Orotate Phosphoribosyltransferase Activity Research Articles

Establishment of an overall transformation system for an oil-producing filamentous fungus, Mortierella alpina 1S-4.

Oil-producing fungus Mortierella alpina 1S-4 is an industrial strain. To determine its physiological properties and to clarify the biosynthetic pathways for polyunsaturated fatty acids, a transformation system for this fungus was established using a derivative of it, i.e., a ura5- mutant lacking orotate phosphoribosyl transferase (OPRTase, EC.2.4.2.10) activity. Transformation with a vector containing the homologous ura5 gene as a marker was successfully performed using microprojectile bombardment, other methods frequently used for transformation, such as the protoplasting, lithium acetate, or electroporation methods, not giving satisfactory results. As a result, two types of transformants were obtained: a few stable transformants overexpressing the ura5 gene, and many unstable transformants showing OPRTase activity comparable to that of the wild-type strain. The results of quantitative PCR indicated that the stable transformants could retain the ura5 genes originating from the transformation vector regardless of the culture conditions. On the other hand, unstable transformants easily lost the marker gene under uracil-containing conditions, as expected. In this paper, we report that an overall transformation system for this fungus was successfully established, and propose how to select useful transformants as experimental and industrial strains.

Orotate phosphoribosyltransferase and orotidine 5 ′-monophosphate decarboxylase exist as multienzyme complex in human malaria parasite Plasmodium falciparum

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.

Significance of Orotate Phosphoribosyltransferase Activity in Renal Cell Carcinoma

The anticancer agent 5-fluorouracil (5-FU) is clinically used against various cancers, including renal cell carcinoma (RCC). It is a prodrug and orotate phosphoribosyltransferase (OPRT) is the principal enzyme that directly converts 5-FU to the active anticancer metabolite 5-fluoro-2'-deoxyuridine 5'-monophosphate. In addition, OPRT is the key enzyme in the de novo DNA and RNA synthetic process. Little is known about the significance of OPRT in various cancers, including RCC. We investigated the activity of OPRT in 83 RCCs and evaluated the association between the level of 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. OPRT activity in nonfixed, fresh frozen RCC and normal kidney was determined enzymatically by the 5-FU phosphorylation assay. The sensitivity of RCC cells to 5-FU was assessed by the microculture tetrazolium dye assay. The activity of OPRT was approximately 8.5-fold higher in RCC than in normal kidney. OPRT activity in stage III/IV RCC was 3-fold higher than 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 longer postoperative disease specific survival than those with high activity at 5-year followup. OPRT activity in RCC cells positively correlated with their sensitivity to 5-FU. To our knowledge this is the first study to demonstrate that OPRT activity in RCC was higher than that in normal kidney and OPRT activity positively correlated with RCC stage/grade. In addition, higher OPRT activity in RCC predicted worse prognosis and higher sensitivity to 5-FU. These results suggest that the level of OPRT activity may be used as a prognostic parameter and predictive indicator for 5-FU efficacy in RCC and OPRT may be a molecular therapeutic target in RCC.

Cloning and sequencing of the ura3 and ura5 genes, and isolation and characterization of uracil auxotrophs of the fungus Mortierella alpina 1S-4.

The oil-producing fungus Mortierella alpina 1S-4 is an industrial strain. In order to prepare host strains for a transformation system for this fungus, six uracil auxotrophs were obtained by means of random mutation with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). When the activities of orotate phosphoribosyl transferase (OPRTase, EC 2.4.2.10) and orotidine-5'-phosphate decarboxylase (OMPdecase, EC 4.1.1.23) were examined in the mutants and wild strain, OPRTase activity was found to be completely absent in all mutants, on the other hand, OMPdecase activity was intact. The genomic DNA and cDNA of the ura5 gene encoding OPRTase and the ura3 gene encoding OMPdecase were cloned and sequenced. The Ura5p deduced amino acid sequence of this fungus showed highest similarity to that of Vibrio cholerae classed among prokaryote. Furthermore, the mutational points in the ura5 genes of two selected mutants were identified; a base-replacement and a base-insertion.

Influence of carbon source on pyrimidine synthesis in Pseudomonas mendocina.

The effect of carbon source on the regulation of the de novo pyrimidine biosynthetic enzymes in the bacterium Pseudomonas mendocina was studied. When glucose was the carbon source, orotic acid supplementation of P. mendocina cells produced the greatest depression of aspartate transcarbamoylase, dihydroorotate dehydrogenase and orotate phosphoribosyltransferase activities while P. mendocina cells grown in the presence of uracil caused the maximal decrease in dihydroorotase and OMP decarboxylase activities. After the pyrimidine starvation of an orotate phosphoribosyltransferase mutant strain of P. mendocina grown on glucose, the pyrimidine biosynthetic pathway enzyme activities were generally diminished. With respect to pyrimidine starvation studies, the carbon source glucose appeared to lessen regulation at the level of enzyme synthesis compared to what has been observed when succinate served as the carbon source. The regulation of the pyrimidine biosynthetic pathway by carbon source in P. mendocina appeared to differ from how carbon source influenced the control of pyrimidine biosynthesis in the closely-related species Pseudomonas stutzeri.

Chromosomal imbalances associated with acquired resistance to fluoropyrimidines in human colorectal cancer cells

The chromosomal aberrations underlying the development of resistance to fluoropyrimidines have not yet been identified. To characterise the genomic changes that induce the development of resistance to fluoropyrimidines, we used comparative genomic hybridisation (CGH) to analyse and compare the parent DLD-1 human colorectal cancer cell line and two cell lines, DLD-1/5-FU and DLD-1/FdUrd, which were resistant to 5-fluorouracil (5-FU) and 5-fluoro-2′-deoxyuridine (FdUrd), respectively. Both resistant cell lines showed a genetic aberration derived from the parental cell line DLD-1. Losses of 3p and 3q were also detected as additional genetic changes in the two resistant cell lines. Both resistant cell lines showed decreased orotate phosphoribosyltransferase (OPRT) activity, which is associated with the activity of the uridine monophosphate (UMP) synthase gene (3q13). These results suggested that the loss of 3q might be a genetic change responsible for the decreased OPRT activity and fluoropyrimidine cytotoxic response in cancer cells. Amplification of 18p11.2-p11.3 containing the thymidine synthase (TS) gene (18p11.32) was observed only in the DLD-1/FdUrd-resistant cell line, which overexpresses TS. These findings suggested that 18p amplification represents a genetic change associated with the overexpression of the TS protein. Our results indicate that chromosomal aberrations identified by CGH could explain, at least in part, acquired fluoropyrimidine resistance.

Correlation between clinicopathologic factors and kinetics of metabolic enzymes for 5-fluorouracil given to patients with colon carcinoma by two different dosage regimens.

Using tumor tissue specimens from colon carcinoma patients given 5-fluorouracil (5-FU) by two different administration methods, we investigated the degree of correlation between clinicopathologic factors and the kinetics of metabolic enzymes for 5-FU. Group A patients received 500 mg/day of 5-FU as a rapid infusion over 2 h for 3 days preoperatively, and group B patients received 500 mg/day of 5-FU as a continuous infusion for 3 days preoperatively. The activities of orotate phosphoribosyl transferase (OPRT), thymidine phosphorylase (TP), uridine phosphorylase (UP), and dihydropyrimidine dehydrogenase (DPD) were measured by separation on ion-exchange filter paper, by high-performance liquid chromatography (HPLC) with UV detection and by HPLC with radioactive flow monitoring, respectively, and the [(3)H]-5-fluorodeoxyuridine monophosphate binding site in thymidylate synthetase (TS) was determined as an index of the amount of TS using a radiobinding assay. The TS inhibition rate (TSIR) was calculated from the formula: 1-(TS(free)/TS(total))x100. Finally, 5-FU incorporation into RNA (FRNA) was measured by capillary gas chromatography-mass spectrometry. In group A patients, FRNA showed a positive correlation only with OPRT, and TSIR showed positive correlations with OPRT and TP, but a negative correlation with TS. In group B patients, FRNA showed a negative correlation with DPD, and TSIR showed a negative correlation with TS. No difference in TSIR levels was seen between groups A and B. FRNA was higher in group A than in group B, but the difference was not statistically significant. The method of administration may influence 5-FU metabolism in colon carcinoma patients.

Effect of carbon source on pyrimidine biosynthesis in Pseudomonas alcaligenes ATCC 14909

The effect of carbon source on the regulation of the de novo pyrimidine biosynthetic enzymes in Pseudomonas alcaligenes ATCC 14909 was investigated. The de novo pyrimidine biosynthetic enzymes were measured in extracts of P. alcaligenes ATCC 14909 cells and of cells from an auxotroph deficient for orotate phosphoribosyltransferase activity. Pyrimidine biosynthetic enzyme activities in ATCC 14909 were influenced by pyrimidine supplementation to the culture medium but not by the carbon source present. Pyrimidine limitation of the auxotroph elevated the de novo enzyme activities indicating that this pathway may be controlled at the transcriptional level by a pyrimidine-related compound. Its regulation seemed to be subject to less transcriptional control by a pyrimidine-related compound than what was observed in the closely related species Pseudomonas pseudoalcaligenes.

Profiles of pyrimidine biosynthesis, salvage and degradation in disks of potato (Solanum tuberosum L.) tubers.

In order to obtain general metabolic profiles of pyrimidine ribo- and deoxyribonucleotides in potato (Solanum tuberosum L.) plants, the in situ metabolic fate of various (14)C-labelled precursors in disks from growing potato tubers was investigated. The activities of key enzymes in potato tuber extracts were also studied. The following results were obtained. Of the intermediates in de novo pyrimidine biosynthesis, [(14)C]carbamoylaspartate was converted to orotic acid and [2-(14)C]orotic acid was metabolized to nucleotides and RNA. UMP synthase, a bifunctional enzyme with activities of orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 5'-monophosphate decarboxylase (EC 4.1.1.23), exhibited high activity. The rates of uptake of pyrimidine ribo- and deoxyribonucleosides by the disks were high, in the range 2.0-2.8 nmol (g FW)(-1) h(-1). The pyrimidine ribonucleosides, uridine and cytidine, were salvaged exclusively to nucleotides, by uridine/cytidine kinase (EC 2.7.1.48) and non-specific nucleoside phosphotransferase (EC 2.7.1.77). Cytidine was also salvaged after conversion to uridine by cytidine deaminase (EC 3.5.4.5) and the presence of this enzyme was demonstrated in cell-free tuber extracts. Deoxycytidine, a deoxyribonucleoside, was efficiently salvaged. Since deoxycytidine kinase (EC 2.7.1.74) activity was extremely low, non-specific nucleoside phosphotransferase (EC 2.7.1.77) probably participates in deoxycytidine salvage. Thymidine, which is another pyrimidine deoxyribonucleoside, was degraded and was not a good precursor for nucleotide synthesis. Virtually all the thymidine 5'-monophosphate synthesis from thymidine appeared to be catalyzed by phosphotransferase activity, since little thymidine kinase (EC 2.7.1.21) activity was detected. Of the pyrimidine bases, uracil, but not cytosine, was salvaged for nucleotide synthesis. Since uridine phosphorylase (EC 2.4.2.3) activity was not detected, uracil phosphoribosyltransferase (EC 2.4.2.9) seems to play the major role in uracil salvage. Uracil was degraded by the reductive pathway via beta-ureidopropionate, but cytosine was not degraded. The activities of the cytosine-metabolizing enzymes observed in other organisms, pyrimidine nucleoside phosphorylase (EC 2.4.2.2) and cytosine deaminase (EC 3.5.4.1), were not detected in potato tuber extracts. Operation of the de novo synthesis of deoxyribonucleotides via ribonucleotide reductase and of the salvage pathway of deoxycytidine was demonstrated via the incorporation of radioactivity from both [2-(14)C]cytidine and [2-(14)C]deoxycytidine into DNA. A novel pathway converting deoxycytidine to uracil nucleotides was found and deoxycytidine deaminase (EC 3.5.4.14), an enzyme that may participate in this pathway, was detected in the tuber extracts.

Regulation of pyrimidine synthesis in Pseudomonas mendocina.

Regulation of the de novo pyrimidine biosynthetic enzymes in the bacterium Pseudomonas mendocina was examined when its cells were grown on succinate as a carbon source. When P. mendocina was grown in the presence of orotic acid or uracil, the de novo enzyme activities were depressed with dihydroorotase activity being significantly depressed after uracil addition. Following pyrimidine limitation of a uracil auxotroph of P. mendocina deficient for orotate phosphoribosyltransferase activity, the pyrimidine biosynthetic pathway enzyme activities were affected indicating possible regulation at the level of enzyme synthesis. Of the de novo pathway enzymes assayed, dihydroorotate dehydrogenase exhibited the highest increase in its activity. The regulation of the pyrimidine biosynthetic pathway by pyrimidines in P. mendocina appeared similar to what was previously observed for the taxonomically-related species P. stutzeri.

A Nonradioactive High-Performance Liquid Chromatographic Microassay for Uridine 5′-Monophosphate Synthase, Orotate Phosphoribosyltransferase, and Orotidine 5′-Monophosphate Decarboxylase

A novel nonradioactive, microassay method has been developed to determine simultaneously the two enzymatic activities of orotate phosphoribosyltransferase (OPRTase) and orotidine 5′-monophosphate decarboxylase (ODCase), either as a bifunctional protein (uridine 5′-monophosphate synthase, UMPS) or as separate enzymes. Substrates (orotate for OPRTase or orotidine 5′-monophosphate for ODCase) and a product (UMP) of the enzymatic assay were separated by high-performance liquid chromatography (HPLC) using a reversed-phase column and an ion-pairing system; the amount of UMP was quantified by dual-wavelength uv detection at 260 and 278 nm. This HPLC assay can easily detect picomole levels of UMP in enzymatic reactions using low specific activity UMPS of mammalian cell extracts, which is difficult to do with the other nonradioactive assays that have been described. The HPLC assay is suitable for use in protein purification and for kinetic study of these enzymes.

Thymidylate synthase (TS) and ribonucleotide reductase (RNR) may be involved in acquired resistance to 5-fluorouracil (5-FU) in human cancer xenografts in vivo

A human tumour sub-line resistant to 5-fluorouracil (5-FU) was established by once a day and every 5, with at least 50 administrations of 5-FU to KM12C human colorectal xenografts in nude mice. KM12C tumours treated with 5-FU showed less sensitivity to 5-FU with an inhibition rate (IR) of 7.9%, while non-treated tumours were highly sensitive to 5-FU with an IR of 81.8%. To clarify the mechanism of 5-FU-resistance, the activities of various enzymes and gene expressions involved in the metabolism of 5-FU in both parental and 5-FU-treated KM12C tumours were measured. A 2- to 3-fold increase in thymidylate synthase (TS) activity and 4- to 5-fold decrease in ribonucleotide reductase (RNR) activity were observed in 5-FU-resistant KM12C tumours, while the activities of orotate phosphoribosyltransferase (OPRT) thymidine and uridine phosphorylases (TP,UP) and thymidine kinase (TK) were not markedly changed as a consequence of repeated treatment of KM12C tumours with 5-FU. The expression of TS mRNA was also amplified in accordance with the increased TS activity in a 5-FU-treated tumour sub-line (KM12C/5-FU) compared with that in parental tumours, but changed expressions of both RNR-R1 and RNA-R2 mRNA could not be detected in the 5-FU-resistant tumour sub-line compared with the parental tumours, suggesting possible post-transcriptional regulation of RNR. Moreover, RNR, in addition to TS and OPRT, seemed to be related to the inherent insensitivity to 5-FU in human cancer xenografts. From these results, it may be concluded that RNR activity is one of the acquired or inherent resistant factors, including TS, to 5-FU in human cancer xenografts in vivo.

Different mechanisms of acquired resistance to fluorinated pyrimidines in human colorectal cancer cells.

5-Fluorouracil (5-FU), 5-fluoro-2'-deoxyuridine (FdUrd) and 5-trifluorothymidine (F3(d)Thd) are antimetabolites which are metabolized to their corresponding active forms which inhibit DNA synthesis via inhibition of thymidylate synthase (TS). To investigate ways of overcoming 5-FU-resistance, we established acquired-resistant colorectal cancer cell lines against these three drugs by continuous and step-wise escalation of drugs, and analyzed the cytotoxicity and the mechanism of resistance to the drugs. When cells were incubated with the 3 drugs for 72 h, the resistance ratio to parental DLD-1 human colorectal tumor cells was 65.2 for DLD-1/5-FU, 9.7 for DLD-1/FdUrd and 448.6 for DLD-1/F3(d)Thd cells. DLD-1/5-FU cells did not show any cross-resistance against FdUrd and F(3)dThd. However, DLD-1/FdUrd cells showed 3- and 9-fold increased resistance to 5-FU and F3(d)Thd, respectively, and DLD-1/F3(d)Thd cells also showed about 90-fold resistance to FdUrd. Analysis of enzyme activities and gene expression associated with pyrimidine metabolism indicated that a significant decrease in orotate phosphoribosyltransferase activity in DLD-1/5-FU cells, a 7-fold increase of TS mRNA in DLD-1/FdUrd cells, and a 37-fold decrease in thymidine kinase activity of DLD-1/F3(d)Thd cells were the major mechanisms of drug resistance. These findings were closely associated with the cytotoxicity of 5-FU, FdUrd and F3(d)Thd against the established 5-FU-, FdUrd- or F3(d)Thd-resistant cells. When DLD-1/FdUrd cells expressing increased TS mRNA were treated with FdUrd and F3(d)Thd for only 4 h, the resistance ratios of DLD-1/FdUrd cells to parental DLD-1 cells were markedly different for FdUrd and F3(d)Thd, suggesting that the cytotoxicity with short-time exposure to F3(d)Thd is due to a mechanism other than TS inhibition, although the cytotoxicity of F3(d)Thd in the short-time is low compared to that of long-time exposure. In conclusion, F3(d)Thd, an antimetabolite that inhibits TS activity, may be effective against 5-FU and/or FdUrd-resistance in colorectal cancer cells caused by amplification of TS and/or deletion of orotate phosphoribosyltransferase.

Effect of administration of 5-(phenylselenenyl)acyclouridine, an inhibitor of uridine phosphorylase, on the anti-tumor efficacy of 5-fluoro-2′-deoxyuridine against murine colon tumor C26–10

The effect of co-administration of 5-(phenylselenenyl)acyclouridine (PSAU), a new uridine phosphorylase (UrdPase, EC 2.4.2.3) inhibitor, on the efficacy of 5-fluoro-2′-deoxyuridine (FdUrd) was tested against murine colon C26–10 tumor xenografts. In contrast to our previous results with human tumors, co-administration of PSAU with FdUrd decreased instead of increasing the efficacy of FdUrd against tumor growth. However, co-administration of PSAU with FdUrd (300 mg/kg/day) protected the mice completely from the 83% mortality induced by the same dose of FdUrd alone. Enzyme studies indicated that UrdPase in colon C26–10 tumors is responsible for the catabolism of FdUrd to 5-fluorouracil (FUra), as colon C26–10 tumors do not have thymidine phosphorylase (dThdPase, EC 2.4.2.4). In contrast, colon C26–10 tumors had extraordinarily high UrdPase activity (300 μmol/min/mg protein), which was at least 200-fold higher than the highest UrdPase activity in any of the human xenografts we tested previously. Furthermore, the activities of UrdPase and orotate phosphoribosyltransferase (OPRTase, EC 2.4.2.10) were 192- and 2-fold higher, respectively, while that of dihydrouracil dehydrogenase (EC 1.3.1.2) was 1000-fold lower in the tumor than in the host liver. It is suggested that FdUrd exerts its anticancer effects against colon C26–10 tumors mainly through the catabolism of FdUrd to FUra by UrdPase, which then could be anabolized to 5-fluorouridine 5′-monophosphate (FUMP) by OPRTase and ultimately to other toxic 5-fluorouridine nucleotides, hence inducing the observed FdUrd toxic effects. Co-administration of PSAU with FdUrd inhibited UrdPase and the catabolism of FdUrd to FUra. This would result in the observed reduction of the antitumor efficacy of FdUrd. In addition, the increase in plasma uridine concentration induced by PSAU as well as the catabolism of FUra by the high dihydrouracil dehydrogenase activity in the liver also may have circumvented any residual FUra toxic effects against the host. These results clearly demonstrate that the anticancer efficacy of the combination of UrdPase inhibitors and FdUrd is not general and is dependent largely on the type of tumor under treatment and the mode of FdUrd metabolism in these tumors.

Decreased fluorouracil cytotoxic effect on EB-virus transformed lymphocytes from hereditary orotic aciduria.

5-Fluorouracil (5-FU), used widely for malignancies, phosphorylate mostly by uracil phosphoribosyl transferase (UPRT). Patients with hereditary orotic aciduria lack the orotate phosphoribosyl transferase (OPRT) activity. In the cancer cells, the OPRT activity is paralleled with the UPRT activity. This study shows that the UPRT activity of the hereditary orotic aciduria homozygote decreased about 40% of normal controls. Moreover, we investigated the 5-FU cytotoxic effects on hereditary orotic aciduria (one homozygote, 4 heterozygotes and 7 normal controls), using EB-virus transformed lymphocytes (EB-LC). 5-FU was addded to the culture medium at concentrations ranging from 0 to 10.0 micromol/l. The 5-FU cytotoxic effects on the homozygote were milder than those on controls at each 5-FU concentration. The 5-FU cytotoxic effects in the heterozygotes were at intermediate levels between the homozygote and controls. We speculate that 5-FU cytotoxic effects, both anti-tumor effects and adverse reactions, would be weak when a patient with hereditary orotic aciduria was treated with 5-FU.

Purine and pyrimidine metabolism in cultured white spruce (Picea glauca) cells: Metabolic fate of 14C‐labeled precursors and activity of key enzymes

In order to examine the biosynthesis, interconversion, and degradation of purine and pyrimidine nucleotides in white spruce cells, radiolabeled adenine, adenosine, inosine, uracil, uridine, and orotic acid were supplied exogenously to the cells and the overall metabolism of these compounds was monitored. [8‐14C]adenine and [8‐14C]adenosine were metabolized to adenylates and part of the adenylates were converted to guanylates and incorporated into both adenine and guanine bases of nucleic acids. A small amount of [8‐14C]inosine was converted into nucleotides and incorporated into both adenine and guanine bases of nucleic acids. High adenosine kinase and adenine phosphoribosyltransferase activities in the extract suggested that adenosine and adenine were converted to AMP by these enzymes. No adenosine nucleosidase activity was detected. Inosine was apparently converted to AMP by inosine kinase and/or a non‐specific nucleoside phosphotransferase. The radioactivity of [8‐14C]adenosine, [8‐14C]adenine, and [8‐14C]inosine was also detected in ureide, especially allantoic acid, and CO2. Among these 3 precursors, the radioactivity from [8‐14C]inosine was predominantly incorporated into CO2. These results suggest the operation of a conventional degradation pathway. Both [2‐14C]uracil and [2‐14C]uridine were converted to uridine nucleotides and incorporated into uracil and cytosine bases of nucleic acids. The salvage enzymes, uridine kinase and uracil phosphoribosyltransferase, were detected in white spruce extracts. [6‐14C]orotic acid, an intermediate of the de novo pyrimidine biosynthesis, was efficiently converted into uridine nucleotides and also incorporated into uracil and cytosine bases of nucleic acids. High activity of orotate phosphoribosyltransferase was observed in the extracts. A large proportion of radioactivity from [2‐14C]uracil was recovered as CO2 and β‐ureidopropionate. Thus, a reductive pathway of uracil degradation is functional in these cells. Therefore, white spruce cells in culture demonstrate both the de novo and salvage pathways of purine and pyrimidine metabolism, as well as some degradation of the substrates into CO2.

Circumvention of 5-fluorouracil resistance in human stomach cancer cells by uracil phosphoribosyltransferase gene transduction.

A human stomach cancer cell line with acquired resistance to 5‐fluorouracil (5‐FU), NUGC‐3/5FU/L, has been found to possess reduced ability to convert 5‐FU into active metabolites. We attempted in vitro gene therapy for this 5‐FU‐resistant cell line. NUGC‐3 and NUGC‐3/5FU/L cells were infected with recombinant adenovirus (Ad) containing Escherichia coli uracil phosphoribosyltransferase (UPRT) gene driven by CAG promoter (CA), AdCA‐UPRT, and changes in their 5‐FU metabolism and sensitivity were investigated. Activities of orotate phosphoribosyltransferase increased from 10.2 and 1.56 (nmol/mg protein/30 min) in the uninfected cells of NUGC‐3 and NUGC‐3/5FU/L to 216 and 237, respectively, after the transfection of UPRT gene. The 5‐FU nucleotide level in the acid‐insoluble fraction increased from 7.32 to 15.9 (pmol/mg protein) in NUGC‐3 cells on infection with AdCA‐UPRT, and in NUGC‐3/5FU/L cells it increased from 1.91 to 21.4. The 50% growth‐inhibition concentration (IC50) was 12.7 μmol/liter for NUGC‐3 and much higher than 100 μmol/liter for NUGC‐3/5FU/L, indicating over 8‐fold resistance. NUGC‐3/5FU/L transfected with the UPRT gene showed very high sensitivity to 5‐FU with an IC50 of 3.2 μmol/liter. The high resistance in this metabolic activation‐deficient cell line was thus completely reversed by transduction of an exogenous gene coding for a 5‐FU‐anabolizing enzyme.

Effects of C-terminal deletion on the activity and thermostability of orotate phosphoribosyltransferase from Thermus thermophilus.

To investigate the role of the C-terminal region on the activity and thermostability of orotate phosphoribosyltransferase (OPRTase, EC 2. 4.2.10) from Thermus thermophilus, four C-terminal amino acid-deleted OPRTases (1, 2, 3, and 5 residues deleted) were constructed. The activities of all the mutant OPRTases were lower than that of wild-type OPRTase at all temperatures investigated (50-80 degreesC). V- and EV-OPRTase, mutants with Val and Glu-Val deletions, respectively, showed 63 to 75% of the activity of wild-type OPRTase at the temperatures investigated. EEV- and PLEEV-OPRTase, with Glu-Glu-Val and Pro-Leu-Glu-Glu-Val deletions, respectively, had activities of 22 to 35% of the wild-type. The Km values for orotate of all mutant OPRTases were more than 4-fold higher than that of the wild-type (25 microM). On the other hand, the Km for PRPP of the wild-type was 34 microM, and there were no significant differences between the wild-type and mutant OPRTases. The kcat values of the V- and EV-OPRTases were similar to that of the wild-type, but those of the EEV- and PLEEV-OPRTases were less than 50% that of the wild-type. The optimum temperature of all mutant OPRTases, 70 degreesC, was 10 degreesC lower than that of the wild-type. The remaining activities of wild-type and V-OPRTase after incubation at 90 degreesC for 20 min were 70 and 60% of the non-treated OPRTase activity, respectively. Although the remaining activity of EV-OPRTase was only 14% of the non-treated OPRTase activity, the addition of 200 mM KCl during heat treatment increased it to 70%. Circular dichroism spectroscopy revealed that V- and EV-OPRTase denature more easily than the wild-type OPRTase. The results suggest that the C-terminal valine and glutamic acid residues are important for the activity and thermostability of T. thermophilus OPRTase.

Interallelic complementation at the pyrF locus and the homodimeric nature of orotate phosphoribosyltransferase (OPRTase) in Mucor circinelloides.

Using 5-fluoroorotic acid (5-FOA) as a positive selection system we isolated mutants of Mucor circinelloides altered in the pyrimidine biosynthetic pathway. These mutants were found to be deficient either in orotidine-5'-monophosphate decarboxylase (OMPdecase), or in orotate phosphoribosyltransferase (OPRTase) activity. Complementation tests among mutants lacking OPRTase activity classified them into three groups, thus suggesting the possibility of interallelic complementation. To investigate this hypothesis a cDNA clone corresponding to the OPRTase-encoding gene of M. circinelloides was isolated by direct complementation of E. coli. The genomic copy transformed to prototrophy one member of each of the three classes of OPRTase-deficient mutants. We therefore concluded that they were all altered at the same locus, the pyrF locus. The corresponding alleles were cloned and sequenced. Comparisons of the amino acid sequence of M. circinelloides OPRTase with those of E. coli and S. typhimurium revealed a high degree of similarity in secondary and tertiary structure. As the two bacterial enzymes exist as dimers, a homodimeric quaternary structure of the M. circinelloides mature protein can be assumed. This would also explain the interallelic complementation between some pyrF mutants. The mutations found could affect either the active site or the structure of the dimer interface of the OPRTase.

Increased sensitivity to long-term 5-fluorouracil exposure of human colon cancer HT-29 cells resistant to short-term exposure.

A 5‐fluorouracil (5‐FU)‐resistant subline of human colon cancer HT‐29 cells was developed by repeated 1‐h exposure in vitro to 5‐FU. This subline (HT‐29/5‐FU/S) had 8‐fold resistance to 5‐FU in a 1‐h exposure assay. However, it had rather increased sensitivity to 5‐FU when assayed after a continuous 96‐h exposure to it. Significantly less 5‐fluorouridine‐5′‐triphosphate was produced in the resistant cells, leading to a lower level of 5‐FU incorporation into the cellular RNA. The reduced activity of orotate phosphoribosyltransferase might explain these results. In contrast, the HT‐29/5‐FU/S cells were more sensitive to the inhibition of in situ thymidylate synthase (TS) by 5‐FU than were the parent cells. The lower in situ TS activity may have made HT‐29/5‐FU/S cells more sensitive to TS inhibition by 5‐FU as compared with the parent cells. The fact that HT‐29/5‐FU/S was more resistant to short‐term 5‐FU exposure but more sensitive to long‐term exposure than the parent line confirmed the existence of different modes of action of 5‐FU, depending on the exposure time.