Radiochromatography of 5-3H-uridine derivatives found in trichloracetic and soluble fractions of mouse brain, liver, muscle and blood.

Trichloracetic acid soluble fractions from the mouse brain, liver, muscle and blood were chromatographed in three solvent systems after a subcutaneous injection of 5-3H-uridine (boric acid -- ethanol -- water -- ammonium hydroxide; n-butanol -- acetic acid -- water; IM ammonium acetate -- 95% ethanol). The amount of uridine converted to uracil in the brain and blood highly prevailed over that phosphorylated to UMP. On the contrary, the amount of 3H-;uracil was small in the LIVER AND MUSCLe, the majority of 3H-uridine being converted to UMP.     

Encephalomyocarditis virus replication complexes preferentially utilizing nucleoside diphosphates as substrates for viral RNA synthesis. Nucleotide kinases specifically associated with the complex channel RNA precursor

Replication complexes (RC) of the encephalomyocarditis (EMC) virus were shown previously to contain components that exhibit marked preference for nucleoside diphosphates over nucleoside triphosphates (NTP) as substrates for viral RNA synthesis [Koonin and Agol (1983), Virology 129, 309-318]. These NDP-preferring components have now been found to posses the following properties. When RC preparations were fractionated by sucrose density gradient centrifugation, the fractions containing NDP-preferring components exhibited a considerably higher nucleotide kinase activity as compared to either the fractions containing NTP-preferring components or corresponding fractions from mock-infected cells. When NDP-preferring RC were incubated with ADP and three other NTP, very low concentrations of endogenously generated ATP ensured a greater rate of RNA synthesis than did much higher concentrations of exogenous ATP. When an equimolar mixture of differently labelled UDP and UTP was used as a substrate for NDP-preferring RC, the label from UDP predominated in the newly synthesized RNA, even though the UDP-derived UTP constituted a minor portion of the total UTP pool. When labelled UDP was diluted with unlabelled uridine nucleotides, unlabelled UTP proved to be far less efficient than unlabelled UDP in diminishing the specific radioactivity of UMP incorporated into RNA by NDP-preferring RC. These data are interpreted in the sense that the NTP generated by the built-in nucleotide kinase system are not freed into the external milieu but rather form a separate pool preferentially used for synthesis of viral RNA by NDP-preferring RC. It is suggested that this functional compartmentation of NTP may be significant for the replication of viral RNA in vivo.     

Liver-specific increase of UTP and UDP-sugar concentrations in rats induced by dietary vitamin B6-deficiency and its relation to complex N-glycan structures of liver membrane-proteins

This is the first known report on the influence of vitamin B6-deficiency on the concentration of UDP-sugars and other uracil nucleotides in rats. Animals aged 3 weeks or 2 months were fed a vitamin B6-free diet for periods varying from 3 days to 7 weeks. Nucleotides were quantified by enzymatic-photometry and by SAX-high precision liquid chromatography. In 3 week-old rats, vitamin B6-deficiency resulted in an up to 6.3-fold increase in the concentrations of UTP, UDP, UMP and UDP-sugars and less of CTP in rat liver, while no changes were observed in older rats. In young rats, the concentration of uracil nucleotides started to increase after 1 week diet, with a maximum after 2 weeks. After 5 weeks, the concentrations returned to normal values. In heart, lungs, kidney and brain, concentrations were measured after 2 weeks diet in young rats. In contrast to liver, the heart muscle uracil nucleotide concentrations were decreased by 40%. In kidney, the sum of UTP, UDP and UMP showed a decrease of 40%, whereas UDP-sugars were increased 1.4-fold. In the lungs, nucleotide concentrations were mostly unaffected by vitamin B6-deficiency, despite a 70% increase of UDP-GA. In brain, UDP-Glc, UDP-Gal and the sum of CTP and CDP showed an increase of 30–50%. We became surprised that the increased UDP-sugar concentrations did not influence the structure of liver plasma membrane-N-glycans. Despite the 4 to 6-fold increase of UTP and UDP-sugars, no changes in the complexity or sialylation of these N-glycans could be detected. This study demonstrates that, especially in liver, pyridoxal phosphate is closely involved in the control of uracil nucleotides during a defined period of development. In contrast to in vitro experiments, in vivo N-glycan biosynthesis in liver is regulated by a more complex or higher mechanism than substrate concentrations. Agnes B. Renner and Kathrin Rieger contributed equally.     

Further studies on the diurnal rhythm of uridine incorporation into RNA in the long-day duckweed, Lemna gibba G3

Using the long-day duckweed Lemna gibba G3, the changes in theactivities of RNA synthesis in isolated nuclei and chloroplastsand of the reaction prerequisite for the incorporation of exogenousuridine into RNA were examined. When the duckweed was exposedto either a light-dark cycle or to continuous light, the activityof RNA synthesis in the nuclear and chloroplast fractions changeddiurnally and reached its highest levels during the night phase.The changes coincided with uridine incorporation into RNA invivo. However, the amount of radioactive uridine taken up intothe acid-soluble fraction remained unchanged during the wholeday. The proportion of radioactivity incorporated into phosphorylateduridine compounds as well as UTP+UDP to the radioactivity inthis fraction remained constant. Thus, the diurnal rhythm ofuridine incorporation into RNA was related to the diurnal rhythmof RNA synthesis in isolated nuclei and chloroplasts. The loweractivity of uridine incorporation into RNA under continuousdarkness may be determined by the activity of RNA synthesisin nuclei and chloroplasts as well as the uptake rate of uridineinto the duckweed cells, not by the activity of its phosphorylation. (Received August 30, 1977; )     

Studies on compartmentation of uridine 5'-triphosphate during synthesis of cytoplasmic and plastid ribosomal RNA's in Euglena gracilis

• 1.1. Compartmentation of uridine 5'-triphosphate (UTP) was studied during synthesis of cytoplasmic ribosomal RNA (cyt-rRNA) and plastid ribosomal RNA (pl-rRNA) in photoorganotrophically grown cells of Euglena gracilis Z.
• 2.2. Using the approach of Wiegers et al. (1976) the steady state specific radioactivity of UTP was compared with that ofcyt-20S rRNA, cyt-25S rRNA, pl-16S rRNA and pl-23S rRNA under low and at 100-fold higher specific radioactivity of exogenously fed pHl-uracil.
• 3.3. The equal steady state specific radioactivities of all rRNAs at both feeding conditions argue against compartmentation of UTP during their synthesis.
• 4.4. At high specific radioactivity of exogenous [³H]-uracil the salvage-derived labelled UMP was shown to be diluted 15,000-fold by unlabelled UMP formed de novo, whereas this dilution factor was 100-fold lower at low specific radioactivity of [³H]-uracil indicating inhibition of the de novo synthesis of UMP.
• 5.5. Transport is suggested of uridine nucleotides into chloroplasts by the 15-fold higher specific radioactivity of intracellular [³H]-uracil than that of UTP as well as UMP residues in pl-rRNA.

     

Polyribonucleotide synthesis by subfractions of microsomes from rat liver

1. The microsome fraction of rat liver has been fractionated and the ability of the fractions to incorporate ribonucleotides into polyribonucleotides has been studied. Activity was found in the rough-surfaced vesicle (light) fraction and in the free-ribosome fraction and this latter activity has been examined. 2. The free-ribosome fraction contains ribosome monomers, dimers and trimers together with some higher oligomers and ferritin. In addition to catalysing the incorporation of ribonucleotides into acid-insoluble material it contains diesterase activity. It catalyses the incorporation of UMP from UTP, but not UDP, AMP from ATP and CMP from CTP into polyribonucleotide material, and for UTP the product appears to be a homopolymer not more than eight units long attached to the ends of primer polyribonucleotide strands. 3. The activity could not be removed from the free-ribosome fraction by washing or by isolation in the presence of ethylenediaminetetra-acetic acid. 4. Partially hydrolysed polyuridylic acid but not polyadenylic acid could serve as a primer for the incorporation of UMP, but some activity was always associated with an endogenous primer. 5. Analysis of RNA extracted from the free-ribosome fraction after incubation with [³H]UTP showed the presence of 28s, 18s, 5s and transfer RNA types, but no radioactivity was associated with any of these RNA fractions.     

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.     

Uridine Metabolism in the Goldfish Retina During Optic Nerve Regeneration: Whole Retina Studies

Accumulation of radioactivity from [3H]uridine in incubations of whole goldfish retinas is increased in the ipsilateral retina during a period of regeneration that follows unilateral optic nerve crush. Brief incubations to investigate the nature of enhanced labeling of the acid-soluble fraction showed a peak uptake 4 days following crush, with a gradual decrease to control levels by 21 days following crush. That nucleoside uptake may not mediate the effect is supported by the observation that the rate of uptake of 5'-deoxyadenosine, a nonmetabolizable nucleoside analog, is the same in post-crush (PC) and normal (N) retinal incubations. Following brief incubations of PC and N retinas with [3H]uridine, there is enhanced labeling in PC retinas relative to N retinas of recovered UMP, UDP, UTP, and uridine nucleotide sugars, whereas recovery of labeled uridine itself is slightly decreased. The results suggest that the increased accumulation of radioactivity in PC retinas following incubation with uridine reflects an increase in the activities of retinal uridine kinase and uridine nucleotide kinases.     

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.     

Structural and functional characterization of Escherichia coli UMP kinase in complex with its allosteric regulator GTP

Bacterial UMP kinases are essential enzymes involved in the multistep synthesis of UTP. They are hexamers regulated by GTP (allosteric activator) and UTP (inhibitor). We describe here the 2.8 angstroms crystal structure of Escherichia coli UMP kinase bound to GTP. The GTP-binding site, situated at 15 angstroms from the UMP-binding site and at 24 angstroms from the ATP-binding site, is delineated by two contiguous dimers. The overall structure, as compared with those bound to UMP, UDP, or UTP, shows a rearrangement of its quaternary structure: GTP induces an 11 degrees opening of the UMP kinase dimer, resulting in a tighter dimer-dimer interaction. A nucleotide-free UMP kinase dimer has an intermediate opening. Superposition of our structure with that of archaeal UMP kinases, which are also hexamers, shows that a loop appears to hamper any GTP binding in archeal enzymes. This would explain the absence of activating effect of GTP on this group of UMP kinases. Among GTP-binding residues, the Asp-93 is the most conserved in bacterial UMP kinases. In the previously published structures of E. coli UMP kinase, this residue was shown to be involved in hydrogen bonds between the subunits of a dimer. Its substitution by an alanine decreases the cooperativity for UTP binding and suppresses the reversal by GTP of UTP inhibition. This demonstrates that the previously described mutual exclusion of these two nucleotides is mediated by Asp-93.     

Effect of divalent cations on the inhibition of alkylsulfatase induction and the generation of ATP in Pseudomonas aeruginosa after exposure to exogenous uridine 5'-triphosphate.

In the absence of added Mg2+, alkylsulfatase induction in resting cells of Pseudomonas aeruginosa was inhibited 17% by exogenous 0.05 mM UTP. Under these conditions, the cells converted UTP to ATP and rapid degradation of these nucleotides did not occur. In the presence of 0.73 mM Mg2+, 0.05 mM UTP repressed the synthesis of the enzyme by 71%. Under these conditions, the cells rapidly degraded both ATP derived from UTP as well as residual UTP. In the presence of Mg2+ and 0.1 mM UTP, full repression of alkylsulfatase formation occurred whereas Mg2+-depleted cell suspensions were still capable of synthesizing 47% of the enzyme under these conditions compared with control levels. The inhibition of alkylsulfatase induction was highly specific for UTP. Some inhibition was observed with exogenous uracil, uridine, and pyrophosophate but only at concentrations greater than 1.0 mM. Exogenous UMP and UDP (2mM) had no effect.     

Structure of Escherichia coli UMP kinase differs from that of other nucleoside monophosphate kinases and sheds new light on enzyme regulation

Bacterial UMP kinases are essential enzymes involved in the multistep synthesis of nucleoside triphosphates. They are hexamers regulated by the allosteric activator GTP and inhibited by UTP. We solved the crystal structure of Escherichia coli UMP kinase bound to the UMP substrate (2.3 A resolution), the UDP product (2.6 A), or UTP (2.45 A). The monomer fold, unrelated to that of other nucleoside monophosphate kinases, belongs to the carbamate kinase-like superfamily. However, the phosphate acceptor binding cleft and subunit assembly are characteristic of UMP kinase. Interactions with UMP explain the high specificity for this natural substrate. UTP, previously described as an allosteric inhibitor, was unexpectedly found in the phosphate acceptor site, suggesting that it acts as a competitive inhibitor. Site-directed mutagenesis of residues Thr-138 and Asn-140, involved in both uracil recognition and active site interaction within the hexamer, decreased the activation by GTP and inhibition by UTP. These experiments suggest a cross-talk mechanism between enzyme subunits involved in cooperative binding at the phosphate acceptor site and in allosteric regulation by GTP. As bacterial UMP kinases have no counterpart in eukaryotes, the information provided here could help the design of new antibiotics.     

Compartmentation of uridine 5′-triphosphate occurs during synthesis of cytoplasmic and mitochondrial ribosomal RNAs of cultured tomato cells but not during synthesis of cytoplasmic ribosomal and transfer RNAs

Compartmentation of uridine 5-triphosphate (UTP) was studied during the nucleolar synthesis of cytoplasmic ribosomal RNA (cyt-rRNA) and the synthesis of cytoplasmic transfer RNA (cyt-tRNA) in the nuclear matrix as well as the synthesis of mitochondrial ribosomal RNA (mt-rRNA) in tomato (Lycopersicon esculentum Mill. cv. Lukullus) cell-suspension culture using the approach of Wiegers et al. (Eur. J. Biochem. 64, 535–540, 1976). Before measurements were made, it was ensured that: (i) there was steady-state labeling of all RNAs studied as well as UTP; (ii) there was stability of cyt-tRNA and cyt-rRNA; (iii) there was no label randomization through degradation of [³H]uridine; (iv) there were significant differences in the specific radioactivity of UTP, the final immediate precursor of RNA, after supplying the cells with two different exogenous [³H]uridine concentrations.By comparing the steady-state specific radioactivity of UTP with that of cyt-tRNA and cyt-18S rRNA during constant [³H]uridine supply, we found that the three molecules had equal specific radioactivities which, however, differed significantly from that of the mt-rRNA. With a 20-fold higher uridine concentration, i.e. a 20-fold lower specific radioactivity of exogenous [³H]uridine, the specific radioactivity of cyt-rRNA, cyt-tRNA and UTP decreased proportionally whereas that of mt-RNA increased. These results argue against different UTP pools during synthesis of cyt-rRNA and cyt-tRNA, but indicate compartmentation of UTP during rRNA synthesis in the nucleus and the mitochondria of tomato cells.Abbreviations CMP cytidine 5-monophosphate - cyt-rRNA cytoplasmic ribosomal RNA - cyt-tRNA cytoplasmic transfer RNA - mt-rRNA mitochondrial rRNA - NC nitrocellulose - PAGE polyacrylamide gel electrophoresis - TLC thin-layer chromatography - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol - UDP uridine 5-diphosphate - UMP uridine 5-monophosphate - UTP uridine 5-triphosphate     

Amino acid dependent and independent insulin stimulation of cartilage metabolism

The effects of insulin on embryonic chicken cartilage in organ culture and the dependence of these effects on essential amino acids have been studied. In the presence of all essential amino acids, insulin: (1) increases 2-deoxy-D-glucose and alpha-aminoisobutyric acid uptake; (2) increases [5(-3H] uridine flux into uridine metabolites and the intracellular UTP pool; (3) expands the size of the intracellular UTP pool; (4) does not change the specific activity of the UTP pool; and (5) stimulates RNA, proteoglycan, and total protein synthesis. In lysine (or other essential amino acid)-deficient medium, the effects of insulin are different. While insulin stimulates incorporation of [5(-3)H] uridine into RNA, it does so by increasing the specific activity of the UTP pool without increasing RNA synthesis. Insulin stimulates 2-deoxy-D-glucose and alpha-aminoisobutyric acid uptake but no longer stimulates proteoglycan, total protein, or RNA synthesis or expands the size of the UTP pool. These data indicate that there are amino acid dependent and independent effects of insulin on cartilage. Transport processes are amino acid independent, while synthetic processes are amino acid dependent.     

Changes in Levels of Purine and Pyrimidine Nucleotides During Acute Hypoxia and Recovery in Neonatal Rat Brain

Neonatal rat brains were examined for changes in levels of ATP, ADP, AMP, cyclic AMP, GTP, GDP, UTP, UDP, UMP, and CTP during exposure to 100% nitrogen for 20 min and subsequent recovery in air. During hypoxia, ATP, GTP, UTP, and CTP levels and the GTP/GDP ratio decreased to 38, 50, 26, 21, and 21%, respectively, of control levels. No significant change in cyclic AMP level was observed. The decrease in the total uridine nucleotide pool during hypoxia was markedly greater (to 53% of control levels) than that in the total adenine nucleotide pool (to 92% of control levels). During recovery, ATP and GTP levels were rapidly and almost completely restored. On the other hand, CTP levels returned slowly to control values after a 2-h recovery period. Restoration of the UTP level was slow and incomplete (87% of the control value even after a 3-h recovery period). The GTP/GDP ratio also did not return to normal. These data suggest that hypoxic insult to the neonate may have an effect on the synthesis of nucleotidyl sugars, phospholipids, and proteins in the brain, resulting in significant problems with developmental processes of the brain. The present study also showed that the delayed restorations of the UTP level and the GTP/GDP ratio were not seen in the brains of adult rats subjected to acute severe hypoxic insult.     

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.     

Distribution of 125I-labeled rat growth hormone in regional brain areas and peripheral tissue of the rat.

The uptake of intraperitoneally injected 125I-labeled rat growth hormone into brain and peripheral tissues was measured in normal and hypophysectomized adult rats. A significant level of radioactivity was observed in the seven brain regions examined -- the telencephalon, diencephalon, midbrain, pons-medulla, cerebellum, pineal and pituitary glands. The pineal and pituitary glands, which are outside the blood-brain barrier, contained three to four times the concentration of radioactivity of the other brain regions. Compared to brain, the level of radioactivity was much higher in peripheral tissues (the diaphragm, kidney, serum and liver). For example, the serum contained ten times the level of radioactivity of most brain regions. For a given tissue, however, the normal and hypophysectomized rats showed a comparable amount of 125I-growth hormone. Trichloroacetic acid precipitates from each tissue sample showed that peripheral tissues had a higher proportion of radioactivity (35-48% of total tissue radioactivity) than the brain samples (13-26%). The data support the view that growth hormone, or a metabolite can enter the central nervous system and may directly affect on-going metabolic processes.     

Alteration in de novo pyrimidine biosynthesis during uridine reversal of pyrazofurin-inhibited DNA synthesis

Pyrazofurin, a pyrimidine nucleoside analogue with antineoplastic activity, inhibits cell proliferation and DNA synthesis in cells by inhibiting uridine 5'-phosphate (UMP) synthase. It has been previously shown in concanavalin A (con A)-stimulated guinea pig lymphocytes (23) that pyrazofurin-inhibited DNA synthesis could be selectively reversed by exogenous uridine (Urd). In this report, we have examined possible mechanisms for the Urd reversal with experiments that determine the ability of exogenous Urd to (a) interfere with either the intracellular transport of pyrazofurin, or the conversion of pyrazofurin to its intracellularly active form, pyrazofurin-5'-phosphate; (b) reverse the pyrazofurin block of [14C]orotic acid incorporation into DNA; and (c) alter the pattern of exogenous [3H]Urd incorporation into DNA-thymine (DNA-Thy) and DNA-cytosine (DNA-Cyt) during pyrazofurin inhibition of pyrimidine de novo biosynthesis. The results of these experiments showed that Urd reversal does not occur through altered pyrazofurin transport or intracellular conversion to pyrazofurin-5'-phosphate, nor does it alter the distribution of [3H]Urd in DNA-Thy and DNA-Cyt. Instead, these findings indicate that the primary mechanism for exogenous Urd reversal of pyrazofurin inhibition of DNA synthesis involves the reversal of pyrazofurin inhibition of UMP synthase, thus restoring orotic acid incorporation into lymphocyte DNA through the pyrimidine de novo pathway.     

The relationship between the turnover of UTP and RNA synthesis in cultured cells treated with aflatoxin B1.

Methods have been adapted to measure the specific activity of UTP in cells in monolayer culture. In HeLa cells labelled with [3H]uridine and treated with aflatoxin B1 there was reduced radioactivity in crude acid extracts, but the toxin did not affect the radioactive incorporation into UTP. Using cells in which the UTP was pre-labelled, the subsequent addition of aflatoxin B1 inhibited UTP incorporation into RNA. Accordingly aflatoxin B1 did not inhibit the uptake of uridine or the latter's conversion to UTP but inhibited the incorporation of UTP into RNA.