Metabolism of pyrimidine bases and nucleosides by pyrimidine-nucleoside phosphorylases in cultured human lymphoid cells (original) (raw)
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Febs Letters, 1997
Two cytoplasmic forms of pyrimidine nucleotidase (PN-I and PN-II) have been purified from human erythrocytes to apparent homogeneity and partially characterized. They preferentially hydrolyse pyrimidine 5'-monophosphates and 3'-monophosphates respectively. PN-I and PN-II operate as interconverting activities, capable of transferring the phosphate from the pyrimidine nucleoside monophosphate donor(s) to various nucleoside acceptors, including important drugs like 3'-azido-3'-deoxy-thymidine (AZT), cytosine-ß-D-arabinofuranoside (AraC) and 5-fluoro-2'-deoxy-uridine (5FdUrd), pyrimidine analogues widely used in chemotherapy. Kinetic analysis showed linear behaviour for both PN-I and PN-II. PN-I phosphotransferase activity revealed higher affinity for oxynucleosides with respect to deoxy-nucleosides, whereas the contrary seems to be true for PN-II. These results show for the first time that soluble pyrimidine nucleotidases are endowed with pyrimidine-specific phosphotransferase activity.
Biochemical Genetics, 1980
Purine and pyrimidine metabolism was compared in erythrocytes from three patients from two families with purine nucleoside phosphorylase deficiency and T-cell immunodeficiency, one heterozygote subject for this enzyme deficiency, one patient with a complete deficiency of hypoxanthine-guanine phosphoribosyltransferase, and two normal subjects. The erythrocytes from the heterozygote subject were indistinguishable from the normal erythrocytes. The purine nucleoside phosphorylase deficient erythrocytes had a block in the conversion of inosine to hypoxanthine. The erythrocytes with 0.07%o of normal purine nueleoside phosphorylase activity resembled erythrocytes with hypoxanthine-guanine phosphoribosyltransferase deficiency by having an elevated intracellular concentration of PP-ribose-P, increased synthesis of PP-ribose-P, and an elevated rate of carbon dioxide release from orotic acid during its conversion to UMP. Two hypotheses to account for the associated immunodeficiency--that the enzyme deficiency leads to a block of PP-ribose-P synthesis or inhibition of pyrimidine synthesis--could not be supported by observations in erythrocytes from both enzyme-deficient families.
Pyrimidine 5′‐nucleotidase activity in normal and deficient human lymphoblastoid cells
Journal of Inherited Metabolic Disease, 1990
SummaryTwo distinct pyrimidine 5′‐nucleotidases (UMPH‐1 and UMPH‐2) have previously been detected in human erythrocytes; UMPH‐1 is deficient in a haemolytic anaemia, while UMPH‐2 is unaffected. Only the erythrocyte shows pathological effects in this disorder. Here we have studied lymphoblastoid cell lines from control and UMPH‐1 deficient patients to determine whether UMPH‐1 can be detected in lymphoblastoid cells and whether the deficiency of UMPH‐1 results in any measurable metabolic effects. Both UMPH‐1 and a UMPH‐2‐like activity were found to be present in control lymphoblastoid cells. UMPH‐1 was undetectable in the patients' cells; minor but significant changes were found in the pyrimidine nucleotide and nucleoside pools in the cells.
International Journal of Laboratory Hematology, 2012
Pyrimidine 5 0 -nucleotidase (P5 0 N-1) also known as uridine 5 0 monophosphate hydrolase-1 is the first degradative enzyme of the pyrimidine salvage pathway and catalyzes the dephosphorylation of uridine mono-phosphate (UMP) and cytidine monophosphate (CMP) to their corresponding nucleosides. The enzyme is totally inactive toward purine nucleotidases . P5 0 N-1 also displays phosphotransferase activity specific for pyrimidine nucleosides, suggesting an additional role in nucleotide metabolism
Purine nucleoside modulation of functions of human lymphocytes
Cellular Immunology, 1990
The accumulation of endogenous substrates in patients with adenosine deaminase deficiency or purine nucleoside phosphorylase deficiency is believed to be responsible for the immunodeficiency observed in these patients. To identify the lymphocyte populations that are most susceptible to these substrates, we investigated the effect of their nucleoside analogs on a number of T and B cell functions of human lymphocytes. We found that tubercidin (Tub), 2-chloro 2'deoxyadenosine (2CldA), 2-fluoro adenine arabinoside-5'phosphate (FaraAMP), and 9-0-Darabinosyl guanine (AraGua) inhibited the proliferative responses of human peripheral blood mononuclear cells (PBMC) to polyclonal activators (PHA, OKT3 mab) or to allogeneic PBMC in mixed lymphocyte cultures (MLC). Addition of recombinant IL2 from the beginning of the culture did not alter the inhibition by Tub of the proliferative responses of PBMC. These purine nucleoside analogs also inhibited the proliferative responses of purified human peripheral blood CD4+ and CDS+ T cells to PHA and of purified B cells to SAC. The concentrations of these nucleosides required to achieve a given degree of inhibition of proliferative responses of T lymphocyte subpopulations or B cells was similar, suggesting that these analogs do not exhibit any selectivity for these purified lymphocyte populations. Tub and FaraAMP, respectively, inhibited and enhanced, at the effector phase, both NK cytotoxicity and specific T cell-mediated cytotoxicity. In contrast to these findings, LAK cytotoxicity at the effector phase was not significantly inhibited by Tub, and was not enhanced by FaraAMP. Both analogs inhibited rIL-2-induced proliferative responses of PBMC, but did not affect the generation of LAK cytotoxicity (induction phase) against the K562 targets when added at the beginning of the culture. This suggests that DNA synthesis is not required for LAK cell induction. Both Tub and FaraAMP inhibited immunoglobulin production (IgG and IgM) by PBMC in the PWM-induced system. These results demonstrate that purine nucieoside analogs SignificantIy inhibited a number of functions of human lymphocytes. Although selectivity for T lymphocyte subpopulations and B cells was not observed, a differential effect of Tub and FaraAMP on LAK cytotoxicity versus NK cytotoxicity and specific T cell cytotoxicity was found. o 1990 Academic press, ITIC.
The Biochemical journal, 1990
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 mu...
Antimicrobial Agents and Chemotherapy, 1991
The effects of thymidine and uridine on the phosphorylation of 3'-azido-3'-deoxythymidine (AZT) were studied in various human mononuclear cell preparations. Thymidine suppressed [3H]AZT phosphorylation in the same concentration range (20 to 100 ,uM) in which it antagonizes the anti-human immunodeficiency virus activity of AZT. Uridine, in turn, had no influence on AZT phosphorylation, just as it has no effect on the anti-human immunodeficiency virus activity of AZT. These findings are consistent with a close relationship between the inhibition of AZT phosphorylation and the influence of physiological nucleosides on the antiviral activity of AZT. 200 NOTES on March 14, 2021 by guest http://aac.asm.org/ Downloaded from
A rat model of purine nucleoside phosphorylase deficiency
Immunology, 1986
Purine nucleoside phosphorylase (NP; EC 2.4.2.1) deficiency is associated with selective T-cell dysfunction and normal B-cell immunity. In order to create an in vivo model of this immune deficiency, we administered 8-aminoguanosine to rats. This water-soluble nucleoside was rapidly converted by NP to the more potent inhibitor 8-aminoguanine, which has a Ki of 0.19 microM. The accumulation of inosine in plasma showed that administration of 8-aminoguanosine was effectively inhibiting NP activity. The administration of 8-aminoguanosine with deoxyguanosine produced increased levels of dGTP only in thymus cells, and increased levels of GTP in cells from thymus, spleen and lymph node and in red cells. This correlated with assays of deoxyguanosine kinase, which showed significantly higher activity in thymus cells than in cells from spleen and lymph node. The intraperitoneal injection of 8-aminoguanosine alone or with deoxyguanosine for 8 consecutive days caused significant decreases in the...
Quantitative analysis of the pyrimidine metabolism in pheochromocytoma PC-12 cells
European journal of biochemistry / FEBS, 1995
A detailed quantitative study of pyrimidine metabolism in exponentially growing rat pheochromocytoma PC-12 cells has been performed. The sizes of ribonucleotide pools have been analysed and the pathways and the rates of metabolism of uridine, cytidine and aspartic acid have been determined, based on the incorporation of radioactive label. The fluxes of radioactive label through uridine-cytidine kinase, cytidine deaminase. CTP synthetase, nucleoside monophosphate kinase and nucleoside diphosphate kinase were obtained, as well as the flux through the pyrimidine de novo pathway. Also, the fluxes of radioactive label towards UDP-sugars, CDP-compounds, DNA and RNA were quantified in situ under steady-state conditions in intact PC-12 cells. From these fluxes of radioactivity, distribution ratios at the branch points of the metabolism were obtained. The pyrimidines synthesised via the de novo pathway were preferentially used for the synthesis of UDP-N-acetylhexosamines and UDP-hexoses, whe...
Homogeneous pyrimidine nucleotidase from human erythrocytes: enzymic and molecular properties
The Biochemical journal, 1994
A pyrimidine nucleotidase with unique specificity has been obtained for the first time as an homogeneous protein from the cytosolic fraction of human erythrocytes. Both conventional chromatography and f.p.l.c. techniques have been used in the purification procedure. The final enzyme preparation gave a single protein band of M(r) = 23,500 on SDS/PAGE under both reducing and non-reducing conditions. The native enzyme was eluted at M(r) = 45,000 in gel filtration chromatography on Superose 12, suggesting a dimeric structure. Amino acid analysis was consistent with an acidic isoelectric point and revealed the presence of six half-cystine and two methionine residues per subunit. The enzyme was active on a variety of pyrimidine nucleoside monophosphates, being most active on the 3'-monophosphates. Km values for 3'dUMP, 3'UMP, 5'dUMP, 5'UMP, 5-fluoro-2'dUMP, ranged from 192 microM to 1.15 mM. The enzyme activity was inhibited by both reaction products, orthophosphat...