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At least 19 recordsLinked to original sources
PubMed1975

Role of cytidine triphosphate and cytidine diphosphate in promoting inositol entry into microsomal phosphatidylinositol.

The Mn2+ activated incorporation of myo-inositol-3H into subfractions of phosphatidylinositol in rat liver microsomes was studied in the presence and absence of cytidine triphosphate or cytidine diphosphate choline using phosphate buffer. The distribution of labeled inositol among molecular species of microsomal phosphatidylinositol was also investigated in vivo. In other experiments, the release of radioactivity from microsomes labeled with inositol-3H in the phospholipid was measured after the addition of Mn2+, unlabeled inositol, and cytidine nucleotide. Similar chase experiments were conducted with microsomes containing phosphatidylcholine-14C or phosphatidylethanolamine-14C. The addition of cytidine triphosphate or cytidine diphosphate choline stimulated the rate of inositol-3H entry into microsomal phosphatidylinositol by 3.5 to 4-fold and the monoenoic plus dienoic, trienoic, tetraenoic, and polyenoic species contained 6-7, 6, 78-81, and 7-9%, of radioactivity, respectively. These latter patterns were very similar to those observed among the corresponding molecular species when the Mn2+ stimulated entry of free inositol into phospholipid was studied in the absence of added cytidine nucleotide. In chase experiments, the release of radioactivity from phospholipid in the presence of cytidine trephosphate or cytidine diphosphate choline was greatly enhanced by the addition of free inositol when microsomes containing phosphatidylinositol-3H, but not phosphatidylcholine-14C or phosphatidylethanolamine-14C, were employed. Therefore, under the present conditions, cytidine triphosphate and cytidine diphosphate choline appear to stimulate the entry of inositol into phosphatidylinositol by enhancing the Mn2+ activated exchange reaction in rat liver microsomes. The results suggest further that phosphatidylinositol is the preferred substrate when this reaction is stimulated by cytidine nucleotide.

Animals↗
PubMed1981

Purification and characterization of cytidine 5'-triphosphate:cytidine 5'-monophosphate-3-deoxy-D-manno-octulosonate cytidylyltransferase.

Cytidine 5'-triphosphate:cytidine 5'-monophosphate-3-deoxy-D-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase) was purified 2,300-fold from frozen Escherichia coli B cells. The enzyme catalyzed the formation of CMP-KDO, a very labile product, from CTP and KDO. No other sugar tested could replace KDO as an alternate substrate. Uridine 5'-triphosphate at pH 9.5 and deoxycytidine 5'-triphosphate at pH 8.0 and 9.5 could be used as alternate substrates in place of CTP. CMP-KDO synthetase required Mg2+ at a concentration of 10.0 mM for optimal activity. The pH optimum was determined to be between 9.6 and 9.3 in tris(hydroxymethyl)aminomethane-acetate or sodium-glycine buffer. This enzyme had an isoelectric point between pH 4.15 and 4.4 and appeared to be a single polypeptide chain with a molecular weight of 36,000 to 40,000. The apparent Km values for CTP and KDO in the presence of 10.0 mM Mg2+ were determined to be 2.0 X 10(-4) and 2.9 X 10(-4) M, respectively, at pH 9.5. Uridine 5'-triphosphate and deoxycytidine 5'-triphosphate had apparent Km values of 8.8 X 10(-4) and 3.4 X 10(-4) M. respectively, at pH 9.5.

Cytidine Monophosphate↗
PubMed1988

Cytidylate cyclase activity: identification of cytidine 3',5'-cyclic monophosphate and four novel cytidine cyclic phosphates as biosynthetic products from cytidine triphosphate.

Identification of cytidine 3',5'-cyclic monophosphate (cyclic CMP) as one of the products resulting from the incubation of dialysed cell-free preparations from rat brain, liver and kidney with cytidine 5'-triphosphate (CTP) is described. The non-acidic precipitable products after incubation of the tissue preparations with unlabelled, with 14C-single labelled, and with 14C- and 32P-dual labelled CTP were examined by thin-layer chromatography and high-pressure liquid chromatography, isotopic ratio determination, UV absorbance spectrophotometry, selective hydrolysis with nucleotidase, phosphodiesterase and acid, and by fast atom bombardment mass spectrometry with mass-analysed ion kinetic energy spectrum scanning. In addition to cyclic CMP and unchanged CTP, the products of the reaction were found to include cytidine monophosphate (CMP) and cytidine diphosphate (CDP) together with four novel cytidine compounds identified as cytidine 3',5'-cyclic pyrophosphate, cytidine 2'-monophosphate 3',5'-cyclic monophosphate, cytidine 2'-O-aspartyl-3',5'-cyclic monophosphate and cytidine 2'-O-glutamyl-3',5'-cyclic monophosphate. The evidence presented constitutes conclusive proof of the natural occurrence of cytidylate cyclase activity; the four novel cytidine cyclic phosphates described provide a feasible explanation of the discrepancies in previous reports which have led to the controversy which exists concerning the existence of cytidylate cyclase activity.

Animals↗
PubMed1996

Isolation, characterization and expression of the gene encoding cytidine triphosphate synthetase from Giardia intestinalis.

The cytidine triphosphate synthetase gene from Giardia intestinalis was cloned using a PCR-based strategy. A 519 bp PCR product was obtained from the amplification of genomic DNA using two oligonucleotides derived from the CTP synthetase amino acid consensus sequences DPYINVDPG and KTKPTQ. This product was used to probe restriction endonuclease digested genomic DNA and the respective plasmid mini-libraries. Two genomic clones were obtained one with a 3.6 kb HindIII DNA fragment, containing approximately three-quarters of the 5'-end of the synthetase gene and subsequently, a 5.8 kb PstI DNA fragment which contained the whole gene. The intronless gene has a 1863 bp open reading frame encoding 620 amino acids (M(r) of 68.3 kDa). A well conserved catalytic glutamine aminotransferase (GAT) domain was identified. In addition, three insert sequences were found which are not present in CTP synthetase from other species. Alignment and comparison of the deduced amino acid sequence relative to CTP synthetases from other species revealed a high degree of identity (34%) with a greater resemblance to prokaryotes than eukaryotes. The gene is located on chromosome 6 and the messenger RNA encoding it is estimated to be 1.9 kb. The coding region of G. intestinalis CTP synthetase was generated by PCR and subsequently cloned into the pQE30 vector for expression in E. coli. This construct yielded a soluble and enzymatically active recombinant protein which was purified by a Ni-NTA affinity column. The purified recombinant protein had a subunit molecular weight of 69.5 kDa and a native molecular weight of approximately 274 kDa. Kinetic studies of the partially purified recombinant G. intestinalis CTP synthetase gave apparent K(m) values of 0.1 mM and approximately 0.5 mM for the substrates UTP and L-glutamine respectively in accord with previously reported values for the native enzyme.

Amino Acid Sequence↗
PubMedDate not supplied

Nucleotide variation in the cytidine triphosphate synthetase gene of Giardia duodenalis.

The cytidine triphosphate synthetase genes from three diverse strains of Giardia duodenalis have been sequenced and found to vary significantly from one another. The isolates were chosen as representatives of three demes as determined by several criteria including divergence in the rDNA repeat unit. Inserts in the genes and protein are conserved in length but are the most divergent regions among the three sequences examined. Variation in the rest of the gene occurs primarily in the third base position resulting in many silent mutations. One of the isolates (1709) was found to contain two genes with high sequence homology.

Amino Acid Sequence↗
PubMed1982

Effect of lead chelation therapy with EDTA in children on erythrocyte pyrimidine 5'-nucleotidase and with cytidine triphosphate levels.

Children with elevated whole blood lead levels were treated for 4 days with EDTA. Changes in whole blood lead concentrations, erythrocyte zinc protoporphyrin concentrations, red cell pyrimidine 5'-nucleotidase activities, and erythrocyte cytidine triphosphate (CTP) levels were measured during the course of the EDTA therapy. EDTA treatment decreased blood lead levels by approximately 50% after 4 days. An inverse relationship existed between blood lead levels and erythrocyte pyrimidine 5'-nucleotidase activity. Despite reactivation of pyrimidine 5'-nucleotidase enzyme activity by EDTA, there was no decrease in erythrocyte cytidine triphosphate or in zinc protoporphyrin concentrations. The lack of change in these two parameters during EDTA treatment supports the concept that pyrimidine 5'-nucleotidase inhibition by lead in the reticulocyte cytidine phosphate accumulation in moderate lead poisoning may reflect chronic lead overburden analogous to prolonged elevation of erythrocyte zinc protoporphyrins.

5'-Nucleotidase↗
PubMed2005

Mechanisms of product feedback regulation and drug resistance in cytidine triphosphate synthetases from the structure of a CTP-inhibited complex.

Cytidine triphosphate synthetases (CTPSs) synthesize CTP and regulate its intracellular concentration through direct interactions with the four ribonucleotide triphosphates. In particular, CTP product is a feedback inhibitor that competes with UTP substrate. Selected CTPS mutations that impart resistance to pyrimidine antimetabolite inhibitors also relieve CTP inhibition and cause a dramatic increase in intracellular CTP concentration, indicating that the drugs act by binding to the CTP inhibitory site. Resistance mutations map to a pocket that, although adjacent, does not coincide with the expected UTP binding site in apo Escherichia coli CTPS [EcCTPS; Endrizzi, J. A., et al. (2004) Biochemistry 43, 6447-6463], suggesting allosteric rather than competitive inhibition. Here, bound CTP and ADP were visualized in catalytically active EcCTPS crystals soaked in either ATP and UTP substrates or ADP and CTP products. The CTP cytosine ring resides in the pocket predicted by the resistance mutations, while the triphosphate moiety overlaps the putative UTP triphosphate binding site, explaining how CTP competes with UTP while CTP resistance mutations are acquired without loss of catalytic efficiency. Extensive complementarity and interaction networks at the interfacial binding sites provide the high specificity for pyrimidine triphosphates and mediate nucleotide-dependent tetramer formation. Overall, these results depict a novel product inhibition strategy in which shared substrate and product moieties bind to a single subsite while specificity is conferred by separate subsites. This arrangement allows for independent adaptation of UTP and CTP binding affinities while efficiently utilizing the enzyme surface.

Binding Sites↗
PubMed1990

Characterization of temperature-sensitive cytidine triphosphate synthase mutations in bacteria by high-performance liquid chromatography.

Cytidine triphosphate (CTP) synthase catalyzes the last step in pyrimidine ribonucleotide synthesis, namely the formation of CTP from UTP, ATP, and glutamine. Mutants devoid of CTP synthase activity require cytidine for growth and have been designated pyrG in an obligate cdd background. Using a ts mutation blocked in the conversion of UTP to CTP at 43 degrees C, it was demonstrated that the conversion occurs by growing cells at 33 degrees C or below where UTP and CTP pools are normal. Growth at 43 degrees C shuts off the enzyme, while UTP accumulates and CTP is decreased significantly. By now feeding exogenous cytidine the CTP pool can be restored to the level found at the permissive temperature. Intracellular nucleoside triphosphates (CTP and UTP) were separated on a Partisil SAX10 cartridge, using a linear gradient of low buffer (7 mM ammonium dihydrogenphosphate, pH 3.8) to high buffer (250 mM ammonium dihydrogenphosphate, pH 4.5 with 500 mM potassium chloride). Nucleoside triphosphates were also separated after enzymatic conversion of UTP to CTP in solution by cell extracts using ion-pair reversed-phase chromatography on a C18 cartridge eluted with a mixture of 95% buffer A (25 mM ammonium dihydrogenphosphate with 1 mM tetrabutylammonium phosphate, pH 7.0) and 5% buffer B (15% aqueous acetonitrile). Using the two different separation techniques, it was possible to monitor the level of UTP and CTP inside cells as well as the enzymatic conversion of UTP to CTP by the enzyme CTP synthase.

Bacteria↗
PubMed1982

Repression of cytidine triphosphate synthetase in Salmonella typhimurium by pyrimidines during uridine nucleotide depletion.

Regulation of the synthesis of cytidine triphosphate (CTP) synthetase (EC 6.3.4.2) was investigated in Salmonella typhimurium. CTP synthetase appeared to be repressed only when intracellular concentrations of uridine nucleotides were significantly lowered. Under such nucleotide pool conditions, a cytidine compound and, to a lesser degree, a thymidine compound appeared as putative repressing metabolites of enzyme synthesis.

Carbon-Nitrogen Ligases↗
PubMed2004

Crystal structure of Escherichia coli cytidine triphosphate synthetase, a nucleotide-regulated glutamine amidotransferase/ATP-dependent amidoligase fusion protein and homologue of anticancer and antiparasitic drug targets.

Cytidine triphosphate synthetases (CTPSs) produce CTP from UTP and glutamine, and regulate intracellular CTP levels through interactions with the four ribonucleotide triphosphates. We solved the 2.3-A resolution crystal structure of Escherichia coli CTPS using Hg-MAD phasing. The structure reveals a nearly symmetric 222 tetramer, in which each bifunctional monomer contains a dethiobiotin synthetase-like amidoligase N-terminal domain and a Type 1 glutamine amidotransferase C-terminal domain. For each amidoligase active site, essential ATP- and UTP-binding surfaces are contributed by three monomers, suggesting that activity requires tetramer formation, and that a nucleotide-dependent dimer-tetramer equilibrium contributes to the observed positive cooperativity. A gated channel that spans 25 A between the glutamine hydrolysis and amidoligase active sites provides a path for ammonia diffusion. The channel is accessible to solvent at the base of a cleft adjoining the glutamine hydrolysis active site, providing an entry point for exogenous ammonia. Guanine nucleotide binding sites of structurally related GTPases superimpose on this cleft, providing insights into allosteric regulation by GTP. Mutations that confer nucleoside drug resistance and release CTP inhibition map to a pocket that neighbors the UTP-binding site and can accommodate a pyrimidine ring. Its location suggests that competitive feedback inhibition is affected via a distinct product/drug binding site that overlaps the substrate triphosphate binding site. Overall, the E. coli structure provides a framework for homology modeling of other CTPSs and structure-based design of anti-CTPS therapeutics.

Amino Acid Sequence↗
PubMed1995

Cytidine triphosphate (CTP) synthetase activity during cell cycle progression in normal and malignant T-lymphocytic cells.

The role of cytidine triphosphate (CTP) synthetase (EC 6.3.4.2.) in the pyrimidine ribonucleotide metabolism of MOLT-3 human T-ALL cell line cells and normal human T lymphocytes during the cell cycle traverse was studied. Highly pure G1-phase samples and samples enriched in S-phase cells were obtained by counterflow centrifugation. The activity of CTP synthetase in situ, measured in pulse-chase experiments, was similar in the G1-phase and S-phase MOLT-3 cells. In contrast, in S-phase T lymphocytes, an increased activity of CTP synthetase was observed compared with G1-phase T lymphocytes. Nevertheless, the MOLT-3 samples showed an increased activity of CTP synthetase in comparison with either G1-phase or S-phase enriched samples of normal T lymphocytes. Therefore, the increased activity of CTP synthetase of MOLT-3 cells is a cell cycle-independent feature, whereas among normal T lymphocytes, the increase in activity of CTP synthetase that arises after a growth stimulus is more prominent in the S-phase.

Carbon-Nitrogen Ligases↗
PubMed2000

In vitro inhibition of cytidine triphosphate synthetase activity by cyclopentenyl cytosine in paediatric acute lymphocytic leukaemia.

Cytidine triphosphate (CTP) synthetase is a key enzyme for the synthesis of cytosine (deoxy)ribonucleotides, catalysing the conversion of uridine triphosphate (UTP) into CTP, and has a high activity in several malignancies. In this preclinical study, the enzyme activity and mRNA expression of the enzyme and (deoxy)ribonucleotide concentrations were analysed in leukaemic cells of 57 children suffering from acute lymphocytic leukaemia (ALL). In addition, in vitro experiments were performed with the CTP synthetase inhibitor cyclopentenyl cytosine (CPEC). A significantly higher activity of CTP synthetase (6.5 +/- 3.9 nmol CTP/mg/h) was detected in ALL cells than in lymphocytes of healthy controls (1.8 +/- 0.9 nmol CTP/mg/h, P < 0.001) that was independent of white blood cell (WBC) count, blast percentage, age, gender or type of ALL. The enzyme activity was not correlated with the CTP synthetase mRNA expression. The activity of CTP synthetase in ALL cells compared with non-malignant CD34+ bone marrow controls (5.6 +/- 2.4 nmol CTP/mg/h) was not statistically different. In vitro treatment of ALL cells with CPEC induced a dose-dependent decrease of the CTP concentration. The lowest concentration of CPEC (0.63 microM) induced a depletion of CTP of 41 +/- 20% and a depletion of dCTP of 27 +/- 21%. The degree of CTP depletion of ALL cells after treatment with CPEC was positively correlated with the activity of CTP synthetase. The inhibition of CTP synthetase in situ was confirmed by flux studies using radiolabelled uridine. From these results, it can be expected that CPEC has a cytostatic effect on lymphoblasts of children with ALL.

Adult↗
PubMed1999

Cytidine triphosphate synthase activity and mRNA expression in normal human blood cells.

Cytidine triphosphate (CTP) synthase is one of the key enzymes in pyrimidine nucleotide anabolic pathways. The activity of this enzyme is elevated in various malignancies including acute lymphocytic leukemia (ALL). In this study we investigated the activity of CTP synthase in various human blood cells isolated from healthy volunteers by density centrifugation and elutriation centrifugation. We also investigated the mRNA expression of CTP synthase in lymphocytes and monocytes. The highest activity of CTP synthase was found in thrombocytes (6.48 nmol CTP x mg(-1) x h(-1)), followed by that of monocytes (2.23), lymphocytes (1.69), granulocytes (0.52) and erythrocytes (0.42). The activity of CTP synthase in whole blood samples was at an intermediate level (1.27). The mRNA expression of CTP synthase in monocytes was comparable to that observed in lymphocytes.

Blood Platelets↗
PubMed1988

Purification of cytidine-triphosphate synthetase from rat liver, and demonstration of monomer, dimer and tetramer.

Cytidine-triphosphate synthetase (UTP: ammonia ligase (ADP-forming), EC 6.3.4.2) has been purified over 31,000-fold to homogeneity with 17% recovery from rat liver cytosol, using high-performance liquid chromatography (HPLC) techniques. The presence of CTP synthetase monomer, dimer and tetramer has been demonstrated in the ammonium sulfate fraction of rat liver cytosol. By gel-permeation HPLC, the molecular weights of the three molecular forms of the enzyme have been estimated as 240,000 (tetramer), 120,000 (dimer) and 60,000 (monomer). By gel-permeation chromatography on Bio-Gel A-1.5m column, the molecular weights of dimer and monomer were estimated as 100,000 and 50,000, respectively. The molecular weight of the monomeric subunit is determined to be 66,000 by SDS-polyacrylamide gel electrophoresis. Monomers isolated fresh from 0-30 (NH4)2SO4 fraction of rat liver cytosol are enzymatically active. Purified rat liver CTP synthetase exhibited sigmoidal kinetic plots as a function of the substrate UTP in the presence of the end-product, CTP. Partially purified CTP synthetase usually forms an inactive coagulum on freezing and subsequent thawing. Incubation of CTP synthetase dimer at 25 degrees C for 1 h in the presence of UTP, ATP and Mg2+ resulted in optimum conversion to tetramer with least inactivation. The purified tetramer dissociates to dimers when UTP, ATP and Mg2+ are removed by dialysis.

Adenosine Triphosphate↗
PubMed1998

Molecular cloning and characterization of the Plasmodium falciparum cytidine triphosphate synthetase gene.

Using degenerate oligonucleotides derived from conserved amino acid regions of cytidine triphosphate synthetase, a fragment of the gene from the malarial parasite, Plasmodium falciparum, was isolated by polymerase chain reaction (PCR). This fragment was used as a probe in the isolation of genomic clones containing the entire pfCTP synthetase coding region (2580 bp). The gene encodes the largest CTP synthetase found in any organism to date due to the presence of two additional sequences which are part of the continuous open reading frame and are not introns as their presence in the mRNA was confirmed by reverse transcriptase-PCR. These features distinguish the parasite enzyme from that of the host making it an attractive target for structure based drug design.

Amino Acid Sequence↗
PubMed2000

Cyclopentenyl cytosine inhibits cytidine triphosphate synthetase in paediatric acute non-lymphocytic leukaemia: a promising target for chemotherapy.

Cytidine triphosphate (CTP) synthetase is a key enzyme in the anabolic pathways of cytosine and uracil ribonucleotide metabolism. The enzyme catalyses the conversion of uridine triphosphate (UTP) into CTP, and has a high activity in various malignancies, which has led to the development of inhibitors of CTP synthetase for therapeutic purposes. We studied both CTP synthetase activity and ribonucleotide concentrations in leukaemic cells of 12 children suffering from acute non-lymphocytic leukaemia (ANLL), and performed incubation experiments with cyclopentenyl cytosine (CPEC), a nucleoside analogue that is capable of inhibiting CTP synthetase. The CTP synthetase activity in ANLL cells (5.1+/-2.3 nmol CTP/mg/h) was significantly higher compared with granulocytes of healthy controls (0.6+/-0.4 nmol CTP/mg/h, P=0.0002), but was not different from the CTP synthetase activity in non-malignant CD34+ bone marrow cells (5. 6+/-2.4 nmol CTP/mg/h). Major shifts were observed in the various ribonucleotide concentrations in ANLL cells compared with granulocytes: the absolute amount of ribonucleotides was increased with a substantial rise of the CTP (2.4 versus 0.4 pmol/microg protein, P=0.0007) and UTP (8.7 versus 1.6 pmol/microg protein, P=0. 0007) concentrations in ANLL cells compared with granulocytes. Treatment of ANLL cells in vitro with CPEC induced a major depletion (77% with 2.5 microM of CPEC) in the concentration of CTP, whilst the concentrations of the other ribonucleotides remained unchanged. Therefore, the high activity of CTP synthetase in acute non-lymphocytic leukaemic cells can be inhibited by CPEC, which provides a key to a new approach for the treatment of ANLL.

Adolescent↗