Some regulatory properties of purine biosynthesis de novo in long-term cultures of epithelial-like rat liver cells

De novo synthesis of purine nucleotides and some regulatory properties of this pathway were studied in cultured epithelial-like rat liver cells. It was found that the physiological 5-phosphoribosyl 1-pyrophosphate (P-Rib-PP) concentration in these cells is limiting for purine synthesis de novo. Increase of P-Rib-PP availability, achieved by activation of P-Rib-PP synthetase at high P_i concentration, resulted in acceleration of purine synthesis. The effects of increasing cellular ribose 5-phosphate (Rib-5-P availability, by methylene blue-induced acceleration of the oxidative pentose phosphate pathway, on P-Rib-PP availability and on the rate of the novo purine synthesis were also studied. It was found that at the P_i concentration prevailing in the tissue at extracellular physiological P_i concentration, Rib-5-P availability is saturating for P-Rib-PP generation and therefore also for purine synthesis.     

Effects of fructose on synthesis and degradation of purine nucleotides in isolated rat hepatocytes

The effects of fructose on purine nucleotide synthesis and degradation were studied in isolated rat hepatocytes. Incubation of the hepatocytes with fructose resulted in deceleration of the rate of de novo purine synthesis, gauged by the rate of incorporation of precusor [14C]formate into total purines produced, and in acceleration of purine nucleotide degradation, as measured by the rate of conversion of prelabelled purine nucleotides into end-product allantoin. These effects were found to be associated with decreases in cellular content of ATP and Pi and in the metabolic availability of 5-phosphoribosyl 1-pyrophosphate. The results support the suggestion that the fructose-induced acceleration of purine degradation is mediated through activation of AMP deaminase. However, the results also suggest that decreased reutilization of hypoxanthine for IMP synthesis, due to the decreased PP-Rib-P availability, is an additional mechanism for the acceleration of purine degradation. The decreased PP-Rib-P availability is also suggested to be the main mechanism for the fructose-induced deceleration of purine synthesis.     

Effects of glycerol and fructose on purine synthesis de novo and on PP-ribose-P availability in rat liver cells

Incubation of freshly isolated rat liver cells with glycerol resulted in an initial decrease, followed by an increase in purine synthesis de novo and in PP-ribose-P availability. The magnitude of these effects was dependent on the concentration of glycerol; as it increased, the initial period of latency or inhibition was prolonged, and the extent of the subsequent stimulation was greater. The intracellular Pi concentration and the [14C]ATP/[14C]ADP ratio were also initially decreased in these cells, and they too returned subsequently to normal values. All these changes were similar to those induced by fructose under the same conditions. The increase in PP-ribose-P availability always preceded that in purine synthesis de novo, indicating that, under most circumstances, PP-ribose-P availability is limiting for purine synthesis de novo. Finally, PP-ribose-P synthesis in these cells varied in parallel with the intracellular Pi concentration and with the ATP/ADP and ATP/AMP ratios.     

Characterization of purine nucleotide metabolism in primary rat muscle cultures

The synthesis and metabolic fate of purine nucleotides were studied, employing labeled precursors, in primary rat muscle cultures. The cultures were found to produce purine nucleotides, by de novo and salvage pathways, both exhibiting dependence on cellular availability of substrate 5-phosphoribosyl-1-pyrophosphate (PPRibP). Depletion of cellular PPRibP decelerated the rate of purine synthesis, whereas increasing PPRibP generation by high Pi concentration in the incubation medium, accelerated purine synthesis. Ribose accelerated purine synthesis, indicating that ribose 5-phosphate availability in the cultured muscle is limiting for PPRibP synthesis. The study in the muscle cultures of the metabolic fate if IMP formed from [14C]formate and that of nucleotides formed from labeled purine bases, revealed that the main flow in the nucleotide interconversions pathways is from AMP to IMP. The flow from IMP to GMP and to AMP appeared to be of a lesser magnitude and virtually no flow could be detected from GMP to IMP. The greatest proportion of radioactivity of purine nucleotides following synthesis by either de novo or salvage pathways, accumulated in IMP, reflecting the relative rates of flows between the various nucleotides and probably also a relatively low, or inhibited activity of the IMP nucleotidase. The results suggest that primary muscle cultures are a plausible model for the study of the role of purine metabolism in muscle work.     

Acceleration of purine synthesis in mouse liver by glycogenolytic hormones.

Administration (ip) into fed mice of glucagon, epinephrine, vasopressin, oxytocin, angiotensin II, and dibutyryl cyclic AMP (dbcAMP) resulted in a rapid (within 2.5 to 15 min) elevation of PRPP content (two- to threefold) and in acceleration of the rate of de novo purine synthesis (twofold). Inhibition of the epinephrine-stimulated glycogenolysis by 2,5-anhydromannitol diminished markedly the acceleration effect of the hormone on the rate of purine synthesis. Administration of the hormones caused a rapid rise in the liver content of glucose 6-phosphate (G6P) by 15-70% but did not increase the ribose 5-phosphate (R5P) content. Liver ATP content was not affected. The hormones did not cause direct activation of PRPP synthetase, as gauged by the specific activity of the enzyme, its Km for substrates R5P and ATP, and its sensitivity to inhibition by ADP and GDP. The hormones did not increase the liver content of the enzyme activators Pi and Mg2+. The results suggest that the glycogenolytic hormones accelerate purine synthesis by a metabolic mechanism associated with the enhancement of glycogenolysis. PRPP synthesis is probably enhanced by the glycogenolysis-induced alterations in the cellular content of some metabolites other than R5P.     

Stimulation of ribose-5-phosphate and 5-phosphoribosyl-1-pyrophosphate generation by pyrroline-5-carboxylate in mouse liver in vivo: evidence for a regulatory role of ribose-5-phosphate availability in nucleotide synthesis.

Pyrroline-5-carboxylase (P5C), a physiological stimulator of hexose-monophosphate-pentose pathway activity, was found before to increase 5-phosphoribosyl-1-pyrophosphate (PRPP) generation and nucleotide synthesis in human erythrocytes and cultured fibroblasts. We now report the stimulation of PRPP generation by P5C also in mouse liver in vivo. In addition we demonstrated a simultaneous elevation in ribose-5-phosphate (R5P) concentration, which was relatively smaller and transient. The demonstrated effect of P5C on liver R5P and PRPP content in vivo provides strong evidence for the physiological role of R5P availability in the regulation of PRPP and purine production.     

Regulation of purine de novo synthesis in cultured human fibroblasts: the role of P-ribose-PP.

Procedures for assaying the rate of purine de novo synthesis in cultured fibroblast cells have been compared. These were (i) the incorporation of [(14)C]-glycine or [(14)C]formate in alpha-N-formylglycinamide ribonucleotide (an intermediate in the purine synthetic pathway) and (ii) the incorporation of [(14)C]-formate into newly synthesised cellular purines and purines excreted by the cell into the medium. Fibroblast cells, derived from patients with a deficiency of hypoxanthine phosphoribosyltransferase (HPRT-) (EC 2.4.2.8) and increased rates of purine de novo synthesis, were compared with fibroblasts from healthy subjects (HPRT+). Fetal calf serum, which was used to supplement the assay and cell growth medium, was found to contain sufficient quantities of the purine base hypoxanthine to inhibit purine de novo synthesis in HPRT+ cells. This inhibition was the basis of differentiation between HPRT- and HPRT+ cells. In the absence of added purine base, both cell types had similar capacities for purine de novo synthesis. This result contrasts with the increased rates of purine de novo synthesis reported for a number of human HPRT- cells in culture but conforms recent studies made on human HPRT- lymphoblast cells. The intracellular concentration and utilisation of 5-phosphoribosyl-1-pyrophosphate (P-Rib-PP), a substrate and potential controlling factor for purine de novo synthesis, were determined in HPRT- and HPRT+ cells. The rate of utilisation of P-Rib-PP in the salvage of free purine bases was far greater than that in purine de novo synthesis. Although HPRT- cells had a 3-fold increase in P-Rib-PP content, the rate of P-Rib-PP generation was similar to HPRT+ cells. Thus, in fibroblasts, the concentration of P-Rib-PP appears to be critical in the control of de novo purine synthesis and its preferential utilisation in the HPRT reaction limits its availability for purine de novo synthesis. In vivo, HPRT+ cells, in contrast to HPRT- cells, may be operating purine de novo synthesis at a reduced rate because of their ability to reutilise hypoxanthine.     

Overproduction disease in man due to enzyme feedback resistance mutation. Purine overproduction in gout due to excessive activity of mutant feedback-resistant phosphoribosylpyrophosphate synthetase.

Physiologically superactive phosphoribosylpyrophosphate (PRPP) synthetase, due to feedback resistance mutation, was found in a family with excessive purine production, gout and uric acid lithiasis. The superactivity of the mutant enzyme was manifest in the propositus' erythrocytes and cultured fibroblasts, in increased generation, content and metabolic availability of PRPP, leading in the fibroblasts to acceleration of the rate of purine synthesis de novo. One of the propositus' two siblings was similarly affected, but the propositus' father, his second brother and four sons, were all clinically and biochemically normal. The mother was clinically normal and normouricemic, but hyperuricosuric. Cultured fibroblasts from her skin exhibited variability in PRPP content and availability and in the rate of purine synthesis de novo. The mother's cultures were found to contain a mosaicism of two cell populations, one with normal and the other with mutant PRPP synthetase, indicating an X-linked pattern of inheritance of the PRPP synthetase abnormality in this gouty family.     

Characterization of the Alterations in Purine Nucleotide Metabolism in Hypoxanthine-Guardne Phosphoribosyltransferase-Deficient Rat Neuroma Cell Line

Abstract: A rat neuroma cell line (B103 4C), deficient of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), was utilized as a model tissue in search for the biochemical basis of the Lesch-Nyhan syndrome (LNS). The HGPRT-deficient neurons exhibited the following properties: an almost complete absence of uptake of guanine and of hypoxanthine into intact cell nucleotides (0.92% and 0.69% of normal, respectively); a significant increase in the availability of 5'-phosphoribosyl-1-pyrophosphate; a three- to fourfold acceleration of the rate of de novo nucleotide synthesis; a normal excretion of xanthine, but 15-fold increase in the excretion of hypoxanthine into the culture media; a normal cellular purine nucleotide content, including the absence of 5-amino-4-imidazole carboxamide nucleotides (Z-nucleotides), but enhanced turnover of adenine nucleotides (loss of 86% of the radioactivity of the prelabeled pool in 24 h, in comparison to 73% in the normal line), and an elevated UTP content. The results suggest that, under physiological conditions, guanine salvage does not occur in the normal neurons, but that hypoxanthine salvage is of great importance in the homeostasis of the adenine nucleotide pool. The finding of the normal profile of purine nucleotides in the HGPRT-deficient neurons indicates that the lack of hypoxanthine salvage is adequately compensated by the enhanced de novo nucleotide synthesis. These results did not furnish evidence in support of the possibility that GTP or ATP depletion, or Z-nucleotide accumulation, occurs in HGPRT-deficient neurons and that these are etiological factors causing the neurological abnormalities in LNS. On the other hand, the results point to the possibility that elevated hypoxanthine concentration in the brain may have an etiological role in the pathogenesis of LNS.     

Sugar-free growth of mammalian cells on some ribonucleosides but not on others

It was shown earlier that a variety of vertebrate cells could grow indefinitely in sugar-free medium supplemented with either uridine or cytidine at greater than or equal to 1 mM. In contrast, most purine nucleosides do not support sugar-free growth for one of the following reasons. The generation of ribose-1-P from nucleoside phosphorylase activity is necessary to provide all essential functions of sugar metabolism. Some nucleosides, e.g. xanthosine, did not support growth because they are poor substrates for this enzyme. De novo pyrimidine synthesis was inhibited greater than 80% by adenosine or high concentrations of inosine, e.g. 10 mM, which prevented growth on these nucleosides; in contrast, pyrimidine synthesis was inhibited only marginally on 1 mM inosine or guanosine, but normal growth was only seen on 1 mM inosine, not on guanosine. The inhibition of de novo adenine nucleotide synthesis prevented growth on guanosine, since guanine nucleotides could not be converted to adenine nucleotides. Guanine nucleotides were necessary for this inhibition of purine synthesis, since a mutant blocked in their synthesis grew normally on guanosine. De novo purine synthesis was severely inhibited by adenosine, inosine, or guanosine, but in contrast to guanosine, adenosine and inosine could provide all purine requirements by direct nucleotide conversions.     

Feedback inhibition of amidophosphoribosyltransferase regulates the rate of cell growth via purine nucleotide, DNA, and protein syntheses

To clarify the contributions of amidophosphoribosyltransferase (ATase) and its feedback regulation to the rates of purine de novo synthesis, DNA synthesis, protein synthesis, and cell growth, mutated human ATase (mhATase) resistant to feedback inhibition by purine ribonucleotides was engineered by site-directed mutagenesis and expressed in CHO ade (-)A cells (an ATase-deficient cell line of Chinese hamster ovary fibroblasts) and in transgenic mice (mhATase-Tg mice). In Chinese hamster ovary transfectants with mhATase, the following parameters were examined: ATase activity and its subunit structure, the metabolic rates of de novo and salvage pathways, DNA and protein synthesis rates, and the rate of cell growth. In mhATase-Tg mice, ATase activity in the liver and spleen, the metabolic rate of the de novo pathway in the liver, serum uric acid concentration, urinary excretion of purine derivatives, and T lymphocyte proliferation by phytohemagglutinin were examined. We concluded the following. 1) ATase and its feedback inhibition regulate not only the rate of purine de novo synthesis but also DNA and protein synthesis rates and the rate of cell growth in cultured fibroblasts. 2) Suppression of the de novo pathway by the salvage pathway is mainly due to the feedback inhibition of ATase by purine ribonucleotides produced via the salvage pathway, whereas the suppression of the salvage pathway by the de novo pathway is due to consumption of 5-phosphoribosyl 1-pyrophosphate by the de novo pathway. 3) The feedback inhibition of ATase is more important for the regulation of the de novo pathway than that of 5-phosphoribosyl 1-pyrophosphate synthetase. 4) ATase superactivity leads to hyperuricemia and an increased bromodeoxyuridine incorporation in T lymphocytes stimulated by phytohemagglutinin.     

Alterations in purine nucleotide metabolism during muscle differentiation in vitro

Pathways of purine nucleotide metabolism affecting the availability of ATP in the muscle tissue were studied in differentiating rat muscle cultures. The rate of de novo purine nucleotide synthesis and of AMP deamination were found to increase markedly with cell differentiation, but the rate of IMP dephosphorylation was similarly low in both myoblasts and contracting fibers. The above differentiation-associated alterations in purine nucleotide metabolism conform with the greater need for ATP as a source of energy in the contracting myotubes.     

Abnormal purine and pyrimidine nucleotide content in primary astroglia cultures from hypoxanthine-guanine phosphoribosyltransferase-deficient transgenic mice

Lesch-Nyhan syndrome is a pediatric metabolic-neurological syndrome caused by the X-linked deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). The cause of the metabolic consequences of HGPRT deficiency has been clarified, but the connection between the enzyme deficiency and the neurological manifestations is still unknown. In search for this connection, in the present study, we characterized purine nucleotide metabolism in primary astroglia cultures from HGPRT-deficient transgenic mice. The HGPRT-deficient astroglia exhibited the basic abnormalities in purine metabolism reported before in neurons and various other HGPRT-deficient cells. The following abnormalities were found: absence of detectable uptake of guanine and of hypoxanthine into intact cell nucleotides; 27.8% increase in the availability of 5-phosphoribosyl-1-pyrophosphate; 9.4-fold acceleration of the rate of de novo nucleotide synthesis; manyfold increase in the excretion into the culture media of hypoxanthine (but normal excretion of xanthine); enhanced loss of label from prelabeled adenine nucleotides (loss of 71% in 24 h, in comparison with 52.7% in the normal cells), due to 4.2-fold greater excretion into the media of labeled hypoxanthine. In addition, the HGPRT-deficient astroglia were shown to contain lower cellular levels of ADP, ATP, and GTP, indicating that the accelerated de novo purine synthesis does not compensate adequately for the deficiency of salvage nucleotide synthesis, and higher level of UTP, probably due to enhanced de novo synthesis of pyrimidine nucleotides. Altered nucleotide content in the brain may have a role in the pathogenesis of the neurological deficit in Lesch-Nyhan syndrome.     

Regulation of purine nucleotide synthesis in human B lymphoblasts with both hypoxanthine-guanine phosphoribosyltransferase deficiency and phosphoribosylpyrophosphate synthetase superactivity.

Human B lymphoblast lines severely deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) were selected for resistance to 6-thioguanine from cloned normal and phosphoribosylpyrophosphate (PP-Rib-P) synthetase-superactive cell lines and were compared with their respective parental cell lines with regard to growth and PP-Rib-P and purine nucleotide metabolism. During blockade of purine synthesis de novo with 6-methylthioinosine or aminopterin, inhibition of growth of all HGPRT-deficient cell lines was refractory to addition of Ade at concentrations which restored substantial growth to parental cell lines. Ade-resistant inhibition of growth of parental lines by 6-methylthioinosine, however, occurred during Ado deaminase inhibition. Insufficient generation of IMP (and ultimately guanylates) to support growth of lymphoblasts lacking HGPRT activity and blocked in purine synthesis de novo best explained these findings, implying that a major route of interconversion of AMP to IMP involves the reaction sequence: AMP----Ado----Ino----Hyp----IMP. PP-Rib-P generation and purine nucleoside triphosphate pools were unchanged by introduction of HGPRT deficiency into normal lymphoblast lines, in agreement with the view that accelerated purine synthesis de novo in this deficiency results from increased availability of PP-Rib-P for the pathway. Cell lines with dual enzyme defects did not differ from PP-Rib-P synthetase-superactive parental lines in rates of PP-Rib-P and purine synthesis despite 5-6-fold increases in PP-Rib-P concentrations, excretion of nearly 50% of newly synthesized purines, and diminished GTP concentrations. Fixed rates of purine synthesis de novo in PP-Rib-P synthetase-superactive cells appeared to reflect saturation of the rate-limiting amidophosphoribosyltransferase reaction for PP-Rib-P. In combination with accelerated purine excretion, increased channeling of newly formed purines into adenylates, and impaired conversion of AMP to IMP, fixed rates of purine synthesis de novo may condition cell lines with defects in HGPRT and PP-Rib-P synthetase to depletion of GTP with consequent growth retardation.     

Purine salvage to adenine nucleotides in different skeletal muscle fiber types.

Rates of purine salvage of adenine and hypoxanthine into the adenine nucleotide (AdN) pool of the different skeletal muscle phenotype sections of the rat were measured using an isolated perfused hindlimb preparation. Tissue adenine and hypoxanthine concentrations and specific activities were controlled over a broad range of purine concentrations, ranging from 3 to 100 times normal, by employing an isolated rat hindlimb preparation perfused at a high flow rate. Incorporation of [(3)H]adenine or [(3)H]hypoxanthine into the AdN pool was not meaningfully influenced by tissue purine concentration over the range evaluated (approximately 0.10-1.6 micromol/g). Purine salvage rates were greater (P < 0.05) for adenine than for hypoxanthine (35-55 and 20-30 nmol x h(-1) x g(-1), respectively) and moderately different (P < 0.05) among fiber types. The low-oxidative fast-twitch white muscle section exhibited relatively low rates of purine salvage that were approximately 65% of rates in the high-oxidative fast-twitch red section of the gastrocnemius. The soleus muscle, characterized by slow-twitch red fibers, exhibited a high rate of adenine salvage but a low rate of hypoxanthine salvage. Addition of ribose to the perfusion medium increased salvage of adenine (up to 3- to 6-fold, P < 0.001) and hypoxanthine (up to 6- to 8-fold, P < 0.001), depending on fiber type, over a range of concentrations up to 10 mM. This is consistent with tissue 5-phosphoribosyl-1-pyrophosphate being rate limiting for purine salvage. Purine salvage is favored over de novo synthesis, inasmuch as delivery of adenine to the muscle decreased (P < 0.005) de novo synthesis of AdN. Providing ribose did not alter this preference of purine salvage pathway over de novo synthesis of AdN. In the absence of ribose supplementation, purine salvage rates are relatively low, especially compared with the AdN pool size in skeletal muscle.     

Methenyltetrahydrofolate synthetase prevents the inhibition of phosphoribosyl 5-aminoimidazole 4-carboxamide ribonucleotide formyltransferase by 5-formyltetrahydrofolate polyglutamates

Methenyltetrahydrofolate synthetase (EC 6.3.3.2) catalyzes the irreversible ATP and Mg2+-dependent transformation of 5-formyltetrahydrofolate (N5-HCO-H4-pteroylglutamic acid (PteGlu] to 5,10-methenyltetrahydrofolate. The physiological function of this reaction remains unknown even though it is potentially involved in the intracellular metabolism of the large doses of N5-HCO-H4-PteGlu (leucovorin) administered to cancer patients. We have tried to elucidate methenyltetrahydrofolate synthetase's physiological role by examining the consequences of its inhibition in MCF-7 human breast cancer cells by the folate analog 5-formyltetrahydrohomofolate (fTHHF), a potent competitive inhibitor with a Ki of 1.4 microM. fTHHF inhibited MCF-7 cell growth with an IC50 of 2.0 microM during 72-h exposures, and this effect was fully reversible by hypoxanthine but not thymidine, indicating specific inhibition of de novo purine synthesis. A correlation was observed between increases in intracellular N5-HCO-H4-PteGlu concentrations following fTHHF and cell growth inhibition. De novo purine synthesis was inhibited at the second folate-dependent enzyme, phosphoribosyl aminoimidazole-carboxamide formyltransferase (AICAR transferase; EC 2.1.2.3), as determined by aminoimidazole carboxamide rescue and azaserine inhibition studies. N5-HCO-H4-PteGlu pentaglutamate was a potent inhibitor of purified MCF-7 cell AICAR transferase with a Ki of 3.0 microM while the monoglutamate was not an inhibitor up to 10 microM and fTHHF was only weakly inhibitory with a Ki of 16 microM. These findings suggest that methenyltetrahydrofolate synthetase activity is needed to prevent de novo purine synthesis inhibition by N5-HCO-H4-PteGlu polyglutamates.     

The 6S- and 6R-diastereomers of 5, 10-dideaza-5, 6, 7, 8-tetrahydrofolate are equiactive inhibitors of de novo purine synthesis

The diasteromers of 5,10-dideaza-5,6,7,8-tetrahydrofolate (DDATHF) differing in chirality about carbon 6 were resolved and studied as inhibitors of folate-dependent processes in mouse leukemia cells. Both diastereomers of DDATHF were found to be potent inhibitors of leukemia cell growth due to effects on de novo purine synthesis. Cell growth inhibition by these compounds was prevented by 5-formyltetrahydrofolate in a dose-dependent manner. This indicated that the effects of the DDATHF diastereomers were due to inhibition of folate-dependent processes. Metabolite reversal experiments indicated that 5'-phosphoribosylglycinamide formyltransferase was the major site of action of these compounds in mouse cells. Another site in de novo purine synthesis was affected at higher concentrations of diastereomer B in L1210 cells. Low concentrations of both diastereomers were found to inhibit pure L1210 5'-phosphoribosylglycinamide formyltransferase competitively with the folate substrate. The two diastereomers were also efficient substrates for mouse liver folylpolyglutamate synthetase. We conclude that the 6R- and 6S-diastereomers of DDATHF are remarkably similar and equiactive antimetabolites inhibitory to de novo purine synthesis and that the biochemical processes involved in their cytotoxicity display little stereochemical specificity.     

Mechanisms of accelerated purine nucleotide synthesis in human fibroblasts with superactive phosphoribosylpyrophosphate synthetases

Concentrations and rates of synthesis of phosphoribosylpyrophosphate (PP-Rib-P) and purine nucleotides were compared in fibroblasts cultured from 5 males with PP-Rib-P synthetase superactivity, 3 normal individuals, and 2 children with severe hypoxanthine-guanine phosphoribosyltransferase deficiency. Although all cell strains with PP-Rib-P synthetase superactivity showed increased PP-Rib-P concentration and generation, increased rates of PP-Rib-P-dependent purine synthetic pathways, and increased purine and pyrimidine nucleoside triphosphate concentrations, two subgroups were discernible. Three fibroblast strains with isolated catalytic defects in PP-Rib-P synthetase showed milder increases in PP-Rib-P concentration (2.5-fold normal) and generation (1.6- to 2.1-fold) and in rates of purine synthesis de novo (1.6- to 2.2-fold) and purine nucleoside triphosphate pools (1.5-fold) than did cells from 2 individuals with combined kinetic defects in PP-Rib-P synthetase, both with purine nucleotide inhibitor-resistance. Values for these processes in the latter two strains were, respectively, 5- to 6-fold, 2.6- to 3.2-fold, 4- to 7-fold, and 1.7- to 2.2-fold those of normal cells. In contrast to cells with catalytic defects, these cells also excreted an abnormally high proportion of labeled purines and resisted purine base-mediated inhibition of PP-Rib-P and purine nucleotide synthesis. Hypoxanthine-guanine phosphoribosyltransferase-deficient cells showed normal regulation of PP-Rib-P synthesis and normal nucleoside triphosphate pools despite increased rates of purine synthesis de novo and of purine excretion. Cells with PP-Rib-P synthetase superactivity thus synthesize purine nucleotides at increased rates as a consequence of increased PP-Rib-P production, despite increased purine nucleotide concentrations. These and additional findings provide evidence that regulation of purine synthesis de novo is effected at both the PP-Rib-P synthetase and amidophosphoribosyltransferase reactions.     

De novo purine synthesis in skeletal muscle

Myoblast and primary muscle cultures from rat were found to contain the complete pathway of de novo purine nucleotide synthesis. Quantitative assessment of the pathway in skeletal muscle in mice in vivo, revealed a more intensive purine production in muscle than in liver. Skeletal muscle is thus a major site of de novo purine production in the mammalian body.     

A possible role for 5-phosphoribosyl 1-pyrophosphate in the stimulation of uterine purine nucleotide synthesis in response to oestradiol-17β

1. It has been reported that the rate of purine nucleotide synthesis de novo in the immature rat uterus is doubled at 6h after administration of oestradiol-17beta. The present work confirms an increased incorporation of glycine and adenine into uterine nucleotides between 2 and 6h after hormone treatment and investigates the mechanism of this response. 2. Activation of regulatory enzymes is unlikely to promote increased nucleotide synthesis: the activities of 5-phosphoribosyl 1-pyrophosphate amidotransferase (EC 2.4.2.14) and adenine phosphoribosyltransferase (EC 2.4.2.7) are the same in uterine extracts from control and oestrogen-treated rats. 3. Therefore it was proposed that oestradiol might promote an increased supply of a rate-limiting substrate. The low oestrogen-sensitive rate of AMP synthesis from adenine and endogenous 5-phosphoribosyl 1-pyrophosphate in the intact uterus compared with the high, oestrogen-insensitive rate in uterine extracts supplemented with 5-phosphoribosyl 1-pyrophosphate is evidence that the supply of 5-phosphoribosyl 1-pyrophosphate limits purine nucleotide formation and may increase after hormone treatment. This proposal is supported by the decrease in AMP synthesis in the whole tissue in the presence of guanine and 7-amino-3-(beta-d-ribofuranosyl)pyrazolo[3,4-d]pyrimidine (formycin). These compounds do not inhibit adenine uptake or adenine phosphoribosyltransferase activity, but they both decrease the availability of 5-phosphoribosyl 1-pyrophosphate, the former by promoting its utilization by hypoxanthine/guanine phosphoribosyltransferase (EC 2.4.2.8) and the latter by inhibiting its synthesis from ribose 5-phosphate and ATP by ribose 5-phosphate pyrophosphokinase (EC 2.7.6.1). 4. It is unlikely that the increased availability of 5-phosphoribosyl 1-pyrophosphate results from hormonal stimulation of ribose 5-phosphate formation. Methylene Blue and phenazine methosulphate both increase ribose 5-phosphate without altering the supply of 5-phosphoribosyl 1-pyrophosphate. 5. The activity of ribose 5-phosphate pyrophosphokinase is low in uterine extracts and increases rapidly in response to oestradiol. Therefore the hormonal activation of the routes of purine nucleotide synthesis both de novo and from preformed precursors may be due, at least in part, to an increased availability of the common rate-limiting substrate 5-phosphoribosyl 1-pyrophosphate, mediated by activation of ribose 5-phosphate pyrophosphokinase.