The effect of various concentrations of nucleobases, nucleosides or glutamine on the incorporation of [3H]thymidine into DNA in rat mesenteric-lymph-node lymphocytes stimulated by phytohaemagglutinin.

1. The rate of [3H]thymidine incorporation into DNA was measured in phytohaemagglutinin-stimulated lymph-node lymphocytes of the rat. 2. Addition of nucleobases or nucleosides to culture medium that already contained 0.2 mM-glutamine had a small stimulatory effect on incorporation. At lower concentrations of glutamine, adenosine (even at 1 microM) caused a marked increase in the rate of incorporation. 3. In the absence of added glutamine, addition of nucleosides or nucleobases markedly increased the rate of incorporation: nucleosides were more effective than nucleobases; and the rate of proliferation in the presence of 10 microM-adenosine plus 10 microM-uridine was similar to that in the presence of optimal concentrations of glutamine. 4. The rate of incorporation was dramatically decreased by an inhibitor of the pathway of pyrimidine nucleotide synthesis de novo. Addition of the pyrimidine nucleosides completely overcame the inhibition; addition of the pyrimidine nucleobases was much less effective. 5. These results indicate that, for proliferation of lymphocytes, glutamine is not essential and can be partially or totally replaced by nucleosides and, to some extent, by nucleobases.     

Comparative studies on pyrimidine metabolism in excised cotyledons of Pinus radiata during shoot formation in vitro

Changes in the pattern of pyrimidine nucleotide metabolism were investigated in Pinus radiata cotyledons cultured under shoot-forming (SF; +N(6)-benzyladenine) and non-shoot-forming (NSF, -N(6)-benzyladenine) conditions, as well as in cotyledons unresponsive (OLD) to N(6)-benzyladenine. This was carried out by following the metabolic fate of externally supplied (14)C-labeled orotic acid, intermediate of the de novo pathway, and (14)C-labeled uridine and uracil, substrates of the salvage pathway. Nucleic acid synthesis was also investigated by following the metabolic fate of (14)C-labeled thymidine during shoot bud formation and development. The de novo synthesis of pyrimidine nucleotides was operative under both SF and NSF conditions, and the activity of orotate phosphoribosyltransferase (OPRT), a key enzyme of the de novo pathway, was higher in SF tissue. Utilization of both uridine and uracil for nucleotide and nucleic acid synthesis clearly indicated that the salvage pathway of pyrimidine metabolism is also operative during shoot organogenesis. In general, uridine was a better substrate for the synthesis of salvage products than uracil, possibly due to the higher activity of uridine kinase (UK), compared to uracil phosphoribosyltransferase (UPRT). Incorporation of uridine into the nucleic acid fraction of OLD cotyledons was lower than that observed for their responsive (day 0) counterparts. Similarly, uracil utilization for nucleic acid synthesis was lower in NSF cotyledons, compared to that observed for SF tissue after 10 days in culture. This difference was ascribed to higher UPRT activity measured in the latter. Thus, there was an apparent difference in the utilization of nucleotides derived from uracil and uridine for nucleotide synthesis. The increased ability to produce pyrimidine nucleotides via the salvage pathway during shoot bud formation may be required in support of nucleic acid synthesis occurring during the process. Studies on thymidine metabolism confirmed this notion.     

Regulation of Escherichia coli pyrC by the purine regulon repressor protein.

The purine regulon repressor, PurR, was identified as a component of the Escherichia coli regulatory system for pyrC, the gene that encodes dihydroorotase, an enzyme in de novo pyrimidine nucleotide synthesis. PurR binds to a pyrC control site that resembles a pur regulon operator and represses expression by twofold. Mutations that increase binding of PurR to the control site in vitro concomitantly increase in vivo regulation. There are completely independent mechanisms for regulation of pyrC by purine and pyrimidine nucleotides. Cross pathway regulation of pyrC by PurR may provide one mechanism to coordinate synthesis of purine and pyrimidine nucleotides.     

Effect of plasma concentrations of uridine on pyrimidine biosynthesis in cultured L1210 cells

The concentration of uridine in the media of cultured L1210 cells was maintained within the concentration range found in plasma (1 to 10 microM) to determine if such concentrations are sufficient to satisfy the pyrimidine requirements of a population of dividing cells and to determine if cells utilize de novo and/or salvage pathways when exposed to plasma concentrations of uridine. When cells were incubated in the presence of N-(phosphonacetyl)-L-aspartate to block de novo biosynthesis, plasma concentrations of uridine maintained normal cell growth. De novo pyrimidine biosynthesis, as determined by [14C]sodium bicarbonate incorporation into uracil nucleotides, was affected by the low concentrations of uridine found in the plasma. Below 1 microM uridine, de novo biosynthesis was not affected; between 3 and 5 microM uridine, de novo biosynthesis was inhibited by approximately 50%; and above 12 microM uridine, de novo biosynthesis was inhibited by greater than 95%. Inhibition of de novo biosynthesis correlated with an increase in the uracil nucleotide pool. The de novo pathway was much more sensitive to the uracil nucleotide pool size than was the salvage pathway, such that when de novo biosynthesis was inhibited by greater than 95% the uracil nucleotide pool continued to expand and the cells continued to take up [14C]uridine. Thus, the pyrimidine requirements of cultured L1210 cells can be met by concentrations of uridine found in the plasma and, when exposed to such physiologic concentrations, L1210 cells decrease their dependency on de novo biosynthesis and utilize their salvage pathway. Circulating uridine, therefore, may be of physiologic importance and could be an important determinant in anti-pyrimidine chemotherapy.     

Synthesis of pyrimidine nucleotides in the heart: uridine and cytidine kinase activity

These experiments were designed to determine through the study of uridine and cytidine kinase activity, the precise mechanisms of plasma nucleoside salvage leading to pyrimidine nucleotide synthesis in the rat heart. The kinetic parameters were: Km = 10 microM, V = 4 nmol g-1 min-1 for cytidine kinase activity and Km = 43 microM and V = 18 nmol g-1 min-1 for uridine kinase activity. Competing activity as concerns the two nucleosides was shown to occur, suggesting that in the rat myocardium as in other cells, one and the same enzyme phosphorylates both uridine and cytidine. UTP and CTP were shown to exert a potent inhibitory action on nucleoside phosphorylation; two factors thus exert a joint influence on the control of pyrimidine nucleotide synthesis in the rat heart: the extracellular concentration of precursor and the intracellular level of UTP and CTP. The kinetic parameters for kinase activities are discussed, taking into account the actual concentration of plasmatic nucleosides. Comparison of these data with respectively those for incorporation of nucleosides into the pyrimidine nucleotides of isolated rat heart and with nucleotide turnover rates in vivo suggests that, under physiological conditions, the utilization of plasma cytidine is crucial to the synthesis of myocardial pyrimidine synthesis.     

Pyrimidine metabolism and sugar nucleotide synthesis in rat liver.

With radioactive precursors, the labelling kinetics of the soluble pyrimidine nucleotides and of RNA were measured in rat liver to determine the contribution of the metabolic flows through synthesis de novo and the salvage pathway. To separate and quantify all pyrimidine nucleotides, an h.p.l.c. technique was developed using anion-exchange chromatography and reversed-phase chromatography. The concentrations of cytidine nucleotides were in the range of 30-45 nmol/g wet weight, and the concentrations of the uridine phosphates and of the UDP-sugars were approx. 6 and 20 times higher respectively. After a single injection of [14C]orotic acid and of [3H]cytidine, the specific radioactivities were determined as a function of time. The 14C/3H ratio was calculated and gave a good indication of the involvement of the different flows. It could be concluded that UTP derived from synthesis de novo and from the salvage pathway is not completely mixed before being utilized. The flow of the salvage pathway is relatively more directed to RNA synthesis in the nucleus and that of synthesis de novo to cytoplasmic processes. For CTP it could also be concluded that the flow of the salvage pathway was relatively more directed to RNA synthesis in the nucleus. Because of the nuclear localization of the enzyme CMP-NeuAc (N-acetylneuraminate) synthase, special attention was paid to CMP-NeuAc. However, a conclusion about a location about the synthesis of CMP-NeuAc could not unequivocally be drawn, because of the small differences in 14C/3H ratio and the different values for the CDP-lipids.     

Characterization of pyrimidine metabolism in the cellular slime mold, Dictyostelium discoideum

The arginine-independent, de novo biosynthetic pathway of pyrimidines in Dictyostelium discoideum is initiated by a class II carbamoyl-phosphate synthetase (EC 6.3.5.5) specific for pyrimidine biosynthesis which utilized L-glutamine as its N donor and was partially inhibited by both UTP and CTP. The second step in the de novo pathway was provided by an unregulated aspartate transcarbamoylase (EC 2.1.3.2) which primarily appeared as a multimeric enzyme of 105 kilodaltons. The next enzyme, dihydroorotase (EC 3.5.2.3), was approximately 90-100 kilodaltons. Although the early enzymatic activities of the pyrimidine pathway appeared to reside in independent protein complexes, various unstable molecular species were observed. These structural variants may represent proteolytic fragments of a multienzyme complex. In addition to de novo synthesis, the amoeba demonstrated the capacity for salvage utilization of uracil, uridine, and cytidine. Upon starvation on a solid substratum, axenically grown amoebas began a concerted developmental program accompanied by a restructuring of nucleotide metabolism. The absolute levels of the ribonucleotide pools droppedby 98% within 30 h; however, both the adenylate energy charge and the GTP/ATP ratios were maintained for 50 h after the initiation of development. The maintenance of these metabolic energy parameters required the tight cell-cell contact necessary for development, and the capacity for pyrimidine metabolism was maintained throughout developmental morphogenesis.     

Regulation of pyrimidine nucleotide biosynthesis in Pseudomonas synxantha

Regulation of pyrimidine nucleotide biosynthesis in Pseudomonas synxantha ATCC 9890 was investigated and the pyrimidine biosynthetic pathway enzyme activities were affected by pyrimidine supplementation in cells grown on glucose or succinate as a carbon source. In pyrimidine-grown ATCC 9890 cells, the activities of four de novo enzymes could be depressed which indicated possible repression of enzyme synthesis. To learn whether the pathway was repressible, pyrimidine limitation experiments were conducted using an orotate phosphoribosyltransferase (pyrE) mutant strain identified in this study. Compared to excess uracil growth conditions for the succinate-grown mutant strain cells, pyrimidine limitation of this strain caused dihydroorotase activity to increase about 3-fold while dihydroorotate dehydrogenase and orotidine 5'-monophosphate decarboxylase activities rose about 2-fold. Regulation of de novo pathway enzyme synthesis by pyrimidines appeared to be occurring. At the level of enzyme activity, aspartate transcarbamoylase activity in P. synxantha ATCC 9890 was strongly inhibited in vitro by pyrophosphate, UTP, ADP, ATP, CTP and GTP under saturating substrate concentrations.     

Pyrimidine de novo synthesis during the life cycle of the intraerythrocytic stage of Plasmodium falciparum

The 6 enzymes involved in de novo synthesis of pyrimidines were measured in Plasmodium falciparum isolated by saponin lysis from RBC's nonsynchronized and synchronized in vitro cultures. The total activities were found to be dependent on the stage of the P. falciparum cycle. In parasites isolated from synchronized cultures, the highest activities for all enzymes were found at about 27 hr after synchronization in the late trophozoite stage, or just before schizont formation. Merozoites and ring forms contained little de novo activity. The first enzyme of the pathway, carbamyl phosphate synthetase (CPS-II) preferentially utilized glutamine. Ammonia was a poor substrate. CPS-II was unstable in the absence of the cryoprotectants, dimethylsulfoxide and glycerol. The apparent Km for MgATP--was 3.8 +/- 0.7 mM and the enzyme in all morphological forms of P. falciparum (ring, mature trophozoites and schizonts) was inhibited by UTP. The activity of the fourth enzyme of the pathway, dihydroorotate dehydrogenase, appeared to be linked to the cell's respiratory chain; inhibitors of mammalian electron transport such as cyanide, amytal, antimycin A, thenoyltrifluoroacetone and ubiquinone analogs also inhibited the P. falciparum enzyme. The demonstration of the variation of activity of the pyrimidine enzymes correlates with the increased synthesis of nucleic acids in the late trophozoite stage. These observations provide a basis for the testing of the effectiveness of pyrimidine analogs as potential antimetabolites against various forms of the parasite.     

Effects of methotrexate on nucleotide pools in normal human T cells and the CEM T cell line

It has been proposed that the clinical utility of methotrexate (MTX) in the treatment of rheumatoid arthritis may be due, in part, to inhibition of 5-amino imidazole-4-carboxamide ribonucleotide formyltransferase (AICARFT) by polyglutamated forms of MTX. AICARFT is the second folate dependent enzyme in de novo purine biosynthesis. In this study, the effects of MTX on de novo purine biosynthesis as well as total nucleotide pools were evaluated in both the human T cell line, CEM, and phytohemagglutinin-activated normal human T lymphocytes. De novo synthesized purines were metabolically labeled with 14C-glycine after MTX treatment and analyzed by HPLC. In normal T cells, MTX produced a dose-dependent reduction in de novo adenosine and guanosine pools with maximal effects (>50%) at 1 microM MTX. In CEM cells, de novo purine synthesis was almost completely blocked by 1 microM MTX. Total purine pools were also reduced in both cell types after MTX treatment. Since 1 microM MTX caused almost complete growth inhibition in CEM cells, we evaluated whether growth could be reconstituted with exogenous purine bases and pyrimidine nucleosides which can be utilized via salvage pathways. The combination of hypoxanthine and thymidine substantially reversed growth inhibition with 1 microM MTX in CEM cells. Taken together, these results demonstrate that MTX inhibits de novo nucleotide synthesis in T cells and suggest that AICARFT inhibition may be one aspect of the multi-site mechanism of MTX action in the treatment of rheumatoid arthritis.     

Critical importance of the de novo pyrimidine biosynthesis pathway for Trypanosoma cruzi growth in the mammalian host cell cytoplasm

The intracellular parasitic protist Trypanosoma cruzi is the causative agent of Chagas disease in Latin America. In general, pyrimidine nucleotides are supplied by both de novo biosynthesis and salvage pathways. While epimastigotes-an insect form-possess both activities, amastigotes-an intracellular replicating form of T. cruzi-are unable to mediate the uptake of pyrimidine. However, the requirement of de novo pyrimidine biosynthesis for parasite growth and survival has not yet been elucidated. Carbamoyl-phosphate synthetase II (CPSII) is the first and rate-limiting enzyme of the de novo biosynthetic pathway, and increased CPSII activity is associated with the rapid proliferation of tumor cells. In the present study, we showed that disruption of the T. cruzi cpsII gene significantly reduced parasite growth. In particular, the growth of amastigotes lacking the cpsII gene was severely suppressed. Thus, the de novo pyrimidine pathway is important for proliferation of T. cruzi in the host cell cytoplasm and represents a promising target for chemotherapy against Chagas disease.     

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.     

Purine nucleotide metabolism in phytohemagglutinin-induced human T lymphocytes

The comprehensive studies of purine nucleotide metabolism were done in nonstimulated and phytohemagglutinin (PHA)-stimulated human peripheral blood T lymphocytes. Nonstimulated lymphocytes synthesize nucleotides in two alternative pathways: via biosynthesis de novo and salvage pathways. Although synthesis of triphosphonucleosides in unstimulated lymphocytes was the predominant pathway, interconversion of monophosphonucleosides was also active. Exposure of cells to PHA affects differently various pathways of nucleotide metabolism. The most marked changes observed were rapid activation of purine salvage within minutes after exposure to PHA, and significant increase of 5-phosphoribosyl-1-pyrophosphate levels. In addition, significant increases were found in de novo purine biosynthesis, nucleotide interconversions, and RNA and DNA synthesis, whereas catabolism of nucleotides remained unchanged. These results indicate that PHA activation of T lymphocytes causes a rapid synthesis of nucleotides which may be required immediately for increases in energy metabolism and later as the precursors of nucleic acid synthesis.     

Contribution of the bacterial endosymbiont to the biosynthesis of pyrimidine nucleotides in the deep-sea tube worm Riftia pachyptila

The deep-sea tube worm Riftia pachyptila (Vestimentifera) from hydrothermal vents lives in an intimate symbiosis with a sulfur-oxidizing bacterium. That involves specific interactions and obligatory metabolic exchanges between the two organisms. In this work, we analyzed the contribution of the two partners to the biosynthesis of pyrimidine nucleotides through both the "de novo" and "salvage" pathways. The first three enzymes of the de novo pathway, carbamyl-phosphate synthetase, aspartate transcarbamylase, and dihydroorotase, were present only in the trophosome, the symbiont-containing tissue. The study of these enzymes in terms of their catalytic and regulatory properties in both the trophosome and the isolated symbiotic bacteria provided a clear indication of the microbial origin of these enzymes. In contrast, the succeeding enzymes of this de novo pathway, dihydroorotate dehydrogenase and orotate phosphoribosyltransferase, were present in all body parts of the worm. This finding indicates that the animal is fully dependent on the symbiont for the de novo biosynthesis of pyrimidines. In addition, it suggests that the synthesis of pyrimidines in other tissues is possible from the intermediary metabolites provided by the trophosomal tissue and from nucleic acid degradation products since the enzymes of the salvage pathway appear to be present in all tissues of the worm. Analysis of these salvage pathway enzymes in the trophosome strongly suggested that these enzymes belong to the worm. In accordance with this conclusion, none of these enzyme activities was found in the isolated bacteria. The enzymes involved in the production of the precursors of carbamyl phosphate and nitrogen assimilation, glutamine synthetase and nitrate reductase, were also investigated, and it appears that these two enzymes are present in the bacteria.     

Pyrimidine nucleotide synthesis in the rat kidney in early diabetes.

Early renal hypertrophy of diabetes is associated with increases in the tissue content of RNA, DNA, and sugar nucleotides involved in the formation of carbohydrate-containing macromolecules. We have previously reported an increase in the activity of enzymes of the de novo and salvage pathways of purine synthesis in early diabetes; the present communication explores the changes in the pathways of pyrimidine synthesis. Measurements have been made of key enzymes of the de novo and salvage pathways at 3, 5, and 14 days after induction of diabetes with streptozotocin (STZ), phosphoribosyl pyrophosphate (PPRibP), and some purine and pyrimidine bases. Carbamoyl-phosphate synthetase II, the rate-limiting enzyme of the de novo route, did not increase in the first 5 days after STZ treatment, the period of most rapid renal growth; a significant rise was seen at 14 days (+38%). Dihydroorotate dehydrogenase, a mitochondrial enzyme, showed the most marked rise (+147%) at 14 days. The conversion of orotate to UMP, catalyzed by the enzymes of complex II, was increased at 3 days (+42%), a rise sustained to 14 days. The salvage route enzyme, uracil phosphoribosyltransferase (UPRTase), showed a pattern of change similar to complex II. The effect of the decreased concentration of PPRibP on the activities of CPSII, for which it is an allosteric activator, and on activities of OPRTase and UPRTase, for which it is an essential substrate, is discussed with respect to the relative Ka and Km values for PPRibP and the possibility of metabolite channeling.     

Changes in the de novo, salvage, and degradation pathways of pyrimidine nucleotides during tobacco shoot organogenesis.

Pyrimidine nucleotide metabolism was studied in tobacco callus cultured for 21days under shoot-forming (SF) and non-shoot-forming (NSF) conditions by following the metabolic fate of orotic acid, a precursor of the de novo pathway, and uridine and uracil, intermediates of the salvage and degradation pathways respectively. Nucleic acid synthesis was also investigated by measuring the incorporation of labeled thymidine into different cellular components. Our results indicate that with respect to nucleotide metabolism, the organogenic process in tobacco can be divided in two "metabolic phases": a de novo phase followed by a salvage phase. The initial stages of meristemoid formation during tobacco organogenesis (up to day 8) are characterized by a heavy utilization of orotic acid into nucleotides and nucleic acids. Utilization of this intermediate for the de novo synthesis of nucleotides, which is limited in NSF tissue, is mainly due to the activity of orotate phosphoribosyltransferase (OPRT), which increases in tissue cultured under SF conditions. After day 8, nucleotide synthesis during shoot growth seems to be mainly due to the salvage activity of both uridine and uracil. Both intermediates are preferentially utilized in SF tissue for the formation of nucleotides and nucleic acids through the activities of their respective salvage enzymes: uridine kinase (URK), and uracil phosphoribosyltransferase (UPRT). Metabolic studies on thymidine indicate that in SF tissue maximal nucleic acid synthesis occurs at day 4, in support of the initiation of meristemoid formation. Overall these results suggest that the organogenic process in tobacco is underlined by precise fluctuations in pyrimidine metabolism which delineate structural events culminating in shoot formation.     

Response of the pyrimidine pathway of Escherichia coli K 12 to exogenous adenine and uracil.

The effect of exogenous adenine or uracil upon the de novo pathway for synthesis of pyrimidine nucleotides in Escherichia coli K12 was investigated. Parameters studied were levels of the enzymes carbamoyl phosphate synthase (EC 2.7.2.9), aspartate carbamoyltransferase (EC 2.1.3.2) and orotate phosphoribosyltransferase (EC 2.4.2.10) and the intermediates carbamoyl phosphate, aspartate and orotate, together with the contributions of exogenous uracil and aspartate to intracellular pyrimidine nucleotide. Taken with earlier data [Bagnara, A.S. & Finch, L. R. (1974) Eur. J. Biochem- 41, 421--430] on contents of UTP, CTP and 5-phosphoribosyl 1-diphosphate in cultures of this strain after the addition of adenine or uracil, the results obtained provide new insights into the regulatory mechanisms operating on the pathway in vivo. These insights enable evaluation of the contributions of such factors as limitation for a substrate, feed-back allosteric control by end products and enzyme repression/depression mechanisms. The evidence presented indicates that depressed levels of orotate phosphoribosyltransferase in E. coli K12 result in the wasteful ultilization of asparatate for excess synthesis of pyrimidine nucleotide precursors during balanced growth of the strain in minimal medium. Exogenous adenine increases the excessive accumulation of these precursors by lowering the intracellular content of 5-phosphoribosyl 1-diphosphate (Bagnara and Finch, 1974). This causes a decrease in the conversion of orotate to orotidine 5'-monophosphate, thus lowering the utilization or orotate and its precursors for synthesis of pyrimidine nucleotides. Further, since the contents of these nucleotide end products are thereby decreased (Bagnara nad Finch, 1974), theri feed-back on the early steps in the pathway is diminished and the production of the precursors is increased. It is postulated that growth of E. coli K12 under these conditions is limited by a compound that is metabolically related to precursors to aspartate.     

Purine metabolism in trypanosomatids.

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 fring 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 reductase. 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.     

De novo pyrimidine biosynthesis in isolated rat hepatocytes. Quantitative aspects of the regulation by UTP

Quantitative aspects of de novo pyrimidine biosynthesis in rat hepatocytes were monitored. A reduction of intracellular UTP contents by different concentrations of D-galactosamine led to a dose-dependent increase of 14CO2 incorporation into the sum of all acid-soluble uracil nucleotides. In controls the rate of de novo synthesis which was calculated from the incorporation rate of 14CO2 into the sum of all acid-soluble uracil nucleotides was 0.014 mumol X h-1 X g-1 compared to 0.056 mumol X h-1 X g-1 wet weight of liver in situations of a maximally stimulated de novo synthesis. Incubation of hepatocytes with uridine led to a dose-dependent reduction of 14CO2 incorporation to less than 25% of the amount incorporated in the controls. Alterations of the CTP content had no influence on the 14CO2 incorporation. In the presence of high D-galactosamine concentrations the increase of the total amount of acid-soluble uracil nucleotides exceeded the rate of the de novo synthesis derived from the incorporation of 14CO2 into the sum of the acid-soluble uracil nucleotide pool. It was also greater than the increase of the total amount of intra- and extracellular orotate after acidic hydrolysis--even in the presence of 6-azauridine, which stimulated de novo pyrimidine biosynthesis by itself.