The Experts below are selected from a list of 273 Experts worldwide ranked by ideXlab platform
Larry D. Barnes - One of the best experts on this subject based on the ideXlab platform.
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the diadenosine hexaphosphate hydrolases fromschizosaccharomyces pombe andsaccharomyces cerevisiae are homologues of the human diphosphoinositol polyphosphate phosphohydrolase overlapping substrate specificities in a mutt type protein
Journal of Biological Chemistry, 1999Co-Authors: Stephen T Safrany, Stephen W Ingram, Larry D. Barnes, Alexander G Mclennan, Jared L Cartwright, John R Falck, Stephen B ShearsAbstract:Abstract Aps1 from Schizosaccharomyces pombe(Ingram, S. W., Stratemann, S. A., and Barnes, L. D. (1999) Biochemistry 38, 3649–3655) and YOR163w fromSaccharomyces cerevisiae (Cartwright, J. L., and McLennan, A. G. (1999) J. Biol. Chem. 274, 8604–8610) have both previously been characterized as MutT family hydrolases with high specificity for diadenosine hexa- and pentaphosphates (Ap6A and Ap5A). Using purified recombinant preparations of these enzymes, we have now discovered that they have an important additional function, namely, the efficient hydrolysis of diphosphorylated inositol polyphosphates. This overlapping specificity of an enzyme for two completely different classes of substrate is not only of enzymological significance, but in addition, this finding provides important new information pertinent to the structure, function, and evolution of the MutT motif. Moreover, we report that the human protein previously characterized as a diphosphorylated inositol phosphate phosphohydrolase represents the first example, in any animal, of an enzyme that degrades Ap6A and Ap5A, in preference to other diadenosine polyphosphates. The emergence of Ap6A and Ap5A as extracellular effectors and intracellular ion-channel ligands points not only to diphosphorylated inositol phosphate phosphohydrolase as a candidate for regulating signaling by diadenosine polyphosphates, but also suggests that diphosphorylated inositol phosphates may competitively inhibit this process.
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fhit a putative tumor suppressor in humans is a dinucleoside 5 5 p1 p3 triphosphate hydrolase
Biochemistry, 1996Co-Authors: Larry D. Barnes, Preston N Garrison, Zurab Siprashvili, Stephen W Ingram, Angela K. Robinson, Andrzej Guranowski, Carlo M. Croce, Masataka Ohta, Kay HuebnerAbstract:Human Fhit (fragile histidine triad) protein, encoded by the FHIT putative tumor suppressor gene, is a typical dinucleoside 5‘,5‘ ‘‘-P1,P3-triphosphate (Ap3A) hydrolase (EC 3.6.1.29) on the basis of its enzymatic properties we report here. Ap3A is the preferred substrate among ApnA (n = 3−6), and AMP is always one of the reaction products. Mn2+ and Mg2+ are equally stimulatory, while Zn2+ is inhibitory with Ap3A as the substrate. Values of the Km for Ap3A and Ap4A are 1.3 and 4.6 μM, respectively. Values of the specificity constant, kcat/Km, for Ap3A and Ap4A are 2.0 × 106 and 6.7 × 103 s-1 M-1, respectively, for a glutathione S-transferase (GST)−Fhit fusion protein. Site-directed mutagenesis of FHIT demonstrated that all four conserved histidines are required for full activity, and the central histidine of the triad is absolutely essential for Ap3A hydrolase activity. This putative tumor suppressor is the first evidence for a connection between dinucleotide oligophosphate metabolism and tumorigenesis. Al...
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cloning of the schizosaccharomyces pombe gene encoding diadenosine 5 5 p1 p4 tetraphosphate Ap4A asymmetrical hydrolase sequence similarity with the histidine triad hit protein family
Biochemical Journal, 1995Co-Authors: Y Huang, P N Garrison, Larry D. BarnesAbstract:Abstract Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) asymmetric hydrolase (EC 3.6.1.17) is a specific catabolic enzyme of Ap4A found in Schizosaccharomyces pombe. We have previously described the partial purification of Ap4A hydrolase from S. pombe [Robinson, de la Pena and Barnes (1993) Biochim. Biophys. Acta 1161, 139-148]. We determined the sequence of the N-terminal 20 amino acids of Ap4A hydrolase and designed two degenerate PCR primers based on the sequence. The 60 bp DNA fragment obtained by PCR, which is specific to Ap4A hydrolase, was used to isolate the Ap4A hydrolase gene, aph1, from S. pombe by screening a genomic DNA library in a multicopy plasmid. Ap4A hydrolase activity from the crude supernatant of a positive S. pombe transformant was about 25-fold higher than the control. There was no detectable stimulation of enzymic activity by phosphate. The aph1 gene from S. pombe contains three introns. The intron boundaries were confirmed by sequencing the cDNA of the aph1 gene from a S. pombe cDNA library. The deduced open reading frame of the aph1 gene codes for 182 amino acids. Two regions of significant local similarity were identified between the Ap4A hydrolase and the histidine triad (HIT) protein family [Seraphin (1992) DNA Sequence 3, 177-179]. HIT proteins are present in prokaryotes, yeast, plants and mammals. Their functions are unknown, except that the bovine protein inhibits protein kinase C in vitro. All four histidine residues which are conserved among the HIT proteins, including the HxHxH putative Zn(2+)-binding motif, are conserved in the Ap4A hydrolase. In addition, there are two regions of similarity between the Ap4A phosphorylases I and II from Saccharomyces cerevisiae and Ap4A hydrolase from S. pombe. These regions overlap with the HIT protein similarity regions. The aph1 gene from S. pombe is the first asymmetrical Ap4A hydrolase gene to be cloned and sequenced.
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isolation and characterization of diadenosine tetraphosphate Ap4A hydrolase from schizosaccharomyces pombe
Biochimica et Biophysica Acta, 1993Co-Authors: Angela K. Robinson, Carlos E De La Pena, Larry D. BarnesAbstract:An enzyme that catalyzes the asymmetric hydrolysis of Ap4A has been partially purified from the fission yeast, Schizosaccharomyces pombe. The crude supernatant fraction from log-phase cells was fractionated by (NH4)2SO4 precipitation followed by chromatography on DEAE-cellulose, Red A dye-ligand and QAE-Sepharose resins. Two peaks of Ap4A hydrolase activity, designated major and minor, were separated on the Red A dye-ligand resin. Both the major and minor Ap4A hydrolase have an apparent molecular mass of 49 kDa based on gel filtration chromatography. On a SDS polyacrylamide gel, a protein of 22 kDa exhibited Ap4A hydrolase activity. Both forms of the enzyme have a Km value in the range of 22 to 36 microM for Ap4A. Both forms of the enzyme asymmetrically hydrolyze Ap4A to AMP and ATP as determined by HPLC. Ap4A is the optimal substrate among several nucleotides and dinucleoside polyphosphates tested at 10 microM. A divalent metal cation is required for activity. Concentrations of Pi below 30 mM stimulate Ap4A hydrolase while higher concentrations inhibit the activity. Pi is not a substrate for this Ap4A-degradative enzyme. Fluoride, from 50 microM to 20 mM, has no significant effect on Ap4A hydrolase activity.
Andrew D Miller - One of the best experts on this subject based on the ideXlab platform.
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biotin c10 appch2ppa is an effective new chemical proteomics probe for diadenosine polyphosphate binding proteins
Bioorganic & Medicinal Chemistry Letters, 2014Co-Authors: Ameruddin M Azhar, Ahmed Kamal, Andrew D Miller, Michael Wright, Judith NagyAbstract:Here we report on the synthesis of a synthetic, stable biotin-c10-AppCH2ppA conjugate involving an unusual Cannizzaro reaction step. This conjugate is used to bind prospective Ap4A binding proteins from Escherichia coli bacterial cell lyzates. Following binding, identities of these proteins are then determined smoothly by a process of magnetic bio-panning and electrospray mass spectrometry. Protein hits appear to be a definitive set of stress protein related targets. While this hit list may not be exclusive, and may vary with the nature of sampling conditions and organism status, nevertheless hits do appear to correspond with bona fide Ap4A-binding proteins. Therefore these hits represent a sound basis on which to construct new hypotheses concerning the cellular importance of Ap4A to bacterial cells and the potential biological significance of Ap4A-protein binding interactions.
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diadenosine polyphosphate analog controls postsynaptic excitation in ca3 ca1 synapses via a nitric oxide dependent mechanism
Journal of Pharmacology and Experimental Therapeutics, 2006Co-Authors: Sergei Melnik, Mike Wright, Andrew D Miller, J A Tanner, Timur Tsintsadze, Vera Tsintsadze, Natalia LozovayaAbstract:Previously, we have described the modulatory effect of diadenosine polyphosphates Ap4A and Ap5A on synaptic transmission in the rat hippocampal slices mediated by presynaptic receptors ([Klishin et al., 1994][1]). In contrast, we now describe how nonhydrolyzable Ap4A analog diadenosine-5′,5′′′- P 1, P 4-[β,β′-methylene]tetraphosphate (AppCH2ppA) at low micromolar concentrations exerts strong nondesensitizing inhibition of orthodromically evoked field potentials (OFPs) without affecting the amplitude of excitatory postsynaptic currents and antidromically evoked field potentials, as recorded in hippocampal CA1 zone. The effects of AppCH2ppA on OFPs are eliminated by a P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) but not mimicked by purinoceptor agonists α,β-methylene-ATP and adenosine 5′- O -(3-thio)-triphosphate, indicating that a P2-like receptor is involved but not one belonging to the conventional P2X/P2Y receptor classes. Diadenosine polyphosphate receptor (P4) antagonist Ip4I (diinosine tetraphosphate) was unable to modulate AppCH2ppA effects. Thus, the PPADS-sensitive P2-like receptor for AppCH2ppA seems to control selectively dendritic excitation of the CA1 neurons. The specific nitric oxide (NO)-scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide is shown to significantly attenuate AppCH2ppA-mediated inhibitory effects, indicating that NO is involved in the cascade of events initiated by AppCH2ppA. Further downstream mediation by adenosine A1 receptors is also demonstrated. Hence, AppCH2ppA-mediated effects involve PPADS-sensitive P2-like receptor activation leading to the production of NO that stimulates intracellular synthesis of adenosine, causing in turn postsynaptic A1 receptor activation and subsequent postsynaptic CA1 dendritic inhibition. Such spatially selective postsynaptic dendritic inhibition may influence dendritic electrogenesis in pyramidal neurons and consequently mediate control of neuronal network activity. [1]: #ref-42
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the duality of lysu a catalyst for both Ap4A and ap3a formation
FEBS Journal, 2006Co-Authors: Mike Wright, J A Tanner, Nonlawat Boonyalai, Alison D Hindley, Andrew D MillerAbstract:Heat shock inducible lysyl-tRNA synthetase of Escherichia coli (LysU) is known to be a highly efficient diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) synthase. However, we use an ion-exchange HPLC technique to demonstrate that active LysU mixtures actually have a dual catalytic activity, initially producing Ap4A from ATP, before converting that tetraphosphate to a triphosphate. LysU appears to be an effective diadenosine 5',5'''-P1,P3-triphosphate (Ap3A) synthase. Mechanistic investigations reveal that Ap3A formation requires: (a) that the second step of Ap4A formation is slightly reversible, thereby leading to a modest reappearance of adenylate intermediate; and (b) that phosphate is present to trap the intermediate (either as inorganic phosphate, as added ADP, or as ADP generated in situ from inorganic phosphate). Ap3A forms readily from Ap4A in the presence of such phosphate-based adenylate traps (via a 'reverse-trap' mechanism). LysU is also clearly demonstrated to exist in a phosphorylated state that is more physically robust as a catalyst of Ap4A formation than the nonphosphorylated state. However, phosphorylated LysU shows only marginally improved catalytic efficiency. We note that Ap3A effects have barely been studied in prokaryotic organisms. By contrast, there is a body of literature that describes Ap3A and Ap4A having substantially different functions in eukaryotic cells. Our data suggest that Ap3A and Ap4A biosynthesis could be linked together through a single prokaryotic dual 'synthase' enzyme. Therefore, in our view there is a need for new research into the effects and impact of Ap3A alone and the intracellular [Ap3A]/[Ap4A] ratio on prokaryotic organisms.
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novel fluorescent labelled affinity probes for diadenosine 5 5 p1 p4 tetraphosphate Ap4A binding studies
Bioorganic & Medicinal Chemistry Letters, 2006Co-Authors: Mike Wright, Andrew D MillerAbstract:Tandem synthetic-biosynthetic procedures were used to prepare two novel fluorescent labelled affinity probes for diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A)-binding studies. These compounds (dial-mant-Ap4A and azido-mant-Ap4A) are shown to clearly distinguish known Ap4A-binding proteins from Escherichia coli (LysU and GroEL) and a variety of other control proteins. Successful labelling of chaperonin GroEL appears to be allosteric with respect to the well-characterized adenosine 5'-triphosphate (ATP)-binding site, suggesting that GroEL possesses a distinct Ap4A-binding site.
Alexander G Mclennan - One of the best experts on this subject based on the ideXlab platform.
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re evaluation of diadenosine tetraphosphate Ap4A from a stress metabolite to bona fide secondary messenger
Frontiers in Molecular Biosciences, 2020Co-Authors: Freya Ferguson, Alexander G Mclennan, Nigel J Jones, Michael D Urbaniak, Nikki A CopelandAbstract:Cellular homeostasis requires adaption to environmental stress. In response to various environmental and genotoxic stresses, all cells produce dinucleoside polyphosphates (NpnNs), the best studied of which is diadenosine tetraphosphate (Ap4A). Despite intensive investigation, the precise biological roles of these molecules have remained elusive. However, recent studies have elucidated distinct and specific signaling mechanisms for these nucleotides in prokaryotes and eukaryotes. This review summarizes these key discoveries and describes the mechanisms of Ap4A and Ap4N synthesis, the mediators of the cellular responses to increased intracellular levels of these molecules and the hydrolytic mechanisms required to maintain low levels in the absence of stress. The intracellular responses to dinucleotide accumulation are evaluated in the context of the "friend" and "foe" scenarios. The "friend (or alarmone) hypothesis" suggests that ApnN act as bona fide secondary messengers mediating responses to stress. In contrast, the "foe" hypothesis proposes that ApnN and other NpnN are produced by non-canonical enzymatic synthesis as a result of physiological and environmental stress in critically damaged cells but do not actively regulate mitigating signaling pathways. In addition, we will discuss potential target proteins, and critically assess new evidence supporting roles for ApnN in the regulation of gene expression, immune responses, DNA replication and DNA repair. The recent advances in the field have generated great interest as they have for the first time revealed some of the molecular mechanisms that mediate cellular responses to ApnN. Finally, areas for future research are discussed with possible but unproven roles for intracellular ApnN to encourage further research into the signaling networks that are regulated by these nucleotides.
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nudt2 disruption elevates diadenosine tetraphosphate Ap4A and down regulates immune response and cancer promotion genes
PLOS ONE, 2016Co-Authors: Andrew S Marriott, Alexander G Mclennan, Nikki A Copeland, Olga Vasieva, Yongxiang Fang, Nigel J JonesAbstract:Regulation of gene expression is one of several roles proposed for the stress-induced nucleotide diadenosine tetraphosphate (Ap4A). We have examined this directly by a comparative RNA-Seq analysis of KBM-7 chronic myelogenous leukemia cells and KBM-7 cells in which the NUDT2 Ap4A hydrolase gene had been disrupted (NuKO cells), causing a 175-fold increase in intracellular Ap4A. 6,288 differentially expressed genes were identified with P < 0.05. Of these, 980 were up-regulated and 705 down-regulated in NuKO cells with a fold-change ≥ 2. Ingenuity® Pathway Analysis (IPA®) was used to assign these genes to known canonical pathways and functional networks. Pathways associated with interferon responses, pattern recognition receptors and inflammation scored highly in the down-regulated set of genes while functions associated with MHC class II antigens were prominent among the up-regulated genes, which otherwise showed little organization into major functional gene sets. Tryptophan catabolism was also strongly down-regulated as were numerous genes known to be involved in tumor promotion in other systems, with roles in the epithelial-mesenchymal transition, proliferation, invasion and metastasis. Conversely, some pro-apoptotic genes were up-regulated. Major upstream factors predicted by IPA® for gene down-regulation included NFκB, STAT1/2, IRF3/4 and SP1 but no major factors controlling gene up-regulation were identified. Potential mechanisms for gene regulation mediated by Ap4A and/or NUDT2 disruption include binding of Ap4A to the HINT1 co-repressor, autocrine activation of purinoceptors by Ap4A, chromatin remodeling, effects of NUDT2 loss on transcript stability, and inhibition of ATP-dependent regulatory factors such as protein kinases by Ap4A. Existing evidence favors the last of these as the most probable mechanism. Regardless, our results suggest that the NUDT2 protein could be a novel cancer chemotherapeutic target, with its inhibition potentially exerting strong anti-tumor effects via multiple pathways involving metastasis, invasion, immunosuppression and apoptosis.
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diadenosine 5 5 p1 p4 tetraphosphate Ap4A is synthesized in response to dna damage and inhibits the initiation of dna replication
DNA Repair, 2015Co-Authors: Andrew S Marriott, Alexander G Mclennan, Nikki A Copeland, Ryan Cunningham, Mark C Wilkinson, Nigel J JonesAbstract:The level of intracellular diadenosine 5', 5'''-P(1),P(4)-tetraphosphate (Ap4A) increases several fold in mammalian cells treated with non-cytotoxic doses of interstrand DNA-crosslinking agents such as mitomycin C. It is also increased in cells lacking DNA repair proteins including XRCC1, PARP1, APTX and FANCG, while >50-fold increases (up to around 25 μM) are achieved in repair mutants exposed to mitomycin C. Part of this induced Ap4A is converted into novel derivatives, identified as mono- and di-ADP-ribosylated Ap4A. Gene knockout experiments suggest that DNA ligase III is primarily responsible for the synthesis of damage-induced Ap4A and that PARP1 and PARP2 can both catalyze its ADP-ribosylation. Degradative proteins such as aprataxin may also contribute to the increase. Using a cell-free replication system, Ap4A was found to cause a marked inhibition of the initiation of DNA replicons, while elongation was unaffected. Maximum inhibition of 70-80% was achieved with 20 μM Ap4A. Ap3A, Ap5A, Gp4G and ADP-ribosylated Ap4A were without effect. It is proposed that Ap4A acts as an important inducible ligand in the DNA damage response to prevent the replication of damaged DNA.
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Ap4A induces apoptosis in human cultured cells
FEBS Letters, 1999Co-Authors: Amalia Vartanian, Alexander G Mclennan, Ivan Alexandrov, Igor Prudowski, L L KisselevAbstract:Diadenosine oligophosphates (ApnA) have been proposed as intracellular and extracellular signaling molecules in animal cells. The ratio of diadenosine 5′,5‴-P1,P3-triphosphate to diadenosine 5′,5‴-P1,P4-tetraphosphate (Ap3A/Ap4A) is sensitive to the cellular status and alters when cultured cells undergo differentiation or are treated with interferons. In cells undergoing apoptosis induced by DNA topoisomerase II inhibitor VP16, the concentration of Ap3A decreases significantly while that of Ap4A increases. Here, we have examined the effects of exogenously added Ap3A and Ap4A on apoptosis and morphological differentiation. Penetration of ApnA into cells was achieved by cold shock. Ap4A at 10 μM induced programmed cell death in human HL60, U937 and Jurkat cells and mouse VMRO cells and this effect appeared to require Ap4A breakdown as hydrolysis-resistant analogues of Ap4A were inactive. On its own, Ap3A induced neither apoptosis nor cell differentiation but did display strong synergism with the protein kinase C activators 12-deoxyphorbol-13-O-phenylacetate and 12-deoxyphorbol-13-O-phenylacetate-20-acetate in inducing differentiation of HL60 cells. We propose that Ap4A and Ap3A are physiological antagonists in determination of the cellular status: Ap4A induces apoptosis whereas Ap3A is a co-inductor of differentiation. In both cases, the mechanism of signal transduction remains unknown.
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the diadenosine hexaphosphate hydrolases fromschizosaccharomyces pombe andsaccharomyces cerevisiae are homologues of the human diphosphoinositol polyphosphate phosphohydrolase overlapping substrate specificities in a mutt type protein
Journal of Biological Chemistry, 1999Co-Authors: Stephen T Safrany, Stephen W Ingram, Larry D. Barnes, Alexander G Mclennan, Jared L Cartwright, John R Falck, Stephen B ShearsAbstract:Abstract Aps1 from Schizosaccharomyces pombe(Ingram, S. W., Stratemann, S. A., and Barnes, L. D. (1999) Biochemistry 38, 3649–3655) and YOR163w fromSaccharomyces cerevisiae (Cartwright, J. L., and McLennan, A. G. (1999) J. Biol. Chem. 274, 8604–8610) have both previously been characterized as MutT family hydrolases with high specificity for diadenosine hexa- and pentaphosphates (Ap6A and Ap5A). Using purified recombinant preparations of these enzymes, we have now discovered that they have an important additional function, namely, the efficient hydrolysis of diphosphorylated inositol polyphosphates. This overlapping specificity of an enzyme for two completely different classes of substrate is not only of enzymological significance, but in addition, this finding provides important new information pertinent to the structure, function, and evolution of the MutT motif. Moreover, we report that the human protein previously characterized as a diphosphorylated inositol phosphate phosphohydrolase represents the first example, in any animal, of an enzyme that degrades Ap6A and Ap5A, in preference to other diadenosine polyphosphates. The emergence of Ap6A and Ap5A as extracellular effectors and intracellular ion-channel ligands points not only to diphosphorylated inositol phosphate phosphohydrolase as a candidate for regulating signaling by diadenosine polyphosphates, but also suggests that diphosphorylated inositol phosphates may competitively inhibit this process.
Andrzej Guranowski - One of the best experts on this subject based on the ideXlab platform.
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diadenosine polyphosphates ap3a and Ap4A behave as alarmones triggering the synthesis of enzymes of the phenylpropanoid pathway in arabidopsis thaliana
FEBS Open Bio, 2011Co-Authors: Malgorzata Pietrowskaborek, Katarzyna Nuc, Malgorzata Zielezinska, Andrzej GuranowskiAbstract:It is known that cells under stress accumulate various dinucleoside polyphosphates, compounds suggested to function as alarmones. In plants, the phenylpropanoid pathways yield metabolites protecting these organisms against various types of stress. Observations reported in this communication link these two phenomena and provide an example of a metabolic “addressee” for an “alarm” signaled by diadenosine triphosphate (Ap3A) or diadenosine tetraphosphate (Ap4A). In response to added Ap3A or Ap4A, seedlings of Arabidopsis thaliana incubated in full nutrition medium increased both the expression of the genes for and the specific activity of phenylalanine ammonia-lyase and 4-coumarate:coenzyme A ligase, enzymes that control the beginning of the phenylpropanoid pathway. Neither adenine mononucleotides (AMP, ADP or ATP) nor adenosine evoked such effects. Reactions catalyzed in vitro by these enzymes were not affected by Ap3A or Ap4A.
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novel diadenosine polyphosphate analogs with oxymethylene bridges replacing oxygen in the polyphosphate chain potential substrates and or inhibitors of Ap4A hydrolases
FEBS Journal, 2009Co-Authors: Andrzej Guranowski, Elzbieta Starzynska, Malgorzata Pietrowskaborek, Dominik Rejman, George M BlackburnAbstract:Dinucleoside polyphosphates (NpnN′s; where N and N′ are nucleosides and n = 3–6 phosphate residues) are naturally occurring compounds that may act as signaling molecules. One of the most successful approaches to understand their biological functions has been through the use of NpnN′ analogs. Here, we present the results of studies using novel diadenosine polyphosphate analogs, with an oxymethylene group replacing one or two bridging oxygen(s) in the polyphosphate chain. These have been tested as potential substrates and/or inhibitors of the symmetrically acting Ap4A hydrolase [bis(5′-nucleosyl)-tetraphosphatase (symmetrical); EC 3.6.1.41] from E. coli and of two asymmetrically acting Ap4A hydrolases [bis(5′-nucleosyl)-tetraphosphatase (asymmetrical); EC 3.6.1.17] from humans and narrow-leaved lupin. The six chemically synthesized analogs were: ApCH2OpOCH2pA (1), ApOCH2pCH2OpA (2), ApOpCH2OpOpA (3), ApCH2OpOpOCH2pA (4), ApOCH2pOpCH2OpA (5) and ApOpOCH2pCH2OpOpA (6). The eukaryotic asymmetrical Ap4A hydrolases degrade two compounds, 3 and 5, as anticipated in their design. Analog 3 was cleaved to AMP (pA) and β,γ-methyleneoxy-ATP (pOCH2pOpA), whereas hydrolysis of analog 5 gave two molecules of α,β-oxymethylene ADP (pCH2OpA). The relative rates of hydrolysis of these analogs were estimated. Some of the novel nucleotides were moderately good inhibitors of the asymmetrical hydrolases, having Ki values within the range of the Km for Ap4A. By contrast, none of the six analogs were good substrates or inhibitors of the bacterial symmetrical Ap4A hydrolase.
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metabolism of diadenosine tetraphosphate Ap4A and related nucleotides in plants review with historical and general perspective
Frontiers in Bioscience, 2004Co-Authors: Andrzej GuranowskiAbstract:This review presents our knowledge of potential biochemical conversions of minor mononucleotides, such as adenosine-5'-tetraphosphate (p4A) and adenosine-5'-pentaphosphate (p5A), and dinucleotides, such as diadenosine-5',5"'-P1,P3-triphosphate (Ap3A) and diadenosine-5',5"'-P1,P4-tetraphosphate (Ap4A), in plants. Although the occurrence of p4A, Ap3A and/or Ap4A has been demonstrated in various bacteria, fungi and animals, identification of these compounds in plants has not been reported as yet. However, the ubiquity of both the compounds and enzymes that can synthesize them (certain ligases and transferases), the demonstration that certain plant ligases can synthesize pnAs and ApnNs in vitro, and the existence in plants of specific and nonspecific degradative enzymes strongly suggest that these various pnNs and NpnN's do indeed occur and play a biological role in plant cells. In fact, some of the plant enzymes involved in the synthesis and degradation of these minor mono- and dinucleotides have been studied even more thoroughly than their counterparts from other organisms.
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analogs of diadenosine tetraphosphate Ap4A
Acta Biochimica Polonica, 2003Co-Authors: Andrzej GuranowskiAbstract:This review summarizes our knowledge of analogs and derivatives of diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A), the most extensively studied member of the dinucleoside 5',5"'-P1,Pn-polyphosphate (NpnN) family. After a short discussion of enzymes that may be responsible for the accumulation and degradation of Np4)N's in the cell, this review focuses on chemically and/or enzymatically produced analogs and their practical applications. Particular attention is paid to compounds that have aided the study of enzymes involved in the metabolism of Ap4A (Np4N'). Certain Ap4A analogs were alternative substrates of Ap4A-degrading enzymes and/or acted as enzyme inhibitors, some other helped to establish enzyme mechanisms, increased the sensitivity of certain enzyme assays or produced stable enzyme:ligand complexes for structural analysis.
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fhit a putative tumor suppressor in humans is a dinucleoside 5 5 p1 p3 triphosphate hydrolase
Biochemistry, 1996Co-Authors: Larry D. Barnes, Preston N Garrison, Zurab Siprashvili, Stephen W Ingram, Angela K. Robinson, Andrzej Guranowski, Carlo M. Croce, Masataka Ohta, Kay HuebnerAbstract:Human Fhit (fragile histidine triad) protein, encoded by the FHIT putative tumor suppressor gene, is a typical dinucleoside 5‘,5‘ ‘‘-P1,P3-triphosphate (Ap3A) hydrolase (EC 3.6.1.29) on the basis of its enzymatic properties we report here. Ap3A is the preferred substrate among ApnA (n = 3−6), and AMP is always one of the reaction products. Mn2+ and Mg2+ are equally stimulatory, while Zn2+ is inhibitory with Ap3A as the substrate. Values of the Km for Ap3A and Ap4A are 1.3 and 4.6 μM, respectively. Values of the specificity constant, kcat/Km, for Ap3A and Ap4A are 2.0 × 106 and 6.7 × 103 s-1 M-1, respectively, for a glutathione S-transferase (GST)−Fhit fusion protein. Site-directed mutagenesis of FHIT demonstrated that all four conserved histidines are required for full activity, and the central histidine of the triad is absolutely essential for Ap3A hydrolase activity. This putative tumor suppressor is the first evidence for a connection between dinucleotide oligophosphate metabolism and tumorigenesis. Al...
Maria Teresa Mirasportugal - One of the best experts on this subject based on the ideXlab platform.
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suramin a powerful inhibitor of neural ecto diadenosine polyphosphate hydrolase
British Journal of Pharmacology, 1996Co-Authors: Jesús Mateo, Pedro Rotllan, Maria Teresa MirasportugalAbstract:The neural ecto-diadenosine polyphosphate hydrolase (ecto-ApnAase) from plasma membranes of Torpedo synaptic terminals is inhibited by suramin. This study was carried out by discontinuous h.p.l.c. and continuous fluorometric methods. The concentration-dependence studies showed a non-competitive mechanism for suramin in the Dixon plot, with a Ki value of 1.79 +/- 0.03 microM with respect to epsilon-(Ap3A) as the substrate and 1.69 +/- 0.05 microM and 1.86 +/- 0.06 microM for epsilon-(Ap4A) and epsilon-(Ap5A) respectively. These results indicate that suramin could be a base compound inhibiting ecto-ApnAase and providing an alternative way of studying the pharmacology of diadenosine polyphosphate receptors.
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the diadenosine polyphosphate receptors p2d purinoceptors
Ciba Foundation Symposium 198 - P2 Purinoceptors: Localization Function and Transduction Mechanisms, 1996Co-Authors: Maria Teresa Mirasportugal, Jesús Mateo, Enrique Castro, Jesus PintorAbstract:Diadenosine polyphosphates-Ap4A, Ap5A and Ap6A-are co-stored in neurosecretory vesicles together with ATP and aminergic compounds. They are released from neural cells and synaptic terminals in a Ca(2+)-dependent process. Ligand binding and displacement experiments carried out with [3H]Ap4A on isolated chromaffin cells and synaptosomal preparations result in curvilinear Scatchard plots with Kd values close to 0.1 nM for the high-affinity binding sites. Displacement curves with two steps are obtained for homologous and heterologous nucleotide ligands; the lowest-affinity step exhibits Ki values in the micromolar range for ApnA compounds. The high-affinity binding sites were named P2D purinoceptors on the basis of their binding characteristics. Single-cell studies in neurochromaffin cells indicate the presence of P2X purinoceptors in noradrenergic cells that do not respond to Ap4A and in which noradrenaline secretion can be induced by influx of extracellular Ca2+. P2Y receptors that respond to ATP analogues and ApnAs are present in endothelial cells from adrenal medulla. Those cells that express P2U purinoceptors are unresponsive to ApnAs. Ectodiadenosine polyphosphate hydrolases with Km values of 0.3 to 2 microM are present in both neural and endothelial cells from adrenal medulla. In midbrain synaptic terminals diadenosine polyphosphates induce Ca2+ entry from the extracellular medium. The fact that the synaptic response is not cross-desensitized by ATP and its non-hydrolysable analogues, the non-blocking effect of suramin, and the differential effect of Ca2+ channel blockers, together suggest that there are different receptors for nucleotides and dinucleotides in rat brain synaptosomes, which we have called P4 purinoceptors on the basis of functional studies.
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ca2 stores mobilization by diadenosine tetraphosphate Ap4A through a putative p2y purinoceptor in adrenal chromaffin cells
British Journal of Pharmacology, 1992Co-Authors: Enrique Castro, Jesus Pintor, Maria Teresa MirasportugalAbstract:Abstract 1. Diadenosine tetraphosphate (Ap4A) evoked a concentration-dependent increase in cytosolic [Ca2+] in resting chromaffin cells. The EC50 value for this action was 28.2 +/- 6.6 microM. This effect was also produced by diadenosine pentaphosphate (Ap5A) with an EC50 of 50 +/- 7 microM. 2. In contrast with this effect, pretreatment with Ap4A or Ap5A induced a 30% reduction in Ca2+ entry following 10 microM dimethylphenylpiperazinium. 3. The elevation in cytosolic [Ca2+] induced by Ap4A was persistent in approximately 100 nM external [Ca2+] and was sensitive to depletion of internal Ca2+ stores by a bradykinin prepulse or whole cell depletion in Ca2+. 4. The effect of Ap4A was mimicked and desensitized by the agonist adenosine 5'-O-(2-thiodiphosphate), and blocked by the P2Y-receptor antagonist, cibachrome blue. The P2X-receptor agonist alpha,beta-methylene adenosine 5'-triphosphate was inactive both by itself or in combination with Ap4A. This is compatible with a P2Y-purinoceptor-mediated action.
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di 1 n6 ethenoadenosine 5 5 p1 p4 tetraphosphate a fluorescent enzymatically active derivative of Ap4A
FEBS Letters, 1991Co-Authors: P Rotilan, Magdalena Torres, Jesus Pintor, A Ramos, Maria Teresa MirasportugalAbstract:Di(1,N6-ethenoadenosine) 5′, 5‴-P1, P4-tetraphosphate, ϵ-(Ap4A), a fluorescent analog of Ap4A has been synthesized by reaction of 2-chloroacetaldehyde with Ap4A. At neutral pH this Ap4A analog presents characteristic maxima at 265 and 274 nm, shoulders at ca 260 and 310 nm and moderate fluorescence (λexc 307 nm, λem 410 nm). Enzymatic hydrolysis of the phosphate backbone produced a slight hyperchromic effect but a notorious increase of the fluorescence emission. Cytosolic extracts from adrenochromaffin tissue as well as cultured chromaffin cells were able to split ϵ(Ap4A) and catabolize the resulting ϵ-nucleotide moieties up to ϵ-Ado.