Order Of ATP Research Articles

Cleft-type imidazoliums for sensing of sulfate and polyphosphate anions with AIE emission

Two novel tetraphenylethylene (TPE) appended cleft-type imidazoliums linked through 1,2-phenyl (1) or 1,2-phenylmethyl (2) spacer were designed and synthesized for anion recognition. In aqueous solution, probe 1 can recognize SO42− and polyphosphate (PPi, ADP, and ATP) anions in the order of ATP > SO42− ~ PPi > ADP, in which the TPE-based flurophore gives a large fluorescence enhancement for increasing the aggregation. In contrast, probe 2 with a relatively flexible linker only responds toward polyphosphate anions ATP, PPi and ADP. In addition, probe 1 showed a higher anion binding affinity than that of 2, attributing to more rigid frame characteristics. Furthermore, probe 1 was also applied to image ATP and SO42− level in living cells and C. elegans.

Apyrase with anti-platelet aggregation activity from the nymph of the camel tick Hyalomma dromedarii.

Apyrase is one of the essential platelet aggregation inhibitors in hematophagous arthropods due to its ability to hydrolyze ATP and ADP molecules. Here, an apyrase (TNapyrase) with antiplatelet aggregation activity was purified and characterized from the nymphs of the camel tick Hyalomma dromedarii through anion exchange and gel filtration columns. The homogeneity of TNapyrase was confirmed by native-PAGE, SDS-PAGE as well as with isoelectric focusing. Purified TNapyrase had a molecular mass of 25kDa and a monomer structure. TNapyrase hydrolyzed various nucleotides in the order of ATP > PPi > ADP > UDP > 6GP. The Km value was 1.25mM ATP and its optimum activity reached at pH 8.4. The influence of various ions on TNapyrase activity showed that FeCl2, FeCl3 and ZnCl2 are activators of TNapyrase. EDTA inhibited TNapyrase activity competitively with a single binding site on the molecule and Ki value of 2mM. Finally, TNapyrase caused 70% inhibition of ADP-stimulated platelets aggregation and is a possible target for antibodies in future tick vaccine studies.

Crystal structure and substrate specificity of plant adenylate isopentenyltransferase from Humulus lupulus: distinctive binding affinity for purine and pyrimidine nucleotides

Cytokinins are important plant hormones, and their biosynthesis most begins with the transfer of isopentenyl group from dimethylallyl diphosphate (DMAPP) to the N6-amino group of adenine by either adenylate isopentenyltransferase (AIPT) or tRNA–IPT. Plant AIPTs use ATP/ADP as an isopentenyl acceptor and bacterial AIPTs prefer AMP, whereas tRNA–IPTs act on specific sites of tRNA. Here, we present the crystal structure of an AIPT–ATP complex from Humulus lupulus (HlAIPT), which is similar to the previous structures of Agrobacterium AIPT and yeast tRNA–IPT. The enzyme is structurally homologous to the NTP-binding kinase family of proteins but forms a solvent-accessible channel that binds to the donor substrate DMAPP, which is directed toward the acceptor substrate ATP/ADP. When measured with isothermal titration calorimetry, some nucleotides displayed different binding affinities to HlAIPT with an order of ATP > dATP ∼ ADP > GTP > CTP > UTP. Two basic residues Lys275 and Lys220 in HlAIPT interact with the β and γ-phosphate of ATP. By contrast, the interactions are absent in Agrobacterium AIPT because they are replaced by the acidic residues Asp221 and Asp171. Despite its structural similarity to the yeast tRNA–IPT, HlAIPT has evolved with a different binding strategy for adenylate.

Extracellular ATP effects on calcium signaling in cultured human non-pigmented ciliary body epithelium

Purpose. To determine the effects of extracellular ATP on calcium signaling in cultured human non-pigmented ciliary body epithelium (HNPE). Methods. Intracellular calcium (Ca i 2+) was measured using spectrofluorescence video microscopy in isolated HNPE cells loaded with the fluorescent dye Fura-2. Results. Nucleotides caused a transient oscillatory increase in Ca i 2+ with a potency order of ATP = UTP > ADP > AMP > a,ß-methylene-ATP. Treatment with thapsigargin (100 nM), an inhibitor of endoplasmic Ca 2+ -ATPase pumps, produced a sustained increase in Ca i 2+. Subsequent exposure to ATP caused a rapid reduction in Ca i 2+ and this effect was reduced by pre-exposure to vanadate and to a lesser extent in sodium free solution. Prolonged exposure to ATP in the presence of thapsigargin caused a transient spike increase in Ca i 2+ which was prevented by exposure to low extracellular Ca 2+ (1 nmol/l), verapamil, nifedipine or the microfilament disrupting agent, cytochalasin B. Conclusions. These results provide evidence for ATP mobilisation of Ca 2+ from intracellular stores via P2Y2 receptor activation in HNPE cells. ATP also primarily activates a vanadate-sensitive Ca 2+ -ATPase pump, in addition to having a smaller effect on the Na + /Ca 2+ exchanger in terminating the calcium signal. Capacitative calcium entry, possibly via an L-type Ca 2+ channel, is implicated in generating a calcium signal following emptying of intracellular stores and is sensitive to cytoskeleton disruption. ATP can thus regulate a potent intracellular signal for secretion, suggest-ing that purinergic receptors may provide a therapeutic target in glaucoma.

Difference in Structure of the Gene Encoding H+-ATPase between Streptococcus bovis and Ruminococcus albus

The full sequences of the genes encoding H+-ATPase of Streptococcus bovis and Ruminococcus albus were determined by the inverse PCR. The atp operon of S. bovis was approximately 6.5kbp and consisted of atp E (c-subunit), B (a), F (b), H (δ), A (α), G (γ), D (β), and C (e) in this order. The subunit order of atp E and B was reversed in S. bovis, when compared to the arrangement in R. albus and many other bacteria. In R. albus, two open reading frames (ORF), which potentially encode 13.7 and 14.9kDa proteins, were found in the area upstream of atp B. This area corresponds to the region of atp I (inhibitor protein) in Escherichia coli. There was no ORF that potentially encodes atp I in S. bovis. The δ-subunit signature found in many bacteria was not present in S. bovis, whereas the γ-subunit signature was not found in R. albus. The absence of the γ-subunit signature may partly explain that the H+-ATPase of R. albus is less stable and active than that of S. bovis, since this motif sequence is considered to be important for catalytic activity and the assembly of subunits. There were many differences between S. bovis and R. albus in deduced amino acid sequences of both structural and regulatory regions, but it is difficult to explain at present how the differences affect the function of H+-ATPase, or the regulation of the synthesis of the enzyme.

ATP responses in the embryo chick ciliary ganglion cells.

The ATP responses in ciliary neurons acutely dissociated from chick ciliary ganglia were investigated using the nystatin perforated patch recording mode. ATP induced a transient inward current at a holding potential of -50 mV in a concentration-dependent manner. The half-maximum effective concentration of ATP was 8.2 microM and the Hill coefficient was 0.9. The current-voltage relationship of the ATP response revealed a strong inward rectification at potentials more positive than -30 mV and the reversal potential was near 0 mV. The relative potencies of purinoceptor agonists were in the order of ATP > 2-methylthio-ATP > ADP. Neither adenosine, AMP nor alpha, beta-methylene-ATP induced any response. The ATP-induced inward current was blocked by suramin, a selective P2 purinoceptor antagonist, in a concentration-dependent manner. The half-maximum inhibitory concentration was 4.5 microM. The cytosolic Ca2+ concentration was increased by ATP and suramin inhibited the increase in a concentration-dependent manner. These results suggest that ATP operates non-selective cation channels by acting on P2y purinoceptors and has a role in the excitation of ciliary neurons.

Nucleotide binding of the C-terminal domains of the major histocompatibility complex-encoded transporter expressed in Drosophila melanogaster cells

The C-terminal domains of the mouse transporter associated with antigen processing (TAP) were expressed as soluble proteins in Drosophila melanogaster cells and labeled by [α- 32P]8-azido-ATP after UV-irradiation. The relative potencies of the nucleotides in preventing azido-ATP labeling were in the order of ATP > GTP > CTP > ITP > UTP for both the TAP1 and TAP2 C-terminal domains, suggesting ATP to be the natural substrate of the transporter. Our data provide the first evidence that the individual C-terminal domain of either TAP1 or TAP2 can be expressed as a functional ATP-binding protein.

ATP-induced inward current in neurons freshly dissociated from the tuberomammillary nucleus

1. Neurons in the tuberomammillary nucleus (TMN), which are considered to be histaminergic, were dissociated and their response to extracellularly applied ATP was investigated in the nystatin-perforated patch recording mode under voltage-clamp condition. 2. ATP induced a sustained inward current that was slowly desensitized at a holding potential of -60 mV. 3. The ATP response increased in a concentration-dependent manner. The half-maximum concentration (EC50) was 44 microM and the Hill coefficient was 1.8. 4. The potency of ATP analogues was in the order of ATP > or = 2-methylthio-ATP >> alpha, beta-methylene ATP > or = ADP. Neither adenosine nor AMP induced any response. The results suggest that the purinergic receptor in TMN neurons is P2y. 5. The current-voltage relationship for the 100 microM ATP showed a significant inward rectification at a potential more positive than -20 mV in an external solution with 150 mM Na+, but a significant rectification current was not observed in an external solution with 150 mM Cs+. The change in the reversal potential of the ATP response (EATP) to a 10-fold change of extracellular Na+ concentration was 56 mV, indicating that the ATP-induced current is highly selective for Na+ over Cl-. 6. The permeability ratio for cations was Na+:Li+:K+:Rb+: Cs+:Ca2+ = 2.16:1.36:1.68:1.54:1:2.55, indicating that the ATP-induced current is passing through the ligand-gated nonselective cation channel. 7. These results suggest that ATP has an excitatory effect on the TMN neurons by opening nonselective cation channels.

Binding kinetics of ATP gamma S35 on cultured primary tracheal surface epithelial cells.

Extracellular ATP can stimulate mucin release from primary hamster tracheal surface epithelial (HTSE) cells via a P2 purinoceptor-mediated mechanism, based on agonist potency studies of mucin release (Br. J. Pharmacol. 1991; 103:1053-1056). In the present study, we examined the kinetics of ATP binding to the surface of intact HTSE cells at 4 degrees C using ATP gamma S35 as a radioligand. We found that ATP gamma S35 bound to HTSE cells in a saturable, reversible manner, reaching an equilibrium at about 30 min. Scatchard analysis of equilibrium binding suggested the presence of two binding sites with Kd values of 0.47 and 9.4 microM. Competitive binding experiments, based on the ability of nucleotides and ATP analogs to block ATP gamma S35 revealed a rank order of ATP > ADP > alpha,beta-methylene ATP > 2-methylthio ATP > or = beta, gamma-methylene ATP. Neither AMP nor adenosine could inhibit the ATP gamma S35 binding. A comparison between the ability of nucleotides to compete with ATP gamma S35 binding and their ability to induce mucin release revealed a rather poor correlation (r2 = 0.67) with all of the above nucleotides but a good correlation (r2 = 0.96) without 2-methylthio ATP, indicating the presence of heterogenous ATP binding sites on the HTSE cell surface. UTP, a pyrimidine nucleotide, which is almost equipotent with ATP in its ability to stimulate mucin release, was much less potent than ATP in its ability to displace the ATP gamma S35 binding in these HTSE cells.

ATP-gated current in dissociated rat nucleus solitarii neurons.

1. The excitatory response of extracellularly applied ATP was investigated in freshly dissociated rat nucleus tractus solitarii neurons under whole-cell configuration using the "concentration-clamp" technique. 2. At a holding potential of -70 mV, 100 microM ATP evoked inward current that was slowly desensitized in the continuous presence of ATP. The ATP-gated current increased in a concentration-dependent manner over the concentration range between 10 microM and 1 mM. The half-maximum concentration was 31 microM and the Hill coefficient was 1.2. 3. The potency of ATP analogues for the purinergic receptor was in the order of ATP = 2-methylthio-ATP much greater than ADP greater than alpha,beta-methylene ATP. Neither adenosine nor AMP evoked any responses. The order was consistent with a P2y receptor subtype. 4. The current-voltage relationship for the 100 microM ATP response showed a clear inward rectification at positive potentials beyond -50 mV. The reversal potential of the ATP-gated current was +13 mV. 5. The time constants of activation and inactivation of the ATP-gated current solution were dependent on the extracellular ATP concentration, and both kinetics became faster at higher ATP concentrations. 6. The ATP-gated current was also elicited in an external solution containing Ca2+ as a permeable cation. The inactivation kinetics in an external solution containing 75 mM Ca2+ were faster than those in an external solution with 150 mM Na+. 7. Calculated relative permeability ratios were PNa/PCs = 1.64 ([Na+]o = 30-150 mM), PCa/PCs = 2.17 ([Ca2+]o = 2 mM). Anions were not measurably permeable in this preparation.

AN ANALYSIS OF THE ACTION OF ATP AND RELATED COMPOUNDS ON MEMBRANE CURRENT AND TENSION COMPONENTS INBULLFROG ATRIAL MUSCLE

The effect of adenosine compounds (ATP, ADP, AMP and adenosine) on membrane potential, current and contractile tension on the bullfrog atrium were studied under voltage clamped and unclamped conditions. The compounds produced immediate positive and late negative inotropic effects in unclamped conditions. The positive inotropic effect and the potency of drugs appeared less marked in the order of ATP, ADP, AMP, and adenosine. Under voltage clamped conditions, only the energy rich compounds, ATP and ADP, produced an enhancement of calcium inward current (Ica) and Ica-dependent phasic tension, while AMP and adenosine elicited a negative inotropic effect. The delayed outward current was initially depressed but later augmented epecially in case of ATP and ADP where Ica was enhanced. All adenosine compounds, however, inhibited the Ica-independent tonic tension. This effect, appearing nonspecific, was ascribable to the action of common structure of these compounds, purine-riboside moiety.

Interaction between synthetic ATP analogues and actomyosin systems. II

The following compounds were synthesized chemically as analogues of ATP; 9-(2′,3′- di-O- acetyl)- D- erythrityladenine 4′-triphosphate (erytrityladenine-TP), 9-β- D-glucopyranosyladenine 6′-triphosphate (glucosyladenine-TP), I-β- D-ribofuranosyl 4-amino-benzimidazole 5′-triphosphate (4-aminobenzimidazoleriboside-TP) and N 6-monomethylcytidine 5′-triphosphate (methyl CTP). The reactions of these analogues with myosin B or myofibrils were investigated. The strength of binding to myosin B, as judged by the change in light scattering of myosin B solution, was in the decreasing order of ATP >/ methyl CTP > glucosyladenine-TP > 4-aminobenzimidazole-riboside-TP > erythrityladenine-TP. The velocity at the steady state of P i-liberation by myosin B was in the order of methyl CTP > ATP > erthrityladenine-TP > 4-aminobenzimidazole-riboside-TP > glucosyladenine-TP in 0.6 M KCl-7 mM Ca 2+, and in the order of ATP > methyl CTP > glucosyladenine-TP > erythrityladenine-TP > >4-aminobenziimidazole-riboside-TP in 0.075 M KCl-2 mM Mg 2+, respectively. The contracting ability of the analogues for myofibrils was in the order of ATP > 4-aminobenzimidazole-riboside-TP ≈ methyl CTP > erthyrityladenine-TP. Glucosyl-adenine-TP did not cause myofibrils to contract.