Reinvestigation of 4-Thiothymidine-5′-triphosphate Synthesis

Abstract

The 4-Thiothymidine-5′-triphosphate 1 (S⁴TTP) was known to be a substrate for polymerase, however a commercial sample of this compound failed to be incorporated into DNA. Mass spectrometry combined to alkaline phosphatase digestion and ³¹P-NMR showed that this sample was in fact 5′-chloro-5′-deoxy-4-thiothymidine-3′-triphosphate 2 . The desired S⁴TTP was synthesized by two alternate routes, was fully characterized and was shown to be incorporated in a DNA polymerase assay.     

ATPace™: injectable adenosine 5′-triphosphate

Carbon monoxide (CO) is produced endogenously by heme oxygenase (HO) enzymes. HO-1 is highly expressed in many inflammatory disease states, where it is broadly protective. The protective effects of HO-1 expression can be largely mimicked by the exogenous application of CO and CO-releasing molecules (CORMs). Despite a dearth of pharmacological tools for their study, molecular methodologies have identified P2X4 receptors as a potential anti-nociceptive drug target. P2X4 receptors are up-regulated in animal models of inflammatory pain, and their knock-down reduces pain behaviours. In these same animal models, HO-1 expression is anti-nociceptive, and we therefore investigated whether P2X4 was a target for CO and tricarbonyldichlororuthenium (II) dimer (CORM-2). Using conventional whole-cell and perforated-patch recordings of heterologously expressed human P2X4 receptors, we demonstrate that CORM-2, but not CO gas, is an inhibitor of these channels. We also investigated the role of soluble guanylate cyclase and mitochondria-derived reactive oxygen species using pharmacological inhibitors but found that they were largely unable to affect the ability of CORM-2 to inhibit P2X4 currents. A control breakdown product of CORM-2 was also without effect on P2X4. These results suggest that P2X4 receptors are not a molecular target of endogenous CO production and are, therefore, unlikely to be mediating the anti-nociceptive effects of HO-1 expression in inflammatory pain models. However, these results show that CORM-2 is an effective antagonist at human P2X4 receptors and represents a useful pharmacological tool for the study of these receptors given the current dearth of antagonists.     

Adenosine 5′-triphosphate sulphurylase from Saccharomyces cerevisiae

1. ATP sulphurylase from Saccharomyces cerevisiae was purified 140-fold by using heat treatment, DEAE-cellulose chromatography and Sepharose 6B gel filtration. 2. The enzyme was stable at -15 degrees C, optimum reaction velocity was between pH7.0 and 9.0, and the activation energy was 62kJ/mol (14.7kcal/mol). 3. The substrate was shown to be the MgATP(2-) complex, free ATP being inhibitory. 4. Double-reciprocal plots from initial-velocity studies were intersecting and the K(m) of each substrate was determined at infinite concentration of the other (K(m) MgATP(2-), 0.07mm; MoO(4) (2-), 0.17mm). 5. Radio-isotopic exchange between the substrate pairs, adenosine 5'-[(35)S]sulphatophosphate and SO(4) (2-), (35)SO(4) (2-) and adenosine 5'-sulphatophosphate, occurred only in the presence of either MgATP(2-) or PP(i). This suggests, along with the initial-velocity data, a sequential reaction mechanism in which both substrates bind before any product is released. 6. The enzyme reaction was specific for ATP and was not inhibited by l-cysteine, l-methionine, SO(3) (2-), S(2)O(3) (2-) (all 2mm) nor by p-chloromercuribenzoate (1mm). 7. Competitive inhibition of the enzyme with respect to MoO(4) (2-) was produced by SO(4) (2-) (K(i)=2.0mm) and non-competitive inhibition by sulphide (K(i)=3.4mm). 8. Adenosine 5'-sulphatophosphate inhibited strongly and concentrations as low as 0.02mm altered the normal hyperbolic velocity-substrate curves with both MgATP(2-) and MoO(4) (2-) to sigmoidal forms.     

ATPace™: injectable adenosine 5′-triphosphate

ATPace?, a novel injectable formulation of adenosine 5′-triphosphate (ATP), is developed by Cordex Pharma, Inc. (Cordex) as a diagnostic and therapeutic drug for the management of cardiac bradyarrhythmias. Extracellular ATP exerts multiple effects in various cell types by activating cell-surface receptors known as P2 receptors. In the heart, ATP suppresses the automaticity of cardiac pacemakers and atrioventricular (AV) nodal conduction via adenosine, the product of its degradation by ecto-enzymes, as well as by triggering a cardio-cardiac vagal reflex. ATP, given as a rapid intravenous bolus injection, has been used since the late 1940s as a highly effective and safe therapeutic agent for the acute termination of reentrant paroxysmal supraventricular tachycardia (PSVT) involving the AV node. In addition, preliminary studies have shown that ATP can also be used as a diagnostic agent for the identification of several cardiac disorders including sinus node dysfunction (sick sinus syndrome), dual AV nodal pathways, long QT syndrome, and bradycardic syncope. The US Food and Drug Administration has approved Cordex formulation for ATP as an Investigational New Drug and two pathways for its marketing approval; one therapeutic, i.e., acute termination of paroxysmal PSVT, and the other diagnostic, i.e., the identification of patients with bradycardic syncope who can benefit from pacemaker therapy. The scientific rationale for the development of ATPace? is discussed.     

Inhibition of a novel subspecies of DNA polymerase α by 2′-deoxy-2′-azidoadenosine 5′-triphosphate

DNA polymerase α₁, a subspecies of DNA polymerase α of Ehrlich ascites tumor cells, was associated with a novel RNA polymerase activity and utilized poly(dT) and single-stranded circular fd DNA as a template without added primer in the presence of ribonucleoside triphosphates and a specific stimulating factor. DNA synthesis in the above system was inhibited by the ATP analogue, 2′-deoxy-2′-azidoadenosine 5′-triphosphate more than the DNA synthesis with poly(dT)·oligo(rA) by DNA polymerase α₁ and RNA synthesis by mouse RNA polymerases I and II. Kinetic analysis showed that the analogue inhibited DNA polymerase α₁ activity on poly(dT) competitively with respect to ATP, suggesting that the analogue inhibited RNA synthesis by the associated RNA polymerase activity.     

Guanosine-5′-triphosphate hydrolysis and tubulin polymerization

Summary GTP hydrolysis associated with polymerization is a distinctive feature of microtubule assembly. This reaction may be fundamentally linked to the dynamic properties of microtubules in vivo. Kinetic analysis of the connection between microtubule assembly and associated GTP hydrolysis indicates that these two events are kinetically uncoupled, GTP hydrolysis occurring after tubulin incorporation in the microtubule. As a consequence, the combination of the diffusionnal incorporation of GTP in microtubules at steady-state and of subsequent GTP hydrolysis results in the formation of a steady-state GTP cap at microtubule ends. The interplay between GTP and GDP at microtubule ends is examined. Inhibition by GDP of steady-state GTP hydrolysis at microtubule ends and of microtubule elongation is understood within a tight reversible binding of GDP at microtubule ends generating inactive elongation sites. Nucleotides are freely exchangeable at microtubule ends. This result indicates that the nature of the nucleotide present at microtubule ends must be considered in a model for microtubule assembly.These data are pooled in order to define the general features of a model describing microtubule assembly and treadmilling in terms somewhat different from previously proposed models.     

Effects of Exogenous Adenosine 5′-triphosphate 3′-diphosphate on Growth and Sporulation of Bacillus subtilis

Exogenous adenosine 5′-triphosphate 3′-diphosphate (pppApp) had interesting effects on the cell cycle of B. subtilis IFO 3027. The growth rate was reduced by the addition of 1 mm pppApp, and the vegetative cell form was significantly changed. Moreover, the sporulation frequency was increased by 100 times or more as compared with the culture without pppApp. The sporulation process seemed to be stimulated around t₀. pppGpp and ppGpp also showed the same effects as pppApp. Among these effects, depression in growth rate was restored by Mg²⁺ and Ca²⁺, and stimulation of sporulation was inhibited by Mg²⁺, Ca²⁺ and certain carbon sources, such as glucose and glycerol. On the other hand, casamino acids or monovalent cations showed no influence on the pppApp effects. pppApp was not incorporated into cells in experiments with radioactive pppApp.     

Sequencing Reactions Using 6-Thio-2′-Deoxyguanosine-5′-triphosphate

Abstract

6-Thio-2′-Deoxyguanosine-5′-Triphosphate (6SdGTP) was employed as a substrate replacing dGTP in sequencing reactions with Sequenase? and Thermo Sequenase?.     

Photoaffinity labeling of ribulose-bisphosphate carboxylase/oxygenase with 8-azidoadenosine 5′-triphosphate

Photoaffinity labeling with [³²P] 8-azidoadenosine 5-triphosphate (8-N₃ATP) was used to identify putative binding sites on tobacco (Nicotiana tabacum L. and N. rustica L.) leaf ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase, EC 4.1.1.39). Incorporation of ³²P was observed in polypeptides corresponding to both RuBPCase subunits when desalted leaf and chloroplast extracts, and purified RuBPCase were irradiated with ultraviolet light in the presence of [³²P] 8-N₃ATP. ³²P-labeling was dependent upon ultraviolet irradiation and occurred with [³²P] 8-N₃ATP labeled in the -position, indicating covalent incorporation of the photoprobe. Both [³²P] 8-N₃ATP and [³²P] 8-N₃GTP were incorporated to a similar extent into the 53-kilodalton (kDa) large subunit (LSu), but incorporation of [³²P] 8-N₃GTP into the 14-kDa small subunit (SSu) of RuBPCase was <5% of that measured with [³²P] 8-N₃ATP. Distinct binding sites for 8-N₃ATP on the two subunits were indicated by different apparent K _D values, 3 and 18 M for the SSu and LSu, respectively, and differences in the response of photoaffinity labeling to Mg²⁺, anions and enzyme activation. Active-site-directed compounds, including the non-gaseous substrate ribulose 1,5-bisphosphate, the reaction intermediate analog 2-carboxyarabinitol-1,5-bisphosphate and several phosphorylated effectors afforded protection to the LSu site against photoincorporation but provided almost no protection to the SSu. These results indicate that 8-N₃ATP binds to the active-site region of the LSu and a distinct site on the SSu of RuBPCase. Experiments conducted with intact pea (Pisum sativum L.) and tobacco chloroplasts showed that the SSu was not photolabeled with [³²P] 8-N₃ATP in organello or in undesalted chloroplast lysates but was photolabeled when lysates were ultrafiltered or desalted. These results indicate that 8-N₃ATP binds to a site on the SSu that has physiological significance.Abbreviations kDa kilodalton - LSu large subunit - 8-N₃ATP 8-azidoadenosine 5-triphosphate - RuBP ribulose-1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase - SSu small subunit Kentucky Agricultural Experiment Station Journal Article No. 89-3-150The authors acknowledge the technical assistance of J.C. Anderson. This work was supported in part by National Institute of Health grant GM 35766 to B.E.H.     

Dynamic redistribution of paxillin in bovine osteoblasts stimulated with adenosine 5′-triphosphate

Exposure to extracellular 5′-adenosine triphosphate (ATP) is known to induce membrane blebbing. In this study, we investigated the subcellular distribution of the cytoskeletal adaptor protein paxillin in primary bovine osteoblasts upon stimulation with ATP. Cells expressing a fusion protein of green fluorescent protein (GFP) and paxillin were followed by time-lapse video-microscopy after stimulation with 100?μM ATP. Within 100?s, GFP-paxillin became incorporated in numerous de novo formed focal aggregates localized at the cell periphery. The assembly of individual paxillin-containing aggregates occurred with a mean half-life time of <60?s, whereas their disassembly lasted twice as long. Despite the ongoing presence of ATP, the formation of paxillin aggregates was self-limiting within 25?min. Paxillin clustering was preceded by a transient rise in cytoplasmic calcium transients, which peaked already 20?s after adding ATP. The high mobility of paxillin was confirmed by measuring the dissociation rate of GFP-paxillin at mature focal adhesions, demonstrating the presence of a highly mobile fraction with a mean recovery half-life of 8.2?±?1.2?s, followed by a slower phase (53?±?20?s). Thus, both the exchange of paxillin at mature focal adhesions and the increase in intracellular calcium concentrations upon ATP stimulation are very rapid processes, which override the time course of ATP-induced paxillin membrane clustering by one to two orders of magnitude. Our data demonstrate that the transient recruitment of paxillin in membrane protuberances is based on the high intracytoplasmic mobility of unbound paxillin molecules and their rapid focal accumulation.     

Pleiotropic mutants of Chinese hamster cells with altered cytidine 5′-triphosphate synthetase

Following chemical mutagenesis and multiple-step indirect selection, four clones of Chinese hamster V79 cells were isolated which exhibited auxotrophy for thymidine, deoxycytidine, or deoxyuridine but not for cytidine or uridine. All were resistant to uridine, 3-deazauridine, 5-fluorouridine, thymidine, and cytosine arabinoside at concentrations that were toxic to wild-type V79 cells. The cytidine 5-triphosphate (CTP) and deoxycytidine 5-triphosphate (dCTP) pools in the mutants were expanded, but the uridine 5-triphosphate (UTP) pool either decreased or remained unchanged relative to the wild-type level. Furthermore, since the parental cells appear to be deficient in dCMP deaminase activity and CTP (or one of its metabolites) has been shown to inhibit uridine 5-diphosphate (UDP) reduction, an elevated CTP level should lead to the observed thymidine auxotrophy. It also explains the joint resistance of mutant clones to thymidine and cytosine arabinoside. The change in the ratio of intracellular dCTP to thymidine 5-triphosphate (dTTP) may be responsible for the elevation in the rates of spontaneous mutations in these mutants.This work was supported by Grant GM 30608 from the U.S. Public Health Service.     

Light-dependent quenching of chlorophyll fluorescence in pea chloroplasts induced by adenosine 5′-triphosphate

Addition of ATP to chloroplasts causes a reversible 25–30% decrease in chlorophyll fluorescence. This quenching is light-dependent, uncoupler insensitive but inhibited by DCMU and electron acceptors and has a half-time of 3 minutes. Electron donors to Photosystem I can not overcome the inhibitory effect of DCMU, suggesting that light activation depends on the reduced state of plastoquinone. Fluorescence emission spectra recorded at ?196°C indicate that ATP treatment increases the amount of excitation energy transferred to Photosystem I. Examination of fluorescence induction curves indicate that ATP treatment decreases both the initial (F_o) and variable (F_v) fluorescence such that the ratio of F_v to the maximum (F_m) yield is unchanged. The initial sigmoidal phase of induction is slowed down by ATP treatment and is quenched 3-fold more than the exponential slow phase, the rate of which is unchanged. A plot of F_v against area above the induction curve was identical plus or minus ATP. Thus ATP treatment can alter quantal distribution between Photosystems II and I without altering Photosystem II-Photosystem II interaction. The effect of ATP strongly resembles in its properties the phosphorylation of the light-harvesting complex by a light activated, ATP-dependent protein kinase found in chloroplast membranes and could be the basis of physiological mechanisms which contribute to slow fluorescence quenching in vivo and regulate excitation energy distribution between Photosystem I and II. It is suggested that the sensor for this regulation is the redox state of plastoquinone.     

A specific effect of deoxycytidine 5′-triphosphate on DNA chain initiation in vitro

Increased levels of dCTP increase the frequency of initiation of discontinuous DNA intermediates in a cellophane disc in vitro system. Increased levels of other ribo- and deoxyribonucleoside triphosphates show no effect.     

Enhancement of antibody production against rabies virus by uridine 5′-triphosphate in mice

Extracellular nucleotides such as adenosine 5′-triphospate (ATP) and uridine 5′-triphosphate (UTP) interact with P2 purinergic receptors on the surface of phagocytic cells and induce various physiological reactions. In this study, the production of antibody in mice immunized with an inactivated rabies vaccine containing these nucleotides was investigated. Injection of inactivated rabies vaccine with UTP, but not with ATP, induced significantly higher serum antibody production in mice. The enhancement of antibody production by UTP was inhibited by an anti-P2Y4 receptor antibody. In an air pouch experiment, UTP treatment increased the number of monocytes and macrophages infiltrating the pouch and up-regulated the gene expression of IL-4 and IL-13 in the regional lymph nodes. These results suggested that UTP admixed with rabies vaccine activates Th2 cells and induces a humoral immune response. Furthermore, the survival rate of mice immunized with a rabies vaccine admixed with UTP before rabies virus challenge was slightly higher than that of control mice. In conclusion, UTP can act as a vaccine adjuvant to enhance antibody production against the rabies virus in mice.     

Inactivation of DNA polymerases by adenosine 2′,3′-riboepoxide 5′-triphosphate allows estimation of the primers affinity

Template-primer dependent inactivation of human DNA polymerase and Klenow fragment of E. coli DNA polymerase I by adenosine 2,3-riboepoxide 5-triphosphate was used for quantitative analysis of the K_d values for oligonucleotide primers of different length. The K_d values are smaller by a factor of 2.5 than the K_m values for the same primers determined in the reaction of DNA polymerization in the case of DNA polymerase . The K_d and K_m values are nearly the same for Klenow fragment. Such approach to the determination of K_m/K_d ratio can likely be used for detailed quantitative analysis of DNA polymerases.Abbreviations epATP adenosine 2,3-riboepoxide 5-triphosphate - KF Klenow fragment of E. coli DNA polymerase I - Pol I E. coli DNA polymerase I - Pol human placenta DNA polymerase      

Analysis of DNA Sequencing Reaction Products Made with 7-Halo-7-deaza-2′-deoxyguanosine-5′-triphosphate

Abstract

7-Chloro- and 7- Iodo-7-Deaza-2′-Deoxyguanosine-5′-Triphosphates were employed as substrates replacing dGTP, dITP, or 7-Deaza-2′-Deoxyguanosine-5′-Triphosphate in sequencing reactions with Thermo Sequenase^TM. Analysis of the reaction products by denaturing gel electrophoresis indicates DNA containing these halogenated analogs can form strong secondary structures.     

Optimization of adenosine 5′-triphosphate extraction for the measurement of␣acidogenic biomass utilizing whey wastewater

A set of experiments was carried out to maximize adenosine 5′-triphosphate (ATP) extraction efficiency from acidogenic culture using whey wastewater. ATP concentrations at different microbial concentrations increased linearly as microbial concentration decreased. More than 50% of ATP was extracted from the sample of 39 mg volatile suspended solids (VSS)/l compared to the sample of 2.8 g VSS/l. The ATP concentrations of the corresponding samples were 0.74±0.06 and 0.49±0.05 mg/l, respectively. For low VSS concentrations ranging from 39 to 92 mg/l, the extracted ATP concentration did not vary significantly at 0.73±0.01 mg ATP/l. Response surface methodology with a central composite in cube design for the experiments was used to locate the optimum for maximal ATP extraction with respect to boiling and bead beating treatments. The overall designed intervals were from 0 to 15 min and from 0 to 3 min for boiling and bead beating, respectively. The extracted ATP concentration ranged from 0.01 to 0.74 mg/l within the design boundary. The following is a partial cubic model where η is the concentration of ATP and x _k is the corresponding variable term (k=boiling time and bead beating time in order): η=0.629+0.035x ₁–0.818x ₂–0.002x ₁ x ₂–0.003x ₁ ² +0.254x ₂ ² +0.002x ₁ ² x ₂. This model successfully approximates the response of ATP concentration with respect to the boiling- and bead beating-time. The condition for maximal ATP extraction was 5.6 min boiling without bead beating. The maximal ATP concentration using the model was 0.74 mg/l, which was identical to the experimental value at optimum condition for ATP extraction.