Synthesis of ribonucleotides and their participation in ribonucleic acid synthesis by Coxiella burnetii.

Synthesis of ribonucleic acid (RNA) by the deoxyribonucleic acid-dependent RNA polymerase of Coxiella burnetii required adenosine, uridine, guanosine, and cytidine 5'-triphosphates. Cell-free preparations of this obligate intracellular procaryotic parasite had competence to phosphorylate ribonucleoside mono- and diphosphates in the presence of exogenous adenosine and guanosine 5'-triphosphates to the corresponding di- and triphosphates. C. burnetii contained about 2 nmol of adenosine 5'-triphosphate per mg of protein, which could serve as a approximately P donor for in vivo synthesis of nucleoside triphosphates. The latter were then used as substrates in the synthesis of RNA in a coordinated metabolic system with C. burnetii RNA polymerase. It is suggested that during infection the rickettsiae might obtain the nucleotides necessary for RNA synthesis from the vacuoles in which C. burnetii proliferates.     

Phosphorylation of mononucleotides and formation of cytidine 5''-diphosphate-choline and sugar nucleotides by respiration-deficient mutants of yeasts.

Respiration-deficient mutants (Rho-, petite) of Saccharomyces carlsbergensis were obtained by treatment with trypaflavin (euflavine). Dried cells of these mutants phosphorylated mononucleotides to their triphosphates and further formed not only cytidine 5'-diphosphate-choline, but also sugar nucleotides, such as uridine 5'-diphosphate-glucose, guanosine 5'-diphosphate-mannose, etc. The activities were the same or slightly greater than those of the wild strain. These results showed that energy (adenosine 5'-triphosphate) necessary for phosphorylation of mononucleotides was sufficiently supplied by the glycolysis system.     

High-performance liquid chromatographic assay for identification and quantitation of nucleotides in lymphocytes and malignant lymphoblasts

A method for the identification and quantitation of nucleotide pools in lymphocytes and leukemic blasts is described. Separation of these metabolites was performed by anion-exchange high-performance liquid chromatography using a pH and concentration gradient consisting of several linear steps.The mono-, di- and triphosphates of adenosine, cytidine, guanosine, inosine, uridine and xanthosine could conveniently be separated together with NAD⁺, cyclic AMP, NADP⁺ and uridinediphosphoglucose (UDPG).In addition, data on the accuracy and precision of the method are given and its potentials for use in the analysis of nucleotide pools in leukemic lymphoblasts are illustrated.     

Thymidine kinase activation in unfertilized sea urchin eggs by homogenization and fertilization

Kinetics of in vivo phosphorylation of ³H-thymidine taken up by sea urchin eggs was compared between unfertilized and fertilized eggs. The percentage of phosphorylated ³H-thymidine in the total acid-soluble radioactivity in the cell increased with increasing incubation time within the first several minutes of incubation in the unfertilized eggs, while nearly 100% of phosphorylation of thymidine was observed without regards to the incubation time and in spite of a tremendous increase in the net uptake of thymidine in the fertilized eggs, suggesting possible activation of thymidine kinase occurring soon after fertilization.In contrast to the in vivo finding, the thymidine kinase activity in unfertilized egg homogenates was found in general to be almost as large as that in fertilized egg homogenates. However, when the enzyme activity was assayed within a short period (30 min) after homogenization of unfertilized eggs, the activity was found to increase more or less with time after homogenization, reaching a level equal to that in fertilized egg homogenates. This enzyme activation after homogenization was especially marked in case of Pseudocentrotus eggs and sometimes amounted to a several fold increase.Preliminary investigations revealed possible involvement of some redox reaction(s) in the thymidine kinase activation during and/or after homogenization of unfertilized sea urchin eggs.     

Separation of common nucleotides,mono-, di- and triphosphates,by capillary electrophoresis

A capillary electrophoretic procedure for the separation of eleven nucleotides, 5′-mono-, di- and triphosphates of adenosine, guanosine, cytidine and uridine, has been developed. All eleven analytes can be separated in a fused-silica capillary (63 cm to the detector, I.D. 75 μm) at 20 kV in a 0.02 mol l⁻¹ phosphate-borate buffer (pH 8.0–9.0) with a separation factor ⩾1. The values of the Offord parameter calculated for individual nucleotides predict that monophosphates will migrate faster than triphosphates, and in turn triphosphates will precede diphosphates. By analogy, faster electroosmotic mobility (lower electromigration) of purine nucleotides (AP, GP) can be explained by a more voluminous structure of purine derivatives (two aromatic rings as compared to pyrimidines). Generally speaking, all compounds separated follow the Offord equation assuming that the triphosphate derivatives are ionized to the third degree forming HL³⁻ anions. This assumption is in agreement with the current knowledge about protolytic equilibria of polyphosphates. The only exception to this rule is faster migration of guanosine-5′-triphosphate (GTP) preceding uridine-5′-monophosphate (UMP) which is ascribed in part to the larger molecule of GTP and the two additional OH-groups bound to the pyrimidine ring of UMP.     

Effects of Cyclic Nucleotides and Nucleoside Triphosphates on Stalk Formation in Caulobacter crescentus

Extensive stalk elongation in Skl mutants of Caulobacter crescentus occurs when they are grown in complete medium. This stalk elongation is less pronounced in synthetic medium with glucose as the sole carbon source than in complex peptone yeast extract medium. Addition of exogenous nucleoside triphosphates (adenosine triphosphate [ATP], guanosine triphosphate [GTP], cytidine triphosphate, and uridine triphosphate) inhibits stalk elongation of the Skl mutants, whereas cyclic guanosine 3',5'-monophosphate (GMP) stimulates stalk elongation in the Skl strains grown in synthetic glucose medium. Cyclic GMP also produces stalk elongation in wild-type C. crescentus and concurrently produces a cell division defect resulting in cellular filament formation. Under conditions tested, cyclic adenosine 3',5'-monophosphate and dibutyryl cyclic adenosine monophosphate did not enhance stalk elongation. Endogenous ATP and GTP levels in the mutants are significantly lower than corresponding nucleotide concentrations of the parent wild-type strains. Control of syntheses resulting in stalk formation in C. crescentus appears to be related to intracellular concentrations of nucleotides, with cyclic GMP as a prominent candidate for an important regulatory role in this aspect of morphogenesis.     

Physiology of Rickettsiae VI. Host-independent Synthesis of Polyribonucleotides by Coxiella burnetii

Preparations of purified and disrupted suspensions of Coxiella burnetii are able to incorporate ribonucleotides into polymers in the presence of adenosine, guanosine, cytidine, and uridine triphosphates. Nucleotide incorporation requires the presence of all four ribonucleoside triphosphates. The reaction is enhanced by the addition of phosphoenolpyruvate and pyruvic kinase, and exogenous deoxyribonucleic acid, and is inhibited by deoxyribonuclease and actinomycin D. Incorporation is maximal between pH 7.0 and 8.0, and at 37 C. The synthesized polymer is relatively insensitive to deoxyribonuclease and is sensitive to ribonuclease and dilute alkaline hydrolysis. The data indicate the presence of an autonomous deoxyribonucleic acid-dependent ribonucleic acid polymerase in the rickettsial agent.     

Pyrimidine salvage pathways in adult Schistosoma mansoni

Adult Schistosoma mansoni can utilize radiolabelled cytidine, uridine, uracil, orotate, deoxycytidine and thymidine for the synthesis of its nucleic acids. In this respect, cytidine is the most efficiently utilized pyrimidine precursor. Cytosine, thymine and orotidine are transported into the parasites but not metabolized. High performance liquid chromatography analysis of the nucleobase, nucleoside and nucleotide pools from in vivo metabolic studies and assays of enzyme activities in cell-free extracts indicate the presence of nucleoside and nucleotide kinases which phosphorylate the various nucleosides to their respective nucleoside mono-, di- and triphosphates. Uridine, thymidine and deoxyuridine can also be cleaved to their respective nucleobases by uridine phosphorylase. Uracil can be converted directly to UMP by orotate phosphoribosyltransferase or by the sequential actions of uridine phosphorylase and uridine kinase. Nucleoside 5'-monophosphates were dephosphorylated by active phosphohydrolases. All enzymes tested were found in the cytosol fraction with the exception of the phosphohydrolases which were associated mainly with the particulate fraction. No deamination of cytosine, cytidine, deoxycytidine, CMP or dCMP was detected either in vivo or in vitro. The active metabolism of cytidine and absence of deamination and phosphorolysis of cytidine derivatives in schistosomes raise the possibility of using cytidine analogues for the selective treatment of schistosomiasis.     

Stimulation of protein phosphorylation during fertilization-induced maturation of Urechis caupo oocytes

Protein phosphorylation was studied during fertilization of Urechis caupo oocytes both in vivo, by measuring [³²P]phosphate incorporation into ³²P preloaded oocytes and in vitro, by measuring endogenous protein kinase and phosphatase activities in homogenates. During fertilization (and maturation) the rate of protein phosphorylation is dramatically increased. No change in the [³²P]phosphate uptake, or the nucleotide levels was observed at fertilization, so the increase cannot be attributed to changes in substrate availability. In vitro enzyme assays showed changes in protein kinase activity which approximately mirrored the changes in the in vivo phosphorylation pattern. As there were no changes in protein phosphatase activity, these results suggest the phosphorylation change results from an increase in protein kinase activity. The pattern of change, investigated by SDS-polyacrylamide gel electrophoresis, shows that proteins that were phosphorylated in the unfertilized egg become phosphorylated to a greater degree after fertilization. One protein (48 kd) undergoes an increase followed by a decrease of its phosphorylation level.     

Dinucleosidetetraphosphatase from Ehrlich ascites tumour cells: inhibition by adenosine, guanosine and uridine 5'-tetraphosphates

1. An enzyme has been partially purified from Ehrlich ascites tumour cells which specifically hydrolyses dinucleosidetetraphosphates, with Km values of around 2 microM. The products of the hydrolysis are the corresponding nucleoside tri- and monophosphates. Dinucleoside Tri- and diphosphates were not substrates of the reaction. 2. The enzyme requires Mg2+ or Mn2+, is maximally active at a pH value of approx. 7.5 and has a mol, wt of 19,800 as estimated by filtration on Sephadex G-75. Nucleoside mono-, di- and triphosphates were competitive inhibitors of the reaction with Ki values in the 0.1 mM range. 3. Particularly relevant is the inhibition of this enzyme by adenosine and guanosine 5'tetraphosphates. In the course of this investigation, the presence of uridine 5'-tetraphosphate was detected in a commercial preparation of UTP. Adenosine, guanosine and uridine 5'-tetraphosphates were very strong inhibitors of the reaction with Ki values in the nM range.     

Regulation of protein phosphorylation in Dictyostelium discoideum

We have examined the phosphorylation of the cyclic adenosine 3':5' monophosphate (cAMP) cell surface chemotactic receptor and a 36 kDa membrane-associated protein (p36) in Dictyostelium discoideum. The activity of CAR-kinase, the enzyme responsible for the phosphorylation of the cAMP receptor, was studied in plasma membrane preparations. It was found that, as in intact cells, the receptor was rapidly phosphorylated in membranes incubated with [gamma 32P] adenosine triphosphate (ATP) but only in the presence of cAMP. This phosphorylation was not observed in membranes prepared from cells which did not display significant cAMP binding activity. cAMP could induce receptor phosphorylation at low concentrations, while cyclic guanosine 3':5' monophosphate (cGMP) could elicit receptor phosphorylation only at high concentrations. Neither ConA, Ca2+, or guanine nucleotides had an effect on CAR-kinase. It was also observed that 2-deoxy cAMP but not dibutyryl cAMP induced receptor phosphorylation. The data suggest that the ligand occupied form of the cAMP receptor is required for CAR-kinase activity. Although the receptor is rapidly dephosphorylated in vivo, we were unable to observe its dephosphorylation in vitro. In contrast, p36 was rapidly dephosphorylated. Also, unlike the cAMP receptor, the phosphorylation of p36 was found to be regulated by the addition of guanine nucleotides. Guanosine diphosphate (GDP) enhanced the phosphorylation while guanosine triphosphate (GTP) decreased the radiolabeling of p36 indicating that GTP can compete with ATP for the nucleotide triphosphate binding site of p36 kinase. Thus was verified using radiolabeled GTP as the phosphate donor. Competition experiments with GTP gamma S, ATP, GTP, CTP, and uridine triphosphate (UTP) indicated that the phosphate donor site of p36 kinase is relatively non-specific.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural basis for the specificity, catalysis, and regulation of human uridine-cytidine kinase

Uridine-cytidine kinase (UCK) catalyzes the phosphorylation of uridine and cytidine and activates pharmacological ribonucleoside analogs. Here we present the crystal structures of human UCK alone and in complexes with a substrate, cytidine, a feedback inhibitor, CTP or UTP, and with phosphorylation products, CMP and ADP, respectively. Free UCK takes an alpha/beta mononucleotide binding fold and exists as a homotetramer with 222 symmetry. Upon inhibitor binding, one loop region was loosened, causing the UCK tetramer to be distorted. Upon cytidine binding, a large induced fit was observed at the uridine/cytidine binding site, which endows UCK with a strict specificity for pyrimidine ribonucleosides. The first UCK structure provided the structural basis for the specificity, catalysis, and regulation of human uridine-cytidine kinase, which give clues for the design of novel antitumor and antiviral ribonucleoside analogs that inhibit RNA synthesis.     

Nucleoside auxotrophy in Drosophila: An autosomal locus yielding mutants supplementable by purine and pyrimidine ribonucleosides

Summary Two allelic auxotrophic mutants at a locus close to the bw locus (2–104.5) of Drosophila melanogaster are described. The mutants respond to dietary ribonucleosides (uridine, cytidine, adenosine, guanosine and inosine) but less well to bases or pyrimidine precursors. This phenotype is unique to these mutants.We suggest that the mutants are defective in phosphoribosyl pyrophosphate biosynthesis.     

Synthesis of diorganotin(IV) complexes with nucleosides and their Characterization by Mössbauer and infrared spectroscopy

A number of new complexes of R₂Sn^IV with adenosine, guanosine, inosine, cytidine and uridine were synthesized by reaction of ribonucleosides with diorganotin oxide in hot methanol. The complexes were characterized by infrared and ¹¹⁹Sn Mössbauer spectroscopy as O(2′), O(3′) (diorganostannylene) nucleosides; the occurence of dimers with three-co-ordinate oxygen atoms is inferred on the basis of spectroscopic data.     

Changes of uridine permeability during the maturation of tunicate eggs.

The time course of uridine uptake by eggs and embryos of the tunicate Ascidia callosa was studied using 5-min pulses of [³H]uridine at intervals from the unfertilized egg to the 16-cell embryo. The unfertilized egg is permeable to uridine, but 5 min after fertilization uptake begins to drop, reaching a minimum of 30% of the unfertilized rate about 30 min after fertilization. At 45 min after fertilization, permeability begins to increase, reaching a plateau about 3 hr after fertilization at the two-cell stage. The initial decrease in permeability occurs at first polar body production; the increase at 45 min is coincident with the formation of the second polar body. Substrate concentration experiments up to 200 μM show strict concentration dependence for uridine uptake. The inhibitors p-chloromercuribenzoate (PCMB), dinitrophenol (DNP), and thymidine have little, if any effect on permeability. Cold (?1°C) and Na⁺-free sea water inhibit uptake 60% during all three developmental stages. The changes in permeability may be indicative of temporary reorganization of the plasma membrane during the fertilization-initiated completion of meiosis.     

Compartmentation of thymidine kinase in unfertilized sea urchin eggs and possible release of thymidine kinase from particulates in activated eggs

The thymidine kinase activity of homogenates of unfertilized eggs of the sea urchin, Hemicentrotus pulcherrimus, in 1 M NaCl was always lower than that of homogenates of the unfertilized eggs in hypotonic media or homogenates of the fertilized or ammonia-activated eggs in 1 M NaCl by 30–50%. Sonication of the unfertilized egg homogenates in 1 M NaCl resulted in the elevation of thymidine kinase activity up to a level in the fertilized or ammonia-activated egg homogenates which is not affected by sonication. Differential centrifugation of unfertilized egg homogenates in 1 M NaCl revealed that the latent thymidine kinase is associated with the 1500g pellet or even with the 200g pellet. Exposure of the 1500g pellet to sonication, hypotonic media, 0.3% Triton X-100 in 1 M NaCl, and 2 M propyleneglycol resulted in the elevation of thymidine kinase, which was eventually shown to be no longer bound to the pellet fraction. Latent thymidine kinase was not detected in the 1500g pellet prepared from the fertilized egg homogenate in 1 M NaCl. These findings seem to suggest that thymidine kinase in unfertilized eggs may be sequestered, at least partly, in some large intracellular structures but may be released from them upon fertilization or ammonia activation, in accordance with our earlier observation on the apparent activation of thymidine kinase afer fertilization.     

Altered in vitro phosphorylation of specific proteins accompanies fertilization of Strongylocentrotus purpuratus eggs

Protein phosphorylation in eggs of Strongylocentrotus purpuratus was examined by incubation of egg homogenates with γ[³²P]ATP. Individual phosphorylated proteins were detected by autoradiography after electrophoresis of the disaggregated proteins on SDS-polyacrylamide slab gels. Nearly all of the radioactivity was labile to treatment with pronase, but not to ribonuclease or hydroxylamine, suggesting it to be in the form of protein phosphoesters. The pH dependence for phosphorylation was broad, with cyclic 3′,5′-adenosine monophosphate (cAMP)-dependent phosphorylation optimal at pH 7.7. Phosphorylation of several protein species at pH 7.7 was altered in homogenates of fertilized eggs, when compared to that of unfertilized eggs. These relative increase or decreases in intensity detected by autoradiography were not accompanied by corresponding changes in protein staining of the gels, suggesting that the differences were not due to major shifts in overall protein composition. Most of the alterations in phosphorylation were evident in homogenates made within 5 min after fertilization and were stable until the first cell division. The alterations were also found with homogenates of eggs activated with the divalent ionophore A23187, and some, but not all were present following treatment with ammonia, under conditions that induce a partial metabolic activation of eggs. The results suggest that fertilization promotes alterations in the availability of phosphorylation sites in egg homogenates, or changes in the activity of the egg kinases toward specific protein substrates, that may play a role in the activation of egg metabolism.     

Calcium-mediated inactivation of the MAP kinase pathway in sea urchin eggs at fertilization.

We have evaluated the regulation of a 43-kDa MAP kinase in sea urchin eggs. Both MAP kinase and MEK (MAP kinase kinase) are phosphorylated and active in unfertilized eggs while both are dephosphorylated and inactivated after fertilization, although with distinct kinetics. Reactivation of MEK or the 43-kDa MAP kinase prior to or during the first cell division was not detected. Confocal immunolocalization microscopy revealed that phosphorylated (active) MAP kinase is present primarily in the nucleus of the unfertilized egg, with some of the phosphorylated form in the cytoplasm as well. Incubation of unfertilized eggs in the MEK inhibitor U0126 (0.5 microM) resulted in the inactivation of MEK and MAP kinase within 30 min. Incubation in low concentrations of U0126 (sufficient to inactivate MEK and MAP kinase) after fertilization had no effect on progression through the embryonic cell cycle. Microinjection of active mammalian MAP kinase phosphatase (MKP-3) resulted in inactivation of MAP kinase in unfertilized eggs, as did addition of MKP-3 to lysates of unfertilized eggs. Incubation of unfertilized eggs in the Ca(2+) ionophore A23187 led to inactivation of MEK and MAP kinase with the same kinetics as observed with sperm-induced egg activation. This suggests that calcium may be deactivating MEK and/or activating a MAP kinase-directed phosphatase. A cell-free system was used to evaluate the activation of phosphatase separately from MEK inactivation. Unfertilized egg lysates were treated with U0126 to inactivate MEK and then Ca(2+) was added. This resulted in increased MAP kinase phosphatase activity. Therefore, MAP kinase inactivation at fertilization in sea urchin eggs likely is the result of a combination of MEK inactivation and phosphatase activation that are directly or indirectly responsive to Ca(2+).     

Effects of purine and pyrimidine derivatives on uptake of tritium-labelled uridine by Closterium acerosum

Uracil, adenosine, cytidine, guanidine, and thymidine were potentinhibitors of uptake of tritium-labelled uridine by Closteriumacerosum. Ribonudeoside monophosphates, ribonudeoside triphosphates,and deoxyribonucleoside triphosphates weakly inhibited uptake,but adenosine triphosphate and cyclic adenosine monophosphatedid not. Ribose-5-phosphate had a slight inhibitory effect onthe uptake of uridine. (Received January 19, 1976; )     

Adenosine triphosphate levels in steelhead (Oncorhynchus mykiss) eggs: an examination of turnover, localization and role

The dynamics of energy production and utilization in fish eggs before and shortly after fertilization may be critical for embryo survival. Therefore, the current study examined the turnover of adenosine triphosphate (ATP) as well as examined the possible role and localization of ATP in unfertilized steelhead (Oncorhynchus mykiss) eggs and early embryos. The mean ATP level in unfertilized steelhead eggs was 1.92+/-0.10 (mean+/-S.E.M., n=17) nmol ATP per egg. Exposure of the unfertilized egg to 10 degrees C water (water activation) and fertilization resulted in comparable and substantial decreases (approx. 20-50%) in egg ATP levels within 3 min. This suggests that the energy expended at fertilization is used in response to water activation rather than fertilization per se. Unfertilized eggs maintained in ovarian fluid for 9 days at 10 degrees C under air showed a progressive decline of fertility that reached zero after 6 days. In contrast, no significant changes were seen in ATP levels throughout this 9 days period. Thus, fertility does not positively correlate with egg ATP levels in stored eggs. In the unfertilized egg, the ATP stored in the yolk accounted for approximately 1.5% of the total egg ATP. After fertilization, the concentration of ATP in the yolk increased approximately seven-fold, with the yolk and blastoderm each now accounting for approximately 20% of the total remaining ATP. Finally, to estimate the changes in oxidative metabolism following fertilization, the cyanide (KCN)-sensitive decline in total ATP was determined for unfertilized eggs and 1 day embryos. In the presence of KCN, ATP levels declined to approximately 50% within 24 h in both unfertilized eggs as well as embryos; the rates of ATP decline were not different. Therefore, there was not a discernible increase in ATP generation by oxidative phosphorylation at the time of fertilization.