Methods in Enzymology, Volume 51: Purine and Pyrimidine Nucleotide Metabolism

1979 ◽  
Vol 7 (1) ◽  
pp. 259-259
Author(s):  
H. ANNE SIMMONDS
Keyword(s):  
Nucleotide Metabolism ◽  
Pyrimidine Nucleotide

Purine and pyrimidine nucleotide metabolism in higher plants

2003 ◽  
Vol 160 (11) ◽  
pp. 1271-1295 ◽  
Author(s):  
Claudio Stasolla ◽  
Riko Katahira ◽  
Trevor A. Thorpe ◽  
Hiroshi Ashihara
Keyword(s):  
Higher Plants ◽  
Nucleotide Metabolism ◽  
Pyrimidine Nucleotide

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

1982 ◽  
Vol 206 (1) ◽  
pp. 139-146 ◽  
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.

scholarly journals Genomic analysis of a riboflavin-overproducing Ashbya gossypii mutant isolated by disparity mutagenesis

2019 ◽  
Author(s):  
Tatsuya Kato ◽  
Junya Azegami ◽  
Ami Yokomori ◽  
Hideo Dohra ◽  
Hesham A. El Enshasy ◽  
...  
Keyword(s):  
Genomic Analysis ◽  
Tca Cycle ◽  
Nucleotide Metabolism ◽  
Repair System ◽  
Ashbya Gossypii ◽  
Riboflavin Production ◽  
Dna Mismatch ◽  
Pyrimidine Nucleotide ◽  
Dna Mismatch Repair System ◽  
Genes Encoding

Abstract Background: Ashbya gossypii naturally overproduces riboflavin and has been utilized for industrial riboflavin production. To improve riboflavin production, various approaches have been developed. In this study, to investigate the change in metabolism of a riboflavin-overproducing mutant, namely, the W122032 strain (MT strain) that was isolated by disparity mutagenesis, genomic analysis was carried out. Results: In the genomic analysis, 33 homozygous and 1377 heterozygous mutations were detected. Among these heterozygous mutations, the proportion of mutated reads in each gene was different, ranging from 21 to 75%. These results suggest that the MT strain exhibits a mixture of diploidy, triploidy and tetraploidy, or contains multiple nuclei containing different mutations. We tried to isolate haploid spores from the MT strain to prove it ploidy, but these spores were never isolated. This result indicate MT strain does not produce its spores. Homozygous and heterozygous mutations were found in genes encoding enzymes involved in amino acid metabolism, the TCA cycle, purine and pyrimidine nucleotide metabolism and the DNA mismatch repair system. Especially, genes encoding enzymes in mitochondria had many homozygous and heterozygous mutations intensively. These results suggest that the overproduction of riboflavin in the MT strain may be associated with mitochondrial dysfunction. Conclusion: This study provides new insights into riboflavin production in A. gossypii and the usefulness of disparity mutagenesis for the creation of new types of mutants for metabolic engineering.

PNC2 (SLC25A36) deficiency associated with the hyperinsulinism/hyperammonemia syndrome

2021 ◽  
Author(s):  
Maher A Shahrour ◽  
Francesco Massimo Lasorsa ◽  
Vito Porcelli ◽  
Imad Dweikat ◽  
Maria Antonietta Di Noia ◽  
...  
Keyword(s):  
Glutamate Dehydrogenase ◽  
Hela Cells ◽  
Nucleotide Metabolism ◽  
Congenital Hyperinsulinism ◽  
Inner Mitochondrial Membrane ◽  
Pathogenic Variants ◽  
Pyrimidine Nucleotide ◽  
Biochemical Assays ◽  
Whole Exome ◽  
Repeat Domain

Abstract Context The hyperinsulinism/hyperammonemia (HI/HA) syndrome, the second most common form of congenital hyperinsulinism, has been associated to dominant mutations in GLUD1, coding for the mitochondrial enzyme glutamate dehydrogenase, that increase enzyme activity by reducing its sensitivity to allosteric inhibition by GTP. Objective To identify the underlying genetic aetiology in two siblings who presented with the biochemical features of HI/HA syndrome but did not carry pathogenic variants in GLUD1, and to determine the functional impact of the newly identified mutation. Main Outcome Measures The patients were investigated by whole exome sequencing. Yeast complementation studies and biochemical assays on the recombinant mutated protein were performed. The consequences of stable slc25a36 silencing in HeLa cells were also investigated. Results A homozygous splice site variant was identified in solute carrier family 25, member 36 (SLC25A36), encoding the pyrimidine nucleotide carrier 2 (PNC2), a mitochondrial nucleotide carrier that transports pyrimidine as well as guanine nucleotides across the inner mitochondrial membrane. The mutation leads to a 26 aa in-frame deletion in the first repeat domain of the protein which abolished transport activity. Furthermore, knockdown of slc25a36 expression in HeLa cells caused a marked reduction in the mitochondrial GTP content which likely leads to an hyperactivation of glutamate dehydrogenase in our patients. Conclusions We report for the first time a mutation in PNC2/SLC25A36 leading to HI/HA and provide functional evidence of the molecular mechanism responsible for this phenotype. Our findings underscore the importance of mitochondrial nucleotide metabolism and expand the role of mitochondrial transporters in insulin secretion.

scholarly journals Severe pyridine nucleotide depletion in fibroblasts from Lesch–Nyhan patients

2002 ◽  
Vol 366 (1) ◽  
pp. 265-272 ◽  
Author(s):  
Lynette D. FAIRBANKS ◽  
Gabriella JACOMELLI ◽  
Vanna MICHELI ◽  
Tina SLADE ◽  
H. Anne SIMMONDS
Keyword(s):  
Dna Repair ◽  
Nicotinic Acid ◽  
Neuronal Damage ◽  
Pyridine Nucleotide ◽  
Culture Conditions ◽  
Nucleotide Metabolism ◽  
Atp Depletion ◽  
Early Developmental Stage ◽  
Pyrimidine Nucleotide ◽  
Nad Depletion

The relationship between a complete deficiency of the purine enzyme hypoxanthine-guanine phosphoribosyltransferase and the neurobehavioural abnormalities in Lesch—Nyhan disease remains an enigma. In vitro studies using lymphoblasts or fibroblasts have evaluated purine and pyrimidine metabolism with conflicting results. This study focused on pyridine nucleotide metabolism in control and Lesch—Nyhan fibroblasts using radiolabelled salvage precursors to couple the extent of uptake with endocellular nucleotide concentrations. The novel finding, highlighted by specific culture conditions, was a marked NAD depletion in Lesch—Nyhan fibroblasts. ATP and GTP were also 50% of the control, as reported in lymphoblasts. A 6-fold greater incorporation of [14C]nicotinic acid into nicotinic acid— adenine dinucleotide by Lesch—Nyhan fibroblasts, with no unmetabolized substrate (20% in controls), supported disturbed pyridine metabolism, NAD depletion being related to utilization by poly(ADP-ribose) polymerase in DNA repair. Although pyrimidine nucleotide concentrations were similar to controls, Lesch—Nyhan cells showed reduced [14C]cytidine/uridine salvage into UDP sugars. Incorporation of [14C]uridine into CTP by both was minimal, with more than 50% [14C]cytidine metabolized to UTP, indicating that fibroblasts, unlike lymphoblasts, lack active CTP synthetase, but possess cytidine deaminase. Restricted culture conditions may be neccesary to mimic the situation in human brain cells at an early developmental stage. Cell type may be equally important. NAD plus ATP depletion in developing brain could restrict DNA repair, leading to neuronal damage/loss by apoptosis, and, with GTP depletion, affect neurotransmitter synthesis and basal ganglia dopaminergic neuronal systems. Thus aberrant pyridine nucleotide metabolism could play a vital role in the pathophysiology of Lesch—Nyhan disease.

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