Pyrimidine biosynthesis and its regulation in the developing rat brain.

—Measurements of the incorporation of [¹⁴C]NaHCO₃ into orotic acid, uridine nucleotides and RNA in tissue minces establish the occurrence of the complete orotate pathway for the de novo biosynthesis of pyrimidines in rat brain. Selective inhibition of the incorporation of various radiolabelled precursors into orotic acid by uridine demonstrates the operation of a feedback control mechanism in brain minces and indicates carbamoylphosphate synthetase to be the site of inhibition; purine nucleosides were similarly found to inhibit the de novo biosynthesis of pyrimidines. The activity of the orotate pathway, as assessed by the rate of incorporation of [¹⁴C]NaHCO₃ into orotic acid, was found to be very high in fetal brain and to decline rapidly with neurological development; the mature rat brain exhibits less than 1% of the activity of the fetal brain at 18 days of gestation. Comparative studies on the ability of minces of the brain and several extraneural tissues to utilize [¹⁴C]NaHCO₃ and [¹⁴C]aspartate as precursors of orotic acid lead us to speculate that variations in the ability of tissues to synthesize orotic acid de novo are determined by similar variations in their ability to synthesize carbamoylphosphate.     

Aspartate Carbamyltransferase : Site of End-Product Inhibition of the Orotate Pathway in Intact Cells of Cucurbita pepo

Lovatt et al. (1979 Plant Physiol 64: 562-569) have previously demonstrated that end-product inhibition functions as a mechanism regulating the activity of the orotic acid pathway in intact cells of roots excised from 2-day-old squash plants (Cucurbita pepo L. cv Early Prolific Straightneck). Uridine (0.5 millimolar final concentration) or one of its metabolites inhibited the incorporation of NaH¹⁴CO₃, but not [¹⁴C]carbamylaspartate or [¹⁴C]orotic acid, into uridine nucleotides (ΣUMP). Thus, regulation of de novo pyrimidine biosynthesis was demonstrated to occur at one or both of the first two reactions of the orotic acid pathway, those catalyzed by carbamylphosphate synthetase (CPSase) and aspartate carbamyltransferase (ACTase). The results of the present study provide evidence that ACTase alone is the site of feedback control by added uridine or one of its metabolites. Evidence demonstrating regulation of the orotic acid pathway by end-product inhibition at ACTase, but not at CPSase, includes the following observations: (a) addition of uridine (0.5 millimolar final concentration) inhibited the incorporation of NaH¹⁴CO₃ into ΣUMP by 80% but did not inhibit the incorporation of NaH¹⁴CO₃ into arginine; (b) inhibition of the orotate pathway by added uridine was not reversed by supplying exogenous ornithine (5 millimolar final concentration), while the incorporation of NaH¹⁴CO₃ into arginine was stimulated more than 15-fold when both uridine and ornithine were added; (c) incorporation of NaH¹⁴CO₃ into arginine increased, with or without added ornithine when the de novo pyrimidine pathway was inhibited by added uridine; and (d) in assays employing cell-free extracts prepared from 2-day-old squash roots, the activity of ACTase, but not CPSase, was inhibited by added pyrimidine nucleotides.     

Regulation of pyrimidine biosynthesis by purine and pyrimidine nucleosides in slices of rat tissue

Evidence of the primary sites for the regulation of de novo pyrimidine biosynthesis by purine and pyrimidine nucleosides has been obtained in tissue slices through measurements of the incorporation of radiolabeled precursors into an intermediate and end product of the pathway. Both purine and pyrimidine nucleosides inhibited the incorporation of [¹⁴C]-NaHCO₃ into orotic acid and uridine nucleotides, and the inhibition was found to be reversible upon transferring the tissue slices to a medium lacking nucleoside. The ammonia-stimulated incorporation of [¹⁴C]NaHCO₃ into orotic acid, which is unique to liver slices, was sensitive to inhibition by pyrimidine nucleosides at physiological levels of ammonia, but this regulatory mechanism was lost at toxic levels of ammonia. Adenosine, but not uridine, was found to have the additional effects of inhibiting the conversion of [¹⁴C]orotic acid to UMP and depleting the tissue slices of PRPP. Since PRPP is required as an activator of the first enzyme of the de novo pathway, CPSase II, and a substrate of the fifth enzyme, OPRTase, these results indicate that adenosine inhibits the incorporation of [¹⁴C]NaHCO₃ into orotic acid and the incorporation of [¹⁴C]orotic acid into UMP by depriving CPSase II and OPRTase, respectively, of PRPP. Uridine or its metabolites, on the other hand, appear to control the de novo biosynthesis of pyrimidines through end product inhibition of an early enzyme, most likely CPSase II. We found no evidence of end product inhibition of the conversion of orotic acid to UMP in tissue slices.     

Biosynthesis of pyrimidine nucleotides in cultured root callus ofCucurbita pepo

Summary Callus cultures derived from roots of summer squash (Cucurbita pepo L. c.v. Early Prolific Straightneck) grown in the dark at 27° C on Murashige and Skoog medium supplemented per liter with 30 g sucrose, 100 mg myo-inositol, 10 mg indole-butyric acid, 2 mg glycine, 1 mg thiamin, 0.5 mg nicotinic acid, 0.5 mg pyridoxine, and 2 g Gelrite were capable of synthesizing pyrimidine nucleotides both de novo and through salvage of existing pyrimidine nucleotides and bases. Evidence that the de novo biosynthesis of pyrimidine nucleotides proceeded via the orotate pathway in this tissue included: (a) demonstration of the incorporation of NaH¹⁴CO₃ and [¹⁴C₆]orotic acid into uridine nucleotides (ΣUMP), and (b) demonstration that the addition of 6-azauridine blocked the incorporation of these two precursors into ΣUMP. The synthesis of pyrimidine nucleotides through the salvage of existing pyrimidine bases and ribosides was demonstrated by measuring the incorporation of [¹⁴C₂]uracil and [¹⁴C₂]uridine into ΣUMP. Salvage of both [¹⁴C₂]uracil and [¹⁴C₂]uridine was sensitive to inhibition by 6-azauridine or one of its metabolites. The orotic acid pathway for the de novo biosynthesis of pyrimidine nucleotides was demonstrated to be sensitive to end-product inhibition. Uridine, or one of its metabolites, inhibited the incorporation of NaH¹⁴CO₃, but not [¹⁴C₆]orotic acid, into ΣUMP. Evidence is presented suggesting that Aspartate carbomoyltransferase is the site of feedback control. This work was supported by the Citrus Research Center and Agricultural Experiment Station of the University of California, Riverside, CA. Submitted in partial fulfillment of the requirements of the University of California for the Master of Science degree in botany (F-F.L.)     

Purine and pyrimidine metabolism in cultured white spruce (Picea glauca) cells: Metabolic fate of 14C-labeled precursors and activity of key enzymes

In order to examine the biosynthesis, interconversion, and degradation of purine and pyrimidine nucleotides in white spruce cells, radiolabeled adenine, adenosine, inosine, uracil, uridine, and orotic acid were supplied exogenously to the cells and the overall metabolism of these compounds was monitored. [8‐¹⁴C]adenine and [8‐¹⁴C]adenosine were metabolized to adenylates and part of the adenylates were converted to guanylates and incorporated into both adenine and guanine bases of nucleic acids. A small amount of [8‐¹⁴C]inosine was converted into nucleotides and incorporated into both adenine and guanine bases of nucleic acids. High adenosine kinase and adenine phosphoribosyltransferase activities in the extract suggested that adenosine and adenine were converted to AMP by these enzymes. No adenosine nucleosidase activity was detected. Inosine was apparently converted to AMP by inosine kinase and/or a non‐specific nucleoside phosphotransferase. The radioactivity of [8‐¹⁴C]adenosine, [8‐¹⁴C]adenine, and [8‐¹⁴C]inosine was also detected in ureide, especially allantoic acid, and CO₂. Among these 3 precursors, the radioactivity from [8‐¹⁴C]inosine was predominantly incorporated into CO₂. These results suggest the operation of a conventional degradation pathway. Both [2‐¹⁴C]uracil and [2‐¹⁴C]uridine were converted to uridine nucleotides and incorporated into uracil and cytosine bases of nucleic acids. The salvage enzymes, uridine kinase and uracil phosphoribosyltransferase, were detected in white spruce extracts. [6‐¹⁴C]orotic acid, an intermediate of the de novo pyrimidine biosynthesis, was efficiently converted into uridine nucleotides and also incorporated into uracil and cytosine bases of nucleic acids. High activity of orotate phosphoribosyltransferase was observed in the extracts. A large proportion of radioactivity from [2‐¹⁴C]uracil was recovered as CO₂ and β‐ureidopropionate. Thus, a reductive pathway of uracil degradation is functional in these cells. Therefore, white spruce cells in culture demonstrate both the de novo and salvage pathways of purine and pyrimidine metabolism, as well as some degradation of the substrates into CO₂.     

Two pathways of pyrimidine nucleotide biosynthesis in birds

Dillerent chicken tissues are shown to display a clearly pronounced specificity relative to [2-14C] orotic acid and [5-3H]uridine as precursors of synthesis of the pool and RNA pyrimidine nucleotides. The fraction of pyrimidine nucleotides synthetized relative to the reserve pathway (uridine utilization) decreases in the series: kidneys greater than duodenum mucosa greater than lungs greater than liver greater than pancreas greater than bone marrow greater than brain greater than spleen. The results of [2-14C]orotic acid and [53H]uridine incorporation into UMP and CMP of the liver and spleen tissues RNA are interpreted in terms of the concept on existence of separate pools of pyrimidine phosphates--RNA precursors.     

Pyrimidine metabolism during somatic embryo development in white spruce (Picea glauca)

Pyrimidine metabolism was investigated at various stages ofsomatic embryo development of white spruce (Picea glauca). The contribution of thede novo and the salvage pathways of pyrimidine biosynthesis to nucleotide and nucleic acid formation and the catabolism of pyrimidine was estimated by the exogenously supplied [6-¹⁴C]orotic acid, an intermediate of thede novo pathway, and with [2-¹⁴C]uridine and [2-¹⁴C]uracil, substrates of the salvage pathways. Thede novo pathway was very active throughout embryo development. More than 80 percnt; of [6-¹⁴C]orotic acid taken up by the tissue was utilized for nucleotide and nucleic acid synthesis in all stages of this process. The salvage pathways of uridine and uracil were also operative. Relatively high nucleic acid biosynthesis from uridine was observed, whereas the contribution of uracil salvage to the pyrimidine nucleotide and nucleic acid synthesis was extremely limited. A large proportion of uracil was degraded as ¹⁴CO₂, probably via β-ureidopropionate. Among the enzymes of pyrimidine metabolism, orotate phosphoribosyltransferase was high during the initial phases of embryo development, after which it gradually declined. Uridine kinase, responsible for the salvage of uridine, showed an opposite pattern, since its activity increased as embryos developed. Low activities of uracil phosphoribosyltransferase and non-specific nucleoside phosphotransferase were also detected throughout the developmental period. These results suggest that the flux of thede novo and salvage pathways of pyrimidine nucleotide biosynthesisin vivo is roughly controlled by the amount of these enzymes. However, changing patterns of enzyme activity during embryo development that were measuredin vitro did not exactly correlate with the flux estimated by the radioactive precursors. Therefore, other fine control mechanisms, such as the fluctuation of levels of substrates and/or effectors may also participate to the real control of pyrimidine metabolism during white spruce somatic embryo development.     

Pyrimidine biosynthesis and its regulation in the estrogen-stimulated chick oviduct.

Studies on the incorporation of radio-labeled precursors into orotic acid and the pyrimidine nucleotides of RNA have established the occurrence of the orotate pathway for the de novo biosynthesis of pyrimidines in the chick oviduct. Measurements of the rate of incorporation of precursors into orotic acid in minces of oviduct revealed the activity of the orotate pathway to be accelerated in response to estrogen-stimulated nucleic acid synthesis and tissue growth. These data indicate that extrahepatic tissues of avian species meet their requirements for pyrimidine nucleotides through de novo synthesis rather than depend upon the liver or other exogenous sources for a supply of preformed pyrimidines. An examination of the influence of pyrimidine and purine nucleosides on the incorporation of radio-labeled precursors into orotic acid yielded evidence that pyrimidine biosynthesis in the chick is quite sensitive to inhibition by both purines and pyrimidines; the data indicate the reaction catalyzed by carbamoylphosphate synthetase to be the site of inhibition in both cases.     

Changs in pyrimidine nucleotide biosynthesis during germination of white spruce (picea glauca) somatic embryos

Summary Changes in pyrimidine metabolism were investigated in germinating white spruce somatic embryos by following the metabolic fate of [2-¹⁴C]uracil and [2-¹⁴C]uridine, intermediate metabolites of the salvage pathway and [6-¹⁴C]orotic acid, a central metabolite of the de novo. nucleotide biosynthesis. An active uridine salvage was found to be responsible for the enlargement of the nucleotide pool at the inception of germination. Uridine kinase, which catalyzes the conversion of uridine to uridine monophosphate (UMP), was found to be very active in partially dried embryos and during the early phases of imbibition. The contribution of uracil to the nucleotide pool was negligible since a large amount of radioactivity from [2-¹⁴C]uracil was recovered in degradation products. As germination progressed, the decline of the uridine salvage pathway was concomitant with an increase of the de novo biosynthetic pathway. The central enzyme of the de novo pathway, orotate phosphoribosyltransferase, showed increased activity and contributed to the larger amount of orotate being anabolized. These results suggest that although both the salvage and de novo pathways operate in germinating white spruce somatic embryos, their contribution to the enlargement of the nucleotide pool appears tightly regulated as germination progresses.     

Effect of short-term salt stress on the metabolic profiles of pyrimidine, purine and pyridine nucleotides in cultured cells of the mangrove tree, Bruguiera sexangula

To investigate the short‐term (3 h) effect of salt on the metabolism of purine, pyrimidine and pyridine nucleotides in mangrove (Bruguiera sexangula) cells, we examined the uptake and overall metabolism of radiolabelled intermediates involved in the de novo pathways and substrates of salvage pathways for nucleotide biosynthesis in the presence and absence of 100 mM NaCl. Uptake by the cells of substrates for the salvage pathways was much faster than uptake of intermediates of the de novo pathways. The activity of the de novo pyrimidine biosynthesis estimated by [2‐¹⁴C]orotate metabolism was not significantly affected by the salt. About 20–30% of [2‐¹⁴C]uridine, [2‐¹⁴C]uracil and more than 50% of [2‐¹⁴C]cytidine were salvaged for pyrimidine nucleotide biosynthesis. However, substantial quantities of these compounds were degraded to ¹⁴CO₂ via β‐ureidopropionate (β‐UP), and degradation of β‐UP was increased by the salt. The activities of the de novo pathway, estimated by [2‐¹⁴C] 5‐aminoimidazole‐4‐carboxamide ribonucleoside, and the salvage pathways from [8‐¹⁴C]adenosine and [8‐¹⁴C]guanosine for the purine nucleotide biosynthesis were not influenced by the salt. Most [8‐¹⁴C]hypoxanthine was catabolised to ¹⁴CO₂, and other purine compounds are also catabolised via xanthine. Purine catabolism was stimulated by the salt. [³H]Quinolinate, [carbonyl‐¹⁴C]nicotinamide and [carboxyl‐¹⁴C]nicotinic acid were utilised for the biosynthesis of pyridine nucleotides. The salvage pathways for pyridine nucleotides were significantly stimulated by the salt. Trigonelline was synthesised from all pyridine precursors that were examined; its synthesis was also stimulated by the salt. We discuss the physiological role of the salt‐stimulated reactions of nucleotide metabolism.     

De Novo Purine Biosynthesis in Intact Cells of Cucurbita pepo

The capacity of intact cells of roots excised from summer squash plants (Cucurbita pepo L. cv Early Prolific Straightneck) to synthesize purine nucleotides de novo was investigated. Evidence that purine nucleotides are synthesized de novo included: (a) demonstration of the incorporation of [1-¹⁴C]glycine, [2-¹⁴C]glycine, NaH¹⁴CO₃, and H¹⁴COONa into total adenine nucleotides; (b) observation that the addition of azaserine or aminopterin, known inhibitors of de novo purine synthesis in other organisms, blocked the incorporation of these precursors into adenine nucleotides; and (c) demonstration that the purine ring synthesized from these precursors was labeled in a manner consistent with the pathway for de novo purine biosynthesis found in microorganisms and animal tissues. Under optimal conditions, the activity of this pathway in roots excised from 2-day-old squash plants was 244 ± 13 nanomoles (mean ± standard error, n = 17) NaH¹⁴CO₃ incorporated into ∑Ade (the sum of the adenine nucleotides, nucleoside and free base) per gram tissue during the 3-hour incubation period.

The possible occurrence of alternative enzymic reactions for the first steps of de novo purine biosynthesis was also investigated. No conclusive evidence was obtained to support the operation of alternative enzymic reactions in the intact cell of C. pepo.

     

Orotic acid biosynthesis in arginine-deficient rats.

Arginine deficiency is associated with a mild orotic aciduria. Liver slices from rats fed a purified l-amino acid diet with (control) and without arginine supplementation were used for studies of [¹⁴C]bicarbonate incorporation into orotic acid. The nanomoles of orotic acid synthesized in isolated liver slices from both control and arginine-deficient animals increased linearly with time. Orotic acid biosynthesis was significantly greater in liver slices than slices of heart, muscle, kidney, and minced spleen. The order of orotate biosynthesis from [¹⁴C]bicarbonate was liver > spleen = kidney > muscle > heart. Arginine deficiency resulted in a significant stimulation of liver orotic acid biosynthesis. This stimulation in pyrimidine biosynthesis can account for a major portion of the orotic aciduria. Orotic acid synthesis from spleens isolated from arginine-deficient rats was also enhanced compared with controls. Although the rate of orotic acid biosynthesis is small relative to liver production, the spleen may contribute slightly to increased orotic aciduria in the arginine-deficient rat. Arginine supplementation in vitro to livers from rats fed either the control of arginine-deficient diet resulted in a significant reduction in synthesis of orotic acid. Dietary arginine may play a key role in regulating mitochondrial carbamoyl phosphate utilization into both pyrimidine and urea biosynthesis.     

Pyrimidine biosynthesis in rat brain

—Studies on the incorporation of [¹⁴C]NaHCO₃ into both orotic acid and RNA in tissue slices reveal the occurrence of the complete orotate pathway for the de novo biosynthesis of pyrimidines in the rat brain. A comparison of the rates of incorporation of bicarbonate into orotic acid and RNA in tissue slices of brain and liver indicate the brain to be one-fourth to one-half as active as the liver in the de novo biosynthesis of pyrimidines. The results of this study favor the proposal that the adult rat brain can meet its needs for pyrimidines through de novo synthesis and is not dependent upon salvage activity and an extraneural supply of pyrimidines.     

Pyrimidine metabolism in cotyledons of germinating alaska peas

Cotyledons from Pisum sativum L. cv. Alaska seeds were excised 12, 36, 108, 132, and 156 hours after imbibition in aerated distilled water. They were then incubated under aseptic conditions for 6 hours in solutions containing either uridine-2-¹⁴C or orotic acid-6-¹⁴C. Uridine was more extensively degraded to ¹⁴CO₂ at all germination stages than was orotate, and these rates remained essentially constant at each stage. Incorporation of each compound into RNA increased about 2-fold from the 12th to the 156th hour, although the total RNA present decreased slightly over this interval. Paper chromatography of soluble labeled metabolites produced from orotate showed that the capacity to metabolize this pyrimidine increased markedly as germination progressed. Radioactivity in uridine-5′-P, uridine diphosphate-hexoses, and uridine diphosphate increased most, while smaller or less consistent increases in uridine, uracil, uridine triphosphate, and an unidentified UDPX compound were also observed. The data suggest that orotate metabolism was initially limited by orotidine-5′-phosphate pyrophosphorylase or by 5-phosphoribosyl-1-pyrophosphate. Incorporation of uridine into RNA appeared to be limited at the earliest germination periods by conversion of uridine-5′-P to uridine diphosphate. Thus, during the 1st week of germination the orotic acid pathway and a salvage pathway converting uridine into RNA become activated.     

Profiles of pyrimidine biosynthesis,salvage and degradation in disks of potato (Solanum tuberosum L.) tubers

In order to obtain general metabolic profiles of pyrimidine ribo- and deoxyribonucleotides in potato (Solanum tuberosum L.) plants, the in situ metabolic fate of various (14)C-labelled precursors in disks from growing potato tubers was investigated. The activities of key enzymes in potato tuber extracts were also studied. The following results were obtained. Of the intermediates in de novo pyrimidine biosynthesis, [(14)C]carbamoylaspartate was converted to orotic acid and [2-(14)C]orotic acid was metabolized to nucleotides and RNA. UMP synthase, a bifunctional enzyme with activities of orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 5'-monophosphate decarboxylase (EC 4.1.1.23), exhibited high activity. The rates of uptake of pyrimidine ribo- and deoxyribonucleosides by the disks were high, in the range 2.0-2.8 nmol (g FW)(-1) h(-1). The pyrimidine ribonucleosides, uridine and cytidine, were salvaged exclusively to nucleotides, by uridine/cytidine kinase (EC 2.7.1.48) and non-specific nucleoside phosphotransferase (EC 2.7.1.77). Cytidine was also salvaged after conversion to uridine by cytidine deaminase (EC 3.5.4.5) and the presence of this enzyme was demonstrated in cell-free tuber extracts. Deoxycytidine, a deoxyribonucleoside, was efficiently salvaged. Since deoxycytidine kinase (EC 2.7.1.74) activity was extremely low, non-specific nucleoside phosphotransferase (EC 2.7.1.77) probably participates in deoxycytidine salvage. Thymidine, which is another pyrimidine deoxyribonucleoside, was degraded and was not a good precursor for nucleotide synthesis. Virtually all the thymidine 5'-monophosphate synthesis from thymidine appeared to be catalyzed by phosphotransferase activity, since little thymidine kinase (EC 2.7.1.21) activity was detected. Of the pyrimidine bases, uracil, but not cytosine, was salvaged for nucleotide synthesis. Since uridine phosphorylase (EC 2.4.2.3) activity was not detected, uracil phosphoribosyltransferase (EC 2.4.2.9) seems to play the major role in uracil salvage. Uracil was degraded by the reductive pathway via beta-ureidopropionate, but cytosine was not degraded. The activities of the cytosine-metabolizing enzymes observed in other organisms, pyrimidine nucleoside phosphorylase (EC 2.4.2.2) and cytosine deaminase (EC 3.5.4.1), were not detected in potato tuber extracts. Operation of the de novo synthesis of deoxyribonucleotides via ribonucleotide reductase and of the salvage pathway of deoxycytidine was demonstrated via the incorporation of radioactivity from both [2-(14)C]cytidine and [2-(14)C]deoxycytidine into DNA. A novel pathway converting deoxycytidine to uracil nucleotides was found and deoxycytidine deaminase (EC 3.5.4.14), an enzyme that may participate in this pathway, was detected in the tuber extracts.     

Formation of vicine and convicine by Vicia faba

Methods are described for the quantitative extraction and separation of the pyrimidine glucosides, vicine and convicine. The contents of these two substances in germinating seeds and young seedlings of Vicia faba remain constant for the first 2 weeks. Net synthesis and accumulation of vicine and convicine occurs in developing seeds. That the synthesis occurs within the pod and the pyrimidine glucosides are not translocated into them, was shown by injection of ¹⁴C-labelled precursors into the pods. [1-¹⁴C]- and [2-¹⁴C]-acetate were weakly incorporated but much greater incorporation was observed with [U-¹⁴C]-aspartic acid and [6-¹⁴C]-orotic acid. The results indicate that the orotic acid pathway is involved in the formation of the pyrimidine ring of both vicine and convicine.     

Circadian variations in pyrimidine nucleotide synthesis in rat liver

The biosynthesis of pyrimidine components in rat liver varies with the time of the day. The concentrations of both the cytidine and the uridine components of the acid-soluble extract are lowest in the morning hours and highest around midnight. The utilization of [2-¹⁴C]orotic acid for the synthesis of the pyrimidine components of the acid-soluble extract, RNA, and DNA has a similar character. Analogous changes also are seen in the uptake of [U-¹⁴C]cytidine and its utilization for the synthesis of RNA cytosine.     

Liver growth, biosynthesis of cytidine nucleotides and level of cytochrome P-450 in rat liver after administration of alpha-hexachlorocyclohexane.

The biosynthesis of cytidine nucleotides and the level of microsomal cytochrome P-450 in intact and regenerating rat liver after repeated administration of alpha-hexachlorocyclohexane (alpha-HCH) were compared. In alpha-HCH treated animals the utilization of [2-14C] orotic acid for the synthesis of cytidine nucleotides is suppressed. In 24-h regenerating liver the incorporation of labelled orotic acid into cytidine nucleotides is markedly activated; the degree of activation is lower in regenerating livers of alpha-HCH treated animals. The changes in the level of cytochrome P-450 vary inversely with the changes in the utilization of [2-14C] orotic acid for the synthesis of cytidine nucleotides. The activity of cytidine triphosphate synthetase of liver cytosol increases shortly after the administration of alpha-HCH; uridine-cytidine kinase is enhanced in the later stages of the drug action. Within 15-45 min after the administration of alpha-HCH the uptake of [U-14 C] cytidine into the liver and its incorporation into RNA cytosine are increased. After the administration of the drug the uptake of [2-14 C] uridine and its incorporation into RNA uracil is also enhanced whereas its utilization for the synthesis of cytidine nucleotides of the acid-soluble extract as well as for the RNA cytosine are suppressed.     

Interaction between the urea cycle and the orotate pathway: studies with isolated hepatocytes

Two enzymes catalyze the synthesis of carbamylphosphate (CP) in the liver. One is intramitochondrial and utilizes ammonia to make CP for ureagenesis; the second is cytoplasmic and utilizes glutamine to produce CP for pyrimidine biosynthesis. The extent to which the metabolic independence of the two pathways is abridged by the use of a common precursor was examined with measurements of the incorporation of [14C]NaHCO3 into orotic acid, uridine nucleotides, and urea in isolated hepatocytes. Pyrimidine synthesis was markedly stimulated by physiological concentrations of ammonia, and the stimulation was antagonized by ornithine. At intracellular concentrations of ornithine and levels of ammonia found in the portal circulation, some 90% of pyrimidine synthesis was ammonia-dependent. When the glutamine-dependent activity was released from feedback inhibition with galactosamine, the ammonia-dependent incorporation still accounted for 2/3 of pyrimidine synthesis. These results do not support the widely held view that the cytoplasmic enzyme is the sole source of CP for pyrimidine biosynthesis in the liver. They suggest instead that the bulk of the CP incorporated into hepatic pyrimidines is of mitochondrial origin. However, an experiment with intact animals failed to provide decisive evidence on this interpretation. Pyrimidine biosynthesis was sharply inhibited by the addition of uridine, but ureagenesis was unaffected. When physiological levels of ammonia were provided, the sensitivity of pyrimidine biosynthesis to uridine was lost. Although inhibition of the ammonia-dependent enzyme by pyrimidines has been observed with cell-free preparations, it was not evident in the intact cell. Thus, to the extent that the CP consumed in pyrimidine biosynthesis is of mitochondrial origin, feedback control of the orotate pathway appears to be thwarted.     

Effect of Inorganic Phosphate on the Biosynthesis of Purine and Pyrimidine Nucleotides in Suspension-Cultured Cells of Catharanthus roseus

The levels of purine and pyrimidine nucleotides in suspensioncultures of Catharanthus roseus were determined 24 h after stationary-phasecells were transferred to fresh complete (‘+Pi’)or phosphate-deficient (‘–Pi’) Murashige-Skoogmedium. The levels of ATP, GTP, UTP and CTP were from approx.3 to 5-fold greater in the cells grown in ‘+Pi’medium than in the cells grown in ‘–Pi’ medium.The levels of almost all other nucleotides were slightly higherin the cells in ‘+Pi’ medium. The rates of de novoand salvage biosynthesis of purine and pyrimidine nucleotideswere estimated from the rates of incorporation of radioactivityfrom [¹⁴C]formate, [2–¹⁴C]glycine, NaH¹⁴CO₃, [6–¹⁴C]orotate,[8–¹⁴C]adenine, [8–¹⁴C]adenosine, [2–¹⁴C]uraciland [2–¹⁴C]uridine. The results indicated that the activityof both the de novo and the salvage pathway was higher in thecells in ‘+Pi’ medium than in the cells in ‘–Pi’medium. The rate of degradation estimated from the rate of releaseof ¹⁴CO₂ from labelled purines and pyrimidines indicated thatdegradation of uridine was significantly reduced in the cellsin ‘+Pi’ medium, but no significant difference wasfound in the degradation of adenine, adenosine and uracil. Thepossible role of Pi in the control of the biosynthesis of nucleotidesand in the degradation of uridine is discussed. Catharanthus roseus, Madagascar periwinkle, suspension culture, inorganic phosphate, nucleotides, purines, pyrimidines, biosynthesis, degradation