guanosine-triphosphate and deoxyguanosine-triphosphate

Topics: Antimetabolites, Antineoplastic; Deoxyguanine Nucleotides; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Inosine Monophosphate; Leukemia; Neoplasms; Purines; Ribavirin; Signal Transduction

PNP, encoded by 6 exons on human chromosome 14q13, is a homotrimetic enzyme of approximately 96 k dalton. This enzyme reversibly catalyzes the phosphorolysis of purine nucleoside to their respective purine bases and the corresponding pentose-1-phosphate. The ultimate degradative product of purine nucleoside is uric acid, which is a depleted product with the absence of PNP activity. This leads to accumulation of deoxy-guanosine triphosphate, which inhibits the enzyme ribonucleoside reductase resulting in DNA synthesis block and T cell proliferation. Basic and brief knowledge of PNP biochemistry and functional physiology is summarized for the understanding of clinical features of PNP deficiency.

Topics: Animals; Deoxyguanine Nucleotides; DNA; Guanosine Triphosphate; Humans; Lymphocyte Activation; Purine Nucleosides; Purine-Nucleoside Phosphorylase; T-Lymphocytes

Here, we reported a spontaneous reaction between anticancer drug doxorubicin and GTP or dGTP. Incubation of doxorubicin with GTP or dGTP at 37 °C or above yields a covalent product: the doxorubicin-GTP or -dGTP conjugate where a covalent bond is formed between the C14 position of doxorubicin and the 2-amino group of guanine. Density functional theory calculations show the feasibility of this spontaneous reaction. Fluorescence imaging studies demonstrate that the doxorubicin-GTP and -dGTP conjugates cannot enter nuclei although they rapidly accumulate in human SK-OV-3 and NCI/ADR-RES cells. Consequently, the doxorubicin-GTP and -dGTP conjugates are less cytotoxic than doxorubicin. We also demonstrate that doxorubicin binds to ATP, GTP, and other nucleotides with a dissociation constant (

Topics: Adenosine Triphosphate; Antineoplastic Agents; Deoxyguanine Nucleotides; Doxorubicin; Guanosine Triphosphate; Humans

The substrate specificity of recombinant full-length diguanylate cyclase (DGC) of Thermotoga maritima with mutant allosteric site was investigated. It has been originally shown that the enzyme could use GTP closest analogues - 2'-deoxyguanosine-5'-triphosphate (dGTP) and 9-β-D-arabinofuranosyl-guanine-5'-triphosphate (araGTP) as the substrates. The first demonstrations of an enzymatic synthesis of bis-(3'-5')-cyclic dimeric deoxyguanosine monophosphate (c-di-dGMP) and the previously unknown bis-(3'-5')-cyclic dimeric araguanosine monophosphate (c-di-araGMP) using DGC of T. maritima in the form of inclusion bodies have been provided.

Topics: Arabinonucleotides; Bacterial Proteins; Cyclic GMP; Deoxyguanine Nucleotides; Escherichia coli Proteins; Guanosine Triphosphate; Phosphorus-Oxygen Lyases; Thermotoga maritima

Cytosolic nucleotidase II (cN-II) from Legionella pneumophila (Lp) catalyzes the hydrolysis of GMP and dGMP displaying sigmoidal curves, whereas catalysis of IMP hydrolysis displayed a biphasic curve in the initial rate versus substrate concentration plots. Allosteric modulators of mammalian cN-II did not activate LpcN-II although GTP, GDP and the substrate GMP were specific activators. Crystal structures of the tetrameric LpcN-II revealed an activator-binding site at the dimer interface. A double mutation in this allosteric-binding site abolished activation, confirming the structural observations. The substrate GMP acting as an activator, partitioning between the allosteric and active site, is the basis for the sigmoidicity of the initial velocity versus GMP concentration plot. The LpcN-II tetramer showed differences in subunit organization upon activator binding that are absent in the activator-bound human cN-II structure. This is the first observation of a structural change induced by activator binding in cN-II that may be the molecular mechanism for enzyme activation.. The coordinates and structure factors reported in this paper have been submitted to the Protein Data Bank under the accession numbers 2BDE and 4G63. The accession number of GMP complexed LpcN-II is 4OHF.. LpcN-II and LpcN-II bind by molecular sieving (View interaction) LpcN-II and LpcN-II bind by x-ray crystallography (View interaction) [Structured digital abstract was added on 5 March 2014 after original online publication].

Topics: 5'-Nucleotidase; Allosteric Regulation; Bacterial Proteins; Catalytic Domain; Crystallography, X-Ray; Deoxyguanine Nucleotides; Enzyme Activation; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Kinetics; Legionella pneumophila; Models, Molecular; Mutagenesis, Site-Directed; Nitrophenols; Organophosphorus Compounds; Protein Conformation; Protein Structure, Quaternary; Protein Structure, Tertiary; Species Specificity; Substrate Specificity

A dogma for DNA polymerase catalysis is that the enzyme binds DNA first, followed by MgdNTP. This mechanism contributes to the selection of correct dNTP by Watson-Crick base pairing, but it cannot explain how low-fidelity DNA polymerases overcome Watson-Crick base pairing to catalyze non-Watson-Crick dNTP incorporation. DNA polymerase X from the deadly African swine fever virus (Pol X) is a half-sized repair polymerase that catalyzes efficient dG:dGTP incorporation in addition to correct repair. Here we report the use of solution structures of Pol X in the free, binary (Pol X:MgdGTP), and ternary (Pol X:DNA:MgdGTP with dG:dGTP non-Watson-Crick pairing) forms, along with functional analyses, to show that Pol X uses multiple unprecedented strategies to achieve the mutagenic dG:dGTP incorporation. Unlike high fidelity polymerases, Pol X can prebind purine MgdNTP tightly and undergo a specific conformational change in the absence of DNA. The prebound MgdGTP assumes an unusual syn conformation stabilized by partial ring stacking with His115. Upon binding of a gapped DNA, also with a unique mechanism involving primarily helix αE, the prebound syn-dGTP forms a Hoogsteen base pair with the template anti-dG. Interestingly, while Pol X prebinds MgdCTP weakly, the correct dG:dCTP ternary complex is readily formed in the presence of DNA. H115A mutation disrupted MgdGTP binding and dG:dGTP ternary complex formation but not dG:dCTP ternary complex formation. The results demonstrate the first solution structural view of DNA polymerase catalysis, a unique DNA binding mode, and a novel mechanism for non-Watson-Crick incorporation by a low-fidelity DNA polymerase.

Topics: African Swine Fever; African Swine Fever Virus; Animals; Base Pairing; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; DNA; DNA Polymerase beta; DNA-Directed DNA Polymerase; Guanosine Triphosphate; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Protein Conformation; Swine

The HIV-1 restriction factor sterile α-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) is a tetrameric protein that catalyzes the hydrolysis of all dNTPs to the deoxynucleoside and tripolyphosphate, which effectively depletes the dNTP substrates of HIV reverse transcriptase. Here, we establish that SAMHD1 is activated by GTP binding to guanine-specific activator sites (A1) as well as coactivation by substrate dNTP binding to a distinct set of nonspecific activator sites (A2). Combined activation by GTP and dNTPs results in a long-lived tetrameric form of SAMHD1 that persists for hours, even after activating nucleotides are withdrawn from the solution. These results reveal an ordered model for assembly of SAMHD1 tetramer from its inactive monomer and dimer forms, where GTP binding to the A1 sites generates dimer and dNTP binding to the A2 and catalytic sites generates active tetramer. Thus, cellular regulation of active SAMHD1 is not determined by GTP alone but instead, the levels of all dNTPs and the generation of a persistent tetramer that is not in equilibrium with free activators. The significance of the long-lived activated state is that SAMHD1 can remain active long after dNTP pools have been reduced to a level that would lead to inactivation. This property would be important in resting CD4(+) T cells, where dNTP pools are reduced to nanomolar levels to restrict infection by HIV-1.

Topics: Catalytic Domain; Deoxyguanine Nucleotides; Deoxyguanosine; Deoxyribonucleotides; Deoxyuracil Nucleotides; Enzyme Activation; Guanosine Triphosphate; HIV-1; Humans; Immunity, Innate; Kinetics; Models, Molecular; Monomeric GTP-Binding Proteins; Protein Multimerization; Protein Structure, Quaternary; SAM Domain and HD Domain-Containing Protein 1; Substrate Specificity; Thionucleosides

SAMHD1 (SAM domain- and HD domain-containing protein 1) is a dGTP-dependent dNTP triphosphohydrolase that converts dNTPs into deoxyribonucleosides and triphosphates. Therefore, SAMHD1 expression, particularly in non-dividing cells, can restrict retroviral infections such as HIV and simian immunodeficiency virus by limiting cellular dNTPs, which are essential for reverse transcription. It has previously been established that dGTP acts as both an activator and a substrate of this enzyme, suggesting that phosphohydrolase activity of SAMHD1 is regulated by dGTP availability in the cell. However, we now demonstrate biochemically that the NTP GTP is equally capable of activating SAMHD1, but GTP is not hydrolyzed by the enzyme. Activation of SAMHD1 phosphohydrolase activity was tested under physiological concentrations of dGTP or GTP found in either dividing or non-dividing cells. Because GTP is 1000-fold more abundant than dGTP in cells, GTP was able to activate the enzyme to a greater extent than dGTP, suggesting that GTP is the primary activator of SAMHD1. Finally, we show that SAMHD1 has the ability to hydrolyze base-modified nucleotides, indicating that the active site of SAMHD1 is not restrictive to such modifications, and is capable of regulating the levels of non-canonical dNTPs such as dUTP. This study provides further insights into the regulation of SAMHD1 with regard to allosteric activation and active site specificity.

Topics: Deoxyguanine Nucleotides; Enzyme Activation; Guanosine Triphosphate; HIV; Humans; Monomeric GTP-Binding Proteins; SAM Domain and HD Domain-Containing Protein 1

The deoxyguanosine (GdR) analog guanine-ß-d-arabinofuranoside (araG) has a specific toxicity for T lymphocytes. Also GdR is toxic for T lymphocytes, provided its degradation by purine nucleoside phosphorylase (PNP) is prevented, by genetic loss of PNP or by enzyme inhibitors. The toxicity of both nucleosides requires their phosphorylation to triphosphates, indicating involvement of DNA replication. In cultured cells we found by isotope-flow experiments with labeled araG a rapid accumulation and turnover of araG phosphates regulated by cytosolic and mitochondrial kinases and deoxynucleotidases. At equilibrium their partition between cytosol and mitochondria depended on the substrate saturation kinetics and cellular abundance of the kinases leading to higher araGTP concentrations in mitochondria. dGTP interfered with the allosteric regulation of ribonucleotide reduction, led to highly imbalanced dNTP pools with gradual inhibition of DNA synthesis and cell-cycle arrest at the G1-S boundary. AraGTP had no effect on ribonucleotide reduction. AraG was in minute amounts incorporated into nuclear DNA and stopped DNA synthesis arresting cells in S-phase. Both nucleosides eventually induced caspases and led to apoptosis. We used high, clinically relevant concentrations of araG, toxic for nuclear DNA synthesis. Our experiments do not exclude an effect on mitochondrial DNA at low araG concentrations when phosphorylation occurs mainly in mitochondria.

Topics: Animals; Apoptosis; Arabinonucleosides; Arabinonucleotides; Biocatalysis; Caspases; Cell Cycle; Cell Line; Cell Line, Tumor; Cell Proliferation; CHO Cells; Cricetinae; Cricetulus; Cytosol; Deoxycytidine Kinase; Deoxyguanine Nucleotides; Deoxyguanosine; Deoxyribonucleotides; DNA; DNA Replication; Fibroblasts; G1 Phase; Guanosine Triphosphate; Humans; Hypoxanthine Phosphoribosyltransferase; Kinetics; Mitochondria; Phosphotransferases (Alcohol Group Acceptor); Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Purine-Nucleoside Phosphorylase; S Phase

To study if mycophenolic acid (MPA), the active metabolite of mycophenolate mofetil (MMF), indeed inhibits T-cell proliferation in kidney transplant recipients by lowering intracellular deoxyguanosine triphosphate (dGTP) and guanosine triphosphate (GTP) levels. Blood was drawn from 11 kidney transplant recipients. Ex vivo T-cell proliferation was measured by stimulation with phytohemagglutin (PHA) and anti-CD3 monoclonal antibody (mAb). Plasma MPA levels and intracellular dGTP and GTP in peripheral blood mononuclear cells were measured. MMF induces a significant decrease in T-lymphocyte proliferation at all time points (i.e. 24 h, 10 days and 8 weeks) after stimulation with both PHA (P = 0.001, 0.002 and 0.013 respectively) and anti-CD3 mAb (P = 0.004, 0.004 and 0.005 respectively). There was no significant change in intracellular dGTP (P = 0.31, 0.16 and 0.35) or GTP levels (P = 0.99, 0.32 and 0.49) between baseline and day 1, day 10 or week 8. All MPA levels were above the minimal required concentration for the inhibition of lymphocyte proliferation. MMF inhibits T-lymphocyte proliferation in kidney transplant recipients without lowering intracellular dGTP or GTP levels. This suggests another mechanism underlying its immunosuppressive capacity.

Topics: Adult; Cell Proliferation; Deoxyguanine Nucleotides; Female; Guanosine Triphosphate; Humans; Kidney Transplantation; Male; Middle Aged; Mycophenolic Acid; T-Lymphocytes

The mechanism of DNA polymerase beta-catalyzed nucleotidyl transfer consists of chemical steps involving primer 3' OH deprotonation, nucleophilic attack, and pyrophosphate leaving-group elimination, preceded by dNTP binding which induces a large-amplitude conformational change for Watson-Crick nascent base pairs. Ambiguity in the nature of the rate-limiting step and active-site structural differences between correct and incorrect base-paired transition states remain obstacles to understanding DNA replication fidelity. Analogues of dGTP where the beta-gamma bridging oxygen is replaced with fluorine-substituted methylene groups have been shown to probe the contribution of leaving-group elimination to the overall catalytic rate (Biochemistry 46, 461-471). Here, the analysis is expanded substantially to include a broad range of halogen substituents with disparate steric and electronic properties. Evaluation of linear free energy relationships for incorporation of dGTP analogues opposite either template base C or T reveals a strong correlation of log(kpol) to leaving group pKa. Significantly different kpol behavior is observed with a subset of the analogues, with magnitude dependent on the identity of the nascent base pair. This observation, and the absence of an analogous effect on ground state analogue binding (Kd values), points to active-site structural differences at the chemical transition state. Reduced catalysis with bulky halo-containing substrates is manifested in the fidelity of T-G incorporation, where the CCl2-bridging analogue shows a 27-fold increase in fidelity over the natural dGTP. Solvent pH and deuterium isotope-effect data are also used to evaluate mechanistic differences between correct and mispaired incorporation.

Topics: Base Pair Mismatch; Catalysis; Catalytic Domain; Deoxyguanine Nucleotides; Deuterium Oxide; Diphosphonates; DNA; DNA Polymerase beta; Guanosine Triphosphate; Halogens; Humans; Hydrogen-Ion Concentration; Kinetics; Models, Chemical; Models, Molecular; Recombinant Proteins; Substrate Specificity; Thermodynamics

Non homologous end-joining (NHEJ)-mediated repair of DNA double-strand breaks in prokaryotes requires Ku and a specific multidomain DNA ligase (LigD). We present crystal structures of the primase/polymerisation domain (PolDom) of Mycobacterium tuberculosis LigD, alone and complexed with nucleotides. The PolDom structure combines the general fold of the archaeo-eukaryotic primase (AEP) superfamily with additional loops and domains that together form a deep cleft on the surface, likely used for DNA binding. Enzymatic analysis indicates that the PolDom of LigD, even in the absence of accessory domains and Ku proteins, has the potential to recognise DNA end-joining intermediates. Strikingly, one of the main signals for the specific and efficient binding of PolDom to DNA is the presence of a 5'-phosphate group, located at the single/double-stranded junction at both gapped and 3'-protruding DNA molecules. Although structurally unrelated, Pol lambda and Pol mu, the two eukaryotic DNA polymerases involved in NHEJ, are endowed with a similar capacity to bind a 5'-phosphate group. Other properties that are beneficial for NHEJ, such as the ability to generate template distortions and realignments of the primer, displayed by Pol lambda and Pol mu, are shared by the PolDom of bacterial LigD. In addition, PolDom can perform non-mutagenic translesion synthesis on termini containing modified bases. Significantly, ribonucleotide insertion appears to be a recurrent theme associated with NHEJ, maximised in this case by the deployment of a dedicated primase, although its in vivo relevance is unknown.

Topics: Base Sequence; Binding Sites; Deoxyguanine Nucleotides; DNA Ligases; DNA Repair; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Guanosine Triphosphate; Molecular Sequence Data; Mycobacterium tuberculosis; Protein Structure, Tertiary; Ribonucleotides; Structure-Activity Relationship; Transferases; X-Ray Diffraction

The coexistence effects of multiple kinds of oxidized deoxyribonucleotides were examined using an SV40 origin-dependent in vitro replication system with a HeLa extract. Oxidized dGTP and dATP, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP) and 2-hydroxy-2'-deoxyadenosine 5'-triphosphate (2-OH-dATP), were used in this study. The mutation frequency synergistically increased when the two oxidized deoxyribonucleotides were together in the reaction. 2-OH-dATP enhanced the mutagenicity of 8-OH-dGTP, since the induced mutations were A.T --> C.G transversions. The contribution of the highly error-prone DNA polymerase eta was unlikely, since similar results were observed with an XP-V cell extract. The possible involvement of 2-hydroxyadenine in the complementary (template) strand was excluded on the basis of experiments using plasmids containing 2-hydroxyadenine as templates in the reactions with 8-OH-dGTP. 2-OH-dATP suppressed hydrolysis of 8-OH-dGTP, suggesting that the inhibition of the MTH1 protein played the major role in the enhancement. These results highlight the importance of specific hydrolysis of 8-OH-dGTP for the suppression of its induced mutation.

Topics: Adenosine Triphosphate; Cell Extracts; Cell-Free System; Cells, Cultured; Deoxyguanine Nucleotides; DNA Mutational Analysis; DNA Repair Enzymes; DNA Replication; Guanosine Triphosphate; HeLa Cells; Humans; Models, Genetic; Mutagenesis; Mutation; Oxygen; Phosphoric Monoester Hydrolases

8-OxoGua (8-oxo-7,8-dihydroguanine) is produced in nucleic acids as well as in nucleotide pools of cells, by reactive oxygen species normally formed during cellular metabolic processes. MutT protein of Escherichia coli specifically degrades 8-oxoGua-containing deoxyribo- and ribonucleoside triphosphates to corresponding nucleoside monophosphates, thereby preventing misincorporation of 8-oxoGua into DNA and RNA, which would cause mutation and phenotypic suppression, respectively. Here, we report that the MutT protein has additional activities for cleaning up the nucleotide pools to ensure accurate DNA replication and transcription. It hydrolyzes 8-oxo-dGDP to 8-oxo-dGMP with a K(m) of 0.058 microM, a value considerably lower than that for its normal counterpart, dGDP (170 microM). Furthermore, the MutT possesses an activity to degrade 8-oxo-GDP to the related nucleoside monophosphate, with a K(m) value 8000 times lower than that for GDP. These multiple enzyme activities of the MutT protein would facilitate the high fidelity of DNA and RNA syntheses.

Topics: Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; DNA Replication; DNA, Bacterial; Escherichia coli Proteins; Guanine; Guanosine Triphosphate; Hydrolysis; Kinetics; Multienzyme Complexes; Phosphoric Monoester Hydrolases; Pyrophosphatases; RNA, Bacterial; Thymine Nucleotides; Transcription, Genetic

Transcription initiation as catalyzed by T7 RNA polymerase consists primarily of promoter binding, strand separation, nucleotide binding, and synthesis of the first phosphodiester bond. The promoter strand separation process occurs at a very fast rate, but promoter opening is incomplete in the absence of the initiating NTPs. In this paper, we investigate how initiating NTPs affect the kinetics and thermodynamics of open complex formation. Transient state kinetic studies show that the open complex, ED(o), is formed via an intermediate ED(c), and the conversion of ED(c) to ED(o) occurs with an unfavorable equilibrium constant. In the presence of the initiating NTP that base-pairs with the template at position +2, the process of open complex formation is nearly complete. Our studies reveal that the nucleotide that drives open complex formation needs to be a triphosphate and to be correctly base-paired with the template. These results indicate that the melted template DNA in the open complex is positioned to bind the +2 NTP. The addition of +1 NTP alone does not stabilize the open complex; nor is it required for +2 NTP binding. However, there appears to be cooperativity in initiating NTP binding in that the binding of +2 NTP facilitates +1 NTP binding. The dissection of the initiation pathway provides insights into how open complex formation steps that are sensitive to the promoter sequence upstream from the initiation start site modulate the affinity of initiating NTPs and allow transcription initiation to be regulated by initiating NTP concentration.

Topics: Bacteriophage T7; Base Sequence; Binding Sites; Deoxyguanine Nucleotides; DNA-Directed RNA Polymerases; DNA, Viral; Escherichia coli; Guanosine Triphosphate; Kinetics; Magnesium; Promoter Regions, Genetic; Ribonucleotides; Thermodynamics; Transcription, Genetic; Viral Proteins

GTP cyclohydrolase II catalyzes the hydrolytic release of formate and pyrophosphate from GTP producing 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate, the first committed intermediate in the biosynthesis of riboflavin. The enzyme was shown to contain one zinc ion per subunit. Replacement of cysteine residue 54, 65 or 67 with serine resulted in proteins devoid of bound zinc and unable to release formate from the imidazole ring of GTP or from the intermediate analog, 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-triphosphate. However, the mutant proteins retained the capacity to release pyrophosphate from GTP and from the formamide-type intermediate analog. The data suggest that the enzyme catalyzes an ordered reaction in which the hydrolytic release of pyrophosphate precedes the hydrolytic attack of the imidazole ring. Ring opening and formate release are both dependent on a zinc ion acting as a Lewis acid, which activates the two water molecules involved in the sequential hydrolysis of two carbon-nitrogen bonds.

Topics: Amino Acid Sequence; Catalytic Domain; Deoxyguanine Nucleotides; Diphosphates; Escherichia coli; GTP Cyclohydrolase; Guanosine Triphosphate; Molecular Sequence Data; Mutagenesis, Site-Directed; Riboflavin; Sequence Homology, Amino Acid; Spectrophotometry, Ultraviolet; Structure-Activity Relationship; Zinc

Topics: Animals; Antigens, CD19; B-Lymphocytes; Biomarkers; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Deoxyguanine Nucleotides; Deoxyguanosine; Guanosine; Guanosine Triphosphate; Inosine; Mice; Mice, Knockout; Purine-Nucleoside Phosphorylase

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

Using HPLC methods, we measured the concentrations of nucleosides and nucleotides for a patient with no purine nucleoside phosphorylase (PNP; EC 2.4.2.1) enzymatic activity. Concentrations of inosine and guanosine were abnormally high in urine and plasma, whereas guanosine diphosphate (GDP) and guanosine triphosphate (GTP) concentrations in erythrocytes were depleted. The unusual presence of deoxyribonucleosides (deoxyinosine and deoxyguanosine) and deoxyribonucleotides (dGDP and dGTP) was also notable. Thus, HPLC represents an accurate and useful tool for the study of purine metabolic disorders.

Topics: Chromatography, High Pressure Liquid; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Erythrocytes; Guanosine; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Infant; Inosine; Male; Purine-Nucleoside Phosphorylase; Purine-Pyrimidine Metabolism, Inborn Errors

BCX-34 inhibits RBC PNP in vitro from humans, rats, and mice with IC50S ranging from 5 to 36 nM. BCX-34 also, in the presence but not in the absence of deoxyguanosine, inhibits human CCRF-CEM T-cell proliferation with an IC50 of 0.57 microM but not rat or mouse T-cell proliferation up to 30 microM. Inhibition of human T-cell proliferation is accompanied by an accumulation of intracellular dGTP with an associated reduction in GTP. These nucleotide changes do not occur in BC16A mouse T-cells and explain why proliferation is not inhibited by PNP inhibitors in this case. Reduction in intracellular GTP is not essential for the antiproliferative action of BCX-34. Oral bioavailability of BCX-34 in rats is 76%. BCX-34 is orally active in elevating plasma inosine in rats (2-fold at 30 mg/kg), in suppressing ex vivo RBC PNP activity in rats (98% at 3 h. 100 mg/kg), and in suppressing ex vivo skin PNP in mice (39% at 3 h, 100 mg/kg). The results demonstrate that BCX-34 inhibits human PNP and T-cell proliferation, is orally bioavailable in rodents, and pharmacologically active in vivo in rodents after oral dosing with no apparent side effects or toxicity. BCX-34 may, therefore, be useful in treating human T-cell proliferative inflammatory disorders.

Topics: Animals; Cells, Cultured; Deoxyguanine Nucleotides; Guanine; Guanosine Triphosphate; Humans; Immunosuppressive Agents; Lymphocyte Activation; Lymphoma, T-Cell; Mice; Purine-Nucleoside Phosphorylase; Rats; T-Lymphocytes; Tumor Cells, Cultured

The assembly of chick brain microtubule protein in a NaCl-supplemented buffer has been examined with respect to nucleation and the subsequent elongation as a function of the nucleotide (GTP vs 2'dGTP), and the protein and nucleotide concentrations. The kinetics suggest that unassembled tubulin can exist in two conformational states (termed Tu1,GTP and Tu2,GIP when GTP is bound to the exchangeable site), with Tu1,GTP contributing to nucleation and Tu2,GTP participating in elongation. The extent of self-nucleation is proposed to be determined, in part, by the rate constant governing this conformational change. This analysis contrasts with that of earlier studies, which concluded that the number of subunits interacting to form an effective nucleus could be estimated from the dependency of self-nucleation on the initial concentration of unassembled tubulin.

Topics: Animals; Chickens; Deoxyguanine Nucleotides; Guanosine Triphosphate; Kinetics; Microtubules; Protein Binding; Protein Conformation; Tubulin

EICAR (5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide) is a cytostatic agent that inhibits murine leukemia L1210 and human lymphocyte CEM cells at a 50% inhibitory concentration of 0.80-1.4 microM, respectively. EICAR causes a rapid and marked inhibition of inosinate (IMP) dehydrogenase (EC 1.1.1.205) activity in intact L1210 and CEM cells reflected by a concentration-dependent accumulation of IMP and depletion of GTP and dGTP levels. EICAR 5'-monophosphate is a potent inhibitor of purified L1210 cell IMP dehydrogenase (Ki/Km 0.06). Inhibition of IMP dehydrogenase by EICAR 5'-monophosphate is competitive with respect to IMP. L1210 cells that were selected for resistance to the cytostatic action of EICAR proved to be adenosine kinase-deficient. Also, studies with other mutant L1210 and CEM cell lines revealed that adenosine kinase, as well as an alternative pathway, may be responsible for the conversion of EICAR to its 5'-monophosphate. Purified 2'-deoxycytidine kinase, 2'-deoxyguanosine kinase, cytosolic 5'-nucleotidase, and nicotinamide dinucleotide (NAD) pyrophosphorylase do not seem to be markedly involved in the metabolism of EICAR.

Topics: Adenosine; Animals; Antineoplastic Agents; Cell Division; Deoxyguanine Nucleotides; Guanine; Guanosine; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Leukemia L1210; Lymphocytes; Mice; Mycophenolic Acid; Purine Nucleotides; Ribavirin; Ribonucleosides; Ribonucleotides; Tumor Cells, Cultured

The solution dynamics of normal and transforming p21ras proteins in both the GTP- and GDP-bound forms were examined with time-resolved fluorescence spectroscopy. The fluorescent 2'(3')-O-(N-methylanthraniloyl) derivatives (mant derivatives) of GTP, dGTP, and GDP and the aminocoumarin and fluorescein derivatives of GTP and GDP were synthesized and used as reporter groups. The fluorescence lifetimes at 5 degrees C of the mant nucleotide derivatives increased from approximately 4 ns in solution to approximately 9 ns when bound to p21ras. At 30 degrees C, there was a 7.8% difference in lifetime between normal p21ras.mantGTP and p21ras.mantGDP, but no difference between similar complexes of the [Asp-12]p21ras protein. These data are consistent with steady-state fluorescence intensity differences among p21ras.mantGTP, p21ras.mantGDP, and the free nucleotides. Rotational correlation times for the mantGTP- and mantGDP-bound p21 proteins, N-ras, K-ras, and H-ras, were similar at 26 ns (5 degrees C), which is significantly longer than the 15-ns rotational correlation time predicted for a globular 21,000-Da protein. The p21-bound fluorescein and aminocoumarin nucleotide derivatives reported correlation times of 19 and 29 ns, respectively. Global analysis of the three fluorophore.p21 complexes with linked protein rotational correlation functions were best fit with a common rotational correlation time of 28 ns. Gel permeation chromatography of the GDP and mantGDP complexes of normal p21N-ras also showed greater apparent molecular weights than were expected in both cases, demonstrating that the high rotational correlation times obtained from time-resolved fluorescence measurements were not a result of the introduction of the fluorophore.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Chemical Phenomena; Chemistry, Physical; Chromatography, Gel; Coumarins; Deoxyguanine Nucleotides; Fluorescein-5-isothiocyanate; Fluorescence; Fluorescence Polarization; Fluorescent Dyes; Guanosine Diphosphate; Guanosine Triphosphate; Macromolecular Substances; Proto-Oncogene Proteins p21(ras); Rotation; Solutions; Spectrometry, Fluorescence; Time Factors

The ability of deoxycytidine kinase (dCK) to phosphorylate 2'-deoxycytidine (dCyd) and its analogs in the presence of eight nucleoside triphosphates (NTPs), simulating the cellular milieu, was investigated. Using highly purified dCK from MOLT-4 T lymphoblasts, Km and Vmax values were determined for the phosphorylation of dCyd in the presence of cellular concentrations of the eight endogenous NTPs. The results demonstrated that the efficiency of dCyd phosphorylation was greatest in the presence of all eight nucleotides, relative to ATP alone, according to relative Vmax/Km values. UTP was a better phosphate donor than ATP but was less efficient than the NTP mixture. The greater efficacy of the NTP mixture, compared with ATP alone, was due in large part to the presence of UTP, although the results suggested that the presence of other nucleotide(s) also enhanced dCyd phosphorylation. Previous results demonstrated that dCTP was a potent competitive or noncompetitive (with respect to dCyd) inhibitor of dCK, with a Ki value of approximately 1 microM. In contrast, the results presented here demonstrated that, in the presence of either the NTP mixture or UTP, inhibition of dCK was uncompetitive with respect to dCyd, with a Ki value of approximately 60 microM. Furthermore, the results demonstrated that the clinically relevant nucleoside analogs 1-beta-D-arabinofuranosylcytosine, 2',2'-difluoro-2'-deoxycytidine (dFdC), and 9-beta-D-arabinofuranosyl-2-fluoroadenine also preferred UTP or the NTP mixture, compared with ATP alone, as a phosphate donor. Of the three nucleoside analogs tested, dFdC was the most efficient dCK substrate. These data indicate that the preferred phosphate donor for dCK is UTP or a combination of UTP and another nucleotide. Furthermore, the dCTP concentration in intact cells, which is typically 10-20 microM, is not sufficient to cause substantial inhibition of dCK, due to the presence of UTP. Strategies to increase cellular dCK activity should focus on optimizing UTP concentrations.

Topics: Adenosine Triphosphate; Deoxycytidine; Deoxycytidine Kinase; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Guanosine Triphosphate; Humans; Kinetics; Nucleotides; Phosphates; Phosphorylation; Substrate Specificity; T-Lymphocytes; Uridine Triphosphate

Tubulin, widely recognized as a GTP/GDP-binding protein, has been isolated in its polymerized state from rat PC12 cells and embryonic chick dorsal root ganglion neurons by Triton X-100 detergent extraction of the cytoskeletal fraction. Perchloric acid extraction and deproteinization of this fraction permitted subsequent analysis of nucleotide identity and content by high performance liquid chromatography. PC12 cells grown in the absence of nerve growth factor (NGF) contained ADP, ATP, GDP, and GTP at levels consistent with the actin and tubulin content of the cytoskeletal fraction. Microtubules from PC12 cells cultured in the presence of NGF contain an additional nucleotide that we have identified as dGTP. Analysis of whole cell nucleotide extracts from PC12 cells grown in the absence or presence of NGF revealed no evidence for the presence of dGTP at 4 and 14 days, respectively. We have determined that embryonic chick dorsal root ganglion neurons also contain this deoxyribonucleotide, and we found virtually no ADP or ATP in the extracted dorsal root ganglion cytoskeletal fraction. On the basis of metabolic labeling studies with [14C] guanine, we have inferred that the presence of dGTP in NGF-treated PC12 cells probably arises either from binding to the nonexchangeable nucleotide site of tubulin undergoing dynamic assembly/disassembly or from binding to the exchangeable site of tubulin subsequently incorporated into highly stabilized microtubules.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Chromatography, High Pressure Liquid; Deoxyguanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Mice; Microtubules; Nerve Growth Factors; Neurites; Neurons; PC12 Cells

PD 116124 (8-amino-2'-nordeoxyguanosine; 2,8-diamino-1,9-dihydro-9- ([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)-6H-purin-6-one) is a competitive, reversible inhibitor of human purine nucleoside phosphorylase with an apparent inhibition constant of 0.41 microM. In a cell line system using human MOLT-4 and CEM T lymphoblasts and human MGL-8 and NC-37 B lymphoblasts, PD 116124 failed to inhibit [3H]thymidine uptake at concentrations up to 500 microM. However, in the presence of 10 microM 2'-deoxyguanosine (dGuo), a noninhibitory dGuo concentration by itself, PD 116124 produced potent inhibition of growth of both T cell lines but not of either B cell line. Significant elevation of intracellular 2'-deoxyguanosine triphosphate was observed in both inhibited T cell lines but not in either B cell line. Greater and more sustained accumulation of 2'-deoxyguanosine triphosphate was observed in T lymphoblasts cultured with PD 116124 plus dGuo than with dGuo only. PD 116124 was only weakly inhibitory in human mixed lymphocyte cultures (IC50 approximately equal to 1420 microM), but in the presence of 10 microM dGuo, the IC50 for PD 116124 was reduced to 108.7 microM. Administration of PD 116124 p.o. to normal male Wistar rats caused dose-dependent elevation of plasma inosine up through 500 mg/kg. Maximal inosine elevation occurred at 30 min after dosing, and elevation was significant even 24 hr after dosing. Guanosine was also elevated, although not in a dose-dependent manner. Administration of PD 116124 i.v. produced marked and statistically significant elevation of both inosine and guanosine.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Arthritis, Experimental; B-Lymphocytes; Biological Availability; Cells, Cultured; Deoxyguanine Nucleotides; Deoxyguanosine; Guanosine Triphosphate; Humans; Inosine; Kinetics; Lymphocyte Culture Test, Mixed; Lymphocytes; Male; Nucleosides; Purine-Nucleoside Phosphorylase; Rats; Rats, Inbred Strains; T-Lymphocytes

CI-972 (2,6-diamino-3,5-dihydro-7-(3-thienylmethyl)-4H-pyrrolo[3, 2-d]pyrimidin-4-one monohydrochloride, monohydrate) is a novel inhibitor of PNP (Ki = 0.83 microM) under development as a T cell-selective immunosuppressive agent. CI-972 inhibited proliferation (3H-thymidine uptake) of human MOLT-4 (T cell) but not MGL-8 (B cell) lymphoblasts with respective IC50s of 3.0 and greater than 50 microM when tested with 10 microM 2'-deoxyguanosine. Without addition of exogenous 2'-deoxyguanosine, CI-972 was not inhibitory to any human T or B lymphoblastoid cell line tested. 2'-Deoxycytidine (10 microM), but not hypoxanthine or adenine, restored MOLT-4 cell growth. Inhibition of 3H-thymidine uptake in MOLT-4 cells correlated with accumulation of dGTP, while alterations in guanine nucleotides were not observed. 2'-Deoxycytidine (10 microM) also blocked dGTP accumulation in MOLT-4 cells. CI-972 showed activity in vivo over a broad dose range: At 5-150 mg/kg p.o., CI-972 produced dose-dependent elevation of plasma inosine one hr after administration to rats (mean maximum of 2.62 vs. 0.06 microM in controls). Guanosine was also significantly elevated in a concentration-dependent manner, although the effect was not as impressive. Plasma nucleosides remained statistically-significantly elevated for up to four hr following a single oral dose of CI-972.

Topics: Animals; Cell Line; Cells, Cultured; Deoxyguanine Nucleotides; Guanosine Triphosphate; Humans; Lymphocytes; Male; Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidines; Rats; Rats, Inbred Strains; Thiophenes; Thymidine

CI-972 (2,6-diamino-3,5-dihydro-7-(3-thienylmethyl)-4H-pyrrolo[3,2- d]pyrimidin-4-one monohydrochloride, monohydrate) is a competitive inhibitor of PNPase (E.C. 2.4.2.1., Ki = 0.83 microM) entering clinical trials as a T cell-selective immunosuppressive agent. Neither CI-972 (less than or equal to 50 microM) nor dGuo (less than or equal to 10 microM) inhibited [3H]Thd uptake by human MOLT-4 (T cell) or MGL-8 (B cell) lymphoblasts, but in the presence of 10 microM dGuo, the IC50 for CI-972 decreased to 3.0 microM for MOLT-4 but remained at greater than 50 microM for MGL-8. Inhibition of MOLT-4 growth was associated with an increase in dGTP that was dependent on CI-972 concentration and inhibited by 2'-deoxycytidine. Growth could not be restored by hypoxanthine or adenine. No alterations in GTP pools were noted in MOLT-4, and neither GTP nor dGTP were altered in MGL-8.

Topics: Adenine; Cell Division; Deoxycytidine; Deoxyguanine Nucleotides; DNA Replication; Guanosine Triphosphate; Humans; Hypoxanthine; Hypoxanthines; Kinetics; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Purine-Nucleoside Phosphorylase; Pyrimidines; T-Lymphocytes; Thiophenes

Topics: B-Lymphocytes; Cell Line; Deoxyguanine Nucleotides; Guanosine Triphosphate; Humans; Immunosuppressive Agents; Intracellular Fluid; Leukocytes, Mononuclear; Lymphocyte Activation; Mycophenolic Acid; Prodrugs; T-Lymphocytes

Escherichia coli encodes a dGTP triphosphohydrolase (dGTPase) that cleaves dGTP to deoxyguanosine and tripolyphosphate. dGTP is hydrolyzed with a Michaelis constant (Km) of 5 microM and a maximal velocity (Vmax) of 1.8 mumols/min/mg. The ribonucleotide GTP is a poor substrate with a much lower affinity. It is hydrolyzed with a Km of 150 microM and Vmax of 0.07 mumols/min/mg. Bacteriophage T7 encodes a specific inhibitor of dGTPase, the gene 1.2 protein, that forms a tight complex with the enzyme. The enzyme-inhibitor complex binds dGTP with a dissociation constant (KD) of 1.5 microM, but the bound dGTP is not hydrolyzed. It remains stably bound to the complex with a half-life of approximately 5 min. In contrast, dGTP is unable to bind to gene 1.2 protein alone, and dGTP bound to dGTPase alone is quickly hydrolyzed and released. Surprisingly, the dGTPase-gene 1.2 protein complex has a higher affinity for GTP than for dGTP. GTP is stably bound to the dGTPase-gene 1.2 protein complex with a half-life greater than 30 min and KD of 0.8 microM; GTP is not stably bound to either dGTPase or gene 1.2 protein alone. Both GTP and dGTP bind to and stabilize the dGTPase-gene 1.2 protein complex, inhibiting its dissociation. Although the presence of dGTP induces conformation changes in dGTPase so that it is unable to associate with the gene 1.2 protein, saturating concentrations of GTP have no such effect. The enzyme efficiently associates with its inhibitor in the presence of GTP. These results indicate that E. coli dGTPase and gene 1.2 protein interact to form a high affinity GTP-binding site. dGTP is most effective in preventing the association of the enzyme with the inhibitor whereas GTP is most effective in preventing the dissociation of the enzyme-inhibitor complex.

Topics: Binding Sites; Cations, Divalent; Deoxyguanine Nucleotides; Escherichia coli; GTP-Binding Proteins; Guanosine Triphosphate; Half-Life; Hydrolysis; Kinetics; Manganese; Nucleotides; Phosphoric Monoester Hydrolases; Protein Conformation; T-Phages; Viral Proteins

Cell-mediated immunity in human subjects is affected adversely as a result of zinc deficiency. The mechanism by which a deficiency of zinc may affect lymphocyte proliferation and functions, is not well understood at present. Nucleoside phosphorylase (NPase), a purine catabolic pathway enzyme, is zinc dependent, and a congenital deficiency of this enzyme is known to affect adversely cell-mediated immunity. This effect has been related to an accumulation of toxic nucleotides in lymphocytes as a result of NPase deficiency. Inasmuch as the effect of zinc deficiency on the activity of NPase and the levels of nucleotides in human lymphocytes has not been previously reported, we assayed these parameters in human subjects with zinc deficiency before and after zinc supplementation. A mild deficiency of zinc was diagnosed in those having decreased zinc in two out of three cell lineages (less than 42 micrograms in granulocytes, less than 48 micrograms in lymphocytes, and less than 1.70 microgram in platelets, per 10(10) cells). In comparison with five subjects with sufficient zinc, six subjects with zinc deficiency showed a decrease in the activity of NPase (p = 0.01), an increase in adenosine diphosphate (ADP) level (p = 0.008), a decreased adenosine triphosphate (ATP)-to-ADP ratio (p = 0.0001), and an increase in both guanosine triphosphate (GTP) (p = 0.02) and deoxyadenosine triphosphate (dGTP) (p = 0.04 in the lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Blood Platelets; Deoxyguanine Nucleotides; Granulocytes; Guanosine Triphosphate; Humans; Lymphocytes; Nucleotides; Pentosyltransferases; Purine-Nucleoside Phosphorylase; Zinc

Two alternative pathways for the synthesis of dGTP and its incorporation into DNA were studied: guanine (Gua)----GMP----GDP----dGDP----dGTP----DNA and dG----dGMP----dGDP----dGTP----DNA. To determine the contribution of each pathway to DNA synthesis independently of each other, [14C]Gua and [3H]dG tracer experiments were performed in a double-mutant S-49 mouse T-lymphoma cell line, dGuo-L, with purine nucleoside phosphorylase (EC 2.4.2.1)-deficiency and dGTP-feedback-resistant ribonucleotide reductase (RR, EC 1.17.4.1). In this cell line, dGTP pools can be selectively elevated by exogenous dG without affect RR and DNA synthesis. Although [3H]dG, but not [14C]Gua (up to 200 microM), readily expanded the cellular dGTP pool in a dose-dependent fashion in asynchronous cells, only a small fraction of the Gua flux into DNA was derived from [3H]dG, with the major fraction coming from [14C]Gua. H.p.l.c. analysis of G1- and partially enriched S-phase cells revealed that [3H]dGTP only accumulates in G1- but not in S-phase cells because of a rapid turnover of the dGTP pool during DNA synthesis. These results fail to provide evidence for cellular dGTP compartmentation and suggest that the pathway dG----dGMP----dGDP----dGTP alone has insufficient capacity to maintain DNA synthesis.

Topics: Animals; Carbon Radioisotopes; Deoxyguanine Nucleotides; Deoxyguanosine; DNA; Guanine; Guanosine Triphosphate; Mice; Ribonucleotide Reductases; Tritium; Tumor Cells, Cultured

PD 119,229 [8-amino-9-(2-thienylmethyl)guanine] is a novel and potent inhibitor of human erythrocyte purine nucleoside phosphorylase (PNP) with a Ki of 0.067 microM. In a cell line assay using human MOLT-4 (T cell) and MGL-8 (B cell) lymphoblasts, PD 119,229 alone had no effect on the growth of either cell line at the highest concentration tested (100 microM). However, in the presence of a nontoxic concentration of 2'-deoxyguanosine (10 microM), the IC50 values of PD 119,229 for MOLT-4 and MGI-8 40-fold either cell line at the highest concentration tested (100 microM). However, in the presence of a nontoxic concentration of 2'-deoxyguanosine (10 microM), the IC50 values of PD 119,229 for MOLT-4 and MGI-8 were 0.9 and greater than 100 microM, respectively. The inhibition of growth of MOLT-4 was accompanied by a 40-fold increase in dGTP and a two-fold reduction in GTP, while no alteration in nucleotide profile was noted in MGL-8. Both the inhibition of growth of MOLT-4 and the accumulation of dGTP were substantially prevented by coaddition of 2'-deoxycytidine.

Topics: Cell Division; Cell Line; Deoxyguanine Nucleotides; Deoxyguanosine; Guanine; Guanosine Triphosphate; Humans; Kinetics; Lymphocytes; Pentosyltransferases; Purine-Nucleoside Phosphorylase

Purine nucleoside phosphorylase (NP; EC 2.4.2.1) deficiency is associated with selective T-cell dysfunction and normal B-cell immunity. In order to create an in vivo model of this immune deficiency, we administered 8-aminoguanosine to rats. This water-soluble nucleoside was rapidly converted by NP to the more potent inhibitor 8-aminoguanine, which has a Ki of 0.19 microM. The accumulation of inosine in plasma showed that administration of 8-aminoguanosine was effectively inhibiting NP activity. The administration of 8-aminoguanosine with deoxyguanosine produced increased levels of dGTP only in thymus cells, and increased levels of GTP in cells from thymus, spleen and lymph node and in red cells. This correlated with assays of deoxyguanosine kinase, which showed significantly higher activity in thymus cells than in cells from spleen and lymph node. The intraperitoneal injection of 8-aminoguanosine alone or with deoxyguanosine for 8 consecutive days caused significant decreases in the number of thymus cells (P less than 0.001) and in lymph node and spleen lymphocytes (P less than 0.01). These data showed that the administration of 8-aminoguanosine to rats provided an animal model of NP deficiency that will allow studies of the specific regulation of T-cell function.

Topics: Animals; Deoxyguanine Nucleotides; Deoxyguanosine; Disease Models, Animal; Erythrocytes; Female; Guanosine; Guanosine Triphosphate; Lymphoid Tissue; Male; Pentosyltransferases; Phosphotransferases; Phosphotransferases (Alcohol Group Acceptor); Purine-Nucleoside Phosphorylase; Rats; Rats, Inbred Lew

Topics: B-Lymphocytes; Cell Cycle; Cell Differentiation; Cells, Cultured; Deoxycytidine; Deoxyguanine Nucleotides; Deoxyguanosine; Guanosine; Guanosine Triphosphate; Humans; Hypoxanthine; Hypoxanthine Phosphoribosyltransferase; Hypoxanthines; In Vitro Techniques; Lymphocyte Activation; Purine-Nucleoside Phosphorylase; T-Lymphocytes

The catabolism of deoxy-GTP and GTP was compared in purine-nucleoside phosphorylase-deficient mouse T lymphoblasts. It was found that guanine ribonucleotides and deoxyribonucleotides are degraded by distinct pathways in cells cultured under both physiological and induced catabolic conditions. In T lymphoblasts, cultured under physiological conditions, 50% of the GMP formed during GTP catabolism was dephosphorylated and 50% was deaminated, whereas in the presence of the catabolic inducer deoxyglucose 90% of the GMP formed was dephosphorylated and only 10% was deaminated. These results indicate that GTP catabolism in lymphoblasts proceeds by alternative pathways, either via GMP dephosphorylation or via GMP reductive deamination, and physiological conditions determine with pathway will be used. In contrast, deoxy-GTP catabolism proceeds exclusively via deoxy-GMP dephosphorylation under both physiological and induced catabolic conditions. The lack of deoxy-GMP deamination may contribute to the accumulation of cytotoxic levels of deoxyguanosine found in purine-nucleoside phosphorylase-deficient patients.

Topics: Animals; Deoxyguanine Nucleotides; Guanosine Monophosphate; Guanosine Triphosphate; Mice; Models, Biological; Pentosyltransferases; Purine-Nucleoside Phosphorylase; T-Lymphocytes

An intact cell assay system based upon Tween-80 permeabilization was used to investigate the regulation of ribonucleotide reductase activity in Chinese hamster ovary cells. Models used to explain the regulation of the enzyme have been based upon work carried out with cell-free extracts, although there is concern that the properties of such a complex enzyme would be modified by extraction procedures. We have used the intact cell assay system to evaluate, within whole cells, the current model of ribonucleotide reductase regulation. While some of the results agree with the proposals of the model, others do not. Most significantly, it was found that ribonucleotide reductase within the intact cell could simultaneously bind the nucleoside triphosphate activators for both CDP and ADP reductions. According to the model based upon studies with cell-free preparations, the binding of one of these nucleotides should exclude the binding of others. Also, studies on intracellular enzyme activity in the presence of combinations of nucleotide effectors indicate that GTP and perhaps dCTP should be included in a model for ribonucleotide reductase regulation. For example, GTP has the unique ability to modify through activation both ADP and CDP reductions, and synergistic effects were obtained for the reduction of CDP by various combinations of ATP and dCTP. In general, studies with intact cells suggest that the in vivo regulation of ribonucleotide reductase is more complex than predicted from enzyme work with cell-free preparations. A possible mechanism for the in vivo regulation of ribonucleotide reductase, which combines observations of enzyme activity in intact cells and recent reports of independent substrate-binding subunits in mammalian cells is discussed.

Topics: Adenosine Triphosphate; Animals; Binding, Competitive; Cell Line; Chemical Phenomena; Chemistry; Cricetinae; Cricetulus; Cytidine Diphosphate; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Female; Guanosine Triphosphate; Nucleotides; Ovary; Ribonucleotide Reductases; Substrate Specificity

Interactions of tubulin with a number of guanine nucleotides modified at the 2' and 3' ribose hydroxyls were examined. Deoxy analogs of GTP were equal or superior to GTP in supporting tubulin polymerization, but analogs bearing either methyl or phosphate groups on the hydroxyls had significantly reduced ability to support polymerization. These substituted GTP analogs were hydrolyzed at the 5'-gamma-phosphate position, although less rapidly than GTP, at rates exceeding those of polymerization. GTP hydrolysis, however, was closely coupled to polymerization. Moreover, the partially active GTP analogs were not effective inhibitors of GTP-dependent polymerization. These data indicate that the substituted GTP analogs have reduced affinity for tubulin at the exchangeable site because of steric factors. No deoxy or substituted GDP analog was as effective as GDP itself in inhibiting GTP-supported tubulin polymerization. Furthermore, there was no apparent relationship between the ability of nucleoside 5'-triphosphates to support polymerization and that of nucleoside 5'-diphosphates to inhibit the reaction. These findings suggest that GTP and GDP may actually bind to different, mutually exclusive sites rather than to a single exchangeable site.

Topics: Animals; Binding Sites; Brain; Cattle; Deoxyguanine Nucleotides; Dideoxynucleotides; Glutamates; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Hydrolysis; Substrate Specificity; Tubulin; Vinblastine

Incubation of mouse T lymphoma (S-49) cells with the inosinate dehydrogenase inhibitor mycophenolic acid produced a depletion of both GTP and dGTP, and resulted in growth inhibition, partial reduction in RNA synthesis, and drastic inhibition of DNA synthesis. Similar results suggested to others that the depletion of dGTP is primarily responsible for toxicity. However, guanosine was as effective as deoxyguanosine at preventing mycophenolic acid toxicity although deoxyguanosine was more effective at elevating dGTP levels. Moreover, in hypoxanthine-guanine phosphoribosyltransferase-deficient mutants of S-49 (6MPR-3-3) deoxyguanosine was unable to prevent mycophenolic acid toxicity or to re-establish normal DNA synthesis, although it returned cellular dGTP but not GTP levels to normal. No other nucleotide levels changed in a way which could account for the toxicity. Incubation of cells with a combination of deoxyadenosine, deoxycytidine, and erythro-9-(2-hydroxy-3-nonyl)adenine produced a selective depletion of dGTP to levels similar to that produced by mycophenolic acid, but did not affect cell growth. Studies with cells synchronized by centrifugal elutriation show that the toxicity of mycophenolic acid is specific to the S-phase of the cell cycle. Addition of actinomycin D at a concentration that inhibited RNA synthesis increased the availability of GTP and re-established normal DNA synthesis in mycophenolic acid-treated S-49 cells. These results suggest that the depletion of GTP rather than that of dGTP produces toxic effects in S-49 cells and that GTP is required for DNA synthesis.

Topics: Animals; Cell Division; Cell Line; Deoxyguanine Nucleotides; DNA, Neoplasm; Guanosine Triphosphate; Hypoxanthine Phosphoribosyltransferase; Kinetics; Lymphoma; Mice; Mutation; Mycophenolic Acid; Neoplasms, Experimental

The nucleoside triphosphate pools of Escherichia coli minicells are different from those in parental cells. The growth phase in which minicells accumulate significantly affects the pool sizes.

Topics: Adenosine Triphosphate; Cytidine Triphosphate; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Escherichia coli; Guanosine Triphosphate; Nucleotides; Thymine Nucleotides; Uridine Triphosphate