Quantitative analysis of the pyrimidine metabolism in pheochromocytoma PC-12 cells (original) (raw)
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Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1987
The anabolism of pyrimidine ribo-and deoxyribonucleosides from uracil and thymine was investigated in phytohemagglutinin-stimulated human peripheral blood lymphocytes and in a Burkitt's lymphoma-derived cell line (Raji). We studied the ability of these cells to synthesize pyrimidine nucleosides by ribo-and deoxyribosyl transfer between pyrimidine bases or nucleosides and the purine nucleosides inosine and deoxyinosine as donors of ribose 1-phosphate and deoxyribose 1-phosphate, respectively: these reactions involve the activities of purine-nucleoside phosphorylase, and of the two pyrimidine-nucleoside phosphorylases (uridine phosphorylase and thymidine phosphorylase). The ability of the cells to synthesize uridine was estimated from their ability to grow on uridine precursors in the presence of an inhibitor of pyrimidine de novo synthesis (pyrazofurin). Their ability to synthesize thymidine and deoxyuridine was estimated from the inhibition of the incorporation of radiolabelled thymidine in cells cultured in the presence of unlabelled precursors. In addition to these studies on intact cells, we determined the activities of purine-and pyrimidine-nucleoside phosphorylases in cell extracts. Our results show that Raji cells efficiently metabolize preformed uridine, deoxyuridine and thymidine, are unable to salvage pyrimidine bases, and possess a low uridine phosphorylase activity and markedly decreased (about 1% of peripheral blood lymphocytes) thymidine phosphorylase activity. Lymphocytes have higher pyrimidine-nucleoside phosphorylases activities, they can synthesize deoxyuridine and thymidine from bases, but at high an non-physiological concentrations of precursors. Neither type of cell is able to salvage uracil into uridine. These results suggest that pyrimidinenucleoside phosphorylases have a catabolic, rather than an anabolic, role in human lymphoid cells. The facts that, compared to peripheral blood lymphocytes, lymphoblasts possess decreased pyrimidine-nucleoside phosphorylases activities, and, on the other hand, more efficiently salvage pyrimidine nucleosides, are consistent with a greater need of these rapidly proliferating cells for pyrimidine nucleotides.
Clinical Chemistry and Laboratory Medicine, 1991
Deoxynucleoside 5'-triphosphates (dNTPs) can be determined in cell extracts by high performance liquid chromatography after prior selective degradation of ribonucleoside S'-triphosphates with sodium periodate and methylamine. When the method is used for the evaluation of deoxynucleoside triphosphates in 1-ß-D-arabinofuranosylcytosine triphosphate (ara-CTP)-containing cell extracts, an additional peak coeluting with thymidine triphosphate (dTTP) is observed. This peak is due to the formation of a carboxylic acid derivative of ara-CTP by periodate oxidation, and it can lead to considerable overestimation of dTTP. Formation of this peak can be avoided by using alkäline reaction conditions (pH 7.5) and by changing the sequence of addition of the reagents used in the periodation procedure. By employing this modified protocol, cellular dNTP and ara-CTP levels can be monitored in extracts of leukaemic blasts during cytosine arabinoside treatment in two separate HPLC runs. base. Subsequent ß-elirhination leads to removal of The measurement of deoxyribonucleoside-5'-triphos-the phosphate groups from the molecule and cleavage phates (dNTPs) has gaiiied widespread importance in of the N-glycosidic bond (6). Ribonucleotides are thus the study of anticancer and antiviral agents. An in-converted to the respective bases, which elute with direct assay üsing the respective endogerious deoxy-the void volume of the anion exchange column. nucleoside äs a limiting factor in a DNA-polyrneräse catalysed reaction has been described (1-3). This Pyrimidine dNTP levels have been shown to influence method caniiot be used, however, when the chemo^ the sensitivity of cells towards l-ß-/)-arabinofuranotherapeutic agents themselves or their metäbolites in-sylcytosine (ara-C) (7-9). The determination of the terfere with the polymerase reaction. An alternative biologically active metabolite, l-ß-Z)-arabinofuranomethod uses high performance liquid chromatogra-sylcytosine triphosphate (ara-CTP), and of cellular phy to separate the deoxyribonucleotides (4, 5). Since dNTP levels in extracts prepared from blasts of the this method does not separate ribonucleotides same patient is, therefore, important, when studying (rNTPs) from deoxyribonucleotides, it is necessary to the mutual interference of drug and dNTP levels. first chemically.degrade the rNTPs, using periodate Although published high performance liquid chroin the presence of methylsunine to cleave the 2'-3' matography methods yield highly accurate and reprocarbon bond of ribose; the resulting dialdehyde de-ducible results for the determination of dNTPs in cell rivative reacts with the methylamine to form a Schiff extracts, samples containing ara-CTP cannol be an
Pyrimidine Synthesis in Tissue Culture
Journal of Neurochemistry, 1968
Myelinated cerebellar tissue culture (organ culture) was used to assess the salvage and de novo pathways of pyrimidine synthesis in mammalian brain. Radioactive orotic acid and carbamyl aspartic acid were readily incorporated into UMP and into perchloric acid-insoluble RNA. The incorporation was effectively blocked by azauridine. Neither radioactive sodium bicarbonate or citrulline was incorporated into UMP or blocked by azauridine.
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.
Pyrimidine nucleotide synthesis in the rat kidney in early diabetes
Biochemical Medicine and Metabolic Biology, 1991
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 nova 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
Inhibition of pyrimidine de novo synthesis by DUP-785 (NSC 368390)
Investigational New Drugs, 1987
The mechanism of action of NSC 368390 (DUP-785, 6-fluoro-2-(2'-fluoro-1, l'-biphenyl-4-yl)-3-methyl-4-quinoline carboxylic acid sodium salt) was studied using three different approaches. First, we studied growth inhibition by DUP-785 in L1210 leukemia cells and M5 melanoma cells. The concentrations causing 50% growth inhibition after 48 hr of culture were 5.8 and 0.6 tzM, respectively. DUP-785 had to be present continuously throughout culture. Growth inhibition by 25 ~M DUP-785 could be prevented by addition of 1 mM uridine or orotic acid to cultures of these cell lines; in M5 cells cytidine was also able to prevent growth inhibition. Dihydro-orotic acid (DHO) and carbamyl-aspartate were not able to prevent growth inhibition by DUP-785. Second, we studied accumulation of orotic acid and of orotidine induced by incubation with 1 ~M pyrazofurin, an inhibitor of the orotate phosphoribosyl-transferase-orotidine-monophosphate decarboxylase complex. Addition of DUP-785 to the culture medium prevented the orotic acid accumulation. Furthermore, DUP-785 prevented accumulation of H14CO3 into orotic acid of pyrazofurin-treated L1210 cells. Third, we measured the effect of DUP-785 on DHO-dehydrogenase (DHO-DH), since the results indicated that this enzyme was affected by DUP-785. DHO-DH was assayed in isolated rat liver mitochondria. The Km for L-DHO was about 12 ~M. DUP-785 appeared to be a potent inhibitor of DHO-DH with an apparent Ki of about 0.1/xM and an apparent Ki' of about 0.8 #M. The mode of inhibition appeared to be linear mixed type. After exposure of L1210 cells to 25 ~M DUP-785 for 2 hr DHO-DH was almost completely inhibited. After suspension in fresh medium without drug, DHO-DH activity was recovered to about 60~ after 24 hr. In conclusion, DUP-785 is a potent inhibitor of pyrimidine de novo biosynthesis, by inhibition of the mitochondrial enzyme DHO-DH.
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...
Purine and pyrimidine metabolism in human gliomas: relation to chromosomal aberrations
British journal of cancer, 1994
Chromosomal aberrations in human gliomas are principally numerical. In tumours of low malignancy, karyotypes are frequently normal, but occasionally an excess of chromosome 7 and a loss of sex chromosome are observed. In highly malignant tumours, the most frequent aberrations are gain of chromosome 7, loss of chromosome 10 and less frequently losses or deletions of chromosomes 9, 22, 6, 13 and 14 or gains of chromosomes 19 and 20. To understand the meaning of these chromosome imbalances, the relationships between chromosome abnormalities and metabolic disturbances were studied. The losses or deletions observed affected principally chromosomes carrying genes encoding enzymes involved in purine metabolism. The activities of ten enzymes were measured: adenosine kinase, adenine phosphoribosyltransferase, adenylate kinase, methylthioadenosine phosphorylase, hypoxanthine phosphoribosyltransferase, adenylosuccinate lyase, inosine monophosphate dehydrogenase, adenosine deaminase, nucleoside...