inosine-triphosphate and xanthosine-5--triphosphate

Nucleotides function in a variety of biological reactions; however, they can undergo various chemical modifications. Such modified nucleotides may be toxic to cells if not eliminated from the nucleotide pools. We performed a screen for modified-nucleotide binding proteins and identified human nucleoside diphosphate linked moiety X-type motif 16 (NUDT16) protein as an inosine triphosphate (ITP)/xanthosine triphosphate (XTP)/GTP-binding protein. Recombinant NUDT16 hydrolyzes purine nucleoside diphosphates to the corresponding nucleoside monophosphates. Among 29 nucleotides examined, the highest k(cat)/K(m) values were for inosine diphosphate (IDP) and deoxyinosine diphosphate (dIDP). Moreover, NUDT16 moderately hydrolyzes (deoxy)inosine triphosphate ([d]ITP). NUDT16 is mostly localized in the nucleus, and especially in the nucleolus. Knockdown of NUDT16 in HeLa MR cells caused cell cycle arrest in S-phase, reduced cell proliferation, increased accumulation of single-strand breaks in nuclear DNA as well as increased levels of inosine in RNA. We thus concluded that NUDT16 is a (deoxy)inosine diphosphatase that may function mainly in the nucleus to protect cells from deleterious effects of (d)ITP.

Topics: Acid Anhydride Hydrolases; Amino Acid Sequence; Cell Nucleus; Cell Proliferation; DNA Breaks, Single-Stranded; Gene Knockdown Techniques; Guanosine Triphosphate; HeLa Cells; Humans; Inosine Nucleotides; Inosine Triphosphate; Molecular Sequence Data; Pyrophosphatases; Ribonucleotides

It has been shown by structural analysis that YjjX, a hypothetical protein in E. coli, is an ITPase/XTPase and suggest that it may play dual roles in prokaryotic translational regulation and oxidative cell stress response.

Topics: Bacterial Proteins; Binding Sites; Crystallography, X-Ray; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation; Inosine Triphosphate; Methanococcus; Peptide Elongation Factor Tu; Protein Binding; Protein Folding; Protein Structure, Quaternary; Protein Structure, Secondary; Ribonucleotides; Structure-Activity Relationship

Inosine triphosphate (ITP) and xanthosine triphosphate (XTP) are formed upon deamination of ATP and GTP as a result of exposure to chemical mutagens and oxidative damage. Nucleic acid synthesis requires safeguard mechanisms to minimize undesired lethal incorporation of ITP and XTP. Here, we present the crystal structure of YjjX, a protein of hitherto unknown function. The three-dimensional fold of YjjX is similar to those of Mj0226 from Methanococcus janschii, which possesses nucleotidase activity, and of Maf from Bacillus subtilis, which can bind nucleotides. Biochemical analyses of YjjX revealed it to exhibit specific phosphatase activity for inosine and xanthosine triphosphates and have a possible interaction with elongation factor Tu. The enzymatic activity of YjjX as an inosine/xanthosine triphosphatase provides evidence for a plausible protection mechanism by clearing the noncanonical nucleotides from the cell during oxidative stress in E. coli.

Topics: Adenosine Triphosphate; Antigens, Neoplasm; Bacterial Proteins; Crystallography, X-Ray; Dimerization; Escherichia coli; Escherichia coli Proteins; Guanosine Triphosphate; Inosine Triphosphatase; Inosine Triphosphate; Kinetics; Methanococcus; Mitochondrial Proteins; Models, Molecular; Peptide Elongation Factor Tu; Peptide Mapping; Protein Folding; Protein Structure, Secondary; Pyrophosphatases; Ribonucleotides; Spectrum Analysis, Raman; Static Electricity; Structure-Activity Relationship; Sulfates

The G(s)-proteins G(salpha-short) (G(salphaS)) and G(salpha-long) (G(salphaL)), and the olfactory G(s) protein (G(alphaolf)) mediate activation of adenylyl cyclase by the beta(2)-adrenoceptor (beta(2)AR). Early studies showed that the purine nucleotides GTP, ITP, and XTP differentially support receptor-mediated adenylyl cyclase activation in various native membrane systems, but those findings have remained unexplained thus far. We systematically analyzed the effects of GTP, ITP, and XTP on the coupling of the beta(2)AR to G(salphaS), G(salphaL), and G(alphaolf), respectively, using fusion proteins expressed in Sf9 insect cells. Fusion proteins ensure defined receptor/G-protein stoichiometry and efficient coupling. At all three fusion proteins, GTP, ITP, and XTP exhibited unique profiles with respect to their potency and efficacy at disrupting high-affinity agonist binding and supporting adenylyl cyclase activation by partial and full agonists. Our data can be interpreted in two ways: (i) GTP, ITP, and XTP may stabilize different active conformations in various G(s)-proteins, or (ii) GTP, ITP, and XTP may differ from one another in the kinetics of interaction with various G(s)-proteins. Regardless of which of the two explanations is correct, our present data demonstrate that GTP, ITP, and XTP are highly efficient regulators of signal transduction mediated through a specific G-protein. Also discussed is the possibility that G-protein activation by ITP and XTP may be of relevance in Lesch-Nyhan syndrome, a defect of the purine salvage pathway associated with abnormalities in various neurotransmitter systems.

Topics: Adenylyl Cyclases; Alternative Splicing; Animals; Cell Membrane; Cells, Cultured; GTP-Binding Protein alpha Subunits; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Guanosine Triphosphate; Heterotrimeric GTP-Binding Proteins; Hydrogen Bonding; Inosine Triphosphate; Insecta; Isoproterenol; Kinetics; Recombinant Fusion Proteins; Ribonucleotides; Transfection

A hypothetical 21.0 kDa protein (ORF O197) from Escherichia coli K-12 was cloned, purified, and characterized. The protein sequence of ORF O197 (termed EcO197) shares a 33.5% identity with that of a novel NTPase from Methanococcus jannaschii. The EcO197 protein was purified using Ni-NTA affinity chromatography, protease digestion, and gel filtration column. It hydrolyzed nucleoside triphosphates with an O6 atom-containing purine base to nucleoside monophosphate and pyrophosphate. The EcO197 protein had a strong preference for deoxyinosine triphosphate (dITP) and xanthosine triphosphate (XTP), while it had little activity in the standard nucleoside triphosphates (dATP, dCTP, dGTP, and dTTP). These aberrant nucleotides can be produced by oxidative deamination from purine nucleotides in cells; they are potentially mutagenic. The mutation protection mechanisms are caused by the incorporation into DNA of unwelcome nucleotides that are formed spontaneously. The EcO197 protein may function to eliminate specifically damaged purine nucleotide that contains the 6-keto group. This protein appears to be the first eubacterial dITP- and XTPhydrolyzing enzyme that has been identified.

Topics: Acid Anhydride Hydrolases; Amino Acid Sequence; Base Sequence; Cloning, Molecular; DNA, Bacterial; Escherichia coli; Escherichia coli Proteins; Hydrolysis; Inosine Triphosphate; Methanococcus; Molecular Sequence Data; Nucleoside-Triphosphatase; Ribonucleotides; Sequence Homology, Amino Acid

ITP and dITP exist in all cells. dITP is potentially mutagenic, and the levels of these nucleotides are controlled by inosine triphosphate pyrophosphatase (EC ). Here we report the cloning, expression, and characterization of a 21.5-kDa human inosine triphosphate pyrophosphatase (hITPase), an enzyme whose activity has been reported in many animal tissues and studied in populations but whose protein sequence has not been determined before. At the optimal pH of 10.0, recombinant hITPase hydrolyzed ITP, dITP, and xanthosine 5'-triphosphate to their respective monophosphates whereas activity with other nucleoside triphosphates was low. K(m) values for ITP, dITP, and xanthosine 5'-triphosphate were 0.51, 0.31, and 0.57 mm, respectively, and k(cat) values were 580, 360, and 640 s(-1), respectively. A divalent cation was absolutely required for activity. The gene encoding the hITPase cDNA sequence was localized by radiation hybrid mapping to chromosome 20p in the interval D20S113-D20S97, the same interval in which the ITPA inosine triphosphatase gene was previously localized. A BLAST search revealed the existence of many similar sequences in organisms ranging from bacteria to mammals. The function of this ubiquitous protein family is proposed to be the elimination of minor potentially mutagenic or clastogenic purine nucleoside triphosphates from the cell.

Topics: Amino Acid Sequence; Blotting, Northern; Chromatography, Gel; Chromosomes, Human, Pair 20; Cloning, Molecular; Databases, Factual; DNA, Complementary; Escherichia coli; Humans; Hydrogen-Ion Concentration; Hydrolysis; Inosine Triphosphatase; Inosine Triphosphate; Kinetics; Models, Biological; Molecular Sequence Data; Pyrophosphatases; Radiation Hybrid Mapping; Recombinant Proteins; Ribonucleotides; RNA; Sequence Homology, Amino Acid; Substrate Specificity; Tissue Distribution; Transcription, Genetic

We have determined the binding affinity for binding of the four purine nucleoside triphosphates GTP, ITP, XTP, and ATP to E-site nucleotide- and nucleoside diphosphate kinase-depleted tubulin. The relative binding affinities are 3000 for GTP, 10 for ITP, 2 for XTP, and 1 for ATP. Thus, the 2-exocyclic amino group in GTP is important in determining the nucleotide specificity of tubulin and may interact with a hydrogen bond acceptor group in the protein. The 6-oxo group also makes a contribution to the high affinity for GTP. NMR ROESY experiments indicate that the four nucleotides have different average conformations in solution. ATP and XTP are characterized by a high anti conformation, ITP by a medium anti conformation, and GTP by a low anti conformation. Possibly, the preferred solution conformation contributes to the differences in affinities. When the tubulin E-site is saturated with nucleotide, there appears to be little difference in the ability of the four nucleotides to stimulate assembly. The critical protein concentration is essentially identical in reactions using the four nucleotides. All four of the nucleotides were hydrolyzed during the assembly reaction, and the NDPs were incorporated into the microtubule. We also examined the binding of two gamma-phosphoryl-modified GTP photoaffinity analogues, p(3)-1, 4-azidoanilido-GTP and p(3)-1,3-acetylanilido-GTP. These analogues are inhibitors of the assembly reaction and bind to tubulin with affinities that are 15- and 50-fold lower, respectively, than the affinty for GTP. The affinity of GTP is less sensitive to substitutions at the gamma-phosphoryl position that to changes in the purine ring.

Topics: Adenosine Triphosphate; Guanosine Triphosphate; Inosine Triphosphate; Molecular Conformation; Nuclear Magnetic Resonance, Biomolecular; Purine Nucleotides; Ribonucleotides; Tubulin

We synthesized 27 GTP analogues with modification or substitution at positions C2, C6, C8 and ribose moiety to investigate their effect on microtubule (Mt) assembly. It was found that C2 and C6 are both functional for the analogues supporting Mt assembly. It was surprising to find that 2-amino- ATP (n2ATP) substantially supports assembly, and that the appearance of the assembled Mts was indistinguishable from those assembled in the standard GTP assembly buffer solution. Furthermore, 2-amino dATP and dGTP are even more potent than GTP in supporting assembly. The substitution of oxo group at C6 with reactive thiol largely reduced the activity of the analogue to support assembly. When free rotation of the glycosidic linkage of GTP was blocked by the introduction of sulfur atom between C8 and C2' of ribose moiety, it resulted in total suppression of assembly. Purine nucleoside triphosphate was found to support assembly better than GTP, and even more efficient was 2-amino purine nucleoside triphosphate. Interestingly, their deoxy-type analogues were totally inhibitory. Although 2-amino 8-hydroxy ATP and other analogues supported assembly much better than did GTP, their diphosphate analogues were totally incapable of supporting assembly. Finally, bulky fluorescent probes were introduced at C3' of ribose moiety (Mant-8-Br-GTP or Mant-GTP) to visualize the fluorescent signal in assembled Mts. Even in this case, the number of most protofilaments was found to be 14, consistent with that found in Mts assembled in GTP standard buffer solution.

Topics: Adenine Nucleotides; Animals; Brain; Deoxyguanine Nucleotides; Dimerization; Guanosine Diphosphate; Guanosine Triphosphate; Hydrogen Bonding; Inosine Triphosphate; Microscopy, Electron; Microtubule-Associated Proteins; Microtubules; Polymers; Protein Conformation; Purines; Ribonucleotides; Ribose; Swine; Tubulin

The aim of our study was to examine the effects of different purine nucleotides [GTP, ITP, and xanthosine 5'-triphosphate (XTP)] on receptor/G protein coupling. As a model system, we used a fusion protein of the beta(2)-adrenergic receptor and the alpha subunit of the G protein G(s). GTP was more potent and efficient than ITP and XTP at inhibiting ternary complex formation and supporting adenylyl cyclase (AC) activation. We also studied the effects of several beta(2)-adrenergic receptor ligands on nucleotide hydrolysis and on AC activity in the presence of GTP, ITP, and XTP. The efficacy of agonists at promoting GTP hydrolysis correlated well with the efficacy of agonists for stimulating AC in the presence of GTP. This was, however, not the case for ITP hydrolysis and AC activity in the presence of ITP. The efficacy of ligands at stimulating AC in the presence of XTP differed considerably from the efficacies of ligands in the presence of GTP and ITP, and there was no evidence for receptor-regulated XTP hydrolysis. Our findings support the concept of multiple ligand-specific receptor conformations and demonstrate the usefulness of purine nucleotides as tools to study conformational states of receptors.

Topics: Adenosine Triphosphate; Adenylyl Cyclases; Adrenergic beta-Agonists; Adrenergic beta-Antagonists; Animals; Cell Membrane; Cells, Cultured; GTP Phosphohydrolases; GTP-Binding Protein alpha Subunits, Gs; Guanosine Triphosphate; Hydrolysis; Inosine Triphosphatase; Inosine Triphosphate; Insecta; Isoproterenol; Kinetics; Ligands; Propanolamines; Protein Binding; Protein Conformation; Purine Nucleotides; Pyrophosphatases; Receptors, Adrenergic, beta-2; Recombinant Fusion Proteins; Ribonucleotides

G-proteins mediate signal transfer from receptors to effector systems. In their guanosine 5'-triphosphate (GTP)-bound form, G-protein alpha-subunits activate effector systems. Termination of G-protein activation is achieved by the high-affinity GTPase [E.C. 3.6.1.-] of their alpha-subunits. Like GTP, inosine 5'-triphosphate (ITP) and xanthosine 5'-triphosphate (XTP) can support effector system activation. We studied the interactions of GTP, ITP, and XTP with the retinal G-protein, transducin (TD), and with G-proteins in HL-60 leukemia cell membranes. TD hydrolyzed nucleoside 5'-triphosphates (NTPs) in the order of efficacy GTP > ITP > XTP. NTPs eluted TD from rod outer segment disk membranes in the same order of efficacy. ITP and XTP competitively inhibited TD-catalyzed GTP hydrolysis. In HL-60 membranes, the chemoattractants N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and leukotriene B4 (LTB4) effectively activated GTP and ITP hydrolysis by Gi-proteins. fMLP and LTB4 were at least 10-fold more potent activators of ITPase than of GTPase. Complement C5a effectively activated the GTPase of Gi-proteins but was only a weak stimulator of ITPase. The potency of C5a to activate GTP and ITP hydrolysis was similar. The fMLP-stimulated GTPase had a lower Km value than the fMLP-stimulated ITPase, whereas the opposite was true for the Vmax values. fMLP, C5a, and LTB4 did not stimulate XTP hydrolysis. Collectively, our data show that GTP, ITP, and XTP bind to G-proteins with different affinities, that G-proteins hydrolyze NTPs with different efficacies, and that chemoattractants stimulate GTP and ITP hydrolysis by Gi-proteins in a receptor-specific manner. On the basis of our results and the data in the literature, we put forward the hypothesis that GTP, ITP, and XTP act as differential signal amplifiers and signal sorters at the G-protein level.

Topics: Animals; Cattle; Cell Membrane; Complement C5a; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine Triphosphate; HL-60 Cells; Humans; Hydrolysis; Inosine Triphosphate; Kinetics; Leukotriene B4; Macromolecular Substances; N-Formylmethionine Leucyl-Phenylalanine; Ribonucleotides; Rod Cell Outer Segment; Substrate Specificity; Transducin

The effect of GTP analogues on catecholamine secretion and [3H]arachidonic acid release from digitonin-permeabilized adrenal chromaffin cells was examined. Several GTP analogues stimulated Ca2(+)-independent exocytosis, with the order of efficacy being XTP greater than ITP greater than guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) greater than guanosine 5'-[gamma-thio]triphosphate (GTP[S]). The stimulatory effect of the GTP analogues appeared to be due to activation of a conventional GTP-binding protein, as it was inhibited by guanosine 5'-[beta-thio]diphosphate (GDP[S]). In contrast, Ca2(+)-dependent exocytosis was only partially inhibited by high doses of GDP[S]. GTP did not stimulate Ca2(+)-independent exocytosis, but instead was found to inhibit secretion caused by micromolar Ca2+. Arachidonic acid (100 microM) also stimulated Ca2(+)-independent catecholamine secretion. Determination of the effect of GTP analogues on release of free [3H]arachidonic acid into the medium showed that it was stimulated by GTP[S] but inhibited by GTP, p[NH]ppG, ITP and XTP. The inhibition of [3H]arachidonic acid release by XTP was not prevented by GDP[S]. These results demonstrate that activation of a GTP-binding protein by certain GTP analogues can induce Ca2(+)-independent secretion in adrenal chromaffin cells and that the effect of GTP analogues on Ca2(+)-independent secretion can be dissociated from generation of arachidonic acid.

Topics: Adrenal Medulla; Animals; Arachidonic Acid; Arachidonic Acids; Calcium; Catecholamines; Cattle; Cell Membrane Permeability; Chromaffin System; Digitonin; Exocytosis; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Guanylyl Imidodiphosphate; Inosine Triphosphate; Ribonucleotides; Thionucleotides