Metabolic Requirement of Cucurbita pepo for Boron

Lateral roots of intact summer squash seedlings (Cucurbita pepo L.) were used to quantify the effects of boron deficiency on DNA synthesis, protein synthesis, and respiration. The temporal relationship between changes in these metabolic activities and the cessation of root elongation caused by boron deprivation was determined. Transferring 5-day-old squash seedlings to a hydroponic culture medium without boron for 6 hours resulted in a 62% reduction in net root elongation and a 30% decrease in the incorporation of [³H]thymidine into DNA by root tips (apical 5-millimeter segments). At this time, root tips from both boron-deficient and boron-sufficient plants exhibited nearly identical rates of incorporation of [¹⁴C]leucine into protein and respiration as measured by O₂ consumption. After an additional 6 hours of boron deprivation, root elongation had nearly ceased. Concomitantly, DNA synthesis in root apices was 66% less than in the boron-sufficient control plants and protein synthesis was reduced 43%. O₂ consumption remained the same for both treatments. The decline and eventual cessation of root elongation correlated temporally with the decrease in DNA synthesis, but preceded changes in protein synthesis and respiration. These results suggest that boron is required for continued DNA synthesis and cell division in root meristems.     

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.

     

Increased Arginine Biosynthesis during Phosphorus Deficiency : A Response to the Increased Ammonia Content of Leaves

The accumulation of arginine in leaves of four citrus rootstock cultivars during P deficiency has been demonstrated to be due to increased de novo synthesis rather than decreased catabolism or increased protein degradation (E Rabe, CJ Lovatt, 1984, Plant Physiol 76: 747-752). In this report, we provide evidence (a) that the increased activity of the arginine biosynthetic pathway observed for citrus rootstocks grown under P-deficient conditions for 7 months is due to an increase in the concentration of ammonia in leaves of P-deficient plants and (b) that ammonia accumulation and removal through arginine systhesis are early responses to phosphorus deficiency for both a woody perennial, rough lemon (Citrus limon), and an herbaceous annual, summer squash (Cucurbita pepo). Transferring 5-day-old squash plants to a phosphorus-deficient nutrient solution for only 10 days resulted in a 2-fold increase in the concentration of nitrate in the youngest fully expanded leaves (YFE). Concomitantly, the specific activity of nitrate reductase doubled and the ammonia content of P-deficient YFE leaves increased to a concentration significantly greater that of leaves from healthy control plants (P < 0.05). Consistent with increased availability of ammonia, the incorporation of NaH¹⁴CO₃ into arginine plus urea doubled during phosphorus deficiency and arginine accumulated. Despite the accumulation of nitrate and ammonia in YFE leaves during phosphorus deficiency, the total nitrogen content of these leaves was less than that of the healthy control plants. Similar results were obtained for rough lemon. Nitrate content of the YFE leaves increased 1.5- and 3.0-fold in plants deprived of phosphorus for 6 and 12 weeks, respectively. Ammonia content of the leaves increased as P deficiency progressed to 1.4 ± 0.08 mg (± se, n = 4) per gram dry weight, a level 1.8-fold greater than that of the P-sufficient control plants. During P deficiency de novo arginine biosynthesis in rough lemon increased 10-fold. Immersing the petiole of YFE leaves from P-sufficient squash and rough lemon plants in 50 millimolar NH₄⁺ for 3 hours resulted in the accumulation of ammonia in the leaves, and a 4-fold increase in the incorporation of NaH¹⁴CO₃ into arginine plus urea. Taken together, these results provide strong evidence that the accumulation of nitrate and ammonia in leaves is an early response of both woody and herbaceous plants to P deprivation. The data are consistent with the hypothesis that increased de novo arginine biosynthesis in leaves during P deficiency is in response to ammonia content of the leaves.     

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.     

Key role of uridine kinase and uridine phosphorylase in the homeostatic regulation of purine and pyrimidine salvage in brain

Uridine, the major circulating pyrimidine nucleoside, participating in the regulation of a number of physiological processes, is readily uptaken into mammalian cells. The balance between anabolism and catabolism of intracellular uridine is maintained by uridine kinase, catalyzing the first step of UTP and CTP salvage synthesis, and uridine phosphorylase, catalyzing the first step of uridine degradation to β-alanine in liver. In the present study we report that the two enzymes have an additional role in the homeostatic regulation of purine and pyrimidine metabolism in brain, which relies on the salvage synthesis of nucleotides from preformed nucleosides and nucleobases, rather than on the de novo synthesis from simple precursors. The experiments were performed in rat brain extracts and cultured human astrocytoma cells. The rationale of the reciprocal regulation of purine and pyrimidine salvage synthesis in brain stands (i) on the inhibition exerted by UTP and CTP, the final products of the pyrimidine salvage pathway, on uridine kinase and (ii) on the widely accepted idea that pyrimidine salvage occurs at the nucleoside level (mostly uridine), while purine salvage is a 5-phosphoribosyl-1-pyrophosphate (PRPP)-mediated process, occurring at the nucleobase level. Thus, at relatively low UTP and CTP level, uptaken uridine is mainly anabolized to uridine nucleotides. On the contrary, at relatively high UTP and CTP levels the inhibition of uridine kinase channels uridine towards phosphorolysis. The ribose-1-phosphate is then transformed into PRPP, which is used for purine salvage synthesis.     

Effect of boron on cell elongation and division in squash roots

This work establishes that cessation of root elongation of intact squash (Cucurbita pepo L.) plants is an early result of boron deficiency. Root elongation is slowed by 6 hours and is virtually stopped as early as 24 hours after boron is first withheld from the nutrient solution. As root elongation ceased, cell elongation progressed distally into the region normally occupied by the apical meristem and eventually the meristem became indistinguishable. Differentiation was determined by use of an elongation index in which cell length was compared to cell width. This index ranged from a low of 0.8 in boron-sufficient root meristems to a high of 3 in root meristems grown in a boron-deficient nutrient solution for 98 hours. It is concluded that a continuous supply of boron is not essential for cell elongation but is required for maintenance of meristematic activity. Boron may act as a regulator of cell division in this tissue.     

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.     

Cellulose production is coupled to sensing of the pyrimidine biosynthetic pathway via c‐di‐GMP production by the DgcQ protein of Escherichia coli

Production of cellulose, a stress response‐mediated process in enterobacteria, is modulated in Escherichia coli by the activity of the two pyrimidine nucleotide biosynthetic pathways, namely, the de novo biosynthetic pathway and the salvage pathway, which relies on the environmental availability of pyrimidine nitrogenous bases. We had previously reported that prevalence of the salvage over the de novo pathway triggers cellulose production via synthesis of the second messenger c‐di‐GMP by the DgcQ (YedQ) diguanylate cyclase. In this work, we show that DgcQ enzymatic activity is enhanced by UTP, whilst being inhibited by N‐carbamoyl‐aspartate, an intermediate of the de novo pathway. Thus, direct allosteric control by these ligands allows full DgcQ activity exclusively in cells actively synthesizing pyrimidine nucleotides via the salvage pathway. Inhibition of DgcQ activity by N‐carbamoyl‐aspartate appears to be favoured by protein‐protein interaction between DgcQ and PyrB, a subunit of aspartate transcarbamylase, which synthesizes N‐carbamoyl‐aspartate. Our results suggest that availability of pyrimidine bases might be sensed, somehow paradoxically, as an environmental stress by E. coli. We hypothesize that this link might have evolved since stress events, leading to extensive DNA/RNA degradation or lysis of neighbouring cells, can result in increased pyrimidine concentrations and activation of the salvage pathway.     

Essentiality of Boron for Dinitrogen Fixation in Anabaena sp. PCC 7119

The relationship between the requirement for boron and the form of N supplied in nutrient media to cyanobacterium Anabaena sp. PCC 7119 was investigated. When cells were grown in a medium which contained nitrate or ammonium-N, boron deficiency in the nutrient media did not inhibit growth or change cell composition. However, when cells were dependent on N₂ fixation, the lack of boron inhibited growth (i.e. growth ceased after 96 hours under these conditions). Additionally, boron-deficient cells showed a significant decrease in their content of phycobiliproteins and chlorophyll and accumulated carbohydrates within 24 hours of removing boron from the nutrient media. Inhibition of photosynthetic O₂ evolution accompanied the decrease in photosynthetic pigments. Boron deficiency symptoms were relieved when either boron or combined N was added to boron-deficient cultures. The degree of recovery depended upon the age of the cultures. Assays of nitrogenase activity showed that, after 2 hours of growth, nitrogenase activity of boron-deficient cells was inhibited by 40%. After 24 hours a total inactivation of nitrogenase activity was observed in boron-deficient cells. These results strongly suggest an involvement of boron in N₂ fixation in cyanobacteria.     

Salt Stress Causes Acceleration of Purine Catabolism and Inhibition of Pyrimidine Salvage in Zea mays Root Tips

Nucleotide metabolism was studied in apical 5.0 mm root tipsof corn plants (Zea mays L., cv. Pioneer 3906) hydroponicallycultured for 7 d and then salinized for 19 d at a rate calculatedto reduce the osmotic potential (_o) of the solutions by O.1MPad^–1 to a final _o = -0.4 MPa. Saline treatments withtwo different molar ratios of Ca₂₊/Na⁺ were employed, viz.,0–03 (2.5 mol m^–3 CaCl₂ + 86.5 mol m^–3 NaCl)for the NaCl treatment and 0.73 (31.5 mol m^–3 CaCl₂ +43.1 mol m^–3 NaCl) for the NaCl + CaCl₂ treatment. Bothsalt treatments reduced root growth by more than 30%. The capacityof roots to provide purine nucleotides either by de novo synthesisor by re-utilization of existing bases, e.g. salvage of hypoxanthineto adenine nucleotides, was not affected by either salt treatment.However, catabolism of hypoxanthine was accelerated more than3.5-fold by both salt treatments, demonstrating an increasedcapacity for purine catabolism which would shift the normal1: 1 ratio of synthesis: degradation of purine nucleotides observedfor the roots of healthy control plants to less than 0.2 duringsalt stress. The ratio of pyrimidine nucleotide synthesis: degradationwas also reduced. In this case, the unfavourable shift towardnucleotide degradation resulted because both salt treatmentsreduced salvage capacity by more than 25%, but had no compensatingeffect on de novo synthesis or catabolism of pyrimidines. Key words: Salinity, osmotic potential, nucleotide metabolism     

Substrate Inhibition of Uracil Phosphoribosyltransferase by Uracil Can Account for the Uracil Growth Sensitivity of Leishmania donovani Pyrimidine Auxotrophs

The pathogenic protozoan parasite Leishmania donovani is capable of both de novo pyrimidine biosynthesis and salvage of pyrimidines from the host milieu. Genetic analysis has authenticated L. donovani uracil phosphoribosyltransferase (LdUPRT), an enzyme not found in mammalian cells, as the focal enzyme of pyrimidine salvage because all exogenous pyrimidines that can satisfy the requirement of the parasite for pyrimidine nucleotides are funneled to uracil and then phosphoribosylated to UMP in the parasite by LdUPRT. To characterize this unique parasite enzyme, LdUPRT was expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. Kinetic analysis revealed apparent K_m values of 20 and 99 μm for the natural substrates uracil and phosphoribosylpyrophosphate, respectively, as well as apparent K_m values 6 and 7 μm for the pyrimidine analogs 5-fluorouracil and 4-thiouracil, respectively. Size exclusion chromatography revealed the native LdUPRT to be tetrameric and retained partial structure and activity in high concentrations of urea. L. donovani mutants deficient in de novo pyrimidine biosynthesis, which require functional LdUPRT for growth, are hypersensitive to high concentrations of uracil, 5-fluorouracil, and 4-thiouracil in the growth medium. This hypersensitivity can be explained by the observation that LdUPRT is substrate-inhibited by uracil and 4-thiouracil, but 5-fluorouracil toxicity transpires via an alternative mechanism. This substrate inhibition of LdUPRT provides a protective mechanism for the parasite by facilitating purine and pyrimidine nucleotide pool balance and by sparing phosphoribosylpyrophosphate for consumption by the nutritionally indispensable purine salvage process.     

The Crystal Structure and Activity of a Putative Trypanosomal Nucleoside Phosphorylase Reveal It to be a Homodimeric Uridine Phosphorylase

Purine nucleoside phosphorylases (PNPs) and uridine phosphorylases (UPs) are closely related enzymes involved in purine and pyrimidine salvage, respectively, which catalyze the removal of the ribosyl moiety from nucleosides so that the nucleotide base may be recycled. Parasitic protozoa generally are incapable of de novo purine biosynthesis; hence, the purine salvage pathway is of potential therapeutic interest. Information about pyrimidine biosynthesis in these organisms is much more limited. Though all seem to carry at least a subset of enzymes from each pathway, the dependency on de novo pyrimidine synthesis versus salvage varies from organism to organism and even from one growth stage to another. We have structurally and biochemically characterized a putative nucleoside phosphorylase (NP) from the pathogenic protozoan Trypanosoma brucei and find that it is a homodimeric UP. This is the first characterization of a UP from a trypanosomal source despite this activity being observed decades ago. Although this gene was broadly annotated as a putative NP, it was widely inferred to be a purine nucleoside phosphorylase. Our characterization of this trypanosomal enzyme shows that it is possible to distinguish between PNP and UP activity at the sequence level based on the absence or presence of a characteristic UP-specificity insert. We suggest that this recognizable feature may aid in proper annotation of the substrate specificity of enzymes in the NP family.     

Regulation of Pyrimidine Biosynthesis in Intact Cells of Cucurbita pepo

The occurrence of the complete orotic acid pathway for the biosynthesis de novo of pyrimidine nucleotides was demonstrated in the intact cells of roots excised from summer squash (Cucurbita pepo L. cv. Early Prolific Straightneck). Evidence that the biosynthesis of pyrimidine nucleotides proceeds via the orotate pathway in C. pepo included: (a) demonstration of the incorporation of [¹⁴C]NaHCO₃, [¹⁴C]carbamylaspartate, and [¹⁴C]orotic acid into uridine nucleotides; (b) the isolation of [¹⁴C]orotic acid when [¹⁴C]NaHCO₃ and [¹⁴C]carbamylaspartate were used as precursors; (c) the observation that 6-azauridine, a known inhibitor of the pathway, blocked the incorporation of early precursors into uridine nucleotides while causing a concomitant accumulation of orotic acid; and (d) demonstration of the activities of the component enzymes of the orotate pathway in assays employing cell-free extracts.     

Purine Metabolism in Trypanosomatids*

SYNOPSIS. Purine nucleotide biosynthesis was studied in culture forms of Trypanosoma cruzi strain Y, Crithidia deanei (a reduviid trypanosomatid with an endosymbiote) and an aposymbiotic strain of C. deanei (obtained by curing C. deanei with chloramphenicol). Trypanosoma cruzi was found to synthesize purine nucleotides only from the preformed bases adenine and guanine (“salvage” pathway), adenine being incorporated into both adenine and guanine nucleotides. Similar results were obtained with guanine, indicating that this flagellate has a system for the interconversion of purine nucleotides. Crithidia deanei was able to synthesize purine and pyrimidine nucleotides from glycine (“de novo” pathway) and purine nucleotides from adenine and guanine (“salvage” pathway). Adenine was incorporated into both adenine and guanine nucleotides, while guanine was incorporated into guanine nucleotides only, indicating the presence of a metabolic block at the level of GMP reducaase. The aposymbiotic C. deanei strain was unable to utilize glycine for the synthesis of purine nucleotides, although glycine was utilized for synthesizing pyrimidine nucleotides. These results suggest that the endosymbiote is implicated in the de novo purine nucleotide pathway of the C. deanei-endosymbiote complex. The incorporation of adenine and guanine by aposymbiotic C. deanei strain followed a pattern similar to that observed for C. deanei.     

Pyrimidine Biosynthesis Is Not an Essential Function for Trypanosoma brucei Bloodstream Forms

Background

African trypanosomes are capable of both pyrimidine biosynthesis and salvage of preformed pyrimidines from the host, but it is unknown whether either process is essential to the parasite.

Methodology/Principal Findings

Pyrimidine requirements for growth were investigated using strictly pyrimidine-free media, with or without single added pyrimidine sources. Growth rates of wild-type bloodstream form Trypanosoma brucei brucei were unchanged in pyrimidine-free medium. The essentiality of the de novo pyrimidine biosynthesis pathway was studied by knocking out the PYR6-5 locus that produces a fusion product of orotate phosphoribosyltransferase (OPRT) and Orotidine Monophosphate Decarboxylase (OMPDCase). The pyrimidine auxotroph was dependent on a suitable extracellular pyrimidine source. Pyrimidine starvation was rapidly lethal and non-reversible, causing incomplete DNA content in new cells. The phenotype could be rescued by addition of uracil; supplementation with uridine, 2′deoxyuridine, and cytidine allowed a diminished growth rate and density. PYR6-5⁻/⁻ trypanosomes were more sensitive to pyrimidine antimetabolites and displayed increased uracil transport rates and uridine phosphorylase activity. Pyrimidine auxotrophs were able to infect mice although the infection developed much more slowly than infection with the parental, prototrophic trypanosome line.

Conclusions/Significance

Pyrimidine salvage was not an essential function for bloodstream T. b. brucei. However, trypanosomes lacking de novo pyrimidine biosynthesis are completely dependent on an extracellular pyrimidine source, strongly preferring uracil, and display reduced infectivity. As T. brucei are able to salvage sufficient pyrimidines from the host environment, the pyrimidine biosynthesis pathway is not a viable drug target, although any interruption of pyrimidine supply was lethal.     

OBSERVATIONS ON PURINE METABOLISM IN RAT BRAIN

Both de novo and preformed base or salvage pathways are simultaneously operative in the biosynthesis of purine nucleotides in rat brain per se. A preferential utilization of de novo precursors is demonstrated.     

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.)