Pyrimidine nucleotide metabolism in rat hepatocytes: evidence for compartmentation of nucleotide pools

Pyrimidine nucleotide metabolism in rat hepatocytes was studied by measurement of the labelling kinetics of the various intermediates after double labelling with [14C]orotic acid and [3H]cytidine, the precursors for the de novo and the salvage pathways respectively. For the uridine nucleotides, differences were found for the 14C/3H ratios in the UDP-sugars, in UMP (of RNA) and in their precursor UTP, suggesting the existence of separated flows of the radioactive precursors through the de novo and the salvage pathways. Higher ratios in the UDP-sugars, which are synthesized in the cytoplasm, and a lower ratio in UMP (of RNA) relative to the 14C/3H ratio in UTP indicated that UTP derived from orotic acid is preferentially used for the cytoplasmic biosynthesis of the UDP-sugars. Uridine, derived from cytidine, is preferentially used for the nuclear-localized synthesis of RNA. In contrast to these findings, the 14C/3H ratios in the cytidine derivatives CMP-NeuAc and CMP (of RNA), and in the liponucleotides CDP-choline and CDP-ethanolamine, were all lower than that in the precursor CTP. This indicates a preferential utilization of the salvage-derived CTP for the synthesis of the liponucleotides as well as for RNA and CMP-NeuAc. Similar conclusions could be drawn from experiments in which the intracellular amounts of several uridine- and cytidine-nucleotide-containing derivatives were increased by preincubating the hepatocytes with unlabelled pyrimidine nucleotides or ethanolamine. Based on these data, we propose a refined model for the intracellular compartmentation of pyrimidine nucleotide biosynthesis in which three pools of UTP are distinguished: a pool of de novo-derived molecules and a pool of salvage-derived molecules, both of which are channelled to the site of utilization; in addition an ‘overflow’ pool exists, consisting of molecules having escaped from channelling. An overflow pool could also be distinguished for CTP, but no discrimination between de novo and salvage-derived molecules could be made.

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Changes in activities of the de novo and salvage pathways of pyrimidine nucleotide biosynthesis during germination of black gram (Phaseolus mungo) seeds

Zeitschrift für Pflanzenphysiologie ◽

10.1016/s0044-328x(77)80195-5 ◽

1977 ◽

Vol 81 (3) ◽

pp. 199-211 ◽

Cited By ~ 47

Author(s):

Hiroshi Ashihara

Keyword(s):

De Novo ◽

Black Gram ◽

Nucleotide Biosynthesis ◽

Phaseolus Mungo ◽

Pyrimidine Nucleotide ◽

Salvage Pathways

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Uridylate trapping, induction of UTP deficiency, and stimulation of pyrimidine synthesis de novo by d-galactosone

Biochemical Journal ◽

10.1042/bj2060139 ◽

1982 ◽

Vol 206 (1) ◽

pp. 139-146 ◽

Cited By ~ 14

Author(s):

Dietrich O. R. Keppler ◽

Christa Schulz-Holstege ◽

Joachim Fauler ◽

Karl A. Reiffen ◽

Friedhelm Schneider

Keyword(s):

Rat Liver ◽

De Novo ◽

Combined Treatment ◽

Hepatoma Cells ◽

Nucleotide Metabolism ◽

Phosphate Deficiency ◽

Initial Time ◽

Pyrimidine Synthesis ◽

Pyrimidine Nucleotide ◽

Time Period

d-Galactosone (d-lyxo-2-hexosulose) is phosphorylated and metabolized to the uridine diphosphate derivative in AS-30D hepatoma cells and rat liver. These reactions were catalysed in vitro by galactokinase and hexose-1-phosphate uridylyltransferase. Nucleotide analyses by high-performance liquid chromatography and enzymic assays revealed that this galactose analogue interferes with cellular pyrimidine nucleotide metabolism leading to a deficiency of UTP. [14C]Uridine labelling of hepatoma cells indicated a division of [14C]uridylate from UTP into UDP-galactosone; the latter was formed at a rate of more than 1.7mmol×h−1×(kg AS-30D or liver wet wt.)−1. As a consequence of UTP deficiency, d-galactosone (1mmol/1 or 1mmol/kg body wt.) strongly enhanced the rate of pyrimidine synthesis de novo as evidenced by incorporation of 14CO2 into uridylate and by an expansion of the uridylate pool. This resulted in a doubling of the total acid-soluble uridylate pool within 70min in the hepatoma cells and within 110min in rat liver. Combined treatment of hepatoma cells with d-galactosone and N-(phosphonoacetyl)-l-aspartate, an inhibitor of aspartate carbamoyltransferase, prevented the expansion of the uridylate pool and led to a synergistic reduction of UTP to 10% of the content in control cells. Hepatic UTP deficiency was selective with respect to other nucleotide 5′-triphosphates but was associated with reduced contents of UDP-glucose, UDP-glucuronate, and UDP-N-acetylhexosamines. Isolation of the UDP derivative of d-galactosone revealed an extremely alkali-labile UDP-sugar, probably an isomerization product of UDP-galactosone, that was degraded by elimination of UDP with a half-life of 45min at pH7.5 and 37°C. The instability of UDP-galactosone may contribute in vivo to limit the time period of severe uridine phosphate deficiency in addition to the compensatory role of pyrimidine synthesis de novo. During the initial time period, however, d-galactosone is effective as a powerful uridylate-trapping sugar analogue.

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Salvage Pathways of Pyrimidine Nucleotide Biosynthesis

Plant Nucleotide Metabolism ‐ Biosynthesis, Degradation, and Alkaloid Formation ◽

10.1002/9781119476139.ch9 ◽

2020 ◽

pp. 137-148

Keyword(s):

Nucleotide Biosynthesis ◽

Pyrimidine Nucleotide ◽

Salvage Pathways

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Regulation of purine nucleotide biosynthesis: in yeast and beyond

Biochemical Society Transactions ◽

10.1042/bst0340786 ◽

2006 ◽

Vol 34 (5) ◽

pp. 786-790 ◽

Cited By ~ 31

Author(s):

R.J. Rolfes

Keyword(s):

Saccharomyces Cerevisiae ◽

De Novo ◽

Purine Nucleotide ◽

De Novo Synthesis ◽

Normal Functioning ◽

Purine Nucleotides ◽

Yeast Saccharomyces Cerevisiae ◽

Nucleotide Biosynthesis ◽

Salvage Pathways ◽

Pool Sizes

Purine nucleotides are critically important for the normal functioning of cells due to their myriad of activities. It is important for cells to maintain a balance in the pool sizes of the adenine-containing and guanine-containing nucleotides, which occurs by a combination of de novo synthesis and salvage pathways that interconvert the purine nucleotides. This review describes the mechanism for regulation of the biosynthetic genes in the yeast Saccharomyces cerevisiae and compares this mechanism with that described in several microbial species.

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Studies on the incorporation of precursors into purine and pyrimidine nucleotides via ‘de novo’ and ‘salvage’ pathways in normal lymphocytes and lymphoblastic cell-line cells

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research ◽

10.1016/0167-4889(89)90088-8 ◽

1989 ◽

Vol 1012 (2) ◽

pp. 148-155 ◽

Cited By ~ 52

Author(s):

Yolanda M.T. Marijnen ◽

Dirk de Korte ◽

Willem A. Haverkort ◽

Engelbertus J.S. den Breejen ◽

Albert H. van Gennip ◽

...

Keyword(s):

Cell Line ◽

De Novo ◽

Pyrimidine Nucleotides ◽

Purine And Pyrimidine Nucleotides ◽

Salvage Pathways

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Inhibition of De Novo Pyrimidine Nucleotide Synthesis By the Novel DHODH Inhibitor PTC299 Induces Differentiation and/or Death of AML Cells

Blood ◽

10.1182/blood-2019-124569 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5152-5152

Author(s):

Marla Weetall ◽

Kensuke Kojima ◽

Sujan Piya ◽

Christopher Trotta ◽

John Baird ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

De Novo ◽

Salvage Pathway ◽

De Novo Synthesis ◽

Dna Breaks ◽

Advisory Committees ◽

Pyrimidine Nucleotides ◽

Nucleotide Synthesis ◽

Pyrimidine Nucleotide

Background: Pyrimidine nucleotides are generated either by de novo synthesis or the salvage pathway in which pyrimidine nucleotides are obtained from the diet. Resting cells typically acquire adequate pyrimidine nucleotides from the salvage pathway. Rapidly proliferating cells, however, are dependent on the de novo synthesis of pyrimidine nucleotides. PTC299 is an inhibitor of dihydroorotate dehydrogenase (DHODH), a rate limiting enzyme for de novo pyrimidine nucleotide synthesis that had previously been in clinical trials for treatment of solid tumors. Results: Using 15N-labelled glutamine, we show that PTC299 reduces de novo pyrimidine nucleotide synthesis in PTC299-sensitive AML cell lines resulting in a depletion of total pyrimidine nucleotides. In parallel to reduction in pyrimidine nucleotides, PTC 299 leads to accumulation of DHO, the substrate of DHODH and unexpectedly, an accumulation of N-carbamoyl aspartate the metabolite above DHO in the de novo pyrimidine nucleotide synthesis pathway. PTC299 was broadly active against leukemia and lymphoma lines, with 80% of the AML lines tested showing sensitivity. Treatment of AML cell lines with PTC299 induced differentiation as shown by increased CD14 and/or reduced proliferation. Using isogenic AML lines, we show that PTC299 reduces the proliferation of both p53 wildtype and p53 deficient leukemia calls with similar potency as measured by the concentration of PTC299 required to reduce cell number by 50% (CC50). In cells expressing wildtype p53, PTC299 increases p53 activation. However, p53- wildtype cells undergo increased apoptosis whereas p53-deficience cells undergo necrosis. PTC299 induced a G1/S cell cycle arrest, also independent of p53 status. PTC299 increased H2A.X (a marker of double stranded DNA breaks) in both p53 wildtype and p53 deficient cells. These data suggest that the depletion of nucleotides results in stalling at the replication fork, and subsequent DNA-breaks. Conclusion: De novo pyrimidine nucleotide synthesis is critical for AML survival and proliferation. Depletion of nucleotides results in reduced proliferation, triggering either differentiation and/or cell death. Disclosures Weetall: PTC Therapeutics: Employment. Trotta:PTC Therapeutics: Employment. Baird:PTC Therapeutics: Employment. O'Keefe:PTC Therapeutics: Employment. Furia:PTC Therapeutics: Employment. Borthakur:PTC Therapeutics: Consultancy; Janssen: Research Funding; AbbVie: Research Funding; Argenx: Membership on an entity's Board of Directors or advisory committees; NKarta: Consultancy; AstraZeneca: Research Funding; Xbiotech USA: Research Funding; Incyte: Research Funding; GSK: Research Funding; Oncoceutics, Inc.: Research Funding; Novartis: Research Funding; Agensys: Research Funding; BMS: Research Funding; Oncoceutics: Research Funding; Cantargia AB: Research Funding; Bayer Healthcare AG: Research Funding; Eisai: Research Funding; FTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; BioTheryX: Membership on an entity's Board of Directors or advisory committees; Polaris: Research Funding; Merck: Research Funding; Cyclacel: Research Funding; Eli Lilly and Co.: Research Funding; BioLine Rx: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Arvinas: Research Funding; Tetralogic Pharmaceuticals: Research Funding; Strategia Therapeutics: Research Funding. Spiegel:PTC Therapeutics: Consultancy.

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