Orotate Phosphoribosyltransferase Activity Research Articles

P1-165: Messenger rna expressions of nucleoside-metabolizing enzymes in tumor cells according to histologic type in patients with non-small-cell lung cancer

UFT including 5-Fluorouracil (5-Fu) and Tegafur has been reported to be effective as adjuvant chemotherapy for an early-stage group of primary lung cancer, mainly for adenocarcinoma (AD), not for squamous cell carcinoma (SQ). The effectiveness is also thought to depend on activities of nucleoside-metabolizing enzymes in the tumor cells such as thymidylate synthase (TS), dihydropyridimine dehydrogenase (DPD), thymidine phosphorylase (TP), and orotate phosphoribosyl transferase (OPRT). Therefore, we investigated m-RNA expressions of these nucleo-side-metabolizing enzymes in surgically resected specimens and compared them between AD and SQ. groups. We studied 51 patients with AD and 49 patients with SQ who underwent surgery from April 2001 to June 2005.The resected specimens were fixed in formalin and stained with hematoxylin and eosin to identify tumor cells. Thus, only tumor cells were collected and the m-RNA expressions of TS, DPD, TP and OPRT were quantified using Danenberg tumor profiling technique. These m-RNA expressions obtained were compared between the AD and SQ groups. Pathological stage did not differ between the AD and SQ groups. Since mean age and gender popula-tion were significantly different between the two groups, the data were adjusted by analysis of covariance. m-RNA expressions of the 4 enzymes in the AD and SQ groups were as follows: Tabled 1TSDPDTPOPRTAD (n=51)1.60±0.862.29±1.229.52±6.300.85±0.53SQ (n=49)4.33±3.351.52±1.2016.27±11.842.26±1.14p-value<0.00010.0020.007<0.001 Open table in a new tab m-RNA expressions of TS, DPD, TP and OPRT were significantly different between the AD and SQ groups. The m-RNA expressions of the 4 enzymes were not different between pathological stages or gender, respectively. 5-Fu is theoretically more effective to tumor cells when the tumor cells have low activity of DPD, TP and OPRT. It is also reported that tumor cells with low TS activity is more sensitive to 5-Fu. Our study suggested that UFT may be more effective to AD than SQ because AD tumor cells have low TS activity and because additive Tegafur sufficiently suppresses high DPD activity of AD tumor cells. m-RNA expressions of TS, DPD, TP and OPRT were significantly different between the AD and SQ groups. These results may provide some insight of mechanism in which UFT is more effective to AD than SQ in primary lung cancer.

Expression of orotate phosphoribosyl transferase in human pancreatic cancer: Implication for the efficacy of uracil and tegafur-based adjuvant chemotherapy

The enzyme orotate phosphoribosyl transferase (OPRT) is involved in the metabolism of the anticancer drug 5-fluorouracil (5-FU), and is a key enzyme for conversion of 5-FU to its active form in tumor tissue. Little is known regarding the significance of OPRT in human pancreatic cancer. The present study was designed to assess the association between the activity of OPRT in the tumor, and the clinicopathological status and prognosis of human resectable pancreatic cancer, especially regarding its relevance to the efficacy of adjuvant chemotherapy with uracil and tegafur (UFT), cyclophosphamide (CPA) and/or gemcitabine (GEM). The present study included 99 resectable pancreatic cancers, which were all invasive ductal tubular carcinomas. OPRT was immunostained with a rabbit anti-human OPRT polyclonal antibody. OPRT was positively stained in 54 (54.5%) of 99 pancreatic cancers. The post-surgical survival rate of the OPRT (+) pancreatic cancers was significantly higher than that of the OPRT (-) ones. In the OPRT (+) group, the survival rate of the patients, who received adjuvant chemotherapy (ACT) with UFT, CPA or GEM, was significantly higher than that of the patients without ACT; however, in the OPRT (-) group, there was no difference in the survival between the ACT (+) and (-) groups. Multivariate analyses demonstrated that for all patients, primary tumor, status of nodal involvement (pN), residual tumor, level of dissection and CPA were significant variables for the prognosis: in OPRT (+) groups, primary tumor, nodal involvement, GEM and CPA were significant variables. In contrast, in the OPRT (-) group, pN was the only significant variable. The present study is the first report on the significance of OPRT in human pancreatic cancer, and the results indicate that the expression of OPRT may be useful to predict the response to adjuvant chemotherapy in human pancreatic cancer.

Enhanced orotate phosphoribosyltransferase activity in renal cell carcinoma and its prognostic significance

15590 Background: 5-Fluorouracil ( 5-FU ) is an anticancer agent clinically used against various cancers including renal cell carcinoma ( RCC ). 5-FU is a prodrug and orotate phosphoribosyltransferase ( OPRT ) is the principal enzyme which directly converts 5-FU to an active anticancer metabolite, 5-fluoro-2’-deoxyuridine 5’-monophosphate. Furthermore, OPRT is the key enzyme in the de novo DNA and RNA synthetic process, which converts orotic acid to orotidine 5’-phosphate. Little is known about the significance of OPRT in a variety of cancers including RCC. We investigated OPRT activity in 83 RCC and evaluated the association between OPRT activity and the stage/grade of RCC. The relationship between OPRT activity in RCC cells and their sensitivity to 5-FU was also examined. Methods: OPRT activity in non-fixed fresh frozen RCC and normal kidney were determined enzymatically by the 5-FU phosphorylation assay. The sensitivity of RCC cells to 5-FU was assessed by the microculture tetrazolium dye assay. Results: OPRT activity was approximately 8.5-fold higher in RCC compared to normal kidney. OPRT activity in T3/4 RCC was 3-fold higher than that in T1/2 RCC. OPRT activity in M1 RCC was 2.5-fold higher than that in M0 RCC. In addition, OPRT activity in Stage III/IV RCC was 3-fold higher than that in Stage I/II RCC. The level of OPRT activity in Grade 3 RCC was 3-fold higher than that in Grade 1/2 cancer. Patients with RCC with low OPRT activity had a longer postoperative disease-specific survival than those with high activity in the 5-year follow-up. OPRT activity in RCC cells positively correlated with their sensitivity to 5-FU. Conclusions: The present study has demonstrated that OPRT activity in RCC was higher than that in normal kidney, and that OPRT activity positively correlated with the stage/grade of RCC. Moreover, higher OPRT activity in RCC predicted worse prognosis and higher sensitivity to 5-FU. These results suggest that OPRT activity may be used as both a prognostic parameter and a predictive indicator for 5-FU efficacy in RCC. No significant financial relationships to disclose.

1097: Prognostic Significance of Orotate Phosphoribosyl Transferase Activity in Bladder Cancer

1097: Prognostic Significance of Orotate Phosphoribosyl Transferase Activity in Bladder Cancer

636: Prognostic Significance of Orotate Phosphoribosyltransferase Activity in Renal Cell Carcinoma

636: Prognostic Significance of Orotate Phosphoribosyltransferase Activity in Renal Cell Carcinoma

Upregulation of Enzymes Metabolizing 5-Fluorouracil in Colorectal Cancer

Background: The purpose of this study was to compare the activities of various enzymes, participating in the metabolism of 5-fluorouracil, between colorectal cancer and nontumor tissues and to investigate the association of the enzyme activities with clinicopathological backgrounds. Methods: Activities of seven enzymes involved in nucleic acid metabolism – orotate phosphoribosyltransferase (OPRT), ribonucleotide reductase (RNR), thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase, uridine phosphorylase and thymidine kinase (TK) – were measured in tumor and nontumor tissues from 28 patients who were operated on for colorectal cancers. Results: OPRT, thymidylate synthase, RNR, thymidine phosphorylase, uridine phosphorylase and thymidine kinase activities were significantly higher in tumor areas than in nontumor areas. OPRT showed the highest T/N ratio (the ratio of each enzyme activity in tumor areas to that in nontumor areas). The T/N ratio of RNR activity showed a tendency to be associated with lymph node metastasis and Dukes classification. Conclusion: The results suggest that OPRT is a main enzyme participating in the phosphorylation of 5-fluorouracil and has an important role in tumor growth. The T/N ratio of RNR may be predictive of tumor progression.

Comparative studies on pyrimidine metabolism in excised cotyledons of Pinus radiata during shoot formation in vitro

Changes in the pattern of pyrimidine nucleotide metabolism were investigated in Pinus radiata cotyledons cultured under shoot-forming (SF; +N(6)-benzyladenine) and non-shoot-forming (NSF, -N(6)-benzyladenine) conditions, as well as in cotyledons unresponsive (OLD) to N(6)-benzyladenine. This was carried out by following the metabolic fate of externally supplied (14)C-labeled orotic acid, intermediate of the de novo pathway, and (14)C-labeled uridine and uracil, substrates of the salvage pathway. Nucleic acid synthesis was also investigated by following the metabolic fate of (14)C-labeled thymidine during shoot bud formation and development. The de novo synthesis of pyrimidine nucleotides was operative under both SF and NSF conditions, and the activity of orotate phosphoribosyltransferase (OPRT), a key enzyme of the de novo pathway, was higher in SF tissue. Utilization of both uridine and uracil for nucleotide and nucleic acid synthesis clearly indicated that the salvage pathway of pyrimidine metabolism is also operative during shoot organogenesis. In general, uridine was a better substrate for the synthesis of salvage products than uracil, possibly due to the higher activity of uridine kinase (UK), compared to uracil phosphoribosyltransferase (UPRT). Incorporation of uridine into the nucleic acid fraction of OLD cotyledons was lower than that observed for their responsive (day 0) counterparts. Similarly, uracil utilization for nucleic acid synthesis was lower in NSF cotyledons, compared to that observed for SF tissue after 10 days in culture. This difference was ascribed to higher UPRT activity measured in the latter. Thus, there was an apparent difference in the utilization of nucleotides derived from uracil and uridine for nucleotide synthesis. The increased ability to produce pyrimidine nucleotides via the salvage pathway during shoot bud formation may be required in support of nucleic acid synthesis occurring during the process. Studies on thymidine metabolism confirmed this notion.

Relationship between p53 status and 5-fluorouracil sensitivity in 3 cell lines

Mouse lymphoma L5178Ytk +/− (MOLY) cells and human lymphoblastoid TK6 and WTK-1 cells are widely used to detect mutagens in vitro. MOLY and WTK-1 cells have a p53 mutation, while TK6 cells, which were derived from the same parental line as WTK-1 cells, do not. In this study, we tested the clastogen 5-fluorouracil (5-FU) in the Tk assay and the in vitro micronucleus (MN) assay in MOLY, TK6, and WTK-1 cells to clarify whether differential responses were related to p53 gene status. We also determined the effect of 5-FU on the frequency of apoptotic cells and on cell cycle distribution in each cell line. Furthermore, we measured the activity of the 5-FU metabolizing enzymes (thymidylate synthetase (TS), dihydrouracil dehydrogenase (DPD), orotate phosphoribosyl transferase (OPRT), and thymidine phosphorylase (TP)) in each cell line. We treated MOLY cells with 1.0–8.0 μg/mL 5-FU for 3 h and TK6 and WTK-1 cells with 1.56–25 and 3.13–50 μg/mL, respectively, for 4 h. In MOLY cells, the mutation frequency (MF) and MN frequency increased. In WTK-1 cells, the MN frequency but not the MF increased. In TK6 cells, neither the MF nor the MN frequency increased. Furthermore, the IC 50 of 5-FU was lower in MOLY cells than in the human cells. The response to 5-FU treatment differed in other ways as well. At the same level of cytotoxicity, the frequency of apoptotic cell was highest in TK6 cells. The cell cycle was delayed just after treatment in MOLY cells while the delay appeared 24 h later in TK6 and WTK-1 cells. Nothing in our analysis, however, revealed marked differences between the cell lines that could account for the severe cytotoxic and mutagenic responses that 5-FU elicited only in MOLY cells. 5-FU is phosphorylated by OPRT and TP and detoxified by DPD. MOLY cells have higher OPRT activity and markedly lower DPD and TP activity than TK6 and WTK-1 cells. The content of TS, however, the target enzyme of 5-FU, was similar in all cell lines, suggesting that 5-FU was more readily phosphorylated and less readily detoxified in MOLY cells than in TK6 and WTK-1 cells. MOLY cells were more sensitive to 5-FU than WTK-1 cells even though both have a mutated p53 gene, suggesting that the different responses to 5-FU were due to differences in 5-FU metabolism rather than the p53 status.

Prognostic impact of orotate phosphoribosyl transferase among 5‐fluorouracil metabolic enzymes in resectable colorectal cancers treated by oral 5‐fluorouracil‐based adjuvant chemotherapy

Orotate phosphoribosyl transferase (OPRT) is the main enzyme that involves in phosphoribosylation of 5-fluorouracil (5-FU), an essential step that leads to tumor growth inhibition. In our study, the prognostic relevance of OPRT, thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) in resectable colorectal cancer (CRC) patients treated by oral 5-FU were compared to further clarify the prognostic value of OPRT. Tumor tissue was collected from 90 CRC patients and the patients were followed for 5.2 years (Median). TS, DPD and OPRT activities in the extract of tumor tissue were determined enzymatically. The cut-off value of OPRT (0.147 nmol/(min mg), TS (0.044 pmol/mg) and DPD (72.10 pmol/(min mg) were determined by maximal chi(2) method. Among these 5-FU metabolic enzymes, only high OPRT group demonstrated significantly better disease-free survival (DFS) (p = 0.0152) and better overall survival (p = 0.0078). In Cox regression analysis, node status (p < 0.0005) and OPRT (p = 0.044) were significant factors for DFS. OPRT activity in tumor tissue was a predictor of prognosis in resectable CRC patients treated by oral 5-FU-based adjuvant chemotherapy, and was useful to pick-up high risk patients independent from known prognosis factors.

Prognostic impact of orotate phosphoribosyl transferase activity in resectable colorectal cancers treated by 5-fluorouracil-based adjuvant chemotherapy

Phosphoribosylation of 5-fluorouracil (5-FU) is an essential step which leads to tumor growth inhibition and orotate phosphoribosyl transferase (OPRT) is the main enzyme that involves in this conversion of 5-FU to 5-fluorouridine monophosphate. This retrospective study was aimed to evaluate the correlation between tumor OPRT activity and the clinical outcome in colorectal cancer (CRC) patients treated by oral 5-FU-based adjuvant chemotherapy. Surgical specimen was obtained from resectable 124 CRC patients who were subsequently treated by oral 5-FU-based adjuvant chemotherapy. OPRT activity in the extract of tumor tissue was enzymatically determined. The cut-off value of intratumor OPRT activity against disease free survival was determined by maximal chi2 method. The disease free survival and overall survival in each group were calculated using the Kaplan-Meier method. Patients were divided into two groups by determined cut-off value of intratumor OPRT (0.147 nmol/min/mg protein) (high group: n = 102, low group: n = 22). Five-year DFS (P = 0.035) and OS (P = 0.020) were significantly better for high OPRT group. This study demonstrated that an assay of tumor OPRT contributes to the determination of 5-FU-based adjuvant chemotherapy outcome and application in clinical practice should be included in tumor analysis prior to 5-FU-based adjuvant chemotherapy.

Impact of orotate phosphoribosyl transferase activity as a predictor of lymph node metastasis in gastric cancer

Orotate phosphoribosyl transferase (OPRT) is an essential nucleotide metabolic enzyme for cell proliferation and also a key enzyme for conversion of 5-FU to its active form in tumor tissue. The association between tumor OPRT activity and pathophysiological status, including lymph node metastasis [pN+], and the impact of OPRT for predicting pN+ were investigated in gastric cancer. The lymph node status of 73 resectable gastric cancer patients was analyzed preoperatively by computed tomography (CT), ultrasonography and magnetic resonance, and the OPRT activity of collected tumor tissue was measured. Then these data were compared with pathological observation of a surgical lymph node specimen. OPRT activity in the tumor tissue decreased as the depth of invasion increased. An OPRT test demonstrated superior sensitivity and comparable accuracy and sensitivity for predicting pN+, against current imaging diagnoses. Furthermore, the analysis of node negative patients by CT revealed that 80% of false negative patients were retrieved by this OPRT test. Thus, OPRT activity in tumor tissue was a powerful predictor of pN+ in resectable gastric cancer, and the preoperative OPRT test, when it becomes possible, would provide a basis for accurate evaluation of disease status, which is indispensable for the planning of personalized therapy.

Preparation of anti-orotate phosphoribosyltransferase antibody and its application to immunochemical detection in human tumor cells

Orotate phosphoribosyltransferase (OPRT) is a key enzyme in the anabolism of 5-fluorouracil (5-FU), and its expression in tumors is thought to increase the efficacy of 5-FU against the tumor. To detect the OPRT protein by immunoblot and/or immunohistochemical methods, we prepared highly specific antibody to the peptides contained in the human OPRT amino acid sequence. The anti-OPRT polyclonal antibodies, obtained by immunizing rabbits with the OPRT peptides, had a high specificity for the OPRT protein in human tumor xenografts when it was analyzed by immunoblotting, and furthermore, there was a positive correlation between OPRT activity and protein content in 12 human tumors (R2 = 0.632). These results suggest that immunohistochemical detection of tumoral OPRT protein expression with our anti-OPRT antibodies may provide a useful method for predicting the clinical response to 5-FU-based chemotherapy.

Comparison of orotate phosphoribosyl transferase (OPRT), thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) activity as prognostic factors in resectable colorectal cancers (CRC)

3643 Background: Phosphoribosylation of 5-FU is an essential step leads to tumor growth inhibition and OPRT is the main enzyme that involves in this conversion of 5-FU to FUMP. We have reported the...

Regulation of pyrimidine synthesis in Pseudomonas resinovorans

To investigate the regulation of de novo pyrimidine biosynthesis in the bacterium Pseudomonas resinovorans ATCC 14235. The pyrimidine biosynthetic pathway enzymes were measured in cell extracts from P. resinovorans ATCC 14235 and from an auxotroph lacking orotate phosphoribosyltransferase activity. Pyrimidine biosynthetic pathway enzyme activities in ATCC 14235 were affected by the addition of pyrimidine bases to the culture medium. The de novo enzyme activities of the phosphoribosyltransferase mutant strain increased after pyrimidine starvation indicating possible repression of the pathway by a pyrimidine-related compound. Aspartate transcarbamoylase activity in ATCC 14235 was inhibited in vitro by ATP, UTP and pyrophosphate. Pyrimidine biosynthesis in P. resinovorans was regulated at the level of enzyme synthesis and at the level of activity for aspartate transcarbamoylase. Its regulation of enzyme synthesis seemed to be similar to what has been observed in the taxonomically related species Pseudomonas oleovorans. This study found that pyrimidine biosynthesis is regulated in P. resinovorans. This could prove helpful to future studies investigating polyhydroxyalkanoate production by the bacterium.

Effect of carbon source on pyrimidine formation in Pseudomonas fluorescens ATCC 13525

The effect of carbon source on the regulation of the de novo pyrimidine biosynthetic enzymes in the type strain of Pseudomonas fluorescens was investigated. The de novo pyrimidine biosynthetic enzyme activities were measured in P. fluorescens ATCC 13525 cells and in cells from an auxotroph deficient for orotate phosphoribosyltransferase activity where glucose or succinate served as the carbon source. Pyrimidine supplementation to the culture medium affected the biosynthetic enzyme activities in ATCC 13525 cells. Pyrimidine Limitation of the mutant strain indicated transcriptional regulation of the biosynthetic pathway by pyrimidines that was influenced by carbon source. Transcriptional regulation of pyrimidine synthesis in P. fluorescens appeared to be more highly regulated than was observed for the taxonomically related species Pseudomonas aeruginosa or Pseudomonas putida.

Heat-Assisted Stretching of Paraffin Sections on Hot Plate Weakens Immunoreactivity of Orotate Phosphoribosyltransferase

Orotate phosphoribosyltransferase (OPRT) is the key enzyme for the phosphorylation of 5-fluorouracil (5-FU), the rate-limiting step for acquiring its anti-tumor effect. Since high enzyme activities and mRNA levels of OPRT are said to be associated with 5-FU chemosensitivity of cancer cells, the immunohistochemical demonstration of OPRT can be expected to contribute to the selection of patients who suffer from 5-FU-sensitive cancer. During a study for establishing the appropriate immunostaining condition using rabbit antiserum in formalin-fixed, paraffin-embedded sections of human cancer, we unexpectedly uncovered the fact that heat-assisted stretching of paraffin sections on a hot plate just after sectioning was critical for preserving OPRT immunoreactivity; namely, stretching sections briefly at 70°C or higher significantly reduced the immunoreactivity. Overnight drying of the sections in an oven at 37°C or 60°C did not influence the immunoreactivity, but pretreatments, including 0.2% trypsin, 0.002% proteinase K, and pressure cooking in 10 mM citrate buffer, pH 6.0 and pH 7.0, and 1 mM ethylenediaminetetraacetic acid solution, pH 8.0, seriously deteriorated the OPRT epitopes. The immunoreactivity was relatively resistant to overfixation, though weakened after fixation in formalin for four weeks. Xenografts with high OPRT enzyme activities showed distinct positive cytoplasmic staining, but those with low OPRT activities were equivocal. OPRT expression in routinely processed cancer tissues also provided valuable data.

Glutathione-induced growth of embryogenic tissue of white spruce correlates with changes in pyrimidine nucleotide metabolism

Exogenous applications of reduced glutathione (GSH) and oxidized glutathione (GSSG) promote growth of embryogenic tissue of white spruce during a 7-day subculture period. Compared to control tissue, a statistically significant increase in fresh weight, as well as RNA and DNA content, was observed in the presence of GSH and GSSG during the last days in culture. The effects of these two metabolites on pyrimidine nucleotide metabolism was investigated by following the metabolic fate of 14C-orotic acid, a precursor of the de novo synthesis, and 14C-uridine and 14C-uracil, intermediates of the respective salvage and degradation pathways. Compared to control embryos, GSH-treated tissue was able to utilize a larger fraction of supplied orotic acid for UMP production, possibly due to the increased activity of orotate phosphoribosyltransferase (OPRT). The activity of this enzyme increased markedly in tissue cultured with GSH during the last days in culture. Salvage of uridine for nucleotide and nucleic acid synthesis was observed in all treatments, especially in GSSG-treated tissue at day 7. The increased salvage activity in this tissue correlated with the increase in activities of the two uridine salvage enzymes, uridine kinase (URK) and nucleoside phosphotransferase measured with uridine (NPT(uridine)). Compared to control tissue, tissue treated with either GSH or GSSG was able to utilize a large fraction of uracil for nucleotide synthesis, denoting a better ability to divert this precursor from degradation. Nucleotide and nucleic acid analyses revealed that in both GSH and GSSG-treated tissue, the endogenous levels of UTP, CTP, as well as those of RNA and DNA, were increased compared to those of control tissue. Overall, the results from this study suggest that GSH and GSSG can induce growth of embryogenic tissue of white spruce through distinct metabolic changes of pyrimidine nucleotides.

Prognostic impact of orotate phosphoribosyl transferase (OPRT) activity in resectable colorectal cancers (CRC) treated by 5-FU-based adjuvant chemotherapy

3574 Background: Dihydropyrimidine dehydrogenase and thymidylate synthase are 5-FU metabolism and target enzyme, and has been studied as prognostic factors in CRC. Meanwhile, phosphoribosylation of 5-FU is an essential step leads to tumor growth inhibition and OPRT is the main enzyme that involves in this conversion of 5-FU to FUMP. The aim of this study was to evaluate the prognostic relevance of OPRT activity in CRC patients treated by 5-FU-based adjuvant chemotherapy. Methods: Surgical specimen was obtained from resectable CRC patients who were subsequently treated by 5-FU-based adjuvant chemotherapy. OPRT activity in the extract of tumor tissue was determined by the method of Laskin et al. Disease-free survival (DFS) and overall survival (OS) were calculated using Kaplan-Meier estimate. Results:During 1999 to 2003, tumor tissue was collected from 124 CRC patients and then the patients were followed for 3.7 years (Median). Patients were divided into 2 groups by the cut-off value of tumor OPRT (0.147 nmol/min/mg protein) determined by maximal χ2 method (high group (≥0.147): n=102, low group (<0.147): n=22). There were no significant imbalances in T and N value between 2 groups. 5-year DFS for high and low group were 75.7% and 56.9% (p=0.05, log-rank test) and OS were 84.6% and 58.4% (p=0.01), respectively. Conclusions: Lower OPRT activity in tumor tissue was associated with poor survival in resectable CRC patients treated by 5-FU-based adjuvant chemotherapy and this research clarified that OPRT assay is indispensable for the determination of 5-FU-based adjuvant chemotherapy indication. Thus, the application of OPRT assay to routine clinical practice should be considered prior to 5-FU-based adjuvant chemotherapy. No significant financial relationships to disclose.

Prognostic impact of orotate phosphoribosyl transferase (OPRT) activity in resectable colorectal cancers (CRC) treated by 5-FU-based adjuvant chemotherapy

3574 Background: Dihydropyrimidine dehydrogenase and thymidylate synthase are 5-FU metabolism and target enzyme, and has been studied as prognostic factors in CRC. Meanwhile, phosphoribosylation of...

Orotate phosphoribosyltransferase and orotidine 5$prime;-monophosphate decarboxylase exist as multienzyme complex in human malaria parasite Plasmodium falciparum*1

Plasmodium falciparum , the causative agent of the most lethal form of human malaria, totally depends on de novo pyrimidine biosynthetic pathway. Orotate phosphoribosyltransferase (OPRT) and orotidine 5 ′ -monophosphate decarboxylase (OMPDC), the fifth and sixth enzymes in the pathway catalyzing formation of uridine 5 ′ -monophosphate (UMP), remain largely uncharacterized in the protozoan parasite. In this study, we achieved purification of OPRT and OMPDC to near homogeneity from P. falciparum cultivated in vitro. The OPRT and OMPDC activities were co-eluted in all chromatographic columns during purification, suggesting the purified proteins exist as a multienzyme complex with a molecular mass of 140 ± 8 kDa and contain two subunits each of OPRT and OMPDC. Monomeric forms of OPRT and OMPDC had molecular masses of 32 ± 3 and 38 ± 3 kDa, respectively, in agreement with those of proteins predicted from P. falciparum genome database. Interestingly, kinetic parameters and inhibitory constants of both OPRT and OMPDC activities were found to be different to those of the bifunctional human red cell UMP synthase. Our evidence provides the first example of OPRT and OMPDC existing as a multienzyme complex.