Conformational restraint is a critical determinant of unnatural nucleotide recognition by protein kinases

This report describes the synthesis of N(4)-(benzyl) AICAR triphosphate, a conformationally restrained analogue of N(4)-(benzyl) ribavirin triphosphate. Both of these nucleotides were evaluated as phosphodonors for wild-type p38MAP kinase and T106G p38MAP kinase, a designed mutant with expanded nucleotide specificity. The conformationally restrained nucleotide, N(4)-(benzyl) AICAR triphosphate, is orthogonal to (not accepted as a substrate by) wild-type p38MAP kinase, in contrast to N(4)-(benzyl) ribavirin triphosphate. Furthermore, N(4)-(benzyl) AICAR triphosphate, is accepted as a substrate by T106G p38MAP kinase, in contrast to N(4)-(benzyl) ribavirin triphosphate. We hypothesize that the presence of an internal hydrogen bond in N(4)-(benzyl) AICAR and its absence in N(4)-(benzyl) ribavirin triphosphate is the main determinant for their differing structure-activity relationships.     

The levels of soluble nucleotides in wheat aleurone tissue

The content of soluble nucleotides in aleurone layers isolated from mature wheat (Triticum aestivum var. Olympic) grain was investigated. The most abundant nucleotides were adenosine triphosphate, uridine triphosphate, and uridine diphosphoglucose. Smaller amounts of guanosine triphosphate, cytidine triphosphate, adenosine diphosphate, and nicotinamide adenine dinucleotide were also identified. The levels of some of these nucleotides were increased after incubation of the tissue under certain conditions.     

ITPase Activity in Dry Blood Spots is Comparable with That in Fresh Erythrocytes

Inosine triphosphate pyrophosphohydrolase (ITPase) catalyzing the pyrophosphohydrolysis of inosine triphosphate, deoxyinosine triphosphate and xanthosine triphosphate is involved in the metabolism and tolerance of thiopurine drugs. ITPase activity plays an important role in the prediction of toxicity to thiopurine therapy. Activities in dry blood spots were compared with fresh erythrocytes. Samples were incubated with inosine triphosphate, then inosine monophosphate was determined by a capillary electrophoresis method. Calculated enzyme activities obtained from dry blood spots were in good accordance with activity in fresh erythrocytes.     

ITPase activity in dry blood spots is comparable with that in fresh erythrocytes

Inosine triphosphate pyrophosphohydrolase (ITPase) catalyzing the pyrophosphohydrolysis of inosine triphosphate, deoxyinosine triphosphate and xanthosine triphosphate is involved in the metabolism and tolerance of thiopurine drugs. ITPase activity plays an important role in the prediction of toxicity to thiopurine therapy. Activities in dry blood spots were compared with fresh erythrocytes. Samples were incubated with inosine triphosphate, then inosine monophosphate was determined by a capillary electrophoresis method. Calculated enzyme activities obtained from dry blood spots were in good accordance with activity in fresh erythrocytes.     

Effects of Alkaline Phosphatase Activity on Nucleotide Measurements in Aquatic Microbial Communities

Alkaline phosphatase (APase) activity was detected in aquatic microbial assemblages from the subtropics to Antarctica. The occurrence of APase in environmental nucleotide extracts was shown to significantly affect the measured concentrations of cellular nucleotides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, guanosine triphosphate, uridine triphosphate, and cytidine triphosphate), adenylate energy charge, and guanosine triphosphate/adenosine triphosphate ratios, when conventional methods of nucleotide extraction were employed. Under the reaction conditions specified in this report, the initial rate of hydrolysis of adenosine triphosphate was directly proportional to the activity of APase in the sample extracts and consequently can be used as a sensitive measure of APase activity. A method was devised for obtaining reliable nucleotide measurements in naturally occurring microbial populations containing elevated levels of APase activity. The metabolic significance of APase activity in microbial cells is discussed, and it is concluded that the occurrence and regulation of APase in nature is dependent upon microscale inorganic phosphate limitation of the autochthonous microbial communities.     

Determination of basal and stimulated levels of inositol triphosphate in [32P]orthophosphate-labeled platelets

Previous studies indicated that thrombin-stimulation of platelets prelabeled with [3H]inositol or [32P]orthophosphate results in an increase of radioactive inositol triphosphate, a substance thought to modulate the levels of free intracellular calcium. In the present study, we improved the method of resolution of inositol triphosphate from other compounds that are also labeled with [32P]orthophosphate using a combination of enzyme treatment and electrophoresis. We have further demonstrated that the specific activities of metabolic ATP and phosphatidylinositol diphosphate (the precursor of inositol triphosphate) are identical in [32P]orthophosphate-labeled platelets. It follows that the amount of inositol triphosphate is proportional to its radioactivity in the metabolic compartment of the cells. Using this protocol, the concentration of inositol triphosphate in resting and thrombin-stimulated platelets were determined to be 1-4 and 10-30 pmol/10(8) cells, respectively.     

Thymidine Phosphorylation in Arachis hypogaea by Combined Activities of Adenosine Triphosphate Phosphohydrolase and Nucleoside Phosphotransferase

Extracts from hypocotyls of germinating peanuts (Arachis hypogaea L.) stimulated the formation of thymidine monophosphate from thymidine and adenosine triphosphate in the presence of magnesium ions. Such extracts were incapable of incorporating isotopic phosphorus from gamma-labelled adenosine triphosphate into thymidine during the synthesis of thymidine monophosphate but were competent in transferring phosphorus from alpha-labelled adenosine triphosphate to thymidine. The apparent thymidine kinase activity thus appeared to result from the combined activities of an adenosine triphosphatase (adenosine triphosphate pyrophosphohydrolase, E. C. 3.6.1.3) and a nucleoside phosphotransferase (E. C. 2.7.1.77). The latter two enzymes could be largely separated by using DEAE-Sephadex chromatography.     

Synthesis and enzymatic incorporation of photolabile dUTP analogues into DNA and their applications for DNA labeling

Two novel photolabile nucleotide triphosphate (NTP) analogues were synthesized through Sonogashira coupling and their enzymatic incorporation into DNA was evaluated with three different DNA polymerases (Taq, Vent exo- and T4) by polymerase chain reaction. Both nucleotide triphosphate analogues were recognized by these DNA polymerases as substrates for primer extension. Light irradiation of PCR products removed the photolabile group and released the amino and carboxyl moieties. Further site-specific dual-labeling for oligodeoxynucleotides (ODNs) and random labeling for a long DNA construct with fluorophores were successfully achieved with incorporation of the photolabile amine modified deoxyuridine triphosphate (dUnTP).     

Ring opening is not rate-limiting in the GTP cyclohydrolase I reaction

GTP cyclohydrolase I catalyzes a mechanistically complex ring expansion affording dihydroneopterin triphosphate and formate from GTP. Single turnover quenched flow experiments were performed with the recombinant enzyme from Escherichia coli. The consumption of GTP and the formation of 5-formylamino-6-ribosylamino-2-amino-4(3H)-pyrimidinone triphosphate, formate, and dihydroneopterin triphosphate were determined by high pressure liquid chromatography analysis. A kinetic model comprising three consecutive unimolecular steps was used for interpretations where the first intermediate, 5-formylamino-6-ribosylamino-2-amino-4(3H)-pyrimidinone 5'-triphosphate, was formed in a reversible reaction. The rate constant k(1) for the reversible opening of the imidazole ring of GTP was 0.9 s(-1), the rate constant k(3) for the release of formate from 5-formylamino-6-ribosylamino-2-amino-4(3H)-pyrimidinone triphosphate was 2.0 s(-1), and the rate constant k(4) for the formation of dihydroneopterin triphosphate was 0.03 s(-1). Thus, the hydrolytic opening of the imidazole ring of GTP is rapid by comparison with the overall reaction.     

Mitochondrial deoxyribonucleoside triphosphate pools in thymidine kinase 2 deficiency

Deficiency of mitochondrial thymidine kinase (TK2) is associated with mitochondrial DNA (mtDNA) depletion and manifests by severe skeletal myopathy in infancy. In order to elucidate the pathophysiology of this condition, mitochondrial deoxyribonucleoside triphosphate (dNTP) pools were determined in patients' fibroblasts. Despite normal mtDNA content and cytochrome c oxidase (COX) activity, mitochondrial dNTP pools were imbalanced. Specifically, deoxythymidine triphosphate (dTTP) content was markedly decreased, resulting in reduced dTTP:deoxycytidine triphosphate ratio. These findings underline the importance of balanced mitochondrial dNTP pools for mtDNA synthesis and may serve as the basis for future therapeutic interventions.     

Differential recognition of Toxoplasma gondii recombinant nucleoside triphosphate hydrolase isoforms by naturally infected human sera

Toxoplasma gondii possesses a highly active nucleoside triphosphate hydrolase, which has been shown to be an immunodominant antigen in mice and humans. Two isoforms (I and II) which exhibit different activities with respect to hydrolysis of ATP exist. Past studies suggest that all strains of T. gondii contain the less active nucleoside triphosphate hydrolase II, whilst only virulent strains contain the nucleoside triphosphate hydrolase I isoform. In order to further investigate the correlation between nucleoside triphosphate hydrolase isoform and biological significance, we cloned and expressed as glutathione S-transferase fusion proteins the full-length nucleoside triphosphate hydrolase I and II isoforms and two truncations of the nucleoside triphosphate hydrolase I isoform in Escherichia coli. We then used ELISAs with the full-length recombinant nucleoside triphosphate hydrolases as antigens to examine 188 naturally infected T. gondii-positive sera and 83 T. gondii-negative sera for antibody reactivity. All positive sera reacted to T. gondii whole tachyzoite lysate antigen, 31 sera reacted to both nucleoside triphosphate hydrolase isoforms, three sera reacted specifically to nucleoside triphosphate hydrolase I and two sera reacted to only nucleoside triphosphate hydrolase II. Immunoblot analysis of the five sera reacting to either nucleoside triphosphate hydrolase I or II revealed both quantitative and qualitative differences in reactivity to the two isoforms. Comparative immunoblot analysis using the truncations of the nucleoside triphosphate hydrolase I isoform, and one of these positive sera identified a presumptive differential epitope between the nucleoside triphosphate hydrolase I and II isoforms within an 81-aa region (aa 445–526) at the C-terminus of the nucleoside triphosphate hydrolase I isoform. This differential reactivity was further localised to the 12-residue region of greatest variability between the two isoforms (residues 488–499) using synthetic peptides. This is the first report where naturally infected human sera have been used to identify a differential epitope. Because this region is essential for substrate binding, an antibody response to this region may play some role in inhibition of this highly active enzyme.     

Protection of Vaccinia from Heat Inactivation by Nucleotide Triphosphates

The presence of adenosine triphosphate, guanosine triphosphate, cytosine triphosphate, or uridine triphosphate reduced the rate of inactivation of vaccinia when heated at 50 C. The virus-associated nucleoside triphosphate phosphohydrolases (adenosine triphosphatase, guanosine triphosphatase, cytosine triphosphatase, and uridine triphosphatase) and ribonucleic acid polymerase were also protected from heat inactivation by these compounds. These obervations are best explained by postulating that ribonucleoside triphosphates bind to enzymes in the virus particle, and that these enzyme-substrate complexes are more resistant to thermal denaturation than are the enzymes without their substrates. The kinetics of heat inactivation of the vaccinia ATP phosphohydrolase activity is biphasic, suggesting that there are two proteins in the vaccinia particle that have this enzyme activity but they have different kinetics of heat inactivation. Any of the vaccinia-associated nucleotide phosphohydrolase activities are protected from heat inactivation by the presence of any one of the respective nucleoside triphosphates. This observation suggests that there is a single enzymatic site in vaccinia that is able to react with any ribonucleoside triphosphate.     

Dinitrophenol-stimulated adenosine triphosphatase activity in extracts of Desulfovibrio gigas

A dinitrophenol (DNP)-stimulated adenosine triphosphatase (ATPase) has been found in both the soluble and particulate fractions of the anaerobic sulfate-reducing bacterium, Desulfovibrio gigas. As the soluble ATPase was labile to storage, only the particulate enzyme was studied in detail. It was optimally stimulated by DNP at 4 mm, and activity was insensitive to inhibition by ouabain. The ATPase was stimulated by both Ca(2+) and Mg(2+), but the magnitude of the stimulation was dependent upon pH. In the presence of Ca(2+) the optimum pH was 6.5, whereas, in the presence of Mg(2+) the pH optimum was 8.0. However, under optimal conditions the activity was the same with either Mg(2+) or Ca(2+). Both adenosine triphosphate and guanosine triphosphate were hydrolyzed, but activity toward guanosine triphosphate was only one-tenth that observed with adenosine triphosphate.     

Kinetics and mechanism of DNA repair. Preparation, purification and some properties of caged dideoxynucleoside triphosphates.

Caged dideoxyribosylthymine triphosphate, dideoxyadenosine triphosphate and arabinosylcytosine triphosphate were prepared in high yield by reaction with 1-(2-nitrophenyl)diazoethane at pH 4 and room temperature for 24 h. Synthesis of caged alpha-32P-labelled dideoxyadenosine triphosphate (approx. 5000 Ci/mmol) in 85% yield was achieved by a modification of the method used for the synthesis of the unlabelled compounds. ATP was shown to be an excellent buffer in the synthesis of alpha-32P-labelled material, and in caged form to be an effective carrier in h.p.l.c. purification. Preparative h.p.l.c. was used to achieve purification of unlabelled caged compounds to greater than 98% purity and 32P-labelled material to 97% purity. Photolysis of unlabelled and 32P-labelled caged compounds by using XeF-excimer laser irradiation at 351 nm was characterized by using difference spectrophotometry and h.p.l.c. analysis. The stability of caged dideoxyadenosine [a-32P]triphosphate in the presence of cultured mammalian cells was evaluated; the adenosine derivative is essentially stable for 1 h.     

Probing the substrate recognition mechanism of the human MTH1 protein by nucleotide analogs

To examine the substrate recognition mechanism of the human MTH1 protein, which hydrolyzes 2-hydroxy-dATP, 8-hydroxy-dATP, and 8-hydroxy-dGTP, ten nucleotide analogs (8-bromo-dATP, 8-bromo-dGTP, deoxyisoinosine triphosphate, 8-hydroxy-dITP, 2-aminopurine-deoxyriboside triphosphate, 2-amino-dATP, deoxyxanthosine triphosphate, deoxyoxanosine triphosphate, dITP, and dUTP) were incubated with the MTH1 protein. Of these, the former five nucleotides were hydrolyzed with various efficiencies. The fact that the syn-oriented brominated nucleotides were hydrolyzed suggests that the MTH1 protein binds to deoxynucleotides adopting the syn-conformation. However, 8-hydroxy-dITP, which lacks the 2-amino group of 8-hydroxy-dGTP, was degraded with tenfold less efficiency as compared with 8-hydroxy-dGTP. In addition, deoxyisoinosine triphosphate, lacking the 6-amino group of 2-hydroxy-dATP, was hydrolyzed as efficiently as 8-hydroxy-dGTP, but less efficiently than 2-hydroxy-dATP. These results clarify the effects of the anti/syn conformation and the functional groups on the 2 and 6 positions of the purine ring on the recognition by the human MTH1 protein.     

Synthesis of Non-Nucleoside Triphosphate Analogues,a New Type of Substrates for Terminal Deoxynucleotidyl Transferase

Abstract

A series of non-nucleoside triphosphate analogues were synthesized. In place of the nucleoside fragment, substituents bearing aromatic groups were introduced; the triphosphate component was replaced at α, β, or γ-positions by phosphonates. α-[2-N-(9-Fluorenylmethoxycarbonyl)aminoethylphosphonyl]-β,γ-difluoromethylenediphosphonate (IIc) revealed the best substrate properties toward terminal deoxynucleotidyl transferase.     

Membrane-associated thiamin triphosphatase. II. Activation by divalent cations.

Activation of membrane-associated thiamin triphosphatase from rat brain requires a divalent cation (Mg2+, Ca2+, or Mn2+). The optimum concentration of Mg2+ necessary for maximal enzyme activity varies with substrate concentration; conversely, the maximal rate of hydrolysis attainbale by increasing thiamin triphosphate concentration is directly proportional to [Mg2+] for all levels of Mg2+ below that of the substrate. Under appropriate conditions, the Km of the thiamin triphosphatase for Mg2+ and for thiamin triphosphate are shown to be identical. Dissociation constants (Kd) for the binding of Mg2+ to thiamin triphosphate, thiamin diphosphate, and thiamin were determined; kinetic data re-expressed in terms of [Mg2+-thiamin triphosphate] conform to simple single substrate predictions, suggesting that the true enzyme substrate may be the Mg2+-thiamin triphosphate complex. Excess free Mg2+ inhibits thiamin triphosphatase activity competitively while excess free thiamin triphosphate in concentrations up to 10 times Km has no effect on the membrane-bound enzyme.     

Effect of ATP analogs of DNA synthesis in isolated nuclei

Optimal synthesis of DNA in Ehrlich ascites cell nuclei is shown to be dependent upon the presence of both ATP and ADP. ATP can be replaced only by dATP. An ATP regenerating system is less effective than ATP alone or ATP in combination with ADP. ATP does not stimulate DNA synthesis primarily by maintenance of deoxyribonucleotide triphosphate levels. When the inhibition of DNA synthesis by high ATP levels is taken into account, the ATP analogs adenosine 5'-(alpha,beta-methylene)triphosphate, adenosine 5'-(beta, gamma-methylene)-triphosphate, and adenosine 5'-(beta, gamma-imino)triphosphate can neither substitute for ATP nor inhibit the ATP stimulation of DNA synthesis. Adenosine 5'-(3-thio)triphosphate, however, is a competitive inhibitor of DNA synthesis.