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The structural and functional role of the [gamma]-[epsilon] rotor in Escherichia coli F₀F₁ ATP synthaseLin, Shin-Kai. January 2008 (has links)
Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. [gamma] and [epsilon] in title are the Greek letters. Includes bibliographical references. Also available online through Digital Dissertations.
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Salivary gland P2 nucleotide receptors structure and function studies /Landon, Linda A. Neighbors January 1998 (has links)
Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.
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Salivary gland P2 nucleotide receptors structure and function studies /Landon, Linda A. Neighbors January 1998 (has links)
Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / Includes bibliographical references.
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The sympathetic cotransmitters neuropeptide Y and ATP in the regulation of the vascular smooth muscle cell mitogenic effects, receptors and second messengers : aspects on clinical patophysiology /Erlinge, David. January 1994 (has links)
Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.
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ATP mimics as glutamine synthetase inhibitors : an exploratory synthetic studySalisu, Sheriff Tomilola January 2008 (has links)
Using a mechanism-based approach to drug discovery, efforts have been directed towards developing novel ATP mimics that can act as GS inhibitors. The purine-based systems, adenosine, adenine and allopurinol, were identified as possible scaffolds for potential ATP mimics, while various meta-disubstituted benzenoid compounds, 3-aminobenzonitrile, 3-aminophenol, resorcinol, 3-aminobenzyl alcohol, 3-hydroxybenzoic acid and 3-aminobenzoic acid have been explored as adenine analogues. These compounds were treated with different alkylating and acylating agents. Allylation of all the substrates was achieved using allyl bromide and N-9 alkylation of protected allopurinol was effected using a number of specially prepared Baylis-Hillman adducts. Acylation of the benzenoid precursors with chloroacetyl chloride, acetoxyacetyl chloride, acryloyl chloride and specially prepared 2,3,4,5,6-pentaacetylgluconoyl chloride afforded the corresponding mono- and /or diacylated products in varying yields (4-96%). Elaboration of the alkylated and acylated products has involved the reaction of hydroxy systems with diethyl chloro phosphate and chloro derivatives with triethyl phosphite in Arbuzov-type reactions to afford phosphorylated products. In all cases, products were fully characterized using 1- and 2-D NMR analysis and, where appropriate, high-resolution mass spectrometry. The application of Modgraph and ChemWindow NMR prediction programmes has been explored and the resulting data have been compared with experimental chemical shift assignments to confirm chemical structures and, in some cases, to establish the position of allylation or acylation. Experimental assignments were found to be generally comparable with the Modgraph data, but not always with the ChemWindow values. The docking of selected products in the 'active-site' of GS and their structural homology with ATP, both in their free and bound conformations have been studied using the ACCELERYS Cerius² platform. All the selected ATP mimics exhibit some form of interaction with the 'active-site' residues, and a number of them appear to be promising GS ligands.
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Studies of the human lymphocyte P2Z receptor and its activation of phospholipase D.Gargett, Caroline Eve, mikewood@deakin.edu.au January 1997 (has links)
Extracellular adenosine 5′-triphosphate (ATP) is an agonist for the P2Z receptor of human leukaemic lymphocytes and opens a Ca <sup>2+</sup>-selective ion channel, which also conducts Ba<sup>2+</sup>, Sr<sup>2+</sup> and the small fluorescent dye, ethidium<sup>+</sup>. A wide range of receptor agonists, many of which raise cytosolic [Ca<sup>2+</sup>] activate phospholipase D (PLD). In the present study, it was shown that both ATP and 3′-O-(4-benzoylbenzoyl)-ATP (BzATP) stimulated PLD activity in a concentration-dependent manner, and the inhibitory effects of suramin, oxidised ATP, extracellular Na<sup>+</sup> and Mg<sup>2+</sup> suggested that the effect of these agonists is mediated by P2Z receptors.
The role of divalent cations in ATP-stimulated PLD activity was investigated. Several agonists (eg ATP, thapsigargin, ionomycin) stimulated a rise in cytosolic [Ca<sup>2+</sup>] in human lymphocytes, but only ATP and ionomycin stimulated PLD activity. When Ca<sup>2+</sup> influx was prevented by EGTA, the majority of ATP-stimulated and all of ionomycin-stimulated PLD activity was inhibited. Preloading cells with the Ca<sup>2+</sup> chelator, BAPTA, reduced cytosolic [Ca<sup>2+</sup>] and, paradoxically, ATP-stimulated PLD activity was potentiated. ATP-stimulated PLD activity was supported by both Ba<sup>2+</sup> and Sr<sup>2+</sup> when they were substituted for extracellular Ca<sup>2+</sup>. Furthermore, both ATP-stimulated PLD activity and ATP-stimulated <sup>133</sup>Ba<sup>2+</sup> influx showed a linear dependence on extracellular [Ba<sup>2+</sup>]. Thus it was concluded that ATP stimulated PLD activity in direct proportion to the influx of divalent cations through the P2Z ion channel and this PLD activity was insensitive to changes in bulk cytosolic [Ca<sup>2+</sup>].
The calmodulin (Ca<sup>2+</sup>/CaM) inhibitor, trifluoperazine (TFP) inhibited ionomycin- and ATP-stimulated PLD activity and ATP-stimulated apoptosis, but had no effect on PLD activity already activated by ATP. However, TFP inhibited ATP-stimulated Ca<sup>2+</sup>, Ba<sup>2+</sup> and ethidium<sup>+</sup> fluxes, at concentrations below those which inhibit Ca<sup>2+</sup>/CaM, suggesting that TFP inhibits the P2Z receptor.
Similarly, the isoquinolinesulphonamide, KN-62, a selective inhibitor of Ca<sup>2+</sup>/CaM-dependent protein kinase II (CaMKII), also prevented ATP-stimulated apoptosis, but had no effect on pre-activated PLD. In addition, KN-62, and an analogue, KN-04, which has no effect on CaMKII, potently inhibited ATP-stimulated Ba<sup>2+</sup> influx (IC<sub>50</sub> 12.7 ± 1.5 and 17.3 ± 2.7 nM, respectively), ATP-stimulated ethidium<sup>+</sup> uptake (IC<sub>50</sub> 13.1 ± 2.6 and 37.2 ± 8.9 nM, respectively), ATP-stimulated phospholipase D activity (50% inhibition 5.9 ± 1.2 and 9.7 ± 2.8 nM, respectively) and ATP-induced shedding of the surface adhesion molecule, L-selectin (IC<sub>50</sub> 31.5 ± 4.5 and 78.7 ± 10.8 nM, respectively). They did not inhibit phorbol ester- or ionomycin-stimulated PLD activity or phorbol ester-induced L-selectin shedding. Neither KN-62 nor KN-04 (both 500 nM) have any effect on UTP-stimulated Ca<sup>2+</sup> transients in fura-2-loaded human neutrophils, a response which is mediated by the P2Y<sub>2</sub> receptor, neither did they inhibit ATP-stimulated contractile responses mediated by the P2X<sub>1</sub> receptor of guinea pig urinary bladder. Thus, KN-62 and KN-04 are almost equipotent as P2Z inhibitors with IC<sub>50</sub>s in the nanomolar, indicating that their actions cannot be due to CaMKII inhibition, but rather that they are potent and direct inhibitors of the P2Z receptor.
Extracellular ATP-induced shedding of L-selectin from lymphocytes into the medium is a Ca<sup>2+</sup>-independent response. L-selectin is either cleaved by a metalloproteinase or a PLD with specificity for glycosylphosphatidylinositol (GPI). The novel hydroxamic acid-based zinc chelator, Ro-31-9790 blocks ATP-induced L-selectin shedding, but was without effect on ATP-induced Ba<sup>2+</sup> influx or ATP-stimulated PLD activity. Furthermore, another zinc chelator, 1,10-phenanthroline, an inhibitor of a GPI-PLD, potentiated rather than inhibited ATP-stimulated PLD activity, suggesting that ATP-induced L-selectin shedding and ATP-stimulated PLD activity are independent of each other.
Although extracellular ATP is the natural ligand for the lymphocyte P2Z receptor, it is less potent than BzATP in stimulating Ba<sup>2+</sup> influx. Concentration-response curves for BzATP- and ATP-stimulated ethidium<sup>+</sup> influx gave EC<sub>50</sub>s 15.4 ± 1.4 µM and 85.6 ± 8.8 µM, respectively. The maximal response to ATP was only 69.8 ± 1.9% of that for BzATP. Hill coefficients were 3.17 ± 0.24 and 2.09 ± 0.45 for BzATP and ATP respectively, suggesting greater positive cooperativity for BzATP than for ATP in opening the P2Z-operated ion channel. A rank order of agonist potency of BzATP > ATP = 2MeSATP > ATPγS was observed for agonist-stimulated ethidium<sup>+</sup> influx, while maximal influxes followed a rank order of BzATP > ATP > 2MeSATP > ATPγS. When ATP (300 -1000 µM) was added simultaneously with 30 µM BzATP (EC<sub>90</sub>), it reduced both ethidium<sup>+</sup> and Ba<sup>2+</sup> fluxes by 30 - 40% relative to values observed with BzATP alone. KN-62, previously shown to be a specific inhibitor of the lymphocyte P2Z receptor, was a less potent antagonist of BzATP-induced fluxes than ATP, when maximal concentrations of both agonists (50 and 500 µM respectively) were used. However, when BzATP (18 µM) was used at a concentration equiactive with a maximally effective ATP concentration, KN-62 showed the same inhibitory potency for both agonists. The ecto-ATPase antagonist, ARL-67156, inhibited both ATP- and BzATP-stimulated Ba<sup>2+</sup> influx, suggesting that the lower efficacy of ATP compared with BzATP was not due to preferential hydrolysis of ATP. Thus, the natural ligand, ATP, is a partial agonist for the P2Z receptor while BzATP is a full agonist. Moreover the competitive studies show that only a single class of P2-receptor (P2Z class) is expressed on human leukaemic lymphocytes.
Both ATP- and BzATP-stimulated PLD activity were significantly inhibited (P < 0.05) when cells were suspended in iso-osmotic choline Cl medium. Choline<sup>+</sup> was found to be a permeant for the P2Z ion channel, since ATP induced a large uptake of [<sup>14</sup>C]choline<sup>+</sup> (60 to 150 µmol/ml intracellular water) during a 5 min incubation, which remained in the cells for several hours, and ATP was used to load cells with these levels of choline<sup>+</sup>. Intracellular choline<sup>+</sup> inhibited ATP-, BzATP-, PMA- and ionomycin-stimulated PLD activity. Brief exposure of lymphocytes to ATP increased the subsequent basal rate of ethidium<sup>+</sup> uptake, and this was prevented by intracellular choline<sup>+</sup>. It is proposed that P2Z-mediated Ca<sup>2+</sup> influx in lymphocytes activates PLD leading to significantly changes of the phospholipid composition of the plasma membrane, which subsequently produces a permeability lesion, which in turn contributes to cell death.
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Effect of visible and near-infrared light on adenosine triphosphate (atp)Amat Genís, Albert 18 April 2005 (has links)
L' ATP es una molecula clau en el metabolisme cel.lular, actuant com a donador d'energia lliure i acoplant reaccions endergoniques i exergoniques. L'ATP es sintetitzat a la mitocondria en un proces anomenat fosforil.lacio oxidativa despres d'una serie de reaccions a la cadena de citocroms que es troba en la membrana interna de l'organel.la. La font d'energia necessaria per aquesta sintesi s'obte en les cel.lules animals dels nutrients de l'ingestio, i de la llum solar en les plantes. Existeix una via alternativa de sintesi d'ATP extramitocondrial, la glicolisi, que s'inicia amb la fosforilacio de la glucosa per l'enzim hexokinasa.Interaccio llum-materiaL'energia electromagnetica l'ona de la qual oscil.la en una longitud d'ona de nanometres es anomenada llum. En aquestes frequencies, l'energia promou excitacio electronica de certs atoms i molecules. Existeix una interaccio diferent de la llum amb molecules, produida per el camp electromagnetic que per definicio la llum provoca en qualsevol medi. El camp electric resultant desplaca els electrons dels enllacos quimics produint una polaritzacio del medi sense que existeixi absorcio de l'energia. Aquest es un mecanisme interactiu que existeix sempre, i es l'unic que es dona en molecules que son transparents (no absorbeixen) per a una determinada frequencia de la llum, com es el cas de l'ATP per l'energia visible i infravermella propera. Experiments, resultats i discussioQuan l'ATP es excitat amb fotons ultraviolats, es produiex una fluorescencia en longituts d'ona visibles. L'io magnesi s'ha utilitzat per estudiar com la llum visible i infravermella propera produiex un desplacament de carregues electriques a la molecula d'ATP. La construccio d'un interferometre de Michelson ha servit per observar l'interaccio no absortiva de la llum i l'ATP. La mesura directa de l'index de refraccio d'una solucio d'ATP dona informacio sobre les caracteristiques electriques del medi. L'observacio de que aquest index canvia despres d'irradiar la solucio amb longituds d'ona visibles i infravermelles properes, confirmen que la llum provoca canvis electrics significatius en l'ATP. En aquest treball tambe s'ha estudiat el comportament bioquimic de l'ATP irradiat quant forma part de dues reaccions quimiques diferents: la de la luciferina-luciferasa i la de la hexoquinasa. En tots dos casos, l'us d'aquest ATP irradiat ha produit una alteracio dels parametres cinetics estudiats, V0 i k en la reaccio de la luciferina-luciferasa, i km i vmax en la reaccio de l'hexoquinasa.ConclusionsAquesta interaccio no absortiva de la llum amb l'ATP es la primera descrita per a una biomolecula. El mecanisme aporta noves dades per explicar els efectes observats en el metabolisme cel.lular despres de l'irradiacio d'organismes, teixits i cultius cel.lulars amb llum visible i infravermella propera. / ATP is a key molecule in cellular metabolism. In this thesis, I examined the effects of visible (635 and 655 nm) and near-infrared (810 and 830 nm) light on ATP in solution. I also examined were the biochemical behavior of light-exposed ATP in the luciferine-luciferase reaction and hexokinase reaction, the initial step in glycolysis that begins extra mithocondrial ATP synthesis. Irradiated groups in the luciferine-luciferase reaction showed an improvement in the kinetic parameters V0 and k, and more ATP molecules reacted with the enzyme when they were excited by light. When irradiated ATP was added to the hexokinase reaction, the experimental groups showed significant differences in the Michaelis-Menten kinetic parameters (km for ATP and vmax) and the rate of product synthesis was greater. Changes in both reactions were wavelength and dose dependant. When ATP was excited with UV photons, it fluoresced. This fluorescence decreased when Mg2+ was added, probably because the ion binds the phosphates, which are the part of the molecule responsible for light emission. Irradiating the ATP-Mg2+ solution with 655 nm and 830 nm light increased the fluorescence resulting from a displacement of charges in the phosphor-oxygen bond that repels Mg2+. The refraction of light in an ATP solution was observed by the Michelson interferometer and by directly measuring the refractive index. The refractive index changed after red and near-infrared light interaction due to a change in the electrical permittivity of the medium. Since ATP in water is transparent to visible and near-infrared light, and is therefore not a chromophore for those wavelengths, I conclude that the observed light interaction with ATP is not due to photon absorption but to the electromagnetic disturbance produced by the light, which leads to a polarization of the dielectric molecule that is ATP. This interaction of visible and near-infrared electromagnetic energy with ATP offers new perspectives for explaining light interaction at subcellular level.
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Mechanisms involved in the release of ATP from skeletal myoblasts at low pHLu, Lin, 鹿琳 January 2012 (has links)
Lactic acid, which induces pH depression, leads to ATP efflux from muscle to extracellular space: it was reported that CFTR was involved in this process. However, the mechanism by which lactic acid activated CFTR and brought about the ATP release is still unknown. This study was performed to investigate (1) what channels may be involved or even conduct ATP release, and (2) how lactic acid activated CFTR.
Expression of the possible channels that may conduct ATP release in L6 cells was investigated using RT-PCR: ClC-2, ClC-3, ClC-7, CACC, VDAC, connexin 40, connexin 43 and pannexin 3 were expressed in L6.
Incubation of cultured L6 cells with lactic acid (10 mM) increased the extracellular ATP from 0.6 ± 0.06 to 1.1 ± 0.09 nM (P ? 0.05), indicating that lactic acid stimulated ATP efflux in vitro. The non-specific chloride channel inhibitor, DIDS, failed to abolish the lactic-acid-induced ATP release, suggesting that DIDS-sensitive chloride channels were not involved in the ATP efflux. Among the non-specific inhibitors of connexin channels, gadolinium inhibited acidosis-induced ATP efflux, but carbenoxolone failed to inhibit it, and so the role of connexins remains uncertain. The specific inhibitor of CFTR, CFTRinh-172, and the non-specific open-channel blocker of CFTR, glibenclamide, both abolished the acidosis-induced ATP release, but another specific inhibitor of CFTR, GlyH-101, which blocks CFTR from the external side, failed to abolish the ATP release, suggesting that acidosis-induced ATP is dependent on CFTR-activation, but does not involve ATP moving through the CFTR chloride channel.
We hypothesize that, at low pH, the Na+/H+ exchanger (NHX) extruded H+ out of the cell and the resulting intracellular Na+ was transported out by Ca2+/Na+ exchanger (NCX); the localized increase in Ca2+ activated adenyl cyclase (AC), thus elevating intracellular cAMP; cAMP-activated-PKA then phosphorylated CFTR, which regulated an ATP release channel. KT-5720, an inhibitor of PKA, abolished the acidosis-induced ATP release, and forskolin, an agent that elevates cAMP, stimulated it, suggesting that the cAMP/PKA pathway was involved. The specific inhibitor of NCX, SN-6 and KB-R7943, both abolished the acidosis-induced ATP release, supporting a role for NCX in mediating this process. However, amiloride, the non-specific inhibitor of NHX failed to abolish ATP efflux.
The whole cell Cl- currents were studied in L6 cells: lactic acid increased the whole cell currents from 2.33 ± 0.10 to 3.54 ± 0.34 nA (P ? 0.05), and this lactic-acid-induced increase in Cl- current could be inhibited by CFTRinh-172, suggesting that the CFTR Cl- channel was opened at low pH. Moreover, forskolin increased whole cell Cl- currents, which supported a role for the cAMP/PKA pathway in the lactic-acid-induced increase in CFTR current.
These data confirm that CFTR is involved in the lactic-acid-induced ATP release from L6 cells. The roles of the NCX and cAMP/PKA pathway in activating CFTR at low pH are supported, but further studies are required to determine whether the NHX is involved in CFTR activation and whether connexins participate in ATP release. / published_or_final_version / Physiology / Master / Master of Philosophy
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Cystic fibrosis transmembrane conductance regulator is involved in therelease of ATP from contracting skeletal muscleCai, Weisong., 蔡蔚松. January 2012 (has links)
Contracting skeletal muscle releases ATP into the interstitial space where it is subsequently broken down to adenosine by the action of ecto-5’-nucleotidase. Both ATP and adenosine are vasodilators that contribute to the exercise hyperaemia. However, the mechanism for the release of ATP from muscle during exercise remains unknown. Cystic fibrosis transmembrane conductance regulator (CFTR) is involved in ATP release from muscle at low intracellular pH: this study was performed to investigate whether CFTR was involved in the ATP release from skeletal muscle during contractions.
Experiments were performed in rats anaesthetised with sodium pentobarbitone and breathing spontaneously. A microdialysis probe was placed in one gastrocnemius muscle: ATP was determined in interstitial microdialysate samples using a bioluminescence assay. The sciatic nerve was stimulated to induce two bouts of muscle contractions, separated by a recovery period of 40 mins; one of the inhibitors was administered prior to the second bout of contractions.
Muscle contractions elevated the interstitial ATP by 1500 to 3000%. In the control experiments, no drug was given: both the contractile force and the increase in interstitial ATP were reproducible in repeated contraction bouts. Infusion of a specific inhibitor of CFTR, CFTRinh-172, did not alter the contractile force, but significantly lowered the interstitial ATP during muscle contractions, suggesting that CFTR was involved in the contraction-induced ATP release. Similarly, infusion of the Protein Kinase A inhibitor, KT5720, significantly reduced interstitial ATP during muscle contractions without altering contractile force, suggesting that CFTR in skeletal muscle is activated through the cAMP/PKA pathway. The increase in interstitial ATP during muscle contraction was also inhibited by the Na/H exchanger inhibitor, amiloride, or the Na/Ca exchanger inhibitor, SN6. It has been also shown that two gap junction hemichannel inhibitors, gadolinium and carbenoxolone, could attenuate the increase of ATP during muscle contraction.
These data suggest that CFTR, activated through the cAMP/protein kinase A pathway, is involved in the ATP release during muscle contraction, and that activation of the Na/H exchanger and Na/Ca exchanger was also required, indicating that the signal transduction mechanism for CFTR activation during muscle contractions may be similar to that which is reported to occur at low pH. The preliminary data showed that the gap junction hemichannels might mediate the ATP release from skeletal muscle cells during muscle contraction. / published_or_final_version / Physiology / Master / Master of Philosophy
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Phosphate interactions with proteinsFairbrother, Wayne J. January 1989 (has links)
Proton nuclear magnetic resonance (NMR) spectroscopy has been used to investigate the interaction of yeast phosphoglycerate kinase (PGK) with its phosphate containing substrates, ATP and 3-phosphoglycerate (3-PG). The application of one-dimensional and, for the first time, two-dimensional proton NMR techniques to this large protein has enabled specific resonance assignments to be made. Assignment has been aided by the investigation of specifically deuterated protein and site-specific mutant forms of the protein, including the isolated N- and C-domains. The effects of ATP and 3-PG binding on the proton NMR spectrum of yeast PGK have been characterised and the assigned resonances used as local probes of structural and dynamic changes. Two binding sites have been determined for the nucleotide substrate, ATP, the occupancies of which are dependent on Mg<sup>2+</sup> concentration. One site corresponds to the catalytic site determined crystallographically. A single binding site was found for 3-PG. This binding was shown to cause highly specific conformational changes throughout the N-domain and the interdomain region, which involve the relative movement of at least three α-helices. Investigation of 3-PG binding to several site-specific mutant forms of yeast PGK revealed a critical role for arginine 168 in the propagation of these changes. The general binding of anions to yeast PGK was investigated using the paramagnetic probes [Cr(CN)<sub>6</sub>]<sup>3-</sup> and [Fe(CN)<sub>6</sub>]<sup>3-</sup>, and the diamagnetic anion [Co(CN)<sub>6</sub>]<sup>3-</sup>. The primary anion binding site was determined from [Cr(CN)<sub>6</sub>]<sup>3-</sup> broadening data and found to share some side-chains involved in 3-PG binding, namely histidine 62 and arginine 168. Evidence for a secondary anion site was found. The anion binding data is discussed in view of the complex activation/inhibition effects of anions on the catalytic activity. Investigation of the isolated N- and C-domains showed that both can fold independently and confirmed that the C-domain is a nucleotide binding domain. It appears that the presence of the interdomain residues and/or the C-terminal peptide are necessary for 3-PG binding to the N-domain. This work shows that the specificity of the substrates is in binding, as expected, but also in the motions induced in the protein as a whole.
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