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1.
Thymidine phosphorylase (dThdPase) is an enzyme involved in pyrimidine nucleoside metabolism, but little is known about its physiological functions. We purified dThdPase from human placenta and used it for antibody preparation. The purified material appears as a single band at 55,000 dalton on sodium dodecylsulfate-polyacrylamide gel electrophoresis. We obtained a specific antibody raised in rabbits that detected a single polypeptide with a molecular weight of 55,000 dalton in the post nuclear homogenates of several human tissues, on immunoblotting. Using the same technique, dThdPase was highly expressed in the liver, lung, spleen, lymph nodes and peripheral lymphocytes. Immunohistochemical staining revealed that macrophage-like cells contained a much higher amount of dThdPase than parenchymal cells in the liver and lung. dThdPase was found to be highly expressed in T- and B-cell-type malignant lymphoma cells, but low in lymphoblastic and myeloblastic leukemia cells. We also found that carcinomas in the stomach, colon and ovary contained higher amounts of this enzyme than non-neoplastic regions of the tissues. These data suggest that dThdPase plays a role in proliferation and/or differentiation of leukocytes and in cancer proliferation. 相似文献
2.
Abstract Seven as-triazine-3,5-dione acyclonucleosides were synthesized and evaluated as inhibitors of orotate phosphoribosyltransferase (OPRTase, EC 2.4.2.10), orotidine 5′-monophosphate decarboxylase (ODCase, EC 4.1.2.23), uridine phosphorylase (UrdPase, EC 2.4.2.3), and thymidine phosphorylase (dThdPase, EC 2.4.2.4). 相似文献
3.
The activity of thymidine kinase, thymidine phosphorylase, adenosine deaminase and 5'-nucleotidase of AMP was studied in tissues of 39 healthy females, as well as blood serum and lymphocytes of 60 healthy females, as well as in 50 patients with fibrocavernous mastopathy aged as 23-70. Comparative determination of adenosine metabolism enzymes activity in lymphocytes was carried out simultaneously with studying some immunological indexes in the organism of the same-aged healthy females and ones with mastopathy. It was revealed that age-related changes in the activity of thymidine kinase in blood serum reflected the analogous changes in enzyme activity in tissues of the healthy women. A direct correlation was established between thymidine kinase activity and age both in the healthy females and those with mastopathy. A significant decrease in activity of thymidine phosphorylase was demonstrated in blood serum of the patients with mastopathy in the age 46-60. Determined 4-fold increase in the activity of adenosine deaminase in serum was accompanied by decreased enzyme activity in lymphocytes and decreased Lymphocyte Blast Transformation Index in the same age range. Changes of immunological status are more expressed in T-system of immunity. The revealed metabolic changes in DNA-precursors metabolism in the patients with mastopathy aged as 46-60 might be one of the reasons of increased risk of oncological disease in this age group. 相似文献
4.
The presence of thymidine phosphorylase was observed in healthy, adenomatous and tumoral prostatic cells. In healthy and adenomatous tissues the enzyme activity was recovered as a single peak after ion exchange chromatography on DEAE-Sephadex gel. On the contrary, two forms of thymidine phosphorylase were found in prostatic cancers, one of them, with high activity appeared consequently as a characteristic feature of prostatic tumoral cells. 相似文献
5.
Uridine and thymidine phosphorylases have been purified to homogeneity from crude extracts of Lactobacillus casei. Both enzymes had an apparent molecular mass of about 80 kDa. Uridine phosphorylase consisted of four identical subunits while thymidine phosphorylase was composed of two identical ones. The sequence of 23 amino-acid residues from its N-terminal end was analyzed. Uridine phosphorylase had a Km of 5.0 x 10(-3) M for uridine and 1.24 x 10(-1) M for phosphate, while thymidine phosphorylase had a Km of 1.32 x 10(-1) M for thymidine and 1.0 x 10(-1) M for phosphate. Uridine phosphorylase was equally active with uridine and 5-methyluridine, but had a low activity towards thymidine. Its activity was inhibited competitively by 3-O-methyl-alpha D-glucopyranoside, on the other hand thymidine phosphorylase activity was not affected by this compound. Thymidine phosphorylase showed specificity towards the deoxyribosyl moiety of the substrate. In addition, it required a nonsubstituted pyrimidine moiety or one which was substituted in position 5. The pattern of the double-reciprocal plots of the initial velocities vs. the concentrations of either one of the substrates, and the product inhibition kinetics, indicated that the catalytic mechanism of both enzymatic reactions is sequential rather than Ping-Pong and that the sequence of the addition of the substrates is random (rapid equilibrium). In the case of the uridine phosphorylase-catalyzed reaction, the products are also released randomly, while in the thymidine phosphorylase-catalyzed reaction deoxyribose 1-phosphate is released after thymine. 相似文献
6.
The activity of metabolic enzymes, adenosine and thymidine, has been studied in the blood serum and lymphocytes of healthy people and oncological patients aged 23-80. An increase in the activity of thymidine kinase (EC 2.7.1.2), an enzyme of thymidine biosynthesis, was observed in the blood serum of oncological patients against a background of a sharp decrease in the activity of thymidine phosphorylase (EC 2.4.2.4), a catabolic enzyme. The revealed enzymic shifts have been observed in breast cancer patients after 36, in patients with the stomach cancer--after 46. It is found that an increase in the activity of adenosine deaminase (EC 3.5.4.4) and 5-nucleotidase of AMP (EC 3.1.3.5) in the blood serum of oncological patients is accompanied by a sharp decrease in the activity of these enzymes in lymphocytes. 相似文献
7.
In non-proliferating cells mitochondrial (mt) thymidine kinase (TK2) salvages thymidine derived from the extracellular milieu for the synthesis of mt dTTP. TK2 is a synthetic enzyme in a network of cytosolic and mt proteins with either synthetic or catabolic functions regulating the dTTP pool. In proliferating cultured cells the canonical cytosolic ribonucleotide reductase (R1-R2) is the prominent synthetic enzyme that by de novo synthesis provides most of dTTP for mt DNA replication. In non-proliferating cells p53R2 substitutes for R2. Catabolic enzymes safeguard the size of the dTTP pool: thymidine phosphorylase by degradation of thymidine and deoxyribonucleotidases by degradation of dTMP. Genetic deficiencies in three of the participants in the network, TK2, p53R2, or thymidine phosphorylase, result in severe mt DNA pathologies. Here we demonstrate the interdependence of the different enzymes of the network. We quantify changes in the size and turnover of the dTTP pool after inhibition of TK2 by RNA interference, of p53R2 with hydroxyurea, and of thymidine phosphorylase with 5-bromouracil. In proliferating cells the de novo pathway dominates, supporting large cytosolic and mt dTTP pools, whereas TK2 is dispensable, even in cells lacking the cytosolic thymidine kinase. In non-proliferating cells the small dTTP pools depend on the activities of both R1-p53R2 and TK2. The activity of TK2 is curbed by thymidine phosphorylase, which degrades thymidine in the cytoplasm, thus limiting the availability of thymidine for phosphorylation by TK2 in mitochondria. The dTTP pool shows an exquisite sensitivity to variations of thymidine concentrations at the nanomolar level. 相似文献
8.
The enzyme thymidine phosphorylase (thymidine: orthophosphate deoxyribosyltransferase, EC 2.4.2.4.), which plays a crucial role in nucleic acid metabolism in both prokaryotic and eukaryotic cells by regulating the availability of thymidine, is present in mammalian blood. Here we describe a simple, rapid HPLC-based micromethod for the assay of blood thymidine phosphorylase. We have arbitarily defined 1 unit of blood thymidine phosphorylase activity as the activity required to produce a 1-nM increment in the plasma concentration of thymine after incubation for 1 h at 37°C with a saturating concentration of exogenous thymidine. In normal adults, whole (peripheral venous) blood thymidine phosphorylase activity with blood cells intact was 64 ± 11 units (mean ± S.D., n =20, range 45–89). The apparent Michaelis constant for thymidine was of the order to 10−4 M but varied nearly 5-fold between different individuals. Activity increased when blood cells were permeabilised or lysed with non-ionic detergents, implying that thymidine phosphorylase is an intracellular enzyme which may be influenced by exogenous as well as intracellular factors. When blood from normal donors was fractionated, thymidine phosphorylase activity consistently co-isolated with platelets. Whole-blood thymidine phosphorylase activity correlated well with platelet parameters. Although thymidine phosphorylase activity was also detected in plasma and serum, the small size and notorious fragility of platelets suggest its platelet origin. Blood from leukaemic donors showed significantly increased thymidine phosphorylase activity compared to normal controls (mean activity ± S.D. was 96 ± 27 units; range 58–140, n = 8). Thymine formation from thymidine was temperature- and pH-depdendent in whole blood. 2′-Deoxyuridine and 3 of its 5-halogenated analogues (but not 3′-azido-3′-deoxythymidine (AZT), were catabolised by blood thymidine phosphorylase, even during blood clotting at room temperature. Assumptions about in vivo concentrations of these compounds should therefore be interpreted cautiously. In the presence of high concentrations of thymine and suitable deoxyribose donors, small amounts of thymidine were formed in some blood samples, so it is conceivable that thymidine catabolism may be reversible in vivo under some circumstances. 相似文献
10.
The purpose of this study was to examine the association of telomerase activity with clinical and histopathological prognostic variables in primary breast cancer (n=64). Telomerase activity in breast cancer was also compared with that in benign (n=10) and non-malignant tissues (n=8; post-lumpectomy tissues histopathologically defined as containing no residual tumor). The parameter was assessed using the Telomerase PCR ELISA kit. Values above OD 0.120 were considered positive. Estrogen and progesterone receptors (ER and PgR) were assayed by the dextran-coated charcoal method and levels >10 fmol/mg cytosol protein were taken as positive. Telomerase activity was detected in 20% and 50% of the patients with benign lesions and primary breast cancer, respectively, and in 50% of post-lumpectomy breast tissues histopathologically defined as containing no residual tumor. Telomerase activity was present in all stages of breast carcinoma and showed a significant inverse correlation with lymph node status (p=0.006), lymphatic invasion (p=0.035) and necrosis (p=0.033). Moreover, when stage II patients were grouped according to nodal involvement, a trend towards significance was observed (p=0.055). No correlation was observed with ER and PgR. The results of our study suggest that telomerase activity might be associated with the presence of cancer cells. Furthermore, telomerase activation may occur early in breast cancer and may be periodically downregulated during subsequent tumor progression. 相似文献
11.
Isoelectric focusing was used as the final step in the isolation of thymidine phosphorylase which was found to have an isoelectric point of 4.1. Analytical acrylamide gel electrophoresis showed the purified enzyme preparation contained one major protein band which stained for thymidine phosphorylase activity and usually a minor, faster migrating band devoid of activity. Inactivation of thymidine phosphorylase alone or in the presence of sensitizers by ultraviolet light, primarily at 253.7 nm, followed first order inactivation kinetics. The rate of inactivation of the enzyme was the same at pH 5 and 7.4 and the addition of various pyrimidine bases and nucleosides enhanced the inactivation rate at both pH values, but to a greater extent at pH 5. Linear plots of inactivation rates versus concentrations of thymidine or thymine were the same. At 7.8 mM thymidine or thymine, 11- and 4.4-fold increases in photoinactivation of thymidine phosphorylase were observed at pH 5 AND 7.4 RESPECTIVELY. Parabolic curves were obtained with increasing concentrations of either 5-iodo-2'-deoxyuridine or 5-iodouracil. 5-Iodouracil at 5.2 mM caused 212- (pH 5) and 100- (pH 7.4) FOLD INCREASES IN THE RATES OF PHOTOINACTIVATION OF THYMIDINE PHOSPHORYLASE. However, 5-iodo-2'-deoxyuridine at 5.0mM only enhanced the photoinactivation of enzyme by factors of 83 (pH 5) and 21 (pH 7.4). Neither 5-bromo-2'-deoxyuridine or 5-bromo-uracil was as potent in sensitizing the enzyme as the iodo analogs. Combinations of 5-iodouracil or 5-iodo-2'-deoxyuridine with thymine resulted in higher inactivation rates than the additive inactivation rates of individual compounds, whereas combinations of either iodo analog with thymidine resulted in lower inactivation rates. Increasing concentrations of phosphate or NaCl lessened the photoinactivation rate of thymidine phosphorylase alone and protected the enzyme from the sensitization caused by the different bases and nucleosides. No quantitative changes in the number of primary amino groups in thymidine phosphorylase was evident as a result of irradiation in the presence or absence of 5-iodouracil or 5-iodo-2'-deoxyuridine. Examination of the irradiated enzyme on Sephadex G-150 indicated that a larger protein species is formed and that 5-iodouracil promotes this process. 相似文献
12.
An angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-Deoxy-D-ribose, a degradation product of thymidine generated by TP, partially prevents hypoxia-induced apoptosis. TP is expressed at higher levels in tumor tissues compared to the adjacent non-neoplastic tissues in a variety of human carcinomas. High expression of TP is associated with an unfavorable prognosis. To investigate the effect of TP on cisplatin-induced apoptosis, human leukemia Jurkat cells were transfected with wild-type or mutant (L148R) TP cDNA. TP inhibited a number of steps in the cisplatin-induced apoptotic pathway, activation of caspases 3 and 9 and mitochondrial cytochrome c release. These findings suggest a mechanism by which TP confers resistance to apoptosis induced by cisplatin. Moreover, mutant TP that has no enzymatic activity also suppressed cisplatin-induced apoptosis. These findings indicate that TP has cytoprotective functions against cytotoxic agents which are independent of its enzymatic activity. 相似文献
13.
Trifluorothymidine (TFT) can be phosphorylated by thymidine kinase (TK) to TFTMP which can inhibit thymidylate synthase (TS), resulting in depletion of thymidine nucleotides. TFT can be degraded by thymidine phosphorylase (TP) which can be inhibited by thymidine phosphorylase inhibitor (TPI). Using the TS in situ Inhibition Assay (TSIA) FM3A breast cancer cells were exposed 4 h or 24 h to TFT and 5-Fluorouracil (5FU). TS activity reduced to 9% (0.1 microM TFT) and 58% (1 microM 5FU) after 4 h exposure and to 6% (TFT) and 21% (5FU) after 24 h exposure. TPI did not affect TS inhibition by TFT. FM3A cells lacking TK or TS activity (FM3A/TK-) were far less sensitive to TFT compared to FM3A cells. Conclusion: TFT can be taken up and activated very rapidly by FM3A cancer cells, probably due to favourable TK enzyme properties, and TPI did not influence this. 相似文献
14.
Thymidine phosphorylase (dThdPase) is an essential enzyme for activation of the oral cytostatic drug capecitabine and its intermediate metabolite, doxifluridine, to 5-fluorouracil in tumors. Methods to estimate dThdPase expression in tumor tissue might be useful to predict the efficacy of capecitabine and doxifluridine in cancer patients. We established a new monoclonal antibody (MAb), 1C6-203, applicable for dThdPase immunohistochemistry and compared its staining characteristics with those of a previously established MAb, 654-1. In 4% paraformaldehyde-fixed colorectal carcinoma, 1C6-203 and 654-1 stained cancer cells in 19/30 and 9/30 patients, respectively. In 10% formalin-fixed colorectal carcinoma, 1C6-203 and 654-1 stained cancer cells in 18/30 and 6/30 patients, respectively. In negative 10% formalin-fixed tissues, microwave treatment improved the positivity of 654-1-stained cancer cells. These results suggest that an epitope recognized by 1C6-203 is resistant to epitope masking by formaldehyde fixation, whereas that for MAb 654-1 is sensitive. Therefore, MAb 1C6-203 might be more suitable than MAb 654-1 for evaluating dThdPase expression in colorectal carcinoma. 相似文献
15.
Trifluorothymidine (TFT) can be phosphorylated by thymidine kinase (TK) to TFTMP which can inhibit thymidylate synthase (TS), resulting in depletion of thymidine nucleotides. TFT can be degraded by thymidine phosphorylase (TP) which can be inhibited by thymidine phosphorylase inhibitor (TPI). Using the TS in situ Inhibition Assay (TSIA) FM3A breast cancer cells were exposed 4 h or 24 h to TFT and 5‐Fluorouracil (5FU). TS activity reduced to 9% (0.1 µM TFT) and 58% (1 µM 5FU) after 4 h exposure and to 6% (TFT) and 21% (5FU) after 24 h exposure. TPI did not affect TS inhibition by TFT. FM3A cells lacking TK or TS activity (FM3A/TK ?) were far less sensitive to TFT compared to FM3A cells. Conclusion: TFT can be taken up and activated very rapidly by FM3A cancer cells, probably due to favourable TK enzyme properties, and TPI did not influence this. 相似文献
16.
Thymidine phosphorylase (TP) is over expressed in several solid tumors and its inhibition can offer unique target suitable for drug discovery in cancer. A series of 1,2,4-triazoles 3a–3l has been synthesized in good yields and subsequently inhibitory potential of synthesized triazoles 3a–3l against thymidine phosphorylase enzyme was evaluated. Out of these twelve analogs five analogues 3b, 3c, 3f, 3l and 3l exhibited a good inhibitory potential against thymidine phosphorylase. Inhibitory potential in term of IC 50 values were found in the range of 61.98 ± 0.43 to 273.43 ± 0.96 μM and 7-Deazaxanthine was taken as a standard inhibitor with IC 50 = 38.68 ± 4.42 μM. Encouraged by these results, more analogues 1,2,4-triazole-3-mercaptocarboxylic acids 4a–4g were synthesized and their inhibitory potential against thymidine phosphorylase was evaluated. In this series, six analogues 4b–4g exhibited a good inhibitory potential in the range of 43.86 ± 1.11–163.43 ± 2.03 μM. Angiogenic response of 1,2,4-triazole acid 4d was estimated using the chick chorionic allantoic membrane (CAM) assay. In the light of these findings, structure activity relationship and molecular docking studies of selected triazoles to determine the key binding interactions was discussed. Docking studies demonstrate that synthesized analogues interacted with active site residues of thymidine phosphorylase enzyme through π-π stacking, thiolate and hydrogen bonding interactions. 相似文献
17.
In Escherichia coli K-12, the rise in activity of thymidine phosphorylase, phosphodeoxyribomutase, and deoxyribose-5-phosphate aldolase caused by exogenous thymidine is dependent on the synthesis of new enzyme protein. Phosphodeoxyribomutase is induced by the purine ribonucleosides adenosine and guanosine, whereas the other two enzymes are not. The mutase activity induced by thymidine and by the purine ribonucleosides has been shown to be the same enzyme by four different criteria. This independent induction of phosphodeoxyribomutase suggests that the gene for this enzyme is in an operon different from the one that may contain the genes for thymidine phosphorylase and deoxyribose-5-phosphate aldolase. 相似文献
18.
Plasma membranes were isolated from rat liver homogenates either by differential centrifugation or by fractionation in discontinuous sucrose density gradients. Both membrane preparations contained about 17% of the total uridine phosphorylase (EC 2.4.2.3) activity and 44% of the total 5'-nucleotidase (EC 3.1.3.5). The enrichment factor for uridine phosphorylase in the fractions prepared by differential centrifugation was about 2.8 and by the gradient method, as much as 11.0; the respective enrichment factors for 5'-nucleotidase were 1.8 and 9.5. Uridine phosphorylase activity of isolated plasma membrane fractions was stimulated 2.5-fold by 0.1% Triton X-100. Unlike the cytosol enzyme, uridine phosphorylase of plasma membranes showed little or no deoxyuridine-cleaving activity. Contamination of the membrane fractions by thymidine phosphorylase (EC 2.4.2.4) of the cytosol was negligible. The other subcellular organelles obtained by either procedure and characterized by marker enzyme activities were found not to contain significant uridine phosphorylase activity; the cytosol fractions contained just over 70% of the total uridine phosphorylase activity with an enrichment of only about 2.8-fold. The activity of the cytosol enzyme was not stimulated by Triton X-100. 相似文献
19.
Substrate-stabilization of uridine phosphorylase (uridine:orthophosphate ribosyltransferase, EC 2.4.2.3), thymidine phosphorylase (thymidine:orthophosphate deoxyribosyltransferase, EC 2.4.2.4) and purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) from Escherichia coli was investigated by heat-inactivation experiments. Nucleoside substrates stabilized uridine phosphorylase and purine-nucleoside phosphorylase, but not thymidine phosphorylase. Aglycone substrates stabilized only uridine phosphorylase. Phosphate or pentose-1-phosphate ester substrates stabilized all three enzymes. The appropriate pentose-1-phosphate ester was a more effective stabilizer than was phosphate with all three enzymes. In previous reports dealing with the kinetic analysis of these phosphorylases, sequential mechanisms were proposed. Each enzyme appeared to have different sequence of substrate addition. The substrate-stabilization patterns reported here are consistent with the proposed mechanisms. 相似文献
20.
Summary A new type of mutant of mutant of Escherichia coli that synthesises thymidine phosphorylase constitutively has been isolated and characterised. The mutation leading to constitutivity is located to the left of the gene specifying deoxyriboaldolase. The mutation is cis dominant in its effect on thymidine phosphorylase activity and therefore believed to be a mutation of the operator-constitutive type. The specific activity of purine nucleoside phosphorylase is not affected by the mutation indicating that the gene specifying this enzyme is located in a different operon from that containing the genes specifying thymidine phosphorylase and deoxyriboaldolase. 相似文献
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