Stimulation of protocollagen proline hydroxylase activity by nucleoside triphosphates.

Activity of purified protocollagen proline hydroxylase was enhanced several fold by addition of nucleoside triphosphates (3 mM) to the assay medium, but nucleoside mono-and diphosphates were almost inactive. Pyrimidine nucleotides were less effective compared with purine nucleotides, among which GTP was the most effective. dATP and ATP analogues such as adenosine 5′-(β,γ-imino) triphosphate (AMP-PNP), adenosine 5′-(β,γ-methylene) triphosphate (AMP-PCP), etc. were inactive. ATP or GTP showed no additive effect on enzyme activity stimulated by dithiothreitol or bovine serum albumin.     

PURINE AND PYRIMIDINE NUCLEOTIDES IN THE BRAIN OF NORMAL AND CONVULSANT RATS

Abstract— Purine and pyrimidine nucleotides were measured in the brain of normal and electroshocked rats after chromatographic separation on ion-exchange resin of mono-, di- and tri-phosphorylated derivatives.
CMP, IMP and NAD did not show any significant quantitative change. Adenine nucleotides showed an abrupt change followed by a rapid return to the control value. GTP was the only purine nucleotide exhibiting a relatively slow return to its starting concentration. The greatest percentage increase after electroshock was observed in UMP, which returned to its control value only after 5 min; UDPCoenzymes (i.e. UDPA plus UDPG) showed a relatively small drop during the development of the seizure and the slowest return to the base line; UTP showed a late transistory increase above the normal level after an initial drop associated with convulsant activity.
Tritiated uridine was injected intracisternally to investigate the turnover of pyrimidine nucleotides. UTP showed the highest specific radioactivity at the earliest time, followed by UMP, UDPCoenzymes and CMP. It was found that convulsant activity is associated with dramatic changes in the specific radioactivity of pyrimidine nucleotides.     

Acetic anhydride requirement in the colorimetric determination of tryptophan

Nucleic acid research frequently necessitates the analytical resolution of nucleic acid derivatives. Thin-layer chromatography (tlc), for its simplicity, short development time, and superior resolving power, is often preferable to other methods (1–3). Although the literature contains a large number of methods that have been devised for the separation of purine and pyrimidine derivatives (4,5) no tlc technique has hitherto been described for the concomitant separation of bases, nucleosides, nucleoside 5′-monophosphates, nucleoside 3′-monophosphates, nucleoside diphosphates, and nucleoside triphosphates.The present communication deals with methods devised for the simultaneous separation of the above-mentioned pyrimidine derivatives. They enable the resolution of either the six uracil derivatives or the six cytosine derivatives, on commercial cellulose tlc sheets. Alternatively, the six pyrimidine derivatives can be separated on cellulose layers 0.75 mm thick. Since formic acid extracts of bacterial cells do not interfere with the separation, these methods can be used for the direct estimation of extracts of biological materials.     

Chromium(III) interactions with nucleotides. III

Some new derivatives of Cr(III) with 5′AMP, 5′ATP, 5′CMP, 5′GMP, 5′IMP and 5′UMP have been obtained by reaction of the starting complexes cis and trans-[Cr(en)₂Cl₂]Cl with the above nucleotides.The complexes were characterized by elemental analysis, conductivity, infrared and electronic spectroscopy, and EPR for the 5′UMP derivative.In all cases, chlorine has been substituted and one ethylenediamine eliminated. The interaction of Cr(III) with the nucleotide seems to occur through the phosphate group and additional interaction through the heterocyclic ring especially for the 5′GMP and 5′IMP derivatives.The 5′UMP complex seems to be a dimer and the other complexes are polymer.     

Synthesis and P2Y receptor activity of nucleoside 5′-phosphonate derivatives

Ribose-based nucleoside 5′-diphosphates and triphosphates and related nucleotides were compared in their potency at the P2Y receptors with the corresponding nucleoside 5′-phosphonate derivatives. Phosphonate derivatives of UTP and ATP activated the P2Y₂ receptor but were inactive or weakly active at P2Y₄ receptor. Uridine 5′-(diphospho)phosphonate was approximately as potent at the P2Y₂ receptor as at the UDP-activated P2Y₆ receptor. These results suggest that removal of the 5′-oxygen atom from nucleotide agonist derivatives reduces but does not prevent interaction with the P2Y₂ receptor. Uridine 5′-(phospho)phosphonate as well as the 5′-methylenephosphonate equivalent of UMP were inactive at the P2Y₄ receptor and exhibited maximal effects at the P2Y₂ receptor that were ?50% of that of UTP suggesting novel action of these analogues.     

Changes in Individual Nucleotides of Developing Soybean Seeds

Perchloric acid extracts of soybean seeds were separated by column chromatography into 17 peaks. Thirteen nucleotides were identified in 11 of these peaks (NAD, CMP, CDP, CTP, AMP, ADP, ATP, GMP, GDP, GTP, UMP, UDP, and UTP). Two of the peaks were identified as ascorbic acid and dehydroascorhic acid. Four peaks remain unidentified although one of these appears to be protein. Monophosphates decreased and triphosphates increased as development proceeded. Diphosphates were initially low and showed no statistically significant difference with maturity. The accumulation of triphosphates in late stages of development is discussed in relation to synthetic activities of the developing seeds.     

A quantitative method for the measurement of cellular guanosine triphosphate pool specific activities

Studies on quantitation of RNA synthesis in eucaryotic cells have frequently used adenosine as the radioactively labeled precursor, largely because of the convenience of the firefly luciferin-luciferase assay in measuring ATP pool specific activity (1,2). This could result in some difficulties if the addition of poly(A) to the 3′ OH end of RNA represents a significant portion of total incorporation, as is the case in sea-urchin embryos (3). In addition, in some cases, the ATP pool may be large enough to prevent the use of adenosine as an effective labeling agent. Hence, a simple and sensitive method for the determination of the specific activity of the other nucleic acid precursor pools would be of value.Although the crystalline luciferase is specific for ATP, extracts of firefly lanterns most commonly used for quantitating ATP (4–9) also exhibit activity with other ribonucleoside triphosphates, adenosine tetraphosphate, ADP, and the deoxyribonucleoside triphosphates. This activity is due to the presence of contaminating enzymes such as nucleoside 5′-diphosphate kinase and adenylate kinase which catalyze the formation of ATP from these nucleotides and trace amounts of ADP, also present in the extracts (10–13). Recently, Manandhar and Van Dyke (14) have reported a procedure for quantitating picomole levels of GTP with a crude extract of firefly lanterns. In the present study, we have adapted their procedure to develop an assay for GTP pool specific activity in Xenopus laevis oocytes microinjected with [8-³H]GTP. Our assay may be extended to the analysis of any nucleoside triphosphate pool, provided that an adequate chromatography system is available for the separation of the extracted nucleotides.     

Nucleotide pools of growing,synchronized and stressed cultures of Saccharomyces cerevisiae

High pressure liquid chromatography has been used to study the acid soluble nucleotide pool of Saccharomyces cerevisiae under different conditions of growth. ATP, ADP, AMP, NAD, GTP, UTP, UDP, CTP, CDP, and UDP-sugars plus UMP could be separated and were found in concentrations higher than 0.1 mumol per g yeast cell dry weight (= detection limit). During glucose-limited continuous culture the levels of individual nucleotides depended on the growth rate, which was most pronounced with pyrimidine (uridine, cytidine) nucleotides. The energy charge (E.C.) remained high (0.9) at all growth rates (0.07-0.3 h-1). During synchronized growth at a constant growth rate (0.11 h-1) almost all nucleotide levels and the E.C. remained at constant values with the only exception of UDP-sugars and UMP of which increased levels were found during the phase of budding. Under conditions of metabolic stress (addition of antimycin A, deoxyglucose plus iodoacetate) pronounced changes in the levels of purine (adenine and guanine) nucleotides and the E.C. were observed. All other nucleotides were less influenced by these conditions. Only under these conditions IMP accumulation was observed. The results strongly argue against the significance of purine nucleotide or E.C. measurements under viable conditions. In contrast, changes in the levels of pyrimidine nucleotides seem to be indicative of changes in the flux through the metabolic pathways where they act as coenzymes.     

Chromium(III) interactions with nucleotides. II

New derivatives were obtained from Cr(urea)₆Cl₃· 3H₂O in an ethyl acetate medium of chromium(III) with uracil, uridine, 5′UMP, 5′CMP, 5′GMP and 5′IMP. The new derivatives were characterized by elemental analysis, electronic and infrared spectroscopy and thermal analysis. These derivatives proved to be outer sphere complexes, in which the nucleotide, the nucleoside or the base interacts with the starting complex through intramolecular hydrogen bonding.Cr(XMP)(OH)·3H₂O (XMP: 5′UMP, 5′CMP, 5′GMP and 5′IMP) complexes were obtained by hydrolysis of the above derivatives of the nucleotides. In these reactions there is a total substitution of the urea molecules. The derivatives obtained by hydrolysis were characterized in solid state by electronic and infrared spectroscopy. These results provide more insight into the biological role of chromium.     

Effect of nucleotide cofactor structure on recA protein-promoted DNA pairing. 1. Three-strand exchange reaction.

The structurally related nucleoside triphosphates, adenosine triphosphate (ATP), purine riboside triphosphate (PTP), inosine triphosphate (ITP), and guanosine triphosphate (GTP), are all hydrolyzed by the recA protein with the same turnover number (17.5 min-1). The S0.5 values for these nucleotides increase progressively in the order ATP (45 microM), PTP (100 microM), ITP (300 microM), and GTP (750 microM). PTP, ITP, and GTP are each competitive inhibitors of recA protein-catalyzed ssDNA-dependent ATP hydrolysis, indicating that these nucleotides all compete for the same catalytic site on the recA protein. Despite these similarities, ATP and PTP function as cofactors for the recA protein-promoted three-strand exchange reaction, whereas ITP and GTP are inactive as cofactors. The strand exchange activity of the various nucleotides correlates directly with their ability to support the isomerization of the recA protein to a strand exchange-active conformational state. The mechanistic deficiency of ITP and GTP appears to arise as a consequence of the hydrolysis of these nucleotides to the corresponding nucleoside diphosphates, IDP and GDP. We speculate the nucleoside triphosphates with S0.5 values greater than 100 microM will be intrinsically unable to sustain the strand exchange-active conformational state of the recA protein during ongoing NTP hydrolysis and will therefore be inactive as cofactors for the strand exchange reaction.     

Identification and biochemical characterization of a unique Mn2+-dependent UMP kinase from Helicobacter pylori

Uridine monophosphate (UMP) kinase converts UMP to the corresponding UDP in the presence of metal ions and ATP and is allosterically regulated by nucleotides such as UTP and GTP. Although the UMP kinase reported to date is Mg²⁺-dependent, we found in this study that the UMP kinase of Helicobacter pylori had a preference for Mn²⁺ over Mg²⁺, which may be related to a conformational difference between the Mn²⁺-bound and Mg²⁺-bound UMP kinase. Similar to previous findings, the UMP kinase activity of H. pylori UMP kinase was inhibited by UTP and activated by GTP. However, a relatively low GTP concentration (0.125 mM) was required to activate H. pylori UMP kinase to a level similar to other bacterial UMP kinases using a higher GTP concentration (0.5 mM). In addition, depending on the presence of either Mg²⁺ or Mn²⁺, a significant difference in the level of GTP activation was observed. It is therefore hypothesized that the Mg²⁺-bound and Mn²⁺-bound H. pylori UMP kinase may be activated by GTP through different mechanisms.     

Multiple effects of guanine nucleotides on human platelet adenylate cyclase

We report that the adenylate cyclase system in human platelets is subject to multiple regulation by guanine nucleotides. Previously it has been reported that GTP is either required for or has little effect on the response of the enzyme to prostaglandin E₁. We have found that when platelet lysates were prepared in the presence of 5 mM EDTA, GTP lowered the basal and prostaglandin E₁-stimulated adenylate cyclase activity when the enzyme was assayed in the presence of Mg²⁺. The basal and prostaglandin E₁-stimulated adenylate cyclase activities were also increased by washing, which presumably removes endogenous GTP. The analog, guanyl-5′-yl-imidodiphosphate mimics the inhibitory effect of GTP on prostaglandin E₁-stimulated adenylate cyclase activity but it stimulates basal enzyme activity. The onset of the inhibitory effect of GTP on the adenylate cyclase system is rapid (1 min) and is maintained at a constant rate during incubation for 10 min. GTP and guanyl-5′-yl-imidodiphosphate were noncompetitive inhibitors of prostaglandin E₁. An increase in the concentration of Mg²⁺ gradually reduces the effect of GTP while having little influence on the effect of guanyl-5′-yl-imidodiphosphate. Neither the substrate concentration nor the pH (7.2–8.5) is related to the inhibitory effect of guanine nucleotides. The inhibition by nucleotides was found to show a specificity for purine nucleotides with the order of potency being guanyl-5′-yl-imidodiphosphate > dGTP > GTP > ITP > XTP > CTP > TTP. The inhibitory effect of GTP is reversible while the effect of guanyl-5′-yl-imidodiphosphate is irreversible. The GTP inhibitory effect was abolished by preparing the lysates in the presence of Ca²⁺. However, the inhibitory effect of guanyl-5′-yl-imidodiphosphate persisted. Substitution of Mn²⁺ for Mg²⁺ in the assay medium resulted in a diminution of the inhibitory effect of GTP on basal activity and converted the inhibitory effect of GTP on prostaglandin E₁-stimulated activity to a stimulatory effect. At a lower concentration of Mn²⁺ (less than 2 mM) guanyl-5′-yl-imidodiphosphate inhibited prostaglandin E₁-stimulated adenylate cyclase activity, but at a higher concentration of Mn²⁺, it caused an increase in enzyme activity exceeding that occuring in the presence of prostaglandin E₁. In the presence of Mn²⁺, dGTP mimics the effect of GTP and is 50% as effective as GTP. Our data suggest that the inhibitory effect of GTP on prostaglandin E₁-stimulated adenylate cyclase is mainly due to its direct effect on the enzyme itself, whereas the stimulatory effect of GTP on prostaglandin E₁-stimulated adenylate cyclase is due to enhancement of the coupling between the prostaglandin E₁ receptor and adenylate cyclase. These studies also indicate that the method of preparation of platelet lysates can profoundly alter the nature of guanine nucleotide regulation of adenylate cyclase.     

Quantification of ribonucleotides and uridine metabolism in ovaries of the kelp fly (Coelopa frigida,Diptera) during final stages of oogenesis

The concentrations of twelve 5′-ribonucleotides (AMP, ADP, ATP, CMP, CDP, CTP, GMP, GDP, GTP, UMP, UDP, UTP) and four ribonucleotide sugars: CDP-glucose (CDPG), UDP-glucose (UDPG), UDP-N-acetyl-glucosamine (UDPGlcNAc), and UDP-glucoronic acid (UDPGlcUA) in homogenates from Coelopa frigida ovarioles were determined by high pressure liquid chromotography. It was possible to separate the ribonucleotides from each other and from the ribonucleotide sugars in the same analysis.The amount of mono- and di-nucleotides per ovariole remains low during the final stages of oogenesis (stages 10–14). Of the mono-, di- and tri-phosphates the amounts of triphosphates per ovariole are the highest in all cases and double during this phase of follicle development. Of all nucleoside triphosphates, ATP reaches the highest concentration with an increase from 30 pmoles per ovariole to 58 pmoles between stage 10 and 14.     

The Isolation and Characterization of the Acid-Soluble Nucleotides from the Tubers of Jerusalem Artichoke (Helianthus tuberosus L.)

The acid-soluble nucleotides were extracted from the tubers of Jerusalem artichoke with percbloric acid, and separated and purified by means of adsorption on and elution from active charcoal, repeated chromatography on columns of Dowex I (Cl^-), followed by paper chromatography. The following nucleotides have been characterized and/or identified: 5′-AMP, 3′-AMP, ADP, ATP, 5′-GMP, 2′-GMP, 3′-GMP, 2′,3′-cyclic GMP, GDP, GTP, 5′-UMP, UDP, UTP, NADP, UDP-glucose, UDP-galactose, UDP-fructose, UDP-N-acetylhexosamine and GDP-mannose.^** Neither cytosine ribonucleotides nor deoxyribonucleotides have been detected. The significance of these observations is discussed.     

Extracellular metabolism of nucleotides in neuroblastoma x glioma NG108-15 cells determined by capillary electrophoresis

1. The metabolism of extracellular nucleotides in NG108-15 cells, a neuroblastoma × glioma hybrid cell line, was studied by means of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC).2. In NG108-15 cells ATP, ADP, AMP, UTP, UDP, and UMP were hydrolyzed to the nucleosides adenosine and uridine indicating the presence of ecto-nucleotidases and ecto-phosphatases. The hydrolysis of the purine nucleotides ATP and ADP was significantly faster than the hydrolysis of the pyrimidine nucleotides UTP and UDP.3. ATP and UTP breakdown appeared to be mainly due to an ecto-nucleotide- diphosphohydrolase. ADP, but not UDP, was initially also phosphorylated to some extent to the corresponding triphosphate, indicating the presence of an adenylate kinase on NG108-15 cells. The alkaline phosphatase (ALP) inhibitor levamisole did not only inhibit the hydrolysis of AMP to adenosine and of UMP to uridine, but also the degradation of ADP and to a larger extent that of UDP. ATP and UTP degradation was only slightly inhibited by levamisole.4. These results underscore the important role of ecto-alkaline phosphatase in the metabolism of adenine as well as uracil nucleotides in NG108-15 cells. Dipyridamole, a potent inhibitor of nucleotide breakdown in superior cervical ganglion cells, had no effect on nucleotide degradation in NG108-15 cells.5. Dipyridamole, which is a therapeutically used nucleoside reuptake inhibitor in humans, reduced the extracellular adenosine accumulation possibly by allosteric enhancement of adenosine reuptake into the cells.     

Acid-soluble purine and pyrimidine derivatives of growing, encysting and encystment-inhibited amoebae

The intracellular acid-soluble purine and pyrimidine derivatives of myxamoebae-swarm cells of Physarum flavicomum were investigated during growth, microcyst formation, and during adenine-inhibition of encystment, using high performance liquid chromatography (HPLC). We also studied the incorporation of exogenous radioactive adenine into the acid soluble purine derivatives and S-adenosyl-sulphur compounds separated by HPLC. The most abundant ribonucleoside monophosphate was AMP in the growing and 15 h encysting cells (NC), while it was UMP in the 15 h adenine-inhibited cells (AIC). ADP was the nucleoside diphosphate present in the greatest quantity in the growing and NC cells but it was CDP in the AIC. The nucleoside triphosphate in highest concentration was ATP, UTP, and GTP in growing, NC, and AIC, respectively. Guanosine was the most abundant nucleoside in all cells. The nucleobase occurring in greatest concentration was cytosine, cytosine and guanine, and adenine in the growing, NC, and AIC, respectively. The AMP content in the 15 h AIC was 2.1-fold higher than that of adenosine. The 15 h NC had the lowest adenylate energy charge, a value of 0.54 +/- 0.02, while the values for growing cells and the AIC were 0.62 +/- 0.02 and 0.76 +/- 0.01, respectively. [14C]-Adenine labelling studies (15 h) revealed the occurrence of purine nucleotide interconversion, as the label was detected not only in adenosine, AMP, ADP, ATP, but also in guanine, guanosine, GMP, GDP, GTP, as well as, in inosine monophosphate and xanthosine monophosphate. The percentage incorporation of the radiolabelled adenine into AMP was higher than into adenosine. An increased intracellular level of guanine nucleotides is associated with the inhibition of encystment. The extracellular adenine, rather than internal adenine sources, appears to be the primary precursor of nucleotide for S-adenosylmethionine synthesis during adenine-inhibition of encystment.     

A flow‐injection chemiluminescent method for the evaluation of the antioxidant activity of 5′‐nucleotides

In the present work, a simple chemiluminescence (CL) method coupled with flow‐injection analysis for the evaluation of antioxidant activity of 5′‐nucleotides (5′‐AMP, 5′‐CMP, 5′‐GMP, 5′‐UMP) was proposed. It is based on inhibition effect of the studied substances on CL emission of luminol–potassium ferricyanide–pyrogallol. Experiments were performed to evaluate the nature of the inhibition by 5′‐nucleotides of the CL reaction and their antioxidant activities. Based on the experimental results, it was observed that 5′‐nucleotides are available antioxidants that could effectively scavenge superoxide anion free radicals in a concentration‐dependent way. This will provide a basis for further development of the use of nucleotides. Copyright © 2009 John Wiley & Sons, Ltd.     

Analysis of Intracellular Nucleotides by Capillary Electrophoresis–Mass Spectrometry

A pilot study using capillary electrophoresis with mass spectrometry for the analysis of nucleotides in human erythrocytes is presented. Erythrocytes were incubated with 5-amino-4-imidazolecarboxamide riboside in order to mimic situation in defect of purine metabolism—AICA-ribosiduria. Characteristic AICA-ribotides together with normal nucleotides were separated by capillary electrophoresis in acetate buffer (20 mmol/L, pH 4.4) and identified on line by mass spectrometry.     

Regulatory effects of purine nucleotide analogs with liver glutamate dehydrogenase.

A total of 26 different purine nucleotides with specific modifications in the base moiety and/or in the polyphosphate chain as well as various combinations of nucleotides were tested as allosteric effectors of beef liver glutamate dehydrogenase (L-glutamate : NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3). The capacity of these nucleotide analogs to activate or to inhibit the glutamate dehydrogenase activity is expressed quantitatively and scaled between the extreme effects of ADP and GTP, respectively. The significance of distinct structural elements for the enzyme-effector interaction is discussed. While the inhibitory GTP site is less specific, accepting many natural and most modified nucleoside triphosphates as inhibitors, the activating ADP site shows a much higher specificity for nucleotides as activators.