Orotic acid biosynthesis in rat liver: Studies on the source of carbamoylphosphate

Tremblay, George C.; Crandall, David E.; Knott, Charlotte E.; Alfant, Marc

doi:10.1016/0003-9861(77)90191-6

Cited by 81 publications

(22 citation statements)

References 39 publications

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“…Thus, in the absence of added NH4C1, very little NaH[14C]03 was incorporated into uridine nucleotides and only a trace of 14C appeared in orotate; addition of 10 mM NH4C1 stimulated incorporation into UMP eightfold and into orotate thirtyfold (not shown). The increased de novo synthesis of pyrimidine nucleotides in response to the addition of ammonium ions to isolated hepatocytes confirms the findings of others using rat liver slices [4], whole rats [5] and rat liver homogenates [S], and could be reversed by addition of 1 mM or 5 mM L-ornithine. The effect of adding a range of concentrations of Orn(PAc) to hepatocytes incubated in 10 mM NH4Cl is shown in Fig.…”

Section: Effect Of Ovn(pac) On the Incorporation Of N U H [ '~C ] O~ supporting

confidence: 88%

“…The excretion of orotic acid in patients with urea cycle defects apparently occurs because mitochondrial carbamoyl phosphate cannot be utilized for citrulline synthesis and becomes available for extramitochondrial synthesis of pyrimidines. Whether carbamoyl phosphate of mitochondrial origin is available for de rzovo pyrimidine biosynthesis in liver under normal physiological conditions depends on the level of ammonium ions and ornithine in normal liver, since the spillage of carbamoyl phosphate from the mitochondria into the pyrimidine pathway is dependent on the ammonium ion concentration and can be reversed by ornithine [4,5].…”

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confidence: 99%

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Effect of the Transition-State Analogue, delta-N-(Phosphonacetyl)-l-ornithine on Citrulline Synthesis in Isolated Rat-Liver Mitochondria and on Urea Synthesis in Isolated Rat Hepatocytes

Hoogenraad¹,

Sutherland²,

Howlett³

1979

Eur J Biochem

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δ‐N‐(Phosphonacetyl)‐l‐ornithine [Orn(PAc)], a transition state analogue for ornithine transcarbamylase, is an effective inhibitor of citrulline synthesis in isolated rat liver mitochondria, and urea synthesis in isolated hepatocytes. In intact mitochondria, the efficacy of inhibition of citrulline synthesis by Orn(PAc) was shown to be dependent on the conditions of incubation. The citrulline biosynthetic capacity of intact mitochondria was reduced to 60% of controls by as little as 0.01 mM inhibitor, and to 20% of controls by 0.05 mM inhibitor in mitochondria treated with 0.5% Triton X‐100. The greater capacity of Orn(PAc) to inhibit citrulline synthesis after treatment of mitochondria with Triton X‐100 was in part due to the elimination of a permeability barrier to reactants of the carbamoyl‐phosphate synthetase (ammonia) reaction. Thus, maximum inhibition by Orn(PAc) was dependent on the relative activities of carbamoyl‐phosphate synthetase (ammonia) and ornithine transcarbamylase, and inhibition could be completely reversed by stimulating ornithine transcarbamylase using ornithine. When added to isolated hepatocytes, Orn(PAc) inhibited urea biosynthesis by up to 20% at 0.5 mM concentration of inhibitor. This inhibition in the conversion of carbamoyl phosphate to citrulline had a marked effect on the utilization of intra‐mitochondrially synthesized carbamoyl phosphate for extramitochondrial synthesis of pyrimidines. There was up to a twofold stimulation in the rate of synthesis of orotate and uridine nucleotides when Orn(PAc) was added to hepatocytes incubated in basal medium (Eagles) supplemented with 10 mM NH4Cl. These data confirm clinical observations that one of the consequences of a reduction in ornithine transcarbamylase activity, such as occurs in certain disease states, is a stimulation in pyrimidine biosynthesis, and further suggests that transition‐state analogues, such as Orn(PAc), may be useful in metabolic modelling studies on the effects of enzymic defects in mammalian tissues.

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Section: Effect Of Ovn(pac) On the Incorporation Of N U H [ '~C ] O~ supporting

confidence: 88%

mentioning

confidence: 99%

Effect of the Transition-State Analogue, delta-N-(Phosphonacetyl)-l-ornithine on Citrulline Synthesis in Isolated Rat-Liver Mitochondria and on Urea Synthesis in Isolated Rat Hepatocytes

Hoogenraad¹,

Sutherland²,

Howlett³

1979

Eur J Biochem

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“…Ornithine at 1 mM and 5 mM inhibited the incorporation of ['4C]bicarbonate into CUMP and into orotic acid by 84% and 92'x respectively when added NH; was 10 mM, and by 22% and 65';;; respectively when added NH4f was 1 mM. These results accord with those of Tremblay et al [S] and Pausch et al [9] and are consistent with ornithine enhancing the utilization of carbainoyl phosphate for citrulline synthesis and thereby reducing the availability of carbamoyl phosphate synthesized by synthetase I for extramitochondrial synthesis of pyrimidines.…”

Section: Ejject C?fornirhine On Stimulution Of Pvrimidine Synthesis supporting

confidence: 90%

“…Changes in the orotic acid excretion of rats brought about by dietary manipulation of urea cycle intermediates [34] would seem to indicate that this contribution could be substantial. Experiments in virro indicate that the stimulation by ammonium ion is apparent at 1 m M and lower [9] (this paper). Tremblay and co-workers conclude that over SO','" of NaH14C03 incorporated into orotic acid is ammoniadependent and ornithine-reversible at physiological ammonium and ornithine concentrations.…”

Section: Discussionmentioning

confidence: 62%

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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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Biochemical basis for D,L,‐beta‐hydroxybutyrate‐induced teratogenesis

Shum

Sadler

1990

Teratology

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Previous investigations have demonstrated that a potential mechanism for D,L,-beta-hydroxybutyrate (BOHB)-induced teratogenesis in neurulating mouse embryos (5-6 somite stage) after 24 hours of exposure in vitro is mediated by an inhibition of the pentose phosphate pathway (PPP) (Hunter, et al. '87). Employing conceptuses of an earlier stage (2-3 somite stage), the biochemistry of BOHB-induced abnormalities was examined further by exposing embryos to 32 mM BOHB for 24 hour and comparing results with controls with respect to the rate of metabolism via the PPP, de novo pyrimidine biosynthesis (PB), and BOHB utilization. Moreover, the capability of these BOHB-exposed embryos to recover from such an insult was also assessed by transferring them to fresh control medium and allowing them to grow for an additional 36 hours. Both controls and BOHB-exposed embryos showed a progressive increase in rate of BOHB utilization between days 9 and 11.5 of gestation in vitro. Exposure to ketone body produced a 100% rate of neural tube defects and a 25.2% decrease in total embryonic protein content. In contrast to results obtained at the 5-6 somite stage, no inhibition of the PPP in whole conceptuses, embryos, or visceral yolk sacs was observed in the group exposed to BOHB at the 2-3 somite stage. Furthermore, a 7.5 mM D-ribose supplement, an intermediate in the PPP, was unable to rescue the younger embryos from BOHB-induced abnormalities and growth retardation. On the other hand, BOHB produced a 34.3% decrease in pyrimidine biosynthesis in the 2-3 somite embryos, but not in the visceral yolk sac. In addition, embryos recovered biochemically after being transferred to control medium, demonstrating a 25.5% overshoot in pyrimidine biosynthesis. Therefore, the mechanism of BOHB-induced teratogenesis appears to differ depending on the stage of embryonic development at the time of initial exposure.

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Orotic acid biosynthesis in rat liver: Studies on the source of carbamoylphosphate

Cited by 81 publications

References 39 publications

Effect of the Transition-State Analogue, delta-N-(Phosphonacetyl)-l-ornithine on Citrulline Synthesis in Isolated Rat-Liver Mitochondria and on Urea Synthesis in Isolated Rat Hepatocytes

Effect of the Transition-State Analogue, delta-N-(Phosphonacetyl)-l-ornithine on Citrulline Synthesis in Isolated Rat-Liver Mitochondria and on Urea Synthesis in Isolated Rat Hepatocytes

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

Biochemical basis for D,L,‐beta‐hydroxybutyrate‐induced teratogenesis

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