three major adducts: N-(dG-8-yl)-6-AC, 5-(dG-N2-yl)-6-AC and N-(dA-8-yl)-6-AC, and 2) the formation of trans-1,2-dihydroxy-1,2-dihydro-6-hydroxylaminochrysene (1,2-DHD-6-NHOH-C) by a combination of nitroreduction and ring oxidation pathways to yield: N-(dG-8-yl)-1,2-DHD-6-AC, 5-(dG-N2-yl)-1,2-DHD-6-AC and N-(dA-8-yl)-1,2-DHD-6-AC. These DNA lesions are likely to cause mutations if they are not removed by cellular defense mechanisms before DNA replication occurs. Here we compared for the first time, in HeLa cell extracts in vitro, the relative nucleotide excision repair (NER) efficiencies of DNA lesions derived from simple nitroreduction and from a combination of nitroreduction and ring oxidation pathways. We show that the N-(dG-8-yl)-1,2-DHD-6-AC adduct is more resistant to NER than the N-(dG-8-yl)-6-AC adduct by a factor of ~2. Furthermore, the N-(dA-8-yl)-6-AC is much more resistant to repair since its NER efficiency is ~ 8-fold lower than that of the N-(dG-8-yl)-6-AC adduct. On the basis of our previous study and the present investigation, lesions derived from 6-NC and benzo[a]pyrene can be ranked from the most to the least resistant lesion as follows: N-(dA-8-yl)-6-AC > N-(dG-8-yl)-1,2-DHD-6-AC > 5-(dG-N2-yl)-6-AC ~ N-(dG-8-yl)-6-AC ~ (+)-7R,8S,9S,10S-benzo[a]pyrene diol epoxide-derived trans-anti-benzo[a]pyrene-N2-dG adduct. The slow repair of the various lesions derived from 6-NC and thus their potential persistence in mammalian tissue, could in part account for the powerful carcinogenicity of 6-NC as compared to B[a]P in the rat mammary gland. PMID:24112095

  9. Nucleotide-induced asymmetry within ATPase activator ring drives σ54–RNAP interaction and ATP hydrolysis

    PubMed Central

    Sysoeva, Tatyana A.; Chowdhury, Saikat; Guo, Liang; Nixon, B. Tracy

    2013-01-01

    It is largely unknown how the typical homomeric ring geometry of ATPases associated with various cellular activities enables them to perform mechanical work. Small-angle solution X-ray scattering, crystallography, and electron microscopy (EM) reconstructions revealed that partial ATP occupancy caused the heptameric closed ring of the bacterial enhancer-binding protein (bEBP) NtrC1 to rearrange into a hexameric split ring of striking asymmetry. The highly conserved and functionally crucial GAFTGA loops responsible for interacting with σ54–RNA polymerase formed a spiral staircase. We propose that splitting of the ensemble directs ATP hydrolysis within the oligomer, and the ring's asymmetry guides interaction between ATPase and the complex of σ54 and promoter DNA. Similarity between the structure of the transcriptional activator NtrC1 and those of distantly related helicases Rho and E1 reveals a general mechanism in homomeric ATPases whereby complex allostery within the ring geometry forms asymmetric functional states that allow these biological motors to exert directional forces on their target macromolecules. PMID:24240239

  10. Domain Interactions in the Yeast ATP Binding Cassette Transporter Ycf1p: Intragenic Suppressor Analysis of Mutations in the Nucleotide Binding Domains

    PubMed Central

    Falcón-Pérez, Juan M.; Martínez-Burgos, Mónica; Molano, Jesús; Mazón, María J.; Eraso, Pilar

    2001-01-01

    The yeast cadmium factor (Ycf1p) is a vacuolar ATP binding cassette (ABC) transporter required for heavy metal and drug detoxification. Cluster analysis shows that Ycf1p is strongly related to the human multidrug-associated protein (MRP1) and cystic fibrosis transmembrane conductance regulator and therefore may serve as an excellent model for the study of eukaryotic ABC transporter structure and function. Identifying intramolecular interactions in these transporters may help to elucidate energy transfer mechanisms during transport. To identify regions in Ycf1p that may interact to couple ATPase activity to substrate binding and/or movement across the membrane, we sought intragenic suppressors of ycf1 mutations that affect highly conserved residues presumably involved in ATP binding and/or hydrolysis. Thirteen intragenic second-site suppressors were identified for the D777N mutation which affects the invariant Asp residue in the Walker B motif of the first nucleotide binding domain (NBD1). Two of the suppressor mutations (V543I and F565L) are located in the first transmembrane domain (TMD1), nine (A1003V, A1021T, A1021V, N1027D, Q1107R, G1207D, G1207S, S1212L, and W1225C) are found within TMD2, one (S674L) is in NBD1, and another one (R1415G) is in NBD2, indicating either physical proximity or functional interactions between NBD1 and the other three domains. The original D777N mutant protein exhibits a strong defect in the apparent affinity for ATP and Vmax of transport. The phenotypic characterization of the suppressor mutants shows that suppression does not result from restoring these alterations but rather from a change in substrate specificity. We discuss the possible involvement of Asp777 in coupling ATPase activity to substrate binding and/or transport across the membrane. PMID:11466279

  11. Interaction of beef-heart mitochondrial F1-ATPase with immobilized ATP in the presence of dimethylsulfoxide.

    PubMed

    Beharry, S; Bragg, P D

    1992-10-01

    Dimethylsulfoxide [Me2SO, 30% (v/v)] promotes the formation of ATP from ADP and phosphate catalyzed by soluble mitochondrial F1-ATPase. The effects of this solvent on the interaction of beef-heart mitochondrial F1 with the immobilized ATP of Agarose-hexane-ATP were studied. In the presence of Me2SO, F1 bound less readily to the immobilized ATP, but once bound was more difficult to elute with exogenous ATP. This suggests that not only was the binding affinity for adenine nucleotide at the first binding site affected but that adenine nucleotide binding affinity at the second and/or third sites, which interact cooperatively with the first site to release bound nucleotide, was also affected. A reduction in the binding of [3H]ADP to these sites was shown. A change in the conformation of F1 in 30% (v/v) Me2SO was demonstrated by crosslinking and by the increased resistance of the enzyme to cold denaturation.

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

    PubMed Central

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

    2013-01-01

    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 Ca2+ like CyPD itself, and that decreasing oligomycin sensitivity-conferring protein expression by RNAi increases the sensitivity of the PTP to Ca2+. Purified dimers of the ATP synthase, which did not contain voltage-dependent anion channel or adenine nucleotide translocator, were reconstituted into lipid bilayers. In the presence of Ca2+, addition of Bz-423 triggered opening of a channel with currents that were typical of the mitochondrial megachannel, which is the PTP electrophysiological equivalent. Channel openings were inhibited by the ATP synthase inhibitor AMP-PNP (γ-imino ATP, a nonhydrolyzable ATP analog) and Mg2+/ADP. These results indicate that the PTP forms from dimers of the ATP synthase. PMID:23530243

  13. Crystal structure of Escherichia coli cytidine triphosphate synthetase, a nucleotide-regulated glutamine amidotransferase/ATP-dependent amidoligase fusion protein and homologue of anticancer and antiparasitic drug targets.

    PubMed

    Endrizzi, James A; Kim, Hanseong; Anderson, Paul M; Baldwin, Enoch P

    2004-06-01

    Cytidine triphosphate synthetases (CTPSs) produce CTP from UTP and glutamine, and regulate intracellular CTP levels through interactions with the four ribonucleotide triphosphates. We solved the 2.3-A resolution crystal structure of Escherichia coli CTPS using Hg-MAD phasing. The structure reveals a nearly symmetric 222 tetramer, in which each bifunctional monomer contains a dethiobiotin synthetase-like amidoligase N-terminal domain and a Type 1 glutamine amidotransferase C-terminal domain. For each amidoligase active site, essential ATP- and UTP-binding surfaces are contributed by three monomers, suggesting that activity requires tetramer formation, and that a nucleotide-dependent dimer-tetramer equilibrium contributes to the observed positive cooperativity. A gated channel that spans 25 A between the glutamine hydrolysis and amidoligase active sites provides a path for ammonia diffusion. The channel is accessible to solvent at the base of a cleft adjoining the glutamine hydrolysis active site, providing an entry point for exogenous ammonia. Guanine nucleotide binding sites of structurally related GTPases superimpose on this cleft, providing insights into allosteric regulation by GTP. Mutations that confer nucleoside drug resistance and release CTP inhibition map to a pocket that neighbors the UTP-binding site and can accommodate a pyrimidine ring. Its location suggests that competitive feedback inhibition is affected via a distinct product/drug binding site that overlaps the substrate triphosphate binding site. Overall, the E. coli structure provides a framework for homology modeling of other CTPSs and structure-based design of anti-CTPS therapeutics. PMID:15157079

  14. The Specialized Hsp70 (HscA) Interdomain Linker Binds to Its Nucleotide-Binding Domain and Stimulates ATP Hydrolysis in Both cis and trans Configurations

    PubMed Central

    2015-01-01

    Proteins from the isc operon of Escherichia coli constitute the machinery used to synthesize iron–sulfur (Fe–S) clusters for delivery to recipient apoproteins. Efficient and rapid [2Fe-2S] cluster transfer from the holo-scaffold protein IscU depends on ATP hydrolysis in the nucleotide-binding domain (NBD) of HscA, a specialized Hsp70-type molecular chaperone with low intrinsic ATPase activity (0.02 min−1 at 25 °C, henceforth reported in units of min–1). HscB, an Hsp40-type cochaperone, binds to HscA and stimulates ATP hydrolysis to promote cluster transfer, yet while the interactions between HscA and HscB have been investigated, the role of HscA’s interdomain linker in modulating ATPase activity has not been explored. To address this issue, we created three variants of the 40 kDa NBD of HscA: NBD alone (HscA386), NBD with a partial linker (HscA389), and NBD with the full linker (HscA395). We found that the rate of ATP hydrolysis of HscA395 (0.45 min–1) is nearly 15-fold higher than that of HscA386 (0.035 min–1), although their apparent affinities for ATP are equivalent. HscA395, which contains the full covalently linked linker peptide, exhibited intrinsic tryptophan fluorescence emission and basal thermostability that were higher than those of HscA386. Furthermore, HscA395 displayed narrower 1HN line widths in its two-dimensional 1H–15N TROSY-HSQC spectrum in comparison to HscA386, indicating that the peptide in the cis configuration binds to and stabilizes the structure of the NBD. The addition to HscA386 of a synthetic peptide with a sequence identical to that of the interdomain linker (L387LLDVIPLS395) stimulated its ATPase activity and induced widespread NMR chemical shift perturbations indicative of a binding interaction in the trans configuration. PMID:25372495

  15. The specialized Hsp70 (HscA) interdomain linker binds to its nucleotide-binding domain and stimulates ATP hydrolysis in both cis and trans configurations.

    PubMed

    Alderson, T Reid; Kim, Jin Hae; Cai, Kai; Frederick, Ronnie O; Tonelli, Marco; Markley, John L

    2014-11-25

    Proteins from the isc operon of Escherichia coli constitute the machinery used to synthesize iron-sulfur (Fe-S) clusters for delivery to recipient apoproteins. Efficient and rapid [2Fe-2S] cluster transfer from the holo-scaffold protein IscU depends on ATP hydrolysis in the nucleotide-binding domain (NBD) of HscA, a specialized Hsp70-type molecular chaperone with low intrinsic ATPase activity (0.02 min(-1) at 25 °C, henceforth reported in units of min(-1)). HscB, an Hsp40-type cochaperone, binds to HscA and stimulates ATP hydrolysis to promote cluster transfer, yet while the interactions between HscA and HscB have been investigated, the role of HscA's interdomain linker in modulating ATPase activity has not been explored. To address this issue, we created three variants of the 40 kDa NBD of HscA: NBD alone (HscA386), NBD with a partial linker (HscA389), and NBD with the full linker (HscA395). We found that the rate of ATP hydrolysis of HscA395 (0.45 min(-1)) is nearly 15-fold higher than that of HscA386 (0.035 min(-1)), although their apparent affinities for ATP are equivalent. HscA395, which contains the full covalently linked linker peptide, exhibited intrinsic tryptophan fluorescence emission and basal thermostability that were higher than those of HscA386. Furthermore, HscA395 displayed narrower (1)H(N) line widths in its two-dimensional (1)H-(15)N TROSY-HSQC spectrum in comparison to HscA386, indicating that the peptide in the cis configuration binds to and stabilizes the structure of the NBD. The addition to HscA386 of a synthetic peptide with a sequence identical to that of the interdomain linker (L(387)LLDVIPLS(395)) stimulated its ATPase activity and induced widespread NMR chemical shift perturbations indicative of a binding interaction in the trans configuration. PMID:25372495

  16. Inhibition of Multidrug Resistance-Linked P-Glycoprotein (ABCB1) Function by 5′-Fluorosulfonylbenzoyl 5′-Adenosine: Evidence for an ATP Analog That Interacts With Both Drug-Substrate- and Nucleotide-Binding Sites†

    PubMed Central

    Ohnuma, Shinobu; Chufan, Eduardo; Nandigama, Krishnamachary; Miller Jenkins, Lisa M.; Durell, Stewart R.; Appella, Ettore; Sauna, Zuben E.; Ambudkar, Suresh V.

    2011-01-01

    5′-fluorosulfonylbenzonyl 5′-adenosine (FSBA) is an ATP analog that covalently modifies several residues in the nucleotide-binding domains (NBDs) of several ATPases, kinases and other proteins. P-glycoprotein (P-gp, ABCB1) is a member of the ATP-binding cassette (ABC) transporter superfamily that utilizes energy from ATP hydrolysis for the efflux of amphipathic anticancer agents from cancer cells. We investigated the interactions of FSBA with P-gp to study the catalytic cycle of ATP hydrolysis. Incubation of P-gp with FSBA inhibited ATP hydrolysis (IC50= 0.21 mM) and the binding of 8-azido[α–32P]ATP (IC50= 0.68 mM). In addition, 14C-FSBA crosslinks to P-gp, suggesting that FSBA-mediated inhibition of ATP hydrolysis is irreversible due to covalent modification of P-gp. However, when the NBDs were occupied with a saturating concentration of ATP prior to treatment, FSBA stimulated ATP hydrolysis by P-gp. Furthermore, FSBA inhibited the photocrosslinking of P-gp with [125I]-Iodoaryl-azidoprazosin (IAAP; IC50 = 0.17 mM). As IAAP is a transport substrate for P-gp, this suggests that FSBA affects not only the NBDs, but also the transport-substrate site in the transmembrane domains. Consistent with these results, FSBA blocked efflux of rhodamine 123 from P-gp-expressing cells. Additionally, mass spectrometric analysis identified FSBA crosslinks to residues within or nearby the NBDs but not in the transmembrane domains and docking of FSBA in a homology model of human P-gp NBDs supports the biochemical studies. Thus, FSBA is an ATP analog that interacts with both the drug-binding and ATP-binding sites of P-gp, but fluorosulfonyl-mediated crosslinking is observed only at the NBDs. PMID:21452853

  17. The substrate specificity of the human ADP/ATP carrier AAC1.

    PubMed

    Mifsud, John; Ravaud, Stéphanie; Krammer, Eva-Maria; Chipot, Chris; Kunji, Edmund R S; Pebay-Peyroula, Eva; Dehez, Francois

    2013-03-01

    The mitochondrial ADP/ATP carrier imports ADP from the cytosol into the mitochondrial matrix for its conversion to ATP by ATP synthase and exports ATP out of the mitochondrion to replenish the eukaryotic cell with chemical energy. Here the substrate specificity of the human mitochondrial ADP/ATP carrier AAC1 was determined by two different approaches. In the first the protein was functionally expressed in Escherichia coli membranes as a fusion protein with maltose binding protein and the effect of excess of unlabeled compounds on the uptake of [(32)P]-ATP was measured. In the second approach the protein was expressed in the cytoplasmic membrane of Lactococcus lactis. The uptake of [(14)C]-ADP in whole cells was measured in the presence of excess of unlabeled compounds and in fused membrane vesicles loaded with unlabeled compounds to demonstrate their transport. A large number of nucleotides were tested, but only ADP and ATP are suitable substrates for human AAC1, demonstrating a very narrow specificity. Next we tried to understand the molecular basis of this specificity by carrying out molecular-dynamics simulations with selected nucleotides, which were placed at the entrance of the central cavity. The binding of the phosphate groups of guanine and adenine nucleotides is similar, yet there is a low probability for the base moiety to be bound, likely to be rooted in the greater polarity of guanine compared to adenine. AMP is unlikely to engage fully with all contact points of the substrate binding site, suggesting that it cannot trigger translocation.

  18. The Coupling of Electron Flow to ATP Synthesis in Pea and Maize Mesophyll Chloroplasts 12

    PubMed Central

    Cole, Richard M.; Macpeek, Wendy A.; Cohen, William S.

    1981-01-01

    The rate of nonphosphorylating electron transport (in the absence of ADP and inorganic phosphate) in well-coupled (ATP/2e− = 0.9-1.1) maize mesophyll chloroplasts is not modulated by external pH (6.5-8.5), low levels of ADP or ATP, or energy transfer inhibitors, e.g. triphenyltin and Hg2+ ions. In contrast nonphosphorylating electron flow in pea chloroplasts is sensitive to alterations in medium pH, and to the presence of adenine nucleotides and energy transfer inhibitors in the assay medium. Although ATP is without effect on the rate of basal electron transport in maize chloroplasts, steady-state proton uptake is stimulated 3- to 5-fold by low levels of ATP. These results suggest that differences may exist in the manner in which the coupling factor complex controls proton efflux from the intrathylakoid space in C3 and C4 mesophyll chloroplasts. PMID:16661966

  19. Structural Models of Zebrafish (Danio rerio) NOD1 and NOD2 NACHT Domains Suggest Differential ATP Binding Orientations: Insights from Computational Modeling, Docking and Molecular Dynamics Simulations

    PubMed Central

    Maharana, Jitendra; Sahoo, Bikash Ranjan; Bej, Aritra; Sahoo, Jyoti Ranjan; Dehury, Budheswar; Patra, Mahesh Chandra; Martha, Sushma Rani; Balabantray, Sucharita; Pradhan, Sukanta Kumar; Behera, Bijay Kumar

    2015-01-01

    Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) and NOD2 are cytosolic pattern recognition receptors playing pivotal roles in innate immune signaling. NOD1 and NOD2 recognize bacterial peptidoglycan derivatives iE-DAP and MDP, respectively and undergoes conformational alternation and ATP-dependent self-oligomerization of NACHT domain followed by downstream signaling. Lack of structural adequacy of NACHT domain confines our understanding about the NOD-mediated signaling mechanism. Here, we predicted the structure of NACHT domain of both NOD1 and NOD2 from model organism zebrafish (Danio rerio) using computational methods. Our study highlighted the differential ATP binding modes in NOD1 and NOD2. In NOD1, γ-phosphate of ATP faced toward the central nucleotide binding cavity like NLRC4, whereas in NOD2 the cavity was occupied by adenine moiety. The conserved ‘Lysine’ at Walker A formed hydrogen bonds (H-bonds) and Aspartic acid (Walker B) formed electrostatic interaction with ATP. At Sensor 1, Arg328 of NOD1 exhibited an H-bond with ATP, whereas corresponding Arg404 of NOD2 did not. ‘Proline’ of GxP motif (Pro386 of NOD1 and Pro464 of NOD2) interacted with adenine moiety and His511 at Sensor 2 of NOD1 interacted with γ-phosphate group of ATP. In contrast, His579 of NOD2 interacted with the adenine moiety having a relatively inverted orientation. Our findings are well supplemented with the molecular interaction of ATP with NLRC4, and consistent with mutagenesis data reported for human, which indicates evolutionary shared NOD signaling mechanism. Together, this study provides novel insights into ATP binding mechanism, and highlights the differential ATP binding modes in zebrafish NOD1 and NOD2. PMID:25811192

  20. Search for interstellar adenine

    NASA Astrophysics Data System (ADS)

    Chakrabarti, Sandip K.; Majumdar, Liton; Das, Ankan; Chakrabarti, Sonali

    2015-05-01

    It is long debated if pre-biotic molecules are indeed present in the interstellar medium. Despite substantial works pointing to their existence, pre-biotic molecules are yet to be discovered with a complete confidence. In this paper, our main aim is to study the chemical evolution of interstellar adenine under various circumstances. We prepare a large gas-grain chemical network by considering various pathways for the formation of adenine. Majumdar et al. (New Astron. 20:15, 2013) proposed that in the absence of adenine detection, one could try to trace two precursors of adenine, namely, HCCN and NH2CN. Recently Merz et al. (J. Phys. Chem. A 118:3637-3644, 2014), proposed another route for the formation of adenine in interstellar condition. They proposed two more precursor molecules. But it was not verified by any accurate gas-grain chemical model. Neither was it known if the production rate would be high or low. Our paper fills this important gap. We include this new pathways to find that the contribution through this pathways for the formation of Adenine is the most dominant one in the context of interstellar medium. We propose that observers may look for the two precursors (C3NH and HNCNH) in the interstellar media which are equally important for predicting abundances of adenine. We perform quantum chemical calculations to find out spectral properties of adenine and its two new precursor molecules in infrared, ultraviolet and sub-millimeter region. Our present study would be useful for predicting abundance of adenine.

  1. Molecular and Subcellular-Scale Modeling of Nucleotide Diffusion in the Cardiac Myofilament Lattice

    PubMed Central

    Kekenes-Huskey, Peter M.; Liao, Tao; Gillette, Andrew K.; Hake, Johan E.; Zhang, Yongjie; Michailova, Anushka P.; McCulloch, Andrew D.; McCammon, J. Andrew

    2013-01-01

    Contractile function of cardiac cells is driven by the sliding displacement of myofilaments powered by the cycling myosin crossbridges. Critical to this process is the availability of ATP, which myosin hydrolyzes during the cross-bridge cycle. The diffusion of adenine nucleotides through the myofilament lattice has been shown to be anisotropic, with slower radial diffusion perpendicular to the filament axis relative to parallel, and is attributed to the periodic hexagonal arrangement of the thin (actin) and thick (myosin) filaments. We investigated whether atomistic-resolution details of myofilament proteins can refine coarse-grain estimates of diffusional anisotropy for adenine nucleotides in the cardiac myofibril, using homogenization theory and atomistic thin filament models from the Protein Data Bank. Our results demonstrate considerable anisotropy in ATP and ADP diffusion constants that is consistent with experimental measurements and dependent on lattice spacing and myofilament overlap. A reaction-diffusion model of the half-sarcomere further suggests that diffusional anisotropy may lead to modest adenine nucleotide gradients in the myoplasm under physiological conditions. PMID:24209858

  2. H+/ATP ratio during ATP hydrolysis by mitochondria: modification of the chemiosmotic theory.

    PubMed

    Brand, M D; Lehninger, A L

    1977-05-01

    The stoichiometry of H+ ejection by mitochondria during hydrolysis of a small pulse of ATP (the H+/ATP ratio) has been reexamined in the light of our recent observation that the stoichiometry of H+ ejection during mitochondrial electron transport (the H+/site ratio) was previously underestimated. We show that earlier estimates of the H+/ATP ratio in intact mitochondria were based upon an invalid correction for scaler H+ production and describe a modified method for determination of this ratio which utilizes mersalyl or N-ethylmaleimide to prevent complicating transmembrane movements of phosphate and H+. This method gives a value for the H+/ATP ratio of 2.0 without the need for questionable corrections, compared with a value of 3.0 for the H+/site ratio also obtained by pulse methods. A modified version of the chemiosmotic theory is presented, in which 3 H+ are ejected per pair of electrons traversing each energy-conserving site of the respiratory chain. Of these, 2 H+ return to the matrix through the ATPase to form ATP from ADP and phosphate, and 1 H+ returns through the combined action of the phosphate and adenine nucleotide exchange carriers of the inner membrane to allow the energy-requiring influx of Pi and ADP3- and efflux of ATP4-. Thus, up to one-third of the energy input into synthesis of extramitochondrial ATP may be required for transport work. Since other methods suggest that the H+/site significantly exceeds 3.0, an alternative possibility is that 4 h+ are ejected per site, followed by return of 3 H+ through the ATPase and 1 H+ through the operation of the proton-coupled membrane transport systems.

  3. Cerulenin-mediated apoptosis is involved in adenine metabolic pathway

    SciTech Connect

    Chung, Kyung-Sook; Sun, Nam-Kyu; Lee, Seung-Hee; Lee, Hyun-Jee; Choi, Shin-Jung; Kim, Sun-Kyung; Song, Ju-Hyun; Jang, Young-Joo; Song, Kyung-Bin; Yoo, Hyang-Sook; Simon, Julian . E-mail: jsimon@fhcrc.org; Won, Misun . E-mail: misun@kribb.re.kr

    2006-10-27

    Cerulenin, a fatty acid synthase (FAS) inhibitor, induces apoptosis of variety of tumor cells. To elucidate mode of action by cerulenin, we employed the proteomics approach using Schizosaccharomyces pombe. The differential protein expression profile of S. pombe revealed that cerulenin modulated the expressions of proteins involved in stresses and metabolism, including both ade10 and adk1 proteins. The nutrient supplementation assay demonstrated that cerulenin affected enzymatic steps transferring a phosphoribosyl group. This result suggests that cerulenin accumulates AMP and p-ribosyl-s-amino-imidazole carboxamide (AICAR) and reduces other necessary nucleotides, which induces feedback inhibition of enzymes and the transcriptional regulation of related genes in de novo and salvage adenine metabolic pathway. Furthermore, the deregulation of adenine nucleotide synthesis may interfere ribonucleotide reductase and cause defects in cell cycle progression and chromosome segregation. In conclusion, cerulenin induces apoptosis through deregulation of adenine nucleotide biosynthesis resulting in nuclear division defects in S. pombe.

  4. ATP/ADP Ratio, the Missed Connection between Mitochondria and the Warburg Effect

    PubMed Central

    Maldonado, Eduardo N.; Lemasters, John J.

    2014-01-01

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

  5. Studies on adenosine triphosphate transphosphorylases. XVIII. Synthesis and preparation of peptides and peptide fragments of rabbit muscle ATP-AMP transphosphorylase (adenylate kinase) and their nucleotide-binding properties.

    PubMed

    Kuby, S A; Hamada, M; Johnson, M S; Russell, G A; Manship, M; Palmieri, R H; Fleming, G; Bredt, D S; Mildvan, A S

    1989-08-01

    Two peptide fragments, derived from the head and tail of rabbit muscle myokinase, were found to possess remarkable and specific ligand-binding properties (Hamada et al., 1979). By initiating systematic syntheses and measurements of equilibrium substrate-binding properties of these two sets of peptides, or portions thereof, which encompass the binding sites for (a) the magnesium complexes of the nucleotide substrates (MgATP2- and MgADP-) and (b) the uncomplexed nucleotide substrates (ADP3- and AMP2-) of rabbit muscle myokinase, some of the requirements for binding of the substrates to ATP-AMP transphosphorylase are being deduced and chemically outlined. One requirement for tight nucleotide binding appears to be a minimum peptide length of 15-25 residues. In addition, Lys-172 and/or Lys-194 may be involved in the binding of epsilon AMP. The syntheses are described as a set of peptides corresponding to residues 31-45, 20-45, 5-45, and 1-45, and a set of peptides corresponding to residues 178-192, 178-194, and 172-194 of rabbit muscle adenylate kinase. The ligand-binding properties of the first set of synthetic peptides to the fluorescent ligands: epsilon MgATP/epsilon ATP and epsilon MgADP/epsilon ADP are quantitatively presented in terms of their intrinsic dissociation constants (K'd) and values of N (maximal number of moles bound per mole of peptide); and compared with the peptide fragment MT-I (1-44) obtained from rabbit muscle myokinase (Kuby et al., 1984) and with the native enzyme (Hamada et al., 1979). In addition, the values of N and K'd are given for the second set of synthetic peptides to the fluorescent ligands epsilon AMP and epsilon ADP as well as for the peptide fragments MT-XII(172-194) and CB-VI(126-194) (Kuby et al., 1984) and, in turn, compared with the native enzyme. A few miscellaneous dissociation constants which had been derived kinetically are also given for comparison (e.g., the Ki for epsilon AMP and the value of KMg epsilon ATP obtained for

  6. The nucleotide exchange factor MGE exerts a key function in the ATP-dependent cycle of mt-Hsp70-Tim44 interaction driving mitochondrial protein import.

    PubMed Central

    Schneider, H C; Westermann, B; Neupert, W; Brunner, M

    1996-01-01

    Import of preproteins into the mitochondrial matrix is driven by the ATP-dependent interaction of mt-Hsp70 with the peripheral inner membrane import protein Tim44 and the preprotein in transit. We show that Mge1p, a co-chaperone of mt-Hsp70, plays a key role in the ATP-dependent import reaction cycle in yeast. Our data suggest a cycle in which the mt-Hsp70-Tim44 complex forms with ATP: Mge1p promotes assembly of the complex in the presence of ATP. Hydrolysis of ATP by mt-Hsp70 occurs in complex with Tim44. Mge1p is then required for the dissociation of the ADP form of mt-Hsp70 from Tim44 after release of inorganic phosphate but before release of ADP. ATP hydrolysis and complex dissociation are accompanied by tight binding of mt-Hsp70 to the preprotein in transit. Subsequently, the release of mt-Hsp70 from the polypeptide chain is triggered by Mge1p which promotes release of ADP from mt-Hsp70. Rebinding of ATP to mt-Hsp70 completes the reaction cycle. Images PMID:8918457

  7. The nucleotide-binding domain of the Zn2+-transporting P-type ATPase from Escherichia coli carries a glycine motif that may be involved in binding of ATP.

    PubMed Central

    Okkeri, Juha; Laakkonen, Liisa; Haltia, Tuomas

    2004-01-01

    In P-type ATPases, the nucleotide-binding (N) domain is located in the middle of the sequence which folds into the phosphorylation (P) domain. The N domain of ZntA, a Zn2+-translocating P-type ATPase from Escherichia coli, is approx. 13% identical with the N domain of sarcoplasmic reticulum Ca2+-ATPase. None of the Ca2+-ATPase residues involved in binding of ATP are found in ZntA. However, the sequence G503SGIEAQV in the N domain of ZntA resembles the motif GxGxxG, which forms part of the ATP-binding site in protein kinases. This motif is also found in Wilson disease protein where several disease mutations cluster in it. In the present work, we have made a set of disease mutation analogues, including the mutants G503S (Gly503-->Ser), G505R and A508F of ZntA. At low [ATP], these mutant ATPases are poorly phosphorylated. The phosphorylation defect of the mutants G503S and G505R can, however, be partially (G503S) or fully (G505R) compensated for by using a higher [ATP], suggesting that these mutations lower the affinity for ATP. In all three mutant ATPases, phosphorylation by P(i) has become less sensitive to the presence of ATP, also consistent with the proposal that the Gly503 motif plays a role in ATP binding. In order to test this hypothesis, we have modelled the N domain of ZntA using the sarcoplasmic reticulum Ca2+-ATPase structure as a template. In the model, the Gly503 motif, as well as the residues Glu470 and His475, are located in the proximity of the ATP-binding site. In conclusion, the mutagenesis data and the molecular model are consistent with the idea that the two loops carrying the residues Glu470, His475, Gly503 and Gly505 play a role in ATP binding and activation. PMID:14510639

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

    PubMed

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

    2013-01-01

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

  9. 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.

  10. The use of nucleotide phosphorothioate diastereomers to define the structure of metal-nucleotide bound to GTP-AMP and ATP-AMP phosphotransferases from beef-heart mitochondria.

    PubMed

    Tomasselli, A G; Marquetant, R; Noda, L H; Goody, R S

    1984-07-16

    The diastereomers of adenosine 5'-O-[1-thio]triphosphate (ATP[alpha S]) and adenosine 5'-O-[2-thio]triphosphate (ATP[beta S]) were utilized to seek unambiguous assignment of Mg2+ coordination to ATP when bound to ATP-AMP phosphotransferase from beef heart mitochondria (AK2). Similarly, the diastereomers of guanosine 5'-O-[thio]triphosphate (GTP[alpha S]) and guanosine 5'-O-[2-thio]triphosphate (GTP[beta S]) were utilized to seek unambiguous assignment of Mg2+ coordination to GTP when bound to GTP-AMP phosphotransferase from beef heart mitochondria (AK3). Furthermore the diastereomers of guanosine 5'-O-[1-thio]diphosphate (GDP-[alpha S]) have been used to assign Mg2+ coordination to GDP when bound to AK3. The ratios (V for isomer Sp)/(V for isomer Rp) obtained in the presence of Mg2+ and Cd2+ are compared to those already published for ATP-AMP phosphotransferases from pig muscle (AK1) [Kalbitzer et al. (1983) Eur. J. Biochem. 133, 221-227] and from baker's yeast (AKy) [Tomasselli and Noda (1983) Eur. J. Biochem. 132, 109-115]. In all cases, coordination of Mg2+ to the beta-phosphate via the pro-R oxygen is present, as shown by reversal of specificity for the diastereomers of ATP [beta S] or GTP [beta S] respectively on changing the metal ion. In contrast, there is no reversal of specificity for the diastereomers of ATP [alpha S] or GTP[alpha S], or for GDP[alpha S] in the case of AK3 for the reverse reaction, indicating that there is no interaction of the metal with the alpha-phosphate group. The observed stereospecificity for the alpha-thiophosphate is consistent with the assumption of an interaction of the pro-R oxygen of the alpha-phosphate group with the enzyme.

  11. The complete nucleotide sequence of the cassava (Manihot esculenta) chloroplast genome and the evolution of atpF in Malpighiales: RNA editing and multiple losses of a group II intron

    PubMed Central

    Wurdack, Kenneth J.; Kanagaraj, Anderson; Lee, Seung-Bum; Saski, Christopher; Jansen, Robert K.

    2008-01-01

    The complete sequence of the chloroplast genome of cassava (Manihot esculenta, Euphorbiaceae) has been determined. The genome is 161,453 bp in length and includes a pair of inverted repeats (IR) of 26,954 bp. The genome includes 128 genes; 96 are single copy and 16 are duplicated in the IR. There are four rRNA genes and 30 distinct tRNAs, seven of which are duplicated in the IR. The infA gene is absent; expansion of IRb has duplicated 62 amino acids at the 3′ end of rps19 and a number of coding regions have large insertions or deletions, including insertions within the 23S rRNA gene. There are 17 intron-containing genes in cassava, 15 of which have a single intron while two (clpP, ycf3) have two introns. The usually conserved atpF group II intron is absent and this is the first report of its loss from land plant chloroplast genomes. The phylogenetic distribution of the atpF intron loss was determined by a PCR survey of 251 taxa representing 34 families of Malpighiales and 16 taxa from closely related rosids. The atpF intron is not only missing in cassava but also from closely related Euphorbiaceae and other Malpighiales, suggesting that there have been at least seven independent losses. In cassava and all other sequenced Malphigiales, atpF gene sequences showed a strong association between C-to-T substitutions at nucleotide position 92 and the loss of the intron, suggesting that recombination between an edited mRNA and the atpF gene may be a possible mechanism for the intron loss. PMID:18214421

  12. Analysis of a nucleotide-binding site of 5-lipoxygenase by affinity labelling: binding characteristics and amino acid sequences.

    PubMed Central

    Zhang, Y Y; Hammarberg, T; Radmark, O; Samuelsson, B; Ng, C F; Funk, C D; Loscalzo, J

    2000-01-01

    5-Lipoxygenase (5LO) catalyses the first two steps in the biosynthesis of leukotrienes, which are inflammatory mediators derived from arachidonic acid. 5LO activity is stimulated by ATP; however, a consensus ATP-binding site or nucleotide-binding site has not been found in its protein sequence. In the present study, affinity and photoaffinity labelling of 5LO with 5'-p-fluorosulphonylbenzoyladenosine (FSBA) and 2-azido-ATP showed that 5LO bound to the ATP analogues quantitatively and specifically and that the incorporation of either analogue inhibited ATP stimulation of 5LO activity. The stoichiometry of the labelling was 1.4 mol of FSBA/mol of 5LO (of which ATP competed with 1 mol/mol) or 0.94 mol of 2-azido-ATP/mol of 5LO (of which ATP competed with 0.77 mol/mol). Labelling with FSBA prevented further labelling with 2-azido-ATP, indicating that the same binding site was occupied by both analogues. Other nucleotides (ADP, AMP, GTP, CTP and UTP) also competed with 2-azido-ATP labelling, suggesting that the site was a general nucleotide-binding site rather than a strict ATP-binding site. Ca(2+), which also stimulates 5LO activity, had no effect on the labelling of the nucleotide-binding site. Digestion with trypsin and peptide sequencing showed that two fragments of 5LO were labelled by 2-azido-ATP. These fragments correspond to residues 73-83 (KYWLNDDWYLK, in single-letter amino acid code) and 193-209 (FMHMFQSSWNDFADFEK) in the 5LO sequence. Trp-75 and Trp-201 in these peptides were modified by the labelling, suggesting that they were immediately adjacent to the C-2 position of the adenine ring of ATP. Given the stoichiometry of the labelling, the two peptide sequences of 5LO were probably near each other in the enzyme's tertiary structure, composing or surrounding the ATP-binding site of 5LO. PMID:11042125

  13. Release of ATP during host cell killing by enteropathogenic E. coli and its role as a secretory mediator.

    PubMed

    Crane, John K; Olson, Ruth A; Jones, Heather M; Duffey, Michael E

    2002-07-01

    Enteropathogenic Escherichia coli (EPEC) causes severe, watery diarrhea in children. We investigated ATP release during EPEC-mediated killing of human cell lines and whether released adenine nucleotides function as secretory mediators. EPEC triggered a release of ATP from all human cell lines tested: HeLa, COS-7, and T84 (colon cells) as measured using a luciferase kit. Accumulation of ATP in the supernatant medium was enhanced if an inhibitor of 5'-ectonucleotidase was included and was further enhanced if an ATP-regenerating system was added. In the presence of the inhibitor/regenerator, ATP concentrations in the supernatant medium reached 1.5-2 microM 4 h after infection with wild-type EPEC strains. In the absence of the inhibitor/regenerator system, extracellular ATP was rapidly broken down to ADP, AMP, and adenosine. Conditioned medium from EPEC-infected cells triggered a brisk chloride secretory response in intestinal tissues studied in the Ussing chamber (rabbit distal colon and T84 cell monolayers), whereas conditioned medium from uninfected cells and sterile filtrates of EPEC bacteria did not. The short-circuit current response to EPEC-conditioned medium was completely reversed by adenosine receptor blockers, such as 8-(p-sulfophenyl)-theophylline and MRS1754. EPEC killing of host cells releases ATP, which is broken down to adenosine, which in turn stimulates secretion via apical adenosine A2b receptors. These findings provide new insight into how EPEC causes watery diarrhea. PMID:12065294

  14. In vivo study of the effect of antiviral acyclic nucleotide phosphonate (R)-9-[2-(phosphonomethoxy)propyl]adenine (PMPA, tenofovir) and its prodrug tenofovir disoproxil fumarate on rat microsomal cytochrome P450.

    PubMed

    Anzenbacherová, E; Anzenbacher, P; Zídek, Z; Buchar, E; Kmonícková, E; Potmesil, P; Nekvindová, J; Veinlichová, A; Holý, A

    2008-01-01

    The total content of rat liver microsomal cytochrome P450 (CYP) significantly decreased after repeated i.p. administration of the antiviral agent tenofovir ((R)-9-[2-(phosphonomethoxy)propyl] adenine) and tenofovir disoproxil at a daily dose 25 mg/kg, although the content of liver microsomal protein did not change. The decrease of the CYP content was accompanied by concomitant increase of the amount of inactive CYP form, cytochrome P420. This effect was confirmed by a parallel study of the activities of selected CYP forms, CYP2E1 (p-nitrophenol hydroxylation) and CYP1A2 (7-ethoxyresorufin deethylation). The activity (expressed relatively to the protein content) of both CYP forms decreased significantly following the decrease of the total CYP. On the other hand, the CYP2E1 activity expressed relatively to the decreasing total CYP content remained unchanged. However, CYP1A2 activity also decreased when calculated relatively to the total native CYP content indicating lower stability of this form. Semiquantitative RT-PCR showed no significant changes in expression of major rat liver microsomal CYP forms after tenofovir treatment. In conclusion, repeated administration of tenofovir in higher doses led to significant decrease of the relative proportion of active liver microsomal CYPs accompanied by a conversion of these enzymes to the inactive form (CYP420) maintaining the sum of CYP proteins unchanged.

  15. Adenine nucleotide binding sites on beef heart F/sub 1/ ATPase: photoaffinity labeling of. beta. -subunit Tyr-368 at a noncatalytic site and. beta. Tyr-345 at a catalytic site

    SciTech Connect

    Cross, R.L.; Cunningham, D.; Miller, C.G.; Xue, Z.; Zhou, J.M.; Boyer, P.D.

    1987-08-01

    2-Azidoadenine (/sup 32/P)nucleotide was bound specifically at catalytic or noncatalytic nucleotide binding sites on beef heart mitochondrial F/sub 1/ ATPase. In both cases, photolysis resulted in nearly exclusive labeling of the ..beta.. subunit. The modified enzyme was digested with trypsin, and labeled peptides were purified by reversed-phase high-pressure liquid chromatography. Amino acid sequence analysis of the major /sup 32/P-labeled tryptic fragments showed ..beta..-subunit Tyr-368 to be present at noncatalytic sites and ..beta.. Tyr-345 to be present at catalytic sites. From the relationship between the degree of inhibition and extent of modification, it is estimated that one-third of the catalytic sites or two-thirds of the noncatalytic sites must be modified to give near-complete inhibition of catalytic activity.

  16. E-NTPDase 3 (ATP diphosphohydrolase) from cardiomyocytes, activity and expression are modulated by thyroid hormone.

    PubMed

    Barreto-Chaves, Maria Luiza M; Carneiro-Ramos, Marcela Sorelli; Cotomacci, Guilherme; Júnior, Marconi Barbosa Coutinho; Sarkis, João José Freitas

    2006-06-01

    Degradation of adenine nucleotides by myocardial cells occurs, in part, by a cascade of surface-located enzymes converting ATP into adenosine that has important implications for the regulation of the nucleotide/nucleoside ratio modulating the cardiac functions. Thyroid hormones have profound effects on cardiovascular system, as observed in hypo- and hyperthyroidism. Combined biochemical parameters and gene expression analysis approaches were used to investigate the influence of tri-iodothyronine (T3) on ATP and ADP hydrolysis by isolated myocytes. Cultures of cardiomyocytes were submitted to increasing doses of T3 for 24h. Enzymatic activity and expression were evaluated. T3 (0.1 nM) caused an increase in ATP and ADP hydrolysis. Experiments with specific inhibitors suggest the involvement of an NTPDase, which was confirmed by an increase in NTPDase 3 messenger RNA (mRNA) levels. Since T3 promotes an increase in the contractile protein, leading to cardiac hypertrophy, it is tempting to postulate that the increase in ATP hydrolysis and the decrease in the extracellular levels signify an important factor for prevention of excessive contractility. PMID:16584835

  17. Endotoxemia alters nucleotide hydrolysis in platelets of rats.

    PubMed

    Vuaden, Fernanda Cenci; Furstenau, Cristina Ribas; Savio, Luiz Eduardo Baggio; Sarkis, João José Freitas; Bonan, Carla Denise

    2009-03-01

    Platelets play a critical role in homeostasis and blood clotting at sites of vascular injury, and also in various ways in innate immunity and inflammation. Platelets are one of the first cells to accumulate at an injured site, and local release of their secretome at some point initiate an inflammatory cascade that attracts leukocytes, activates target cells, stimulates vessel growth and repair. The level of exogenous ATP in the body may be increased in various inflammatory and shock conditions, primarily as a consequence of nucleotide release from platelets, endothelium and blood vessel cells. An increase of ATP release has been described during inflammation and this compound presents proinflammatory properties. ADP is a nucleotide known to induce changes in platelets shape and aggregation, to promote the exposure of fibrinogen-binding sites and to inhibit the stimulation of adenylate cyclase. Adenosine, the final product of the nucleotide hydrolysis, is a vasodilator and an inhibitor of platelet aggregation. There is a group of ecto-enzymes responsible for extracellular nucleotide hydrolysis named ectonucleotidases, which includes the NTPDase (nucleoside triphosphate diphosphohydrolase) family, the NPP (nucleoside pyrophosphatase/phosphodiesterase) family and an ecto-5'-nucleotidase. Therefore, we have aimed to investigate the effect of lipopolysaccharide endotoxin from Escherichia coli on ectonucleotidases in platelets from adult rats in order to better understand the role of extracellular adenine nucleotides and nucleosides in the maintenance of blood homeostasis in inflammatory processes. LPS administered in vitro was not able to alter the ATP, ADP, AMP and rho-Nph-5'-TMP hydrolysis of platelets from untreated rats in all concentrations tested (25-100 microg/ml). There was a significant decrease in ATP, ADP, AMP and rho-Nph-5'-TMP hydrolysis in rat platelets after 48 hours of LPS exposure (2 mg/Kg, i.p.). ATP and ADP hydrolysis has been reduced about 28

  18. Possible prebiotic catalysts formed from adenine and aldehyde

    NASA Astrophysics Data System (ADS)

    Vergne, J.; Dumas, L.; Décout, J.-L.; Maurel, M.-C.

    2000-09-01

    Careful examination of the present metabolism and in vitro selection of various catalytic RNAs strongly support the "RNA World" hypothesis of the origin of life. However, in this scenario, the difficult prebiotic synthesis of ribose and consequently of nucleotides remain a major problem. In order to overcome this problem and obtain nucleoside analogs, we are investigating reactions of the nucleic acid base, adenine 1, with different aldehydes under presumably prebiotic conditions. In the reaction of adenine and pyruvaldehyde 2 in water, we report here the formation in high yield of two isomeric products. These compounds possessing alcohols functions as nucleosides result from condensation of two molecules of pyruvaldehyde on the 6-amino group of one adenine molecule. Their catalytic activities in the model hydrolysis of p-nitrophenylesters appeared interesting in the search of prebiotic catalysts.

  19. Vertical Ionization Energies of Adenine and 9-Methyl Adenine

    NASA Astrophysics Data System (ADS)

    Dolgounitcheva, O.; Zakrzewski, V. G.; Ortiz, J. V.

    2009-07-01

    Vertical ionization energies of 9-H adenine and 9-methyl adenine have been calculated with the following, ab initio, electron propagator methods: the outer valence Green's function (OVGF), partial third-order theory (P3), and the third-order algebraic diagrammatic construction, or ADC(3). Basis set effects have been systematically examined. All methods predict near degeneracy in the π2-n1 and π3-n2 pairs of cationic, adenine final states and larger splittings of the corresponding, cationic states of 9-methyl adenine. P3 results for adenine predict the following order of the first six final states: π1, n1, π2, n2, π3, n3. Coupled-cluster calculations on the first three cationic states of adenine confirm these predictions. OVGF and ADC(3) calculations reverse the order of the second and third states and of the fourth and fifth states. All results confirm previous interpretations of experiments in which the second and third spectral bands correspond to the aforementioned pairs of final states and disagree with a recent reassignment based on time-resolved photoelectron spectra. Lower ionization energies and larger splittings in the methylated molecule are interpreted in terms of phase relationships in the Dyson orbitals. ADC(3) results confirm the qualitative validity of the one-electron approximation for the first six final states of both molecules and disclose its inadequacies for higher ionization energies.

  20. Adenine Phosphoribosyltransferase in Plant Tissues: Some Effects of Kinetin on Enzymic Activity 1

    PubMed Central

    Nicholls, P. B.; Murray, A. W.

    1968-01-01

    Adenine phosphoribosyltransferase activity was measured in extracts of soybean (Glycine max var. Acme) callus and of senescing barley leaves (Hordeum distichon c.v. Prior). The enzyme from soybean callus had Michaelis constants for adenine and 5-phosphoribosyl pyrophosphate of 1.5 and 7.5 μm respectively and was inhibited by AMP and stimulated by ATP. The presence of kinetin was found to considerably increase the activity of adenine phosphoribosyltransferase in extracts of soybean callus and senescing barley leaves. PMID:16656820

  1. ATP synthase superassemblies in animals and plants: two or more are better.

    PubMed

    Seelert, Holger; Dencher, Norbert A

    2011-09-01

    ATP synthases are part of the sophisticated cellular metabolic network and therefore multiple interactions have to be considered. As discussed in this review, ATP synthases form various supramolecular structures. These include dimers and homooligomeric species. But also interactions with other proteins, particularly those involved in energy conversion exist. The supramolecular assembly of the ATP synthase affects metabolism, organellar structure, diseases, ageing and vice versa. The most common approaches to isolate supercomplexes from native membranes by use of native electrophoresis or density gradients are introduced. On the one hand, isolated ATP synthase dimers and oligomers are employed for structural studies and elucidation of specific protein-protein interactions. On the other hand, native electrophoresis and other techniques serve as tool to trace changes of the supramolecular organisation depending on metabolic alterations. Upon analysing the structure, dimer-specific subunits can be identified as well as interactions with other proteins, for example, the adenine nucleotide translocator. In the organellar context, ATP synthase dimers and oligomers are involved in the formation of mitochondrial cristae. As a consequence, changes in the amount of such supercomplexes affect mitochondrial structure and function. Alterations in the cellular power plant have a strong impact on energy metabolism and ultimately play a significant role in pathophysiology. In plant systems, dimers of the ATP synthase have been also identified in chloroplasts. Similar to mammals, a correlation between metabolic changes and the amount of the chloroplast ATP synthase dimers exists. Therefore, this review focusses on the interplay between metabolism and supramolecular organisation of ATP synthase in different organisms.

  2. Kif2C Minimal Functional Domain Has Unusual Nucleotide Binding Properties That Are Adapted to Microtubule Depolymerization*

    PubMed Central

    Wang, Weiyi; Jiang, Qiyang; Argentini, Manuela; Cornu, David; Gigant, Benoît; Knossow, Marcel; Wang, Chunguang

    2012-01-01

    The kinesin-13 Kif2C hydrolyzes ATP and uses the energy released to disassemble microtubules. The mechanism by which this is achieved remains elusive. Here we show that Kif2C-(sN+M), a monomeric construct consisting of the motor domain with the proximal part of the N-terminal Neck extension but devoid of its more distal, unstructured, and highly basic part, has a robust depolymerase activity. When detached from microtubules, the Kif2C-(sN+M) nucleotide-binding site is occupied by ATP at physiological concentrations of adenine nucleotides. As a consequence, Kif2C-(sN+M) starts its interaction with microtubules in that state, which differentiates kinesin-13s from motile kinesins. Moreover, in this ATP-bound conformational state, Kif2C-(sN+M) has a higher affinity for soluble tubulin compared with microtubules. We propose a mechanism in which, in the first step, the specificity of ATP-bound Kif2C for soluble tubulin causes it to stabilize a curved conformation of tubulin heterodimers at the ends of microtubules. Data from an ATPase-deficient Kif2C mutant suggest that, then, ATP hydrolysis precedes and is required for tubulin release to take place. Finally, comparison with Kif2C-Motor indicates that the binding specificity for curved tubulin and, accordingly, the microtubule depolymerase activity are conferred to the motor domain by its N-terminal Neck extension. PMID:22403406

  3. Insights into the Influence of Nucleotides on Actin Family Proteins from Seven Structures of Arp2/3 Complex

    SciTech Connect

    Nolen,B.; Pollard, T.

    2007-01-01

    ATP is required for nucleation of actin filament branches by Arp2/3 complex, but the influence of ATP binding and hydrolysis are poorly understood. We determined crystal structures of bovine Arp2/3 complex cocrystalized with various bound adenine nucleotides and cations. Nucleotide binding favors closure of the nucleotide binding cleft of Arp3, but no large scale conformational changes in the complex. Thus, ATP binding does not directly activate Arp2/3 complex, but is part of a network of interactions that contribute to nucleation. We compared nucleotide-induced conformational changes of residues lining the cleft in Arp3 and actin structures to construct a movie depicting the proposed ATPase cycle for the actin family. Chemical crosslinking stabilized subdomain 1 of Arp2, revealing new electron density for 69 residues in this subdomain. Steric clashes with Arp3 appear to be responsible for intrinsic disorder of subdomains 1 and 2 of Arp2 in inactive Arp2/3 complex.

  4. A Nucleotide-Analogue-Induced Gain of Function Corrects the Error-Prone Nature of Human DNA Polymerase iota

    SciTech Connect

    Ketkar, Amit; Zafar, Maroof K.; Banerjee, Surajit; Marquez, Victor E.; Egli, Martin; Eoff, Robert L.

    2012-10-25

    Y-family DNA polymerases participate in replication stress and DNA damage tolerance mechanisms. The properties that allow these enzymes to copy past bulky adducts or distorted template DNA can result in a greater propensity for them to make mistakes. Of the four human Y-family members, human DNA polymerase iota (hpol{iota}) is the most error-prone. In the current study, we elucidate the molecular basis for improving the fidelity of hpol{iota} through use of the fixed-conformation nucleotide North-methanocarba-2{prime}-deoxyadenosine triphosphate (N-MC-dATP). Three crystal structures were solved of hpol{iota} in complex with DNA containing a template 2{prime}-deoxythymidine (dT) paired with an incoming dNTP or modified nucleotide triphosphate. The ternary complex of hpol{iota} inserting N-MC-dATP opposite dT reveals that the adenine ring is stabilized in the anti orientation about the pseudo-glycosyl torsion angle, which mimics precisely the mutagenic arrangement of dGTP:dT normally preferred by hpol{iota}. The stabilized anti conformation occurs without notable contacts from the protein but likely results from constraints imposed by the bicyclo[3.1.0]hexane scaffold of the modified nucleotide. Unmodified dATP and South-MC-dATP each adopt syn glycosyl orientations to form Hoogsteen base pairs with dT. The Hoogsteen orientation exhibits weaker base-stacking interactions and is less catalytically favorable than anti N-MC-dATP. Thus, N-MC-dATP corrects the error-prone nature of hpol{iota} by preventing the Hoogsteen base-pairing mode normally observed for hpol{iota}-catalyzed insertion of dATP opposite dT. These results provide a previously unrecognized means of altering the efficiency and the fidelity of a human translesion DNA polymerase.

  5. Protein Modification by Adenine Propenal

    PubMed Central

    2015-01-01

    Base propenals are products of the reaction of DNA with oxidants such as peroxynitrite and bleomycin. The most reactive base propenal, adenine propenal, is mutagenic in Escherichia coli and reacts with DNA to form covalent adducts; however, the reaction of adenine propenal with protein has not yet been investigated. A survey of the reaction of adenine propenal with amino acids revealed that lysine and cysteine form adducts, whereas histidine and arginine do not. Nε-Oxopropenyllysine, a lysine–lysine cross-link, and S-oxopropenyl cysteine are the major products. Comprehensive profiling of the reaction of adenine propenal with human serum albumin and the DNA repair protein, XPA, revealed that the only stable adduct is Nε-oxopropenyllysine. The most reactive sites for modification in human albumin are K190 and K351. Three sites of modification of XPA are in the DNA-binding domain, and two sites are subject to regulatory acetylation. Modification by adenine propenal dramatically reduces XPA’s ability to bind to a DNA substrate. PMID:25211669

  6. The two ATP binding sites of cystic fibrosis transmembrane conductance regulator (CFTR) play distinct roles in gating kinetics and energetics.

    PubMed

    Zhou, Zhen; Wang, Xiaohui; Liu, Hao-Yang; Zou, Xiaoqin; Li, Min; Hwang, Tzyh-Chang

    2006-10-01

    Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC (ATP binding cassette) transporter family, is a chloride channel whose activity is controlled by protein kinase-dependent phosphorylation. Opening and closing (gating) of the phosphorylated CFTR is coupled to ATP binding and hydrolysis at CFTR's two nucleotide binding domains (NBD1 and NBD2). Recent studies present evidence that the open channel conformation reflects a head-to-tail dimerization of CFTR's two NBDs as seen in the NBDs of other ABC transporters (Vergani et al., 2005). Whether these two ATP binding sites play an equivalent role in the dynamics of NBD dimerization, and thus in gating CFTR channels, remains unsettled. Based on the crystal structures of NBDs, sequence alignment, and homology modeling, we have identified two critical aromatic amino acids (W401 in NBD1 and Y1219 in NBD2) that coordinate the adenine ring of the bound ATP. Conversion of the W401 residue to glycine (W401G) has little effect on the sensitivity of the opening rate to [ATP], but the same mutation at the Y1219 residue dramatically lowers the apparent affinity for ATP by >50-fold, suggesting distinct roles of these two ATP binding sites in channel opening. The W401G mutation, however, shortens the open time constant. Energetic analysis of our data suggests that the free energy of ATP binding at NBD1, but not at NBD2, contributes significantly to the energetics of the open state. This kinetic and energetic asymmetry of CFTR's two NBDs suggests an asymmetric motion of the NBDs during channel gating. Opening of the channel is initiated by ATP binding at the NBD2 site, whereas separation of the NBD dimer at the NBD1 site constitutes the rate-limiting step in channel closing.

  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 α-helix of unknown function. The mechanism by which calcium binding to the regulatory domain modulates substrate transport in the carrier domain has not been resolved. Here, we present two new crystal structures of the regulatory domain of the human isoform 1. Careful analysis by SEC confirmed that although the regulatory domain crystallised as dimers, full-length ATP-Mg/Pi carrier is monomeric. Therefore, the ATP-Mg/Pi carrier must have a different mechanism of calcium regulation than the architecturally related aspartate/glutamate carrier, which is dimeric. The structure showed that an amphipathic α-helix is bound to the regulatory domain in a hydrophobic cleft of EF-hand 3/4. Detailed bioinformatics analyses of different EF-hand states indicate that upon release of calcium, EF-hands close, meaning that the regulatory domain would release the amphipathic α-helix. We propose a mechanism for ATP-Mg/Pi carriers in which the amphipathic α-helix becomes mobile upon release of calcium and could block the transport of substrates across the mitochondrial inner membrane. PMID:26164100

  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 α-helix of unknown function. The mechanism by which calcium binding to the regulatory domain modulates substrate transport in the carrier domain has not been resolved. Here, we present two new crystal structures of the regulatory domain of the human isoform 1. Careful analysis by SEC confirmed that although the regulatory domain crystallised as dimers, full-length ATP-Mg/Pi carrier is monomeric. Therefore, the ATP-Mg/Pi carrier must have a different mechanism of calcium regulation than the architecturally related aspartate/glutamate carrier, which is dimeric. The structure showed that an amphipathic α-helix is bound to the regulatory domain in a hydrophobic cleft of EF-hand 3/4. Detailed bioinformatics analyses of different EF-hand states indicate that upon release of calcium, EF-hands close, meaning that the regulatory domain would release the amphipathic α-helix. We propose a mechanism for ATP-Mg/Pi carriers in which the amphipathic α-helix becomes mobile upon release of calcium and could block the transport of substrates across the mitochondrial inner membrane. PMID:26164100

  9. Extracellular ATP

    PubMed Central

    Chivasa, Stephen; Tomé, Daniel FA; Murphy, Alex M; Hamilton, John M; Lindsey, Keith; Carr, John P

    2009-01-01

    Living organisms acquire or synthesize high energy molecules, which they frugally conserve and use to meet their cellular metabolic demands. Therefore, it is surprising that ATP, the most accessible and commonly utilized chemical energy carrier, is actively secreted to the extracellular matrix of cells. It is now becoming clear that in plants this extracellular ATP (eATP) is not wasted, but harnessed at the cell surface to signal across the plasma membrane of the secreting cell and neighboring cells to cxontrol gene expression and influence plant development. Identification of the gene/protein networks regulated by eATP-mediated signaling should provide insight into the physiological roles of eATP in plants. By disrupting eATP-mediated signaling, we have identified pathogen defense genes as part of the eATP-regulated gene circuitry, leading us to the discovery that eATP is a negative regulator of pathogen defense in plants.1 Previously, we reported that eATP is a key signal molecule that modulates programmed cell death in plants.2 A complex picture is now emerging, in which eATP-mediated signaling cross-talks with signaling mediated by the major plant defense hormone, salicylic acid, in the regulation of pathogen defense and cell death. PMID:20009563

  10. Crystallographic structure of phosphofructokinase-2 from Escherichia coli in complex with two ATP molecules. Implications for substrate inhibition.

    PubMed

    Cabrera, Ricardo; Ambrosio, Andre L B; Garratt, Richard C; Guixé, Victoria; Babul, Jorge

    2008-11-14

    Phosphofructokinase-1 and -2 (Pfk-1 and Pfk-2, respectively) from Escherichia coli belong to different homologous superfamilies. However, in spite of the lack of a common ancestor, they share the ability to catalyze the same reaction and are inhibited by the substrate MgATP. Pfk-2, an ATP-dependent 6-phosphofructokinase member of the ribokinase-like superfamily, is a homodimer of 66 kDa subunits whose oligomerization state is necessary for catalysis and stability. The presence of MgATP favors the tetrameric form of the enzyme. In this work, we describe the structure of Pfk-2 in its inhibited tetrameric form, with each subunit bound to two ATP molecules and two Mg ions. The present structure indicates that substrate inhibition occurs due to the sequential binding of two MgATP molecules per subunit, the first at the usual site occupied by the nucleotide in homologous enzymes and the second at the allosteric site, making a number of direct and Mg-mediated interactions with the first. Two configurations are observed for the second MgATP, one of which involves interactions with Tyr23 from the adjacent subunit in the dimer and the other making an unusual non-Watson-Crick base pairing with the adenine in the substrate ATP. The oligomeric state observed in the crystal is tetrameric, and some of the structural elements involved in the binding of the substrate and allosteric ATPs are also participating in the dimer-dimer interface. This structure also provides the grounds to compare analogous features of the nonhomologous phosphofructokinases from E. coli.

  11. Structural basis of AMPK regulation by adenine nucleotides and glycogen

    SciTech Connect

    Li, Xiaodan; Wang, Lili; Zhou, X. Edward; Ke, Jiyuan; de Waal, Parker W.; Gu, Xin; Tan, M. H. Eileen; Wang, Dongye; Wu, Donghai; Xu, H. Eric; Melcher, Karsten

    2014-11-21

    AMP-activated protein kinase (AMPK) is a central cellular energy sensor and regulator of energy homeostasis, and a promising drug target for the treatment of diabetes, obesity, and cancer. Here we present low-resolution crystal structures of the human α1β2γ1 holo-AMPK complex bound to its allosteric modulators AMP and the glycogen-mimic cyclodextrin, both in the phosphorylated (4.05 Å) and non-phosphorylated (4.60 Å) state. In addition, we have solved a 2.95 Å structure of the human kinase domain (KD) bound to the adjacent autoinhibitory domain (AID) and have performed extensive biochemical and mutational studies. Altogether, these studies illustrate an underlying mechanism of allosteric AMPK modulation by AMP and glycogen, whose binding changes the equilibria between alternate AID (AMP) and carbohydrate-binding module (glycogen) interactions.

  12. Structural basis of AMPK regulation by adenine nucleotides and glycogen

    DOE PAGES

    Li, Xiaodan; Wang, Lili; Zhou, X. Edward; Ke, Jiyuan; de Waal, Parker W.; Gu, Xin; Tan, M. H. Eileen; Wang, Dongye; Wu, Donghai; Xu, H. Eric; et al

    2014-11-21

    AMP-activated protein kinase (AMPK) is a central cellular energy sensor and regulator of energy homeostasis, and a promising drug target for the treatment of diabetes, obesity, and cancer. Here we present low-resolution crystal structures of the human α1β2γ1 holo-AMPK complex bound to its allosteric modulators AMP and the glycogen-mimic cyclodextrin, both in the phosphorylated (4.05 Å) and non-phosphorylated (4.60 Å) state. In addition, we have solved a 2.95 Å structure of the human kinase domain (KD) bound to the adjacent autoinhibitory domain (AID) and have performed extensive biochemical and mutational studies. Altogether, these studies illustrate an underlying mechanism of allostericmore » AMPK modulation by AMP and glycogen, whose binding changes the equilibria between alternate AID (AMP) and carbohydrate-binding module (glycogen) interactions.« less

  13. Factors influencing the rate of non-enzymatic activation of carboxylic and amino acids by ATP

    NASA Technical Reports Server (NTRS)

    Mullins, D. W., Jr.; Lacey, J. C., Jr.

    1981-01-01

    The nonenzymatic formation of adenylate anhydrides of carboxylic and amino acids is discussed as a necessary step in the origin of the genetic code and protein biosynthesis. Results of studies are presented which have shown the rate of activation to depend on the pKa of the carboxyl group, the pH of the medium, temperature, the divalent metal ion catalyst, salt concentration, and the nature of the amino acid. In particular, it was found that of the various amino acids investigated, phenylalanine had the greatest affinity for the adenine derivatives adenosine and ATP. Results thus indicate that selective affinities between amino acids and nucleotides were important during prebiotic chemical evolution, and may have played a major role in the origin of protein synthesis and genetic coding.

  14. Different effects of guanine nucleotides (GDP and GTP) on protein-mediated mitochondrial proton leak.

    PubMed

    Woyda-Ploszczyca, Andrzej M; Jarmuszkiewicz, Wieslawa

    2014-01-01

    In this study, we compared the influence of GDP and GTP on isolated mitochondria respiring under conditions favoring oxidative phosphorylation (OXPHOS) and under conditions excluding this process, i.e., in the presence of carboxyatractyloside, an adenine nucleotide translocase inhibitor, and/or oligomycin, an FOF1-ATP synthase inhibitor. Using mitochondria isolated from rat kidney and human endothelial cells, we found that the action of GDP and GTP can differ diametrically depending on the conditions. Namely, under conditions favoring OXPHOS, both in the absence and presence of linoleic acid, an activator of uncoupling proteins (UCPs), the addition of 1 mM GDP resulted in the state 4 (non-phosphorylating respiration)-state 3 (phosphorylating respiration) transition, which is characteristic of ADP oxidative phosphorylation. In contrast, the addition of 1 mM GTP resulted in a decrease in the respiratory rate and an increase in the membrane potential, which is characteristic of UCP inhibition. The stimulatory effect of GDP, but not GTP, was also observed in inside-out submitochondrial particles prepared from rat kidney mitochondria. However, the effects of GDP and GTP were more similar in the presence of OXPHOS inhibitors. The importance of these observations in connection with the action of UCPs, adenine nucleotide translocase (or other carboxyatractyloside-sensitive carriers), carboxyatractyloside- and purine nucleotide-insensitive carriers, as well as nucleoside-diphosphate kinase (NDPK) are considered. Because the measurements favoring oxidative phosphorylation better reflect in vivo conditions, our study strongly supports the idea that GDP cannot be considered a significant physiological inhibitor of UCP. Moreover, it appears that, under native conditions, GTP functions as a more efficient UCP inhibitor than GDP and ATP.

  15. Binding of ATP to the progesterone receptor.

    PubMed Central

    Moudgil, V K; Toft, D O

    1975-01-01

    The possible interaction of progesterone--receptor complexes with nucleotides was tested by affinity chromatography. The cytosol progesterone receptor from hen oviduct was partially purified by ammonium sulfate precipitation before use. When progesterone was bound to the receptor, the resulting complex could be selectively adsorbed onto columns of ATP-Sepharose. This interaction was reversible and of an ionic nature since it could be disrupted by high-salt conditions. A competitive binding assay was used to test the specificity of receptor binding to several other nucleotides, including ADP, AMP, and cAMP. A clear specificity for binding ATP was evident from these studies. When ATP was added to receptor preparations, the nucleotide did not affect the sedimentation properties or hormone binding characteristics of the receptor. Although the function of ATP remains unknown, these studies indicate a role of this nucleotide in some aspect of hormone receptor activity. PMID:165493

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

    PubMed Central

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

    2013-01-01

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

  17. The structural basis of ATP as an allosteric modulator.

    PubMed

    Lu, Shaoyong; Huang, Wenkang; Wang, Qi; Shen, Qiancheng; Li, Shuai; Nussinov, Ruth; Zhang, Jian

    2014-09-01

    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

  18. 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

  19. Comparative study of spontaneous deamination of adenine and cytosine in unbuffered aqueous solution at room temperature

    NASA Astrophysics Data System (ADS)

    Wang, Shiliang; Hu, Anguang

    2016-06-01

    Adenine in unbuffered nanopure water at a concentration of 2 mM is completely deaminated (>99%) to hypoxanthine at room temperature in ca. 10 weeks, with an estimated half-life (t1/2) less than 10 days, about six orders of magnitude faster than previously reported. Cytosine is not deaminated under the same condition, even after 3 years. This is in contrast to previous observations that cytosine deaminates 20-40 times faster than adenine free base, in nucleoside, in nucleotide and in single-stranded DNA in buffered neutral aqueous solutions.

  20. Intramolecular interactions in aminoacyl nucleotides: Implications regarding the origin of genetic coding and protein synthesis

    NASA Technical Reports Server (NTRS)

    Lacey, J. C., Jr.; Mullins, D. W., Jr.; Watkins, C. L.; Hall, L. M.

    1986-01-01

    Cellular organisms store information as sequences of nucleotides in double stranded DNA. This information is useless unless it can be converted into the active molecular species, protein. This is done in contemporary creatures first by transcription of one strand to give a complementary strand of mRNA. The sequence of nucleotides is then translated into a specific sequence of amino acids in a protein. Translation is made possible by a genetic coding system in which a sequence of three nucleotides codes for a specific amino acid. The origin and evolution of any chemical system can be understood through elucidation of the properties of the chemical entities which make up the system. There is an underlying logic to the coding system revealed by a correlation of the hydrophobicities of amino acids and their anticodonic nucleotides (i.e., the complement of the codon). Its importance lies in the fact that every amino acid going into protein synthesis must first be activated. This is universally accomplished with ATP. Past studies have concentrated on the chemistry of the adenylates, but more recently we have found, through the use of NMR, that we can observe intramolecular interactions even at low concentrations, between amino acid side chains and nucleotide base rings in these adenylates. The use of this type of compound thus affords a novel way of elucidating the manner in which amino acids and nucleotides interact with each other. In aqueous solution, when a hydrophobic amino acid is attached to the most hydrophobic nucleotide, AMP, a hydrophobic interaction takes place between the amino acid side chain and the adenine ring. The studies to be reported concern these hydrophobic interactions.

  1. Impaired mitochondrial Ca{sup 2+} homeostasis in respiratory chain-deficient cells but efficient compensation of energetic disadvantage by enhanced anaerobic glycolysis due to low ATP steady state levels

    SciTech Connect

    Kleist-Retzow, Juergen-Christoph von ||. E-mail: juergen-christoph.vonkleist@uk-koeln.de; Hue-Tran Hornig-Do; Schauen, Matthias; Eckertz, Sabrina; Tuan Anh Duong Dinh; Stassen, Frank; Lottmann, Nadine; Bust, Maria; Galunska, Bistra; Wielckens, Klaus; Hein, Wolfgang; Beuth, Joseph; Braun, Jan-Matthias; Fischer, Juergen H.; Ganitkevich, Vladimir Y. |; Maniura-Weber, Katharina; Wiesner, Rudolf J. |

    2007-08-15

    Energy-producing pathways, adenine nucleotide levels, oxidative stress response and Ca{sup 2+} homeostasis were investigated in cybrid cells incorporating two pathogenic mitochondrial DNA point mutations, 3243A > G and 3302A > G in tRNA{sup Leu(UUR)}, as well as Rho{sup 0} cells and compared to their parental 143B osteosarcoma cell line. All cells suffering from a severe respiratory chain deficiency were able to proliferate as fast as controls. The major defect in oxidative phosphorylation was efficiently compensated by a rise in anaerobic glycolysis, so that the total ATP production rate was preserved. This enhancement of glycolysis was enabled by a considerable decrease of cellular total adenine nucleotide pools and a concomitant shift in the AMP + ADP/ATP ratios, while the energy charge potential was still in the normal range. Further important consequences were an increased production of superoxide which, however, was neither escorted by major changes in the antioxidative defence systems nor was it leading to substantial oxidative damage. Most interestingly, the lowered mitochondrial membrane potential led to a disturbed intramitochondrial calcium homeostasis, which most likely is a major pathomechanism in mitochondrial diseases.

  2. 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,…

  3. Bound anionic states of adenine

    SciTech Connect

    Haranczyk, Maciej; Gutowski, Maciej S; Li, Xiang; Bowen, Kit H

    2007-03-20

    Anionic states of nucleic acid bases are involved in DNA damage by low-energy electrons and in charge transfer through DNA. Previous gas phase studies of free, unsolvated nucleic acid base parent anions probed only dipole-bound states, which are not present in condensed phase environments, but did not observe valence anionic states, which for purine bases, are thought to be adiabatically unbound. Contrary to this expectation, we have demonstrated that some thus far ignored tautomers of adenine, which result from enamine-imine transformations, support valence anionic states with electron vertical detachment energies as large as 2.2 eV, and at least one of these anionic tautomers is adiabatically bound. Moreover, we predict that the new anionic tautomers should also dominate in solutions and should be characterized by larger values of electron vertical detachment energy than the canonical valence anion. All of the new-found anionic tautomers might be formed in the course of dissociative electron attachment followed by a hydrogen atom attachment to a carbon atom, and they might affect the structure and properties of DNA and RNA exposed to low-energy electrons. The discovery of these valence anionic states of adenine was facilitated by the development of: (i) a new experimental method for preparing parent anions of nucleic acid bases for photoelectron experiments, and (ii) a new combinatorial/ quantum chemical approach for identification of the most stable tautomers of organic molecules. The computational portion of this work was supported by the: (i) Polish State Committee for Scientific Research (KBN) Grants: DS/8000-4-0140-7 (M.G.) and N204 127 31/2963 (M.H.), (ii) European Social Funds (EFS) ZPORR/2.22/II/2.6/ARP/U/2/05 (M.H.), and (iii) US DOE Office of Biological and Environmental Research, Low Dose Radiation Research Program (M.G.). M.H. holds the Foundation for Polish Science (FNP) award for young scientists. The calculations were performed at the Academic

  4. Purification and characterization of TnsC, a Tn7 transposition protein that binds ATP and DNA.

    PubMed Central

    Gamas, P; Craig, N L

    1992-01-01

    The bacterial transposon Tn7 encodes five transposition genes tnsABCDE. We report a simple and rapid procedure for the purification of TnsC protein. We show that purified TnsC is active in and required for Tn7 transposition in a cell-free recombination system. This finding demonstrates that TnsC participates directly in Tn7 transposition and explains the requirement for tnsC function in Tn7 transposition. We have found that TnsC binds adenine nucleotides and is thus a likely site of action of the essential ATP cofactor in Tn7 transposition. We also report that TnsC binds non-specifically to DNA in the presence of ATP or the generally non-hydrolyzable analogues AMP-PNP and ATP-gamma-S, and that TnsC displays little affinity for DNA in the presence of ADP. We speculate that TnsC plays a central role in the selection of target DNA during Tn7 transposition. Images PMID:1317955

  5. Mitochondrial diaphorases as NAD⁺ donors to segments of the citric acid cycle that support substrate-level phosphorylation yielding ATP during respiratory inhibition.

    PubMed

    Kiss, Gergely; Konrad, Csaba; Pour-Ghaz, Issa; Mansour, Josef J; Németh, Beáta; Starkov, Anatoly A; Adam-Vizi, Vera; Chinopoulos, Christos

    2014-04-01

    Substrate-level phosphorylation mediated by succinyl-CoA ligase in the mitochondrial matrix produces high-energy phosphates in the absence of oxidative phosphorylation. Furthermore, when the electron transport chain is dysfunctional, provision of succinyl-CoA by the α-ketoglutarate dehydrogenase complex (KGDHC) is crucial for maintaining the function of succinyl-CoA ligase yielding ATP, preventing the adenine nucleotide translocase from reversing. We addressed the source of the NAD(+) supply for KGDHC under anoxic conditions and inhibition of complex I. Using pharmacologic tools and specific substrates and by examining tissues from pigeon liver exhibiting no diaphorase activity, we showed that mitochondrial diaphorases in the mouse liver contribute up to 81% to the NAD(+) pool during respiratory inhibition. Under these conditions, KGDHC's function, essential for the provision of succinyl-CoA to succinyl-CoA ligase, is supported by NAD(+) derived from diaphorases. Through this process, diaphorases contribute to the maintenance of substrate-level phosphorylation during respiratory inhibition, which is manifested in the forward operation of adenine nucleotide translocase. Finally, we show that reoxidation of the reducible substrates for the diaphorases is mediated by complex III of the respiratory chain.

  6. Mitochondrial diaphorases as NAD⁺ donors to segments of the citric acid cycle that support substrate-level phosphorylation yielding ATP during respiratory inhibition.

    PubMed

    Kiss, Gergely; Konrad, Csaba; Pour-Ghaz, Issa; Mansour, Josef J; Németh, Beáta; Starkov, Anatoly A; Adam-Vizi, Vera; Chinopoulos, Christos

    2014-04-01

    Substrate-level phosphorylation mediated by succinyl-CoA ligase in the mitochondrial matrix produces high-energy phosphates in the absence of oxidative phosphorylation. Furthermore, when the electron transport chain is dysfunctional, provision of succinyl-CoA by the α-ketoglutarate dehydrogenase complex (KGDHC) is crucial for maintaining the function of succinyl-CoA ligase yielding ATP, preventing the adenine nucleotide translocase from reversing. We addressed the source of the NAD(+) supply for KGDHC under anoxic conditions and inhibition of complex I. Using pharmacologic tools and specific substrates and by examining tissues from pigeon liver exhibiting no diaphorase activity, we showed that mitochondrial diaphorases in the mouse liver contribute up to 81% to the NAD(+) pool during respiratory inhibition. Under these conditions, KGDHC's function, essential for the provision of succinyl-CoA to succinyl-CoA ligase, is supported by NAD(+) derived from diaphorases. Through this process, diaphorases contribute to the maintenance of substrate-level phosphorylation during respiratory inhibition, which is manifested in the forward operation of adenine nucleotide translocase. Finally, we show that reoxidation of the reducible substrates for the diaphorases is mediated by complex III of the respiratory chain. PMID:24391134

  7. Mitochondrial ATP transporter Ant2 depletion impairs erythropoiesis and B lymphopoiesis

    PubMed Central

    Cho, J; Seo, J; Lim, C H; Yang, L; Shiratsuchi, T; Lee, M-H; Chowdhury, R R; Kasahara, H; Kim, J-S; Oh, S P; Lee, Y J; Terada, N

    2015-01-01

    Adenine nucleotide translocases (ANTs) transport ADP and ATP through mitochondrial inner membrane, thus playing an essential role for energy metabolism of eukaryotic cells. Mice have three ANT paralogs, Ant1 (Slc25a4), Ant2 (Slc25a5) and Ant4 (Slc25a31), which are expressed in a tissue-dependent manner. While knockout mice have been characterized with Ant1 and Ant4 genes, which resulted in exercise intolerance and male infertility, respectively, the role of the ubiquitously expressed Ant2 gene in animal development has not been fully demonstrated. Here, we generated Ant2 hypomorphic mice by targeted disruption of the gene, in which Ant2 expression is largely depleted. The mice showed apparently normal embryonic development except pale phenotype along with a reduced birth rate. However, postnatal growth was severely retarded with macrocytic anemia, B lymphocytopenia, lactic acidosis and bloated stomach, and died within 4 weeks. Ant2 depletion caused anemia in a cell-autonomous manner by maturation arrest of erythroid precursors with increased reactive oxygen species and premature deaths. B-lymphocyte development was similarly affected by Ant2 depletion, and splenocytes showed a reduction in maximal respiration capacity and cellular ATP levels as well as an increase in cell death accompanying mitochondrial permeability transition pore opening. In contrast, myeloid, megakaryocyte and T-lymphocyte lineages remained apparently intact. Erythroid and B-cell development may be particularly vulnerable to Ant2 depletion-mediated mitochondrial dysfunction and oxidative stress. PMID:25613378

  8. Mitochondrial ATP transporter Ant2 depletion impairs erythropoiesis and B lymphopoiesis.

    PubMed

    Cho, J; Seo, J; Lim, C H; Yang, L; Shiratsuchi, T; Lee, M-H; Chowdhury, R R; Kasahara, H; Kim, J-S; Oh, S P; Lee, Y J; Terada, N

    2015-09-01

    Adenine nucleotide translocases (ANTs) transport ADP and ATP through mitochondrial inner membrane, thus playing an essential role for energy metabolism of eukaryotic cells. Mice have three ANT paralogs, Ant1 (Slc25a4), Ant2 (Slc25a5) and Ant4 (Slc25a31), which are expressed in a tissue-dependent manner. While knockout mice have been characterized with Ant1 and Ant4 genes, which resulted in exercise intolerance and male infertility, respectively, the role of the ubiquitously expressed Ant2 gene in animal development has not been fully demonstrated. Here, we generated Ant2 hypomorphic mice by targeted disruption of the gene, in which Ant2 expression is largely depleted. The mice showed apparently normal embryonic development except pale phenotype along with a reduced birth rate. However, postnatal growth was severely retarded with macrocytic anemia, B lymphocytopenia, lactic acidosis and bloated stomach, and died within 4 weeks. Ant2 depletion caused anemia in a cell-autonomous manner by maturation arrest of erythroid precursors with increased reactive oxygen species and premature deaths. B-lymphocyte development was similarly affected by Ant2 depletion, and splenocytes showed a reduction in maximal respiration capacity and cellular ATP levels as well as an increase in cell death accompanying mitochondrial permeability transition pore opening. In contrast, myeloid, megakaryocyte and T-lymphocyte lineages remained apparently intact. Erythroid and B-cell development may be particularly vulnerable to Ant2 depletion-mediated mitochondrial dysfunction and oxidative stress. PMID:25613378

  9. Myoadenylate deaminase deficiency. Functional and metabolic abnormalities associated with disruption of the purine nucleotide cycle.

    PubMed Central

    Sabina, R L; Swain, J L; Olanow, C W; Bradley, W G; Fishbein, W N; DiMauro, S; Holmes, E W

    1984-01-01

    by the combined increases of adenosine, inosine, hypoxanthine, and IMP. Studies performed in vitro with muscle samples from seven MDD and seven non-MDD subjects demonstrated that ATP catabolism was associated with a fivefold greater increase in IMP in non-MDD muscle. There were significant increases in AMP and ADP content of the muscle from MDD patients following ATP catabolism in vitro, while there was no detectable increase in AMP or ADP in non-MDD muscle. Adenosine content of MDD muscle increased following ATP catabolism, but there was no detectable increase in adenosine content of non-MDD muscle following ATP catabolism in vitro. These studies demonstrate that AMP deaminase deficiency leads to reduced entry of adenine nucleotides into the purine nucleotide cycle during exercise. We postulate that the resultant disruption of the purine nucleotide cycle accounts for the muscle dysfunction observed in these patients. Images PMID:6707201

  10. Running out of time: the decline of channel activity and nucleotide activation in adenosine triphosphate-sensitive K-channels

    PubMed Central

    Proks, Peter; Puljung, Michael C.; Vedovato, Natascia; Sachse, Gregor; Mulvaney, Rachel; Ashcroft, Frances M.

    2016-01-01

    KATP channels act as key regulators of electrical excitability by coupling metabolic cues—mainly intracellular adenine nucleotide concentrations—to cellular potassium ion efflux. However, their study has been hindered by their rapid loss of activity in excised membrane patches (rundown), and by a second phenomenon, the decline of activation by Mg-nucleotides (DAMN). Degradation of PI(4,5)P2 and other phosphoinositides is the strongest candidate for the molecular cause of rundown. Broad evidence indicates that most other determinants of rundown (e.g. phosphorylation, intracellular calcium, channel mutations that affect rundown) also act by influencing KATP channel regulation by phosphoinositides. Unfortunately, experimental conditions that reproducibly prevent rundown have remained elusive, necessitating post hoc data compensation. Rundown is clearly distinct from DAMN. While the former is associated with pore-forming Kir6.2 subunits, DAMN is generally a slower process involving the regulatory sulfonylurea receptor (SUR) subunits. We speculate that it arises when SUR subunits enter non-physiological conformational states associated with the loss of SUR nucleotide-binding domain dimerization following prolonged exposure to nucleotide-free conditions. This review presents new information on both rundown and DAMN, summarizes our current understanding of these processes and considers their physiological roles. This article is part of the themed issue ‘Evolution brings Ca2+ and ATP together to control life and death’. PMID:27377720

  11. Progesterone-adenine hybrids as bivalent inhibitors of P-glycoprotein-mediated multidrug efflux: design, synthesis, characterization and biological evaluation.

    PubMed

    Zeinyeh, Waël; Mahiout, Zahia; Radix, Sylvie; Lomberget, Thierry; Dumoulin, Axel; Barret, Roland; Grenot, Catherine; Rocheblave, Luc; Matera, Eva-Laure; Dumontet, Charles; Walchshofer, Nadia

    2012-10-01

    Bivalent ligands were designed on the basis of the described close proximity of the ATP-site and the putative steroid-binding site of P-glycoprotein (ABCB1). The syntheses of 19 progesterone-adenine hybrids are described. Their abilities to inhibit P-glycoprotein-mediated daunorubicin efflux in K562/R7 human leukemic cells overexpressing P-glycoprotein were evaluated versus progesterone. The hybrid with a hexamethylene linker chain showed the best inhibitory potency. The efficiency of these progesterone-adenine hybrids depends on two main factors: (i) the nature of the linker and (ii) its attachment point on the steroid skeleton.

  12. Mapping of the nucleotide-binding sites in the ADP/ATP carrier of beef heart mitochondria by photolabeling with 2-azido[alpha-32P]adenosine diphosphate.

    PubMed

    Dalbon, P; Brandolin, G; Boulay, F; Hoppe, J; Vignais, P V

    1988-07-12

    2-Azido[alpha-32P]adenosine diphosphate (2-azido[alpha-32P]ADP) has been used to photolabel the ADP/ATP carrier in beef heart mitochondria. In reversible binding assays carried out in the dark, this photoprobe was found to inhibit ADP/ATP transport in beef heart mitochondria and to bind to two types of specific sites of the ADP/ATP carrier characterized by high-affinity binding (Kd = 20 microM) and low-affinity binding (Kd = 400 microM). In contrast, it was unable to bind to specific carrier sites in inverted submitochondrial particles. Upon photoirradiation of beef heart mitochondria in the presence of 2-azido[alpha-32P]ADP, the ADP/ATP carrier was covalently labeled. After purification, the photolabeled carrier protein was cleaved chemically by acidolysis or cyanogen bromide and enzymatically with the Staphylococcus aureus V8 protease. In the ADP/ATP carrier protein, which is 297 amino acid residues in length, two discrete regions extending from Phe-153 to Met-200 and from Tyr-250 to Met-281 were labeled by 2-azido[alpha-32P]ADP. The peptide fragments corresponding to these regions were sequenced, and the labeled amino acids were identified. As 2-azido-ADP is not transported into mitochondria and competes against transport of externally added ADP, it is concluded that the two regions of the carrier which are photolabeled are facing the cytosol. Whether the two photolabeled regions are located in a single peptide chain of the carrier or in different peptide chains of an oligomeric structure is discussed.

  13. Mapping of the nucleotide-binding sites in the ADP/ATP carrier of beef heart mitochondria by photolabeling with 2-azido(. cap alpha. -/sup 32/P)adenosine diphosphate

    SciTech Connect

    Dalbon, P.; Brandolin, G.; Boulay, F.; Hoppe, J.; Vignais, P.V.

    1988-07-12

    2-Azido(..cap alpha..-/sup 32/P)adenosine diphosphate (2-azido(..cap alpha..-/sup 32/P)ADP) has been used to photolabel the ADP/ATP carrier in beef heart mitochondria. In reversible binding assays carried out in the dark, this photoprobe was found to inhibit ADP/ATP transport in beef heart mitochondria and to bind to two types of specific sites of the ADP/ATP carrier characterized by high-affinity binding (K/sub d/ = 20 ..mu..M) and low-affinity binding (K/sub d/ = 400 ..mu..M). In contrast, it was unable to bind to specific carrier sites in inverted submitochondrial particles. Upon photoirradiation of beef heart mitochondria in the presence of 2-azido(..cap alpha..-/sup 32/P)ADP, the ADP/ATP carrier was covalently labeled. After purification, the photolabeled carrier protein was cleaved chemically by acidolysis or cyanogen bromide and enzymatically with the Staphylococcus aureus V8 protease. In the ADP/ATP carrier protein, which is 297 amino acid residues in length, two discrete regions extending from Phe-153 to Met-200 and from Tyr-250 to Met-281 were labeled by 2-azido(..cap alpha..-/sup 32/P)ADP. The peptide fragments corresponding to these regions were sequenced, and the labeled amino acids were identified. As 2-azido-ADP is not transported into mitochondria and competes against transport of externally added ADP, it is concluded that the two regions of the carrier which are photolabeled are facing the cytosol. Whether the two photolabeled regions are located in a single peptide chain of the carrier or in different peptide chains of an oligomeric structure is discussed.

  14. 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

  15. 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

  16. Photophysical deactivation pathways in adenine oligonucleotides.

    PubMed

    Spata, Vincent A; Matsika, Spiridoula

    2015-12-14

    In this work we study deactivation processes in adenine oligomers after absorption of UV radiation using Quantum Mechanics combined with Molecular Mechanics (QM/MM). Correlated electronic structure methods appropriate for describing the excited states are used to describe a π-stacked dimer of adenine bases incorporated into (dA)20(dT)20. The results of these calculations reveal three different types of excited state minima which play a role in deactivation processes. Within this set of minima there are minima where the excited state is localized on one adenine (monomer-like) as well as minima where the excited state is delocalized on two adenines, forming different types of excimers and bonded excimers of varying but inter-related character. The proximity of their energies reveals that the minima can decay into one another along a flat potential energy surface dependent on the interbase separation. Additionally, analysis of the emissive energies and other physical properties, including theoretical anisotropy calculations, and comparison with fluorescence experiments, provides evidence that excimers play an important role in long-lived signals in adenine oligonucleotides while the subpicosecond decay is attributed to monomer-like minima. The necessity for a close approach of the nucleobases reveals that the deactivation mechanism is tied to macro-molecular motion. PMID:26536353

  17. ATP and related purines stimulate motility, spatial congregation, and coalescence in red algal spores.

    PubMed

    Huidobro-Toro, Juan P; Donoso, Verónica; Flores, Verónica; Santelices, Bernabé

    2015-04-01

    Adenosine 5'-triphosphate (ATP) is a versatile extracellular signal along the tree of life, whereas cAMP plays a major role in vertebrates as an intracellular messenger for hormones, transmitters, tastants, and odorants. Since red algal spore coalescence may be considered analogous to the congregation process of social amoeba, which is stimulated by cAMP, we ascertained whether exogenous applications of ATP, cAMP, adenine, or adenosine modified spore survival and motility, spore settlement and coalescence. Concentration-response studies were performed with carpospores of Mazzaella laminarioides (Gigartinales), incubated with and without added purines. Stirring of algal blades released ADP/ATP to the cell media in a time-dependent manner. 10-300 μM ATP significantly increased spore survival; however, 1,500 μM ATP, cAMP or adenine induced 100% mortality within less than 24 h; the exception was adenosine, which up to 3,000 μM, did not alter spore survival. ATP exposure elicited spore movement with speeds of 2.2-2.5 μm · s(-1) . 14 d after 1,000 μM ATP addition, spore abundance in the central zone of the plaques was increased 2.7-fold as compared with parallel controls. Likewise, 1-10 μM cAMP or 30-100 μM adenine also increased central zone spore abundance, albeit these purines were less efficacious than ATP; adenosine up to 3,000 μM did not influence settlement. Moreover, 1,000 μM ATP markedly accelerated coalescence, the other purines caused a variable effect. We conclude that exogenous cAMP, adenine, but particularly ATP, markedly influence red algal spore physiology; effects are compatible with the expression of one or more membrane purinoceptor(s), discarding adenosine receptor participation. PMID:26986520

  18. ATP and related purines stimulate motility, spatial congregation, and coalescence in red algal spores.

    PubMed

    Huidobro-Toro, Juan P; Donoso, Verónica; Flores, Verónica; Santelices, Bernabé

    2015-04-01

    Adenosine 5'-triphosphate (ATP) is a versatile extracellular signal along the tree of life, whereas cAMP plays a major role in vertebrates as an intracellular messenger for hormones, transmitters, tastants, and odorants. Since red algal spore coalescence may be considered analogous to the congregation process of social amoeba, which is stimulated by cAMP, we ascertained whether exogenous applications of ATP, cAMP, adenine, or adenosine modified spore survival and motility, spore settlement and coalescence. Concentration-response studies were performed with carpospores of Mazzaella laminarioides (Gigartinales), incubated with and without added purines. Stirring of algal blades released ADP/ATP to the cell media in a time-dependent manner. 10-300 μM ATP significantly increased spore survival; however, 1,500 μM ATP, cAMP or adenine induced 100% mortality within less than 24 h; the exception was adenosine, which up to 3,000 μM, did not alter spore survival. ATP exposure elicited spore movement with speeds of 2.2-2.5 μm · s(-1) . 14 d after 1,000 μM ATP addition, spore abundance in the central zone of the plaques was increased 2.7-fold as compared with parallel controls. Likewise, 1-10 μM cAMP or 30-100 μM adenine also increased central zone spore abundance, albeit these purines were less efficacious than ATP; adenosine up to 3,000 μM did not influence settlement. Moreover, 1,000 μM ATP markedly accelerated coalescence, the other purines caused a variable effect. We conclude that exogenous cAMP, adenine, but particularly ATP, markedly influence red algal spore physiology; effects are compatible with the expression of one or more membrane purinoceptor(s), discarding adenosine receptor participation.

  19. The nucleotide sequence of the uvrD gene of E. coli.

    PubMed Central

    Finch, P W; Emmerson, P T

    1984-01-01

    The nucleotide sequence of a cloned section of the E. coli chromosome containing the uvrD gene has been determined. The coding region for the UvrD protein consists of 2,160 nucleotides which would direct the synthesis of a polypeptide 720 amino acids long with a calculated molecular weight of 82 kd. The predicted amino acid sequence of the UvrD protein has been compared with the amino acid sequences of other known adenine nucleotide binding proteins and a common sequence has been identified, thought to contribute towards adenine nucleotide binding. PMID:6379604

  20. ATP insertion opposite 8-oxo-deoxyguanosine by Pol4 mediates error-free tolerance in Schizosaccharomyces pombe.

    PubMed

    Sastre-Moreno, Guillermo; Sánchez, Arancha; Esteban, Verónica; Blanco, Luis

    2014-09-01

    7,8-Dihydro-8-oxo-deoxyguanosine (8oxodG) is a highly premutagenic DNA lesion due to its ability to mispair with adenine. Schizosaccharomyces pombe lacks homologs for relevant enzymes that repair 8oxodG, which suggests that this lesion could be persistent and must be tolerated. Here we show that SpPol4, the unique PolX in fission yeast, incorporates ATP opposite 8oxodG almost exclusively when all nucleotides (ribos and deoxys) are provided at physiological concentrations. Remarkably, this SpPol4-specific reaction could also occur during the NHEJ of DSBs. In cell extracts, misincorporation of ATP opposite 8oxodG was shown to be SpPol4-specific, although RNase H2 efficiently recognized the 8oxodG:AMP mispair to remove AMP and trigger error-free incorporation of dCTP. These data are the first evidence that ribonucleotides can be used safely for 8oxodG tolerance, suggesting that insertion of the highly abundant ATP substrate could be beneficial to promote efficient and error-free repair of 8oxodG-associated DSBs. Moreover, we demonstrate that purified SpPol4 uses 8oxo-dGTP and 8oxo-GTP as substrates for DNA polymerization, although with poor efficiency compared to the incorporation of undamaged nucleotides opposite either 8oxodG or undamaged templates. This suggests that SpPol4 is specialized in tolerating 8oxodG as a DNA template, without contributing significantly to the accumulation of this lesion in the DNA.

  1. 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

  2. Different responses of astrocytes and neurons to nitric oxide: The role of glycolytically generated ATP in astrocyte protection

    PubMed Central

    Almeida, Angeles; Almeida, Julia; Bolaños, Juan P.; Moncada, Salvador

    2001-01-01

    It was recently proposed that in Jurkat cells, after inhibition of respiration by NO, glycolytically generated ATP plays a critical role in preventing the collapse of mitochondrial membrane potential (Δψm) and thus apoptotic cell death. We have investigated this observation further in primary cultures of rat cortical neurons and astrocytes—cell types that differ greatly in their glycolytic capacity. Continuous and significant (≈85%) inhibition of respiration by NO (1.4 μM at 175 μM O2) generated by [(z)-1-[2-aminoethyl]-N-[2-ammonioethyl]amino]diazen-1-ium-1,2 diolate (DETA-NO) initially (10 min) depleted ATP concentrations by ≈25% in both cell types and increased the rate of glycolysis in astrocytes but not in neurons. Activation of glycolysis in astrocytes, as judged by lactate production, prevented further ATP depletion, whereas in neurons, which do not invoke this mechanism, there was a progressive decrease in ATP concentrations over the next 60 min. During this time, there was a persistent mitochondrial hyperpolarization and absence of apoptotic cell death in astrocytes, whereas in the neurons there was a progressive fall in Δψm and increased apoptosis. After glucose deprivation or treatment with inhibitors of the F1F0-ATPase and adenine nucleotide translocase, astrocytes responded to NO with a fall in Δψm and apoptotic cell death similar to the response in neurons. Finally, although treatment of astrocytes with NO partially prevented staurosporin-induced collapse in Δψm and cell death, NO and staurosporin synergized in decreasing Δψm and inducing apoptosis in neurons. These results demonstrate that although inhibition of cellular respiration by NO leads to neurotoxicity, it may also result in initial neuroprotection, depending on the glycolytic capacity of the particular cell. PMID:11742096

  3. The catalase activity of diiron adenine deaminase.

    PubMed

    Kamat, Siddhesh S; Holmes-Hampton, Gregory P; Bagaria, Ashima; Kumaran, Desigan; Tichy, Shane E; Gheyi, Tarun; Zheng, Xiaojing; Bain, Kevin; Groshong, Chris; Emtage, Spencer; Sauder, J Michael; Burley, Stephen K; Swaminathan, Subramanyam; Lindahl, Paul A; Raushel, Frank M

    2011-12-01

    Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn(2+) before induction, the post-translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo-enzyme was prepared and reconstituted with two equivalents of FeSO(4). Inductively coupled plasma mass spectrometry and Mössbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [Fe(II) /Fe(II) ]-ADE catalyzed the conversion of H(2)O(2) to O(2) and H(2)O. The values of k(cat) and k(cat)/K(m) for the catalase activity are 200 s(-1) and 2.4 × 10(4) M(-1) s(-1), respectively. [Fe(II)/Fe(II)]-ADE underwent more than 100 turnovers with H(2)O(2) before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high-spin ferric with g(ave) = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H(2)O(2) by [Fe(II)/Fe(II)]-ADE that involves the cycling of the binuclear metal center between the di-ferric and di-ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS. PMID:21998098

  4. Mitochondrial ADP/ATP exchange inhibition: a novel off-target mechanism underlying ibipinabant-induced myotoxicity.

    PubMed

    Schirris, Tom J J; Ritschel, Tina; Herma Renkema, G; Willems, Peter H G M; Smeitink, Jan A M; Russel, Frans G M

    2015-01-01

    Cannabinoid receptor 1 (CB1R) antagonists appear to be promising drugs for the treatment of obesity, however, serious side effects have hampered their clinical application. Rimonabant, the first in class CB1R antagonist, was withdrawn from the market because of psychiatric side effects. This has led to the search for more peripherally restricted CB1R antagonists, one of which is ibipinabant. However, this 3,4-diarylpyrazoline derivative showed muscle toxicity in a pre-clinical dog study with mitochondrial dysfunction. Here, we studied the molecular mechanism by which ibipinabant induces mitochondrial toxicity. We observed a strong cytotoxic potency of ibipinabant in C2C12 myoblasts. Functional characterization of mitochondria revealed increased cellular reactive oxygen species generation and a decreased ATP production capacity, without effects on the catalytic activities of mitochondrial enzyme complexes I-V or the complex specific-driven oxygen consumption. Using in silico off-target prediction modelling, combined with in vitro validation in isolated mitochondria and mitoplasts, we identified adenine nucleotide translocase (ANT)-dependent mitochondrial ADP/ATP exchange as a novel molecular mechanism underlying ibipinabant-induced toxicity. Minor structural modification of ibipinabant could abolish ANT inhibition leading to a decreased cytotoxic potency, as observed with the ibipinabant derivative CB23. Our results will be instrumental in the development of new types of safer CB1R antagonists.

  5. Mitochondrial ADP/ATP exchange inhibition: a novel off-target mechanism underlying ibipinabant-induced myotoxicity

    PubMed Central

    Schirris, Tom J. J.; Ritschel, Tina; Herma Renkema, G.; Willems, Peter H. G. M.; Smeitink, Jan A. M.; Russel, Frans G. M.

    2015-01-01

    Cannabinoid receptor 1 (CB1R) antagonists appear to be promising drugs for the treatment of obesity, however, serious side effects have hampered their clinical application. Rimonabant, the first in class CB1R antagonist, was withdrawn from the market because of psychiatric side effects. This has led to the search for more peripherally restricted CB1R antagonists, one of which is ibipinabant. However, this 3,4-diarylpyrazoline derivative showed muscle toxicity in a pre-clinical dog study with mitochondrial dysfunction. Here, we studied the molecular mechanism by which ibipinabant induces mitochondrial toxicity. We observed a strong cytotoxic potency of ibipinabant in C2C12 myoblasts. Functional characterization of mitochondria revealed increased cellular reactive oxygen species generation and a decreased ATP production capacity, without effects on the catalytic activities of mitochondrial enzyme complexes I–V or the complex specific-driven oxygen consumption. Using in silico off-target prediction modelling, combined with in vitro validation in isolated mitochondria and mitoplasts, we identified adenine nucleotide translocase (ANT)-dependent mitochondrial ADP/ATP exchange as a novel molecular mechanism underlying ibipinabant-induced toxicity. Minor structural modification of ibipinabant could abolish ANT inhibition leading to a decreased cytotoxic potency, as observed with the ibipinabant derivative CB23. Our results will be instrumental in the development of new types of safer CB1R antagonists. PMID:26416158

  6. ATP regulation in bioproduction.

    PubMed

    Hara, Kiyotaka Y; Kondo, Akihiko

    2015-12-10

    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.

  7. Adenine auxotrophy--be aware: some effects of adenine auxotrophy in Saccharomyces cerevisiae strain W303-1A.

    PubMed

    Kokina, Agnese; Kibilds, Juris; Liepins, Janis

    2014-08-01

    Adenine auxotrophy is a commonly used genetic marker in haploid yeast strains. Strain W303-1A, which carries the ade2-1 mutation, is widely used in physiological and genetic research. Yeast extract-based rich medium contains a low level of adenine, so that adenine is often depleted before glucose. This could affect the cell physiology of adenine auxotrophs grown in rich medium. The aim of our study was to assess the effects of adenine auxotrophy on cell morphology and stress physiology. Our results show that adenine depletion halts cell division, but that culture optical density continues to increase due to cell swelling. Accumulation of trehalose and a coincident 10-fold increase in desiccation stress tolerance is observed in adenine auxotrophs after adenine depletion, when compared to prototrophs. Under adenine starvation, long-term survival of W303-1A is lower than during carbon starvation, but higher than during leucine starvation. We observed drastic adenine-dependent changes in cell stress physiology, suggesting that results may be biased when adenine auxotrophs are grown in rich media without adenine supplementation.

  8. When Too Much ATP Is Bad for Protein Synthesis.

    PubMed

    Pontes, Mauricio H; Sevostyanova, Anastasia; Groisman, Eduardo A

    2015-08-14

    Adenosine triphosphate (ATP) is the energy currency of living cells. Even though ATP powers virtually all energy-dependent activities, most cellular ATP is utilized in protein synthesis via tRNA aminoacylation and guanosine triphosphate regeneration. Magnesium (Mg(2+)), the most common divalent cation in living cells, plays crucial roles in protein synthesis by maintaining the structure of ribosomes, participating in the biochemistry of translation initiation and functioning as a counterion for ATP. A non-physiological increase in ATP levels hinders growth in cells experiencing Mg(2+) limitation because ATP is the most abundant nucleotide triphosphate in the cell, and Mg(2+) is also required for the stabilization of the cytoplasmic membrane and as a cofactor for essential enzymes. We propose that organisms cope with Mg(2+) limitation by decreasing ATP levels and ribosome production, thereby reallocating Mg(2+) to indispensable cellular processes.

  9. Thermodynamics of proton transport coupled ATP synthesis.

    PubMed

    Turina, Paola; Petersen, Jan; Gräber, Peter

    2016-06-01

    The thermodynamic H(+)/ATP ratio of the H(+)-ATP synthase from chloroplasts was measured in proteoliposomes after energization of the membrane by an acid base transition (Turina et al. 2003 [13], 418-422). The method is discussed, and all published data obtained with this system are combined and analyzed as a single dataset. This meta-analysis led to the following results. 1) At equilibrium, the transmembrane ΔpH is energetically equivalent to the transmembrane electric potential difference. 2) The standard free energy for ATP synthesis (reference reaction) is ΔG°(ref)=33.8±1.3kJ/mol. 3) The thermodynamic H(+)/ATP ratio, as obtained from the shift of the ATP synthesis equilibrium induced by changing the transmembrane ΔpH (varying either pH(in) or pH(out)) is 4.0±0.1. The structural H(+)/ATP ratio, calculated from the ratio of proton binding sites on the c-subunit-ring in F(0) to the catalytic nucleotide binding sites on the β-subunits in F(1), is c/β=14/3=4.7. We infer that the energy of 0.7 protons per ATP that flow through the enzyme, but do not contribute to shifting the ATP/(ADP·Pi) ratio, is used for additional processes within the enzyme, such as activation, and/or energy dissipation, due e.g. to internal uncoupling. The ratio between the thermodynamic and the structural H(+)/ATP values is 0.85, and we conclude that this value represents the efficiency of the chemiosmotic energy conversion within the chloroplast H(+)-ATP synthase.

  10. NMR studies of the MgATP binding site of adenylate kinase and of a 45-residue peptide fragment of the enzyme.

    PubMed

    Fry, D C; Kuby, S A; Mildvan, A S

    1985-08-13

    Proton NMR was used to study the interaction of beta,gamma-bidentate Cr3+ATP and MgATP with rabbit muscle adenylate kinase, which has 194 amino acids, and with a synthetic peptide consisting of residues 1-45 of the enzyme, which has previously been shown to bind MgepsilonATP [Hamada, M., Palmieri, R. H., Russell, G. A., & Kuby, S. A. (1979) Arch. Biochem. Biophys. 195, 155-177]. The peptide is globular and binds Cr3+ATP competitively with MgATP with a dissociation constant, KD(Cr3+ATP) = 35 microM, comparable to that of the complete enzyme [KI(Cr3+ATP) = 12 microM]. Time-dependent nuclear Overhauser effects (NOE's) were used to measure interproton distances on enzyme- and peptide-bound MgATP. The correlation time was measured directly for peptide-bound MgATP by studying the frequency dependence of the NOE's at 250 and 500 MHz. The H2' to H1' distance so obtained (3.07 A) was within the range established by X-ray and model-building studies of nucleotides (2.9 +/- 0.2 A). Interproton distances yielded conformations of enzyme- and peptide-bound MgATP with indistinguishable anti-glycosyl torsional angles (chi = 63 +/- 12 degrees) and 3'-endo/O1'-endo ribose puckers (sigma = 96 +/- 12 degrees). Enzyme- and peptide-bound MgATP molecules exhibited different C4'-C5' torsional angles (gamma) of 170 degrees and 50 degrees, respectively. Ten intermolecular NOE's from protons of the enzyme and four such NOE's from protons of the peptide to protons of bound MgATP were detected, which indicated proximity of the adenine ribose moiety to the same residues on both the enzyme and the peptide. Paramagnetic effects of beta,gamma-bidentate Cr3+ATP on the longitudinal relaxation rates of protons of the peptide provided a set of distances to the side chains of five residues, which allowed the location of the bound Cr3+ atom to be uniquely defined. Distances from enzyme-bound Cr3+ATP to the side chains of three residues of the protein agreed with those measured for the peptide. The mutual

  11. Trichomonas vaginalis NTPDase and ecto-5'-nucleotidase hydrolyze guanine nucleotides and increase extracellular guanosine levels under serum restriction.

    PubMed

    Menezes, Camila Braz; Durgante, Juliano; de Oliveira, Rafael Rodrigues; Dos Santos, Victor Hugo Jacks Mendes; Rodrigues, Luiz Frederico; Garcia, Solange Cristina; Dos Santos, Odelta; Tasca, Tiana

    2016-05-01

    Trichomonas vaginalis is the aethiologic agent of trichomoniasis, the most common non-viral sexually transmitted disease in the world. The purinergic signaling pathway is mediated by extracellular nucleotides and nucleosides that are involved in many biological effects as neurotransmission, immunomodulation and inflammation. Extracellular nucleotides can be hydrolyzed by a family of enzymes known as ectonucleotidases including the ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) family which hydrolyses nucleosides triphosphate and diphosphate as preferential substrates and ecto-5'-nucleotidase which catalyzes the conversion of monophosphates into nucleosides. In T. vaginalis the E-NTPDase and ecto-5'-nucleotidase activities upon adenine nucleotides have already been characterized in intact trophozoites but little is known concerning guanine nucleotides and nucleoside. These enzymes may exert a crucial role on nucleoside generation, providing the purine sources for the synthesis de novo of these essential nutrients, sustaining parasite growth and survival. In this study, we investigated the hydrolysis profile of guanine-related nucleotides and nucleoside in intact trophozoites from long-term-grown and fresh clinical isolates of T. vaginalis. Knowing that guanine nucleotides are also substrates for T. vaginalis ectoenzymes, we evaluated the profile of nucleotides consumption and guanosine uptake in trophozoites submitted to a serum limitation condition. Results show that guanine nucleotides (GTP, GDP, GMP) were substrates for T. vaginalis ectonucleotidases, with expected kinetic parameters for this enzyme family. Different T. vaginalis isolates (two from the ATCC and nine fresh clinical isolates) presented a heterogeneous hydrolysis profile. The serum culture condition increased E-NTPDase and ecto-5'-nucleotidase activities with high consumption of extracellular GTP generating enhanced GDP, GMP and guanosine levels as demonstrated by HPLC, with final

  12. Trichomonas vaginalis NTPDase and ecto-5'-nucleotidase hydrolyze guanine nucleotides and increase extracellular guanosine levels under serum restriction.

    PubMed

    Menezes, Camila Braz; Durgante, Juliano; de Oliveira, Rafael Rodrigues; Dos Santos, Victor Hugo Jacks Mendes; Rodrigues, Luiz Frederico; Garcia, Solange Cristina; Dos Santos, Odelta; Tasca, Tiana

    2016-05-01

    Trichomonas vaginalis is the aethiologic agent of trichomoniasis, the most common non-viral sexually transmitted disease in the world. The purinergic signaling pathway is mediated by extracellular nucleotides and nucleosides that are involved in many biological effects as neurotransmission, immunomodulation and inflammation. Extracellular nucleotides can be hydrolyzed by a family of enzymes known as ectonucleotidases including the ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) family which hydrolyses nucleosides triphosphate and diphosphate as preferential substrates and ecto-5'-nucleotidase which catalyzes the conversion of monophosphates into nucleosides. In T. vaginalis the E-NTPDase and ecto-5'-nucleotidase activities upon adenine nucleotides have already been characterized in intact trophozoites but little is known concerning guanine nucleotides and nucleoside. These enzymes may exert a crucial role on nucleoside generation, providing the purine sources for the synthesis de novo of these essential nutrients, sustaining parasite growth and survival. In this study, we investigated the hydrolysis profile of guanine-related nucleotides and nucleoside in intact trophozoites from long-term-grown and fresh clinical isolates of T. vaginalis. Knowing that guanine nucleotides are also substrates for T. vaginalis ectoenzymes, we evaluated the profile of nucleotides consumption and guanosine uptake in trophozoites submitted to a serum limitation condition. Results show that guanine nucleotides (GTP, GDP, GMP) were substrates for T. vaginalis ectonucleotidases, with expected kinetic parameters for this enzyme family. Different T. vaginalis isolates (two from the ATCC and nine fresh clinical isolates) presented a heterogeneous hydrolysis profile. The serum culture condition increased E-NTPDase and ecto-5'-nucleotidase activities with high consumption of extracellular GTP generating enhanced GDP, GMP and guanosine levels as demonstrated by HPLC, with final

  13. Nucleotide release by airway epithelia.

    PubMed

    Lazarowski, Eduardo R; Sesma, Juliana I; Seminario, Lucia; Esther, Charles R; Kreda, Silvia M

    2011-01-01

    The purinergic events regulating the airways' innate defenses are initiated by the release of purines from the epithelium, which occurs constitutively and is enhanced by chemical or mechanical stimulation. While the external triggers have been reviewed exhaustively, this chapter focuses on current knowledge of the receptors and signaling cascades mediating nucleotide release. The list of secreted purines now includes ATP, ADP, AMP and nucleotide sugars, and involves at least three distinct mechanisms reflecting the complexity of airway epithelia. First, the constitutive mechanism involves ATP translocation to the ER/Golgi complex as energy source for protein folding, and fusion of Golgi-derived vesicles with the plasma membrane. Second, goblet cells package ATP with mucins into granules, which are discharged in response to P2Y(2)R activation and Ca(2+)-dependent signaling pathways. Finally, non-mucous cells support a regulated mechanism of ATP release involving protease activated receptor (PAR)-elicited G(12/13) activation, leading to the RhoGEF-mediated exchange of GDP for GTP on RhoA, and cytoskeleton rearrangement. Together, these pathways provide fine tuning of epithelial responses regulated by purinergic signaling events. PMID:21560042

  14. Metabolic fate of 14C-labelled nicotinamide and adenine in germinating propagules of the mangrove Bruguiera gymnorrhiza.

    PubMed

    Yin, Yuling; Watanabe, Shin; Ashihara, Hiroshi

    2012-01-01

    We studied the metabolic fate of [carbonyl-14C]nicotinamide and [8-(14)C]adenine in segments taken from young and developing leaves, stem, hypocotyls, and roots of a shoot-root type emerging propagule of the mangrove plant Bruguiera gymnorrhiza. Thin-layer chromatography was used together with a bioimaging analyser system. During 4 h of incubation, incorporation of radioactivity from [carbonyl-14C]nicotinamide into NAD and trigonelline was found in all parts of the propagules; the highest incorporation rates into NAD and trigonelline were found in newly emerged stem and young leaves, respectively. Radioactivity from [8-(14)C]adenine was distributed mainly in the salvage products (adenine nucleotides and RNA), and incorporation was less in catabolites (allantoin, allantoic acid, and CO2). Adenine salvage activity was higher in young leaves and stem than in hypocotyls and roots. Over a short time, the effect of 500 mM NaCl on nicotinamide and adenine metabolism indicated that NaCl inhibits both salvage and degradation activities in roots. PMID:22888538

  15. The catalase activity of diiron adenine deaminase

    SciTech Connect

    Kamat S. S.; Swaminathan S.; Holmes-Hampton, G. P.; Bagaria, A.; Kumaran, D.; Tichy, S. E.; Gheyi, T.; Zheng, X.; Bain, K.; Groshong, C.; Emtage, S.; Sauder, J. M.; Burley, S. K.; Lindahl, P. A.; Raushel, F. M.

    2011-12-01

    Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn{sup 2+} before induction, the post-translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo-enzyme was prepared and reconstituted with two equivalents of FeSO{sub 4}. Inductively coupled plasma mass spectrometry and Moessbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [Fe{sup II}/Fe{sup II}]-ADE catalyzed the conversion of H{sub 2}O{sub 2} to O{sub 2} and H{sub 2}O. The values of k{sub cat} and k{sub cat}/K{sub m} for the catalase activity are 200 s{sup -1} and 2.4 x 10{sup 4} M{sup -1} s{sup -1}, respectively. [Fe{sup II}/Fe{sup II}]-ADE underwent more than 100 turnovers with H{sub 2}O{sub 2} before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high-spin ferric with g{sub ave} = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H{sub 2}O{sub 2} by [Fe{sup II}/Fe{sup II}]-ADE that involves the cycling of the binuclear metal center between the di-ferric and di-ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS.

  16. The adsorption of nucleotides and polynucleotides on montmorillonite clay

    NASA Astrophysics Data System (ADS)

    Ferris, James P.; Ertem, Gözen; Agarwal, Vipin K.

    1989-03-01

    The binding of adenine derivatives to Na+-montmorillonite increases in the order 5'-AMP, 3'-AMP, 5'-ADPadenine. With the exception of cytosine, cytosine derivatives bind less strongly than the corresponding adenine derivatives in the order 5'-CMPadenine analogs. It is concluded that the adenine ring in adenine derivatives is protonated by the acidic montmorillonite surface and binding is a consequence of the electrostatic interaction between the protonated base and the negative charges on the surface of the montmorillonite. Different binding trends were observed with Cu2+-montmorillonite with AMP binding more strongly than adenosine and UMP binding more strongly than uridine. It is concluded that ligation to the Cu2+ is a major force in the binding of nucleotides to Cu2+-montmorillonite. RNA homopolymers exhibit strong adsorption to Na+- and Cu2+-montmorillonite and are not readily washed from the clay. Factors contributing to the binding are discussed. Watson-Crick hydrogen bonding of 5'-AMP to poly(U) and 5'-GMP to poly(C) was observed when the homopolymers are bound to the surface of the clay. No association of 5'-UMP to poly(U) bound to clay was detected. The possible role of montmorillonite clays in the prebiotic formation of RNA is discussed.

  17. Muscle interstitial ATP and norepinephrine concentrations in the human leg during exercise and ATP infusion.

    PubMed

    Mortensen, Stefan P; González-Alonso, José; Nielsen, Jens-Jung; Saltin, Bengt; Hellsten, Ylva

    2009-12-01

    ATP has been proposed to play multiple roles in local skeletal muscle blood flow regulation by inducing vasodilation and modulating sympathetic vasoconstrictor activity, but the mechanisms remain unclear. Here we evaluated the effects of arterial ATP infusion and exercise on leg muscle interstitial ATP and norepinephrine (NE) concentrations to gain insight into the interstitial and intravascular mechanisms by which ATP causes muscle vasodilation and sympatholysis. Leg hemodynamics and muscle interstitial nucleotide and NE concentrations were measured during 1) femoral arterial ATP infusion (0.42 +/- 0.04 and 2.26 +/- 0.52 micromol/min; mean +/- SE) and 2) one-leg knee-extensor exercise (18 +/- 0 and 37 +/- 2 W) in 10 healthy men. Arterial ATP infusion and exercise increased leg blood flow (LBF) in the experimental leg from approximately 0.3 l/min at baseline to 4.2 +/- 0.3 and 4.6 +/- 0.5 l/min, respectively, whereas it was reduced or unchanged in the control leg. During arterial ATP infusion, muscle interstitial ATP, ADP, AMP, and adenosine concentrations remained unchanged in both legs, but muscle interstitial NE increased from approximately 5.9 nmol/l at baseline to 8.3 +/- 1.2 and 8.7 +/- 0.7 nmol/l in the experimental and control leg, respectively (P < 0.05), in parallel to a reduction in arterial pressure (P < 0.05). During exercise, however, interstitial ATP, ADP, AMP, and adenosine concentrations increased in the contracting muscle (P < 0.05), but not in inactive muscle, whereas interstitial NE concentrations increased similarly in both active and inactive muscles. These results suggest that the vasodilatory and sympatholytic effects of intraluminal ATP are mainly mediated via endothelial purinergic receptors. Intraluminal ATP and muscle contractions appear to modulate sympathetic nerve activity by inhibiting the effect of NE rather than blunting its local concentration. PMID:19797688

  18. ATP metabolism in rat liver chronically treated with ethanol and high fat

    SciTech Connect

    Miyamoto, K.; French, S.W.

    1986-03-01

    Five pairs of Wistar male rats weighing about 350 g were continuously infused with a liquid diet in which 25-35% of total calories was derived from fat, plus ethanol or isocaloric dextrose through gastrostomy cannulas for 3 wks to 3.5 mos. Mean ethanol intake was 12.9 +/- 0.7 g/kg B.W. (55% of total calories). High blood alcohol levels (BAL, 342 +/- 151 mg/dl) were maintained. The liver showed severe steatosis (4+) in all the ethanol-fed rats (ER). Two had mild focal mononuclear cell infiltration, one had mild fibrosis and one had spotty necrosis. Mild steatosis (1+) was seen in 4 out of 5 pair-fed control rats (CR). Serum ALT was significantly higher in ER (129 +/- 44 U) compared with Cr (59 +/- 30 U) or rats fed chow ad lib (NR) (48 +/- 26 U). Biopsied liver tissue was used to measure the concentration of adenine nucleotides by HPLC (6 pairs). There was a significant decrease of ATP in ER (1.7 +/- 0.3 ..mu..mol/g liver) as compared to CR (2.5 +/- 0.5 ..mu..mol/g) or NR (2.8 +/- 0.2 ..mu..mol/g, n = 6). There was no significant change in the ADP or AMP content, however. The total adenylate pool of the liver was also significantly reduced in ER when compared to that of CR or NR (3.2 +/- 0.4, 4.0 +/- 0.5 and 4.3 +/- 0.2 ..mu..mol/g liver, respectively). Adeynlate energy charge (E.C.) of the ER livers (0.71 +/- 0.05) was significantly reduced compared to NR (0.77 +/- 0.02) but not with CR (0.75 +/- 0.06). The results indicate that ethanol decreases the level of ATP as well as the biological mechanism to compensate for the lowered level.

  19. 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.

  20. Fluorescent xDNA nucleotides as efficient substrates for a template-independent polymerase

    PubMed Central

    Jarchow-Choy, Sarah K.; Krueger, Andrew T.; Liu, Haibo; Gao, Jianmin; Kool, Eric T.

    2011-01-01

    Template independent polymerases, and terminal deoxynucleotidyl transferase (TdT) in particular, have been widely used in enzymatic labeling of DNA 3′-ends, yielding fluorescently-labeled polymers. The majority of fluorescent nucleotides used as TdT substrates contain tethered fluorophores attached to a natural nucleotide, and can be hindered by undesired fluorescence characteristics such as self-quenching. We previously documented the inherent fluorescence of a set of four benzo-expanded deoxynucleoside analogs (xDNA) that maintain Watson–Crick base pairing and base stacking ability; however, their substrate abilities for standard template-dependent polymerases were hampered by their large size. However, it seemed possible that a template-independent enzyme, due to lowered geometric constraints, might be less restrictive of nucleobase size. Here, we report the synthesis and study of xDNA nucleoside triphosphates, and studies of their substrate abilities with TdT. We find that this polymerase can incorporate each of the four xDNA monomers with kinetic efficiencies that are nearly the same as those of natural nucleotides, as measured by steady-state methods. As many as 30 consecutive monomers could be incorporated. Fluorescence changes over time could be observed in solution during the enzymatic incorporation of expanded adenine (dxATP) and cytosine (dxCTP) analogs, and after incorporation, when attached to a glass solid support. For (dxA)n polymers, monomer emission quenching and long-wavelength excimer emission was observed. For (dxC)n, fluorescence enhancement was observed in the polymer. TdT-mediated synthesis may be a useful approach for creating xDNA labels or tags on DNA, making use of the fluorescence and strong hybridization properties of the xDNA. PMID:20947563

  1. Imaging Adenosine Triphosphate (ATP).

    PubMed

    Rajendran, Megha; Dane, Eric; Conley, Jason; Tantama, Mathew

    2016-08-01

    Adenosine triphosphate (ATP) is a universal mediator of metabolism and signaling across unicellular and multicellular species. There is a fundamental interdependence between the dynamics of ATP and the physiology that occurs inside and outside the cell. Characterizing and understanding ATP dynamics provide valuable mechanistic insight into processes that range from neurotransmission to the chemotaxis of immune cells. Therefore, we require the methodology to interrogate both temporal and spatial components of ATP dynamics from the subcellular to the organismal levels in live specimens. Over the last several decades, a number of molecular probes that are specific to ATP have been developed. These probes have been combined with imaging approaches, particularly optical microscopy, to enable qualitative and quantitative detection of this critical molecule. In this review, we survey current examples of technologies available for visualizing ATP in living cells, and identify areas where new tools and approaches are needed to expand our capabilities. PMID:27638696

  2. Imaging Adenosine Triphosphate (ATP).

    PubMed

    Rajendran, Megha; Dane, Eric; Conley, Jason; Tantama, Mathew

    2016-08-01

    Adenosine triphosphate (ATP) is a universal mediator of metabolism and signaling across unicellular and multicellular species. There is a fundamental interdependence between the dynamics of ATP and the physiology that occurs inside and outside the cell. Characterizing and understanding ATP dynamics provide valuable mechanistic insight into processes that range from neurotransmission to the chemotaxis of immune cells. Therefore, we require the methodology to interrogate both temporal and spatial components of ATP dynamics from the subcellular to the organismal levels in live specimens. Over the last several decades, a number of molecular probes that are specific to ATP have been developed. These probes have been combined with imaging approaches, particularly optical microscopy, to enable qualitative and quantitative detection of this critical molecule. In this review, we survey current examples of technologies available for visualizing ATP in living cells, and identify areas where new tools and approaches are needed to expand our capabilities.

  3. ß-nicotinamide adenine dinucleotide is an enteric inhibitory neurotransmitter in human and non-human primate colons

    PubMed Central

    Hwang, Sung Jin; Durnin, Leonie; Dwyer, Laura; Rhee, Poong-Lyul; Ward, Sean M.; Koh, Sang Don; Sanders, Kenton M.; Mutafova-Yambolieva, Violeta N.

    2010-01-01

    Background & Aims An important component of enteric inhibitory neurotransmission is mediated by a purine neurotransmitter, such as adenosine 5’-triphosphate (ATP), binding to P2Y1 receptors and activating small conductance K+ channels. In murine colon ß-nicotinamide adenine dinucleotide (ß-NAD) is released with ATP and mimics the pharmacology of inhibitory neurotransmission better than ATP. Here ß-NAD and ATP were compared as possible inhibitory neurotransmitters in human and monkey colons. Methods A small-volume superfusion assay and HPLC with fluorescence detection were used to evaluate spontaneous and nerve-evoked overflow of ß-NAD, ATP and metabolites. Postjunctional responses to nerve stimulation, ß-NAD and ATP were compared using intracellular membrane potential and force measurements. Effects of ß-NAD on smooth muscle cells (SMCs) were recorded by patch clamp. P2Y receptor transcripts and proteins were assayed by RT-PCR. Results In contrast to ATP, overflow of ß-NAD evoked by electrical field stimulation correlated with stimulation frequency and was diminished by neurotoxins, tetrodotoxin and ω-conotoxin GVIA. Inhibitory junction potentials and responses to exogenous ß-NAD, but not ATP, were blocked by P2Y receptor antagonists suramin, PPADS, MRS2179 and MRS2500. ß-NAD activated non-selective cation currents in SMCs, but failed to activate outward currents. Conclusions ß-NAD meets the criteria for a neurotransmitter better than ATP in human and monkey colons and therefore may contribute to neural regulation of colonic motility. SMCs are unlikely targets for inhibitory purine neurotransmitters because dominant responses of SMCs were activation of net inward, rather than outward, current. PMID:20875415

  4. Classification of pseudo pairs between nucleotide bases and amino acids by analysis of nucleotide-protein complexes.

    PubMed

    Kondo, Jiro; Westhof, Eric

    2011-10-01

    Nucleotide bases are recognized by amino acid residues in a variety of DNA/RNA binding and nucleotide binding proteins. In this study, a total of 446 crystal structures of nucleotide-protein complexes are analyzed manually and pseudo pairs together with single and bifurcated hydrogen bonds observed between bases and amino acids are classified and annotated. Only 5 of the 20 usual amino acid residues, Asn, Gln, Asp, Glu and Arg, are able to orient in a coplanar fashion in order to form pseudo pairs with nucleotide bases through two hydrogen bonds. The peptide backbone can also form pseudo pairs with nucleotide bases and presents a strong bias for binding to the adenine base. The Watson-Crick side of the nucleotide bases is the major interaction edge participating in such pseudo pairs. Pseudo pairs between the Watson-Crick edge of guanine and Asp are frequently observed. The Hoogsteen edge of the purine bases is a good discriminatory element in recognition of nucleotide bases by protein side chains through the pseudo pairing: the Hoogsteen edge of adenine is recognized by various amino acids while the Hoogsteen edge of guanine is only recognized by Arg. The sugar edge is rarely recognized by either the side-chain or peptide backbone of amino acid residues.

  5. Expression of Vesicular Nucleotide Transporter in Rat Odontoblasts

    PubMed Central

    Ikeda, Erina; Goto, Tetsuya; Gunjigake, Kaori; Kuroishi, Kayoko; Ueda, Masae; Kataoka, Shinji; Toyono, Takashi; Nakatomi, Mitsushiro; Seta, Yuji; Kitamura, Chiaki; Nishihara, Tatsuji; Kawamoto, Tatsuo

    2016-01-01

    Several theories have been proposed regarding pain transmission mechanisms in tooth. However, the exact signaling mechanism from odontoblasts to pulp nerves remains to be clarified. Recently, ATP-associated pain transmission has been reported, but it is unclear whether ATP is involved in tooth pain transmission. In the present study, we focused on the vesicular nucleotide transporter (VNUT), a transporter of ATP into vesicles, and examined whether VNUT was involved in ATP release from odontoblasts. We examined the expression of VNUT in rat pulp by RT-PCR and immunostaining. ATP release from cultured odontoblast-like cells with heat stimulation was evaluated using ATP luciferase methods. VNUT was expressed in pulp tissue, and the distribution of VNUT-immunopositive vesicles was confirmed in odontoblasts. In odontoblasts, some VNUT-immunopositive vesicles were colocalized with membrane fusion proteins. Additionally P2X3, an ATP receptor, immunopositive axons were distributed between odontoblasts. The ATP release by thermal stimulation from odontoblast-like cells was inhibited by the addition of siRNA for VNUT. These findings suggest that cytosolic ATP is transported by VNUT and that the ATP in the vesicles is then released from odontoblasts to ATP receptors on axons. ATP vesicle transport in odontoblasts seems to be a key mechanism for signal transduction from odontoblasts to axons in the pulp. PMID:27006518

  6. ATP: The crucial component of secretory vesicles.

    PubMed

    Estévez-Herrera, Judith; Domínguez, Natalia; Pardo, Marta R; González-Santana, Ayoze; Westhead, Edward W; Borges, Ricardo; Machado, José David

    2016-07-12

    The colligative properties of ATP and catecholamines demonstrated in vitro are thought to be responsible for the extraordinary accumulation of solutes inside chromaffin cell secretory vesicles, although this has yet to be demonstrated in living cells. Because functional cells cannot be deprived of ATP, we have knocked down the expression of the vesicular nucleotide carrier, the VNUT, to show that a reduction in vesicular ATP is accompanied by a drastic fall in the quantal release of catecholamines. This phenomenon is particularly evident in newly synthesized vesicles, which we show are the first to be released. Surprisingly, we find that inhibiting VNUT expression also reduces the frequency of exocytosis, whereas the overexpression of VNUT drastically increases the quantal size of exocytotic events. To our knowledge, our data provide the first demonstration that ATP, in addition to serving as an energy source and purinergic transmitter, is an essential element in the concentration of catecholamines in secretory vesicles. In this way, cells can use ATP to accumulate neurotransmitters and other secreted substances at high concentrations, supporting quantal transmission.

  7. Extracellular nucleotides regulate cellular functions of podocytes in culture.

    PubMed

    Fischer, K G; Saueressig, U; Jacobshagen, C; Wichelmann, A; Pavenstädt, H

    2001-12-01

    Extracellular nucleotides are assumed to be important regulators of glomerular functions. This study characterizes purinergic receptors in podocytes. The effects of purinergic agonists on electrophysiological properties and the intracellular free Ca(2+) concentration of differentiated podocytes were examined with the patch-clamp and fura 2 fluorescence techniques. mRNA expression of purinergic receptors was investigated by RT-PCR. Purinergic agonists depolarized podocytes. Purinergic agonists similarly increased intracellular free Ca(2+) concentration of podocytes. The rank order of potency of various nucleotides on membrane voltage and free cytosolic calcium concentration was UTP approximately UDP > [adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S)] > ATP > 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) > 2'- and 3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP) > ADP-beta-S. alpha,beta-Me-ATP was without effect. In the presence of UTP, BzATP did not cause an additional depolarization of podocytes. Incubation of cells with ATP or BzATP did not induce lactate dehydrogenase release. In RT-PCR studies, mRNAs of the P2Y(1), P2Y(2), P2Y(6), and P2X(7) receptors were detected within glomeruli and podocytes. The data indicate that extracellular nucleotides modulate podocyte function mainly by an activation of both P2Y(2) and P2Y(6) receptors.

  8. Extracellular nucleotides regulate cellular functions of podocytes in culture.

    PubMed

    Fischer, K G; Saueressig, U; Jacobshagen, C; Wichelmann, A; Pavenstädt, H

    2001-12-01

    Extracellular nucleotides are assumed to be important regulators of glomerular functions. This study characterizes purinergic receptors in podocytes. The effects of purinergic agonists on electrophysiological properties and the intracellular free Ca(2+) concentration of differentiated podocytes were examined with the patch-clamp and fura 2 fluorescence techniques. mRNA expression of purinergic receptors was investigated by RT-PCR. Purinergic agonists depolarized podocytes. Purinergic agonists similarly increased intracellular free Ca(2+) concentration of podocytes. The rank order of potency of various nucleotides on membrane voltage and free cytosolic calcium concentration was UTP approximately UDP > [adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S)] > ATP > 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) > 2'- and 3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP) > ADP-beta-S. alpha,beta-Me-ATP was without effect. In the presence of UTP, BzATP did not cause an additional depolarization of podocytes. Incubation of cells with ATP or BzATP did not induce lactate dehydrogenase release. In RT-PCR studies, mRNAs of the P2Y(1), P2Y(2), P2Y(6), and P2X(7) receptors were detected within glomeruli and podocytes. The data indicate that extracellular nucleotides modulate podocyte function mainly by an activation of both P2Y(2) and P2Y(6) receptors. PMID:11704558

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

    PubMed Central

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

    2015-01-01

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

  10. Sites of Adsorption of Adenine, Uracil, and Their Corresponding Derivatives on Sodium Montmorillonite

    NASA Astrophysics Data System (ADS)

    Perezgasga, L.; Serrato-Díaz, A.; Negrón-Mendoza, A.; Gal'N, L. De Pablo; Mosqueira, F. G.

    2005-04-01

    Clay minerals are considered important to chemical evolution processes due to their properties, ancient origin, and wide distribution. To extend the knowledge of their role in the prebiotic epoch, the adsorption sites of adenine, adenosine, AMP, ADP, ATP, Poly A, uracil, uridine, UMP, UDP, UTP and Poly U on sodium montmorillonite are investigated. X-ray diffraction, ultraviolet and infrared spectroscopy studies indicate that these molecules distribute into the interlamellar channel and the edge of the clay crystals. Monomers are adsorbed predominantly in the interlamellar channel, whereas polymers adsorb along the crystal edges. Such behavior is discussed mainly in terms of bulk pH, pKa of the adsorbate, and Van der Waals interactions.

  11. Structural basis for discriminative regulation of gene expression by adenine- and guanine-sensing mRNAs.

    PubMed

    Serganov, Alexander; Yuan, Yu-Ren; Pikovskaya, Olga; Polonskaia, Anna; Malinina, Lucy; Phan, Anh Tuân; Hobartner, Claudia; Micura, Ronald; Breaker, Ronald R; Patel, Dinshaw J

    2004-12-01

    Metabolite-sensing mRNAs, or "riboswitches," specifically interact with small ligands and direct expression of the genes involved in their metabolism. Riboswitches contain sensing "aptamer" modules, capable of ligand-induced structural changes, and downstream regions, harboring expression-controlling elements. We report the crystal structures of the add A-riboswitch and xpt G-riboswitch aptamer modules that distinguish between bound adenine and guanine with exquisite specificity and modulate expression of two different sets of genes. The riboswitches form tuning fork-like architectures, in which the prongs are held in parallel through hairpin loop interactions, and the internal bubble zippers up to form the purine binding pocket. The bound purines are held by hydrogen bonding interactions involving conserved nucleotides along their entire periphery. Recognition specificity is associated with Watson-Crick pairing of the encapsulated adenine and guanine ligands with uridine and cytosine, respectively. PMID:15610857

  12. Structural Basis for Discriminative Regulation of Gene Expression by Adenine- and Guanine-Sensing mRNAs

    PubMed Central

    Serganov, Alexander; Yuan, Yu-Ren; Pikovskaya, Olga; Polonskaia, Anna; Malinina, Lucy; Phan, Anh Tuân; Hobartner, Claudia; Micura, Ronald; Breaker, Ronald R.; Patel, Dinshaw J.

    2015-01-01

    Summary Metabolite-sensing mRNAs, or “riboswitches,” specifically interact with small ligands and direct expression of the genes involved in their metabolism. Ribo-switches contain sensing “aptamer” modules, capable of ligand-induced structural changes, and downstream regions, harboring expression-controlling elements. We report the crystal structures of the add A-riboswitch and xpt G-riboswitch aptamer modules that distinguish between bound adenine and guanine with exquisite specificity and modulate expression of two different sets of genes. The riboswitches form tuning fork-like architectures, in which the prongs are held in parallel through hairpin loop interactions, and the internal bubble zippers up to form the purine binding pocket. The bound purines are held by hydrogen bonding interactions involving conserved nucleotides along their entire periphery. Recognition specificity is associated with Watson-Crick pairing of the encapsulated adenine and guanine ligands with uri-dine and cytosine, respectively. PMID:15610857

  13. What is adenine doing in photolyase?

    PubMed

    Acocella, Angela; Jones, Garth A; Zerbetto, Francesco

    2010-03-25

    The short answer to the title question is that it acts as an electrostatic bouncer that shoves the charge flow from flavin toward the DNA lesion that photolyase repairs. This explanation is provided by an explicit time-dependent quantum mechanical approach, which is used to investigate the electron transfer process that triggers the repair mechanism. The transfer occurs from the flavin photolyase cofactor to the cyclobutane ring of DNA, previously formed by light-induced cycloaddition of adjacent pyrimidine bases. The electron wave function dynamics accurately accounts for the previously proposed mechanism of transfer via the terminal methyl group of the flavin moiety present in the catalytic electron-donor cofactor, FADH(-), which also contains adenine. This latter moiety, which has often been assumed to be present mainly for structural reasons, instantaneously modifies the interaction between acceptor and donor by a variation of the electrostatic interactions so that the presence of its local atomic charges is necessary to trigger the transfer. In principle, knowledge of the details of the electron transfer dynamics and of the important role of polarization effects can be exploited to improve the efficiency of the repair mechanism in artificial systems.

  14. Ultraviolet Photostability of Adenine on Gold and Silicon Surfaces

    NASA Astrophysics Data System (ADS)

    Mateo-Martí, Eva; Pradier, Claire-Marie; Martín-Gago, Jose-Angel

    2009-08-01

    The adenine molecule is a DNA nucleobase, an essential component of genetic material. Because of the important role of DNA nucleobases in terrestrial biochemistry, we have studied the molecular adsorption, orientation, and chemical binding of adenine on metallic and semiconducting surfaces, such as gold and silicon, respectively, and their stability toward ultraviolet radiation by X-ray photoelectron spectroscopy (XPS) and reflection absorption infrared spectroscopy (RAIRS) techniques. We have exposed the adenine surface system to UV radiation (200-400 nm) under a high-vacuum environment (10-7 mbar) to study the photostability and photochemistry of adenine on different surfaces. After 10 or 24 hours of exposure under interplanetary space conditions, UV radiation induces desorption and partial dissociation of the molecule, which is dependant on the nature of the surface. The electronic excitations, induced in the material by UV absorption, play a major role in the photodestruction of the absorbed molecules on the solid surfaces.

  15. Adenine adlayers on Cu(111): XPS and NEXAFS study

    SciTech Connect

    Tsud, Nataliya; Bercha, Sofiia; Ševčíková, Klára; Matolín, Vladimír; Acres, Robert G.; Prince, Kevin C.

    2015-11-07

    The adsorption of adenine on Cu(111) was studied by photoelectron and near edge x-ray absorption fine structure spectroscopy. Disordered molecular films were deposited by means of physical vapor deposition on the substrate at room temperature. Adenine chemisorbs on the Cu(111) surface with strong rehybridization of the molecular orbitals and the Cu 3d states. Annealing at 150 °C caused the desorption of weakly bonded molecules accompanied by formation of a short-range ordered molecular adlayer. The interface is characterized by the formation of new states in the valence band at 1.5, 7, and 9 eV. The present work complements and refines existing knowledge of adenine interaction with this surface. The coverage is not the main parameter that defines the adenine geometry and adsorption properties on Cu(111). Excess thermal energy can further rearrange the molecular adlayer and, independent of the initial coverage, the flat lying stable molecular adlayer is formed.

  16. 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 Central

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

    2007-01-01

    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. 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 4 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

  17. The detection of micromolar pericellular ATP pool on lymphocyte surface by using lymphoid ecto-adenylate kinase as intrinsic ATP sensor.

    PubMed

    Yegutkin, Gennady G; Mikhailov, Andrey; Samburski, Sergei S; Jalkanen, Sirpa

    2006-08-01

    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 [alpha-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 gamma-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.

  18. Raw coffee based dietary supplements contain carboxyatractyligenin derivatives inhibiting mitochondrial adenine-nucleotide-translocase.

    PubMed

    Lang, Roman; Fromme, Tobias; Beusch, Anja; Lang, Tatjana; Klingenspor, Martin; Hofmann, Thomas

    2014-08-01

    Capsules, powders and tablets containing raw coffee extract are advertised to the consumer as antioxidant rich dietary supplements as part of a healthy diet. We isolated carboxyatractyligenin (4), 2-O-β-d-glucopyranosyl carboxyatractyligenin (6) and 3'-O-β-d-glucopyranosyl-2'-O-isovaleryl-2β-(2-desoxy-carboxyatractyligenin)-β-d-glucopyranoside (8) from green coffee and found strong inhibitory effects on phosphorylating respiration in isolated mitochondria similar to the effects of the known phytotoxin carboxyatractyloside. LC-MS/MS analysis of commercial green coffee based dietary supplements revealed the occurrence of carboxyatractyligenin, 3'-O-β-d-glucopyranosyl-2'-O-isovaleryl-2β-(2-desoxy-carboxyatractyligenin)-β-d-glucopyranoside, and 2-O-β-d-glucopyranosyl carboxyatractyligenin in concentrations up to 4.0, 5.7, and 41.6μmol/g, respectively. These data might help to gain first insight into potential physiological side-effects of green coffee containing dietary supplement.

  19. Vertical Singlet Excitations on Adenine Dimer: A Time Dependent Density Functional Study

    NASA Astrophysics Data System (ADS)

    Crespo-Hernández, Carlos E.; Marai, Christopher N. J.

    2007-12-01

    The condense phase, excited state dynamics of the adenylyl(3'→5')adenine (ApA) dinucleotide has been previously studied using transient absorption spectroscopy with femtosecond time resolution (Crespo-Hernández et al. Chem. Rev. 104, 1977-2019 (2004)). An ultrafast and a long-lived component were observed with time constants of <1 ps and 60±16 ps, respectively. Comparison of the time constants measured for the dinucleotide with that for the adenine nucleotide suggested that the fast component observed in ApA could be assigned to monomer dynamics. The long-lived component observed in ApA was assigned to an excimer state that originates from a fraction of base stacked conformations present at the time of excitation. In this contribution, supermolecule calculations using the time dependent implementation of density functional theory is used to provide more insights on the origin of the initial Franck-Condon excitations. Monomer-like, localized excitations are observed for conformations having negligible base stacking interactions, whereas delocalized excitations are predicted for conformations with significant vertical base-base overlap.

  20. Nucleotide Salvage Deficiencies, DNA Damage and Neurodegeneration

    PubMed Central

    Fasullo, Michael; Endres, Lauren

    2015-01-01

    Nucleotide balance is critically important not only in replicating cells but also in quiescent cells. This is especially true in the nervous system, where there is a high demand for adenosine triphosphate (ATP) produced from mitochondria. Mitochondria are particularly prone to oxidative stress-associated DNA damage because nucleotide imbalance can lead to mitochondrial depletion due to low replication fidelity. Failure to maintain nucleotide balance due to genetic defects can result in infantile death; however there is great variability in clinical presentation for particular diseases. This review compares genetic diseases that result from defects in specific nucleotide salvage enzymes and a signaling kinase that activates nucleotide salvage after DNA damage exposure. These diseases include Lesch-Nyhan syndrome, mitochondrial depletion syndromes, and ataxia telangiectasia. Although treatment options are available to palliate symptoms of these diseases, there is no cure. The conclusions drawn from this review include the critical role of guanine nucleotides in preventing neurodegeneration, the limitations of animals as disease models, and the need to further understand nucleotide imbalances in treatment regimens. Such knowledge will hopefully guide future studies into clinical therapies for genetic diseases. PMID:25923076

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

    SciTech Connect

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

    2013-10-08

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

  2. N-Sulfomethylation of guanine, adenine and cytosine with formaldehyde-bisulfite. A selective modification of guanine in DNA.

    PubMed

    Hayatsu, H; Yamashita, Y; Yui, S; Yamagata, Y; Tomita, K; Negishi, K

    1982-10-25

    When guanine-, adenine- and cytosine-nucleosides and nucleotides were treated with formaldehyde and then with bisulfite, stable N-sulfomethyl compounds were formed. N2-Sulfomethylguanine, N6-sulfomethyladenine, N4-sulfomthylcytosine and N6-sulfomethyl-9-beta-D-arabinofuranosyladenine were isolated as crystals and characterized. A guanine-specific sulfomethylation was brought about by treatment and denatured single-stranded DNA with formaldehyde and then with bisulfite at pH 7 and 4 degrees C. Since native double-stranded DNA was not modified by this treatment, this new method of modification is expected to be useful as a conformational probe for polynucleotides. PMID:7177848

  3. N-Sulfomethylation of guanine, adenine and cytosine with formaldehyde-bisulfite. A selective modification of guanine in DNA.

    PubMed Central

    Hayatsu, H; Yamashita, Y; Yui, S; Yamagata, Y; Tomita, K; Negishi, K

    1982-01-01

    When guanine-, adenine- and cytosine-nucleosides and nucleotides were treated with formaldehyde and then with bisulfite, stable N-sulfomethyl compounds were formed. N2-Sulfomethylguanine, N6-sulfomethyladenine, N4-sulfomthylcytosine and N6-sulfomethyl-9-beta-D-arabinofuranosyladenine were isolated as crystals and characterized. A guanine-specific sulfomethylation was brought about by treatment and denatured single-stranded DNA with formaldehyde and then with bisulfite at pH 7 and 4 degrees C. Since native double-stranded DNA was not modified by this treatment, this new method of modification is expected to be useful as a conformational probe for polynucleotides. PMID:7177848

  4. N-Sulfomethylation of guanine, adenine and cytosine with formaldehyde-bisulfite. A selective modification of guanine in DNA.

    PubMed

    Hayatsu, H; Yamashita, Y; Yui, S; Yamagata, Y; Tomita, K; Negishi, K

    1982-10-25

    When guanine-, adenine- and cytosine-nucleosides and nucleotides were treated with formaldehyde and then with bisulfite, stable N-sulfomethyl compounds were formed. N2-Sulfomethylguanine, N6-sulfomethyladenine, N4-sulfomthylcytosine and N6-sulfomethyl-9-beta-D-arabinofuranosyladenine were isolated as crystals and characterized. A guanine-specific sulfomethylation was brought about by treatment and denatured single-stranded DNA with formaldehyde and then with bisulfite at pH 7 and 4 degrees C. Since native double-stranded DNA was not modified by this treatment, this new method of modification is expected to be useful as a conformational probe for polynucleotides.

  5. Prolonged nonhydrolytic interaction of nucleotide with CFTR's NH2-terminal nucleotide binding domain and its role in channel gating.

    PubMed

    Basso, Claudia; Vergani, Paola; Nairn, Angus C; Gadsby, David C

    2003-09-01

    CFTR, the protein defective in cystic fibrosis, functions as a Cl- channel regulated by cAMP-dependent protein kinase (PKA). CFTR is also an ATPase, comprising two nucleotide-binding domains (NBDs) thought to bind and hydrolyze ATP. In hydrolyzable nucleoside triphosphates, PKA-phosphorylated CFTR channels open into bursts, lasting on the order of a second, from closed (interburst) intervals of a second or more. To investigate nucleotide interactions underlying channel gating, we examined photolabeling by [alpha32P]8-N3ATP or [gamma32P]8-N3ATP of intact CFTR channels expressed in HEK293T cells or Xenopus oocytes. We also exploited split CFTR channels to distinguish photolabeling at NBD1 from that at NBD2. To examine simple binding of nucleotide in the absence of hydrolysis and gating reactions, we photolabeled after incubation at 0 degrees C with no washing. Nucleotide interactions under gating conditions were probed by photolabeling after incubation at 30 degrees C, with extensive washing, also at 30 degrees C. Phosphorylation of CFTR by PKA only slightly influenced photolabeling after either protocol. Strikingly, at 30 degrees C nucleotide remained tightly bound at NBD1 for many minutes, in the form of nonhydrolyzed nucleoside triphosphate. As nucleotide-dependent gating of CFTR channels occurred on the time scale of seconds under comparable conditions, this suggests that the nucleotide interactions, including hydrolysis, that time CFTR channel opening and closing occur predominantly at NBD2. Vanadate also appeared to act at NBD2, presumably interrupting its hydrolytic cycle, and markedly delayed termination of channel open bursts. Vanadate somewhat increased the magnitude, but did not alter the rate, of the slow loss of nucleotide tightly bound at NBD1. Kinetic analysis of channel gating in Mg8-N3ATP or MgATP reveals that the rate-limiting step for CFTR channel opening at saturating [nucleotide] follows nucleotide binding to both NBDs. We propose that ATP

  6. The isolation and characterization of the Escherichia coli DNA adenine methylase (dam) gene.

    PubMed Central

    Brooks, J E; Blumenthal, R M; Gingeras, T R

    1983-01-01

    The E. coli dam (DNA adenine methylase) enzyme is known to methylate the sequence GATC. A general method for cloning sequence-specific DNA methylase genes was used to isolate the dam gene on a 1.14 kb fragment, inserted in the plasmid vector pBR322. Subsequent restriction mapping and subcloning experiments established a set of approximate boundaries of the gene. The nucleotide sequence of the dam gene was determined, and analysis of that sequence revealed a unique open reading frame which corresponded in length to that necessary to code for a protein the size of dam. Amino acid composition derived from this sequence corresponds closely to the amino acid composition of the purified dam protein. Enzymatic and DNA:DNA hybridization methods were used to investigate the possible presence of dam genes in a variety of prokaryotic organisms. PMID:6300769

  7. Production and characterization of reduced NAADP (nicotinic acid-adenine dinucleotide phosphate).

    PubMed Central

    Billington, Richard A; Thuring, Jan W; Conway, Stuart J; Packman, Len; Holmes, Andrew B; Genazzani, Armando A

    2004-01-01

    The pyridine nucleotide NAADP (nicotinic acid-adenine dinucleotide phosphate) has been shown to act as a Ca2+-releasing intracellular messenger in a wide variety of systems from invertebrates to mammals and has been implicated in a number of cellular processes. NAADP is structurally very similar to its precursor, the endogenous coenzyme NADP and while much is known about the reduced form of NADP, NADPH, it is not known whether NAADP can also exist in a reduced state. Here we report that NAADP can be reduced to NAADPH by endogenous cellular enzymes and that NAADPH is functionally inert at the NAADP receptor. These data suggest that NAADPH could represent a mechanism for rapidly inactivating NAADP in cells. PMID:14606955

  8. Characterization of SCaMC-3-like/slc25a41, a novel calcium-independent mitochondrial ATP-Mg/Pi carrier.

    PubMed

    Traba, Javier; Satrústegui, Jorgina; del Arco, Araceli

    2009-02-15

    The SCaMCs (small calcium-binding mitochondrial carriers) constitute a subfamily of mitochondrial carriers responsible for the ATP-Mg/P(i) exchange with at least three paralogues in vertebrates. SCaMC members are proteins with two functional domains, the C-terminal transporter domain and the N-terminal domain which harbours calcium-binding EF-hands and faces the intermembrane space. In the present study, we have characterized a shortened fourth paralogue, SCaMC-3L (SCaMC-3-like; also named slc25a41), which lacks the calcium-binding N-terminal extension. SCaMC-3L orthologues are found exclusively in mammals, showing approx. 60% identity to the C-terminal half of SCaMC-3, its closest paralogue. In mammalian genomes, SCaMC-3 and SCaMC-3L genes are adjacent on the same chromosome, forming a head-to-tail tandem array, and show identical exon-intron boundaries, indicating that SCaMC-3L could have arisen from an SCaMC-3 ancestor by a partial duplication event which occurred prior to mammalian radiation. Expression and functional data suggest that, following the duplication event, SCaMC-3L has acquired more restrictive functions. Unlike the broadly expressed longer SCaMCs, mouse SCaMC-3L shows a limited expression pattern; it is preferentially expressed in testis and, at lower levels, in brain. SCaMC-3L transport activity was studied in yeast deficient in Sal1p, the calcium-dependent mitochondrial ATP-Mg/P(i) carrier, co-expressing SCaMC-3L and mitochondrial-targeted luciferase, and it was found to perform ATP-Mg/P(i) exchange, in a similar manner to Sal1p or other ATP-Mg/P(i) carriers. However, metabolite transport through SCaMC-3L is calcium-independent, representing a novel mechanism involved in adenine nucleotide transport across the inner mitochondrial membrane, different to ADP/ATP translocases or long SCaMC paralogues.

  9. Nucleotide-dependent displacement and dynamics of the α-1 helix in kinesin revealed by site-directed spin labeling EPR

    SciTech Connect

    Yasuda, Satoshi; Yanagi, Takanori; Yamada, Masafumi D.; Ueki, Shoji; Maruta, Shinsaku; Inoue, Akio; Arata, Toshiaki

    2014-01-17

    Highlights: •Dipolar EPR detects the distance between the spin-labeled kinesin α-1 and α-2 helices. •The distance has at least two populations: 1.5 nm (in crystal form: 20%) and >2.5 nm. •The short distance conformer was populated 40% in the apo state with microtubules. •ATP analog or ADP binding caused the 1.5 nm distance to be less populated (∼20%). •The α-1 helix moves closer to the neck-linker (away from α-2) to facilitate docking. -- Abstract: In kinesin X-ray crystal structures, the N-terminal region of the α-1 helix is adjacent to the adenine ring of the bound nucleotide, while the C-terminal region of the helix is near the neck-linker (NL). Here, we monitor the displacement of the α-1 helix within a kinesin monomer bound to microtubules (MTs) in the presence or absence of nucleotides using site-directed spin labeling EPR. Kinesin was doubly spin-labeled at the α-1 and α-2 helices, and the resulting EPR spectrum showed dipolar broadening. The inter-helix distance distribution showed that 20% of the spins have a peak characteristic of 1.4–1.7 nm separation, which is similar to what is predicted from the X-ray crystal structure, albeit 80% were beyond the sensitivity limit (>2.5 nm) of the method. Upon MT binding, the fraction of kinesin exhibiting an inter-helix distance of 1.4–1.7 nm in the presence of AMPPNP (a non-hydrolysable ATP analog) and ADP was 20% and 25%, respectively. In the absence of nucleotide, this fraction increased to 40–50%. These nucleotide-induced changes in the fraction of kinesin undergoing displacement of the α-1 helix were found to be related to the fraction in which the NL undocked from the motor core. It is therefore suggested that a shift in the α-1 helix conformational equilibrium occurs upon nucleotide binding and release, and this shift controls NL docking onto the motor core.

  10. Mechanism of nucleotide sensing in group II chaperonins

    PubMed Central

    Pereira, Jose H; Ralston, Corie Y; Douglas, Nicholai R; Kumar, Ramya; Lopez, Tom; McAndrew, Ryan P; Knee, Kelly M; King, Jonathan A; Frydman, Judith; Adams, Paul D

    2012-01-01

    Group II chaperonins mediate protein folding in an ATP-dependent manner in eukaryotes and archaea. The binding of ATP and subsequent hydrolysis promotes the closure of the multi-subunit rings where protein folding occurs. The mechanism by which local changes in the nucleotide-binding site are communicated between individual subunits is unknown. The crystal structure of the archaeal chaperonin from Methanococcus maripaludis in several nucleotides bound states reveals the local conformational changes associated with ATP hydrolysis. Residue Lys-161, which is extremely conserved among group II chaperonins, forms interactions with the γ-phosphate of ATP but shows a different orientation in the presence of ADP. The loss of the ATP γ-phosphate interaction with Lys-161 in the ADP state promotes a significant rearrangement of a loop consisting of residues 160–169. We propose that Lys-161 functions as an ATP sensor and that 160–169 constitutes a nucleotide-sensing loop (NSL) that monitors the presence of the γ-phosphate. Functional analysis using NSL mutants shows a significant decrease in ATPase activity, suggesting that the NSL is involved in timing of the protein folding cycle. PMID:22193720

  11. Theoretical study on absorption and emission spectra of adenine analogues.

    PubMed

    Liu, Hongxia; Song, Qixia; Yang, Yan; Li, Yan; Wang, Haijun

    2014-04-01

    Fluorescent nucleoside analogues have attracted much attention in studying the structure and dynamics of nucleic acids in recent years. In the present work, we use theoretical calculations to investigate the structural and optical properties of four adenine analogues (termed as A1, A2, A3, and A4), and also consider the effects of aqueous solution and base pairing. The results show that the fluorescent adenine analogues can pair with thymine to form stable H-bonded WC base pairs. The excited geometries of both adenine analogues and WC base pairs are similar to the ground geometries. The absorption and emission maxima of adenine analogues are greatly red shifted compared with nature adenine, the oscillator strengths of A1 and A2 are stronger than A3 and A4 in both absorption and emission spectra. The calculated low-energy peaks in the absorption spectra are in good agreement with the experimental data. In general, the aqueous solution and base pairing can slightly red-shift both the absorption and emission maxima, and can increase the oscillator strengths of absorption spectra, but significantly decrease the oscillator strengths of A3 in emission spectra.

  12. DNA adenine hypomethylation leads to metabolic rewiring in Deinococcus radiodurans.

    PubMed

    Shaiwale, Nayana S; Basu, Bhakti; Deobagkar, Deepti D; Deobagkar, Dileep N; Apte, Shree K

    2015-08-01

    The protein encoded by DR_0643 gene from Deinococcus radiodurans was shown to be an active N-6 adenine-specific DNA methyltransferase (Dam). Deletion of corresponding protein reduced adenine methylation in the genome by 60% and resulted in slow-growth phenotype. Proteomic changes induced by DNA adenine hypomethylation were mapped by two-dimensional protein electrophoresis coupled with mass spectrometry. As compared to wild type D. radiodurans cells, at least 54 proteins were differentially expressed in Δdam mutant. Among these, 39 metabolic enzymes were differentially expressed in Δdam mutant. The most prominent change was DNA adenine hypomethylation induced de-repression of pyruvate dehydrogenase complex, E1 component (aceE) gene resulting in 10 fold increase in the abundance of corresponding protein. The observed differential expression profile of metabolic enzymes included increased abundance of enzymes involved in fatty acid and amino acid degradation to replenish acetyl Co-A and TCA cycle intermediates and diversion of phosphoenolpyruvate and pyruvate into amino acid biosynthesis, a metabolic rewiring attempt by Δdam mutant to restore energy generation via glycolysis-TCA cycle axis. This is the first report of DNA adenine hypomethylation mediated rewiring of metabolic pathways in prokaryotes.

  13. 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

    2012-09-03

    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.

  14. 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

  15. Nucleotide-metabolizing enzymes in Chlamydomonas flagella.

    PubMed

    Watanabe, T; Flavin, M

    1976-01-10

    Nucleotides have at least two functions in eukaryotic cilia and flagella. ATP, originating in the cells, is utilized for motility by energy-transducing protein(s) called dynein, and the binding of guanine nucleotides to tubulin, and probably certain transformations of the bound nucleotides, are prerequisites for the assembly of microtubules. Besides dynein, which can be solubulized from Chlamydomonas flagella as a heterogeneous, Mg2+ or Ca2+-activated ATPase, we have purified and characterized five other flagellar enzymes involved in nucleotide transformations. A homogeneous, low molecular weight, Ca2+-specific adenosine triphosphatase was isolated, which was inhibited by Mg2+ and was not specific for ATP. This enzyme was not formed by treating purified dynein with proteases. It was absent from extracts of Tetrahymena cilia. Its function might be an auxiliary energy transducer, or in steering or tactic responses. Two species of adenylate kinase were isolated, one of which was much elevated in regenerating flagella; the latter was also present in cell bodies. A large part of flagellar nucleoside diphosphokinase activity could not be solubilized. Two soluble enzyme species were identified, one of which was also present in cell bodies. Since these enzymes are of interest because they might function in microtubule assembly, we studied the extent to which brain nucleoside diphosphokinase co-polymerizes with tubulin purified by repeated cycles of polymerization. Arginine kinase was not detected in Chlamydomonas flagellar extracts. PMID:397

  16. ATP-dependent DNA binding, unwinding, and resection by the Mre11/Rad50 complex.

    PubMed

    Liu, Yaqi; Sung, Sihyun; Kim, Youngran; Li, Fuyang; Gwon, Gwanghyun; Jo, Aera; Kim, Ae-Kyoung; Kim, Taeyoon; Song, Ok-Kyu; Lee, Sang Eun; Cho, Yunje

    2016-04-01

    ATP-dependent DNA end recognition and nucleolytic processing are central functions of the Mre11/Rad50 (MR) complex in DNA double-strand break repair. However, it is still unclear how ATP binding and hydrolysis primes the MR function and regulates repair pathway choice in cells. Here,Methanococcus jannaschii MR-ATPγS-DNA structure reveals that the partly deformed DNA runs symmetrically across central groove between two ATPγS-bound Rad50 nucleotide-binding domains. Duplex DNA cannot access the Mre11 active site in the ATP-free full-length MR complex. ATP hydrolysis drives rotation of the nucleotide-binding domain and induces the DNA melting so that the substrate DNA can access Mre11. Our findings suggest that the ATP hydrolysis-driven conformational changes in both DNA and the MR complex coordinate the melting and endonuclease activity. PMID:26717941

  17. ATP-dependent DNA binding, unwinding, and resection by the Mre11/Rad50 complex.

    PubMed

    Liu, Yaqi; Sung, Sihyun; Kim, Youngran; Li, Fuyang; Gwon, Gwanghyun; Jo, Aera; Kim, Ae-Kyoung; Kim, Taeyoon; Song, Ok-Kyu; Lee, Sang Eun; Cho, Yunje

    2016-04-01

    ATP-dependent DNA end recognition and nucleolytic processing are central functions of the Mre11/Rad50 (MR) complex in DNA double-strand break repair. However, it is still unclear how ATP binding and hydrolysis primes the MR function and regulates repair pathway choice in cells. Here,Methanococcus jannaschii MR-ATPγS-DNA structure reveals that the partly deformed DNA runs symmetrically across central groove between two ATPγS-bound Rad50 nucleotide-binding domains. Duplex DNA cannot access the Mre11 active site in the ATP-free full-length MR complex. ATP hydrolysis drives rotation of the nucleotide-binding domain and induces the DNA melting so that the substrate DNA can access Mre11. Our findings suggest that the ATP hydrolysis-driven conformational changes in both DNA and the MR complex coordinate the melting and endonuclease activity.

  18. Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide

    NASA Astrophysics Data System (ADS)

    Freudenthal, Bret D.; Beard, William A.; Perera, Lalith; Shock, David D.; Kim, Taejin; Schlick, Tamar; Wilson, Samuel H.

    2015-01-01

    Oxidative stress promotes genomic instability and human diseases. A common oxidized nucleoside is 8-oxo-7,8-dihydro-2'-deoxyguanosine, which is found both in DNA (8-oxo-G) and as a free nucleotide (8-oxo-dGTP). Nucleotide pools are especially vulnerable to oxidative damage. Therefore cells encode an enzyme (MutT/MTH1) that removes free oxidized nucleotides. This cleansing function is required for cancer cell survival and to modulate Escherichia coli antibiotic sensitivity in a DNA polymerase (pol)-dependent manner. How polymerases discriminate between damaged and non-damaged nucleotides is not well understood. This analysis is essential given the role of oxidized nucleotides in mutagenesis, cancer therapeutics, and bacterial antibiotics. Even with cellular sanitizing activities, nucleotide pools contain enough 8-oxo-dGTP to promote mutagenesis. This arises from the dual coding potential where 8-oxo-dGTP(anti) base pairs with cytosine and 8-oxo-dGTP(syn) uses its Hoogsteen edge to base pair with adenine. Here we use time-lapse crystallography to follow 8-oxo-dGTP insertion opposite adenine or cytosine with human pol β, to reveal that insertion is accommodated in either the syn- or anti-conformation, respectively. For 8-oxo-dGTP(anti) insertion, a novel divalent metal relieves repulsive interactions between the adducted guanine base and the triphosphate of the oxidized nucleotide. With either templating base, hydrogen-bonding interactions between the bases are lost as the enzyme reopens after catalysis, leading to a cytotoxic nicked DNA repair intermediate. Combining structural snapshots with kinetic and computational analysis reveals how 8-oxo-dGTP uses charge modulation during insertion that can lead to a blocked DNA repair intermediate.

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

    PubMed Central

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

    2000-01-01

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

  20. Structural basis for removal of adenine mispaired with 8-oxoguanine by MutY adenine DNA glycosylase.

    PubMed

    Fromme, J Christopher; Banerjee, Anirban; Huang, Susan J; Verdine, Gregory L

    2004-02-12

    The genomes of aerobic organisms suffer chronic oxidation of guanine to the genotoxic product 8-oxoguanine (oxoG). Replicative DNA polymerases misread oxoG residues and insert adenine instead of cytosine opposite the oxidized base. Both bases in the resulting A*oxoG mispair are mutagenic lesions, and both must undergo base-specific replacement to restore the original C*G pair. Doing so represents a formidable challenge to the DNA repair machinery, because adenine makes up roughly 25% of the bases in most genomes. The evolutionarily conserved enzyme adenine DNA glycosylase (called MutY in bacteria and hMYH in humans) initiates repair of A*oxoG to C*G by removing the inappropriately paired adenine base from the DNA backbone. A central issue concerning MutY function is the mechanism by which A*oxoG mispairs are targeted among the vast excess of A*T pairs. Here we report the use of disulphide crosslinking to obtain high-resolution crystal structures of MutY-DNA lesion-recognition complexes. These structures reveal the basis for recognizing both lesions in the A*oxoG pair and for catalysing removal of the adenine base. PMID:14961129

  1. RESPIRATORY PATHWAYS IN THE MYCOPLASMA. II. PATHWAY OF ELECTRON TRANSPORT DURING OXIDATION OF REDUCED NICOTINAMIDE ADENINE DINUCLEOTIDE BY MYCOPLASMA HOMINIS.

    PubMed

    VANDEMARK, P J; SMITH, P F

    1964-07-01

    VanDemark, P. J. (University of South Dakota, Vermillion), and P. F. Smith. Respiratory pathways in the Mycoplasma. II. Pathway of electron transport during oxidation of reduced nicotinamide adenine dinucleotide by Mycoplasma hominis. J. Bacteriol. 88:122-129. 1964.-Unlike the flavin-terminated respiratory pathway of the fermentative Mycoplasma, the respiratory chain of the nonfermentative M. hominis strain 07 appears to be more complex, involving quinones and cytochromes in addition to flavins. In addition to reduction by reduced nicotine adenine dinucleotide (NADH) and reduced nicotine adenine dinucleotide phosphate, nonpyridine nucleotide-linked reduction of the respiratory chain of this organism occurred with succinate, lactate, and short-chained acyl coenzyme A derivatives as electron donors. Enzymes catalyzing the oxidation of NADH included an NADH oxidase, a diaphorase, a quinone reductase, and a cytochrome c reductase. The oxidation of NADH was sensitive to a variety of inhibitors, including 10(-4)m Atabrine, 10(-3)m sodium amytal, 10(-5)mp-chloromercuribenzoate, 10(-4)m antimycin A, and 10(-4)m potassium cyanide. The oxidase was resolved by the addition of 5% trichloroacetic acid and reactivated by the addition of flavin adenine dinucleotide but not flavin mononucleotide. The M. hominis sonic extract contained an NADH-coenzyme Q reductase. The oxidation of NADH was stimulated by the addition of either menadione or vitamin K(2) (C(35)). The oxidase was inactivated by extraction with ether or irradiation at 360 mmu. The ether-inactivated enzyme was partially reactivated by the addition of "lipid" extract of the enzyme and coenzyme Q(6). Difference spectra of the cell extracts revealed the presence of "b" and "a" type cytochromes. These cell extracts were found to contain a cyanide-and azide-sensitive cytochrome oxidase and catalase. PMID:14197876

  2. Absence of Ca2+-Induced Mitochondrial Permeability Transition but Presence of Bongkrekate-Sensitive Nucleotide Exchange in C. crangon and P. serratus

    PubMed Central

    Konrad, Csaba; Kiss, Gergely; Torocsik, Beata; Adam-Vizi, Vera; Chinopoulos, Christos

    2012-01-01

    Mitochondria from the embryos of brine shrimp (Artemia franciscana) do not undergo Ca2+-induced permeability transition in the presence of a profound Ca2+ uptake capacity. Furthermore, this crustacean is the only organism known to exhibit bongkrekate-insensitive mitochondrial adenine nucleotide exchange, prompting the conjecture that refractoriness to bongkrekate and absence of Ca2+-induced permeability transition are somehow related phenomena. Here we report that mitochondria isolated from two other crustaceans, brown shrimp (Crangon crangon) and common prawn (Palaemon serratus) exhibited bongkrekate-sensitive mitochondrial adenine nucleotide transport, but lacked a Ca2+-induced permeability transition. Ca2+ uptake capacity was robust in the absence of adenine nucleotides in both crustaceans, unaffected by either bongkrekate or cyclosporin A. Transmission electron microscopy images of Ca2+-loaded mitochondria showed needle-like formations of electron-dense material strikingly similar to those observed in mitochondria from the hepatopancreas of blue crab (Callinectes sapidus) and the embryos of Artemia franciscana. Alignment analysis of the partial coding sequences of the adenine nucleotide translocase (ANT) expressed in Crangon crangon and Palaemon serratus versus the complete sequence expressed in Artemia franciscana reappraised the possibility of the 208-214 amino acid region for conferring sensitivity to bongkrekate. However, our findings suggest that the ability to undergo Ca2+-induced mitochondrial permeability transition and the sensitivity of adenine nucleotide translocase to bongkrekate are not necessarily related phenomena. PMID:22768139

  3. Increase of reduced nicotinamide adenine dinucleotide fluorescence lifetime precedes mitochondrial dysfunction in staurosporine-induced apoptosis of HeLa cells

    NASA Astrophysics Data System (ADS)

    Yu, Jia-Sin; Guo, Han-Wen; Wang, Chih-Hao; Wei, Yau-Huei; Wang, Hsing-Wen

    2011-03-01

    In vivo noninvasive detection of apoptosis represents a new tool that may yield a more definite diagnosis, a more accurate prognosis, and help improve therapies for human diseases. The intrinsic fluorescence of reduced nicotinamide adenine dinucleotide (NADH) may be a potential optical biomarker for the apoptosis detection because NADH is involved in the respiration for the mitochondrial membrane potential (ΔΨ) formation and adenosine-5'-triphosphate (ATP) synthesis, and the depletion of ΔΨ and ATP level is the hallmark of apoptosis. We have previously observed the NADH fluorescence lifetime change is associated with staurosporine (STS)-induced mitochondria-mediated apoptosis. However, its relationship with mitochondrial functions such as ΔΨ, ATP, and oxygen consumption rate is not clear. In this study, we investigated this relationship. Our results indicate that the NADH fluorescence lifetime increased when ΔΨ and ATP levels were equal to or higher than their values of controls and decreased before the depletion of ΔΨ and ATP, and the oxygen consumption rate did not change. These findings suggest that the increased NADH fluorescence lifetime in STS-induced cell death occurred before the depletion of ΔΨ and ATP and activation of caspase 3, and was not simply caused by cellular metabolic change. Furthermore, the NADH fluorescence lifetime change is associated with the pace of apoptosis.

  4. ATP as a cotransmitter in the autonomic nervous system.

    PubMed

    Kennedy, Charles

    2015-09-01

    The role of adenosine 5'-triphosphate (ATP) as a major intracellular energy source is well-established. In addition, ATP and related nucleotides have widespread extracellular actions via the ionotropic P2X (ligand-gated cation channels) and metabotropic P2Y (G protein-coupled) receptors. Numerous experimental techniques, including myography, electrophysiology and biochemical measurement of neurotransmitter release, have been used to show that ATP has several major roles as a neurotransmitter in peripheral nerves. When released from enteric nerves of the gastrointestinal tract it acts as an inhibitory neurotransmitter, mediating descending muscle relaxation during peristalsis. ATP is also an excitatory cotransmitter in autonomic nerves; 1) It is costored with noradrenaline in synaptic vesicles in postganglionic sympathetic nerves innervating smooth muscle preparations, such as the vas deferens and most arteries. When coreleased with noradrenaline, ATP acts at postjunctional P2X1 receptors to evoke depolarisation, Ca(2+) influx, Ca(2+) sensitisation and contraction. 2) ATP is also coreleased with acetylcholine from postganglionic parasympathetic nerves innervating the urinary bladder and again acts at postjunctional P2X1 receptors, and possibly also a P2X1+4 heteromer, to elicit smooth muscle contraction. In both cases the neurotransmitter actions of ATP are terminated by dephosphorylation by extracellular, membrane-bound enzymes and soluble nucleotidases released from postganglionic nerves. There are indications of an increased contribution of ATP to control of blood pressure in hypertension, but further research is needed to clarify this possibility. More promising is the upregulation of P2X receptors in dysfunctional bladder, including interstitial cystitis, idiopathic detrusor instability and overactive bladder syndrome. Consequently, these roles of ATP are of great therapeutic interest and are increasingly being targeted by pharmaceutical companies.

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

    PubMed

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

    2015-11-01

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

  6. Curtains for ATP?

    NASA Astrophysics Data System (ADS)

    The administration's efforts to keep various technology-transfer programs afloat in the budget process appear to be stalled. House Science Committee chair Robert Walker (R-Pa.) advised in early April that the Republican agenda for the pending budget process entails zeroing out the Commerce Department's Advanced Technology Program (ATP), which was funded at 431 million in fiscal year 1995. The ATP would lose about 90 million from its FY 95 budget. Although Walker says that the Republican leadership has no intention to dictate to the subcommittees how cuts should be made, they will be held to the "fairly severe caps" established by the House Budget Committee. In other words, Walker says, if ATP stays, something else will have to go in its place. In addition, a bill to rescind about 223 million from the FY 1995 budget of the Technology Reinvestment Project and another 77 million from TRP's FY 1994 budget, which has not been spent, is heading for the president's signature. Yet Walker says while he supports the merits of technology transfer, "the question is do you have to create government programs to get the technology out?"

  7. Analysis of Electric Properties of DNA Nucleotides

    NASA Astrophysics Data System (ADS)

    Zikic, R.; Zhang, X.-G.; Krstic, P. S.; Wells, J. C.; Fuentes-Cabrera, M.

    2006-05-01

    Calculation of the quantum tunnelling conductance through the DNA nucleotides between gold nanoelectrodes and analysis of the corresponding molecular spectra reveals that the tunneling conductance at low electric bias can be separated into two simple and approximately independent factors. The first is an exponential factor due to the potential barrier between the molecule and the electrode. The second factor is different for each molecule, but follows a universal form that can be expressed in terms of the bending angle of the DNA base relative to the sugar-phosphate group. This factor is also oscillatory indicating interference and resonance effects inside the molecule. Distinguishable conductances of Adenine (A), Cytosine (C), Guanine (G), and Thymine (T) are correlated to their differences in geometric dimensions.

  8. Receptor binding of somatostatin-14 and somatostatin-28 in rat brain: differential modulation by nucleotides and ions.

    PubMed

    Srikant, C B; Dahan, A; Craig, C

    1990-02-01

    The tissue-selective binding of the two principal bioactive forms of somatostatin, somatostatin-14 (SS-14) and somatostatin-28 (SS-28), their ability to modulate cAMP-dependent and -independent regulation of post-receptor events to different degrees and the documentation of specific labelling of SS receptor subtypes with SS-28 but not SS-14 in discrete regions of rat brain suggest the existence of distinct SS-14 and SS-28 binding sites. Receptor binding of SS-14 ligands has been shown to be modulated by nucleotides and ions, but the effect of these agents on SS-28 binding has not been studied. In the present study we investigated the effects of adenine and guanine nucleotides as well as monovalent and divalent cations on rat brain SS receptors quantitated with radioiodinated analogs of SS-14 ([125I-Tyr11]SS14, referred to in this paper as SS-14) and SS-28 ([Leu8, D-Trp22, 125I-Tyr25] SS-28, referred to as LTT* SS-28) in order to determine if distinct receptor sites for SS-14 and SS-28 could be distinguished on the basis of their modulation by nucleotides and ions. GTP as well as ATP exerted a dose-dependent inhibition (over a concentration range of 10(-7)-10(-3) M) of the binding of the two radioligands. The nucleotide inhibition of binding resulted in a decrease the Bmax of the SS receptors, the binding affinity remaining unaltered. GTP (10(-4) M) decreased the Bmax of LTT* SS-28 binding sites to a greater extent than ATP (145 +/- 10 and 228 +/- 16 respectively, compared to control value of 320 +/- 20 pmol mg-1). Under identical conditions GTP was less effective than ATP in reducing the number of T* SS-14 binding sites (Bmax = 227 +/- 8 and 182 +/- 15, respectively, compared to 340 +/- 15 pmol mg-1 in the absence of nucleotides). Monovalent cations inhibited the binding of both radioligands, Li+ and Na+ inhibited the binding of T* SS-14 to a greater extent than K+. The effect of divalent cations on the other hand was varied. At low concentration (2 mM) Mg2+, Ba2

  9. Detection of electronically equivalent tautomers of adenine base: DFT study

    SciTech Connect

    Siddiqui, Shamoon Ahmad; Bouarissa, Nadir; Rasheed, Tabish; Al-Assiri, M.S.; Al-Hajry, A.

    2014-03-01

    Graphical abstract: - Highlights: • DFT calculations have been performed on adenine and its rare tautomer Cu{sup 2+} complexes. • Interaction of A-Cu{sup 2+} and rA-Cu{sup 2+} complexes with AlN modified fullerene (C{sub 60}) have been studied briefly. • It is found that AlN modified C{sub 60} could be used as a nanoscale sensor to detect these two A-Cu{sup 2+} and rA-Cu{sup 2+} complexes. - Abstract: In the present study, quantum chemical calculations were carried out to investigate the electronic structures and stabilities of adenine and its rare tautomer along with their Cu{sup 2+} complexes. Density Functional Theory (B3LYP method) was used in all calculations. The two Cu{sup 2+} complexes of adenine have almost similar energies and electronic structures; hence, their chemical differentiation is very difficult. For this purpose, interactions of these complexes with AlN modified fullerene (C{sub 60}) have been studied. Theoretical investigations reveal that AlN-doped C{sub 60} may serve as a potentially viable nanoscale sensor for detection of the two Cu{sup 2+} complexes of adenine.

  10. Thymine, adenine and lipoamino acid based gene delivery systems.

    PubMed

    Skwarczynski, Mariusz; Ziora, Zyta M; Coles, Daniel J; Lin, I-Chun; Toth, Istvan

    2010-05-14

    A novel class of thymine, adenine and lipoamino acid based non-viral carriers for gene delivery has been developed. Their ability to bind to DNA by hydrogen bonding was confirmed by NMR diffusion, isothermal titration calorimetry and transmission electron microscopy experiments.

  11. PolyAdenine cryogels for fast and effective RNA purification.

    PubMed

    Köse, Kazım; Erol, Kadir; Özgür, Erdoğan; Uzun, Lokman; Denizli, Adil

    2016-10-01

    Cryogels are used effectively for many diverse applications in a variety of fields. The isolation or purification of RNA, one of the potential utilizations for cryogels, is crucial due to their vital roles such as encoding, decoding, transcription and translation, and gene expression. RNA principally exists within every living thing, but their tendency to denaturation easily is still the most challenging issue. Herein, we aimed to develop adenine incorporated polymeric cryogels as an alternative sorbent for cost-friendly and fast RNA purification with high capacity. For this goal, we synthesized the polymerizable derivative of adenine called as adenine methacrylate (AdeM) through the substitution reaction between adenine and methacryloyl chloride. Then, 2-hydroxyethyl methacrylate (HEMA)-based cryogels were prepared in a partially frozen aqueous medium by copolymerization of monomers, AdeM, and HEMA. The cryogels were characterized by using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), surface area measurements, thermogravimetric analysis (TGA), and swelling tests. RNA adsorption experiments were performed via batch system while varying different conditions including pH, initial RNA concentration, temperature, and interaction time. We achieved high RNA adsorption capacity of cryogels, with the swelling ratio around 510%, as 11.86mg/g. The cryogels might be reused at least five times without significant decrease in adsorption capacity.

  12. PolyAdenine cryogels for fast and effective RNA purification.

    PubMed

    Köse, Kazım; Erol, Kadir; Özgür, Erdoğan; Uzun, Lokman; Denizli, Adil

    2016-10-01

    Cryogels are used effectively for many diverse applications in a variety of fields. The isolation or purification of RNA, one of the potential utilizations for cryogels, is crucial due to their vital roles such as encoding, decoding, transcription and translation, and gene expression. RNA principally exists within every living thing, but their tendency to denaturation easily is still the most challenging issue. Herein, we aimed to develop adenine incorporated polymeric cryogels as an alternative sorbent for cost-friendly and fast RNA purification with high capacity. For this goal, we synthesized the polymerizable derivative of adenine called as adenine methacrylate (AdeM) through the substitution reaction between adenine and methacryloyl chloride. Then, 2-hydroxyethyl methacrylate (HEMA)-based cryogels were prepared in a partially frozen aqueous medium by copolymerization of monomers, AdeM, and HEMA. The cryogels were characterized by using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), surface area measurements, thermogravimetric analysis (TGA), and swelling tests. RNA adsorption experiments were performed via batch system while varying different conditions including pH, initial RNA concentration, temperature, and interaction time. We achieved high RNA adsorption capacity of cryogels, with the swelling ratio around 510%, as 11.86mg/g. The cryogels might be reused at least five times without significant decrease in adsorption capacity. PMID:27434154

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

    PubMed Central

    Zalk, Ran; Shoshan-Barmatz, Varda

    2003-01-01

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

  14. ATP-dependent degradation of ubiquitin-protein conjugates.

    PubMed Central

    Hershko, A; Leshinsky, E; Ganoth, D; Heller, H

    1984-01-01

    Previous studies have indicated that the ATP-requiring conjugation of ubiquitin with proteins plays a role in the energy-dependent degradation of intracellular proteins. To examine whether such conjugates are indeed intermediates in protein breakdown, conjugates of 125I-labeled lysozyme with ubiquitin were isolated and incubated with a fraction of reticulocyte extract that lacks the enzymes that carry out ubiquitin-protein conjugation. ATP markedly stimulated degradation of the lysozyme moiety of ubiquitin conjugates to products soluble in trichloroacetic acid. By contrast, free 125I-labeled lysozyme was not degraded under these conditions, unless ubiquitin and the three enzymes required for ubiquitin conjugation were supplemented. Mg2+ was absolutely required for conjugate breakdown. Of various nucleotides, only CTP replaced ATP. Nonhydrolyzable analogs of ATP were not effective. In the absence of ATP, free lysozyme is released from ubiquitin-lysozyme conjugates by isopeptidases present in the extract. Thus, ATP is involved in both the formation and the breakdown of ubiquitin-protein conjugates. Images PMID:6324208

  15. Catalytic Mechanism and Three-Dimensional Structure of Adenine Deaminase

    SciTech Connect

    Kamat, S.S.; Swaminathan, S.; Bagaria, A.; Kumaran, D.; Holmes-Hampton, G. P.; Fan, H.; Sali, A.; Sauder, J. M.; Burley, S. K.; Lindahl, P. A.; Raushel, F. M.

    2011-03-22

    Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine and ammonia. The enzyme isolated from Escherichia coli using standard expression conditions was low for the deamination of adenine (k{sub cat} = 2.0 s{sup -1}; k{sub cat}/K{sub m} = 2.5 x 10{sup 3} M{sup -1} s{sup -1}). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn{sup 2+} prior to induction, the purified enzyme was substantially more active for the deamination of adenine with kcat and kcat/Km values of 200 s{sup -1} and 5 x 10{sup 5} M{sup -1} s{sup -1}, respectively. The apoenzyme was prepared and reconstituted with Fe{sup 2+}, Zn{sup 2+}, or Mn{sup 2+}. In each case, two enzyme equivalents of metal were necessary for reconstitution of the deaminase activity. This work provides the first example of any member of the deaminase subfamily of the amidohydrolase superfamily to utilize a binuclear metal center for the catalysis of a deamination reaction. [Fe{sup II}/Fe{sup II}]-ADE was oxidized to [Fe{sup III}/Fe{sup III}]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [Fe{sup III}/Fe{sup III}]-ADE with dithionite restored the deaminase activity, and thus, the diferrous form of the enzyme is essential for catalytic activity. No evidence of spin coupling between metal ions was evident by electron paramagnetic resonance or Moessbauer spectroscopy. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by X-ray crystallography at 2.2 {angstrom} resolution, and adenine was modeled into the active site on the basis of homology to other members of the amidohydrolase superfamily. On the basis of the model of the adenine-ADE complex and subsequent mutagenesis experiments, the roles for each of the highly conserved residues were proposed. Solvent isotope effects, pH-rate profiles, and solvent viscosity were utilized to propose a chemical reaction mechanism and the

  16. Catalytic Mechanism and Three-Dimensional Structure of Adenine Deaminase

    SciTech Connect

    S Kamat; A Bagaria; D Kumaran; G Holmes-Hampton; H Fan; A Sali; J Sauder; S Burley; P Lindahl; et. al.

    2011-12-31

    Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine and ammonia. The enzyme isolated from Escherichia coli using standard expression conditions was low for the deamination of adenine (k{sub cat} = 2.0 s{sup -1}; k{sub cat}/K{sub m} = 2.5 x 10{sup 3} M{sup -1} s{sup -1}). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn{sup 2+} prior to induction, the purified enzyme was substantially more active for the deamination of adenine with k{sub cat} and k{sub cat}/K{sub m} values of 200 s{sup -1} and 5 x 10{sup 5} M{sup -1} s{sup -1}, respectively. The apoenzyme was prepared and reconstituted with Fe{sup 2+}, Zn{sup 2+}, or Mn{sup 2+}. In each case, two enzyme equivalents of metal were necessary for reconstitution of the deaminase activity. This work provides the first example of any member of the deaminase subfamily of the amidohydrolase superfamily to utilize a binuclear metal center for the catalysis of a deamination reaction. [Fe{sup II}/Fe{sup II}]-ADE was oxidized to [Fe{sup III}/Fe{sup III}]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [Fe{sup III}/Fe{sup III}]-ADE with dithionite restored the deaminase activity, and thus, the diferrous form of the enzyme is essential for catalytic activity. No evidence of spin coupling between metal ions was evident by electron paramagnetic resonance or Moessbauer spectroscopy. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by X-ray crystallography at 2.2 {angstrom} resolution, and adenine was modeled into the active site on the basis of homology to other members of the amidohydrolase superfamily. On the basis of the model of the adenine-ADE complex and subsequent mutagenesis experiments, the roles for each of the highly conserved residues were proposed. Solvent isotope effects, pH-rate profiles, and solvent viscosity were utilized to propose a chemical reaction

  17. 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.

  18. Nucleotide homeostasis and purinergic nociceptive signaling in rat meninges in migraine-like conditions.

    PubMed

    Yegutkin, Gennady G; Guerrero-Toro, Cindy; Kilinc, Erkan; Koroleva, Kseniya; Ishchenko, Yevheniia; Abushik, Polina; Giniatullina, Raisa; Fayuk, Dmitriy; Giniatullin, Rashid

    2016-09-01

    Extracellular ATP is suspected to contribute to migraine pain but regulatory mechanisms controlling pro-nociceptive purinergic mechanisms in the meninges remain unknown. We studied the peculiarities of metabolic and signaling pathways of ATP and its downstream metabolites in rat meninges and in cultured trigeminal cells exposed to the migraine mediator calcitonin gene-related peptide (CGRP). Under resting conditions, meningeal ATP and ADP remained at low nanomolar levels, whereas extracellular AMP and adenosine concentrations were one-two orders higher. CGRP increased ATP and ADP levels in meninges and trigeminal cultures and reduced adenosine concentration in trigeminal cells. Degradation rates for exogenous nucleotides remained similar in control and CGRP-treated meninges, indicating that CGRP triggers nucleotide release without affecting nucleotide-inactivating pathways. Lead nitrate-based enzyme histochemistry of whole mount meninges revealed the presence of high ATPase, ADPase, and AMPase activities, primarily localized in the medial meningeal artery. ATP and ADP induced large intracellular Ca(2+) transients both in neurons and in glial cells whereas AMP and adenosine were ineffective. In trigeminal glia, ATP partially operated via P2X7 receptors. ATP, but not other nucleotides, activated nociceptive spikes in meningeal trigeminal nerve fibers providing a rationale for high degradation rate of pro-nociceptive ATP. Pro-nociceptive effect of ATP in meningeal nerves was reproduced by α,β-meATP operating via P2X3 receptors. Collectively, extracellular ATP, which level is controlled by CGRP, can persistently activate trigeminal nerves in meninges which considered as the origin site of migraine headache. These data are consistent with the purinergic hypothesis of migraine pain and suggest new targets against trigeminal pain.

  19. 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.

  20. Modeling regulation of cardiac KATP and L-type Ca2+ currents by ATP, ADP, and Mg2+.

    PubMed

    Michailova, Anushka; Saucerman, Jeffrey; Belik, Mary Ellen; McCulloch, Andrew D

    2005-03-01

    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. PMID:15738467

  1. 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.

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

    PubMed Central

    O’Mara, Megan L.; Mark, Alan E.

    2014-01-01

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

  3. Deciphering the molecular basis for nucleotide selection by the West Nile virus RNA helicase

    PubMed Central

    Despins, Simon; Issur, Moheshwarnath; Bougie, Isabelle; Bisaillon, Martin

    2010-01-01

    The West Nile virus RNA helicase uses the energy derived from the hydrolysis of nucleotides to separate complementary strands of RNA. Although this enzyme has a preference for ATP, the bias towards this purine nucleotide cannot be explained on the basis of specific protein–ATP interactions. Moreover, the enzyme does not harbor the characteristic Q-motif found in other helicases that regulates binding to ATP. In the present study, we used structural homology modeling to generate a model of the West Nile virus RNA helicase active site that provides instructive findings on the interaction between specific amino acids and the ATP substrate. In addition, we evaluated both the phosphohydrolysis and the inhibitory potential of a collection of 30 synthetic purine analogs. A structure-guided alanine scan of 16 different amino acids was also performed to clarify the contacts that are made between the enzyme and ATP. Our study provides a molecular rationale for the bias of the enzyme for ATP by highlighting the specific functional groups on ATP that are important for binding. Moreover, we identified three new essential amino acids (Arg-185, Arg-202 and Asn-417) that are critical for phosphohydrolysis. Finally, we provide evidence that a region located upstream of motif I, which we termed the nucleotide specificity region, plays a functional role in nucleotide selection which is reminiscent to the role exerted by the Q-motif found in other helicases. PMID:20421212

  4. Dorsal horn neurons release extracellular ATP in a VNUT-dependent manner that underlies neuropathic pain.

    PubMed

    Masuda, Takahiro; Ozono, Yui; Mikuriya, Satsuki; Kohro, Yuta; Tozaki-Saitoh, Hidetoshi; Iwatsuki, Ken; Uneyama, Hisayuki; Ichikawa, Reiko; Salter, Michael W; Tsuda, Makoto; Inoue, Kazuhide

    2016-01-01

    Activation of purinergic receptors in the spinal cord by extracellular ATP is essential for neuropathic hypersensitivity after peripheral nerve injury (PNI). However, the cell type responsible for releasing ATP within the spinal cord after PNI is unknown. Here we show that PNI increases expression of vesicular nucleotide transporter (VNUT) in the spinal cord. Extracellular ATP content ([ATP]e) within the spinal cord was increased after PNI, and this increase was suppressed by exocytotic inhibitors. Mice lacking VNUT did not show PNI-induced increase in [ATP]e and had attenuated hypersensitivity. These phenotypes were recapitulated in mice with specific deletion of VNUT in spinal dorsal horn (SDH) neurons, but not in mice lacking VNUT in primary sensory neurons, microglia or astrocytes. Conversely, ectopic VNUT expression in SDH neurons of VNUT-deficient mice restored PNI-induced increase in [ATP]e and pain. Thus, VNUT is necessary for exocytotic ATP release from SDH neurons which contributes to neuropathic pain. PMID:27515581

  5. Dorsal horn neurons release extracellular ATP in a VNUT-dependent manner that underlies neuropathic pain

    PubMed Central

    Masuda, Takahiro; Ozono, Yui; Mikuriya, Satsuki; Kohro, Yuta; Tozaki-Saitoh, Hidetoshi; Iwatsuki, Ken; Uneyama, Hisayuki; Ichikawa, Reiko; Salter, Michael W.; Tsuda, Makoto; Inoue, Kazuhide

    2016-01-01

    Activation of purinergic receptors in the spinal cord by extracellular ATP is essential for neuropathic hypersensitivity after peripheral nerve injury (PNI). However, the cell type responsible for releasing ATP within the spinal cord after PNI is unknown. Here we show that PNI increases expression of vesicular nucleotide transporter (VNUT) in the spinal cord. Extracellular ATP content ([ATP]e) within the spinal cord was increased after PNI, and this increase was suppressed by exocytotic inhibitors. Mice lacking VNUT did not show PNI-induced increase in [ATP]e and had attenuated hypersensitivity. These phenotypes were recapitulated in mice with specific deletion of VNUT in spinal dorsal horn (SDH) neurons, but not in mice lacking VNUT in primary sensory neurons, microglia or astrocytes. Conversely, ectopic VNUT expression in SDH neurons of VNUT-deficient mice restored PNI-induced increase in [ATP]e and pain. Thus, VNUT is necessary for exocytotic ATP release from SDH neurons which contributes to neuropathic pain. PMID:27515581

  6. Copper-Adenine Complex Catalyst for O2 Production from

    NASA Astrophysics Data System (ADS)

    Vergne, Jacques; Bruston, F.; Calvayrac, R.; Grajcar, L.; Baron, M.-H.; Maurel, M.-C.

    The advent of oxygen-evolving photosynthesis is one of the central event in the development of life on earth. The early atmosphere has been midly reducing or neutral in overall redox balance and water photolysis by UV light can produce hydrogen peroxide. Before oxidation of water, intermediate stages are proposed in which H_2^O_2 was oxidized. The oxidation of H_2^O_2 to oxygen can be carried out by a modestly oxidizing species in which a metal-catalase like enzyme could extract electrons from H_2^O_2 producing the first oxygen-evolving complex. After what, modern photosynthesis with chlorophyll, to help transform H_2^O in O_2 was ready to come to light. In preliminary UV studies we were able to show that [Cu(adenine)2] system, containing copper coordinated to nitrogen activates H_2^O_2 disappearance. This was confirmed with the help of Raman and polarographic studies. Raman spectroscopy shows the formation of [Cu(adenine)2] complex in solution, quantifies H_2^O_2 consumption, polarography quantifies O_2 production. In both cases CuCl_2 addition entails H_2^O_2 disappearance. Without adenine, Cu_2^+ has only a weak catalytic effect. The molar activity of the [Cu(adenine)2] complex is much larger and concentration dependent. We emphasize that Cu(adenine)2 may have mimicked enzyme properties in the first stage of life evolution, in order to split H_2^O_2 into O_2 and H_2^O. Moreover, diluted copper and adenine, in small ephemeral prebiotic ponds , could have preserved biologically active entities from H_2^O_2 damage via dual properties: catalyzing H_2^O_2 disproportionation and also directly acting as a reductant complex. Finally, the present Mars surface is considered to be both reactive and embedded with oxydants. As it has been shown that the depth of diffusion for H_2^O_2 is less than 3 meters, it is important to study all the ways of H_2^O_2 consumption.

  7. BF0801, a novel adenine derivative, inhibits platelet activation via phosphodiesterase inhibition and P2Y12 antagonism.

    PubMed

    Zhang, Si; Hu, Liang; Du, Hongguang; Guo, Yan; Zhang, Yan; Niu, Haixia; Jin, Jianguo; Zhang, Jian; Liu, Junling; Zhang, Xiaohui; Kunapuli, Satya P; Ding, Zhongren

    2010-10-01

    Though antiplatelet drugs are proven beneficial to patients with coronary heart disease and stroke, more effective and safer antiplatelet drugs are still needed. In this study we report the antiplatelet effects and mechanism of BF0801, a novel adenine derivative. BF0801 dramatically inhibited platelet aggregation and ATP release induced by ADP, 2MeSADP, AYPGKF, SFLLRN or convulxin without affecting shape change in vitro . It also potentiated the inhibitory effects of adenosine-based P2Y12 antagonist AR-C69931MX or phosphodiesterase (PDE) inhibitor IBMX on platelet aggregation. The cAMP levels in both resting and forskolin-stimulated platelets were increased by BF0801 suggesting its PDE inhibitor activity, which is further confirmed by the concentration-dependent suppression of BF0801 on the native and recombinant PDE. Similar to AR-C69931MX, BF0801 drastically inhibited 2MeSADP- induced adenylyl cyclase inhibition in platelets indicating its P2Y12 antagonism activity, which is substantiated by the inhibition of BF0801 on the interaction between ADP and P2Y12 receptor expressed in CHO-K1 cells measured by atomic force microscopy. Moreover, we confirmed the antiplatelet effects of BF0801 using platelets from rats intravenously given BF0801. In summary, for the first time we developed a novel adenine derivative bearing dual activities of PDE inhibition and P2Y12 antagonism, which may have therapeutic advantage as a potential antithrombotic drug. PMID:20806121

  8. Reduction of nicotinamide adenine dinucleotide by pyruvate:lipoate oxidoreductase in anaerobic, dark-grown Rhodospirillum rubrum mutant C.

    PubMed Central

    Gorrell, T E; Uffen, R L

    1978-01-01

    Cell extracts from fermentatively grown Rhodospirillum rubrum reduced about 80 nmol of nicotinamide adenine dinucleotide (NAD) per mg of protein per min under anaerobic conditions with sodium pyruvate. The reaction was specific for pyruvate and NAD; NAD phosphate was not reduced. Results indicated that pyruvate-linked NAD reduction occurred via pyruvate:lipoate oxidoreductase. The reaction required catalytic amounts of both coenzyme A and thiamine pyrophosphate. Addition of sodium arsenite inhibited enzyme activity by 90%. Pyruvate:lipoate oxidoreductase was the only system detected in anaerobic, dark-grown R. rubrum cell extracts which operated to produce reduced NAD. The low activity of the enzyme system suggested that it was not quantitatively important in ATP formation. PMID:207677

  9. ATP release through pannexon channels.

    PubMed

    Dahl, Gerhard

    2015-07-01

    Extracellular adenosine triphosphate (ATP) serves as a signal for diverse physiological functions, including spread of calcium waves between astrocytes, control of vascular oxygen supply and control of ciliary beat in the airways. ATP can be released from cells by various mechanisms. This review focuses on channel-mediated ATP release and its main enabler, Pannexin1 (Panx1). Six subunits of Panx1 form a plasma membrane channel termed 'pannexon'. Depending on the mode of stimulation, the pannexon has large conductance (500 pS) and unselective permeability to molecules less than 1.5 kD or is a small (50 pS), chloride-selective channel. Most physiological and pathological stimuli induce the large channel conformation, whereas the small conformation so far has only been observed with exclusive voltage activation of the channel. The interaction between pannexons and ATP is intimate. The pannexon is not only the conduit for ATP, permitting ATP efflux from cells down its concentration gradient, but the pannexon is also modulated by ATP. The channel can be activated by ATP through both ionotropic P2X as well as metabotropic P2Y purinergic receptors. In the absence of a control mechanism, this positive feedback loop would lead to cell death owing to the linkage of purinergic receptors with apoptotic processes. A control mechanism preventing excessive activation of the purinergic receptors is provided by ATP binding (with low affinity) to the Panx1 protein and gating the channel shut. PMID:26009770

  10. Quencher-free molecular aptamer beacons (QF-MABs) for detection of ATP.

    PubMed

    Park, Jung Woo; Park, Yoojin; Kim, Byeang Hyean

    2015-10-15

    We have constructed a simple and efficient system-based on quencher-free molecular aptamer beacons (QF-MABs)-for probing ATP. In the absence of ATP, the fluorescence of a pyrene fluorophore on the loop position (15 nucleotides from the 5' end) of the optimal QF-MAB was quenched by the neighboring nucleobases; in its presence, fluorescence was recovered, due to a conformational change in the secondary structure of the QF-MAB.

  11. Caffeine inhibits glucose transport by binding at the GLUT1 nucleotide-binding site.

    PubMed

    Sage, Jay M; Cura, Anthony J; Lloyd, Kenneth P; Carruthers, Anthony

    2015-05-15

    Glucose transporter 1 (GLUT1) is the primary glucose transport protein of the cardiovascular system and astroglia. A recent study proposes that caffeine uncompetitive inhibition of GLUT1 results from interactions at an exofacial GLUT1 site. Intracellular ATP is also an uncompetitive GLUT1 inhibitor and shares structural similarities with caffeine, suggesting that caffeine acts at the previously characterized endofacial GLUT1 nucleotide-binding site. We tested this by confirming that caffeine uncompetitively inhibits GLUT1-mediated 3-O-methylglucose uptake in human erythrocytes [Vmax and Km for transport are reduced fourfold; Ki(app) = 3.5 mM caffeine]. ATP and AMP antagonize caffeine inhibition of 3-O-methylglucose uptake in erythrocyte ghosts by increasing Ki(app) for caffeine inhibition of transport from 0.9 ± 0.3 mM in the absence of intracellular nucleotides to 2.6 ± 0.6 and 2.4 ± 0.5 mM in the presence of 5 mM intracellular ATP or AMP, respectively. Extracellular ATP has no effect on sugar uptake or its inhibition by caffeine. Caffeine and ATP displace the fluorescent ATP derivative, trinitrophenyl-ATP, from the GLUT1 nucleotide-binding site, but d-glucose and the transport inhibitor cytochalasin B do not. Caffeine, but not ATP, inhibits cytochalasin B binding to GLUT1. Like ATP, caffeine renders the GLUT1 carboxy-terminus less accessible to peptide-directed antibodies, but cytochalasin B and d-glucose do not. These results suggest that the caffeine-binding site bridges two nonoverlapping GLUT1 endofacial sites-the regulatory, nucleotide-binding site and the cytochalasin B-binding site. Caffeine binding to GLUT1 mimics the action of ATP but not cytochalasin B on sugar transport. Molecular docking studies support this hypothesis.

  12. Dynamics and reactivity in Thermus aquaticus N6-adenine methyltransferase.

    PubMed

    Aranda, Juan; Zinovjev, Kirill; Roca, Maite; Tuñón, Iñaki

    2014-11-19

    M.TaqI is a DNA methyltransferase from Thermus aquaticus that catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the N6 position of an adenine, a process described only in prokaryotes. We have used full atomistic classical molecular dynamics simulations to explore the protein-SAM-DNA ternary complex where the target adenine is flipped out into the active site. Key protein-DNA interactions established by the target adenine in the active site are described in detail. The relaxed structure was used for a combined quantum mechanics/molecular mechanics exploration of the reaction mechanism using the string method. According to our free energy calculations the reaction takes place through a stepwise mechanism where the methyl transfer precedes the abstraction of the proton from the exocyclic amino group. The methyl transfer is the rate-determining step, and the obtained free energy barrier is in good agreement with the value derived from the experimental rate constant. Two possible candidates to extract the leftover proton have been explored: a water molecule found in the active site and Asn105, a residue activated by the hydrogen bonds formed through the amide hydrogens. The barrier for the proton abstraction is smaller when Asn105 acts as a base. The reaction mechanisms can be different in other N6-DNA-methyltransferases, as determined from the exploration of the reaction mechanism in the Asn105Asp M.TaqI mutant. PMID:25347783

  13. High resolution dissociative electron attachment to gas phase adenine

    SciTech Connect

    Huber, D.; Beikircher, M.; Denifl, S.; Zappa, F.; Matejcik, S.; Bacher, A.; Grill, V.; Maerk, T. D.; Scheier, P.

    2006-08-28

    The dissociative electron attachment to the gas phase nucleobase adenine is studied using two different experiments. A double focusing sector field mass spectrometer is utilized for measurements requiring high mass resolution, high sensitivity, and relative ion yields for all the fragment anions and a hemispherical electron monochromator instrument for high electron energy resolution. The negative ion mass spectra are discussed at two different electron energies of 2 and 6 eV. In contrast to previous gas phase studies a number of new negative ions are discovered in the mass spectra. The ion efficiency curves for the negative ions of adenine are measured for the electron energy range from about 0 to 15 eV with an electron energy resolution of about 100 meV. The total anion yield derived via the summation of all measured fragment anions is compared with the total cross section for negative ion formation measured recently without mass spectrometry. For adenine the shape of the two cross section curves agrees well, taking into account the different electron energy resolutions; however, for thymine some peculiar differences are observed.

  14. Fragmentation mechanisms of cytosine, adenine and guanine ionized bases.

    PubMed

    Sadr-Arani, Leila; Mignon, Pierre; Chermette, Henry; Abdoul-Carime, Hassan; Farizon, Bernadette; Farizon, Michel

    2015-05-01

    The different fragmentation channels of cytosine, adenine and guanine have been studied through DFT calculations. The electronic structure of bases, their cations, and the fragments obtained by breaking bonds provides a good understanding of the fragmentation process that can complete the experimental approach. The calculations allow assigning various fragments to the given peaks. The comparison between the energy required for the formation of fragments and the peak intensity in the mass spectrum is used. For cytosine and guanine the elimination of the HNCO molecule is a major route of dissociation, while for adenine multiple loss of HCN or HNC can be followed up to small fragments. For cytosine, this corresponds to the initial bond cleavage of N3-C4/N1-C2, which represents the main dissociation route. For guanine the release of HNCO is obtained through the N1-C2/C5-C6 bond cleavage (reverse order also possible) leading to the largest peak of the spectrum. The corresponding energies of 3.5 and 3.9 eV are typically in the range available in the experiments. The loss of NH3 or HCN is also possible but requires more energy. For adenine, fragmentation consists of multiple loss of the HCN molecule and the main route corresponding to HC8N9 loss is followed by the release of HC2N1. PMID:25869111

  15. Regulation of purinergic signaling in biliary epithelial cells by exocytosis of SLC17A9-dependent ATP-enriched vesicles.

    PubMed

    Sathe, Meghana N; Woo, Kangmee; Kresge, Charles; Bugde, Abhijit; Luby-Phelps, Kate; Lewis, Matthew A; Feranchak, Andrew P

    2011-07-15

    ATP in bile is a potent secretogogue, stimulating biliary epithelial cell (BEC) secretion through binding apical purinergic receptors. In response to mechanosensitive stimuli, BECs release ATP into bile, although the cellular basis of ATP release is unknown. The aims of this study in human and mouse BECs were to determine whether ATP release occurs via exocytosis of ATP-enriched vesicles and to elucidate the potential role of the vesicular nucleotide transporter SLC17A9 in purinergic signaling. Dynamic, multiscale, live cell imaging (confocal and total internal reflection fluorescence microscopy and a luminescence detection system with a high sensitivity charge-coupled device camera) was utilized to detect vesicular ATP release from cell populations, single cells, and the submembrane space of a single cell. In response to increases in cell volume, BECs release ATP, which was dependent on intact microtubules and vesicular trafficking pathways. ATP release occurred as stochastic point source bursts of luminescence consistent with exocytic events. Parallel studies identified ATP-enriched vesicles ranging in size from 0.4 to 1 μm that underwent fusion and release in response to increases in cell volume in a protein kinase C-dependent manner. Present in all models, SLC17A9 contributed to ATP vesicle formation and regulated ATP release. The findings are consistent with the existence of an SLC17A9-dependent ATP-enriched vesicular pool in biliary epithelium that undergoes regulated exocytosis to initiate purinergic signaling.

  16. Fluorescence characteristic study of the ternary complex of fluoroquinolone antibiotics and cobalt (II) with ATP

    NASA Astrophysics Data System (ADS)

    Wu, Shuqing; Zhang, Wujuan; Chen, Xingguo; Hu, Zhide; Hooper, Martin; Hooper, Beveley; Zhao, Zhengfeng

    2001-05-01

    The results from the measurement of the fluorescence spectra of fluoroquinolone antibiotics including ofloxacin (OF), norfloxacin (NOR) and ciprofloxacin (CIP) complexed with cobalt (II) and ATP give information concerning the antibiotics-nucleotide interactions. From the fluorescence spectral data, it appears that the fluoroquinolone antibiotic cannot directly complex with ATP but indirectly complex with cobalt (II), which is playing an intermediary role. The interaction of fluoroquinolone antibiotic with the nucleotide occurs mainly through the phosphate group. The conclusion offers a more complete mechanism, which is important for understanding the interaction of these drugs with DNA.

  17. Photoaffinity Labeling of High Affinity Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)-Binding Proteins in Sea Urchin Egg*♦

    PubMed Central

    Walseth, Timothy F.; Lin-Moshier, Yaping; Jain, Pooja; Ruas, Margarida; Parrington, John; Galione, Antony; Marchant, Jonathan S.; Slama, James T.

    2012-01-01

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is a messenger that regulates calcium release from intracellular acidic stores. Recent studies have identified two-pore channels (TPCs) as endolysosomal channels that are regulated by NAADP; however, the nature of the NAADP receptor binding site is unknown. To further study NAADP binding sites, we have synthesized and characterized [32P-5-azido]nicotinic acid adenine dinucleotide phosphate ([32P-5N3]NAADP) as a photoaffinity probe. Photolysis of sea urchin egg homogenates preincubated with [32P-5N3]NAADP resulted in specific labeling of 45-, 40-, and 30-kDa proteins, which was prevented by inclusion of nanomolar concentrations of unlabeled NAADP or 5N3-NAADP, but not by micromolar concentrations of structurally related nucleotides such as NAD, nicotinic acid adenine dinucleotide, nicotinamide mononucleotide, nicotinic acid, or nicotinamide. [32P-5N3]NAADP binding was saturable and displayed high affinity (Kd ∼10 nm) in both binding and photolabeling experiments. [32P-5N3]NAADP photolabeling was irreversible in a high K+ buffer, a hallmark feature of NAADP binding in the egg system. The proteins photolabeled by [32P-5N3]NAADP have molecular masses smaller than the sea urchin TPCs, and antibodies to TPCs do not detect any immunoreactivity that comigrates with either the 45-kDa or the 40-kDa photolabeled proteins. Interestingly, antibodies to TPC1 and TPC3 were able to immunoprecipitate a small fraction of the 45- and 40-kDa photolabeled proteins, suggesting that these proteins associate with TPCs. These data suggest that high affinity NAADP binding sites are distinct from TPCs. PMID:22117077

  18. 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.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  1. Changes in phosphorylation of adenosine phosphate and redox state of nicotinamide-adenine dinucleotide (phosphate) in Geobacter sulfurreducens in response to electron acceptor and anode potential variation.

    PubMed

    Rose, Nicholas D; Regan, John M

    2015-12-01

    Geobacter sulfurreducens is one of the dominant bacterial species found in biofilms growing on anodes in bioelectrochemical systems. The intracellular concentrations of reduced and oxidized forms of nicotinamide-adenine dinucleotide (NADH and NAD(+), respectively) and nicotinamide-adenine dinucleotide phosphate (NADPH and NADP(+), respectively) as well as adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) were measured in G. sulfurreducens using fumarate, Fe(III)-citrate, or anodes poised at different potentials (110, 10, -90, and -190 mV (vs. SHE)) as the electron acceptor. The ratios of CNADH/CNAD+ (0.088±0.022) and CNADPH/CNADP+ (0.268±0.098) were similar under all anode potentials tested and with Fe(III)-citrate (reduced extracellularly). Both ratios significantly increased with fumarate as the electron acceptor (0.331±0.094 for NAD and 1.96±0.37 for NADP). The adenylate energy charge (the fraction of phosphorylation in intracellular adenosine phosphates) was maintained near 0.47 under almost all conditions. Anode-growing biofilms demonstrated a significantly higher molar ratio of ATP/ADP relative to suspended cultures grown on fumarate or Fe(III)-citrate. These results provide evidence that the cellular location of reduction and not the redox potential of the electron acceptor controls the intracellular redox potential in G. sulfurreducens and that biofilm growth alters adenylate phosphorylation.

  2. OmpF, a nucleotide-sensing nanoprobe, computational evaluation of single channel activities

    NASA Astrophysics Data System (ADS)

    Abdolvahab, R. H.; Mobasheri, H.; Nikouee, A.; Ejtehadi, M. R.

    2016-09-01

    The results of highthroughput practical single channel experiments should be formulated and validated by signal analysis approaches to increase the recognition precision of translocating molecules. For this purpose, the activities of the single nano-pore forming protein, OmpF, in the presence of nucleotides were recorded in real time by the voltage clamp technique and used as a means for nucleotide recognition. The results were analyzed based on the permutation entropy of current Time Series (TS), fractality, autocorrelation, structure function, spectral density, and peak fraction to recognize each nucleotide, based on its signature effect on the conductance, gating frequency and voltage sensitivity of channel at different concentrations and membrane potentials. The amplitude and frequency of ion current fluctuation increased in the presence of Adenine more than Cytosine and Thymine in milli-molar (0.5 mM) concentrations. The variance of the current TS at various applied voltages showed a non-monotonic trend whose initial increasing slope in the presence of Thymine changed to a decreasing one in the second phase and was different from that of Adenine and Cytosine; e.g., by increasing the voltage from 40 to 140 mV in the 0.5 mM concentration of Adenine or Cytosine, the variance decreased by one third while for the case of Thymine it was doubled. Moreover, according to the structure function of TS, the fractality of current TS differed as a function of varying membrane potentials (pd) and nucleotide concentrations. Accordingly, the calculated permutation entropy of the TS, validated the biophysical approach defined for the recognition of different nucleotides at various concentrations, pd's and polarities. Thus, the promising outcomes of the combined experimental and theoretical methodologies presented here can be implemented as a complementary means in pore-based nucleotide recognition approaches.

  3. Complete nucleotide sequences of two adjacent early vaccinia virus genes located within the inverted terminal repetition.

    PubMed

    Venkatesan, S; Gershowitz, A; Moss, B

    1982-11-01

    The proximal part of the 10,000-base pair (bp) inverted terminal repetition of vaccinia virus DNA encodes at least three early mRNAs. A 2,236-bp segment of the repetition was sequenced to characterize two of the genes. This task was facilitated by constructing a series of recombinants containing overlapping deletions; oligonucleotide linkers with synthetic restriction sites provided points for radioactive labeling before sequencing by the chemical degradation method of Maxam and Gilbert (Methods Enzymol. 65:499-560, 1980). The ends of the transcripts were mapped by hybridizing labeled DNA fragments to early viral RNA and resolving nuclease S1-protected fragments in sequencing gels, by sequencing cDNA clones, and from the lengths of the RNAs. The nucleotide sequences for at least 60 bp upstream of both transcriptional initiation sites are more than 80% adenine . thymine rich and contain long runs of adenines and thymines with some homology to procaryotic and eucaryotic consensus sequences. The gene transcribed in the rightward direction encodes an RNA of approximately 530 nucleotides with a single open reading frame of 420 nucleotides. Preceding the first AUG, there is a heptanucleotide that can hybridize to the 3' end of 18S rRNA with only one mismatch. The derived amino acid sequence of the protein indicated a molecular weight of 15,500. The gene transcribed in the leftward direction encodes an RNA 1,000 to 1,100 nucleotides long with an open reading frame of 996 nucleotides and a leader sequence of only 5 to 6 nucleotides. The derived amino acid sequence of this protein indicated a molecular weight of 38,500. The 3' ends of the two transcripts were located within 100 bp of each other. Although there are adenine . thymine-rich clusters near the putative transcriptional termination sites, specific AATAAA polyadenylic acid signal sequences are absent.

  4. Unraveling the complexity of the interactions of DNA nucleotides with gold by single molecule force spectroscopy.

    PubMed

    Bano, Fouzia; Sluysmans, Damien; Wislez, Arnaud; Duwez, Anne-Sophie

    2015-12-14

    Addressing the effect of different environmental factors on the adsorption of DNA to solid supports is critical for the development of robust miniaturized devices for applications ranging from biosensors to next generation molecular technology. Most of the time, thiol-based chemistry is used to anchor DNA on gold - a substrate commonly used in nanotechnology - and little is known about the direct interaction between DNA and gold. So far there have been no systematic studies on the direct adsorption behavior of the deoxyribonucleotides (i.e., a nitrogenous base, a deoxyribose sugar, and a phosphate group) and on the factors that govern the DNA-gold bond strength. Here, using single molecule force spectroscopy, we investigated the interaction of the four individual nucleotides, adenine, guanine, cytosine, and thymine, with gold. Experiments were performed in three salinity conditions and two surface dwell times to reveal the factors that influence nucleotide-Au bond strength. Force data show that, at physiological ionic strength, adenine-Au interactions are stronger, asymmetrical and independent of surface dwell time as compared to cytosine-Au and guanine-Au interactions. We suggest that in these conditions only adenine is able to chemisorb on gold. A decrease of the ionic strength significantly increases the bond strength for all nucleotides. We show that moderate ionic strength along with longer surface dwell period suggest weak chemisorption also for cytosine and guanine.

  5. Enlightening energy parasitism by analysis of an ATP/ADP transporter from chlamydiae.

    PubMed

    Trentmann, Oliver; Horn, Matthias; van Scheltinga, Anke C Terwisscha; Neuhaus, H Ekkehard; Haferkamp, Ilka

    2007-09-01

    Energy parasitism by ATP/ADP transport proteins is an essential, common feature of intracellular bacteria such as chlamydiae and rickettsiae, which are major pathogens of humans. Although several ATP/ADP transport proteins have so far been characterized, some fundamental questions regarding their function remained unaddressed. In this study, we focused on the detailed biochemical analysis of a representative ATP/ADP transporter (PamNTT1), from the amoeba symbiont Protochlamydia amoebophila (UWE25) to further clarify the principle of energy exploitation. We succeeded in the purification of the first bacterial nucleotide transporter (NTT) and its functional reconstitution into artificial lipid vesicles. Reconstituted PamNTT1 revealed high import velocities for ATP and an unexpected and previously unobserved stimulating effect of the luminal ADP on nucleotide import affinities. Latter preference of the nucleotide hetero-exchange is independent of the membrane potential, and therefore, PamNTT1 not only structurally but also functionally differs from the well-characterized mitochondrial ADP/ATP carriers. Reconstituted PamNTT1 exhibits a bidirectional orientation in lipid vesicles, but interestingly, only carriers inserted with the N-terminus directed to the proteoliposomal interior are functional. The data presented here comprehensively explain the functional basis of how the intracellular P. amoebophila manages to exploit the energy pool of its host cell effectively by using the nucleotide transporter PamNTT1. This membrane protein mediates a preferred import of ATP, which is additionally stimulated by a high internal (bacterial) ADP/ATP ratio, and the orientation-dependent functionality of the transporter ensures that it is not working in a mode that is detrimental to P. amoebophila. Heterologous expression and purification of high amounts of PamNTT1 provides the basis for its crystallization and detailed structure/function analyses. Furthermore, functional

  6. Energy-dependent dissociation of ATP from high affinity catalytic sites of beef heart mitochondrial adenosine triphosphatase

    SciTech Connect

    Penefsky, H.S.

    1985-11-05

    Incubation of (gamma-TSP)ATP with a molar excess of the membrane-bound form of mitochondrial ATPase (F1) results in binding of the bulk of the radioactive nucleotide in high affinity catalytic sites (Ka = 10(12) M-1). Subsequent initiation of respiration by addition of succinate or NADH is accompanied by a profound decrease in the affinity for ATP. About one-third of the bound radioactive ATP appears to dissociate, that is, the (gamma-TSP)ATP becomes accessible to hexokinase. The NADH-stimulated dissociation of (gamma-TSP)ATP is energy-dependent since the stimulation is inhibited by uncouplers of oxidative phosphorylation and is prevented by respiratory chain inhibitors. The rate of the energy-dependent dissociation of ATP that occurs in the presence of NADH, ADP, and Pi is commensurate with the measured initial rate of ATP synthesis in NADH-supported oxidative phosphorylation catalyzed by the same submitochondrial particles. Thus, the rate of dissociation of ATP from the high affinity catalytic site of submitochondrial particles meets the criterion of kinetic competency under the conditions of oxidative phosphorylation. These experiments provide evidence in support of the argument that energy conserved during the oxidation of substrates by the respiratory chain can be utilized to reduce the very tight binding of product ATP in high affinity catalytic sites and to promote dissociation of the nucleotide.

  7. Influence of hydrogen bonding on the geometry of the adenine fragment

    NASA Astrophysics Data System (ADS)

    Słowikowska, Joanna Maria; Woźniak, Krzysztof

    1996-01-01

    The crystal structures of two adenine derivatives, N(6),9-dimethyl-8-butyladenine (I) and its hydrate (1 : 1) (II), have been determined by single-crystal X-ray diffraction. The geometrical features of both structures are discussed. The influence of protonation, substitution and hydrogen bond formation on the geometry of the adenine fragment was studied, based on data retrieved from the Cambridge Structural Database. Total correlation analysis showed mutual correlation between the structural parameters in the adenine ring system; partial correlation calculations for the adenine nucleoside fragments suggest intercorrelation between the parameters of the hydrogen bonding involved in base pairing and the N(adenine)-C(sugar) bond through the adenine fragment; few such correlations were found for fragments without the sugar substituent.

  8. Generation and Characterization of ATP Analog-specific Protein Kinase Cδ*

    PubMed Central

    Kumar, Varun; Weng, Yi-Chinn; Geldenhuys, Werner J.; Wang, Dan; Han, Xiqian; Messing, Robert O.; Chou, Wen-Hai

    2015-01-01

    To better study the role of PKCδ in normal function and disease, we developed an ATP analog-specific (AS) PKCδ that is sensitive to specific kinase inhibitors and can be used to identify PKCδ substrates. AS PKCδ showed nearly 200 times higher affinity (Km) and 150 times higher efficiency (kcat/Km) than wild type (WT) PKCδ toward N6-(benzyl)-ATP. AS PKCδ was uniquely inhibited by 1-(tert-butyl)-3-(1-naphthyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (1NA-PP1) and 1-(tert-butyl)-3-(2-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (2MB-PP1) but not by other 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) analogs tested, whereas WT PKCδ was insensitive to all PP1 analogs. To understand the mechanisms for specificity and affinity of these analogs, we created in silico WT and AS PKCδ homology models based on the crystal structure of PKCι. N6-(Benzyl)-ATP and ATP showed similar positioning within the purine binding pocket of AS PKCδ, whereas N6-(benzyl)-ATP was displaced from the pocket of WT PKCδ and was unable to interact with the glycine-rich loop that is required for phosphoryl transfer. The adenine rings of 1NA-PP1 and 2MB-PP1 matched the adenine ring of ATP when docked in AS PKCδ, and this interaction prevented the potential interaction of ATP with Lys-378, Glu-428, Leu-430, and Phe-633 residues. 1NA-PP1 failed to effectively dock within WT PKCδ. Other PP1 analogs failed to interact with either AS PKCδ or WT PKCδ. These results provide a structural basis for the ability of AS PKCδ to efficiently and specifically utilize N6-(benzyl)-ATP as a phosphate donor and for its selective inhibition by 1NA-PP1 and 2MB-PP1. Such homology modeling could prove useful in designing molecules to target PKCδ and other kinases to understand their function in cell signaling and to identify unique substrates. PMID:25505183

  9. Examination of tyrosine/adenine stacking interactions in protein complexes.

    PubMed

    Copeland, Kari L; Pellock, Samuel J; Cox, James R; Cafiero, Mauricio L; Tschumper, Gregory S

    2013-11-14

    The π-stacking interactions between tyrosine amino acid side chains and adenine-bearing ligands are examined. Crystalline protein structures from the protein data bank (PDB) exhibiting face-to-face tyrosine/adenine arrangements were used to construct 20 unique 4-methylphenol/N9-methyladenine (p-cresol/9MeA) model systems. Full geometry optimization of the 20 crystal structures with the M06-2X density functional theory method identified 11 unique low-energy conformations. CCSD(T) complete basis set (CBS) limit interaction energies were estimated for all of the structures to determine the magnitude of the interaction between the two ring systems. CCSD(T) computations with double-ζ basis sets (e.g., 6-31G*(0.25) and aug-cc-pVDZ) indicate that the MP2 method overbinds by as much as 3.07 kcal mol(-1) for the crystal structures and 3.90 kcal mol(-1) for the optimized structures. In the 20 crystal structures, the estimated CCSD(T) CBS limit interaction energy ranges from -4.00 to -6.83 kcal mol(-1), with an average interaction energy of -5.47 kcal mol(-1), values remarkably similar to the corresponding data for phenylalanine/adenine stacking interactions. Geometry optimization significantly increases the interaction energies of the p-cresol/9MeA model systems. The average estimated CCSD(T) CBS limit interaction energy of the 11 optimized structures is 3.23 kcal mol(-1) larger than that for the 20 crystal structures.

  10. Evolving nucleotide binding surfaces

    NASA Technical Reports Server (NTRS)

    Kieber-Emmons, T.; Rein, R.

    1981-01-01

    An analysis is presented of the stability and nature of binding of a nucleotide to several known dehydrogenases. The employed approach includes calculation of hydrophobic stabilization of the binding motif and its intermolecular interaction with the ligand. The evolutionary changes of the binding motif are studied by calculating the Euclidean deviation of the respective dehydrogenases. Attention is given to the possible structural elements involved in the origin of nucleotide recognition by non-coded primordial polypeptides.

  11. Approach to the unfolding and folding dynamics of add A-riboswitch upon adenine dissociation using a coarse-grained elastic network model

    NASA Astrophysics Data System (ADS)

    Li, Chunhua; Lv, Dashuai; Zhang, Lei; Yang, Feng; Wang, Cunxin; Su, Jiguo; Zhang, Yang

    2016-07-01

    Riboswitches are noncoding mRNA segments that can regulate the gene expression via altering their structures in response to specific metabolite binding. We proposed a coarse-grained Gaussian network model (GNM) to examine the unfolding and folding dynamics of adenosine deaminase (add) A-riboswitch upon the adenine dissociation, in which the RNA is modeled by a nucleotide chain with interaction networks formed by connecting adjoining atomic contacts. It was shown that the adenine binding is critical to the folding of the add A-riboswitch while the removal of the ligand can result in drastic increase of the thermodynamic fluctuations especially in the junction regions between helix domains. Under the assumption that the native contacts with the highest thermodynamic fluctuations break first, the iterative GNM simulations showed that the unfolding process of the adenine-free add A-riboswitch starts with the denature of the terminal helix stem, followed by the loops and junctions involving ligand binding pocket, and then the central helix domains. Despite the simplified coarse-grained modeling, the unfolding dynamics and pathways are shown in close agreement with the results from atomic-level MD simulations and the NMR and single-molecule force spectroscopy experiments. Overall, the study demonstrates a new avenue to investigate the binding and folding dynamics of add A-riboswitch molecule which can be readily extended for other RNA molecules.

  12. Energy transduction in ATP synthase

    NASA Astrophysics Data System (ADS)

    Elston, Timothy; Wang, Hongyun; Oster, George

    1998-01-01

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

  13. Comparing the catalytic strategy of ATP hydrolysis in biomolecular motors.

    PubMed

    Kiani, Farooq Ahmad; Fischer, Stefan

    2016-07-27

    ATP-driven biomolecular motors utilize the chemical energy obtained from the ATP hydrolysis to perform vital tasks in living cells. Understanding the mechanism of enzyme-catalyzed ATP hydrolysis reaction has substantially progressed lately thanks to combined quantum/classical molecular mechanics (QM/MM) simulations. Here, we present a comparative summary of the most recent QM/MM results for myosin, kinesin and F1-ATPase motors. These completely different motors achieve the acceleration of ATP hydrolysis through a very similar catalytic mechanism. ATP hydrolysis has high activation energy because it involves the breaking of two strong bonds, namely the Pγ-Oβγ bond of ATP and the H-O bond of lytic water. The key to the four-fold decrease in the activation barrier by the three enzymes is that the breaking of the Pγ-Oβγ bond precedes the deprotonation of the lytic water molecule, generating a metaphosphate hydrate complex. The resulting singly charged trigonal planar PγO3(-) metaphosphate is a better electrophilic target for attack by an OaH(-) hydroxyl group. The formation of this OaH(-) is promoted by a strong polarization of the lytic water: in all three proteins, this water is forming a hydrogen-bond with a backbone carbonyl group and interacts with the carboxylate group of glutamate (either directly or via an intercalated water molecule). This favors the shedding of one proton by the attacking water. The abstracted proton is transferred to the γ-phosphate via various proton wires, resulting in a H2PγO4(-)/ADP(3-) product state. This catalytic strategy is so effective that most other nucleotide hydrolyzing enzymes adopt a similar approach, as suggested by their very similar triphosphate binding sites. PMID:27296627

  14. The nucleotide exchange factors of Hsp70 molecular chaperones.

    PubMed

    Bracher, Andreas; Verghese, Jacob

    2015-01-01

    Molecular chaperones of the Hsp70 family form an important hub in the cellular protein folding networks in bacteria and eukaryotes, connecting translation with the downstream machineries of protein folding and degradation. The Hsp70 folding cycle is driven by two types of cochaperones: J-domain proteins stimulate ATP hydrolysis by Hsp70, while nucleotide exchange factors (NEFs) promote replacement of Hsp70-bound ADP with ATP. Bacteria and organelles of bacterial origin have only one known NEF type for Hsp70, GrpE. In contrast, a large diversity of Hsp70 NEFs has been discovered in the eukaryotic cell. These NEFs belong to the Hsp110/Grp170, HspBP1/Sil1, and BAG domain protein families. In this short review we compare the structures and molecular mechanisms of nucleotide exchange factors for Hsp70 and discuss how these cochaperones contribute to protein folding and quality control in the cell. PMID:26913285

  15. The nucleotide exchange factors of Hsp70 molecular chaperones

    PubMed Central

    Bracher, Andreas; Verghese, Jacob

    2015-01-01

    Molecular chaperones of the Hsp70 family form an important hub in the cellular protein folding networks in bacteria and eukaryotes, connecting translation with the downstream machineries of protein folding and degradation. The Hsp70 folding cycle is driven by two types of cochaperones: J-domain proteins stimulate ATP hydrolysis by Hsp70, while nucleotide exchange factors (NEFs) promote replacement of Hsp70-bound ADP with ATP. Bacteria and organelles of bacterial origin have only one known NEF type for Hsp70, GrpE. In contrast, a large diversity of Hsp70 NEFs has been discovered in the eukaryotic cell. These NEFs belong to the Hsp110/Grp170, HspBP1/Sil1, and BAG domain protein families. In this short review we compare the structures and molecular mechanisms of nucleotide exchange factors for Hsp70 and discuss how these cochaperones contribute to protein folding and quality control in the cell. PMID:26913285

  16. Functional interactions between nucleotide binding domains and leukotriene C4 binding sites of multidrug resistance protein 1 (ABCC1).

    PubMed

    Payen, Lea; Gao, Mian; Westlake, Christopher; Theis, Ashley; Cole, Susan P C; Deeley, Roger G

    2005-06-01

    Multidrug resistance protein 1 (MRP1) is a member of the "C" branch of the ATP-binding cassette transporter superfamily. The NH(2)-proximal nucleotide-binding domain (NBD1) of MRP1 differs functionally from its COOH-proximal domain (NBD2). NBD1 displays intrinsic high-affinity ATP binding and little ATPase activity. In contrast, ATP binding to NBD2 is strongly dependent on nucleotide binding by NBD1, and NBD2 is more hydrolytically active. We have demonstrated that occupancy of NBD2 by ATP or ADP markedly decreased substrate binding by MRP1. We have further explored the relationship between nucleotide and substrate binding by examining the effects of various ATP analogs and ADP trapping, as well as mutations in conserved functional elements in the NBDs, on the ability of MRP1 to bind the photoactivatable, high-affinity substrate cysteinyl leukotriene C(4) (LTC(4))(.) Overall, the results support a model in which occupancy of both NBD1 and NBD2 by ATP results in the formation of a low-affinity conformation of the protein. However, nonhydrolyzable ATP analogs (beta,gamma-imidoadenosine 5'-triphosphate and adenylylmethylene diphosphonate) failed to substitute for ATP or adenosine 5'-O-(thiotriphosphate) (ATPgammaS) in decreasing LTC(4) photolabeling. Furthermore, mutations of the signature sequence in either NBD that had no apparent effect on azido-ATP binding abrogated the formation of a low-affinity substrate binding state in the presence of ATP or ATPgammaS. We suggest that the effect of these mutations, and possibly the failure of some ATP analogs to decrease LTC(4) binding, may be attributable to an inability to elicit a conformational change in the NBDs that involves interactions between the signature sequence and the gamma-phosphate of the bound nucleotide.

  17. Modelling proton tunnelling in the adenine-thymine base pair.

    PubMed

    Godbeer, A D; Al-Khalili, J S; Stevenson, P D

    2015-05-21

    The energies of the canonical (standard, amino-keto) and tautomeric (non-standard, imino-enol) charge-neutral forms of the adenine-thymine base pair (A-T and A*-T*, respectively) are calculated using density functional theory. The reaction pathway is then computed using a transition state search to provide the asymmetric double-well potential minima along with the barrier height and shape, which are combined to create the potential energy surface using a polynomial fit. The influence of quantum tunnelling on proton transfer within a base pair H-bond (modelled as the DFT deduced double-well potential) is then investigated by solving the time-dependent master equation for the density matrix. The effect on a quantum system by its surrounding water molecules is explored via the inclusion of a dissipative Lindblad term in the master equation, in which the environment is modelled as a heat bath of harmonic oscillators. It is found that quantum tunnelling, due to transitions to higher energy eigenstates with significant amplitudes in the shallow (tautomeric) side of the potential, is unlikely to be a significant mechanism for the creation of adenine-thymine tautomers within DNA, with thermally assisted coupling of the environment only able to boost the tunnelling probability to a maximum of 2 × 10(-9). This is barely increased for different choices of the starting wave function or when the geometry of the potential energy surface is varied.

  18. Mutants of Neurospora deficient in nicotinamide adenine dinucleotide (phosphate) glycohydrolase.

    PubMed Central

    Nelson, R E; Selitrennikoff, C P; Siegel, R W

    1975-01-01

    A new screening technique has been developed for the rapid identification of Neurospora crassa mutants that are deficient in nicotinamide adenine dinucleotide glycohydrolase (NADase) and nicotinamide adenine dinucleotide phosphate glycohydrolase (NADPase) activities. Using this procedure, five single-gene mutants were isolated whose singular difference from wild type appeared to be the absence of NAD(P)ase (EC 3.2.2.6). All five mutants were found to be genetically allelic and did not complement in heterocaryons. This gene, nada [NAD(P)ase], was localized in linkage group IV. One of the nada alleles was found to specify an enzyme that was critically temperature sensitive and had altered substrate affinity. Mutations at the nada locus did not affect the genetic program for the expression of NAD(P)ase during cell differentiation, nor did they have a general effect on NAD catabolism. Nada mutations did not have simultaneous effects on other glycohydrolase activities. Tests of dominance (in heterocaryons) and in vitro mixing experiments did not provide evidence that nada mutations alter activators or inhibitors of NAD(P)ase. Thus, the nada gene appears to specify only the structure of N. crassa NAD(P)ase. Images PMID:165174

  19. Nonselective enrichment for yeast adenine mutants by flow cytometry

    NASA Technical Reports Server (NTRS)

    Bruschi, C. V.; Chuba, P. J.

    1988-01-01

    The expression of certain adenine biosynthetic mutations in the yeast Saccharomyces cerevisiae results in a red colony color. This phenomenon has historically provided an ideal genetic marker for the study of mutation, recombination, and aneuploidy in lower eukaryotes by classical genetic analysis. In this paper, it is reported that cells carrying ade1 and/or ade2 mutations exhibit primary fluorescence. Based on this observation, the nonselective enrichment of yeast cultures for viable adenine mutants by using the fluorescence-activated cell sorter has been achieved. The advantages of this approach over conventional genetic analysis of mutation, recombination, and mitotic chromosomal stability include speed and accuracy in acquiring data for large numbers of clones. By using appropriate strains, the cell sorter has been used for the isolation of both forward mutations and chromosomal loss events in S. cerevisiae. The resolving power of this system and its noninvasiveness can easily be extended to more complex organisms, including mammalian cells, in which analogous metabolic mutants are available.

  20. Exploitation of the Low Fidelity of Human Immunodeficiency Virus Type 1 (HIV-1) Reverse Transcriptase and the Nucleotide Composition Bias in the HIV-1 Genome To Alter the Drug Resistance Development of HIV

    PubMed Central

    Balzarini, Jan; Camarasa, Maria-José; Pérez-Pérez, Maria-Jesus; San-Félix, Ana; Velázquez, Sonsoles; Perno, Carlo-Federico; De Clercq, Erik; Anderson, John N.; Karlsson, Anna

    2001-01-01

    The RNA genome of the lentivirus human immunodeficiency virus type 1 (HIV-1) is significantly richer in adenine nucleotides than the statistically equal distribution of the four different nucleotides that is expected. This compositional bias may be due to the guanine-to-adenine (G→A) nucleotide hypermutation of the HIV genome, which has been explained by dCTP pool imbalances during reverse transcription. The adenine nucleotide bias together with the poor fidelity of HIV-1 reverse transcriptase markedly enhances the genetic variation of HIV and may be responsible for the rapid emergence of drug-resistant HIV-1 strains. We have now attempted to counteract the normal mutational pattern of HIV-1 in response to anti-HIV-1 drugs by altering the endogenous deoxynucleoside triphosphate pool ratios with antimetabolites in virus-infected cell cultures. We showed that administration of these antimetabolic compounds resulted in an altered drug resistance pattern due to the reversal of the predominant mutational flow of HIV (G→A) to an adenine-to-guanine (A→G) nucleotide pattern in the intact HIV-1-infected lymphocyte cultures. Forcing the virus to change its inherent nucleotide bias may lead to better control of viral drug resistance development. PMID:11390579

  1. 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-1β 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-1β 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-1β secretion, and they suggest that maintenance of the intracellular ATP pools could be a strategy for countering NLRP3-mediated inflammation. PMID:26546608

  2. 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 α-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

  3. 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.

  4. New insights into one of nature's remarkable catalysts, the ATP synthase.

    PubMed

    Boyer, P D

    2001-08-01

    In the August 10, 2001 issue of Cell, Menz et al. report the crystal structure of a novel inhibited form of the bovine mitochondrial F(1)-ATPase at 2 A resolution, with all three catalytic sites occupied by nucleotide, one of which binds ADP in preference to ATP.

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

    PubMed

    Dangelmaier, Carol A; Holmsen, Holm

    2014-01-01

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

  6. Does ATP cross the cell plasma membrane.

    PubMed Central

    Chaudry, I. H.

    1982-01-01

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

  7. Nucleotide Binding Site Communication in Arabidopsis thaliana Adenosine 5;-Phosphosulfate Kinase

    SciTech Connect

    Ravilious, Geoffrey E.; Jez, Joseph M.

    2012-08-31

    Adenosine 5{prime}-phosphosulfate kinase (APSK) catalyzes the ATP-dependent synthesis of adenosine 3{prime}-phosphate 5{prime}-phosphosulfate (PAPS), which is an essential metabolite for sulfur assimilation in prokaryotes and eukaryotes. Using APSK from Arabidopsis thaliana, we examine the energetics of nucleotide binary and ternary complex formation and probe active site features that coordinate the order of ligand addition. Calorimetric analysis shows that binding can occur first at either nucleotide site, but that initial interaction at the ATP/ADP site was favored and enhanced affinity for APS in the second site by 50-fold. The thermodynamics of the two possible binding models (i.e. ATP first versus APS first) differs and implies that active site structural changes guide the order of nucleotide addition. The ligand binding analysis also supports an earlier suggestion of intermolecular interactions in the dimeric APSK structure. Crystallographic, site-directed mutagenesis, and energetic analyses of oxyanion recognition by the P-loop in the ATP/ADP binding site and the role of Asp136, which bridges the ATP/ADP and APS/PAPS binding sites, suggest how the ordered nucleotide binding sequence and structural changes are dynamically coordinated for catalysis.

  8. Major and minor groove conformations of DNA trimers modified on guanine or adenine by 4-aminobiphenyl: Adenine adducts favor the minor groove

    SciTech Connect

    Shapiro, R.; Ellis, S.; Hingerty, B.E.

    1995-01-01

    We have studied the conformational effects of 4-aminobiphenyl modification at C-8 of guanine or adenine on double-stranded DNA trimers. We used sequences with the modified purine at the central base pair and all 16 possible neighboring sequences at the outer pairs. Minimized potential energy calculations were carried out using the molecular mechanics program DUPLEX to survey the conformation space of these adducts, using a total of 1280 starting structures both in the modified guanine series and in the modified adenine series. Conformer families in which the bound 4-aminobiphenyl was located in the DNA major groove, and in the minor groove, were located for both adenine and guanine modification. In the modified guanine series, the major and minor groove families were roughly comparable in energy, and the sequence context determined which was more stable in a particular case. In the modified adenine series, however, the minor groove structure was more that 10 kcal/mol more stable than the major groove for all sequences. As a result, minor groove adducts provided most of the global minima in the adenine-modified series. This result may be relevant to a previous mutagenesis study [Lasko et al. (1988) J. Biol. Chem. 263, 15429-15435] in which the hot spot of most frequent occurrence was located at an adenine, in the sequence GAT. 25 refs., 9 figs., 4 tabs.

  9. Characterization of Nucleotide Misincorporation Patterns in the Iceman's Mitochondrial DNA

    PubMed Central

    Olivieri, Cristina; Ermini, Luca; Rizzi, Ermanno; Corti, Giorgio; Bonnal, Raoul; Luciani, Stefania; Marota, Isolina; De Bellis, Gianluca; Rollo, Franco

    2010-01-01

    Background The degradation of DNA represents one of the main issues in the genetic analysis of archeological specimens. In the recent years, a particular kind of post-mortem DNA modification giving rise to nucleotide misincorporation (“miscoding lesions”) has been the object of extensive investigations. Methodology/Principal Findings To improve our knowledge regarding the nature and incidence of ancient DNA nucleotide misincorporations, we have utilized 6,859 (629,975 bp) mitochondrial (mt) DNA sequences obtained from the 5,350–5,100-years-old, freeze-desiccated human mummy popularly known as the Tyrolean Iceman or Ötzi. To generate the sequences, we have applied a mixed PCR/pyrosequencing procedure allowing one to obtain a particularly high sequence coverage. As a control, we have produced further 8,982 (805,155 bp) mtDNA sequences from a contemporary specimen using the same system and starting from the same template copy number of the ancient sample. From the analysis of the nucleotide misincorporation rate in ancient, modern, and putative contaminant sequences, we observed that the rate of misincorporation is significantly lower in modern and putative contaminant sequence datasets than in ancient sequences. In contrast, type 2 transitions represent the vast majority (85%) of the observed nucleotide misincorporations in ancient sequences. Conclusions/Significance This study provides a further contribution to the knowledge of nucleotide misincorporation patterns in DNA sequences obtained from freeze-preserved archeological specimens. In the Iceman system, ancient sequences can be clearly distinguished from contaminants on the basis of nucleotide misincorporation rates. This observation confirms a previous identification of the ancient mummy sequences made on a purely phylogenetical basis. The present investigation provides further indication that the majority of ancient DNA damage is reflected by type 2 (cytosine→thymine/guanine→adenine) transitions and

  10. Interaction of sulfanilamide and sulfamethoxazole with bovine serum albumin and adenine: spectroscopic and molecular docking investigations.

    PubMed

    Rajendiran, N; Thulasidhasan, J

    2015-06-01

    Interaction between sulfanilamide (SAM) and sulfamethoxazole (SMO) with BSA and DNA base (adenine) was investigated by UV-visible, fluorescence, cyclic voltammetry and molecular docking studies. Stern-Volmer fluorescence quenching constant (Ka) suggests SMO is more quenched with BSA/adenine than that of SAM. The distance r between donor (BSA/adenine) and acceptor (SAM and SMO) was obtained according to fluorescence resonance energy transfer (FRET). The results showed that hydrophobic forces, electrostatic interactions, and hydrogen bonds played vital roles in the SAM and SMO with BSA/adenine binding interaction. During the interaction, sulfa drugs could insert into the hydrophobic pocket, where the non-radioactive energy transfer from BSA/adenine to sulfa drugs occurred with high possibility. Cyclic voltammetry results suggested that when the drug concentration is increased, the anodic electrode potential deceased. The docking method indicates aniline group is interacted with the BSA molecules. PMID:25754395

  11. Interaction of sulfanilamide and sulfamethoxazole with bovine serum albumin and adenine: Spectroscopic and molecular docking investigations

    NASA Astrophysics Data System (ADS)

    Rajendiran, N.; Thulasidhasan, J.

    2015-06-01

    Interaction between sulfanilamide (SAM) and sulfamethoxazole (SMO) with BSA and DNA base (adenine) was investigated by UV-visible, fluorescence, cyclic voltammetry and molecular docking studies. Stern-Volmer fluorescence quenching constant (Ka) suggests SMO is more quenched with BSA/adenine than that of SAM. The distance r between donor (BSA/adenine) and acceptor (SAM and SMO) was obtained according to fluorescence resonance energy transfer (FRET). The results showed that hydrophobic forces, electrostatic interactions, and hydrogen bonds played vital roles in the SAM and SMO with BSA/adenine binding interaction. During the interaction, sulfa drugs could insert into the hydrophobic pocket, where the non-radioactive energy transfer from BSA/adenine to sulfa drugs occurred with high possibility. Cyclic voltammetry results suggested that when the drug concentration is increased, the anodic electrode potential deceased. The docking method indicates aniline group is interacted with the BSA molecules.

  12. Single turnovers of fluorescent ATP bound to bipolar myosin filament during actin filaments sliding.

    PubMed

    Maruta, Takahiro; Kobatake, Takahiro; Okubo, Hiroyuki; Chaen, Shigeru

    2013-01-01

    The nucleotide turnover rates of bipolar myosin thick filament along which actin filament slides were measured by the displacement of prebound fluorescent ATP analog 2'(3')-O-[N-[2-[(Cy3)]amindo]ethyl] carbamoyl]-adenosine 5' triphosphate (Cy3-EDA-ATP) upon flash photolysis of caged ATP. The fluorescence of the thick filament where actin filament slides decayed with two exponential processes. The slower rate constant was the same as that without actin filament. Along bipolar myosin thick filament, actin filaments slide at a fast speed towards the central bare zone (forward), but more slowly away from the bare zone (backward). The displacement rate constant of fluorescent ATP from the myosin filament where actin filament moved forward was 5.0 s(-1), whereas the rate constant where the actin filament slid backward was 1.7 s(-1). These findings suggest that the slow ADP release rate is responsible for the slow backward sliding movement.

  13. Enzyme-mononucleotide interactions: three different folds share common structural elements for ATP recognition.

    PubMed Central

    Denessiouk, K. A.; Lehtonen, J. V.; Johnson, M. S.

    1998-01-01

    Three ATP-dependent enzymes with different folds, cAMP-dependent protein kinase, D-Ala:D-Ala ligase and the alpha-subunit of the alpha2beta2 ribonucleotide reductase, have a similar organization of their ATP-binding sites. The most meaningful similarity was found over 23 structurally equivalent residues in each protein and includes three strands each from their beta-sheets, in addition to a connecting loop. The equivalent secondary structure elements in each of these enzymes donate four amino acids forming key hydrogen bonds responsible for the common orientation of the "AMP" moieties of their ATP-ligands. One lysine residue conserved throughout the three families binds the alpha-phosphate in each protein. The common fragments of structure also position some, but not all, of the equivalent residues involved in hydrophobic contacts with the adenine ring. These examples of convergent evolution reinforce the view that different proteins can fold in different ways to produce similar structures locally, and nature can take advantage of these features when structure and function demand it, as shown here for the common mode of ATP-binding by three unrelated proteins. PMID:10082373

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

    PubMed Central

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

    2012-01-01

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

  15. Radioimmunoassay for cyclic nucleotides

    SciTech Connect

    Chiang, C.S.

    1984-02-21

    An improved radioimmunoassay for the determination of cyclic nucleotides in body fluids which comprises adding a source of divalent cation prior to assay minimizes the effects of both endogenous calcium ion and EDTA used as an anticoagulant in blood plasma samples.

  16. Energy Conversion by Molecular Motors Coupled to Nucleotide Hydrolysis

    NASA Astrophysics Data System (ADS)

    Lipowsky, Reinhard; Liepelt, Steffen; Valleriani, Angelo

    2009-06-01

    Recent theoretical work on the energy conversion by molecular motors coupled to nucleotide hydrolysis is reviewed. The most abundant nucleotide is provided by adenosine triphosphate (ATP) which is cleaved into adenosine diphosphate (ADP) and inorganic phosphate. The motors have several catalytic domains (or active sites), each of which can be empty or occupied by ATP or ADP. The chemical composition of all catalytic domains defines distinct nucleotide states of the motor which form a discrete state space. Each of these motor states is connected to several other states via chemical transitions. For stepping motors such as kinesin, which walk along cytoskeletal filaments, some motor states are also connected by mechanical transitions, during which the motor is displaced along the filament and able to perform mechanical work. The different motor states together with the possible chemical and mechanical transitions provide a network representation for the chemomechanical coupling of the motor molecule. The stochastic motor dynamics on these networks exhibits several distinct motor cycles, which represent the dominant pathways for different regimes of nucleotide concentrations and load force. For the kinesin motor, the competition of two such cycles determines the stall force, at which the motor velocity vanishes and the motor reverses its direction of motion. In general, kinesin is found to be governed by the competition of three distinct chemomechanical cycles. The corresponding network representation provides a unified description for all motor properties that have been determined by single molecule experiments.

  17. The purine nucleotide cycle. A pathway for ammonia production in the rat kidney.

    PubMed Central

    Bogusky, R T; Lowenstein, L M; Lowenstein, J M

    1976-01-01

    Particle-free extracts prepared from kidney cortex of rat catalyze the formation of ammonia via the purine nucleotide cycle. The cycle generates ammonia and fumarate from aspartate, using catalytic amounts of inosine monophosphate, adenylosuccinate, and adenosine monophosphate. The specific activities of the enzymes of the cycle are 1.27+/-0.27 nmol/mg protein per min (SE) for adenoylosuccinate synthetase, 1.38+/-0.16 for adenylosuccinase, and 44.0+/-3.3 for AMP deaminase. Compared with controls, extracts prepared from kidneys of rats fed ammonium chloride for 2 days show a 60% increase in adenylosuccinate synthetase and a threefold increase in adenylosuccinase activity, and a greater and more rapid synthesis of ammonia and adenine nucleotide from aspartate and inosine monophosphate. Extracts prepared from kidneys of rats fed a potassium-deficient diet show a twofold increase in adenylosuccinate synthetase and a threefold increase in adenylosuccinase activity. In such extracts the rate of synthesis of ammonia and adenine nucleotide from aspartate and inosine monophosphate is also increased. These results show that the reactions of the purine nucleotide cycle are present and can operate in extracts of kidney cortex. The operational capacity of the cycle is accelerated by ammonium chloride feeding and potassium depletion, conditions known to increase renal ammonia excretion. Extracts of kidney cortex convert inosine monophosphate to uric acid. This is prevented by addition of allopurinol of 1-pyrophosphoryl ribose 5-phosphate to the reaction mixture. PMID:821968

  18. Nucleotide diversity in gorillas.

    PubMed Central

    Yu, Ning; Jensen-Seaman, Michael I; Chemnick, Leona; Ryder, Oliver; Li, Wen-Hsiung

    2004-01-01

    Comparison of the levels of nucleotide diversity in humans and apes may provide valuable information for inferring the demographic history of these species, the effect of social structure on genetic diversity, patterns of past migration, and signatures of past selection events. Previous DNA sequence data from both the mitochondrial and the nuclear genomes suggested a much higher level of nucleotide diversity in the African apes than in humans. Noting that the nuclear DNA data from the apes were very limited, we previously conducted a DNA polymorphism study in humans and another in chimpanzees and bonobos, using 50 DNA segments randomly chosen from the noncoding, nonrepetitive parts of the human genome. The data revealed that the nucleotide diversity (pi) in bonobos (0.077%) is actually lower than that in humans (0.087%) and that pi in chimpanzees (0.134%) is only 50% higher than that in humans. In the present study we sequenced the same 50 segments in 15 western lowland gorillas and estimated pi to be 0.158%. This is the highest value among the African apes but is only about two times higher than that in humans. Interestingly, available mtDNA sequence data also suggest a twofold higher nucleotide diversity in gorillas than in humans, but suggest a threefold higher nucleotide diversity in chimpanzees than in humans. The higher mtDNA diversity in chimpanzees might be due to the unique pattern in the evolution of chimpanzee mtDNA. From the nuclear DNA pi values, we estimated that the long-term effective population sizes of humans, bonobos, chimpanzees, and gorillas are, respectively, 10,400, 12,300, 21,300, and 25,200. PMID:15082556

  19. Extracellular ATP released by osteoblasts is a key local inhibitor of bone mineralisation.

    PubMed

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

    2013-01-01

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

  20. High-altitude adaptation of Tibetan chicken from MT-COI and ATP-6 perspective.

    PubMed

    Zhao, Xiaoling; Wu, Nan; Zhu, Qing; Gaur, Uma; Gu, Ting; Li, Diyan

    2016-09-01

    The problem of hypoxia adaptation in high altitudes is an unsolved brainteaser in the field of life sciences. As one of the best chicken breeds with adaptability to highland environment, the Tibetan chicken, is genetically different from lowland chicken breeds. In order to gain a better understanding of the mechanism of hypoxic adaptability in high altitude, in the present study, we focused on the MT-COI together with ATP-6 gene to explore the regulatory mechanisms for hypoxia adaptability in Tibet chicken. Here, we sequenced MT-COI of 29 Tibetan chickens and 30 Chinese domestic chickens and ATP-6 gene of 28 Tibetan chickens and 29 Chinese domestic chickens. In MT-COI gene, 9 single nucleotide polymorphisms (SNPs) were detected though none of these was a missense mutation, confirming the fact that MT-COI gene is a largely conservative sequence. In ATP-6 gene, 6 single nucleotide polymorphisms (SNPs) were detected and we found a missense mutation (m.9441G > A) in the ATP-6 gene of Tibetan chicken resulting in an amino acid substitution. Due to the critical role of ATP-6 gene in the proton translocation and energy metabolism, we speculated the possibility of this mutation playing an important role in easier energy conversion and metabolism in Tibetan chickens than Chinese domestic chickens so as to better adapt to the harsh environment of the high-altitude areas. The Median-joining profile also suggested that haplotype Ha2 has the ancestral position to the other haplotypes and has significant relationship with high-altitude adaptation in ATP-6 gene. Therefore, we considered that the polymorphism (m.9441G > A) in the ATP-6 gene may affect the specific functions of ATP-6 enzyme relating to high-altitude adaptation of Tibetan chicken and MT-COI gene is a largely conservative sequence.

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

    PubMed

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

    2008-04-01

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

  2. Role of key residues at the flavin mononucleotide (FMN):adenylyltransferase catalytic site of the bifunctional riboflavin kinase/flavin adenine dinucleotide (FAD) Synthetase from Corynebacterium ammoniagenes.

    PubMed

    Serrano, Ana; Frago, Susana; Velázquez-Campoy, Adrián; Medina, Milagros

    2012-11-08

    In mammals and in yeast the conversion of Riboflavin (RF) into flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) is catalysed by the sequential action of two enzymes: an ATP:riboflavin kinase (RFK) and an ATP:FMN adenylyltransferase (FMNAT). However, most prokaryotes depend on a single bifunctional enzyme, FAD synthetase (FADS), which folds into two modules: the C-terminal associated with RFK activity and the N-terminal associated with FMNAT activity. Sequence and structural analysis suggest that the 28-HxGH-31, 123-Gx(D/N)-125 and 161-xxSSTxxR-168 motifs from FADS must be involved in ATP stabilisation for the adenylylation of FMN, as well as in FAD stabilisation for FAD phyrophosphorolysis. Mutants were produced at these motifs in the Corynebacterium ammoniagenes FADS (CaFADS). Their effects on the kinetic parameters of CaFADS activities (RFK, FMNAT and FAD pyrophosphorilase), and on substrates and product binding properties indicate that H28, H31, N125 and S164 contribute to the geometry of the catalytically competent complexes at the FMNAT-module of CaFADS.

  3. Unraveling the complexity of the interactions of DNA nucleotides with gold by single molecule force spectroscopy

    NASA Astrophysics Data System (ADS)

    Bano, Fouzia; Sluysmans, Damien; Wislez, Arnaud; Duwez, Anne-Sophie

    2015-11-01

    Addressing the effect of different environmental factors on the adsorption of DNA to solid supports is critical for the development of robust miniaturized devices for applications ranging from biosensors to next generation molecular technology. Most of the time, thiol-based chemistry is used to anchor DNA on gold - a substrate commonly used in nanotechnology - and little is known about the direct interaction between DNA and gold. So far there have been no systematic studies on the direct adsorption behavior of the deoxyribonucleotides (i.e., a nitrogenous base, a deoxyribose sugar, and a phosphate group) and on the factors that govern the DNA-gold bond strength. Here, using single molecule force spectroscopy, we investigated the interaction of the four individual nucleotides, adenine, guanine, cytosine, and thymine, with gold. Experiments were performed in three salinity conditions and two surface dwell times to reveal the factors that influence nucleotide-Au bond strength. Force data show that, at physiological ionic strength, adenine-Au interactions are stronger, asymmetrical and independent of surface dwell time as compared to cytosine-Au and guanine-Au interactions. We suggest that in these conditions only adenine is able to chemisorb on gold. A decrease of the ionic strength significantly increases the bond strength for all nucleotides. We show that moderate ionic strength along with longer surface dwell period suggest weak chemisorption also for cytosine and guanine.Addressing the effect of different environmental factors on the adsorption of DNA to solid supports is critical for the development of robust miniaturized devices for applications ranging from biosensors to next generation molecular technology. Most of the time, thiol-based chemistry is used to anchor DNA on gold - a substrate commonly used in nanotechnology - and little is known about the direct interaction between DNA and gold. So far there have been no systematic studies on the direct

  4. Adenosine triphosphate (ATP) reduces amyloid-β 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-β protein (Aβ) levels. Since levels of ATP decrease over the course of the disease and Aβ 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 Aβ (oligomerization motif) and nucleotide binding proteins (Rossmann fold). Second, ATP was demonstrated to protect against Aβ 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 Aβ42. To test this hypothesis, we performed computational and biochemical studies. Our computational studies demonstrate that ATP interacts strongly with Tyr10 and Ser26 of Aβ fibrils in solution. Experimentally, both ATP and ADP reduced Aβ misfolding at physiological intracellular concentrations, with thresholds at ~500 μM and 1 mM respectively. This inhibition of Aβ misfolding is specific; requiring Tyr10 of Aβ 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 Aβ and reduction of Aβ 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 Aβ mirrors lowered extracellular ATP levels. Last, the findings suggest that Aβ conformation change may be a sensor of metabolic dysfunction.

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

    PubMed

    Ho, James; Sielaff, Hendrik; Nadeem, Aftab; Svanborg, Catharina; Grüber, Gerhard

    2015-05-22

    HAMLET (human alpha-lactalbumin made lethal to tumor cells) interacts with multiple tumor cell compartments, affecting cell morphology, metabolism, proteasome function, chromatin structure and viability. This study investigated if these diverse effects of HAMLET might be caused, in part, by a direct effect on the ATP synthase and a resulting reduction in cellular ATP levels. A dose-dependent reduction in cellular ATP levels was detected in A549 lung carcinoma cells, and by confocal microscopy, co-localization of HAMLET with the nucleotide-binding subunits α (non-catalytic) and β (catalytic) of the energy converting F1F0 ATP synthase was detected. As shown by fluorescence correlation spectroscopy, HAMLET binds to the F1 domain of the F1F0 ATP synthase with a dissociation constant (KD) of 20.5μM. Increasing concentrations of the tumoricidal protein HAMLET added to the enzymatically active α3β3γ complex of the F-ATP synthase lowered its ATPase activity, demonstrating that HAMLET binding to the F-ATP synthase effects the catalysis of this molecular motor. Single-molecule analysis was applied to study HAMLET-α3β3γ complex interaction. Whereas the α3β3γ complex of the F-ATP synthase rotated in a counterclockwise direction with a mean rotational rate of 3.8±0.7s(-1), no rotation could be observed in the presence of bound HAMLET. Our findings suggest that direct effects of HAMLET on the F-ATP synthase may inhibit ATP-dependent cellular processes. PMID:25681694

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

    PubMed

    Ho, James; Sielaff, Hendrik; Nadeem, Aftab; Svanborg, Catharina; Grüber, Gerhard

    2015-05-22

    HAMLET (human alpha-lactalbumin made lethal to tumor cells) interacts with multiple tumor cell compartments, affecting cell morphology, metabolism, proteasome function, chromatin structure and viability. This study investigated if these diverse effects of HAMLET might be caused, in part, by a direct effect on the ATP synthase and a resulting reduction in cellular ATP levels. A dose-dependent reduction in cellular ATP levels was detected in A549 lung carcinoma cells, and by confocal microscopy, co-localization of HAMLET with the nucleotide-binding subunits α (non-catalytic) and β (catalytic) of the energy converting F1F0 ATP synthase was detected. As shown by fluorescence correlation spectroscopy, HAMLET binds to the F1 domain of the F1F0 ATP synthase with a dissociation constant (KD) of 20.5μM. Increasing concentrations of the tumoricidal protein HAMLET added to the enzymatically active α3β3γ complex of the F-ATP synthase lowered its ATPase activity, demonstrating that HAMLET binding to the F-ATP synthase effects the catalysis of this molecular motor. Single-molecule analysis was applied to study HAMLET-α3β3γ complex interaction. Whereas the α3β3γ complex of the F-ATP synthase rotated in a counterclockwise direction with a mean rotational rate of 3.8±0.7s(-1), no rotation could be observed in the presence of bound HAMLET. Our findings suggest that direct effects of HAMLET on the F-ATP synthase may inhibit ATP-dependent cellular processes.

  7. Substrate binding stabilizes a pre-translocation intermediate in the ATP-binding cassette transport protein MsbA.

    PubMed

    Doshi, Rupak; van Veen, Hendrik W

    2013-07-26

    ATP-binding cassette (ABC) transporters belong to one of the largest protein superfamilies that expands from prokaryotes to man. Recent x-ray crystal structures of bacterial and mammalian ABC exporters suggest a common alternating access mechanism of substrate transport, which has also been biochemically substantiated. However, the current model does not yet explain the coupling between substrate binding and ATP hydrolysis that underlies ATP-dependent substrate transport. In our studies on the homodimeric multidrug/lipid A ABC exporter MsbA from Escherichia coli, we performed cysteine cross-linking, fluorescence energy transfer, and cysteine accessibility studies on two reporter positions, near the nucleotide-binding domains and in the membrane domains, for transporter embedded in a biological membrane. Our results suggest for the first time that substrate binding by MsbA stimulates the maximum rate of ATP hydrolysis by facilitating the dimerization of nucleotide-binding domains in a state, which is markedly distinct from the previously described nucleotide-free, inward-facing and nucleotide-bound, outward-facing conformations of ABC exporters and which binds ATP. PMID:23766512

  8. The low-affinity ATP binding site of the Escherichia coli SecA dimer is localized at the subunit interface.

    PubMed

    van der Wolk, J P; Boorsma, A; Knoche, M; Schäfer, H J; Driessen, A J

    1997-12-01

    The homodimeric SecA protein is the ATP-dependent force generator in the Escherichia coli precursor protein translocation cascade. SecA contains two essential nucleotide binding sites (NBSs), i.e., NBS1 and NBS2 that bind ATP with high and low affinity, respectively. The photoactivatable bifunctional cross-linking agent 3'-arylazido-8-azidoadenosine 5'-triphosphate (diN3ATP) was used to investigate the spatial arrangement of the nucleotide binding sites of SecA. DiN3ATP is an authentic ATP analogue as it supports SecA-dependent precursor protein translocation and translocation ATPase. UV-induced photo-cross-linking of the diN3ATP-bound SecA results in the formation of stable dimeric species of SecA. D209N SecA, a mutant unable to bind nucleotides at NBS1, was also photo-cross-linked by diN3ATP, whereas no cross-linking occurred with the NBS2 mutant R509K SecA. We concluded that the low-affinity NBS2, which is located in the carboxyl-terminal half of SecA, is the site of crosslinking and that NBS2 binds nucleotides at or near the subunit interface of the SecA dimer. PMID:9398216

  9. Allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP

    PubMed Central

    Qi, Ruifeng; Sarbeng, Evans Boateng; Liu, Qun; Le, Katherine Quynh; Xu, Xinping; Xu, Hongya; Yang, Jiao; Wong, Jennifer Li; Vorvis, Christina; Hendrickson, Wayne A.; Zhou, Lei; Liu, Qinglian

    2013-01-01

    The 70kD heat shock proteins (Hsp70s) are ubiquitous molecular chaperones essential for cellular protein folding and proteostasis. Each Hsp70 has two functional domains: a nucleotide-binding domain (NBD) that binds and hydrolyzes ATP, and a substrate-binding domain (SBD) that binds extended polypeptides. NBD and SBD interact little when in ADP; however, ATP binding allosterically couples the polypeptide- and ATP-binding sites. ATP binding promotes polypeptide release; polypeptide rebinding stimulates ATP hydrolysis. This allosteric coupling is poorly understood. Here we present the crystal structure of an intact Hsp70 from Escherichia coli in an ATP-bound state at 1.96 Å resolution. NBD-ATP adopts a unique conformation, forming extensive interfaces with a radically changed SBD that has its α-helical lid displaced and the polypeptide-binding channel of its β-subdomain restructured. These conformational changes together with our biochemical tests provide a long-sought structural explanation for allosteric coupling in Hsp70 activity. PMID:23708608

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  12. 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

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

    PubMed

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

    2014-10-21

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

  14. 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.

  15. Fine-tuned ATP signals are acute mediators in osteocyte mechanotransduction.

    PubMed

    Kringelbach, Tina M; Aslan, Derya; Novak, Ivana; Ellegaard, Maria; Syberg, Susanne; Andersen, Christina K B; Kristiansen, Kim A; Vang, Ole; Schwarz, Peter; Jørgensen, Niklas R

    2015-12-01

    Osteocytes are considered the primary mechanosensors of bone, but the signaling pathways they apply in mechanotransduction are still incompletely investigated and characterized. A growing body of data strongly indicates that P2 receptor signaling among osteoblasts and osteoclasts has regulatory effects on bone remodeling. Therefore, we hypothesized that ATP signaling is also applied by osteocytes in mechanotransduction. We applied a short fluid pulse on MLO-Y4 osteocyte-like cells during real-time detection of ATP and demonstrated that mechanical stimulation activates the acute release of ATP and that these acute ATP signals are fine-tuned according to the magnitude of loading. ATP release was then challenged by pharmacological inhibitors, which indicated a vesicular release pathway for acute ATP signals. Finally, we showed that osteocytes express functional P2X2 and P2X7 receptors and respond to even low concentrations of nucleotides by increasing intracellular calcium concentration. These results indicate that in osteocytes, vesicular ATP release is an acute mediator of mechanical signals and the magnitude of loading. These and previous results, therefore, implicate purinergic signaling as an early signaling pathway in osteocyte mechanotransduction.

  16. Synthesis and in vivo evaluation of prodrugs of 9-[2-(phosphonomethoxy)ethoxy]adenine.

    PubMed

    Serafinowska, H T; Ashton, R J; Bailey, S; Harnden, M R; Jackson, S M; Sutton, D

    1995-04-14

    A number of esters and amides of the anti-HIV nucleotide analogue 9-[2-(phosphonomethoxy)-ethoxy]adenine (1) have been synthesized as potential prodrugs and evaluated for oral bioavailability in mice. Dialkyl esters 17-20 were prepared via a Mitsunobu coupling of alcohols 8-11 with 9-hydroxypurine 12 whereas (acyloxy)alkyl esters 25-33 and bis-[(alkoxycarbonyl)methyl] and bis(amidomethyl) esters 34-39 were obtained by reaction of 1 with a suitable alkylating agent. Phosphonodichloridate chemistry was employed for the preparation of dialkyl and diaryl esters 42-65, and bis(phosphonoamidates) 66 and 67. Following oral administration to mice, most of the dialkyl esters 17-20 were well-absorbed and then converted to the corresponding monoesters, but minimal further metabolism to 1 occurred. Bis[(pivaloyloxy)methyl] ester 25 displayed an oral bioavailability of 30% that was 15-fold higher than the bioavailability observed after dosing of 1. Methyl substitution at the alpha carbon of the bis[(pivaloyloxy)methyl] ester 25 (33) increased the oral bioavailability of 1 to 74%. Some of the diaryl esters also showed improved absorption properties in comparison with that of 1. In particular, the crystalline hydrochloride salt of diphenyl ester 55 was well-absorbed and efficiently converted to the parent compound with an oral bioavailability of 50%. On the basis of these results as well as the physicochemical properties of the prodrugs and their stability in mouse duodenal contents, the hydrochloride salt of diphenyl ester 55 was identified as the preferred prodrug of 1. PMID:7731022

  17. Nucleotide binding domain 1 of the human retinal ABC transporter functions as a general ribonucleotidase.

    PubMed

    Biswas, E E

    2001-07-27

    Members of the ATP binding cassette (ABC) superfamily are transmembrane proteins that are found in a variety of tissues which transport substances across cell membranes in an energy-dependent manner. The retina-specific ABC protein (ABCR) has been linked through genetic studies to a number of inherited visual disorders, including Stargardt macular degeneration and age-related macular degeneration (ARMD). Like other ABC transporters, ABCR is characterized by two nucleotide binding domains and two transmembrane domains. We have cloned and expressed the 522-amino acid (aa) N-terminal cytoplasmic region (aa 854-1375) of ABCR containing nucleotide binding domain 1 (NBD1) with a purification tag at its amino terminus. The expressed recombinant protein was found to be soluble and was purified using single-step affinity chromatography. The purified protein migrated as a 66 kDa protein on SDS-PAGE. Analysis of the ATP binding and hydrolysis properties of the NBD1 polypeptide demonstrated significant differences between NBD1 and NBD2 [Biswas, E. E., and Biswas, S. B. (2000) Biochemistry 39, 15879-15886]. NBD1 was active as an ATPase, and nucleotide inhibition studies suggested that nucleotide binding was not specific for ATP and all four ribonucleotides can compete for binding. Further analysis demonstrated that NBD1 is a general nucleotidase capable of hydrolysis of ATP, CTP, GTP, and UTP. In contrast, NBD2 is specific for adenosine nucleotides (ATP and dATP). NBD1 bound ATP with a higher affinity than NBD2 (K(mNBD1) = 200 microm vs K(mNBD2) = 631 microm) but was less efficient as an ATPase (V(maxNBD1) = 28.9 nmol min(-)(1) mg(-)(1) vs V(maxNBD2) = 144 nmol min(-)(1) mg(-)(1)). The binding efficiencies for CTP and GTP were comparable to that observed for ATP (K(mCTP) = 155 microm vs K(mGTP) = 183 microm), while that observed for UTP was decreased 2-fold (K(mUTP) = 436 microm). Thus, the nucleotide binding preference of NBD1 is as follows: CTP > GTP > ATP > UTP. These

  18. The Lipid Bilayer Modulates the Structure and Function of an ATP-binding Cassette Exporter.

    PubMed

    Zoghbi, Maria E; Cooper, Rebecca S; Altenberg, Guillermo A

    2016-02-26

    ATP-binding cassette exporters use the energy of ATP hydrolysis to transport substrates across membranes by switching between inward- and outward-facing conformations. Essentially all structural studies of these proteins have been performed with the proteins in detergent micelles, locked in specific conformations and/or at low temperature. Here, we used luminescence resonance energy transfer spectroscopy to study the prototypical ATP-binding cassette exporter MsbA reconstituted in nanodiscs at 37 °C while it performs ATP hydrolysis. We found major differences when comparing MsbA in these native-like conditions with double electron-electron resonance data and the crystal structure of MsbA in the open inward-facing conformation. The most striking differences include a significantly smaller separation between the nucleotide-binding domains and a larger fraction of molecules with associated nucleotide-binding domains in the nucleotide-free apo state. These studies stress the importance of studying membrane proteins in an environment that approaches physiological conditions.

  19. Polyamine/Nucleotide Coacervates Provide Strong Compartmentalization of Mg²⁺, Nucleotides, and RNA.

    PubMed

    Frankel, Erica A; Bevilacqua, Philip C; Keating, Christine D

    2016-03-01

    Phase separation of aqueous solutions containing polyelectrolytes can lead to formation of dense, solute-rich liquid droplets referred to as coacervates, surrounded by a dilute continuous phase of much larger volume. This type of liquid-liquid phase separation is thought to help explain the appearance of polyelectrolyte-rich intracellular droplets in the cytoplasm and nucleoplasm of extant biological cells and may be relevant to protocellular compartmentalization of nucleic acids on the early Earth. Here we describe complex coacervates formed upon mixing the polycation poly(allylamine) (PAH, 15 kDa) with the anionic nucleotides adenosine 5'-mono-, di-, and triphosphate (AMP, ADP, and ATP). Droplet formation was observed over a wide range of pH and MgCl2 concentrations. The nucleotides themselves as well as Mg(2+) and RNA oligonucleotides were all extremely concentrated within the coacervates. Nucleotides present at just 2.5 mM in bulk solution had concentrations greater than 1 M inside the coacervate droplets. A solution with a total Mg(2+) concentration of 10 mM had 1-5 M Mg(2+) in the coacervates, and RNA random sequence (N54) partitioned ∼10,000-fold into the coacervates. Coacervate droplets are thus rich in nucleotides, Mg(2+), and RNA, providing a medium favorable for generating functional RNAs. Compartmentalization of nucleotides at high concentrations could have facilitated their polymerization to form oligonucleotides, which preferentially accumulate in the droplets. Locally high Mg(2+) concentrations could have aided folding and catalysis in an RNA world, making coacervate droplets an appealing platform for exploring protocellular environments. PMID:26844692

  20. Association of poly(N-isopropylacrylamide) containing nucleobase multiple hydrogen bonding of adenine for DNA recognition

    NASA Astrophysics Data System (ADS)

    Yang, Hsiu-Wen; Chen, Jem-Kun; Cheng, Chih-Chia; Kuo, Shiao-Wei

    2013-04-01

    In this study we used the poly(N-isopropylacrylamide) (PNIPAAm) as a medium to generate PNIPAAm-adenine supramolecular complexes. A nucleobase-like hydrogen bonding (NLHB) between PNIPAAm and adenine was found that changed the morphology, crystalline structure, and temperature responsiveness of PNIPAAm microgels relatively to the adenine concentrations. With increasing the adenine concentration, the PNIPAAm-adenine supramolecular complexes gradually altered their morphologies from microgel particles to thin film structures and suppressed the thermodynamical coil-to-globule transition of PNIPAAm because of the NLHB existed between the PNIPAAm amide and ester groups and the adenine amide groups (Cdbnd O⋯Hsbnd N and Nsbnd H⋯Nsbnd R), verified by FTIR spectral analysis. NLHB was also diverse and extensive upon increasing the temperature; therefore, the thermoresponsive behavior of the complexes was altered with the NLBH intensity, evaluated by the inter-association equilibrium constant (Ka) above and below their LCST. Therefore, PNIPAAm can be as a medium to recognize adenine in various concentrations, which could potentially be applied in DNA recognition.

  1. Mechanism of action of ATP on canine pulmonary vagal C fibre nerve terminals.

    PubMed Central

    Pelleg, A; Hurt, C M

    1996-01-01

    1. The effects of extracellular adenosine 5'-triphosphate (ATP) on pulmonary vagal afferent fibres (n = 46) was studied in a canine model in vivo (n = 38). 2. ATP (3-6 mumol kg-1), administered as a rapid bolus into the right atrium, elicited a transient burst of action potentials in cervical vagal fibres, which was not affected by either blockade of ganglionic transmission (hexamethonium) or a drop in arterial blood pressure (nitroglycerine). 3. The fibres with ATP-sensitive terminals were otherwise quiescent with no activity related to either cardiac or respiratory cycles and their conduction velocity was 0.85 +/- 0.13 m s-1 (n = 7). 4. Inflation of the lungs to 2-3 times the tidal volume triggered brief bursts of action potentials in these fibres. 5. Capsaicin (10 micrograms kg-1), given as a rapid bolus into the right atrium, elicited a burst of action potentials in these ATP-sensitive fibres. 6. Smaller amounts of ATP and capsaicin (0.5-3 mumol kg-1 and 1-5 micrograms kg-1, respectively) had similar effects when the two compounds were given into the right pulmonary artery. 7. Adenosine, adenosine 5'-monophosphate, or adenosine 5'-diphosphate did not excite these fibres (n = 30). 8. The non-degradable analogue of ATP alpha,beta-methylene ATP (alpha,beta-mATP) was tenfold more potent than ATP while beta,gamma-methylene ATP (beta,gamma-mATP) was in active. 9. The selective P2x-purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid markedly attenuated the effect of ATP but not of capsaicin. The P2Y-purinoceptor antagonist Reactive Blue 2 was without effect. 10. Pretreatment with pertussis toxin (PTX) did not affect this action of ATP. 11. In the canine lungs ATP activates vagal C fibre nerve terminals. This action is mediated by P2X-purinoceptors and is independent of a PTX-sensitive guanine nucleotide binding protein (G protein). PMID:8745294

  2. Standard role for a conserved aspartate or more direct involvement in deglycosylation? An ONIOM and MD investigation of adenine-DNA glycosylase.

    PubMed

    Kellie, Jennifer L; Wilson, Katie A; Wetmore, Stacey D

    2013-12-01

    8-Oxoguanine (OG) is one of the most frequently occurring forms of DNA damage and is particularly deleterious since it forms a stable Hoogsteen base pair with adenine (A). The repair of an OG:A mispair is initiated by adenine-DNA glycosylase (MutY), which hydrolyzes the sugar-nucleobase bond of the adenine residue before the lesion is processed by other proteins. MutY has been proposed to use a two-part chemical step involving protonation of the adenine nucleobase, followed by SN1 hydrolysis of the glycosidic bond. However, differences between a recent (fluorine recognition complex, denoted as the FLRC) crystal structure and the structure on which most mechanistic conclusions have been based to date (namely, the lesion recognition complex or LRC) raise questions regarding the mechanism used by MutY and the discrete role of various active-site residues. The present work uses both molecular dynamics (MD) and quantum mechanical (ONIOM) models to compare the active-site conformational dynamics in the two crystal structures, which suggests that only the understudied FLRC leads to a catalytically competent reactant. Indeed, all previous computational studies on MutY have been initiated from the LRC structure. Subsequently, for the first time, various mechanisms are examined with detailed ONIOM(M06-2X:PM6) reaction potential energy surfaces (PES) based on the FLRC structure, which significantly extends the mechanistic picture. Specifically, our work reveals that the reaction proceeds through a different route than the commonly accepted mechanism and the catalytic function of various active-site residues (Geobacillus stearothermophilus numbering). Specifically, contrary to proposals based on the LRC, E43 is determined to solely be involved in the initial adenine protonation step and not the deglycosylation reaction as the general base. Additionally, a novel catalytic role is proposed for Y126, whereby this residue plays a significant role in stabilizing the highly charged

  3. 2´,3´-Dialdehyde of ATP, ADP, and adenosine inhibit HIV-1 reverse transcriptase and HIV-1 replication.

    PubMed

    Schachter, Julieta; Valadao, Ana Luiza Chaves; Aguiar, Renato Santana; Barreto-de-Souza, Victor; Rossi, Atila Duque; Arantes, Pablo Ricardo; Verli, Hugo; Quintana, Paula Gabriela; Heise, Norton; Tanuri, Amilcar; Bou-Habib, Dumith Chequer; Persechini, Pedro Muanis

    2014-01-01

    The 2´3´-dialdehyde of ATP or oxidized ATP (oATP) is a compound known for specifically making covalent bonds with the nucleotide-binding site of several ATP-binding enzymes and receptors. We investigated the effects of oATP and other oxidized purines on HIV-1 infection and we found that this compound inhibits HIV-1 and SIV infection by blocking early steps of virus replication. oATP, oxidized ADP (oADP), and oxidized Adenosine (oADO) impact the natural activity of endogenous reverse transcriptase enzyme (RT) in cell free virus particles and are able to inhibit viral replication in different cell types when added to the cell cultures either before or after infection. We used UFLC-UV to show that both oADO and oATP can be detected in the cell after being added in the extracellular medium. oATP also suppresses RT activity and replication of the HIV-1 resistant variants M184V and T215Y. We conclude that oATP, oADP and oADO display anti HIV-1 activity that is at in least in part due to inhibitory activity on HIV-1 RT.

  4. Renoprotective effect of the xanthine oxidoreductase inhibitor topiroxostat on adenine-induced renal injury.

    PubMed

    Kamijo-Ikemori, Atsuko; Sugaya, Takeshi; Hibi, Chihiro; Nakamura, Takashi; Murase, Takayo; Oikawa, Tsuyoshi; Hoshino, Seiko; Hisamichi, Mikako; Hirata, Kazuaki; Kimura, Kenjiro; Shibagaki, Yugo

    2016-06-01

    The aim of the present study was to reveal the effect of a xanthine oxidoreductase (XOR) inhibitor, topiroxostat (Top), compared with another inhibitor, febuxostat (Feb), in an adenine-induced renal injury model. We used human liver-type fatty acid-binding protein (L-FABP) chromosomal transgenic mice, and urinary L-FABP, a biomarker of tubulointerstitial damage, was used to evaluate tubulointerstitial damage. Male transgenic mice (n = 24) were fed a 0.2% (wt/wt) adenine-containing diet. Two weeks after the start of this diet, renal dysfunction was confirmed, and the mice were divided into the following four groups: the adenine group was given only the diet containing adenine, and the Feb, high-dose Top (Top-H), and low-dose Top (Top-L) groups were given diets containing Feb (3 mg/kg), Top-H (3 mg/kg), and Top-L (1 mg/kg) in addition to adenine for another 2 wk. After withdrawal of the adenine diet, each medication was continued for 2 wk. Serum creatinine levels, the degree of macrophage infiltration, tubulointerstitial damage, renal fibrosis, urinary 15-F2t-isoprostane levels, and renal XOR activity were significantly attenuated in the kidneys of the Feb, Top-L, and Top-H groups compared with the adenine group. Serum creatinine levels in the Top-L and Top-H groups as well as renal XOR in the Top-H group were significantly lower than those in the Feb group. Urinary excretion of L-FABP in both the Top-H and Top-L groups was significantly lower than in the adenine and Feb groups. In conclusion, Top attenuated renal damage in an adenine-induced renal injury model. PMID:27029427

  5. Renoprotective effect of the xanthine oxidoreductase inhibitor topiroxostat on adenine-induced renal injury.

    PubMed

    Kamijo-Ikemori, Atsuko; Sugaya, Takeshi; Hibi, Chihiro; Nakamura, Takashi; Murase, Takayo; Oikawa, Tsuyoshi; Hoshino, Seiko; Hisamichi, Mikako; Hirata, Kazuaki; Kimura, Kenjiro; Shibagaki, Yugo

    2016-06-01

    The aim of the present study was to reveal the effect of a xanthine oxidoreductase (XOR) inhibitor, topiroxostat (Top), compared with another inhibitor, febuxostat (Feb), in an adenine-induced renal injury model. We used human liver-type fatty acid-binding protein (L-FABP) chromosomal transgenic mice, and urinary L-FABP, a biomarker of tubulointerstitial damage, was used to evaluate tubulointerstitial damage. Male transgenic mice (n = 24) were fed a 0.2% (wt/wt) adenine-containing diet. Two weeks after the start of this diet, renal dysfunction was confirmed, and the mice were divided into the following four groups: the adenine group was given only the diet containing adenine, and the Feb, high-dose Top (Top-H), and low-dose Top (Top-L) groups were given diets containing Feb (3 mg/kg), Top-H (3 mg/kg), and Top-L (1 mg/kg) in addition to adenine for another 2 wk. After withdrawal of the adenine diet, each medication was continued for 2 wk. Serum creatinine levels, the degree of macrophage infiltration, tubulointerstitial damage, renal fibrosis, urinary 15-F2t-isoprostane levels, and renal XOR activity were significantly attenuated in the kidneys of the Feb, Top-L, and Top-H groups compared with the adenine group. Serum creatinine levels in the Top-L and Top-H groups as well as renal XOR in the Top-H group were significantly lower than those in the Feb group. Urinary excretion of L-FABP in both the Top-H and Top-L groups was significantly lower than in the adenine and Feb groups. In conclusion, Top attenuated renal damage in an adenine-induced renal injury model.

  6. Low background signal platform for the detection of ATP: when a molecular aptamer beacon meets graphene oxide.

    PubMed

    He, Yue; Wang, Zhi-Gang; Tang, Hong-Wu; Pang, Dai-Wen

    2011-11-15

    A novel molecular aptamer beacon (MAB) was designed by integrating a single-labeled hairpin-shaped aptamer and graphene oxide (GO). The hairpin-shaped aptamer was constructed with anti-ATP aptamer and another five nucleotides added to the 5'-end of the aptamer which are complementary to nucleotides at the 3'-end of the aptamer to form a hairpin-shaped probe. This newly designed MAB which acts as a low background signal platform was used for the ATP detection based on long-range resonance energy transfer (LrRET). In the absence of ATP, the adsorption of the dye-labeled hairpin-shaped aptamer on GO makes the dyes close proximity to GO surface resulting in high efficiency quenching of fluorescence of the dyes. Therefore, the fluorescence of the designed MAB is completely quenched by GO, and the system shows very low background. Conversely, and very importantly, upon the adding of ATP, the quenched fluorescence is recovered significantly, and ATP can be detected in a wide range of 5-2500μM with a detection limit of 2μM and good selectivity. Moreover, when the GO-based MAB was used in cellular ATP assays, preeminent fluorescence signals were obtained, thus the platform of GO-based MAB could be used to detect ATP in real-world samples.

  7. Statistical analysis of nucleotide runs in coding and noncoding DNA sequences.

    PubMed

    Sprizhitsky YuA; Nechipurenko YuD; Alexandrov, A A; Volkenstein, M V

    1988-10-01

    A statistical analysis of the occurrence of particular nucleotide runs in DNA sequences of different species has been carried out. There are considerable differences of run distributions in DNA sequences of procaryotes, invertebrates and vertebrates. There is an abundance of short runs (1-2 nucleotides long) in the coding sequences and there is a deficiency of such runs in the noncoding regions. However, some interesting exceptions from this rule exist for the run distribution of adenine in procaryotes and for the arrangement of purine-pyrimidine runs in eucaryotes. The similarity in the distributions of such runs in the coding and noncoding regions may be due to some structural features of the DNA molecule as a whole. Runs of guanine (or cytosine) of three to six nucleotides occur predominantly in noncoding DNA regions in eucaryotes, especially in vertebrates.

  8. N6-methyl-adenine: an epigenetic signal for DNA-protein interactions.

    PubMed

    Wion, Didier; Casadesús, Josep

    2006-03-01

    N(6)-methyl-adenine is found in the genomes of bacteria, archaea, protists and fungi. Most bacterial DNA adenine methyltransferases are part of restriction-modification systems. Certain groups of Proteobacteria also harbour solitary DNA adenine methyltransferases that provide signals for DNA-protein interactions. In gamma-proteobacteria, Dam methylation regulates chromosome replication, nucleoid segregation, DNA repair, transposition of insertion elements and transcription of specific genes. In Salmonella, Haemophilus, Yersinia and Vibrio species and in pathogenic Escherichia coli, Dam methylation is required for virulence. In alpha-proteobacteria, CcrM methylation regulates the cell cycle in Caulobacter, Rhizobium and Agrobacterium, and has a role in Brucella abortus infection.

  9. Cytochrome b nucleotide sequence variation among the Atlantic Alcidae.

    PubMed

    Friesen, V L; Montevecchi, W A; Davidson, W S

    1993-01-01

    Analysis of cytochrome b nucleotide sequences of the six extant species of Atlantic alcids and a gull revealed an excess of adenines and cytosines and a deficit of guanines at silent sites on the coding strand. Phylogenetic analyses grouped the sequences of the common (Uria aalge) and Brünnich's (U. lomvia) guillemots, followed by the razorbill (Alca torda) and little auk (Alle alle). The black guillemot (Cepphus grylle) sequence formed a sister taxon, and the puffin (Fratercula arctica) fell outside the other alcids. Phylogenetic comparisons of substitutions indicated that mutabilities of bases did not differ, but that C was much more likely to be incorporated than was G. Imbalances in base composition appear to result from a strand bias in replication errors, which may result from selection on secondary RNA structure and/or the energetics of codon-anticodon interactions. PMID:7916741

  10. Redox regulation of ATP sulfurylase in microalgae.

    PubMed

    Prioretti, Laura; Lebrun, Régine; Gontero, Brigitte; Giordano, Mario

    2016-09-30

    ATP sulfurylase (ATPS) catalyzes the first step of sulfur assimilation in photosynthetic organisms. An ATPS type A is mostly present in freshwater cyanobacteria, with four conserved cysteine residues. Oceanic cyanobacteria and most eukaryotic algae instead, possess an ATPS-B containing seven to ten cysteines; five of them are conserved, but only one in the same position as ATPS-A. We investigated the role of cysteines on the regulation of the different algal enzymes. We found that the activity of ATPS-B from four different microorganisms was enhanced when reduced and decreased when oxidized. The LC-MS/MS analysis of the ATPS-B from the marine diatom Thalassiosira pseudonana showed that the residue Cys-247 was presumably involved in the redox regulation. The absence of this residue in the ATPS-A of the freshwater cyanobacterium Synechocystis sp. instead, was consistent with its lack of regulation. Some other conserved cysteine residues in the ATPS from T. pseduonana and not in Synechocystis sp.were accessible to redox agents and possibly play a role in the enzyme regulation. Furthermore, the fact that oceanic cyanobacteria have ATPS-B structurally and functionally closer to that from most of eukaryotic algae than to the ATPS-A from other cyanobacteria suggests that life in the sea or freshwater may have driven the evolution of ATPS. PMID:27613093

  11. Redox regulation of ATP sulfurylase in microalgae.

    PubMed

    Prioretti, Laura; Lebrun, Régine; Gontero, Brigitte; Giordano, Mario

    2016-09-30

    ATP sulfurylase (ATPS) catalyzes the first step of sulfur assimilation in photosynthetic organisms. An ATPS type A is mostly present in freshwater cyanobacteria, with four conserved cysteine residues. Oceanic cyanobacteria and most eukaryotic algae instead, possess an ATPS-B containing seven to ten cysteines; five of them are conserved, but only one in the same position as ATPS-A. We investigated the role of cysteines on the regulation of the different algal enzymes. We found that the activity of ATPS-B from four different microorganisms was enhanced when reduced and decreased when oxidized. The LC-MS/MS analysis of the ATPS-B from the marine diatom Thalassiosira pseudonana showed that the residue Cys-247 was presumably involved in the redox regulation. The absence of this residue in the ATPS-A of the freshwater cyanobacterium Synechocystis sp. instead, was consistent with its lack of regulation. Some other conserved cysteine residues in the ATPS from T. pseduonana and not in Synechocystis sp.were accessible to redox agents and possibly play a role in the enzyme regulation. Furthermore, the fact that oceanic cyanobacteria have ATPS-B structurally and functionally closer to that from most of eukaryotic algae than to the ATPS-A from other cyanobacteria suggests that life in the sea or freshwater may have driven the evolution of ATPS.

  12. Nucleotide cleaving agents and method

    DOEpatents

    Que, Jr., Lawrence; Hanson, Richard S.; Schnaith, Leah M. T.

    2000-01-01

    The present invention provides a unique series of nucleotide cleaving agents and a method for cleaving a nucleotide sequence, whether single-stranded or double-stranded DNA or RNA, using and a cationic metal complex having at least one polydentate ligand to cleave the nucleotide sequence phosphate backbone to yield a hydroxyl end and a phosphate end.

  13. The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange.

    PubMed

    Fenyk, Stepan; Dixon, Christopher H; Gittens, William H; Townsend, Philip D; Sharples, Gary J; Pålsson, Lars-Olof; Takken, Frank L W; Cann, Martin J

    2016-01-15

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA.

  14. The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange.

    PubMed

    Fenyk, Stepan; Dixon, Christopher H; Gittens, William H; Townsend, Philip D; Sharples, Gary J; Pålsson, Lars-Olof; Takken, Frank L W; Cann, Martin J

    2016-01-15

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA. PMID:26601946

  15. The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange*

    PubMed Central

    Fenyk, Stepan; Dixon, Christopher H.; Gittens, William H.; Townsend, Philip D.; Sharples, Gary J.; Pålsson, Lars-Olof; Takken, Frank L. W.; Cann, Martin J.

    2016-01-01

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA. PMID:26601946

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

    PubMed

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

    2014-10-17

    ATP has dual roles in the reaction cycle of sarcoplasmic reticulum Ca(2+)-ATPase. Upon binding to the Ca2E1 state, ATP phosphorylates the enzyme, and by binding to other conformational states in a non-phosphorylating modulatory mode ATP stimulates the dephosphorylation and other partial reaction steps of the cycle, thereby ensuring a high rate of Ca(2+) transport under physiological conditions. The present study elucidates the mechanism underlying the modulatory effect on dephosphorylation. In the intermediate states of dephosphorylation the A-domain residues Ser(186) and Asp(203) interact with Glu(439) (N-domain) and Arg(678) (P-domain), respectively. Single mutations to these residues abolish the stimulation of dephosphorylation by ATP. The double mutation swapping Asp(203) and Arg(678) rescues ATP stimulation, whereas this is not the case for the double mutation swapping Ser(186) and Glu(439). By taking advantage of the ability of wild type and mutant Ca(2+)-ATPases to form stable complexes with aluminum fluoride (E2·AlF) and beryllium fluoride (E2·BeF) as analogs of the E2·P phosphoryl transition state and E2P ground state, respectively, of the dephosphorylation reaction, the mutational effects on ATP binding to these intermediates are demonstrated. In the wild type Ca(2+)-ATPase, the ATP affinity of the E2·P phosphoryl transition state is higher than that of the E2P ground state, thus explaining the stimulation of dephosphorylation by nucleotide-induced transition state stabilization. We find that the Asp(203)-Arg(678) and Ser(186)-Glu(439) interdomain bonds are critical, because they tighten the interaction with ATP in the E2·P phosphoryl transition state. Moreover, ATP binding and the Ser(186)-Glu(439) bond are mutually exclusive in the E2P ground state. PMID:25193668

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

    PubMed

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

    2007-01-19

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

  18. DNA methylation on N6-adenine in C. elegans

    PubMed Central

    Greer, Eric Lieberman; Blanco, Mario Andres; Gu, Lei; Sendinc, Erdem; Liu, Jianzhao; Aristizábal-Corrales, David; Hsu, Chih-Hung; Aravind, L.; He, Chuan; Shi, Yang

    2015-01-01

    Summary In mammalian cells, DNA methylation on the 5th position of cytosine (5mC) plays an important role as an epigenetic mark. However, DNA methylation was considered to be absent in C. elegans because of the lack of detectable 5mC as well as homologs of the cytosine DNA methyltransferases. Here, using multiple approaches, we demonstrate the presence of adenine N6-methylation (6mA) in C. elegans DNA. We further demonstrate that this modification increases trans-generationally in a paradigm of epigenetic inheritance. Importantly, we identify a DNA demethylase, NMAD-1, and a potential DNA methyltransferase, DAMT-1, which regulate 6mA levels and crosstalk between methylation of histone H3K4me2 and 6mA, and control the epigenetic inheritance of phenotypes associated with the loss of the H3K4me2 demethylase spr-5. Together, these data identify a DNA modification in C. elegans and raise the exciting possibility that 6mA may be a carrier of heritable epigenetic information in eukaryotes. PMID:25936839

  19. Extracellular nucleotide and nucleoside signaling in vascular and blood disease

    PubMed Central

    Idzko, Marco; Ferrari, Davide; Riegel, Ann-Kathrin

    2014-01-01

    Nucleotides and nucleosides—such as adenosine triphosphate (ATP) and adenosine—are famous for their intracellular roles as building blocks for the genetic code or cellular energy currencies. In contrast, their function in the extracellular space is different. Here, they are primarily known as signaling molecules via activation of purinergic receptors, classified as P1 receptors for adenosine or P2 receptors for ATP. Because extracellular ATP is rapidly converted to adenosine by ectonucleotidase, nucleotide-phosphohydrolysis is important for controlling the balance between P2 and P1 signaling. Gene-targeted mice for P1, P2 receptors, or ectonucleotidase exhibit only very mild phenotypic manifestations at baseline. However, they demonstrate alterations in disease susceptibilities when exposed to a variety of vascular or blood diseases. Examples of phenotypic manifestations include vascular barrier dysfunction, graft-vs-host disease, platelet activation, ischemia, and reperfusion injury or sickle cell disease. Many of these studies highlight that purinergic signaling events can be targeted therapeutically. PMID:25001468

  20. Spin-dependent electron transport in zinc- and manganese-doped adenine molecules

    SciTech Connect

    Simchi, Hamidreza; Esmaeilzadeh, Mahdi Mazidabadi, Hossein

    2014-01-28

    The spin-dependent electron transport properties of zinc- and manganese-doped adenine molecules connected to zigzag graphene leads are studied in the zero bias regime using the non-equilibrium Green's function method. The conductance of the adenine molecule increased and became spin-dependent when a zinc or manganese atom was doped into the molecules. The effects of a transverse electric field on the spin-polarization of the transmitted electrons were investigated and the spin-polarization was controlled by changing the transverse electric field. Under the presence of a transverse electric field, both the zinc- and manganese-doped adenine molecules acted as spin-filters. The maximum spin-polarization of the manganese-doped adenine molecule was greater than the molecule doped with zinc.

  1. Binding of nicotinamide–adenine dinucleotides to diphtheria toxin

    PubMed Central

    Montanaro, L.; Sperti, Simonetta

    1967-01-01

    1. Changes in protein fluorescence have been utilized in determining the stoicheiometry and dissociation constants of the complexes of diphtheria toxin with NADH2, NAD, NADPH2 and NADP. 2. The binding stoicheiometry is 2moles of NADH2 and 1mole of NADPH2/mole of diphtheria toxin. The binding sites for NADH2 appear to be equivalent and independent. 3. The toxin shows a higher affinity for the reduced than for the oxidized forms of the nucleotides. 4. Dissociation constants at 0·01I, pH7 and 25° are 0·7×10−6m for NADH2 and 0·45×10−6m for NADPH2. Dissociation constants increase with increasing ionic strength, indicating that the binding is mainly electrostatic. 5. Bound NADH2 and NADPH2 may be activated to fluoresce by the transfer of energy from the excited aromatic amino acids of the toxin. Activation and emission spectra of bound and free nucleotides are compared. 6. Since NAD and NADH2 are cofactors specifically required for the inhibition of protein synthesis by diphtheria toxin, the possible role of toxin–nucleotide complexes is discussed in this regard. PMID:4384596

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

    PubMed

    Kiani, Farooq Ahmad; Fischer, Stefan

    2014-07-22

    Myosin is a molecular motor responsible for biological motions such as muscle contraction and intracellular cargo transport, for which it hydrolyzes adenosine 5'-triphosphate (ATP). Early steps of the mechanism by which myosin catalyzes ATP hydrolysis have been investigated, but still missing are the structure of the final ADP·inorganic phosphate (Pi) product and the complete pathway leading to it. Here, a comprehensive description of the catalytic strategy of myosin is formulated, based on combined quantum-classical molecular mechanics calculations. A full exploration of catalytic pathways was performed and a final product structure was found that is consistent with all experiments. Molecular movies of the relevant pathways show the different reorganizations of the H-bond network that lead to the final product, whose γ-phosphate is not in the previously reported HPγO4(2-) state, but in the H2PγO4(-) state. The simulations reveal that the catalytic strategy of myosin employs a three-pronged tactic: (i) Stabilization of the γ-phosphate of ATP in a dissociated metaphosphate (PγO3(-)) state. (ii) Polarization of the attacking water molecule, to abstract a proton from that water. (iii) Formation of multiple proton wires in the active site, for efficient transfer of the abstracted proton to various product precursors. The specific role played in this strategy by each of the three loops enclosing ATP is identified unambiguously. It explains how the precise timing of the ATPase activation during the force generating cycle is achieved in myosin. The catalytic strategy described here for myosin is likely to be very similar in most nucleotide hydrolyzing enzymes.

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

    PubMed Central

    Kiani, Farooq Ahmad; Fischer, Stefan

    2014-01-01

    Myosin is a molecular motor responsible for biological motions such as muscle contraction and intracellular cargo transport, for which it hydrolyzes adenosine 5'-triphosphate (ATP). Early steps of the mechanism by which myosin catalyzes ATP hydrolysis have been investigated, but still missing are the structure of the final ADP·inorganic phosphate (Pi) product and the complete pathway leading to it. Here, a comprehensive description of the catalytic strategy of myosin is formulated, based on combined quantum–classical molecular mechanics calculations. A full exploration of catalytic pathways was performed and a final product structure was found that is consistent with all experiments. Molecular movies of the relevant pathways show the different reorganizations of the H-bond network that lead to the final product, whose γ-phosphate is not in the previously reported HPγO42− state, but in the H2PγO4− state. The simulations reveal that the catalytic strategy of myosin employs a three-pronged tactic: (i) Stabilization of the γ-phosphate of ATP in a dissociated metaphosphate (PγO3−) state. (ii) Polarization of the attacking water molecule, to abstract a proton from that water. (iii) Formation of multiple proton wires in the active site, for efficient transfer of the abstracted proton to various product precursors. The specific role played in this strategy by each of the three loops enclosing ATP is identified unambiguously. It explains how the precise timing of the ATPase activation during the force generating cycle is achieved in myosin. The catalytic strategy described here for myosin is likely to be very similar in most nucleotide hydrolyzing enzymes. PMID:25006262

  4. Nicotinamide adenine dinucleotide-dependent and nicotinamide adenine dinucleotide-independent lactate dehydrogenases in homofermentative and heterofermentative lactic acid bacteria.

    PubMed

    Doelle, H W

    1971-12-01

    Three homofermentative (Lactobacillus plantarum B38, L. plantarum B33, Pediococcus pentosaceus B30) and three heterofermentative (Leuconostoc mesenteroides 39, L. oenos B70, Lactobacillus brevis) lactic acid bacteria were examined for the presence or absence of nicotinamide adenine dinucleotide (NAD)-dependent and NAD-independent d- and l-lactate dehydrogenases. Two of the six strains investigated, P. pentosaceus and L. oenos, did not exhibit an NAD-independent enzyme activity capable of reducing dichlorophenol indophenol. The pH optima of the lactic dehydrogenases were determined. The NAD-dependent enzymes from homofermentative strains exhibited optima at pH 7.8 to 8.8, whereas values from 9.0 to 10.0 were noted for these enzymes from heterofermentative organisms. The optima for the NAD-independent enzymes were between 5.8 and 6.6. The apparent Michaelis-Menten constants determined for both NAD and the substrates demonstrated the existence of a greater affinity for d- than l-lactic acid. A comparison of the specific NAD-dependent and NAD-independent lactate dehydrogenase activities revealed a direct correlation of the d/l ratios of these activities with the type of lactic acid produced during the growth of the organism.

  5. Benchmark Thermochemistry for Biologically Relevant Adenine and Cytosine. A Combined Experimental and Theoretical Study.

    PubMed

    Emel'yanenko, Vladimir N; Zaitsau, Dzmitry H; Shoifet, Evgeni; Meurer, Florian; Verevkin, Sergey P; Schick, Christoph; Held, Christoph

    2015-09-17

    The thermochemical properties available in the literature for adenine and cytosine are in disarray. A new condensed phase standard (p° = 0.1 MPa) molar enthalpy of formation at T = 298.15 K was measured by using combustion calorimetry. New molar enthalpies of sublimation were derived from the temperature dependence of vapor pressure measured by transpiration and by the quarz-crystal microbalance technique. The heat capacities of crystalline adenine and cytosine were measured by temperature-modulated DSC. Thermodynamic data on adenine and cytosine available in the literature were collected, evaluated, and combined with our experimental results. Thus, the evaluated collection of data together with the new experimental results reported here has helped to resolve contradictions in the available enthalpies of formation. A set of reliable thermochemical data is recommended for adenine and cytosine for further thermochemical calculations. Quantum-chemical calculations of the gas phase molar enthalpies of formation of adenine and cytosine have been performed by using the G4 method and results were in excellent agreement with the recommended experimental data. The standard molar entropies of formation and the standard molar Gibbs functions of formation in crystal and gas state have been calculated. Experimental vapor-pressure data measured in this work were used to estimate pure-component PC-SAFT parameters. This allowed modeling solubility of adenine and cytosine in water over the temperature interval 278-310 K. PMID:26317826

  6. Benchmark Thermochemistry for Biologically Relevant Adenine and Cytosine. A Combined Experimental and Theoretical Study.

    PubMed

    Emel'yanenko, Vladimir N; Zaitsau, Dzmitry H; Shoifet, Evgeni; Meurer, Florian; Verevkin, Sergey P; Schick, Christoph; Held, Christoph

    2015-09-17

    The thermochemical properties available in the literature for adenine and cytosine are in disarray. A new condensed phase standard (p° = 0.1 MPa) molar enthalpy of formation at T = 298.15 K was measured by using combustion calorimetry. New molar enthalpies of sublimation were derived from the temperature dependence of vapor pressure measured by transpiration and by the quarz-crystal microbalance technique. The heat capacities of crystalline adenine and cytosine were measured by temperature-modulated DSC. Thermodynamic data on adenine and cytosine available in the literature were collected, evaluated, and combined with our experimental results. Thus, the evaluated collection of data together with the new experimental results reported here has helped to resolve contradictions in the available enthalpies of formation. A set of reliable thermochemical data is recommended for adenine and cytosine for further thermochemical calculations. Quantum-chemical calculations of the gas phase molar enthalpies of formation of adenine and cytosine have been performed by using the G4 method and results were in excellent agreement with the recommended experimental data. The standard molar entropies of formation and the standard molar Gibbs functions of formation in crystal and gas state have been calculated. Experimental vapor-pressure data measured in this work were used to estimate pure-component PC-SAFT parameters. This allowed modeling solubility of adenine and cytosine in water over the temperature interval 278-310 K.

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

    SciTech Connect

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

    1987-08-01

    The energy requirement for protein breakdown in Escherichia coli has generally been attributed to the ATP-dependence of protease La, the lon gene product. The authors have partially purified another ATP-dependent protease from lon/sup -/ cells that lack protease La (as shown by immunoblotting). This enzyme hydrolyzes (/sup 3/H)methyl-casein to acid-soluble products in the presence of ATP and Mg/sup 2 +/. ATP hydrolysis appears necessary for proteolytic activity. Since this enzyme is inhibited by diisopropyl fluorophosphate, it appears to be a serine protease, but it also contains essential thiol residues. They propose to name this enzyme protease Ti. It differs from protease La in nucleotide specificity, inhibitor sensitivity, and subunit composition. On gel filtration, protease Ti has an apparent molecular weight of 370,000. It can be fractionated by phosphocellulose chromatography or by DEAE chromatography into two components with apparent molecular weights of 260,000 and 140,000. When separated, they do not show preteolytic activity. One of these components, by itself, has ATPase activity and is labile in the absence of ATP. The other contains the diisopropyl fluorophosphate-sensitive proteolytic site. These results and the similar findings of Katayama-Fujimura et al. indicate that E. coli contains two ATP-hydrolyzing proteases, which differ in many biochemical features and probably in their physiological roles.

  8. Role of ATP binding and hydrolysis in assembly of MacAB-TolC macrolide transporter.

    PubMed

    Lu, Shuo; Zgurskaya, Helen I

    2012-12-01

    MacB is a founding member of the Macrolide Exporter family of transporters belonging to the ATP-Binding Cassette superfamily. These proteins are broadly represented in genomes of both Gram-positive and Gram-negative bacteria and are implicated in virulence and protection against antibiotics and peptide toxins. MacB transporter functions together with MacA, a periplasmic membrane fusion protein, which stimulates MacB ATPase. In Gram-negative bacteria, MacA is believed to couple ATP hydrolysis to transport of substrates across the outer membrane through a TolC-like channel. In this study, we report a real-time analysis of concurrent ATP hydrolysis and assembly of MacAB-TolC complex. MacB binds nucleotides with a low millimolar affinity and fast on- and off-rates. In contrast, MacA-MacB complex is formed with a nanomolar affinity, which further increases in the presence of ATP. Our results strongly suggest that association between MacA and MacB is stimulated by ATP binding to MacB but remains unchanged during ATP hydrolysis cycle. We also found that the large periplasmic loop of MacB plays the major role in coupling reactions separated in two different membranes. This loop is required for MacA-dependent stimulation of MacB ATPase and at the same time, contributes to recruitment of TolC into a trans-envelope complex. PMID:23057817

  9. A sensitive ligase-based ATP electrochemical assay using molecular beacon-like DNA.

    PubMed

    Wang, Yonghong; He, Xiaoxiao; Wang, Kemin; Ni, Xiaoqi

    2010-05-15

    A sensitive and selective ligase-based signal-on electrochemical sensing method for adenosine-5'-triphosphate (ATP) detection had been developed using molecular beacon (MB)-like DNA. In this method, the biotin-tagged MB-like DNA was self-assembled onto a gold electrode to form a stem-loop structure by means of facile gold-thiol chemistry, which resulted in blockage of electronic transmission. It was eT OFF state. In the presence of ATP, two nucleotide fragments which were complementary to the loop of the MB-like DNA could be ligated by the ATP-dependent T4 DNA ligase. Hybridization of the ligated DNA with the MB-like DNA induced a significant conformational change in this surface-confined DNA structure, which in turn released the biotin from the surface allowing free exchange of electrons with the electrode generating a measurable electrochemical signal (eT ON). The resulting change in electron transfer efficiency was readily measured by differential pulse voltammetry at target ATP concentrations as low as 0.05 nM and with linear response range from 0.1 to 1000 nM. Moreover, it was also able to discriminate ATP from its analogues. The proposed method had been successfully applied to the determination of ATP in the Escherichia coli O157:H7 extracts of water samples, and the linear response was found between the concentrations of 10(3) and 10(7) cfu/mL.

  10. Synthesis and Characterization of Oligodeoxyribonucleotides Modified with 2′-Amino-α-L-LNA Adenine Monomers: High-affinity Targeting of Single-Stranded DNA

    PubMed Central

    Andersen, Nicolai K.; Anderson, Brooke A.; Wengel, Jesper

    2014-01-01

    Development of conformationally restricted nucleotide building blocks continues to attract considerable interest due to their successful use within antisense, antigene and other gene-targeting strategies. Locked nucleic acid (LNA) and its diastereomer α-L-LNA are two interesting examples hereof. Oligonucleotides modified with these units display greatly increased affinity toward nucleic acid targets, improved binding specificity and enhanced enzymatic stability relative to unmodified strands. Here, we present the synthesis and biophysical characterization of oligodeoxyribonucleotides (ONs) modified with 2′-amino-α-L-LNA adenine monomers W–Z. The synthesis of target phosphoramidites 1–4 initiates from pentafuranose 5, which upon Vorbrüggen glycosylation, O2′-deacylation, O2′-activation and C2′-azide introduction yields nucleoside 8. A one-pot tandem Staudinger/intramolecular nucleophilic substitution converts 8 into 2′-amino-α-L-LNA adenine intermediate 9, which after a series of non-trivial protecting group manipulations affords key intermediate 15. Subsequent chemoselective N2′-functionalization and O3′-phosphitylation gives targets 1–4 in ~1–3% overall yield over eleven steps from 5. ONs modified with pyrene-functionalized 2′-amino-α-L-LNA adenine monomers X-Z display greatly increased affinity toward DNA targets (ΔTm/modification up to +14 °C). Results from absorption and fluorescence spectroscopy suggest that the duplex stabilization is a result of pyrene intercalation. These characteristics render N2′-pyrene-functionalized 2′-amino-α-L-LNA of considerable interest for DNA-targeting applications. PMID:24304240

  11. Anesthetic propofol overdose causes vascular hyperpermeability by reducing endothelial glycocalyx and ATP production.

    PubMed

    Lin, Ming-Chung; Lin, Chiou-Feng; Li, Chien-Feng; Sun, Ding-Ping; Wang, Li-Yun; Hsing, Chung-Hsi

    2015-05-27

    Prolonged treatment with a large dose of propofol may cause diffuse cellular cytotoxicity; however, the detailed underlying mechanism remains unclear, particularly in vascular endothelial cells. Previous studies showed that a propofol overdose induces endothelial injury and vascular barrier dysfunction. Regarding the important role of endothelial glycocalyx on the maintenance of vascular barrier integrity, we therefore hypothesized that a propofol overdose-induced endothelial barrier dysfunction is caused by impaired endothelial glycocalyx. In vivo, we intraperitoneally injected ICR mice with overdosed propofol, and the results showed that a propofol overdose significantly induced systemic vascular hyperpermeability and reduced the expression of endothelial glycocalyx, syndecan-1, syndecan-4, perlecan mRNA and heparan sulfate (HS) in the vessels of multiple organs. In vitro, a propofol overdose reduced the expression of syndecan-1, syndecan-4, perlecan, glypican-1 mRNA and HS and induced significant decreases in the nicotinamide adenine dinucleotide (NAD+)/NADH ratio and ATP concentrations in human microvascular endothelial cells (HMEC-1). Oligomycin treatment also induced significant decreases in the NAD+/NADH ratio, in ATP concentrations and in syndecan-4, perlecan and glypican-1 mRNA expression in HMEC-1 cells. These results demonstrate that a propofol overdose induces a partially ATP-dependent reduction of endothelial glycocalyx expression and consequently leads to vascular hyperpermeability due to the loss of endothelial barrier functions.

  12. Mutations in the NB-ARC Domain of I-2 That Impair ATP Hydrolysis Cause Autoactivation1[OA

    PubMed Central

    Tameling, Wladimir I.L.; Vossen, Jack H.; Albrecht, Mario; Lengauer, Thomas; Berden, Jan A.; Haring, Michel A.; Cornelissen, Ben J.C.; Takken, Frank L.W.

    2006-01-01

    Resistance (R) proteins in plants confer specificity to the innate immune system. Most R proteins have a centrally located NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4) domain. For two tomato (Lycopersicon esculentum) R proteins, I-2 and Mi-1, we have previously shown that this domain acts as an ATPase module that can hydrolyze ATP in vitro. To investigate the role of nucleotide binding and hydrolysis for the function of I-2 in planta, specific mutations were introduced in conserved motifs of the NB-ARC domain. Two mutations resulted in autoactivating proteins that induce a pathogen-independent hypersensitive response upon expression in planta. These mutant forms of I-2 were found to be impaired in ATP hydrolysis, but not in ATP binding, suggesting that the ATP- rather than the ADP-bound state of I-2 is the active form that triggers defense signaling. In addition, upon ADP binding, the protein displayed an increased affinity for ADP suggestive of a change of conformation. Based on these data, we propose that the NB-ARC domain of I-2, and likely of related R proteins, functions as a molecular switch whose state (on/off) depends on the nucleotide bound (ATP/ADP). PMID:16489136

  13. Mitochondrial Hsp90 is a ligand-activated molecular chaperone coupling ATP binding to dimer closure through a coiled-coil intermediate

    PubMed Central

    Sung, Nuri; Lee, Jungsoon; Kim, Ji-Hyun; Chang, Changsoo; Joachimiak, Andrzej; Lee, Sukyeong; Tsai, Francis T. F.

    2016-01-01

    Heat-shock protein of 90 kDa (Hsp90) is an essential molecular chaperone that adopts different 3D structures associated with distinct nucleotide states: a wide-open, V-shaped dimer in the apo state and a twisted, N-terminally closed dimer with ATP. Although the N domain is known to mediate ATP binding, how Hsp90 senses the bound nucleotide and facilitates dimer closure remains unclear. Here we present atomic structures of human mitochondrial Hsp90N (TRAP1N) and a composite model of intact TRAP1 revealing a previously unobserved coiled-coil dimer conformation that may precede dimer closure and is conserved in intact TRAP1 in solution. Our structure suggests that TRAP1 normally exists in an autoinhibited state with the ATP lid bound to the nucleotide-binding pocket. ATP binding displaces the ATP lid that signals the cis-bound ATP status to the neighboring subunit in a highly cooperative manner compatible with the coiled-coil intermediate state. We propose that TRAP1 is a ligand-activated molecular chaperone, which couples ATP binding to dramatic changes in local structure required for protein folding. PMID:26929380

  14. Phenotype and Genotype Characterization of Adenine Phosphoribosyltransferase Deficiency

    PubMed Central

    Bollée, Guillaume; Dollinger, Cécile; Boutaud, Lucile; Guillemot, Delphine; Bensman, Albert; Harambat, Jérôme; Deteix, Patrice; Daudon, Michel; Knebelmann, Bertrand

    2010-01-01

    Adenine phosphoribosyltransferase (APRT) deficiency is a rare autosomal recessive disorder causing 2,8-dihydroxyadenine stones and renal failure secondary to intratubular crystalline precipitation. Little is known regarding the clinical presentation of APRT deficiency, especially in the white population. We retrospectively reviewed all 53 cases of APRT deficiency (from 43 families) identified at a single institution between 1978 and 2009. The median age at diagnosis was 36.3 years (range 0.5 to 78.0 years). In many patients, a several-year delay separated the onset of symptoms and diagnosis. Of the 40 patients from 33 families with full clinical data available, 14 (35%) had decreased renal function at diagnosis. Diagnosis occurred in six (15%) patients after reaching ESRD, with five diagnoses made at the time of disease recurrence in a renal allograft. Eight (20%) patients reached ESRD during a median follow-up of 74 months. Thirty-one families underwent APRT sequencing, which identified 54 (87%) mutant alleles on the 62 chromosomes analyzed. We identified 18 distinct mutations. A single T insertion in a splice donor site in intron 4 (IVS4 + 2insT), which produces a truncated protein, accounted for 40.3% of the mutations. We detected the IVS4 + 2insT mutation in two (0.98%) of 204 chromosomes of healthy newborns. This report, which is the largest published series of APRT deficiency to date, highlights the underdiagnosis and potential severity of this disease. Early diagnosis is crucial for initiation of effective treatment with allopurinol and for prevention of renal complications. PMID:20150536

  15. Studies on yeast nucleoside triphosphate-nucleoside diphosphate transphosphorylase (nucleoside diphosphokinase). IV. Steady-state kinetic properties with thymidine nucleotides (including 3'-azido-3'-deoxythymidine analogues).

    PubMed

    Kuby, S A; Fleming, G; Alber, T; Richardson, D; Takenaka, H; Hamada, M

    1991-01-01

    A study of the steady-state kinetics of the crystalline brewer's yeast (Saccharomyces carlsbergensis) nucleoside diphosphokinase, with the magnesium complexes of the adenine and thymidine nucleotides as reactants, has led to a postulated kinetic mechanism which proceeds through a substituted enzyme. This agrees with the earlier conclusions of Garces and Cleland [Biochemistry 1969; 8:633-640] who characterized a reaction between the magnesium complexes of the adenine and uridine nucleotides. An advantage of using thymidine nucleotides as reactants is that they permit accurate, rapid and continuous assays of the enzymatic activity in coupled-enzymatic tests. Through measurements of the initial velocities and product inhibition studies, the Michaelis constants, maximum velocities, and inhibition constants could be evaluated for the individual substrates. Competitive substrate inhibition was encountered at relatively high substrate concentrations, which also permitted an evaluation of their ability to act as 'dead-end' inhibitors. The Michaelis constants for the 3'-azido-3'-deoxythymidine (AzT) analogues were also evaluated and, although these values were only somewhat higher than those of their natural substrates, the Km's for the adenine nucleotides as paired substrates were lower and the Vmax's were drastically reduced. The pharmacological implications of these observations are touched upon and extrapolated to the cases where therapeutic doses of AzT may be employed.

  16. Interaction of nucleotides with Asp(351) and the conserved phosphorylation loop of sarcoplasmic reticulum Ca(2+)-ATPase.

    PubMed

    McIntosh, D B; Woolley, D G; MacLennan, D H; Vilsen, B; Andersen, J P

    1999-09-01

    The nucleotide binding properties of mutants with alterations to Asp(351) and four of the other residues in the conserved phosphorylation loop, (351)DKTGTLT(357), of sarcoplasmic reticulum Ca(2+)-ATPase were investigated using an assay based on the 2', 3'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N(3)-ATP) photolabeling of Lys(492) and competition with ATP. In selected cases where the competition assay showed extremely high affinity, ATP binding was also measured by a direct filtration assay. At pH 8.5 in the absence of Ca(2+), mutations removing the negative charge of Asp(351) (D351N, D351A, and D351T) produced pumps that bound MgTNP-8N(3)-ATP and MgATP with affinities 20-156-fold higher than wild type (K(D) as low as 0.006 microM), whereas the affinity of mutant D351E was comparable with wild type. Mutations K352R, K352Q, T355A, and T357A lowered the affinity for MgATP and MgTNP-8N(3)-ATP 2-1000- and 1-6-fold, respectively, and mutation L356T completely prevented photolabeling of Lys(492). In the absence of Ca(2+), mutants D351N and D351A exhibited the highest nucleotide affinities in the presence of Mg(2+) and at alkaline pH (E1 state). The affinity of mutant D351A for MgATP was extraordinarily high in the presence of Ca(2+) (K(D) = 0.001 microM), suggesting a transition state like configuration at the active site under these conditions. The mutants with reduced ATP affinity, as well as mutants D351N and D351A, exhibited reduced or zero CrATP-induced Ca(2+) occlusion due to defective CrATP binding.

  17. Synergistic effect of ATP for RuvA–RuvB–Holliday junction DNA complex formation

    PubMed Central

    Iwasa, Takuma; Han, Yong-Woon; Hiramatsu, Ryo; Yokota, Hiroaki; Nakao, Kimiko; Yokokawa, Ryuji; Ono, Teruo; Harada, Yoshie

    2015-01-01

    The Escherichia coli RuvB hexameric ring motor proteins, together with RuvAs, promote branch migration of Holliday junction DNA. Zero mode waveguides (ZMWs) constitute of nanosized holes and enable the visualization of a single fluorescent molecule under micromolar order of the molecules, which is applicable to characterize the formation of RuvA–RuvB–Holliday junction DNA complex. In this study, we used ZMWs and counted the number of RuvBs binding to RuvA–Holliday junction DNA complex. Our data demonstrated that different nucleotide analogs increased the amount of Cy5-RuvBs binding to RuvA–Holliday junction DNA complex in the following order: no nucleotide, ADP, ATPγS, and mixture of ADP and ATPγS. These results suggest that not only ATP binding to RuvB but also ATP hydrolysis by RuvB facilitates a stable RuvA–RuvB–Holliday junction DNA complex formation. PMID:26658024

  18. Study of sperm cell phosphorylating systems using nucleotide photoaffinity probes

    SciTech Connect

    Khatoon, S.

    1983-01-01

    The major thrust of the research presented in this thesis was to identify specific nucleotide binding proteins and phosphoproteins of rat caput and cauda sperm. Also, the differences in these proteins between caput and cauda sperm were investigated as well as determination of the membrane sidedness of the proteins and their location in either the head or tail/mid-piece region. In addition, the effects of small molecular weight modifers such as cGMP, cAMP and Ca/sup 2 +/ on the detection of binding proteins and phosphorylated proteins was studied. The technique used to identify and locate nucleotide binding proteins was photoaffinity labeling using the proven 8-azidopurine nucleotide analogs of cAMP, ATP and GTP in radioactive form. The first study presented involved the use of (/sup 32/P)8-N /sub 3/cAMP which showed that both caput and cauda sperm contained both type I and type II regulatory subunits (R/sub I/ and R/sub II/, respectively) of the cAMP dependent kinases and that the great majority of the regulatory subunits were located in the tail/mid-piece section and not in the sperm head. The second phase of this study involved the use of (..gamma../sup 32/P)8-azidoadensosine triphosphate ((..gamma../sup 32/P)8-N/sub 3/ATP) and (..gamma../sup 32/P)8-azidoguanosine triphosphate ((..gamma../sup 32/P)8-N/sub 3/GTP) to photolable specific ATP and GTP binding proteins and to phosphorylate specific phosphoproteins. Again, this was done on caput versus cauda sperm and the location of the majority of the photolabeled or phosphorylated proteins was shown to be in the tail/mid-piece fraction. In addition, considerable differences were found in both the phosphorylated and photolabeled proteins of caput versus cauda sperm.

  19. Extracellular ATP Selectively Upregulates Ecto-Nucleoside Triphosphate Diphosphohydrolase 2 and Ecto-5'-Nucleotidase by Rat Cortical Astrocytes In Vitro.

    PubMed

    Brisevac, Dusica; Adzic, Marija; Laketa, Danijela; Parabucki, Ana; Milosevic, Milena; Lavrnja, Irena; Bjelobaba, Ivana; Sévigny, Jean; Kipp, Markus; Nedeljkovic, Nadezda

    2015-11-01

    Extracellular ATP (eATP) acts as a danger-associated molecular pattern which induces reactive response of astrocytes after brain insult, including morphological remodeling of astrocytes, proliferation, chemotaxis, and release of proinflammatory cytokines. The responses induced by eATP are under control of ecto-nucleotidases, which catalyze sequential hydrolysis of ATP to adenosine. In the mammalian brain, ecto-nucleotidases comprise three enzyme families: ecto-nucleoside triphosphate diphosphohydrolases 1-3 (NTPDase1-3), ecto-nucleotide pyrophosphatase/phospodiesterases 1-3 (NPP1-3), and ecto-5'-nucleotidase (eN), which crucially determine ATP/adenosine ratio in the pericellular milieu. Altered expression of ecto-nucleotidases has been demonstrated in several experimental models of human brain dysfunctions. In the present study, we have explored the pattern of NTPDase1-3, NPP1-3, and eN expression by cultured cortical astrocytes challenged with 1 mmol/L ATP (eATP). At the transcriptional level, eATP upregulated expression of NTPDase1, NTPDase2, NPP2, and eN, while, at translational and functional levels, these were paralleled only by the induction of NTPDase2 and eN. Additionally, eATP altered membrane topology of eN, from clusters localized in membrane domains to continuous distribution along the cell membrane. Our results suggest that eATP, by upregulating NTPDase2 and eN and altering the enzyme membrane topology, affects local kinetics of ATP metabolism and signal transduction that may have important roles in the process related to inflammation and reactive gliosis. PMID:26080748

  20. Activation and inhibition of calcium-dependent histamine secretion by ATP ions applied to rat mast cells.

    PubMed Central

    Cockcroft, S; Gomperts, B D

    1979-01-01

    1. The concentration dependence on ATP of mast cell histamine secretion in the presence of various concentrations of Mg2+ and Ca2+ confirms that the agonist form of ATP is the free form of ATP (ATP(free) not bound to divalent cations, i.e. ATP4-. It induces 50% activation at about 1.2 microM, maximal secretion at about 2.7 microM and 50% self-inhibition at about 4.4 microM. 2. The divalent cations Mg2+ and Ca2+ were used to buffer ATP(tree) in the range 1-8 microM in the presence of much higher concentrations of ATP(total). In addition to its effect as a buffer for ATP, Ca2+ is required for secretion. 3. With ATP(free) at 1 microM, the time-course of histamine secretion is characterized by a delay of about 10 min before secretion commences. With increasing concentration of ATP(free) the delay becomes shorter (less than 5 min with ATP(free) at 2 microM). 4. Secretion commences promptly on addition of Ca2+ to cells which have been pretreated with low concentrations of ATP(free) (less than 2 microM). This observation suggests that the delay normally observed represents the time taken for Ca2+ sensitivity to develop (i.e. probably the time taken for Ca2+ channels to open). 5. Late addition of Ca2+ to cells pretreated with higher concentrations of ATP(free) (greater than 2 microM) results in a reduced amount of histamine secretion compared with that which normally occurs. This reduction (which increases with time of exposure to ATP) and the self-inhibition due to higher concentrations of ATP(free) may be two facets of a common inhibitory mechanism. 6. These results are discussed in the light of other experiments which show that mast cells treated with ATP(free) at self-inhibitory concentrations become permeable to phosphorylated metabolites and nucleotides. PMID:93638

  1. Adenilate pool and thylakoid ATP-synthase content in pea leaves under clinorotation.

    PubMed

    Onoiko, E B; Podorvanov, V V; Zolotareva, E K

    2001-07-01

    It is known that plant resistance to stress factors is connected with energy metabolism. The energy stored in the process of photophosphorylation in the form of ATP is used then to support respiration, transpiration, organic compound synthesis, growth and development as well as to restore cell structure after its damage under extremal environmental factors. Transformation of light energy into chemical energy of ATP is catalyzed by thylakoid membrane enzymatic complex of ATPsynthase-CF1CF0. Its activity and amount in the thylakoid membrane depends on plant growth conditions. The aim of this work was investigation of clinorotation effect on light-induced dynamics of adenyl nucleotides (AMP, ADP and ATP) and estimation of CF1CF0 content in thylakoids of pea leaves grown under slow clinorotation and vertical control.

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

    PubMed Central

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

    2015-01-01

    P2 receptors are membrane-bound receptors for extracellular nucleotides such as ATP and UTP. P2 receptors have been classified as ligand-gated ion channels or P2X receptors and G protein-coupled P2Y receptors. Recently, purinergic signaling has begun to attract attention as a potential therapeutic target for a variety of diseases especially associated with gastroenterology. This study determined the ATP and UTP-induced receptor signaling mechanism in feline esophageal contraction. Contraction of dispersed feline esophageal smooth muscle cells was measured by scanning micrometry. Phosphorylation of MLC20 was determined by western blot analysis. ATP and UTP elicited maximum esophageal contraction at 30 s and 10 μM concentration. Contraction of dispersed cells treated with 10 μM ATP was inhibited by nifedipine. However, contraction induced by 0.1 μM ATP, 0.1 μM UTP and 10 μM UTP was decreased by U73122, chelerythrine, ML-9, PTX and GDPβS. Contraction induced by 0.1 μM ATP and UTP was inhibited by Gαi3 or Gαq antibodies and by PLCβ1 or PLCβ3 antibodies. Phosphorylated MLC20 was increased by ATP and UTP treatment. In conclusion, esophageal contraction induced by ATP and UTP was preferentially mediated by P2Y receptors coupled to Gαi3 and G q proteins, which activate PLCβ1 and PLCβ3. Subsequently, increased intracellular Ca2+ and activated PKC triggered stimulation of MLC kinase and inhibition of MLC phosphatase. Finally, increased pMLC20 generated esophageal contraction. PMID:26013385

  3. Dissection of the PHO pathway in Schizosaccharomyces pombe using epistasis and the alternate repressor adenine.

    PubMed

    Estill, Molly; Kerwin-Iosue, Christine L; Wykoff, Dennis D

    2015-05-01

    In Saccharomyces cerevisiae, intracellular phosphate levels are maintained by the PHO pathway, activation of which is assayed by increased phosphatase activity. The PHO pathway of Schizosaccharomyces pombe upregulates phosphatase activity (encoded by pho1 (+)) during low extracellular phosphate levels, but the underlying mechanism is poorly understood. We utilized an alternate repressor of pho1 (+) expression (adenine supplementation) along with epistasis analysis to develop a model of how S. pombe PHO pathway components interact. Analyzing Pho1 activity in S. pombe PHO pathway deletion mutants during adenine starvation, we observed most mutants with a phosphatase defect in phosphate starvation also had a defect in adenine starvation. Pho7, a transcription factor in the PHO pathway, is necessary for an adenine starvation-mediated increase in Pho1 activity. Comparing adenine starvation to phosphate starvation, there are differences in the degree to which individual mutants regulate the two responses. Through epistasis studies, we identified two positive regulatory arms and one repressive arm of the PHO pathway. PKA activation is a positive regulator of Pho1 activity under both environmental conditions and is critical for transducing adenine concentrations in the cell. The synthesis of IP7 also appears critical for the induction of Pho1 activity during adenine starvation, but IP7 is not critical during phosphate starvation, which differs from S. cerevisiae. Finally, Csk1 is critical for repression of pho1 (+) expression during phosphate starvation. We believe all of these regulatory arms converge to increase transcription of pho1 (+) and some of the regulation acts through pho7 (+).

  4. Electrochemical detection of nicotinamide adenine dinucleotide based on molecular beacon-like DNA and E. coli DNA ligase.

    PubMed

    He, Xiaoxiao; Ni, Xiaoqi; Wang, Yonghong; Wang, Kemin; Jian, Lixin

    2011-01-15

    An electrochemical method for nicotinamide adenine dinucleotide (NAD(+)) detection with high sensitivity and selectivity has been developed by using molecular beacon (MB)-like DNA and Escherichia coli DNA ligase. In this method, MB-like DNA labeled with 5'-SH and 3'-biotin was self-assembled onto a gold electrode in its duplex form by means of facile gold-thiol chemistry, which resulted in blockage of electronic transmission. It was eT OFF state. In the presence of NAD(+), E. coli DNA ligase was activated, and the two nucleotide fragments which were complementary to the loop of the MB-like DNA could be ligated by the NAD(+)-dependent E. coli DNA ligase. Hybridization of the ligated DNA with the MB-like DNA induced a large conformational change in this surface-confined DNA structure, which in turn pushed the biotin away from the electrode surface and made the electrons exchange freely with the electrode. Then the generated electrochemical signals can be measured by differential pulse voltammetry (DPV). Under optimized conditions, a linear response to logarithmic concentration of NAD(+) range from 3 nM to 5 μM and a detection limit of 1.8 nM were obtained. Furthermore, the proposed strategy had sufficient selectivity to discriminate NAD(+) from its analogues.

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

    PubMed Central

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

    2011-01-01

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

  6. Conserved mechanisms of microtubule-stimulated ADP release, ATP binding, and force generation in transport kinesins

    PubMed Central

    Atherton, Joseph; Farabella, Irene; Yu, I-Mei; Rosenfeld, Steven S; Houdusse, Anne; Topf, Maya; Moores, Carolyn A

    2014-01-01

    Kinesins are a superfamily of microtubule-based ATP-powered motors, important for multiple, essential cellular functions. How microtubule binding stimulates their ATPase and controls force generation is not understood. To address this fundamental question, we visualized microtubule-bound kinesin-1 and kinesin-3 motor domains at multiple steps in their ATPase cycles—including their nucleotide-free states—at ∼7 Å resolution using cryo-electron microscopy. In both motors, microtubule binding promotes ordered conformations of conserved loops that stimulate ADP release, enhance microtubule affinity and prime the catalytic site for ATP binding. ATP binding causes only small shifts of these nucleotide-coordinating loops but induces large conformational changes elsewhere that allow force generation and neck linker docking towards the microtubule plus end. Family-specific differences across the kinesin–microtubule interface account for the distinctive properties of each motor. Our data thus provide evidence for a conserved ATP-driven mechanism for kinesins and reveal the critical mechanistic contribution of the microtubule interface. DOI: http://dx.doi.org/10.7554/eLife.03680.001 PMID:25209998

  7. 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.

  8. ATP Released by Electrical Stimuli Elicits Calcium Transients and Gene Expression in Skeletal Muscle*

    PubMed Central

    Buvinic, Sonja; Almarza, Gonzalo; Bustamante, Mario; Casas, Mariana; López, Javiera; Riquelme, Manuel; Sáez, Juan Carlos; Huidobro-Toro, Juan Pablo; Jaimovich, Enrique

    2009-01-01

    ATP released from cells is known to activate plasma membrane P2X (ionotropic) or P2Y (metabotropic) receptors. In skeletal muscle cells, depolarizing stimuli induce both a fast calcium signal associated with contraction and a slow signal that regulates gene expression. Here we show that nucleotides released to the extracellular medium by electrical stimulation are partly involved in the fast component and are largely responsible for the slow signals. In rat skeletal myotubes, a tetanic stimulus (45 Hz, 400 1-ms pulses) rapidly increased extracellular levels of ATP, ADP, and AMP after 15 s to 3 min. Exogenous ATP induced an increase in intracellular free Ca2+ concentration, with an EC50 value of 7.8 ± 3.1 μm. Exogenous ADP, UTP, and UDP also promoted calcium transients. Both fast and slow calcium signals evoked by tetanic stimulation were inhibited by either 100 μm suramin or 2 units/ml apyrase. Apyrase also reduced fast and slow calcium signals evoked by tetanus (45 Hz, 400 0.3-ms pulses) in isolated mouse adult skeletal fibers. A likely candidate for the ATP release pathway is the pannexin-1 hemichannel; its blockers inhibited both calcium transients and ATP release. The dihydropyridine receptor co-precipitated with both the P2Y2 receptor and pannexin-1. As reported previously for electrical stimulation, 500 μm ATP significantly increased mRNA expression for both c-fos and interleukin 6. Our results suggest that nucleotides released during skeletal muscle activity through pannexin-1 hemichannels act through P2X and P2Y receptors to modulate both Ca2+ homeostasis and muscle physiology. PMID:19822518

  9. ATP released by electrical stimuli elicits calcium transients and gene expression in skeletal muscle.

    PubMed

    Buvinic, Sonja; Almarza, Gonzalo; Bustamante, Mario; Casas, Mariana; López, Javiera; Riquelme, Manuel; Sáez, Juan Carlos; Huidobro-Toro, Juan Pablo; Jaimovich, Enrique

    2009-12-11

    ATP released from cells is known to activate plasma membrane P2X (ionotropic) or P2Y (metabotropic) receptors. In skeletal muscle cells, depolarizing stimuli induce both a fast calcium signal associated with contraction and a slow signal that regulates gene expression. Here we show that nucleotides released to the extracellular medium by electrical stimulation are partly involved in the fast component and are largely responsible for the slow signals. In rat skeletal myotubes, a tetanic stimulus (45 Hz, 400 1-ms pulses) rapidly increased extracellular levels of ATP, ADP, and AMP after 15 s to 3 min. Exogenous ATP induced an increase in intracellular free Ca(2+) concentration, with an EC(50) value of 7.8 +/- 3.1 microm. Exogenous ADP, UTP, and UDP also promoted calcium transients. Both fast and slow calcium signals evoked by tetanic stimulation were inhibited by either 100 mum suramin or 2 units/ml apyrase. Apyrase also reduced fast and slow calcium signals evoked by tetanus (45 Hz, 400 0.3-ms pulses) in isolated mouse adult skeletal fibers. A likely candidate for the ATP release pathway is the pannexin-1 hemichannel; its blockers inhibited both calcium transients and ATP release. The dihydropyridine receptor co-precipitated with both the P2Y(2) receptor and pannexin-1. As reported previously for electrical stimulation, 500 mum ATP significantly increased mRNA expression for both c-fos and interleukin 6. Our results suggest that nucleotides released during skeletal muscle activity through pannexin-1 hemichannels act through P2X and P2Y receptors to modulate both Ca(2+) homeostasis and muscle physiology. PMID:19822518

  10. SLOW MYOSIN ATP TURNOVER IN THE SUPER-RELAXED STATE IN TARANTULA MUSCLE

    PubMed Central

    Naber, Nariman; Cooke, Roger

    2011-01-01

    We measured the nucleotide turnover rate of myosin in tarantula leg-muscle fibers by observing single turnovers of the fluorescent nucleotide analog, mantATP, as monitored by the decrease in fluorescence when mantATP is replaced by ATP in a chase experiment. We find a multi-exponential process, with approximately two-thirds of the myosin showing a very slow nucleotide turnover time constant, ~30 minutes. This slow-turnover state is termed the super-relaxed state (SRX). If fibers are incubated in mantADP and chased with ADP, the SRX is not seen, indicating that trinucleotide-relaxed myosins are responsible for the SRX. Phosphorylation of the myosin regulatory light chain eliminates the fraction of myosin with the very long lifetime. The data imply that the very long-lived SRX in tarantula fibers is a highly novel adaptation for energy conservation in an animal that spends extremely long periods of time in a quiescent state employing a lie-in-wait hunting strategy. The presence of the SRX measured here correlates well with the binding of myosin heads to the core of the thick filament in a structure known as the “interacting-heads motif” observed previously by electron microscopy. Both the structural array and the long-lived SRX require relaxed filaments or relaxed fibers, both are lost upon myosin phosphorylation, and both appear to be more stable in tarantula than in vertebrate skeletal or vertebrate cardiac preparations. PMID:21763701

  11. Assignment of the Gene for Adenine Phosphoribosyltransferase to Human Chromosome 16 by Mouse-Human Somatic Cell Hybridization

    PubMed Central

    Tischfield, Jay A.; Ruddle, Frank H.

    1974-01-01

    A series of mouse-human hybrids was prepared from mouse cells deficient in adenine phosphoribosyltransferase (EC 2.4.2.7) and normal human cells. The hybrids were made in medium containing adenine and alanosine, an antimetabolite known to inhibit de novo adenylic acid biosynthesis. The mouse cells, unable to utilize exogenous adenine, were killed in this medium, but the hybrids proliferated as a consequence of their retaining the human aprt gene. The hybrids were then exposed to the adenine analogs 2,6-diaminopurine and 2-fluoroadenine to select for cells that had lost this gene. Before exposure to the adenine analogs, the expression of human adenine phosphoribosyltransferase by the hybrids was strongly associated only with the presence of human chromosome 16, and afterwards this was the only human chromosome consistently lost. This observation suggests that the human aprt gene can be assigned to chromosome 16. Images PMID:4129802

  12. Kinetic Validation of the Models for P-Glycoprotein ATP Hydrolysis and Vanadate-Induced Trapping. Proposal for Additional Steps

    PubMed Central

    Lugo, Miguel Ramón; Sharom, Frances Jane

    2014-01-01

    P-Glycoprotein, a member of the ATP-binding cassette (ABC) superfamily, is a multidrug transporter responsible for cellular efflux of hundreds of structurally unrelated compounds, including natural products, many clinically used drugs and anti-cancer agents. Expression of P-glycoprotein has been linked to multidrug resistance in human cancers. ABC transporters are driven by ATP hydrolysis at their two cytoplasmic nucleotide-binding domains, which interact to form a closed ATP-bound sandwich dimer. Intimate knowledge of the catalytic cycle of these proteins is clearly essential for understanding their mechanism of action. P-Glycoprotein has been proposed to hydrolyse ATP by an alternating mechanism, for which there is substantial experimental evidence, including inhibition of catalytic activity by trapping of ortho-vanadate at one nucleotide-binding domain, and the observation of an asymmetric occluded state. Despite many studies of P-glycoprotein ATPase activity over the past 20 years, no comprehensive kinetic analysis has yet been carried out, and some puzzling features of its behaviour remain unexplained. In this work, we have built several progressively more complex kinetic models, and then carried out simulations and detailed analysis, to test the validity of the proposed reaction pathway employed by P-glycoprotein for ATP hydrolysis. To establish kinetic parameters for the catalytic cycle, we made use of the large amount of published data on ATP hydrolysis by hamster P-glycoprotein, both purified and in membrane vesicles. The proposed kinetic scheme(s) include a high affinity priming reaction for binding of the first ATP molecule, and an independent pathway for ADP binding outside the main catalytic cycle. They can reproduce to varying degrees the observed behavior of the protein's ATPase activity and its inhibition by ortho-vanadate. The results provide new insights into the mode of action of P-glycoprotein, and some hypotheses about the nature of the

  13. Functional analysis of a structural model of the ATP-binding site of the KATP channel Kir6.2 subunit

    PubMed Central

    Antcliff, Jennifer F; Haider, Shozeb; Proks, Peter; Sansom, Mark S P; Ashcroft, Frances M

    2005-01-01

    ATP-sensitive potassium (KATP) channels couple cell metabolism to electrical activity by regulating K+ flux across the plasma membrane. Channel closure is mediated by ATP, which binds to the pore-forming subunit (Kir6.2). Here we use homology modelling and ligand docking to construct a model of the Kir6.2 tetramer and identify the ATP-binding site. The model is consistent with a large amount of functional data and was further tested by mutagenesis. Ligand binding occurs at the interface between two subunits. The phosphate tail of ATP interacts with R201 and K185 in the C-terminus of one subunit, and with R50 in the N-terminus of another; the N6 atom of the adenine ring interacts with E179 and R301 in the same subunit. Mutation of residues lining the binding pocket reduced ATP-dependent channel inhibition. The model also suggests that interactions between the C-terminus of one subunit and the ‘slide helix' of the adjacent subunit may be involved in ATP-dependent gating. Consistent with a role in gating, mutations in the slide helix bias the intrinsic channel conformation towards the open state. PMID:15650751

  14. Nuclear genetic defects of mitochondrial ATP synthase.

    PubMed

    Hejzlarová, K; Mráček, T; Vrbacký, M; Kaplanová, V; Karbanová, V; Nůsková, H; Pecina, P; Houštěk, J

    2014-01-01

    Disorders of ATP synthase, the key enzyme of mitochondrial energy provision belong to the most severe metabolic diseases presenting as early-onset mitochondrial encephalo-cardiomyopathies. Up to now, mutations in four nuclear genes were associated with isolated deficiency of ATP synthase. Two of them, ATP5A1 and ATP5E encode enzyme's structural subunits alpha and epsilon, respectively, while the other two ATPAF2 and TMEM70 encode specific ancillary factors that facilitate the biogenesis of ATP synthase. All these defects share a similar biochemical phenotype with pronounced decrease in the content of fully assembled and functional ATP synthase complex. However, substantial differences can be found in their frequency, molecular mechanism of pathogenesis, clinical manifestation as well as the course of the disease progression. While for TMEM70 the number of reported patients as well as spectrum of the mutations is steadily increasing, mutations in ATP5A1, ATP5E and ATPAF2 genes are very rare. Apparently, TMEM70 gene is highly prone to mutagenesis and this type of a rare mitochondrial disease has a rather frequent incidence. Here we present overview of individual reported cases of nuclear mutations in ATP synthase and discuss, how their analysis can improve our understanding of the enzyme biogenesis.

  15. Action of ATP on ventricular automaticity.

    PubMed

    Stark, G; Domanowits, H; Sterz, F; Stark, U; Bachernegg, M; Kickenweiz, E; Decrinis, M; Laggner, A N; Tritthart, H A

    1994-11-01

    ATP is an effective treatment of supraventricular tachycardia when the atrioventricular (AV) node is part of the reentrant circuit. However, the lower a pace-maker in the pacemaker hierarchy, the more sensitive it is to adenosine. Therefore, we investigated the effects of ATP on ventricular automaticity in in vivo and in vitro conditions. Wide and narrow QRS complex tachycardia in 46 patients was treated with 6, 12, and 18 mg ATP as sequential intravenous (i.v.) bolus. ATP terminated tachycardias in 67%. Bolus infusion ATP caused < or = 6.4-s asystole that was self-limited. Perfusion of isolated spontaneously beating guinea pig heart with 100 microM ATP completely suppressed ventricular automaticity. After ATP-infusion was discontinued, the first ventricular beat was evident after 3.1 +/- 0.9 s and sinus node activity recovered with a time constant of 3.0 +/- 1.1 s. Because sinus node and ventricular automaticity recovered within seconds after ATP infusion was discontinued in vitro, recovery in vivo is also likely to be determined by the short half-life (+1/2) of ATP. PMID:7532751

  16. Nucleotide Bias Observed with a Short SELEX RNA Aptamer Library

    PubMed Central

    Thiel, William H.; Bair, Thomas; Thiel, Kristina Wyatt; Dassie, Justin P.; Rockey, William M.; Howell, Craig A.; Liu, Xiuying Y.; Dupuy, Adam J.; Huang, Lingyan; Owczarzy, Richard; Behlke, Mark A.; McNamara, James O.

    2011-01-01

    Systematic evolution of ligands by exponential enrichment (SELEX) is a powerful in vitro selection process used for over 2 decades to identify oligonucleotide sequences (aptamers) with desired properties (usually high affinity for a protein target) from randomized nucleic acid libraries. In the case of RNA aptamers, several highly complex RNA libraries have been described with RNA sequences ranging from 71 to 81 nucleotides (nt) in length. In this study, we used high-throughput sequencing combined with bioinformatics analysis to thoroughly examine the nucleotide composition of the sequence pools derived from several selections that employed an RNA library (Sel2N20) with an abbreviated variable region. The Sel2N20 yields RNAs 51 nt in length, which unlike longer RNAs, are more amenable to large-scale chemical synthesis for therapeutic development. Our analysis revealed a consistent and early bias against inclusion of adenine, resulting in aptamers with lower predicted minimum free energies (ΔG) (higher structural stability). This bias was also observed in control, “nontargeted” selections in which the partition step (against the target) was omitted, suggesting that the bias occurred in 1 or more of the amplification and propagation steps of the SELEX process. PMID:21793789

  17. (19)F-labeling of the adenine H2-site to study large RNAs by NMR spectroscopy.

    PubMed

    Sochor, F; Silvers, R; Müller, D; Richter, C; Fürtig, B; Schwalbe, H

    2016-01-01

    In comparison to proteins and protein complexes, the size of RNA amenable to NMR studies is limited despite the development of new isotopic labeling strategies including deuteration and ligation of differentially labeled RNAs. Due to the restricted chemical shift dispersion in only four different nucleotides spectral resolution remains limited in larger RNAs. Labeling RNAs with the NMR-active nucleus (19)F has previously been introduced for small RNAs up to 40 nucleotides (nt). In the presented work, we study the natural occurring RNA aptamer domain of the guanine-sensing riboswitch comprising 73 nucleotides from Bacillus subtilis. The work includes protocols for improved in vitro transcription of 2-fluoroadenosine-5'-triphosphat (2F-ATP) using the mutant P266L of the T7 RNA polymerase. Our NMR analysis shows that the secondary and tertiary structure of the riboswitch is fully maintained and that the specific binding of the cognate ligand hypoxanthine is not impaired by the introduction of the (19)F isotope. The thermal stability of the (19)F-labeled riboswitch is not altered compared to the unmodified sequence, but local base pair stabilities, as measured by hydrogen exchange experiments, are modulated. The characteristic change in the chemical shift of the imino resonances detected in a (1)H,(15)N-HSQC allow the identification of Watson-Crick base paired uridine signals and the (19)F resonances can be used as reporters for tertiary and secondary structure transitions, confirming the potential of (19)F-labeling even for sizeable RNAs in the range of 70 nucleotides.

  18. The Lys1010-Lys1325 fragment of the Wilson's disease protein binds nucleotides and interacts with the N-terminal domain of this protein in a copper-dependent manner.

    PubMed

    Tsivkovskii, R; MacArthur, B C; Lutsenko, S

    2001-01-19

    Wilson's disease, an autosomal disorder associated with vast accumulation of copper in tissues, is caused by mutations in a gene encoding a copper-transporting ATPase (Wilson's disease protein, WNDP). Numerous mutations have been identified throughout the WNDP sequence, particularly in the Lys(1010)-Lys(1325) segment; however, the biochemical properties and molecular mechanism of WNDP remain poorly characterized. Here, the Lys(1010)-Lys(1325) fragment of WNDP was overexpressed, purified, and shown to form an independently folded ATP-binding domain (ATP-BD). ATP-BD binds the fluorescent ATP analogue trinitrophenyl-ATP with high affinity, and ATP competes with trinitrophenyl-ATP for the binding site; ADP and AMP appear to bind to ATP-BD at the site separate from ATP. Purified ATP-BD hydrolyzes ATP and interacts specifically with the N-terminal copper-binding domain of WNDP (N-WNDP). Strikingly, copper binding to N-WNDP diminishes these interactions, suggesting that the copper-dependent change in domain-domain contact may represent the mechanism of WNDP regulation. In agreement with this hypothesis, N-WNDP induces conformational changes in ATP-BD as evidenced by the altered nucleotide binding properties of ATP-BD in the presence of N-WNDP. Significantly, the effects of copper-free and copper-bound N-WNDP on ATP-BD are not identical. The implications of these results for the WNDP function are discussed.

  19. Regulation of Ca2+ release from mitochondria by the oxidation-reduction state of pyridine nucleotides.

    PubMed

    Lehninger, A L; Vercesi, A; Bababunmi, E A

    1978-04-01

    Mitochondria from normal rat liver and heart, and also Ehrlich tumor cells, respiring on succinate as energy source in the presence of rotenone (to prevent net electron flow to oxygen from the endogenous pyridine nucleotides), rapidly take up Ca(2+) and retain it so long as the pyridine nucleotides are kept in the reduced state. When acetoacetate is added to bring the pyridine nucleotides into a more oxidized state, Ca(2+) is released to the medium. A subsequent addition of a reductant of the pyridine nucleotides such as beta-hydroxybutyrate, glutamate, or isocitrate causes reuptake of the released Ca(2+). Successive cycles of Ca(2+) release and uptake can be induced by shifting the redox state of the pyridine nucleotides to more oxidized and more reduced states, respectively. Similar observations were made when succinate oxidation was replaced as energy source by ascorbate oxidation or by the hydrolysis of ATP. These and other observations form the basis of a hypothesis for feedback regulation of Ca(2+)-dependent substrate- or energy-mobilizing enzymatic reactions by the uptake or release of mitochondrial Ca(2+), mediated by the cytosolic phosphate potential and the ATP-dependent reduction of mitochondrial pyridine nucleotides by reversal of electron transport.

  20. Regulation of Ca2+ release from mitochondria by the oxidation-reduction state of pyridine nucleotides

    PubMed Central

    Lehninger, Albert L.; Vercesi, Anibal; Bababunmi, Enitan A.

    1978-01-01

    Mitochondria from normal rat liver and heart, and also Ehrlich tumor cells, respiring on succinate as energy source in the presence of rotenone (to prevent net electron flow to oxygen from the endogenous pyridine nucleotides), rapidly take up Ca2+ and retain it so long as the pyridine nucleotides are kept in the reduced state. When acetoacetate is added to bring the pyridine nucleotides into a more oxidized state, Ca2+ is released to the medium. A subsequent addition of a reductant of the pyridine nucleotides such as β-hydroxybutyrate, glutamate, or isocitrate causes reuptake of the released Ca2+. Successive cycles of Ca2+ release and uptake can be induced by shifting the redox state of the pyridine nucleotides to more oxidized and more reduced states, respectively. Similar observations were made when succinate oxidation was replaced as energy source by ascorbate oxidation or by the hydrolysis of ATP. These and other observations form the basis of a hypothesis for feedback regulation of Ca2+-dependent substrate- or energy-mobilizing enzymatic reactions by the uptake or release of mitochondrial Ca2+, mediated by the cytosolic phosphate potential and the ATP-dependent reduction of mitochondrial pyridine nucleotides by reversal of electron transport. Images PMID:25436

  1. Serine Metabolism Supports the Methionine Cycle and DNA/RNA Methylation through De Novo ATP Synthesis in Cancer Cells

    PubMed Central

    Maddocks, Oliver D.K.; Labuschagne, Christiaan F.; Adams, Peter D.; Vousden, Karen H.

    2016-01-01

    Summary Crosstalk between cellular metabolism and the epigenome regulates epigenetic and metabolic homeostasis and normal cell behavior. Changes in cancer cell metabolism can directly impact epigenetic regulation and promote transformation. Here we analyzed the contribution of methionine and serine metabolism to methylation of DNA and RNA. Serine can contribute to this pathway by providing one-carbon units to regenerate methionine from homocysteine. While we observed this contribution under methionine-depleted conditions, unexpectedly, we found that serine supported the methionine cycle in the presence and absence of methionine through de novo ATP synthesis. Serine starvation increased the methionine/S-adenosyl methionine ratio, decreasing the transfer of methyl groups to DNA and RNA. While serine starvation dramatically decreased ATP levels, this was accompanied by lower AMP and did not activate AMPK. This work highlights the difference between ATP turnover and new ATP synthesis and defines a vital function of nucleotide synthesis beyond making nucleic acids. PMID:26774282

  2. Serine Metabolism Supports the Methionine Cycle and DNA/RNA Methylation through De Novo ATP Synthesis in Cancer Cells.

    PubMed

    Maddocks, Oliver D K; Labuschagne, Christiaan F; Adams, Peter D; Vousden, Karen H

    2016-01-21

    Crosstalk between cellular metabolism and the epigenome regulates epigenetic and metabolic homeostasis and normal cell behavior. Changes in cancer cell metabolism can directly impact epigenetic regulation and promote transformation. Here we analyzed the contribution of methionine and serine metabolism to methylation of DNA and RNA. Serine can contribute to this pathway by providing one-carbon units to regenerate methionine from homocysteine. While we observed this contribution under methionine-depleted conditions, unexpectedly, we found that serine supported the methionine cycle in the presence and absence of methionine through de novo ATP synthesis. Serine starvation increased the methionine/S-adenosyl methionine ratio, decreasing the transfer of methyl groups to DNA and RNA. While serine starvation dramatically decreased ATP levels, this was accompanied by lower AMP and did not activate AMPK. This work highlights the difference between ATP turnover and new ATP synthesis and defines a vital function of nucleotide synthesis beyond making nucleic acids.

  3. Determination of adenine based on the fluorescence recovery of the L-Tryptophan-Cu(2+) complex.

    PubMed

    Duan, Ruilin; Li, Chunyan; Liu, Shaopu; Liu, Zhongfang; Li, Yuanfang; Yuan, Yusheng; Hu, Xiaoli

    2016-01-01

    A simple and sensitive method for determination of adenine was developed based on fluorescence quenching and recovery of L-Tryptophan (L-Trp). The fluorescence of L-Trp could efficiently quenched by copper ion compared with other common metal ions. Upon addition of adenine (Ade) in L-Trp-Cu(II) system, the fluorescence was reoccurred. Under the optimum conditions, the recovery fluorescence intensity was linearly correlated with the concentration of adenine in the range from 0.34 to 25.0μmolL(-1), with a correlation coefficient (R(2)) of 0.9994. The detection limit (3σ/k) was 0.046μmolL(-1), indicating that this method could applied to detect trace adenine. In this study, amino acids including L-Trp, D-Trp, L-Tyr, D-Tyr, L-Phe, D-Phe were investigated and only L-Trp could well chelated copper ion. Additionally, the mechanism of quench and recovery also were discussed and the method was successfully applied to detect the adenine in DNA with satisfactory results.

  4. Determination of adenine based on the fluorescence recovery of the L-Tryptophan-Cu2+ complex

    NASA Astrophysics Data System (ADS)

    Duan, Ruilin; Li, Chunyan; Liu, Shaopu; Liu, Zhongfang; Li, Yuanfang; Yuan, Yusheng; Hu, Xiaoli

    2016-01-01

    A simple and sensitive method for determination of adenine was developed based on fluorescence quenching and recovery of L-Tryptophan (L-Trp). The fluorescence of L-Trp could efficiently quenched by copper ion compared with other common metal ions. Upon addition of adenine (Ade) in L-Trp-Cu(II) system, the fluorescence was reoccurred. Under the optimum conditions, the recovery fluorescence intensity was linearly correlated with the concentration of adenine in the range from 0.34 to 25.0 μmol L-1, with a correlation coefficient (R2) of 0.9994. The detection limit (3σ/k) was 0.046 μmol L-1, indicating that this method could applied to detect trace adenine. In this study, amino acids including L-Trp, D-Trp, L-Tyr, D-Tyr, L-Phe, D-Phe were investigated and only L-Trp could well chelated copper ion. Additionally, the mechanism of quench and recovery also were discussed and the method was successfully applied to detect the adenine in DNA with satisfactory results.

  5. Adenine: an important drug scaffold for the design of antiviral agents

    PubMed Central

    Wang, Changyuan; Song, Zhendong; Yu, Haiqing; Liu, Kexin; Ma, Xiaodong

    2015-01-01

    Adenine derivatives, in particular the scaffold bearing the acyclic nucleoside phosphonates (ANPS), possess significant antiviral and cytostatic activity. Till now, several effective adenine derivatives have been marketed for the treatment of HIV, HBV, CMV and other virus-infected diseases. These compounds are represented by tenofovir (PMPA), a medicine for both HIV and HBV, and adefovir as an anti-HBV agent. More than this, other analogs, such as GS9148, GS9131, and GS7340, are also well-known anti-viral agents that have been progressed to the clinical studies for their excellent activity. In general, the structures of these compounds include an adenine nucleobase linked to a phosphonate side chain. Considerable structural modifications on the scaffold itself and the peripheral sections were made. The structure-activity relationships (SARs) of this skeleton will provide valuable clues to identify more effective adenine derivatives as antiviral drugs. Here, we systematically summarized the SARs of the adenine derivatives, and gave important information for further optimizing this template. PMID:26579473

  6. Modulator-induced interference in functional cross talk between the substrate and the ATP sites of human P-glycoprotein.

    PubMed

    Maki, Nazli; Moitra, Karobi; Silver, Cara; Ghosh, Pratiti; Chattopadhyay, Apurba; Dey, Saibal

    2006-02-28

    The human P-glycoprotein (Pgp, ABCB1) is an ATP-dependent efflux pump for structurally unrelated hydrophobic compounds, conferring simultaneous resistance to and restricting bioavailability of several anticancer and antimicrobial agents. Drug transport by Pgp requires a coordinated communication between its substrate binding/translocating pathway (substrate site) and the nucleotide binding domains (NBDs or ATP sites). In this study, we demonstrate that certain thioxanthene-based Pgp modulators, such as cis-(Z)-flupentixol and its closely related analogues, effectively disrupt molecular cross talk between the substrate, and the ATP, sites without affecting the basic functional aspects of the two domains, such as substrate recognition, binding, and hydrolysis of ATP and dissociation of ADP following ATP hydrolysis. The allosteric modulator cis-(Z)-flupentixol has no effect on [alpha-(32)P]-8-azido-ATP binding to Pgp under nonhydrolytic conditions or on the K(m) for ATP during ATP hydrolysis. Both hydrolysis of ATP and vanadate-induced [alpha-(32)P]-8-azido-ADP trapping (following [alpha-(32)P]-8-azido-ATP breakdown) by Pgp are stimulated by the modulator. However, the ability of Pgp substrates (such as prazosin) to stimulate ATP hydrolysis and facilitate vanadate-induced trapping of [alpha-(32)P]-8-azido-ADP is substantially affected in the presence of cis-(Z)-flupentixol. Substrate recognition by Pgp as determined by [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) binding both in the presence and in the absence of ATP is facilitated by the modulator, whereas substrate dissociation in response to vanadate trapping is considerably affected in its presence. In the Pgp F983A mutant, which is impaired in modulation by cis-(Z)-flupentixol, the modulator has a minimal effect on substrate-stimulated ATP hydrolysis as well as on substrate dissociation coupled to vanadate trapping. Finally, cis-(Z)-flupentixol has no effect on dissociation of [alpha-(32)P]-8-azido-ADP (or ADP

  7. Identification of ATP-binding regions in the RyR1 Ca²⁺ release channel.

    PubMed

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

    2012-01-01

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

  8. Interplay of Mg2+, ADP, and ATP in the cytosol and mitochondria: unravelling the role of Mg2+ in cell respiration.

    PubMed

    Gout, Elisabeth; Rébeillé, Fabrice; Douce, Roland; Bligny, Richard

    2014-10-28

    In animal and plant cells, the ATP/ADP ratio and/or energy charge are generally considered key parameters regulating metabolism and respiration. The major alternative issue of whether the cytosolic and mitochondrial concentrations of ADP and ATP directly mediate cell respiration remains unclear, however. In addition, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP synthase (EC 3.6.3.14), the cytosolic and mitochondrial Mg(2+) concentrations must be considered as well. Here we developed in vivo/in vitro techniques using (31)P-NMR spectroscopy to simultaneously measure these key components in subcellular compartments. We show that heterotrophic sycamore (Acer pseudoplatanus L.) cells incubated in various nutrient media contain low, stable cytosolic ADP and Mg(2+) concentrations, unlike ATP. ADP is mainly free in the cytosol, but complexed by Mg(2+) in the mitochondrial matrix, where [Mg(2+)] is tenfold higher. In contrast, owing to a much higher affinity for Mg(2+), ATP is mostly complexed by Mg(2+) in both compartments. Mg(2+) starvation used to alter cytosolic and mitochondrial [Mg(2+)] reversibly increases free nucleotide concentration in the cytosol and matrix, enhances ADP at the expense of ATP, decreases coupled respiration, and stops cell growth. We conclude that the cytosolic ADP concentration, and not ATP, ATP/ADP ratio, or energy charge, controls the respiration of plant cells. The Mg(2+) concentration, remarkably constant and low in the cytosol and tenfold higher in the matrix, mediates ADP/ATP exchange between the cytosol and matrix, [MgADP]-dependent mitochondrial ATP synthase activity, and cytosolic free ADP homeostasis. PMID:25313036

  9. Interplay of Mg2+, ADP, and ATP in the cytosol and mitochondria: Unravelling the role of Mg2+ in cell respiration

    PubMed Central

    Gout, Elisabeth; Rébeillé, Fabrice; Douce, Roland; Bligny, Richard

    2014-01-01

    In animal and plant cells, the ATP/ADP ratio and/or energy charge are generally considered key parameters regulating metabolism and respiration. The major alternative issue of whether the cytosolic and mitochondrial concentrations of ADP and ATP directly mediate cell respiration remains unclear, however. In addition, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP synthase (EC 3.6.3.14), the cytosolic and mitochondrial Mg2+ concentrations must be considered as well. Here we developed in vivo/in vitro techniques using 31P-NMR spectroscopy to simultaneously measure these key components in subcellular compartments. We show that heterotrophic sycamore (Acer pseudoplatanus L.) cells incubated in various nutrient media contain low, stable cytosolic ADP and Mg2+ concentrations, unlike ATP. ADP is mainly free in the cytosol, but complexed by Mg2+ in the mitochondrial matrix, where [Mg2+] is tenfold higher. In contrast, owing to a much higher affinity for Mg2+, ATP is mostly complexed by Mg2+ in both compartments. Mg2+ starvation used to alter cytosolic and mitochondrial [Mg2+] reversibly increases free nucleotide concentration in the cytosol and matrix, enhances ADP at the expense of ATP, decreases coupled respiration, and stops cell growth. We conclude that the cytosolic ADP concentration, and not ATP, ATP/ADP ratio, or energy charge, controls the respiration of plant cells. The Mg2+ concentration, remarkably constant and low in the cytosol and tenfold higher in the matrix, mediates ADP/ATP exchange between the cytosol and matrix, [MgADP]-dependent mitochondrial ATP synthase activity, and cytosolic free ADP homeostasis. PMID:25313036

  10. Interplay of Mg2+, ADP, and ATP in the cytosol and mitochondria: unravelling the role of Mg2+ in cell respiration.

    PubMed

    Gout, Elisabeth; Rébeillé, Fabrice; Douce, Roland; Bligny, Richard

    2014-10-28

    In animal and plant cells, the ATP/ADP ratio and/or energy charge are generally considered key parameters regulating metabolism and respiration. The major alternative issue of whether the cytosolic and mitochondrial concentrations of ADP and ATP directly mediate cell respiration remains unclear, however. In addition, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP synthase (EC 3.6.3.14), the cytosolic and mitochondrial Mg(2+) concentrations must be considered as well. Here we developed in vivo/in vitro techniques using (31)P-NMR spectroscopy to simultaneously measure these key components in subcellular compartments. We show that heterotrophic sycamore (Acer pseudoplatanus L.) cells incubated in various nutrient media contain low, stable cytosolic ADP and Mg(2+) concentrations, unlike ATP. ADP is mainly free in the cytosol, but complexed by Mg(2+) in the mitochondrial matrix, where [Mg(2+)] is tenfold higher. In contrast, owing to a much higher affinity for Mg(2+), ATP is mostly complexed by Mg(2+) in both compartments. Mg(2+) starvation used to alter cytosolic and mitochondrial [Mg(2+)] reversibly increases free nucleotide concentration in the cytosol and matrix, enhances ADP at the expense of ATP, decreases coupled respiration, and stops cell growth. We conclude that the cytosolic ADP concentration, and not ATP, ATP/ADP ratio, or energy charge, controls the respiration of plant cells. The Mg(2+) concentration, remarkably constant and low in the cytosol and tenfold higher in the matrix, mediates ADP/ATP exchange between the cytosol and matrix, [MgADP]-dependent mitochondrial ATP synthase activity, and cytosolic free ADP homeostasis.

  11. Switch II mutants reveal coupling between the nucleotide- and actin-binding regions in myosin V.

    PubMed

    Trivedi, Darshan V; David, Charles; Jacobs, Donald J; Yengo, Christopher M

    2012-06-01

    Conserved active-site elements in myosins and other P-loop NTPases play critical roles in nucleotide binding and hydrolysis; however, the mechanisms of allosteric communication among these mechanoenzymes remain unresolved. In this work we introduced the E442A mutation, which abrogates a salt-bridge between switch I and switch II, and the G440A mutation, which abolishes a main-chain hydrogen bond associated with the interaction of switch II with the γ phosphate of ATP, into myosin V. We used fluorescence resonance energy transfer between mant-labeled nucleotides or IAEDANS-labeled actin and FlAsH-labeled myosin V to examine the conformation of the nucleotide- and actin-binding regions, respectively. We demonstrate that in the absence of actin, both the G440A and E442A mutants bind ATP with similar affinity and result in only minor alterations in the conformation of the nucleotide-binding pocket (NBP). In the presence of ADP and actin, both switch II mutants disrupt the formation of a closed NBP actomyosin.ADP state. The G440A mutant also prevents ATP-induced opening of the actin-binding cleft. Our results indicate that the switch II region is critical for stabilizing the closed NBP conformation in the presence of actin, and is essential for communication between the active site and actin-binding region.

  12. Switch II Mutants Reveal Coupling between the Nucleotide- and Actin-Binding Regions in Myosin V

    PubMed Central

    Trivedi, Darshan V.; David, Charles; Jacobs, Donald J.; Yengo, Christopher M.

    2012-01-01

    Conserved active-site elements in myosins and other P-loop NTPases play critical roles in nucleotide binding and hydrolysis; however, the mechanisms of allosteric communication among these mechanoenzymes remain unresolved. In this work we introduced the E442A mutation, which abrogates a salt-bridge between switch I and switch II, and the G440A mutation, which abolishes a main-chain hydrogen bond associated with the interaction of switch II with the γ phosphate of ATP, into myosin V. We used fluorescence resonance energy transfer between mant-labeled nucleotides or IAEDANS-labeled actin and FlAsH-labeled myosin V to examine the conformation of the nucleotide- and actin-binding regions, respectively. We demonstrate that in the absence of actin, both the G440A and E442A mutants bind ATP with similar affinity and result in only minor alterations in the conformation of the nucleotide-binding pocket (NBP). In the presence of ADP and actin, both switch II mutants disrupt the formation of a closed NBP actomyosin.ADP state. The G440A mutant also prevents ATP-induced opening of the actin-binding cleft. Our results indicate that the switch II region is critical for stabilizing the closed NBP conformation in the presence of actin, and is essential for communication between the active site and actin-binding region. PMID:22713570

  13. On the peripheral and central chemoreception and control of breathing: an emerging role of ATP

    PubMed Central

    Gourine, Alexander V

    2005-01-01

    Peripheral and central respiratory chemoreceptors are ultimately responsible for maintenance of constant levels of arterial PO2, PCO2 and [H+], protecting the brain from hypoxia and ensuring that the breathing is always appropriate for metabolism. The aim of this discussion is to shed some light on the potential mechanisms of chemosensory transduction– the process which links chemosensory mechanisms to the central nervous mechanisms controlling breathing. Recent experimental data suggest that the purine nucleotide ATP acts as a common mediator of peripheral and central chemosensory transduction (within the carotid body and the medulla oblongata, respectively). In response to a decrease in PO2 (hypoxia) oxygen-sensitive glomus cells of the carotid body release ATP to activate chemoafferent fibres of the carotid sinus nerve which transmit this information to the brainstem respiratory centres. In response to an increase in PCO2/[H+] (hypercapnia) chemosensitive structures located on the ventral surface of the medulla oblongata rapidly release ATP, which acts locally within the medullary respiratory network. The functional role of ATP released at both sites is similar – to evoke adaptive enhancement in breathing. Understanding the mechanisms of ATP release in response to chemosensory stimulation may prove to be essential for further detailed analysis of cellular and molecular mechanisms underlying respiratory chemosensitivity. PMID:16141266

  14. Iron transport by proteoliposomes containing mitochondrial F(1)F(0) ATP synthase isolated from rat heart.

    PubMed

    Kim, Misun; Song, Eunsook

    2010-04-01

    In this work, we present evidence of Fe(2+) transport by rat heart mitochondrial F(1)F(0) ATP synthase. Iron uptake by the vesicles containing the enzyme was concentration- and temperature-dependent, with an optimum temperature of 37 degrees C. Both ATP and ADP stimulated iron uptake in a concentration-dependent manner, whereas AMP, AMPPCP, and mADP did not. Inhibitors of the enzyme, oligomycin, and resveratrol similarly blocked iron transport. The iron uptake was confirmed by inhibition using specific antibodies against the alpha, beta, and c subunits of the enzyme. Interestingly, slight transport of common divalent and trivalent metal ions such as Mg(+2), Ca(+2), Mn(+2), Zn(+2), Cu(+2), Fe(+3), and Al(+3) was observed. Moreover, Cu(+2), even in the nM range, inhibited iron uptake and attained maximum inhibition of approximately 56%. Inorganic phosphate (Pi) in the medium exerted an opposite effect depending on the type of adenosine nucleotide, which was suppressed with ATP, but enhanced with ADP. A similarly stimulating effect of ATP and ADP with an inverse effect of Pi suggests that the activity of ATPase and ATP synthase may be associated with iron uptake in a different manner, probably via antiport of H(+). PMID:20100539

  15. Urothelial ATP exocytosis: regulation of bladder compliance in the urine storage phase.

    PubMed

    Nakagomi, Hiroshi; Yoshiyama, Mitsuharu; Mochizuki, Tsutomu; Miyamoto, Tatsuya; Komatsu, Ryohei; Imura, Yoshio; Morizawa, Yosuke; Hiasa, Miki; Miyaji, Takaaki; Kira, Satoru; Araki, Isao; Fujishita, Kayoko; Shibata, Keisuke; Shigetomi, Eiji; Shinozaki, Youichi; Ichikawa, Reiko; Uneyama, Hisayuki; Iwatsuki, Ken; Nomura, Masatoshi; de Groat, William C; Moriyama, Yoshinori; Takeda, Masayuki; Koizumi, Schuichi

    2016-01-01

    The bladder urothelium is more than just a barrier. When the bladder is distended, the urothelium functions as a sensor to initiate the voiding reflex, during which it releases ATP via multiple mechanisms. However, the mechanisms underlying this ATP release in response to the various stretch stimuli caused by bladder filling remain largely unknown. Therefore, the aim of this study was to elucidate these mechanisms. By comparing vesicular nucleotide transporter (VNUT)-deficient and wild-type male mice, we showed that ATP has a crucial role in urine storage through exocytosis via a VNUT-dependent mechanism. VNUT was abundantly expressed in the bladder urothelium, and when the urothelium was weakly stimulated (i.e. in the early filling stages), it released ATP by exocytosis. VNUT-deficient mice showed reduced bladder compliance from the early storage phase and displayed frequent urination in inappropriate places without a change in voiding function. We conclude that urothelial, VNUT-dependent ATP exocytosis is involved in urine storage mechanisms that promote the relaxation of the bladder during the early stages of filling. PMID:27412485

  16. Urothelial ATP exocytosis: regulation of bladder compliance in the urine storage phase

    PubMed Central

    Nakagomi, Hiroshi; Yoshiyama, Mitsuharu; Mochizuki, Tsutomu; Miyamoto, Tatsuya; Komatsu, Ryohei; Imura, Yoshio; Morizawa, Yosuke; Hiasa, Miki; Miyaji, Takaaki; Kira, Satoru; Araki, Isao; Fujishita, Kayoko; Shibata, Keisuke; Shigetomi, Eiji; Shinozaki, Youichi; Ichikawa, Reiko; Uneyama, Hisayuki; Iwatsuki, Ken; Nomura, Masatoshi; de Groat, William C.; Moriyama, Yoshinori; Takeda, Masayuki; Koizumi, Schuichi

    2016-01-01

    The bladder urothelium is more than just a barrier. When the bladder is distended, the urothelium functions as a sensor to initiate the voiding reflex, during which it releases ATP via multiple mechanisms. However, the mechanisms underlying this ATP release in response to the various stretch stimuli caused by bladder filling remain largely unknown. Therefore, the aim of this study was to elucidate these mechanisms. By comparing vesicular nucleotide transporter (VNUT)-deficient and wild-type male mice, we showed that ATP has a crucial role in urine storage through exocytosis via a VNUT-dependent mechanism. VNUT was abundantly expressed in the bladder urothelium, and when the urothelium was weakly stimulated (i.e. in the early filling stages), it released ATP by exocytosis. VNUT-deficient mice showed reduced bladder compliance from the early storage phase and displayed frequent urination in inappropriate places without a change in voiding function. We conclude that urothelial, VNUT-dependent ATP exocytosis is involved in urine storage mechanisms that promote the relaxation of the bladder during the early stages of filling. PMID:27412485

  17. ATP-triggered anticancer drug delivery

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  18. Temperature-dependent self-assembly of adenine derivative on HOPG.

    PubMed

    Mu, Zhongcheng; Rubner, Oliver; Bamler, Markus; Blömker, Tobias; Kehr, Gerald; Erker, Gerhard; Heuer, Andreas; Fuchs, Harald; Chi, Lifeng

    2013-08-27

    Temperature-dependent self-assembly formed by the adsorption of the nucleobase adenine derivative on a graphite surface were investigated by in situ scanning tunneling microscopy (STM). The high-resolution STM images reveal two types of structures, α phase and β phase, which are mainly driven by either hydrogen bonding or aromatic π-π interactions between adenine bases, respectively, as well as the interactions of alkyl chains. α-Phase structures can be transformed into β-phase structures by increasing temperature. The reverse is true for decreasing temperature. This reflects structural stabilities resulting from the different interactions. Density functional theory (DFT) calculations were performed to characterize possible arrangements of adjacent adenine moieties systematically in terms of binding energies and structural properties. Via a systematic search algorithm, all possible network structures were determined on a microscopic level. In this way, it is possible to rationalize the structural parameters as found in the STM images.

  19. Deep-UV surface-enhanced resonance Raman scattering of adenine on aluminum nanoparticle arrays.

    PubMed

    Jha, Shankar K; Ahmed, Zeeshan; Agio, Mario; Ekinci, Yasin; Löffler, Jörg F

    2012-02-01

    We report the ultrasensitive detection of adenine using deep-UV surface-enhanced resonance Raman scattering on aluminum nanostructures. Well-defined Al nanoparticle arrays fabricated over large areas using extreme-UV interference lithography exhibited sharp and tunable plasmon resonances in the UV and deep-UV wavelength ranges. Theoretical modeling based on the finite-difference time-domain method was used to understand the near-field and far-field optical properties of the nanoparticle arrays. Raman measurements were performed on adenine molecules coated uniformly on the Al nanoparticle arrays at a laser excitation wavelength of 257.2 nm. With this technique, less than 10 amol of label-free adenine molecules could be detected reproducibly in real time. Zeptomole (~30,000 molecules) detection sensitivity was readily achieved proving that deep-UV surface-enhanced resonance Raman scattering is an extremely sensitive tool for the detection of biomolecules.

  20. Nucleotides regulate the mechanical hierarchy between subdomains of the nucleotide binding domain of the Hsp70 chaperone DnaK.

    PubMed

    Bauer, Daniela; Merz, Dale R; Pelz, Benjamin; Theisen, Kelly E; Yacyshyn, Gail; Mokranjac, Dejana; Dima, Ruxandra I; Rief, Matthias; Žoldák, Gabriel

    2015-08-18

    The regulation of protein function through ligand-induced conformational changes is crucial for many signal transduction processes. The binding of a ligand alters the delicate energy balance within the protein structure, eventually leading to such conformational changes. In this study, we elucidate the energetic and mechanical changes within the subdomains of the nucleotide binding domain (NBD) of the heat shock protein of 70 kDa (Hsp70) chaperone DnaK upon nucleotide binding. In an integrated approach using single molecule optical tweezer experiments, loop insertions, and steered coarse-grained molecular simulations, we find that the C-terminal helix of the NBD is the major determinant of mechanical stability, acting as a glue between the two lobes. After helix unraveling, the relative stability of the two separated lobes is regulated by ATP/ADP binding. We find that the nucleotide stays strongly bound to lobe II, thus reversing the mechanical hierarchy between the two lobes. Our results offer general insights into the nucleotide-induced signal transduction within members of the actin/sugar kinase superfamily. PMID:26240360

  1. The ectoenzyme E-NPP3 negatively regulates ATP-dependent chronic allergic responses by basophils and mast cells.

    PubMed

    Tsai, Shih Han; Kinoshita, Makoto; Kusu, Takashi; Kayama, Hisako; Okumura, Ryu; Ikeda, Kayo; Shimada, Yosuke; Takeda, Akira; Yoshikawa, Soichiro; Obata-Ninomiya, Kazushige; Kurashima, Yosuke; Sato, Shintaro; Umemoto, Eiji; Kiyono, Hiroshi; Karasuyama, Hajime; Takeda, Kiyoshi

    2015-02-17

    Crosslinking of the immunoglobulin receptor FcεRI activates basophils and mast cells to induce immediate and chronic allergic inflammation. However, it remains unclear how the chronic allergic inflammation is regulated. Here, we showed that ecto-nucleotide pyrophosphatase-phosphodiesterase 3 (E-NPP3), also known as CD203c, rapidly induced by FcεRI crosslinking, negatively regulated chronic allergic inflammation. Basophil and mast cell numbers increased in Enpp3(-/-) mice with augmented serum ATP concentrations. Enpp3(-/-) mice were highly sensitive to chronic allergic pathologies, which was reduced by ATP blockade. FcεRI crosslinking induced ATP secretion from basophils and mast cells, and ATP activated both cells. ATP clearance was impaired in Enpp3(-/-) cells. Enpp3(-/-)P2rx7(-/-) mice showed decreased responses to FcεRI crosslinking. Thus, ATP released by FcεRI crosslinking stimulates basophils and mast cells for further activation causing allergic inflammation. E-NPP3 decreases ATP concentration and suppresses basophil and mast cell activity. PMID:25692702

  2. Extracellular ATP Causes ROCK I-dependent Bleb Formation in P2X7-transfected HEK293 CellsV⃞

    PubMed Central

    Morelli, Anna; Chiozzi, Paola; Chiesa, Anna; Ferrari, Davide; Sanz, Juana M.; Falzoni, Simonetta; Pinton, Paolo; Rizzuto, Rosario; Olson, Michael F.; Di Virgilio, Francesco

    2003-01-01

    The P2X7 ATP receptor mediates the cytotoxic effect of extracellular ATP. P2X7-dependent cell death is heralded by dramatic plasma membrane bleb formation. Membrane blebbing is a complex phenomenon involving as yet poorly characterized intracellular pathways. We have investigated the effect of extracellular ATP on HEK293 cells transfected with the cytotoxic/pore-forming P2X7 receptor. Addition of ATP to P2X7-transfected, but not to wt P2X7-less, HEK293 cells caused massive membrane blebbing within 1–2 min. UTP, a nucleotide incapable of activating P2X7, had no early effects on cell shape and bleb formation. Bleb formation triggered by ATP was reversible and required extracellular Ca2+ and an intact cytoskeleton. Furthermore, it was completely prevented by preincubation with the P2X blocker oxidized ATP. It was recently observed that the ROCK protein is a key determinant of bleb formation. Preincubation of HEK293-P2X7 cells with the ROCK blocker Y-27632 completely prevented P2X7-dependent blebbing. Although ATP triggered cleavage of the ROCK I isoform in P2X7-transfected HEK293 cells, the wide range caspase inhibitor z-VAD-fluoromethylketone had no effect. These observations suggest that P2X7-dependent plasma membrane blebbing depends on the activation of the serine/threonine kinase ROCK I. PMID:12857854

  3. DNA with adenine tracts contains poly(dA).poly(dT) conformational features in solution.

    PubMed

    Brahms, S; Brahms, J G

    1990-03-25

    The conformation of DNA's with adenine-thymine tracts exhibiting retardation in electrophoretic migration and considered as curved were investigated in solution by CD and RAMAN spectroscopy. The following curved multimers with adenine tracts but of different flanking sequences d(CA5TGCC)n, d(TCTCTA6TATATA5)n, d(GA4T4C)n yield CD spectroscopic features indicating a non-B structure of the dA.dT tract with similarities to polyd(A).polyd(T). We suggest that adenine-thymine bases in these multimers contain some of the distinctive conformational features of poly(A).polyd(T) probably with large propeller twist found by NMR (Behling and Kearns, 1987) and by X-ray diffraction on oligonucleotides containing a tract of adenines (Nelson et al. 1987, Coll et al; 1987; DiGabriele et al. 1989). Some elements of distinctive CD features of the contiguous adenines run are also observed in the straight multi-9-mer d(CA5GCC)n which lacks in-phase relation to the helical repeat. Despite the presence of the TpA step in the straight multimer d(GT4A4)n, the altered dA.dT conformation is not completely destroyed. Interruption of adenine tract by a guanine in d(CAAGAATGCC)n leads to a B-like conformation and to a normal electrophoretic mobility. The Raman spectra reveal a rearrangement of the sugar-phosphate backbone of dA.dT tract in the multimer d(CA5TGCC)n with respect to that of polydA.polydT. This is reflected in the presence of an unique Raman band associated to C2'-endo sugar with a predominant contribution of C1'-exo puckering which is exhibited by the multimer whereas two distinct Raman bands characterize poly(dA).poly(dT) backbone conformation.

  4. High information throughput analysis of nucleotides and their isotopically enriched isotopologues by direct-infusion FTICR-MS.

    PubMed

    Lorkiewicz, Pawel; Higashi, Richard M; Lane, Andrew N; Fan, Teresa W-M

    2012-01-01

    Fourier transform-ion cyclotron resonance-mass spectrometry (FTICR-MS) is capable of acquiring unmatched quality of isotopologue data for stable isotope resolved metabolomics (SIRM). This capability drives the need for a continuous ion introduction for obtaining optimal isotope ratios. Here we report the simultaneous analysis of mono and dinucleotides from crude polar extracts by FTICR-MS by adapting an ion-pairing sample preparation method for LC-MS analysis. This involves a rapid cleanup of extracted nucleotides on pipet tips containing a C(18) stationary phase, which enabled global analysis of nucleotides and their (13)C isotopologues at nanomolar concentrations by direct infusion nanoelectrospray FTICR-MS with 5 minutes of data acquisition. The resolution and mass accuracy enabled computer-assisted unambiguous assignment of most nucleotide species, including all phosphorylated forms of the adenine, guanine, uracil and cytosine nucleotides, NAD(+), NADH, NADP(+), NADPH, cyclic nucleotides, several UDP-hexoses, and all their (13)C isotopologues. The method was applied to a SIRM study on human lung adenocarcinoma A549 cells grown in [U-(13)C] glucose with or without the anti-cancer agent methylseleninic acid. At m/z resolving power of 400,000, (13)C-isotopologues of nucleotides were fully resolved from all other elemental isotopologues, thus allowing their (13)C fractional enrichment to be accurately determined. The method achieves both high sample and high information throughput analysis of nucleotides for metabolic pathway reconstruction in SIRM investigations.

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

    PubMed

    Fernández-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 μM) and IL-6 mRNA levels were measured using qPCR. ATP evoked a concentration-dependent rise in IL-6 mRNA in both SED and VA mice. VA mice however, had significantly higher ATP sensitivity (pD2 pharmacological values: VA=5.58±0.02 vs. SED=4.95±0.04, p<0.05). Interestingly, in VA mice we observed a positive correlation between the level of physical activity and the IL-6 mRNA increase following fiber stimulation with 10 μM 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.

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

    PubMed

    Fernández-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 μM) and IL-6 mRNA levels were measured using qPCR. ATP evoked a concentration-dependent rise in IL-6 mRNA in both SED and VA mice. VA mice however, had significantly higher ATP sensitivity (pD2 pharmacological values: VA=5.58±0.02 vs. SED=4.95±0.04, p<0.05). Interestingly, in VA mice we observed a positive correlation between the level of physical activity and the IL-6 mRNA increase following fiber stimulation with 10 μM 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

  7. Eliciting the mitochondrial unfolded protein response by nicotinamide adenine dinucleotide repletion reverses fatty liver disease in mice

    PubMed Central

    Gariani, Karim; Menzies, Keir J.; Ryu, Dongryeol; Wegner, Casey J.; Wang, Xu; Ropelle, Eduardo R.; Moullan, Norman; Zhang, Hongbo; Perino, Alessia; Lemos, Vera; Kim, Bohkyung; Park, Young‐Ki; Piersigilli, Alessandra; Pham, Tho X.; Yang, Yue; Ku, Chai Siah; Koo, Sung I.; Fomitchova, Anna; Cantó, Carlos; Schoonjans, Kristina; Sauve, Anthony A.

    2015-01-01

    With no approved pharmacological treatment, nonalcoholic fatty liver disease (NAFLD) is now the most common cause of chronic liver disease in Western countries and its worldwide prevalence continues to increase along with the growing obesity epidemic. Here, we show that a high‐fat high‐sucrose (HFHS) diet, eliciting chronic hepatosteatosis resembling human fatty liver, lowers hepatic nicotinamide adenine dinucleotide (NAD+) levels driving reductions in hepatic mitochondrial content, function, and adenosine triphosphate (ATP) levels, in conjunction with robust increases in hepatic weight, lipid content, and peroxidation in C57BL/6J mice. To assess the effect of NAD+ repletion on the development of steatosis in mice, nicotinamide riboside, a precursor of NAD+ biosynthesis, was added to the HFHS diet, either as a preventive strategy or as a therapeutic intervention. We demonstrate that NR prevents and reverts NAFLD by inducing a sirtuin (SIRT)1‐ and SIRT3‐dependent mitochondrial unfolded protein response, triggering an adaptive mitohormetic pathway to increase hepatic β‐oxidation and mitochondrial complex content and activity. The cell‐autonomous beneficial component of NR treatment was revealed in liver‐specific Sirt1 knockout mice (Sirt1hep−/−), whereas apolipoprotein E‐deficient mice (Apoe −/−) challenged with a high‐fat high‐cholesterol diet affirmed the use of NR in other independent models of NAFLD. Conclusion: Our data warrant the future evaluation of NAD+ boosting strategies to manage the development or progression of NAFLD. (Hepatology 2016;63:1190–1204) PMID:26404765

  8. Identification of widespread adenosine nucleotide binding in Mycobacterium tuberculosis

    SciTech Connect

    Ansong, Charles; Ortega, Corrie; Payne, Samuel H.; Haft, Daniel H.; Chauvigne-Hines, Lacie M.; Lewis, Michael P.; Ollodart, Anja R.; Purvine, Samuel O.; Shukla, Anil K.; Fortuin, Suereta; Smith, Richard D.; Adkins, Joshua N.; Grundner, Christoph; Wright, Aaron T.

    2013-01-24

    The annotation of protein function is almost completely performed by in silico approaches. However, computational prediction of protein function is frequently incomplete and error prone. In Mycobacterium tuberculosis (Mtb), ~25% of all genes have no predicted function and are annotated as hypothetical proteins. This lack of functional information severely limits our understanding of Mtb pathogenicity. Current tools for experimental functional annotation are limited and often do not scale to entire protein families. Here, we report a generally applicable chemical biology platform to functionally annotate bacterial proteins by combining activity-based protein profiling (ABPP) and quantitative LC-MS-based proteomics. As an example of this approach for high-throughput protein functional validation and discovery, we experimentally annotate the families of ATP-binding proteins in Mtb. Our data experimentally validate prior in silico predictions of >250 ATPases and adenosine nucleotide-binding proteins, and reveal 73 hypothetical proteins as novel ATP-binding proteins. We identify adenosine cofactor interactions with many hypothetical proteins containing a diversity of unrelated sequences, providing a new and expanded view of adenosine nucleotide binding in Mtb. Furthermore, many of these hypothetical proteins are both unique to Mycobacteria and essential for infection, suggesting specialized functions in mycobacterial physiology and pathogenicity. Thus, we provide a generally applicable approach for high throughput protein function discovery and validation, and highlight several ways in which application of activity-based proteomics data can improve the quality of functional annotations to facilitate novel biological insights.

  9. Solution structure of a five-adenine bulge loop within a DNA duplex.

    PubMed

    Dornberger, U; Hillisch, A; Gollmick, F A; Fritzsche, H; Diekmann, S

    1999-09-28

    The three-dimensional solution structure of a DNA molecule of the sequence 5'-d(GCATCGAAAAAGCTACG)-3' paired with 5'-d(CGTAGCCGATGC)-3' containing a five-adenine bulge loop (dA(5)-bulge) between two double helical stems was determined by 2D (1)H and (31)P NMR, infrared, and Raman spectroscopy. The DNA in both stems adopt a classical B-form double helical structure with Watson-Crick base pairing and C2'-endo sugar conformation. In addition, the two dG/dC base pairs framing the dA(5)-bulge loop are formed and are stable at least up to 30 degrees C. The five adenine bases of the bulge loop are localized at intrahelical positions within the double helical stems. Stacking on the double helical stem is continued for the first four 5'-adenines in the bulge loop. The total rise (the height) of these four stacked adenines roughly equals the diameter of the double helical stem. The stacking interactions are broken between the last of these four 5'-adenines and the fifth loop adenine at the 3'-end. This 3'-adenine partially stacks on the other stem. The angle between the base planes of the two nonstacking adenines (A10 and A11) in the bulge loop reflects the kinking angle of the global DNA structure. The neighboring cytosines opposite the dA(5)-bulge (being parts of the bulge flanking base pairs) do not stack on one another. This disruption of stacking is characterized by a partial shearing of these bases, such that certain sequential NOEs for this base step are preserved. In the base step opposite the loop, an extraordinary hydrogen bond is observed between the phosphate backbone of the 5'-dC and the amino proton of the 3'-dC in about two-thirds of the conformers. This hydrogen bond probably contributes to stabilizing the global DNA structure. The dA(5)-bulge induces a local kink into the DNA molecule of about 73 degrees (+/-11 degrees ). This kinking angle and the mutual orientation of the two double helical stems agree well with results from fluorescence resonance energy

  10. Copper-catalyzed intramolecular cyclization of N-propargyl-adenine: synthesis of purine-fused tricyclics.

    PubMed

    Li, Ren-Long; Liang, Lei; Xie, Ming-Sheng; Qu, Gui-Rong; Niu, Hong-Ying; Guo, Hai-Ming

    2014-04-18

    A novel protocol to construct fluorescent purine-fused tricyclic products via intramolecular cyclization of N-propargyl-adenine has been developed. With CuBr as the catalyst, a series of purine-fused tricyclic products were obtained in good to excellent yields (19 examples, 75-89% yields). When R2 was a hydrogen atom in N-propargyl-adenines, the reactions only afforded the endocyclic double bond products. When R2 was an aryl group, the electron-donating groups favored the endocyclic double bond products, while the electron-withdrawing groups favored the exocyclic double bond products. PMID:24678722

  11. Labeled nucleotide phosphate (NP) probes

    SciTech Connect

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2009-02-03

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  12. Differential function of the two nucleotide binding domains on cystic fibrosis transmembrane conductance regulator.

    PubMed

    Nagel, G

    1999-12-01

    The genetic disease cystic fibrosis is caused by defects in the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). CFTR belongs to the family of ABC transporters. In contrast to most other members of this family which transport substrates actively across a membrane, the main function of CFTR is to regulate passive flux of substrates across the plasma membrane. Chloride channel activity of CFTR is dependent on protein phosphorylation and presence of nucleoside triphosphates. From electrophysiological studies of CFTR detailed models of its regulation by phosphorylation and nucleotide interaction have evolved. These investigations provide ample evidence that ATP hydrolysis is crucial for CFTR gating. It becomes apparent that the two nucleotide binding domains on CFTR not only diverge strongly in sequence, but also in function. Based on previous models and taking into account new data from pre-steady-state experiments, a refined model for the action of nucleotides at two nucleotide binding domains was recently proposed.

  13. Pathway Engineered Enzymatic de novo Purine Nucleotide Synthesis

    PubMed Central

    Schultheisz, Heather L.; Szymczyna, Blair R.; Scott, Lincoln G.; Williamson, James R.

    2009-01-01

    A general method for isotopic labeling of the purine base moiety of nucleotides and RNA has been developed through biochemical pathway engineering in vitro. A synthetic scheme was designed and implemented utilizing recombinant enzymes from the pentose phosphate and de novo purine synthesis pathways, with regeneration of folate, aspartate, glutamine, ATP, and NADPH cofactors, in a single-pot reaction. Syntheses proceeded quickly and efficiently in comparison to chemical methods with isolated yields up to 66% for 13C, 15N enriched ATP and GTP. The scheme is robust and flexible, requiring only serine, NH4+, glucose and CO2 as stoichiometric precursors in labeled form. Using this approach, U-13C- GTP, U-13C,15N- GTP, 13C2,8- ATP and U-15N- GTP were synthesized on a millimole scale, and the utility of the isotope labeling is illustrated in NMR spectra of HIV-2 transactivation region (TAR) RNA containing 13C 2,8-adenosine and 15N-1,3,7,9,2-guanosine. Pathway engineering in vitro permits complex synthetic cascades to be effected expanding the applicability of enzymatic synthesis. PMID:18707057

  14. Molecular Disruption of the Power Stroke in the ATP-binding Cassette Transport Protein MsbA*

    PubMed Central

    Doshi, Rupak; Ali, Anam; Shi, Wilma; Freeman, Elizabeth V.; Fagg, Lisa A.; van Veen, Hendrik W.

    2013-01-01

    ATP-binding cassette transporters affect drug pharmacokinetics and are associated with inherited human diseases and impaired chemotherapeutic treatment of cancers and microbial infections. Current alternating access models for ATP-binding cassette exporter activity suggest that ATP binding at the two cytosolic nucleotide-binding domains provides a power stroke for the conformational switch of the two membrane domains from the inward-facing conformation to the outward-facing conformation. In outward-facing crystal structures of the bacterial homodimeric ATP-binding cassette transporters MsbA from Gram-negative bacteria and Sav1866 from Staphylococcus aureus, two transmembrane helices (3 and 4) in the membrane domains have their cytoplasmic extensions in close proximity, forming a tetrahelix bundle interface. In biochemical experiments on MsbA from Escherichia coli, we show for the first time that a robust network of inter-monomer interactions in the tetrahelix bundle is crucial for the transmission of nucleotide-dependent conformational changes to the extracellular side of the membrane domains. Our observations are the first to suggest that modulation of tetrahelix bundle interactions in ATP-binding cassette exporters might offer a potent strategy to alter their transport activity. PMID:23306205

  15. Highly Dynamic Interactions Maintain Kinetic Stability of the ClpXP Protease During the ATP-Fueled Mechanical Cycle.

    PubMed

    Amor, Alvaro J; Schmitz, Karl R; Sello, Jason K; Baker, Tania A; Sauer, Robert T

    2016-06-17

    The ClpXP protease assembles in a reaction in which an ATP-bound ring hexamer of ClpX binds to one or both heptameric rings of the ClpP peptidase. Contacts between ClpX IGF-loops and clefts on a ClpP ring stabilize the complex. How ClpXP stability is maintained during the ATP-hydrolysis cycle that powers mechanical unfolding and translocation of protein substrates is poorly understood. Here, we use a real-time kinetic assay to monitor the effects of nucleotides on the assembly and disassembly of ClpXP. When ATP is present, complexes containing single-chain ClpX assemble via an intermediate and remain intact until transferred into buffers containing ADP or no nucleotides. ATP binding to high-affinity subunits of the ClpX hexamer prevents rapid dissociation, but additional subunits must be occupied to promote assembly. Small-molecule acyldepsipeptides, which compete with the IGF loops of ClpX for ClpP-cleft binding, cause exceptionally rapid dissociation of otherwise stable ClpXP complexes, suggesting that the IGF-loop interactions with ClpP must be highly dynamic. Our results indicate that the ClpX hexamer spends almost no time in an ATP-free state during the ATPase cycle, allowing highly processive degradation of protein substrates.

  16. The Tomato R Gene Products I-2 and Mi-1 Are Functional ATP Binding Proteins with ATPase Activity

    PubMed Central

    Tameling, Wladimir I. L.; Elzinga, Sandra D. J.; Darmin, Patricia S.; Vossen, Jack H.; Takken, Frank L. W.; Haring, Michel A.; Cornelissen, Ben J. C.

    2002-01-01

    Most plant disease resistance (R) genes known today encode proteins with a central nucleotide binding site (NBS) and a C-terminal Leu-rich repeat (LRR) domain. The NBS contains three ATP/GTP binding motifs known as the kinase-1a or P-loop, kinase-2, and kinase-3a motifs. In this article, we show that the NBS of R proteins forms a functional nucleotide binding pocket. The N-terminal halves of two tomato R proteins, I-2 conferring resistance to Fusarium oxysporum and Mi-1 conferring resistance to root-knot nematodes and potato aphids, were produced as glutathione S-transferase fusions in Escherichia coli. In a filter binding assay, purified I-2 was found to bind ATP rather than other nucleoside triphosphates. ATP binding appeared to be fully dependent on the presence of a divalent cation. A mutant I-2 protein containing a mutation in the P-loop showed a strongly reduced ATP binding capacity. Thin layer chromatography revealed that both I-2 and Mi-1 exerted ATPase activity. Based on the strong conservation of NBS domains in R proteins of the NBS-LRR class, we propose that they all are capable of binding and hydrolyzing ATP. PMID:12417711

  17. N-type and P/Q-type calcium channels regulate differentially the release of noradrenaline, ATP and β-NAD in blood vessels

    PubMed Central

    Smyth, Lisa M.; Yamboliev, Ilia A.; Mutafova-Yambolieva, Violeta N.

    2009-01-01

    Using HPLC techniques we evaluated the electrical field stimulation-evoked overflow of noradrenaline (NA), adenosine 5′-triphosphate (ATP), and β-nicotinamide adenine dinucleotide (β-NAD) in the presence of low nanomolar concentrations of ω-conotoxin GVIA or ω-agatoxin IVA in the canine mesenteric arteries and veins. ω-conotoxin GVIA abolished the evoked overflow of NA and β-NAD in artery and vein, whereas the evoked overflow of ATP remained unchanged in the presence of ω-conotoxin GVIA. ω-agatoxin IVA significantly reduced the evoked overflow of ATP and β-NAD. The overflow of NA remained largely unaffected by ω-agatoxin IVA, except at 16 Hz in the vein where the overflow of NA was reduced by about 50%. Artery and vein exhibited similar expression levels of the α1B (CaV2.2, N-type) subunit, whereas the vein showed greater levels of the α1A (CaV2.1, P/Q-type) subunit than artery. Therefore, there are at least two release sites for NA, β-NAD and ATP in the canine mesenteric artery and vein: an N-type-associated site releasing primarily NA, β-NAD and some ATP, and a P/Q-type-associated site releasing ATP, β-NAD and some NA. The N-type-mediated mechanisms are equally expressed in artery and vein, whereas the P/Q-type-mediated mechanisms are more pronounced in the vein and may ensure additional neurotransmitter release at higher levels of neural activity. In artery, β-NAD caused a dual effect consisting of vasodilatation or vasoconstriction depending on concentrations, whereas vein responded with vasodilatation only. In contrast, ATP caused vasoconstriction in both vessels. β-NAD and ATP may mediate disparate functions in the canine mesenteric resistive and capacitative circulations. PMID:18824011

  18. Mutations in the Atp1p and Atp3p subunits of yeast ATP synthase differentially affect respiration and fermentation in Saccharomyces cerevisiae.

    PubMed

    Francis, Brian R; White, Karen H; Thorsness, Peter E

    2007-04-01

    ATP1-111, a suppressor of the slow-growth phenotype of yme1Delta lacking mitochondrial DNA is due to the substitution of phenylalanine for valine at position 111 of the alpha-subunit of mitochondrial ATP synthase (Atp1p in yeast). The suppressing activity of ATP1-111 requires intact beta (Atp2p) and gamma (Atp3p) subunits of mitochondrial ATP synthase, but not the stator stalk subunits b (Atp4p) and OSCP (Atp5p). ATP1-111 and other similarly suppressing mutations in ATP1 and ATP3 increase the growth rate of wild-type strains lacking mitochondrial DNA. These suppressing mutations decrease the growth rate of yeast containing an intact mitochondrial chromosome on media requiring oxidative phosphorylation, but not when grown on fermentable media. Measurement of chronological aging of yeast in culture reveals that ATP1 and ATP3 suppressor alleles in strains that contain mitochondrial DNA are longer lived than the isogenic wild-type strain. In contrast, the chronological life span of yeast cells lacking mitochondrial DNA and containing these mutations is shorter than that of the isogenic wild-type strain. Spore viability of strains bearing ATP1-111 is reduced compared to wild type, although ATP1-111 enhances the survival of spores that lacked mitochondrial DNA.

  19. Obligate coupling of CFTR pore opening to tight nucleotide-binding domain dimerization.

    PubMed

    Mihályi, Csaba; Töröcsik, Beáta; Csanády, László

    2016-01-01

    In CFTR, the chloride channel mutated in cystic fibrosis (CF) patients, ATP-binding-induced dimerization of two cytosolic nucleotide binding domains (NBDs) opens the pore, and dimer disruption following ATP hydrolysis closes it. Spontaneous openings without ATP are rare in wild-type CFTR, but in certain CF mutants constitute the only gating mechanism, stimulated by ivacaftor, a clinically approved CFTR potentiator. The molecular motions underlying spontaneous gating are unclear. Here we correlate energetic coupling between residues across the dimer interface with spontaneous pore opening/closure in single CFTR channels. We show that spontaneous openings are also strictly coupled to NBD dimerization, which may therefore occur even without ATP. Coordinated NBD/pore movements are therefore intrinsic to CFTR: ATP alters the stability, but not the fundamental structural architecture, of open- and closed-pore conformations. This explains correlated effects of phosphorylation, mutations, and drugs on ATP-driven and spontaneous activity, providing insights for understanding CF mutation and drug mechanisms. PMID:27328319

  20. Obligate coupling of CFTR pore opening to tight nucleotide-binding domain dimerization

    PubMed Central

    Mihályi, Csaba; Töröcsik, Beáta; Csanády, László

    2016-01-01

    In CFTR, the chloride channel mutated in cystic fibrosis (CF) patients, ATP-binding-induced dimerization of two cytosolic nucleotide binding domains (NBDs) opens the pore, and dimer disruption following ATP hydrolysis closes it. Spontaneous openings without ATP are rare in wild-type CFTR, but in certain CF mutants constitute the only gating mechanism, stimulated by ivacaftor, a clinically approved CFTR potentiator. The molecular motions underlying spontaneous gating are unclear. Here we correlate energetic coupling between residues across the dimer interface with spontaneous pore opening/closure in single CFTR channels. We show that spontaneous openings are also strictly coupled to NBD dimerization, which may therefore occur even without ATP. Coordinated NBD/pore movements are therefore intrinsic to CFTR: ATP alters the stability, but not the fundamental structural architecture, of open- and closed-pore conformations. This explains correlated effects of phosphorylation, mutations, and drugs on ATP-driven and spontaneous activity, providing insights for understanding CF mutation and drug mechanisms. DOI: http://dx.doi.org/10.7554/eLife.18164.001 PMID:27328319

  1. Nucleotide Regulation of the Structure and Dynamics of G-Actin

    PubMed Central

    Saunders, Marissa G.; Tempkin, Jeremy; Weare, Jonathan; Dinner, Aaron R.; Roux, Benoît; Voth, Gregory A.

    2014-01-01

    Actin, a highly conserved cytoskeletal protein found in all eukaryotic cells, facilitates cell motility and membrane remodeling via a directional polymerization cycle referred to as treadmilling. The nucleotide bound at the core of each actin subunit regulates this process. Although the biochemical kinetics of treadmilling has been well characterized, the atomistic details of how the nucleotide affects polymerization remain to be definitively determined. There is increasing evidence that the nucleotide regulation (and other characteristics) of actin cannot be fully described from the minimum energy structure, but rather depends on a dynamic equilibrium between conformations. In this work we explore the conformational mobility of the actin monomer (G-actin) in a coarse-grained subspace using umbrella sampling to bias all-atom molecular-dynamics simulations along the variables of interest. The results reveal that ADP-bound actin subunits are more conformationally mobile than ATP-bound subunits. We used a multiscale analysis method involving coarse-grained and atomistic representations of these simulations to characterize how the nucleotide affects the low-energy states of these systems. The interface between subdomains SD2–SD4, which is important for polymerization, is stabilized in an actin filament-like (F-actin) conformation in ATP-bound G-actin. Additionally, the nucleotide modulates the conformation of the SD1-SD3 interface, a region involved in the binding of several actin-binding proteins. PMID:24739170

  2. Modulation by extracellular ATP of L-type calcium channels in guinea-pig single sinoatrial nodal cell.

    PubMed Central

    Qi, A. D.; Kwan, Y. W.

    1996-01-01

    1. The effects of extracellular adenosine 5'-triphosphate ([ATP]zero) on the L-type Ca2+ channel currents in guinea-pig single sinoatrial nodal (SAN) cells, isolated by enzymatic dissociation, were investigated by use of whole-cell patch-clamp techniques. 2. The application of [ATP]zero (2 microM-1 mM) produced an inhibitory effect on the L-type Ca2+ channel current peak amplitude (10 mM Ba2+ as charge carrier) in a concentration-dependent and reversible manner with an IC50 of 100 microM and a Hill coefficient of 1.83. 3. The presence of the adenosine receptor antagonists, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 0.1 microM) and 8-phenyltheophylline (10 microM) did not affect the [ATP]zero-induced inhibition of the Ca2+ channel currents. Adenosine (100 microM) had little effect on the basal Ca2+ channel currents. Adenosine 500 microM, caused 23% inhibition of the Ca2+ channel current, which was abolished by 0.1 microM DPCPX. 4. The presence of the P2-purinoceptor antagonists, suramin (1, 10 and 100 microM), reactive blue 2 (1 and 10 microM) and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 50 and 100 microM) failed to affect the inhibitory action of [ATP]zero on Ca2+ channel currents. 5. The relative rank order of potency of different nucleotides and nucleosides, at a concentration of 100 microM, on the inhibition of the Ca2+ channel currents is as follows: adenosine 5'-triphosphate (ATP) = alpha,beta-methylene-ATP (alpha,beta MeATP) > > 2-methylthioATP (2-MeSATP) > or = adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) > > uridine 5'-triphosphate (UTP) = adenosine 5'-diphosphate (ADP) > adenosine 5'-monophosphate (AMP) > or = adenosine. 6. These results suggest that [ATP]zero may play an important role in the heart beat by inhibiting the L-type Ca2+ channel currents in single SAN cells. This inhibitory effect is not due to the formation of adenosine resulting from the enzymatic degradation of [ATP]zero. Based on the relative order of inhibitory

  3. Metal-Dependent Regulation of ATP7A and ATP7B in Fibroblast Cultures

    PubMed Central

    Lenartowicz, Malgorzata; Moos, Torben; Ogórek, Mateusz; Jensen, Thomas G.; Møller, Lisbeth B.

    2016-01-01

    Deficiency of one of the copper transporters ATP7A and ATP7B leads to the rare X-linked disorder Menkes Disease (MD) or the rare autosomal disorder Wilson disease (WD), respectively. In order to investigate whether the ATP7A and the ATP7B genes may be transcriptionally regulated, we measured the expression level of the two genes at various concentrations of iron, copper, and insulin. Treating fibroblasts from controls or from individuals with MD or WD for 3 and 10 days with iron chelators revealed that iron deficiency led to increased transcript levels of both ATP7A and ATP7B. Copper deficiency obtained by treatment with the copper chelator led to a downregulation of ATP7A in the control fibroblasts, but surprisingly not in the WD fibroblasts. In contrast, the addition of copper led to an increased expression of ATP7A, but a decreased expression of ATP7B. Thus, whereas similar regulation patterns for the two genes were observed in response to iron deficiency, different responses were observed after changes in the access to copper. Mosaic fibroblast cultures from female carriers of MD treated with copper or copper chelator for 6–8 weeks led to clonal selection. Cells that express the normal ATP7A allele had a selective growth advantage at high copper concentrations, whereas more surprisingly, cells that express the mutant ATP7A allele had a selective growth advantage at low copper concentrations. Thus, although the transcription of ATP7A is regulated by copper, clonal growth selection in mosaic cell cultures is affected by the level of copper. Female carriers of MD are rarely affected probably due to a skewed inactivation of the X-chromosome bearing the ATP7A mutation. PMID:27587995

  4. Metal-Dependent Regulation of ATP7A and ATP7B in Fibroblast Cultures.

    PubMed

    Lenartowicz, Malgorzata; Moos, Torben; Ogórek, Mateusz; Jensen, Thomas G; Møller, Lisbeth B

    2016-01-01

    Deficiency of one of the copper transporters ATP7A and ATP7B leads to the rare X-linked disorder Menkes Disease (MD) or the rare autosomal disorder Wilson disease (WD), respectively. In order to investigate whether the ATP7A and the ATP7B genes may be transcriptionally regulated, we measured the expression level of the two genes at various concentrations of iron, copper, and insulin. Treating fibroblasts from controls or from individuals with MD or WD for 3 and 10 days with iron chelators revealed that iron deficiency led to increased transcript levels of both ATP7A and ATP7B. Copper deficiency obtained by treatment with the copper chelator led to a downregulation of ATP7A in the control fibroblasts, but surprisingly not in the WD fibroblasts. In contrast, the addition of copper led to an increased expression of ATP7A, but a decreased expression of ATP7B. Thus, whereas similar regulation patterns for the two genes were observed in response to iron deficiency, different responses were observed after changes in the access to copper. Mosaic fibroblast cultures from female carriers of MD treated with copper or copper chelator for 6-8 weeks led to clonal selection. Cells that express the normal ATP7A allele had a selective growth advantage at high copper concentrations, whereas more surprisingly, cells that express the mutant ATP7A allele had a selective growth advantage at low copper concentrations. Thus, although the transcription of ATP7A is regulated by copper, clonal growth selection in mosaic cell cultures is affected by the level of copper. Female carriers of MD are rarely affected probably due to a skewed inactivation of the X-chromosome bearing the ATP7A mutation. PMID:27587995

  5. Characterization of an ATP Translocase Identified in the Destructive Plant Pathogen “Candidatus Liberibacter asiaticus”▿

    PubMed Central

    Vahling, Cheryl M.; Duan, Yongping; Lin, Hong

    2010-01-01

    ATP/ADP translocases transport 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. All bacterial ATP/ADP translocases characterized thus far have been found in endosymbionts of protozoa or pathogens of higher-order animals, including humans. A putative ATP/ADP translocase was uncovered during the genomic sequencing of the intracellular plant pathogen “Candidatus Liberibacter asiaticus,” the causal agent of citrus huanglongbing. Bioinformatic analysis of the protein revealed 12 transmembrane helices and predicted an isoelectric point of 9.4, both of which are characteristic of this family of proteins. The “Ca. Liberibacter asiaticus” gene (nttA) encoding the translocase was subsequently expressed in Escherichia coli and shown to enable E. coli to import ATP directly into the cell. Competition assays with the heterologous E. coli system demonstrated that the translocase was highly specific for ATP and ADP but that other nucleotides, if present in high concentrations, could also be taken up and/or block the ability of the translocase to import ATP. In addition, a protein homologous to NttA was identified in “Ca. Liberibacter solanacearum,” the bacterium associated with potato zebra chip disease. This is the first reported characterization of an ATP translocase from “Ca. Liberibacter asiaticus,” indicating that some intracellular bacteria of plants also have the potential to import ATP directly from their environment. PMID:19948801

  6. Populus euphratica APYRASE2 Enhances Cold Tolerance by Modulating Vesicular Trafficking and Extracellular ATP in Arabidopsis Plants1[OPEN

    PubMed Central

    Deng, Shurong; Sun, Jian; Zhao, Rui; Ding, Mingquan; Zhang, Yinan; Sun, Yuanling; Wang, Wei; Tan, Yeqing; Liu, Dandan; Ma, Xujun; Hou, Peichen; Wang, Meijuan; Lu, Cunfu; Shen, Xin; Chen, Shaoliang

    2015-01-01

    Apyrase and extracellular ATP play crucial roles in mediating plant growth and defense responses. In the cold-tolerant poplar, Populus euphratica, low temperatures up-regulate APYRASE2 (PeAPY2) expression in callus cells. We investigated the biochemical characteristics of PeAPY2 and its role in cold tolerance. We found that PeAPY2 predominantly localized to the plasma membrane, but punctate signals also appeared in the endoplasmic reticulum and Golgi apparatus. PeAPY2 exhibited broad substrate specificity, but it most efficiently hydrolyzed purine nucleotides, particularly ATP. PeAPY2 preferred Mg2+ as a cofactor, and it was insensitive to various, specific ATPase inhibitors. When PeAPY2 was ectopically expressed in Arabidopsis (Arabidopsis thaliana), cold tolerance was enhanced, based on root growth measurements and survival rates. Moreover, under cold stress, PeAPY2-transgenic plants maintained plasma membrane integrity and showed reduced cold-elicited electrolyte leakage compared with wild-type plants. These responses probably resulted from efficient plasma membrane repair via vesicular trafficking. Indeed, transgenic plants showed accelerated endocytosis and exocytosis during cold stress and recovery. We found that low doses of extracellular ATP accelerated vesicular trafficking, but high extracellular ATP inhibited trafficking and reduced cell viability. Cold stress caused significant increases in root medium extracellular ATP. However, under these conditions, PeAPY2-transgenic lines showed greater control of extracellular ATP levels than wild-type plants. We conclude that Arabidopsis plants that overexpressed PeAPY2 could increase membrane repair by accelerating vesicular trafficking and hydrolyzing extracellular ATP to avoid excessive, cold-elicited ATP accumulation in the root medium and, thus, reduced ATP-induced inhibition of vesicular trafficking. PMID:26224801

  7. Controlling two-phase self-assembly of an adenine derivative on HOPG via kinetic effects.

    PubMed

    Wang, Can; Jana, Pritam Kumar; Zhang, Haiming; Mu, Zhongcheng; Kehr, Gerald; Blömker, Tobias; Erker, Gerhard; Fuchs, Harald; Heuer, Andreas; Chi, Lifeng

    2014-08-21

    Large-area self-assembled structures of a nucleobase adenine derivative were successfully realized through vacuum deposition. STM images reveal two types of structures, which could be regulated by substrate temperature and the evaporation rate, indicating the relevance of kinetic effects. The results are supported by computer simulations.

  8. The effect of activated charcoal on adenine-induced chronic renal failure in rats.

    PubMed

    Ali, Badreldin H; Alza'abi, Mohamed; Ramkumar, Aishwarya; Al-Lawati, Intisar; Waly, Mostafa I; Beegam, Sumaya; Nemmar, Abderrahim; Brand, Susanne; Schupp, Nicole

    2014-03-01

    Activated charcoal (AC) is a sorbent that has been shown to remove urinary toxins like urea and indoxyl sulfate. Here, the influence of AC on kidney function of rats with experimental chronic renal failure (CRF) is investigated. CRF was induced in rats by feeding adenine (0.75%) for four weeks. As an intervention, AC was added to the feed at concentrations of 10%, 15% or 20%. Adenine treatment impaired kidney function: it lowered creatinine clearance and increased plasma concentrations of creatinine, urea, neutrophil gelatinase-associated lipocalin and vanin-1. Furthermore, it raised plasma concentrations of the uremic toxins indoxyl sulfate, phosphate and uric acid. Renal morphology was severely damaged and histopathological markers of inflammation and fibrosis were especially increased. In renal homogenates, antioxidant indices, including superoxide dismutase and catalase activity, total antioxidant capacity and reduced glutathione were adversely affected. Most of these changes were significantly ameliorated by dietary administration of AC at a concentration of 20%, while effects induced by lower doses of dietary AC on adenine nephrotoxicity were not statistically significant. The results suggest that charcoal is a useful sorbent agent in dietary adenine-induced CRF in rats and that its usability as a nephroprotective agent in human kidney disease should be studied.

  9. Effects of adenine arabinoside on lymphocytes infected with Epstein-Barr virus.

    PubMed Central

    Benz, W C; Siegel, P J; Baer, J

    1978-01-01

    Low concentrations of adenine arabinoside inhibited growth of two Epstein-Barr virus producer cell lines in culture, while not significantly affecting a nonproducer cell line and a B-cell-negative line. These observations were extended to include freshly infected cells. Mitogen-stimulated human umbilical cord blood lymphocytes were unaffected by the drug at concentration levels that inhibited [3H]thymidine incorporation into the DNA of Epstein-Barr virus-stimulated cells. DNA synthesis in Epstein-Barr virus-superinfected Raji cells was also adversely affected by adenine arabinoside. However, these same low concentrations of adenine arabinoside in the triphosphate form produced less effect on DNA synthesis in nuclear systems and DNA polymerase assays than on growth or DNA synthesis in whole cells. Therefore the effects reported here of low concentrations of the drug on whole cells may be only in part related to DNA polymerase inhibition. The work reported here suggests that adenine arabinoside has multiple sites of action in infected cells. PMID:212577

  10. Ameliorative Effect of Chrysin on Adenine-Induced Chronic Kidney Disease in Rats

    PubMed Central

    Ali, Badreldin H.; Adham, Sirin A.; Al Za’abi, Mohammed; Waly, Mostafa I.; Yasin, Javed; Nemmar, Abderrahim; Schupp, Nicole

    2015-01-01

    Chrysin (5, 7- dihydroxyflavone) is a flavonoid with several pharmacological properties that include antioxidant, anti-inflammatory and antiapoptotic activities. in this work, we investigated some effects of three graded oral doses of chrysin (10, 50 and 250 mg/kg) on kidney structure and function in rats with experimental chronic renal disease (CKD) induced by adenine (0.25% w/w in feed for 35 days), which is known to involve inflammation and oxidative stress. Using several indices in plasma, urine and kidney homogenates, adenine was found to impair kidney function as it lowered creatinine clearance and increased plasma concentrations of creatinine, urea, neutrophil gelatinase-associated lipocalin and N-Acetyl-beta-D-glucosaminidase activity. Furthermore, it raised plasma concentrations of the uremic toxin indoxyl sulfate, some inflammatory cytokines and urinary albumin concentration. Renal morphology was severely damaged and histopathological markers of inflammation and fibrosis were especially increased. In renal homogenates, antioxidant indices, including superoxide dismutase and catalase activities, total antioxidant capacity and reduced glutathione were all adversely affected. Most of these adenine – induced actions were moderately and dose -dependently mitigated by chrysin, especially at the highest dose. Chrysin did not cause any overt adverse effect on the treated rats. The results suggest that different doses of chrysin produce variable salutary effects against adenine-induced CKD in rats, and that, pending further pharmacological and toxicological studies, its usability as a possible ameliorative agent in human CKD should be considered. PMID:25909514

  11. Macrophage Trafficking as Key Mediator of Adenine-Induced Kidney Injury

    PubMed Central

    Braga, Tárcio Teodoro; Felizardo, Raphael José Ferreira; Andrade-Oliveira, Vinícius; Hiyane, Meire Ioshie; da Silva, João Santana; Câmara, Niels Olsen Saraiva

    2014-01-01

    Macrophages play a special role in the onset of several diseases, including acute and chronic kidney injuries. In this sense, tubule interstitial nephritis (TIN) represents an underestimated insult, which can be triggered by different stimuli and, in the absence of a proper regulation, can lead to fibrosis deposition. Based on this perception, we evaluated the participation of macrophage recruitment in the development of TIN. Initially, we provided adenine-enriched food to WT and searched for macrophage presence and action in the kidney. Also, a group of animals were depleted of macrophages with the clodronate liposome while receiving adenine-enriched diet. We collected blood and renal tissue from these animals and renal function, inflammation, and fibrosis were evaluated. We observed higher expression of chemokines in the kidneys of adenine-fed mice and a substantial protection when macrophages were depleted. Then, we specifically investigated the role of some key chemokines, CCR5 and CCL3, in this TIN experimental model. Interestingly, CCR5 KO and CCL3 KO animals showed less renal dysfunction and a decreased proinflammatory profile. Furthermore, in those animals, there was less profibrotic signaling. In conclusion, we can suggest that macrophage infiltration is important for the onset of renal injury in the adenine-induced TIN. PMID:25132730

  12. Structural and quantum chemical studies of 8-aryl-sulfanyl adenine class Hsp90 inhibitors.

    PubMed

    Immormino, Robert M; Kang, Yanlong; Chiosis, Gabriela; Gewirth, Daniel T

    2006-08-10

    Hsp90 chaperones play a critical role in modulating the activity of many cell signaling proteins and are an attractive target for anti-cancer therapeutics. We report here the structures of the water soluble 8-aryl-sulfanyl adenine class Hsp90 inhibitors, 1 (PU-H71) and 2 (PU-H64), in complex with the N-terminal domain of human Hsp90alpha. The conformation of 1 when bound to Hsp90 differs from previously reported 8-aryl adenine Hsp90 inhibitors including 3 (PU24FCl). While the binding mode for 3 places the 2'-halide of the 8-aryl group on top of the adenine ring, for 1 and 2, we show that the 2'-halide is rotated approximately 180 degrees away. This difference explains the opposing trends in Hsp90 inhibitory activity for the 2'-halo derivatives of the 3',4',5'-trimethoxy series where Cl > Br > I compared to the 4',5'-methylenedioxy series where I > Br > Cl. We also present quantum chemical calculations of 2 and its analogues that illuminate their basis for Hsp90 inhibition. The calculated conformation of 2 agreed well with the crystallographically observed conformations of 1 and 2. The predictive nature of the calculations has allowed the exploration of additional derivatives based on the 8-aryl adenine scaffold.

  13. SERS, XPS, and DFT Study of Adenine Adsorption on Silver and Gold Surfaces.

    PubMed

    Pagliai, Marco; Caporali, Stefano; Muniz-Miranda, Maurizio; Pratesi, Giovanni; Schettino, Vincenzo

    2012-01-19

    The adsorption of adenine on silver and gold surfaces has been investigated combining density functional theory calculations with surface-enhanced Raman scattering and angle-resolved X-ray photoelectron spectroscopy measurements, obtaining useful insight into the orientation and interaction of the nucleobase with the metal surfaces.

  14. Blau syndrome polymorphisms in NOD2 identify nucleotide hydrolysis and helical domain 1 as signalling regulators.

    PubMed

    Parkhouse, Rhiannon; Boyle, Joseph P; Monie, Tom P

    2014-09-17

    Understanding how single nucleotide polymorphisms (SNPs) lead to disease at a molecular level provides a starting point for improved therapeutic intervention. SNPs in the innate immune receptor nucleotide oligomerisation domain 2 (NOD2) can cause the inflammatory disorders Blau Syndrome (BS) and early onset sarcoidosis (EOS) through receptor hyperactivation. Here, we show that these polymorphisms cluster into two primary locations: the ATP/Mg(2+)-binding site and helical domain 1. Polymorphisms in these two locations may consequently dysregulate ATP hydrolysis and NOD2 autoinhibition, respectively. Complementary mutations in NOD1 did not mirror the NOD2 phenotype, which indicates that NOD1 and NOD2 are activated and regulated by distinct methods. PMID:25093298

  15. Tightly bound nucleotides of the energy-transducing ATPase, and their role in oxidative phosphor