Control of Pyrimidine Biosynthesis in the Perfused Liver. Feedback Inhibition of Glutamine-Dependent Carbamoyl Phosphate Synthetase

Pausch, Jürgen; Wilkening, J.; Nowack, Junko; Decker, Karl

doi:10.1111/j.1432-1033.1975.tb04075.x

Cited by 35 publications

(4 citation statements)

References 29 publications

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“…6) corresponds quantitatively to the expansion of the uridylate pool ( Fig. 5) and is in line with the regulation of glutamine-dependent carbamoyl-phosphate synthetase by UTP (Pausch et al, 1975). This site of regulation of pyrimidine synthesis de novo is also consistent with the labelling pattern of the intermediates of this pathway, all of which remained at low levels (Fig.…”

Section: Stimulation Of Pyrimidine Synthesis De Novo By D-galactosonesupporting

confidence: 83%

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

Keppler

Schulz-Holstege

Fauler

et al. 1982

Biochemical Journal

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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. [(14)C]Uridine labelling of hepatoma cells indicated a division of [(14)C]uridylate from UTP into UDP-galactosone; the latter was formed at a rate of more than 1.7mmolxh(-1)x(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 (14)CO(2) 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 degrees 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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Section: Stimulation Of Pyrimidine Synthesis De Novo By D-galactosonesupporting

confidence: 83%

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

Keppler

Schulz-Holstege

Fauler

et al. 1982

Biochemical Journal

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“…This results, eventually, in cell necrosis and hepatitis-like damage of the liver' 1 '. On the other hand, pyrimidine nucleotide de novo biosynthesis was shown to be enhanced after GalN treatment, due to relief of feedback inhibition of glutaminedependent carbamoyl phosphate synthetase' 6 '. Uridine effectively counteracts the UTP deficiency; it was shown to replenish the GalN-depleted uridine phosphate pool within one hour' 5 ' and to prevent the pathogenic sequence of events when given as late as 3 h after GalN administration' 2 '.…”

mentioning

confidence: 99%

Response of Isolated Rat Hepatocytes to D-Galactosamine and Uridine

Hofmann¹,

Wilkening²,

Nowack³

et al. 1976

Hoppe-Seyler´s Zeitschrift für physiologische Chemie

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Isolated rat hepatocytes preserved for several hours their contents of ATP, CTP, GTP, UTP and UDPG. Leakage of intracellular and membrane-bound enzymes remained in the range observed with the isolated perfused rat liver. D -Galactosamine led to a rapid decrease of the UTP and UDPG contents while ATP and GTP levels remained unaffected. The sum of acidsoluble uracil nucleotides increased immediately after the addition of D-galactosamine, indicating release of the UTP-mediated feed-back inhibition of pyrimidine nucleotide de novo biosynthesis. Addition of undine to a hepatocy te suspension resulted in a rapid increase in the levels of uridylate derivatives; the concentrations of UTP and UDPG, previously depleted by D-galactosamine, were restored within 1 h. Isolated hepatocytes appear suitable for studies on the pathogenic sequence of events elicited by D -galactosamine and on the regulation of pyrimidine biosynthesis. Die Reaktion isolierter Hepatozyten auf Gabe von D-Galaktosamin und UridinZusammenfassung: In isolierten Rattenhepatozyten blieben die Gehalte von ATP, CTP, GTP, UTP und UDPG über mehrere Stunden hinweg weitgehend konstant. Der Austritt intrazellulärer und membrangebundener Enzyme bewegte sich im Bereich des an der isoliert perfundierten Rattenleber Beobachteten. D-Galaktosamin-Zugabe führte zu einer raschen Abnahme der UTP-und UDPG-Spiegel, während der Gehalt an ATP und GTP konstant blieb. Die Summe aller säurelösli-chen Uracilnucleotide nahm unmittelbar nach Gabe von D-Galaktosamin zu; dies deutet darauf hin, daß unter diesen Bedingungen auch in isolierten Hepatozyten die feed-back-Hemmung der

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“…This compares with a rate of synthesis found for unstimulated perfused rat liver by Pausch et al [25] of 0.084 kmol x g wet wt-' x h-' and a D-galactosamine-induced rate of I .78 ymol x g wet wt x h-'. It is possible that the protein catabolic state of isolated hepatocytes may provide sufficient endogenous ammonium ion for some stimulation of' pyrimidine synthesis, particularly if urea cycle intermediates are depleted during cell preparation.…”

Section: Synthc3sis Qf Pyrimidines De Novomentioning

confidence: 82%

Effect of Ammonium Ion on Pyrimidine Synthesis de novo in Isolated Rat Hepatocytes

Barton

Hoogenraad

1981

European Journal of Biochemistry

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Addition of ammonium ions to isolated rat hepatocytes stimulated the rate of synthesis of pyrimidines. Isolation and quantification of pyrimidine nucleotides, orotic acid and the acid-hydrolyzed product of carbamoylaspartic acid by ion-exchange chromatography and high-pressure liquid chromatography show a marked stimulation in the incorporation of ['4C]bicarbonatc in incubations with added ammonium ions. The incorporation into total uridine nucleotides (CUMP) was increased twofold in the presence of 5 m M ammonium ion, and approximately eightyfold into orotic acid. There was a parallel increase in labelling of carbamoylaspartic acid from undetectable to a level similar to that of orotic acid. The specific activity of urea formed during the incubations did not change during incubations or in the presence of ammonium ions confirming that the change in labelling of pyrimidine was not due to a change in the specific activity of precursor. Despite the stimulation in incorporation of label into pyrimidines, there was no increase in the hepatocyte content of CUMP, which was 11.5 pmol/g dry weight, although the orotic acid content increased from 0.09 pmol/g dry weight in the absence of added ammonium ions (but in the presence of 2 mM L-glutamine) to 8.6 pmol/g dry weight with 5 mM ammonium ion.The stimulated incorporation of ['4C]bicarbonate in the presence of 5 mM and 10 mM ammonium ion was shown to be due to a stimulated synthesis of carbamoyl phosphate, since greater than 80 of label in the uracil ring was present at position C-2. Incubation of hepatocytes in basal medium (Eagles) containing 2.5 foetal calf serum and 20 niM bicarbonate showed that there was a significant stimulation of pyrimidine synthesis with 1 mM ammonium ion. The stimulatory effect of ammonium ions on incorporation of bicarbonate into pyrimidines was almost completely reversed by 5 mM L-ornithine and was partially reversed by 1 mM L-ornithine. Evidence for a contribution of the urea cycle carbamoyl phosphate synthetase to pyrimidine synthesis is discussed.

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Control of Pyrimidine Biosynthesis in the Perfused Liver. Feedback Inhibition of Glutamine-Dependent Carbamoyl Phosphate Synthetase

Cited by 35 publications

References 29 publications

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

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

Response of Isolated Rat Hepatocytes to D-Galactosamine and Uridine

Effect of Ammonium Ion on Pyrimidine Synthesis de novo in Isolated Rat Hepatocytes

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