PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “Pyrimidine Phosphorylases”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed1991

Production of 5-methyluridine by immobilized thermostable purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus JTS 859.

5-Methyluridine was produced continuously from thymine and inosine by immobilized enzymes, which consisted of thermostable purine nucleoside phosphorylase and thermostable pyrimidine nucleoside phosphorylase obtained from Bacillus stearothermophilus JTS 859. The process was carried out in a column reactor at 60 degrees C for 17 d without any bacterial contamination under non-aseptical conditions. Half-lives of the activity of the immobilized enzymes were 47 d and 4.5 d at 60 degrees C and 70 degrees C, respectively, although half-life of the crude enzyme was only 14 h at 70 degrees C.

Bacterial Proteins↗
PubMed1998

[A new inhibitor of pyrimidine phosphorylase].

5,5-Bis(hydroxymethyl)-2-oxo-[1-(2-trifluoromethyl)-3,3,3- trifluoropropionamido)-1-trifluoromethyl-2,2,2-trifluoroethyl- 1,3,2-dioxaphosphan (CA-423) is an in vitro inhibitor of the Escherichia coli uridine and thymidine phosphorylases. Unlike widely studied nucleoside analogues, this compound binds to the enzymes irreversibly. Its LD50 in mice was 40 mg/kg. Due to the involvement of pyrimidine phosphorylases in carcinogenesis and the relatively low toxicity of CA-423, it is promising for anticancer therapy.

Animals↗
PubMed1996

Purification and characterization of purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus TH 6-2.

The purine nucleoside phosphorylase (Pu-NPase) and the pyrimidine nucleoside phosphorylase (Py-NPase) have been purified from Bacillus stearothermophilus TH 6-2. The Pu-NPase is a trimer of 30-kDa subunits and the Py-NPase is a dimer of 46-kDa subunits. The isoelectric points of Pu-NPase and Py-NPase were pH 4.3 and 4.6, respectively. The Pu-NPase could catalyze the phosphorolysis of inosine and guanosine, but not adenosine. the Py-NPase could phosphorolyze both uridine and thymidine.

Amino Acid Sequence↗
PubMed2006

Activity and substrate specificity of pyrimidine phosphorylases and their role in fluoropyrimidine sensitivity in colon cancer cell lines.

Thymidine phosphorylase (TP) and uridine phosphorylase (UP) are often upregulated in solid tumors and catalyze the phosphorolysis of natural (deoxy)nucleosides and a wide variety of fluorinated pyrimidine nucleosides. Because the relative contribution of each of the two enzymes to these reactions is still largely unknown, we investigated the substrate specificity of TP and UP in colon cancer cells for the (fluoro)pyrimidine nucleosides thymidine (TdR), uridine (Urd), 5'-deoxy-5-fluorouridine (5'DFUR), and 5FU. Specific inhibitors of TP (TPI) and UP (BAU) were used to determine the contribution of each enzyme in relation to their cytotoxic effect. The high TP expressing Colo320TP1 cells were most sensitive to 5'DFUR and 5FU, with IC50 values of 1.4 and 0.2 microM, respectively, while SW948 and SW1398 were insensitive to 5'DFUR (IC50>150 microM for 5'DFUR). TPI and BAU only moderately affected sensitivity of Colo320, SW948, and SW1398, whereas TPI significantly increased IC(50) for 5'DFUR (50-fold) and 5FU (11-fold) in Colo320TP1 and BAU that in C26A (9-fold for 5'DFUR; p<0.01). In the epithelial skin cell line HaCaT both inhibitors were able to decrease sensitivity to 5'DFUR and 5FU separately. HaCaT might be a model for 5'DFUR toxicity. In the colon cancer cells 5'DFUR degradation varied from 0.4 to 50 nmol 5FU/h/10(6)cells, that of TdR from 0.3 to 103 nmol thymine/h/10(6)cells, that of Urd from 0.8 to 79 nmol uracil/h/10(6)cells, while conversion of 5FU to FUrd was from 0.3 to 46 nmol/h/10(6)cells. SW948 and SW1398 were about equally sensitive to 5'DFUR and 5FU, but SW1398 had higher phosphorylase activity (>65-fold) compared to SW948. In SW948 and HaCaT TPI and BAU inhibited TdR and Urd phosphorolysis (>80%), respectively. Both TP and UP contributed to the phosphorolysis of 5'DFUR and 5FU. In the presence of both inhibitors, still phosphorolysis of 5FU (>40%) was detected in the tumor and HaCaT cell lines, and remarkably, that of all four substrates in SW1398 cells. 5'DFUR phosphorolysis was also measured in situ, where Colo320TP1, SW1398, and HaCaT cells produced significant amounts 5FU from 5'DFUR (>10 nmol/24h/10(6)cells). In Colo320TP1 and in HaCaT cells TPI completely prevented 5FU production, but not in SW1398 cells, where BAU decreased this by 67% (p<0.01). High uracil and dUrd levels were detected in the medium. Uracil accumulation was heavily reduced in the presence of TPI for Colo320TP1 and HaCaT cells, whereas 5FU-induced dUrd production by these cell lines increased (p<0.01). In contrast, for SW1398 cells only BAU was able to reduce uracil levels, and dUrd production remained unchanged. In conclusion, overlapping substrate specificity was found for TP and UP in the cell lines, in which both enzymes were responsible for converting TdR and Urd, and 5'DFUR. 5'DFUR and 5FU were converted to their products in both the colon cancer cells and keratinocytes.

Cell Line, Tumor↗
PubMed1980

Localization of purine and pyrimidine nucleoside phosphorylases in heart, kidney, and liver.

In isolated livers and kidneys perfused with Krebs-Henseleit solution, the relationship of the concentration of adenosine (Ado) to that of its degradation products inosine (Ino) and hypoxanthine (Hyp) in biliary, urinary, and venous effluents were determined. They revealed ratios of Hyp:Ado:Ino, 1.9:1:0.9, 0.7:1:0.6, and 1.3:1:0.5 for guinea pig biliary, guinea pig urinary, and rat urinary effluents, respectively, and their respective venous effluent were 58:1:29, 8.6:1:5.4, and 7.4:1:3.2. The greater proportion of Ino and Hyp in the venous effluents suggests active production in Ino and Hyp at the vessel wall. Purine nucleoside phosphorylase localization was determined histochemically and found most active in the cytoplasm of capillary endothelium and Kupffer cells. Thus, there is agreement between purine analysis and histochemical findings. The reliability of the histochemical technique was also tested by comparing activities of purine nucleoside phosphorylase (a cytoplasmic enzyme) and pyrmidine nucleoside phosphorylase (a nuclear enzyme) that catalyze similar reactions (nucleoside + inorganic phosphate in equilibrium base + ribose-1-phosphate) but with different base specificites and cellular localization, as indicated by cell fractionation studies. The histochemical results show that in contrast to the purine nucleoside phosphorylase, the pyrmidine specific enzyme was most active in the nuclei of endothelial and Kupffer cells. Thus, the technique discriminates between the two enzymes.

Adenosine↗
PubMed1989

[Demonstration of pyrimidine nucleoside phosphorylases in breast cancer].

Two pyrimidine phosphorylase activities have been isolated from the cytosol of cultivated MCF-7 cells of a human breast cancer, by ion exchange chromatography. Both enzymes are responsible for the cleavage of thymidine into thymine and deoxyribose-1-phosphate, for the synthesis of thymidine and for the transfer of deoxyribose from d-uridine to thymine. These activities are likely to participate in the regulation of the pool of pyrimidine nucleosides required for DNA synthesis.

Breast Neoplasms↗
PubMed1999

Cloning, expression and crystallization of pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus.

Pyrimidine nucleoside phosphorylase (PYNP) from B. stearothermophilus has been cloned and purified for crystallization. Crystals of a potential protein-inhibitor complex have been prepared by co-crystallization techniques using the substrate analog pseudouridine. These crystals provide good-quality diffraction images to 2.7 A and belong to space group P21. The asymmetric unit contains the dimer structure of PYNP with unit-cell parameters a = 53.9, b = 71.9, c = 123.3 A and beta = 96.9 degrees.

Amino Acid Sequence↗
PubMed1998

The crystal structure of pyrimidine nucleoside phosphorylase in a closed conformation.

BACKGROUND: Pyrimidine nucleoside phosphorylase (PYNP) catalyzes the reversible phosphorolysis of pyrimidines in the nucleotide synthesis salvage pathway. In lower organisms (e.g. Bacillus stearothermophilus) PYNP accepts both thymidine and uridine, whereas in mammalian and other higher organisms it is specific for thymidine (designated thymidine phosphorylase, TP). PYNP shares 40% sequence similarity (and presumably significant structural similarity) with human TP, which has been implicated as a growth factor in tumor angiogenesis. It is thought that TP undergoes a major conformational change upon substrate binding that consequently produces an active conformation. RESULTS: The crystal structure of PYNP from B. stearothermophilus with the substrate analog pseudouridine in its active site has been solved to 2.1 A resolution. This structure confirms the similarity of PYNP to TP and supports the idea of a closed active conformation, which is the result of rigid body movement of the alpha and alpha/beta domains. The active-site cleft, where the pyrimidine and phosphate substrates bind, is between the two domains. The structure reveals an asymmetric dimer in which one subunit is fully closed and the other is only partially closed. CONCLUSIONS: The closed conformation of PYNP serves as a good model to better understand the domain movement and overall function of TP. Active-site residues are confirmed and a possible mechanism for substrate binding and subsequent domain movement is suggested. Potent inhibitors of TP might have significant therapeutic value in various chemotherapeutic strategies, and the structure of PYNP should provide valuable insight into the rational design of such inhibitors.

Binding Sites↗
PubMed1995

Pyrimidine nucleoside catabolism in Sphingomonas paucimobilis: role of cytidine deaminase and uridine phosphorylase.

Pyrimidine nucleoside catabolism in the human pathogen Sphingomonas paucimobilis was studied. It was observed that S. paucimobilis was only capable of utilizing cytidine or deoxycytidine as a sole nitrogen source when glucose served as the carbon source. Thinlayer chromatographic analyses of cytidine and uridine catabolic products revealed that the enzymes cytidine deaminase and uridine phosphorylase were active in the extracts prepared from S. paucimobilis cells. The levels of cytidine deaminase and cytosine deaminase activities were lowered after growth on cytidine or deoxycytidine as a nitrogen source instead of ammonium sulfate. Uridine phosphorylase activity increased more than 4-fold after growth on deoxycytidine as a nitrogen source while growth on the nitrogen source cytidine caused a depression in phosphorylase activity.

Cytidine Deaminase↗
PubMed1989

Species differences in substrate specificity of pyrimidine nucleoside phosphorylase.

To compare the activity of pyrimidine nucleoside phosphorylase (PNP), an enzyme involved in the metabolism of 5-fluorouracil (5-FU), we used uridine (Urd), deoxyuridine (dUrd), and thymidine (dThd) as substrates and human, rat, and mouse neoplastic and normal tissues. As PNP activity was higher in the tumor tissues than in the normal ones in all species examined, the level of PNP activity is expected to be one critical factor linked to the effectiveness of 5-FU. In rats and mice, the ratio of the activities of Urd, dUrd, and dThd was about 10:7:1, whereas in humans, the ratio was 1:30:20. The main enzyme of PNP is Urd phosphorylase in rodents and dThd phosphorylase in humans. Therefore, when examining the metabolism of 5-FU and its analogues for potential clinical application, human tissues should be used.

Animals↗
PubMed2006

Role of each residue in catalysis in the active site of pyrimidine nucleoside phosphorylase from Bacillus subtilis: a hybrid QM/MM study.

Pyrimidine nucleoside phosphorylase (PYNP) catalyzes the reversible phosphorolysis of pyrimidines in the nucleotide synthesis salvage pathway. We have built a model of a closed active conformation of the three-dimensional structure of PYNP from Bacillus subtilis. Using docking, molecular dynamics, and hybrid quantum-mechanical/molecular-mechanical methods to study the reaction mechanics between PYNP and a substrate, we identified the role of each residue in the active site during the entire catalytic process. The results indicate that the function of His(82), Arg(169), and Lys(188) is to stabilize the uridine in a high-energy conformation by means of electrostatic interactions and that these residues are involved in catalysis. In addition, the function of Asp(162) is likely to activate Lys(188) for phosphorolytic catalysis through polarization effects.

Amino Acids↗
PubMed1983

Structure-activity relationship of ligands of the pyrimidine nucleoside phosphorylases.

Eighty-seven pyrimidine base and nucleoside analogs were evaluated as inhibitors of uridine phosphorylase (UrdPase) and thymidine phosphorylase (dThdPase). These findings, together with an extensive literature review, have allowed construction of structure-activity relationships for the binding of ligands to UrdPase and dThdPase and provide a basis for the rational design of new inhibitors of these enzymes. Additionally, 2,6-pyridinediol and 6-benzyl-2-thiouracil have been identified as being potent inhibitors of UrdPase and dThdPase respectively.

Animals↗
PubMed1996

Molecular cloning and expression of the pyrimidine nucleoside phosphorylase gene from Bacillus stearothermophilus TH 6-2.

The pyrimidine nucleoside phosphorylase (Py-NPase) of Bacillus stearothermophilus TH 6-2 is a dimer of 46-kDa subunits and catalyzes the reversible phosphorolysis of uridine and thymidine. The gene encoding this pyrimidine nucleoside phosphorylase (pyn gene) has been cloned and sequenced from B. stearothermophilus TH 6-2. The pyn gene corresponded to an open reading frame of 1299 nucleotides that translates into a putative 433 amino acid protein with a molecular weight of 46,271. The deduced amino terminal sequence of Py-NPase coincided with that previously found for the purified enzyme. The deduced amino acid sequence of Py-NPase shared significant similarity with those of human and Escherichia coli thymidine phosphorylases. The cloned pyn gene was overexpressed in E. coli cells to produce an active enzyme in large quantities that accounted for approximately 20% of the total protein.

Amino Acid Sequence↗
PubMed1980

Specificity of pyrimidine nucleoside phosphorylases and the phosphorolysis of 5-fluoro-2'-deoxyuridine.

Isoelectric focusing and studies with 1-(2'-deoxy-beta-D-glucopyranosyl)thymine (GPT), a specific inhibitor of uridine phosphorylase activity, were used to determine the substrate specificities of mammalian pyrimidine nucleoside phosphorylases and their cleavage of 5-fluoro-2'-deoxyuridine (FdUrd). Isoelectric focusing profiles for the cytosol fractions from Ehrlich ascites cells and from Novikoff hepatoma cells each consisted essentially of one peak of nucleoside phosphorylase activity [isoelectric points (pl) 5.4 and 5.8, respectively] that cleaved both uridine and thymidine (dThd), as well as FdUrd. By contrast, cytosol fractions from HeLa (S3) cells, mouse liver, and normal human leukocytes each exhibited a major peak of activity (pl 4.6, 6.5, and 4.9, respectively) that cleaved only dThd and FdUrd, while mouse liver exhibited a second peak (pl 5.2) that cleaved primarily uridine. To distinguish clearly between (a) uridine phosphorylases that cleave primarily uridine and that are inhibited by GPT and (b)dThd phosphorylases that cleave only deoxynucleosides and that are not inhibited by GPT, we propose the term "uridine-deoxyuridine phosphorylases" to define those pyrimidine nucleoside phosphorylases that cleave both uridine and dThd and that are inhibited by GPT. On the basis of this definition and studies with GPT in nonfocused cytosol preparations, we conclude that FdUrd is cleaved to 5-fluorouracil by uridine-deoxyuridine phosphorylase activity in Ehrlich ascites cells and in Novikoff hepatoma cells, and by dThd phosphorylases in mouse liver, in normal human leukocytes, and in HeLa (S3) cells.

Animals↗
PubMed1982

Pyrimidine ribonucleoside phosphorylase activity vs 5- and/or 6-substituted uracil and uridine analogues, including conformational aspects.

The pyrimidine ribonucleoside phosphorylase from Salmonella typhimurium phosphorylyses 6-methyluridine, a uridine analogue sterically constrained to the syn conformation about the glycosylic bond, as effectively as uridine itself. In conjunction with the observation that 3-methyluridine is a very poor substrate compared to 5-methyluridine and 5,6-dimethyluridine, it follows that the phosphorolysis reaction involves the initial conversion of uridine, and other 5-substituted uridines (including 5-fluorouridine), to the syn conformation during interaction with the enzyme. Furthermore, and consistent with the foregoing, the enzyme recognizes as substrates, to varying degrees, the N(3)-ribosides of xanthine and uric acid, and will also catalyze the formation of these ribosides from the corresponding purines, which may be considered formally as 5,6-disubstituted uracils. Similar observations are reported for the synthetic 5,6-trimethyleneuridine. The enzyme does not, however, recognize 6-methyluracil and 5,6-tetramethyleneuridine in the reverse, synthetic, reaction. The conformational aspects of these reactions are discussed. Since it was previously shown that 6-methyluridine is an equally effective substrate for the pyrimidine phosphorylase of primary rabbit kidney cells, at least some of these conformational requirements apply to the enzyme from mammalian sources, and are consequently of relevance in the design of chemotherapeutic agents, for which some examples are cited.

Molecular Conformation↗
PubMed1987

Metabolism of pyrimidine bases and nucleosides by pyrimidine-nucleoside phosphorylases in cultured human lymphoid cells.

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 pyrimidine-nucleoside 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.

Cells, Cultured↗