Thirteen Years Experience with Selective Screening for Disorders in Purine and Pyrimidine Metabolism

Purine and pyrimidine disorders represent a heterogeneous group with variable clinical symptoms and low prevalence rate. In the last thirteen years, we have studied urine/plasma specimens from about 1600 patients and we have identified 35 patients: eight patients with adenylosuccinate lyase deficiency, eight patients with hypoxanthine-guanine phosphoribosyltransferase deficiency, one patient with purine nucleoside phosphorylase deficiency, ten patients with xanthine dehydrogenase deficiency, six patients with molybdenum cofactor deficiency and two patients with dihydropyrimidine dehydrogenase deficiency.

Despite low incidence of these diseases, our findings highlight the importance of including the purine and pyrimidine analysis in the selective screening for inborn errors of metabolism in specialized laboratories, where amino acid and organic acid disorders are simultaneously investigated.     

Lesch-Nyhan syndrome: Absence of the mutant enzyme in erythrocytes of a heterozygote for both normal and mutant hypoxanthine-guanine phosphoribosyl transferase

Subjects heterozygous for the Lesch-Nyhan syndrome with a deficiency of the X-linked gene for the enzyme hypoxanthine-guanine phosphoribosyl transferase (PRT) would be expected to have two populations of erythrocytes in roughly equal proportions—one type with the normal enzyme and the other type exhibiting the mutant form of the enzyme. In contrast to this prediction, previous studies utilizing an X-linked gene for another enzyme as a marker for the PRT locus have suggested that erythrocytes from heterozygotes consist largely of cells with the normal form of the enzyme. We have recently described a mutant form of hypoxanthine-guanine phosphoribosyl tranferase with altered kinetic properties which allow it to be measured in artificial mixtures with the normal enzyme. The mutant enzyme could not be detected in erythrocyte lysates from a proven heterozygote for both the normal and this mutant form of the enzyme. This provides additional evidence that either inactivation of the X-chromosome in erythropoietic tissue from the heterozygote for PRT deficiency is not random or that random X-chromosome inactivation is followed by selection against erythrocyte precursors with the mutant enzyme.This study was supported in part by USPHS Research Grant No. AM14362, USPHS Training Grant No. AM05620, and a grant (RR-30) from the General Clinical Research Centers Program of the Division of Research Resources, National Institutes of Health.     

A method for the determination of 5-phosphoribosyl 1-pyrophosphate concentrations in erythrocytes using high-performance liquid chromatography

A method for the measurement of erythrocyte 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P) using HPLC is described. Inosinic acid formed from the enzyme-catalyzed reaction of hypoxanthine and PP-ribose-P using partially purified hypoxanthine-guanine phosphoribosyltransferase is measured after chromatography on an ion-exchange column (Partisil 10 SAX). The average recovery of PP-ribose-P added to erythrocytes was 96.6%. Normal values found were 1.3 +/- 0.6 nmol PP-ribose-P/ml packed RBC (20 individuals). Replication experiments gave a coefficient of variation of 4.4%. Elevated levels in the range 4.4-7.9 nmol PP-ribose-P/ml packed RBC were found in four patients with gout and partial deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase.     

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.     

Guanosine metabolism in novikoff hepatoma cells: Isolation and characterization of guanosine resistant variants

Clones resistant to 0.15% guanosine were isolated from rat hepatoma cells. Analysis of cell extracts from these clones revealed the presence of normal levels of purine nucleoside phosphorylase activity but less than 2% of the parental level of hypoxanthine-guanine phosphoribosyltransferase activity. In addition, the resistant cells transported guanosine and inosine at less than 2% of the rate of sensitive cells. Despite this low rate of transport, the resistant cells were still capable of metabolizing extracellular guanosine and inosine. The ability of the resistant cells to metabolize guanosine and inosine without requiring their direct transport lends support to the existence of a membrane localized form of purine nucleoside phosphorylase which metabolizes extracellular purine nucleosides.     

Escherichia coli K-12 mutants capable of catabolizing purine nucleosides in the absence of purine nucleoside phosphorylase]

Strains of Escherichia coli K-12 defective in purine nucleoside phosphorylase (pup gene) formed on the medium with inosine as the source of carbon and energy phenotypical reversions for the ability of utilizing inosine as source of carbon or purines. The phenotypical suppression of the purine nucleoside phosphorylase deficiency is the result of the mutations (called pnd), which are mapped on the chromosome of E. coli beyond the region of the structural pup-gene location and have phenotypic manifestation distinct from that of pup+ allele: a) pnd mutants divide into some groups for the ability of utilizing several purine nucleosides, including xantosine that cannot be metabolized by pnd+ strains of E. coli; b) pnd mutations do not restore the ability of purine auxotrophs (pur) defective in purine nucleoside phosphorylase (pup) and adenine phosphoribosyltransferase (apt) to grow on the medium with adenine as the sole source of purines. Cell-free extracts of pnd mutants fail to degrade the guanine nucleosides in the absence of phosphate or arsenate ions. These data (and also the ability of pnd mutants to utilize both purine ribonucleosides and deoxyribonucleosides) seem to indicate that the activities induced by pnd mutations are phosphorylase activities.     

Purine salvage enzyme activities in normal and neoplastic human tissues

The enzymatic pattern of five enzymes involved in the purine salvage pathway, namely purine nucleoside phosphorylase (EC 2.4.2.1), adenosine deaminase (EC 3.5.4.4), 5'-nucleotidase (EC 3.1.3.5), alkaline phosphatase (EC 3.1.3.1), and hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) has been evaluated both in human intestinal and breast carcinomas and compared to that of normal tissues. A higher level of hypoxanthine-guanine phosphoribosyltransferase was associated with tumor tissues. This metabolic alteration should lead to an elevated synthesis of nucleotides in cancer cells, might confer selective growth advantages to neoplastic tissues, and account, at least in part, for the difficulties encountered in the chemotherapy of human tumors, by using compounds affecting only the purine de novo biosynthesis.     

Incorporation of purine nucleosides in cultured fibroblasts from a patient with purine nucleoside phosphorylase deficiency and associated T-cell immunodeficiency.

Cultured skin fibroblasts from a patient with T-cell immune deficiency and an absence of purine nucleoside phosphorylase activity in red cells were assayed for their capacity to metabolize inosine and guanosine. The cultured fibroblasts were lacking activity of nucleoside phosphorylase and, compared to normal fibroblasts, could incorporate only 2% and 4% of 14C-inosine and 3H-guanosine, respectively, into acid precipitable material. Autoradiography visually confirmed the failure of the NP deficient cell line to incorporate the nucleosides into nuclear material. The physiological mechanism by which the deficiency of purine nucleoside phosphorylase causes T-cell dysfunction remains unclear.     

Enzymes of the purine interconversion system in chronic lymphatic leukemia: decreased purine nucleoside phosphorylase and adenosine deaminase activity.

Activities of adenosine deaminase (ADA), adenosine kinase (AK), adenine phosphoribosyltransferase (APRT), hypoxanthine guanine phosphoribosyltransferase (HGPRT), and purine nucleoside phosphorylase (PNP), all enzymes of the purine interconversion system, were determined in lymphocytes of 25 patients with chronic lymphatic leukemia (CLL) and in 23 controls. A statistically significant decrease of PNP activities and a reduction of ADA activities at borderline levels were found in the patients, whereas for the other enzymes assayed no deviation from normal values was observed.     

Genetic and physiological characterization of Bacillus subtilis mutants resistant to purine analogs.

Bacillus subtilis mutants defective in purine metabolism have been isolated by selecting for resistance to purine analogs. Mutants resistant to 2-fluoroadenine were found to be defective in adenine phosphoribosyltransferase (apt) activity and slightly impaired in adenine uptake. By making use of apt mutants and mutants defective in adenosine phosphorylase activity, it was shown that adenine deamination is an essential step in the conversion of both adenine and adenosine to guanine nucleotides. Mutants resistant to 8-azaguanine, pbuG mutants, appeared to be defective in hypoxanthine and guanine transport and normal in hypoxanthine-guanine phosphoribosyltransferase activity. Purine auxotrophic pbuG mutants grew in a concentration-dependent way on hypoxanthine, while normal growth was observed on inosine as the purine source. Inosine was taken up by a different transport system and utilized after conversion to hypoxanthine. Two mutants resistant to 8-azaxanthine were isolated: one was defective in xanthine phosphoribosyltransferase (xpt) activity and xanthine transport, and another had reduced GMP synthetase activity. The results obtained with the various mutants provide evidence for the existence of specific purine base transport systems. The genetic lesions causing the mutant phenotypes, apt, pbuG, and xpt, have been located on the B. subtilis linkage map at 243, 55, and 198 degrees, respectively.     

Purification and characterization of human erythrocyte purine nucleoside phosphorylase and its subunits.

Purine nucleoside phosphorylase (EC 2.4.2.1; purine nucleoside:orthophosphate ribosyltransferase) from fresh human erythrocytes has been purified to homogeneity in two steps with an overall yield of 56%. The purification involves DEAE-Sephadex chromatography followed by affinity chromatography on a column of Sepharose/formycin B. This scheme is suitable for purification of the phosphorylase from as little as 0.1 ml of packed erythrocytes. The native enzyme appears to be a trimer with native molecular weight of 93,800 and the subunit molecular weight of 29,700 +/- 1,100. Two-dimensional gel electrophoresis of the purified enzyme under denaturing conditions revealed four major separable subunits (numbered 1 to 4) with the same molecular weight. The apparent isoelectric points of subunits 1 to 4 in 9.5 M urea are 6.63, 6.41, 6.29, and 6.20, respectively. The different subunits are likely the result of post-translational modification of the enzyme and provide an explanation of the complex native isoelectric focusing pattern of purine nucleoside phosphorylase from erythrocytes. Three of the four subunits are detectable in two-dimensional electrophoretic gels of crude hemolysates. Knowing the location of the subunits of purine nucleoside phosphorylase in a two-dimensional electropherogram allows one to characterize the purine nucleoside phosphorylase in crude cell extracts from individuals with variant or mutant purine nucleoside phosphorylase as demonstrated in a subsequent communication. Partial purification of the phosphorylase from 1 ml of erythrocytes on DEAE-Sephadex increases the sensitivity of detection of the subunits to the 0.3% level.     

Simultaneous determination of purine and pyrimidine metabolites in HPRT-deficient cell lines

Genetic mutations in the purine salvage enzyme, hypoxanthine-guanine phosphoribosyltransferase (HPRT), are known to cause Lesch-Nyhan syndrome and Kelley-Seegmiller syndrome. In patients, purine metabolism is different from that of normal persons. We have previously developed a method for simultaneously determining the concentration of purine and pyrimidine nucleosides and nucleotides. This system was applied to determine the concentrations of nucleosides and nucleotides in HPRT-deficient cell lines. The amount of inosine 5'-monophosphate (IMP) was different in Lesch-Nyhan syndrome, Kelley-Seegmiller syndrome, and control cell lines. The difference in the amount of IMP confirmed the mutation of the enzyme.     

Characterization of the subunits of purine nucleoside phosphorylase from cultured normal human fibroblasts

In previous communications we have demonstrated that the subunits of normal human erythrocyte purine nucleoside phosphorylase can be resolved into four major (1–4) and two minor (1p and 2p) components with the same molecular weight but different apparent isoelectric points (and net ionic charge). The existence of subunits with different charge results in a complex isoelectric focusing pattern of the native erythrocytic enzyme. In contrast, the isoelectric focusing pattern of the native enzyme obtained from cultured human fibroblasts is simpler. The multiple native isoenzymes obtained from human erythrocytes and human brain have isoelectric points ranging from 5.0 to 6.4 and from 5.2 to 5.8, respectively, whereas cultured human fibroblasts have two major native isoenzymes with apparent isoelectric points of 5.1 and 5.6.Purine nucleoside phosphorylase has been purified at least a hundredfold from ³⁵S-labeled cultured human fibroblasts. A two-dimensional electrophoretic analysis of the denatured purified normal fibroblast enzyme revealed that it consists mainly of subunit 1 (90%) with small amounts of subunits 2 (10%) and 3 (1%). This accounts for the observed differences between the native isoelectric focusing and the electrophoretic patterns of the erythrocyte and fibroblast enzymes. The purine nucleoside phosphorylase subunit 1 is detectable in the autoradiogram from a two-dimensional electrophoretic analysis of a crude, unpurified extract of ³⁵S-labeled cultured normal human fibroblasts. The fibroblast phosphorylase coincides with the erythrocytic subunit 1 of the same enzyme, and the cultured fibroblasts of a purine nucleoside phosphorylase deficient patient (patient I) lack this protein component, genetically confirming the identity of the purine nucleoside phosphorylase subunit in cultured fibroblasts.This work was supported by a grant from the National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, United States Public Health Service. L. J. G. is supported by a fellowship from the National Institute of Child Health and Human Development. D. W. M. is an Investigator, Howard Hughes Medical Institute.     

Selectivity of febuxostat, a novel non-purine inhibitor of xanthine oxidase/xanthine dehydrogenase

The purine analogue, allopurinol, has been in clinical use for more than 30 years as an inhibitor of xanthine oxidase (XO) in the treatment of hyperuricemia and gout. As consequences of structural similarities to purine compounds, however, allopurinol, its major active product, oxypurinol, and their respective metabolites inhibit other enzymes involved in purine and pyrimidine metabolism. Febuxostat (TEI-6720, TMX-67) is a potent, non-purine inhibitor of XO, currently under clinical evaluation for the treatment of hyperuricemia and gout. In this study, we investigated the effects of febuxostat on several enzymes in purine and pyrimidine metabolism and characterized the mechanism of febuxostat inhibition of XO activity. Febuxostat displayed potent mixed-type inhibition of the activity of purified bovine milk XO, with Ki and Ki' values of 0.6 and 3.1 nM respectively, indicating inhibition of both the oxidized and reduced forms of XO. In contrast, at concentrations up to 100 muM, febuxostat had no significant effects on the activities of the following enzymes of purine and pyrimidine metabolism: guanine deaminase, hypoxanthine-guanine phosphoribosyltransferase, purine nucleoside phosphorylase, orotate phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase. These results demonstrate that febuxostat is a potent non-purine, selective inhibitor of XO, and could be useful for the treatment of hyperuricemia and gout.     

5-Iodoribose 1-phosphate, an analog of ribose 1-phosphate. Enzymatic synthesis and kinetic studies with enzymes of purine, pyrimidine, and sugar phosphate metabolism

The 5'-deoxy-5'-iodo-substituted analogs of adenosine and inosine are cytotoxic to tumor cells that have high activities of 5'-methylthioadenosine phosphorylase and purine nucleoside phosphorylase, respectively (Savarese, T.M., Chu, S-H., Chu, M.Y., and Parks, R. E., Jr. (1984) Biochem. Pharmacol. 34, 361-367). 5-Iodoribose 1-phosphate (5-IRib-1-P), the common intracellular metabolite of these 5'-iodonucleosides, has been synthesized enzymatically from 5'-deoxy-5'-iodoadenosine via adenosine deaminase from Aspergillus oryzae and human erythrocytic purine nucleoside phosphorylase. The purification and chemical properties of 5-IRib-1-P are described. The analog sugar phosphate inhibited purine nucleoside phosphorylase from human erythrocytes, phosphoglucomutase from rabbit muscle, and 5'-methylthioadenosine phosphorylase from Sarcoma 180 cells with Ki values of 26, 100, and 9 microM, respectively. Enzymes that react with 5-phosphoribosyl 1-pyrophosphate (P-Rib-PP), P-Rib-PP amidotransferase, hypoxanthine-guanine phosphoribosyltransferase, adenine phosphoribosyltransferase, and orotate phosphoribosyltransferase-orotidylate decarboxylase from extracts of Sarcoma 180 cells, were inhibited with Ki values of 49, 465, 307, and 275 microM, respectively. 5-IRib-1-P had no effect on P-Rib-PP synthetase. Since the Ki values of the analog sugar phosphate for 5'-methylthioadenosine phosphorylase and P-Rib-PP amidotransferase are much lower than the Km values of the natural substrates, Pi or P-Rib-PP which are reported to be present at nonsaturating concentrations under physiological conditions, these enzymes could be significantly inhibited by 5-IRib-1-P in intact cells.     

The purine and pyrimidine metabolism of Tetrahymena pyriformis

The metabolism of purines and pyrimidines by the ciliated protozoan Tetrahymena was investigated with the use of enzymatic assays and radioactive tracers. A survey of enzymes involved in purine metabolism revealed that the activities of inosine and guanosine phosphorylase (purine nucleoside: orthophosphate ribosyltransferase, E.C. 2.4.2.1) were high, but adenosine phosphorylase activity could not be demonstrated. The apparent K_m for guanosine in the system catalyzing its phosphorolysis was 4.1 ± 0.6 × 10^?3 M. Pyrophosphorylase activities for IMP and GMP (GMP: pyrophosphate phosphoribosyltransferase, E.C. 2.4.2.8), AMP (AMP: pyrophosphate phosphoribosyltransferase, E.C. 2.4.2.7), and 6-mercaptopurine ribonucleotide were also found in this organism; but a number of purine and pyrimidine analogs did not function as substrates for these enzymes. The metabolism of labeled guanine and hypoxanthine by intact cells was consistent with the presence of the phosphorylases and pyrophosphorylases of purine metabolism found by enzymatic studies. Assays for adenosine kinase (ATP: adenosine 5'-phosphotransferase, E.C. 2.7.1.20) inosine kinase, guanosine kinase, xanthine oxidase (xanthine: O₂ oxidoreductase, E.C. 1.2.3.2), and GMP reductase (reduced-NADP: GMP oxidoreductase [deaminating], E.C. 1.6.6.8) were all negative. In pyrimidine metabolism, cytidine-deoxycytidine deaminase (cytidine aminohydrolase, E.C. 3.5.4.5), thymidine phosphorylase (thymidine: orthophosphate ribosyltransferase, E.C. 2.4.2.4), and uridine-deoxyuridine phosphorylase (uridine: orthophosphate ribosyltransferase, E.C. 2.4.2.3) were active; but cytidine kinase, uridine kinase (ATP: uridine 5'-phosphotransferase, E.C. 2.7.1.48), and CMP pyrophosphorylase could not be demonstrated.     

Mechanisms of deoxyguanosine lymphotoxicity. Human thymocytes, but not peripheral blood lymphocytes accumulate deoxy-GTP in conditions simulating purine nucleoside phosphorylase deficiency

This study was designed to simulate purine nucleoside phosphorylase (PNP) deficiency by preincubating with guanosine (Guo) to minimize PNP activity while investigating the metabolism of [14C] deoxyguanosine (dGuo) at physiologic concentrations (10 microM) by unstimulated thymocytes, tonsil-derived T and B lymphocytes, and peripheral blood cells over short time periods. GTP was the principal metabolite formed from dGuo by all cell types with functional PNP and hypoxanthine-guanine phosphoribosyltransferase, confirming formation via degradation to guanine with subsequent salvage by hypoxanthine-guanine phosphoribosyltransferase. Thymocytes also formed a small amount of deoxyguanosine triphosphate (dGTP), presumably through direct phosphorylation by deoxycytidine kinase. Incorporation of dGuo into GTP was effectively inhibited in all instances under PNP deficiency conditions and dGTP levels increased up to 10-fold in thymocytes, but tonsil-derived B or T lymphocytes and unfractionated PBL still accumulated no detectable dGTP. E and platelets formed low amounts of dGTP under these conditions. Preincubation with adenine (50 microM) to reverse any Guo-induced toxicity reduced the incorporation of dGuo into GTP without inhibitor in all cell types with intact adenine phosphoribosyltransferase, but had no effect on dGTP accumulation in thymocytes, with or without inhibitor, thus excluding any indirect formation of dGTP via the de novo route. The rapid metabolism of dGuo to GTP, in the absence of PNP inhibition and subsequent effects of the altered GTP concentrations on cellular metabolism, may account for the differing responses reported by investigators with the use of low dGuo concentrations (enhancing), compared with high (inhibitory), concentrations in mitogen-stimulated lymphocyte studies. The exclusive ability of thymocytes to accumulate significant amounts of dGTP, and inability of B cells to do so, provides a logical explanation for the selective T cell immunodeficiency in PNP deficiency.     

Purine pathway enzymes in a cyst forming strain of Toxoplasma gondii

The activities of purine salvage enzymes in tachyzoites from a cyst-forming strain of Toxoplasma gondii were determined using HPLC. Six enzymes were assayed both in vitro and in vivo: adenosine deaminase, guanine deaminase, purine nucleoside phosphorylase, xanthine oxidase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase. In vitro, the tachyzoites were cultured in the human myelomonocytic cell line THP-1, for 24 h to 96 h. Neither guanine deaminase nor hypoxanthine-guanine phosphoribosyltransferase activity was detected in 24 and 96 h cultures. In vivo, in controls and infected animals, the purine nucleoside phosphorylase and adenosine deaminase activities were the most important activities both in sera and cerebral tissue in comparison with the other activities. It was also noted that the infection modified the enzymatic activities of this purine salvage pathway, in particular, the guanine deaminase cerebral activity of infected mice was 20-fold lower than the value of controls. The treatment of mice with 2',3'-dideoxyinosine, a purine analog, at the dose of 100 mg.kg(-1).d for 30 days, induced an important increase of all enzymatic activities in the brains in comparison with control animals. These data suggest that one target of 2',3'-dideoxyinosine is the purine metabolism.     

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.     

Transport of adenine, hypoxanthine and uracil into Escherichia coli.

Uptake of adenine, hypoxanthine and uracil by an uncA strain of Escherichia coli is inhibited by uncouplers or when phosphate in the medium is replaced by less than 1 mM-arsenate, indicating a need for both a protonmotive force and phosphorylated metabolites. The rate of uptake of adenine or hypoxanthine was not markedly affected by a genetic deficiency of purine nucleoside phosphorylase. In two mutants with undetected adenine phosphoribosyltransferase, the rate of adenine uptake was about 30% of that in their parent strain, and evidence was obtained to confirm that adenine had then been utilized via purine nucleoside phosphorylase. In a strain deficient in both enzymes adenine uptake was about 1% of that shown by wild-type strains. Uptake of hypoxanthine was similarly limited in a strain lacking purine nucleoside phosphorylase, hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase. Deficiency of uracil phosphoribosyltransferase severely limits uracil uptake, but the defect can be circumvented by addition of inosine, which presumably provides ribose 1-phosphate for reversal of uridine phosphorylase. The results indicate that there are porter systems for adenine, hypoxanthine and uracil dependent on a protonmotive force and facilitated by intracellular metabolism of the free bases.