Overexpression of Phosphoribosyl Pyrophosphate Synthase Enhances Resistance of Chlamydomonas to Ionizing Radiation

The enzyme phosphoribosyl pyrophosphate synthase (PRPS) catalyzes the conversion of ribose 5-phosphate into phosphoribosyl diphosphate; the latter is a precursor of purine and pyrimidine nucleotides. Here, we investigated the function of PRPS from the single-celled green alga Chlamydomonas reinhardtii in its response to DNA damage from gamma radiation or the alkylating agent LiCl. CrPRPS transcripts were upregulated in cells treated with these agents. We generated CrPRPS-overexpressing transgenic lines to study the function of CrPRPS. When grown in culture with LiCl or exposed to gamma radiation, the transgenic cells grew faster and had a greater survival rate than wild-type cells. CrPRPS overexpression enhanced expression of genes associated with DNA damage response, namely RAD51, RAD1, and LIG1. We observed, from transcriptome analysis, upregulation of genes that code for key enzymes in purine metabolism, namely ribonucleoside-diphosphate pyrophosphokinase subunit M1, adenylate kinase, and nucleoside-diphosphate kinase. We conclude that CrPRPS may affect DNA repair process via regulation of de novo nucleotide synthesis.

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Repression of the pyr Operon in Lactobacillus plantarum Prevents Its Ability To Grow at Low Carbon Dioxide Levels

Journal of Bacteriology ◽

10.1128/jb.187.6.2093-2104.2005 ◽

2005 ◽

Vol 187 (6) ◽

pp. 2093-2104 ◽

Cited By ~ 17

Author(s):

Hervé Nicoloff ◽

Aram Elagöz ◽

Florence Arsène-Ploetze ◽

Benoît Kammerer ◽

Jan Martinussen ◽

...

Keyword(s):

Carbon Dioxide ◽

Lactobacillus Plantarum ◽

De Novo ◽

Site Directed Mutagenesis ◽

Low Carbon ◽

Wild Type ◽

Carbamoyl Phosphate ◽

Phosphoribosyl Pyrophosphate ◽

Pyrimidine Nucleotides ◽

Transcription Attenuation

ABSTRACT Carbamoyl phosphate is a precursor for both arginine and pyrimidine biosynthesis. In Lactobacillus plantarum, carbamoyl phosphate is synthesized from glutamine, ATP, and carbon dioxide by two sets of identified genes encoding carbamoyl phosphate synthase (CPS). The expression of the carAB operon (encoding CPS-A) responds to arginine availability, whereas pyrAaAb (encoding CPS-P) is part of the pyrR1BCAaAbDFE operon coding for the de novo pyrimidine pathway repressed by exogenous uracil. The pyr operon is regulated by transcription attenuation mediated by a trans-acting repressor that binds to the pyr mRNA attenuation site in response to intracellular UMP/phosphoribosyl pyrophosphate pools. Intracellular pyrimidine triphosphate nucleoside pools were lower in mutant FB335 (carAB deletion) harboring only CPS-P than in the wild-type strain harboring both CPS-A and CPS-P. Thus, CPS-P activity is the limiting step in pyrimidine synthesis. FB335 is unable to grow in the presence of uracil due to a lack of sufficient carbamoyl phosphate required for arginine biosynthesis. Forty independent spontaneous FB335-derived mutants that have lost regulation of the pyr operon were readily obtained by their ability to grow in the presence of uracil and absence of arginine; 26 harbored mutations in the pyrR1-pyrB loci. One was a prototroph with a deletion of both pyrR1 and the transcription attenuation site that resulted in large amounts of excreted pyrimidine nucleotides and increased intracellular UTP and CTP pools compared to wild-type levels. Low pyrimidine-independent expression of the pyr operon was obtained by antiterminator site-directed mutagenesis. The resulting AE1023 strain had reduced UTP and CTP pools and had the phenotype of a high-CO2-requiring auxotroph, since it was able to synthesize sufficient arginine and pyrimidines only in CO2-enriched air. Therefore, growth inhibition without CO2 enrichment may be due to low carbamoyl phosphate pools from lack of CPS activity.

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The Novel Dihydroorotate Dehydrogenase (DHODH) Inhibitor PTC299 Inhibit De Novo Pyrimidine Synthesis with Broad Anti-Leukemic Activity Against Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2020-139940 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 8-9

Author(s):

Sujan Piya ◽

Marla Weetall ◽

Josephine Sheedy ◽

Balmiki Ray ◽

Huaxian Ma ◽

...

Keyword(s):

Dna Damage ◽

Acute Myeloid Leukemia ◽

Cell Lines ◽

Myeloid Leukemia ◽

Research Funding ◽

De Novo ◽

Dihydroorotate Dehydrogenase ◽

Nucleotide Synthesis ◽

Inhibition Of Proliferation ◽

Acute Myeloid

Introduction: Acute myeloid leukemia (AML) is characterized by both aberrant proliferation and differentiation arrest at hematopoietic progenitor stages 1,2. AML relies upon de novo nucleotide synthesis to meet a dynamic metabolic landscape and to provide a sufficient supply of nucleotides and other macromolecules 3,4. Hence, we hypothesized that inhibition of de novo nucleotide synthesis would lead to depletion of the nucleotide pool and pyrimidine starvation in leukemic cells compared to their non-malignant counterparts and impact proliferative and differentiation inhibition pathways. PTC299 is an inhibitor of dihydroorotate dehydrogenase (DHODH), a rate limiting enzyme for de novo pyrimidine nucleotide synthesis that is currently in a clinical trial for the treatment of AML. Aim: We investigated the pre-clinical activity of PTC299 against AML in primary AML blasts and cytarabine-resistant cell lines. To confirm that PTC299 effects are due to inhibition of de novo pyrimidine nucleotide synthesis for leukemic growth, we specifically tested the impact of uridine and orotate rescue. In addition, a comprehensive analysis of alteration of metabolic signaling in PI3K/AKT pathways, apoptotic signatures and DNA damage responses were analyzed by Mass cytometry based proteomic analysis (CyTOF) and immunoblotting. The potential clinical relevance of DHODH inhibition was confirmed in an AML-PDX model. Results: The IC50s for all tested cell lines (at 3 day) and primary blasts (at 5-7 day) were in a very low nanomolar range: OCI-AML3 -4.43 nM, HL60 -59.7 nM and primary samples -18-90 nM. Treatment of AML in cytarabine-resistant cells demonstrated that PTC299 induced apoptosis, differentiation, and reduced proliferation with corresponding increase in Annexin V and CD14 positive cells (Fig.1). PTC299-induced apoptosis and inhibition of proliferation was rescued by uridine and orotate. To gain more mechanistic insights, we used an immunoblotting and mass cytometry (CyTOF) based approach to analyze changes in apoptotic and cell signaling proteins in OCI-AML3 cells. Apoptotic pathways were induced (cleaved PARP, cleaved Caspase-3) and DNA damage responses (TP53, γH2AX) and the PI3/AKT pathway were downregulated in response to PTC299. In isogenic cell lines, p53-wildtype cells were sustained and an increased DNA damage response with corresponding increase in apoptosis in comparison to p53-deficient cells was shown. (Fig.2) In a PDX mouse model of human AML, PTC299 treatment improved survival compared to mice treated with vehicle (median survival 40 days vs. 30 days, P=0.0002) (Fig.3). This corresponded with a reduction in the bone marrow burden of leukemia with increased expression of differentiation markers in mice treated with PTC299 (Fig.3). Conclusion: PTC299 is a novel dihydroorotate dehydrogenase (DHODH) inhibitor that triggers differentiation, apoptosis and/or inhibition of proliferation in AML and is being tested in a clinical trials for the treatment of acute myeloid malignancies. Reference: 1. Thomas D, Majeti R. Biology and relevance of human acute myeloid leukemia stem cells. Blood 2017; 129(12): 1577-1585. e-pub ahead of print 2017/02/06; doi: 10.1182/blood-2016-10-696054 2. Quek L, Otto GW, Garnett C, Lhermitte L, Karamitros D, Stoilova B et al. Genetically distinct leukemic stem cells in human CD34- acute myeloid leukemia are arrested at a hemopoietic precursor-like stage. The Journal of experimental medicine 2016; 213(8): 1513-1535. e-pub ahead of print 2016/07/06; doi: 10.1084/jem.20151775 3. Villa E, Ali ES, Sahu U, Ben-Sahra I. Cancer Cells Tune the Signaling Pathways to Empower de Novo Synthesis of Nucleotides. Cancers (Basel) 2019; 11(5). e-pub ahead of print 2019/05/22; doi: 10.3390/cancers11050688 4. DeBerardinis RJ, Chandel NS. Fundamentals of cancer metabolism. Sci Adv 2016; 2(5): e1600200. e-pub ahead of print 2016/07/08; doi: 10.1126/sciadv.1600200 Disclosures Weetall: PTC Therapeutic: Current Employment. Sheedy:PTC therapeutics: Current Employment. Ray:PTC Therapeutics Inc.: Current Employment. Konopleva:Genentech: Consultancy, Research Funding; Rafael Pharmaceutical: Research Funding; Ablynx: Research Funding; Ascentage: Research Funding; Agios: Research Funding; Kisoji: Consultancy; Eli Lilly: Research Funding; AstraZeneca: Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; AbbVie: Consultancy, Research Funding; Calithera: Research Funding; Cellectis: Research Funding; Amgen: Consultancy; Stemline Therapeutics: Consultancy, Research Funding; Forty-Seven: Consultancy, Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Sanofi: Research Funding. Andreeff:Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees. Borthakur:BioLine Rx: Consultancy; BioTherix: Consultancy; Nkarta Therapeutics: Consultancy; Treadwell Therapeutics: Consultancy; Xbiotech USA: Research Funding; Polaris: Research Funding; AstraZeneca: Research Funding; BMS: Research Funding; BioLine Rx: Research Funding; Cyclacel: Research Funding; GSK: Research Funding; Jannsen: Research Funding; Abbvie: Research Funding; Novartis: Research Funding; Incyte: Research Funding; PTC Therapeutics: Research Funding; FTC Therapeutics: Consultancy; Curio Science LLC: Consultancy; PTC Therapeutics: Consultancy; Argenx: Consultancy; Oncoceutics: Research Funding.

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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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Renal hypertrophy in experimental diabetes. The activity of the ‘de novo’ and salvage pathways of purine [corrected] synthesis

Biochemical Journal ◽

10.1042/bj2490911 ◽

1988 ◽

Vol 249 (3) ◽

pp. 911-914 ◽

Cited By ~ 3

Author(s):

S Kunjara ◽

S J Beardsley ◽

A L Greenbaum

Keyword(s):

Nucleic Acids ◽

De Novo ◽

Experimental Diabetes ◽

Salvage Pathway ◽

Phosphoribosyl Pyrophosphate ◽

Renal Hypertrophy ◽

Early Diabetes ◽

Nucleotide Synthesis ◽

Main Route ◽

Salvage Pathways

Measurements were made of the activity of phosphoribosyl pyrophosphate amidotransferase (PPRibP-At, EC 2.4.2.14) and of adenine (APRT, EC 2.4.2.7) and hypoxanthine (HPRT, EC 2.4.2.8) phosphoribosyltransferases, representing the ‘de novo’ and salvage pathways respectively. PPRibP-At activity increased within 3 days of diabetes, whereas APRT and HPRT increased later. Incorporation of [14C]formate and of [8-14C]adenine into the nucleic acids of kidney slices showed that formate was incorporated earlier, and to a greater extent, than was adenine. These results indicate that, although the ‘de novo’ pathway for nucleotide synthesis is the main route in early diabetes, the salvage pathway assumes greater importance at later stages.

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BIOL-10. DISTRIBUTION AND VULNERABILITY OF TRANSCRIPTIONAL OUTPUTS ACROSS THE GENOME IN MYC-AMPLIFIED MEDULLOBLASTOMA CELLS

Neuro-Oncology ◽

10.1093/neuonc/noab090.017 ◽

2021 ◽

Vol 23 (Supplement_1) ◽

pp. i5-i5

Author(s):

Rui Yang ◽

Wenzhe Wang ◽

Meichen Dong ◽

Kristen Roso ◽

Xuhui Bao ◽

...

Keyword(s):

Tumor Cells ◽

Target Genes ◽

De Novo ◽

Inhibitory Effect ◽

Nucleotide Synthesis ◽

Expression Of Genes ◽

High Level ◽

The Impact

Abstract Myc plays a central role in tumorigenesis by orchestrating the expression of genes essential to numerous cellular processes. While it is well established that Myc functions by binding to its target genes to regulate their transcription, the distribution of the transcriptional output across human genome in Myc-amplified cancer cells, and the susceptibility of such transcriptional outputs to therapeutic interferences remain to be fully elucidated. Here, we analyze the distribution of transcriptional outputs in Myc-amplified medulloblastoma (MB) cells by profiling nascent total RNAs within a temporal context. This profiling reveals a major portion of transcriptional action in these cells was directed at the genes fundamental to cellular infrastructures, including rRNAs and particularly those in the mitochondrial genome (mtDNA). Notably, even when Myc protein was depleted by as much as 80%, the impact on transcriptional outputs across the genome was limited, with notable reduction mostly in genes of involved in ribosomal biosynthesis, genes residing in mtDNA or encoding mitochondria-localized proteins, and those encoding histones. In contrast to the limited direct impact of Myc depletion, we found that the global transcriptional outputs were highly dependent on the activity of Inosine Monophosphate Dehydrogenases (IMPDHs), rate limiting enzymes for de novo guanine nucleotide synthesis and whose expression in tumor cells was positively correlated with Myc’s expression. Blockage of IMPDHs attenuated the global transcriptional outputs with a particularly strong inhibitory effect on the aforementioned infrastructure genes, which was accompanied by the abrogation of MB cell’s proliferation in vitro and in vivo. Together, our findings reveal a real time action of Myc as a transcriptional factor in tumor cells, gain new insight into the pathogenic mechanism underlying Myc-driven tumorigenesis, and support IMPDHs as a therapeutic vulnerability in MB cells empowered by a high level of Myc oncoprotein.

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Antitumor Mechanism of Hydroxycamptothecin via the Metabolic Perturbation of Ribonucleotide and Deoxyribonucleotide in Human Colorectal Carcinoma Cells

Molecules ◽

10.3390/molecules26164902 ◽

2021 ◽

Vol 26 (16) ◽

pp. 4902

Author(s):

Yan Li ◽

Wendi Luo ◽

Huixia Zhang ◽

Caiyun Wang ◽

Caiyuan Yu ◽

...

Keyword(s):

Dna Damage ◽

Colorectal Carcinoma ◽

Dna Synthesis ◽

Topoisomerase I ◽

De Novo ◽

Nucleotide Metabolism ◽

Dependent Manner ◽

Anticancer Effect ◽

Nucleotide Synthesis ◽

Human Colorectal Carcinoma

Hydroxycamptothecin (SN38) is a natural plant extract isolated from Camptotheca acuminate. It has a broad spectrum of anticancer activity through inhibition of DNA topoisomerase I, which could affect DNA synthesis and lead to DNA damage. Thus, the action of SN38 against cancers could inevitably affect endogenous levels of ribonucleotide (RNs) and deoxyribonucleotide (dRNs) that play critical roles in many biological processes, especially in DNA synthesis and repair. However, the exact impact of SN38 on RNs and dRNs is yet to be fully elucidated. In this study, we evaluated the anticancer effect and associated mechanism of SN38 in human colorectal carcinoma HCT 116 cells. As a result, SN38 could decrease the cell viability and induce DNA damage in a concentration-dependent manner. Furthermore, cell cycle arrest and intracellular nucleotide metabolism were perturbed due to DNA damage response, of which ATP, UTP, dATP, and TTP may be the critical metabolites during the whole process. Combined with the expression of deoxyribonucleoside triphosphates synthesis enzymes, our results demonstrated that the alteration and imbalance of deoxyribonucleoside triphosphates caused by SN38 was mainly due to the de novo nucleotide synthesis at 24 h, and subsequently the salvage pathways at 48 h. The unique features of SN38 suggested that it might be recommended as an effective supplementary drug with an anticancer effect.

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Down-Regulation of Phosphoribosyl Pyrophosphate Synthetase 1 Inhibits Neuroblastoma Cell Proliferation

Cells ◽

10.3390/cells8090955 ◽

2019 ◽

Vol 8 (9) ◽

pp. 955

Author(s):

Jifu Li ◽

Junhong Ye ◽

Shunqin Zhu ◽

Hongjuan Cui

Keyword(s):

Cell Proliferation ◽

De Novo ◽

Neuroblastoma Cell ◽

Neuroblastoma Cells ◽

Phosphoribosyl Pyrophosphate ◽

Down Regulation ◽

Nucleotide Synthesis ◽

Phosphoribosyl Pyrophosphate Synthetase

Phosphoribosyl pyrophosphate synthetase 1 (PRPS1) is a key enzyme in de novo nucleotide synthesis and nucleotide salvage synthesis pathways that are critical for purine and pyrimidine biosynthesis. Abnormally high expression of PRPS1 can cause many diseases, including hearing loss, hypotonia, and ataxia, in addition to being associated with neuroblastoma. However, the role of PRPS1 in neuroblastoma is still unclear. In this study, we found that PRPS1 was commonly expressed in neuroblastoma cells and was closely related to poor prognosis for cancer. Furthermore, down-regulation of PRPS1 inhibited neuroblastoma cell proliferation and tumor growth in vitro and in vivo via disturbing DNA synthesis. This study provides new insights into the treatment of neuroblastoma patients and new targets for drug development.

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A Comparative Transcriptome Analysis Reveals Physiological Maturation Properties of Mycelia in Pleurotus tuoliensis

Genes ◽

10.3390/genes10090703 ◽

2019 ◽

Vol 10 (9) ◽

pp. 703 ◽

Cited By ~ 1

Author(s):

Du ◽

ti ◽

Hu ◽

Zou ◽

Ye ◽

...

Keyword(s):

De Novo ◽

Transcriptome Assembly ◽

Nucleoside Diphosphate Kinase ◽

Fungal Species ◽

Glycoside Hydrolase Family ◽

Nucleotide Synthesis ◽

Edible Fungus ◽

Highly Active ◽

Medicinal Value ◽

Pleurotus Species

Pleurotus tuoliensis is a precious edible fungus with extremely high nutritive and medicinal value. The cultivation period of P. tuoliensis is longer than those of other Pleurotus species, which is mainly due to a longer mycelium physiological maturation period (30–60 days). Currently, the molecular processes underlying physiological maturation of the mycelium remain unclear. We performed a comparative transcriptomic analysis of immature and mature mycelia using RNA-seq. De novo transcriptome assembly resulted in identification of 17,030 unigenes. 451 differentially expressed genes—including those encoding nucleoside diphosphate kinase (NDPK), glycoside hydrolase family proteins, exopolygalacturonase, and versatile peroxidases—were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that nucleotide synthesis and energy metabolism are highly active during the physiological maturation of mycelia, and genes related to these pathways were significantly upregulated in mature mycelia. NDPK is predicted to be essential for mycelia maturation. Our findings contribute to a comprehensive understanding of mycelia maturation in a commercially important fungal species. Future efforts will focus on the function of NDPK and the mechanism by which it regulates mycelia maturation.

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The effect of glutamine concentration on the activity of carbamoyl-phosphate synthase II and on the incorporation of [3H]thymidine into DNA in rat mesenteric lymphocytes stimulated by phytohaemagglutinin

Biochemical Journal ◽

10.1042/bj2610979 ◽

1989 ◽

Vol 261 (3) ◽

pp. 979-983 ◽

Cited By ~ 53

Author(s):

Z Szondy ◽

E A Newsholme

Keyword(s):

De Novo ◽

Glutamine Concentration ◽

Carbamoyl Phosphate ◽

Purine Nucleotides ◽

Pyrimidine Nucleotides ◽

Nucleotide Synthesis ◽

Pyrimidine Synthesis ◽

Pyrimidine Nucleotide ◽

Formation De Novo ◽

Phosphate Synthase

The maximum catalytic activities of carbamoyl-phosphate synthase II, a limiting enzyme for pyrimidine nucleotide synthesis, are very much less than those of glutaminase, a limiting enzyme for glutamine utilization, in lymphocytes and macrophages; and the flux through the pathway for pyrimidine formation de novo is only about 0.4% of the rate of glutamine utilization by lymphocytes. The Km of synthase II for glutamine is about 16 microM and the concentration of glutamine necessary to stimulate lymphocyte proliferation half-maximally is about 21 microM. This agreement suggests that the importance of glutamine for these cells is provision of nitrogen for biosynthesis of pyrimidine nucleotides (and probably purine nucleotides). However, the glutamine concentration necessary for half-maximal stimulation of glutamine utilization (glutaminolysis) by the lymphocytes is 2.5 mM. The fact that the rate of glutamine utilization by lymphocytes is markedly in excess of the rate of the pathway for pyrimidine nucleotide synthesis de novo and that the Km and ‘half-maximal concentration’ values are so different, suggests that the glutaminolytic pathway is independent of the use of glutamine nitrogen for pyrimidine synthesis.

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Concentration of phosphoribosyl pyrophosphate in the kidney during development and in experimental diabetic hypertrophy

Biochemical Journal ◽

10.1042/bj2340579 ◽

1986 ◽

Vol 234 (3) ◽

pp. 579-585 ◽

Cited By ~ 13

Author(s):

S Kunjara ◽

M Sochor ◽

A Adeoya ◽

P McLean ◽

A L Greenbaum

Keyword(s):

De Novo ◽

Pentose Phosphate ◽

Short Term ◽

Phosphoribosyl Pyrophosphate ◽

Nucleotide Synthesis ◽

Immature Rat ◽

Adult Rat ◽

Developmental Growth ◽

Kidney Hypertrophy ◽

Salvage Pathways

The effect of developmental growth on the kidney content of phosphoribosyl pyrophosphate PPRibP was studied in rats at ages between the foetal animal and up to 100 days of age. In addition, the effect of short-term diabetes (up to 14 days) on the renal content of PPRibP was studied in immature rats and in adults aged approx. 60 days. The developmental pattern of PPRibP is such that the PPRibP content is lowest in the young rat and increases as the rate of kidney growth slows. In the adult rat, the early kidney hypertrophy of diabetes is accompanied by a fall in PPRibP content and, again, the PPRibP content returns to normal as the rate of kidney hypertrophy diminishes. Induction of diabetes in the immature rat causes a lesser degree of kidney hypertrophy and also a smaller depression of renal PPRibP content. The activity of PPRibP synthetase (EC 2.7.6.1) is not significantly affected by age or diabetes. The changes in PPRibP content are discussed in relation to the generation of ribose 5-phosphate by the pentose phosphate pathway and the utilization of PPRibP for nucleotide synthesis via the ‘de novo’ and salvage pathways.

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