Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.5.1.3 (dihydrofolate reductase)
5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

High speed centrifugal supernatant fractions of homogenates of a number of trypanosomatids were assayed for thymidylate synthase (5,10-methylene-tetrahydrofolate: dUMP C-methyltransferase, EC 2.1.1.45) activity using the method of Lomax and Greenberg (1967) J. Biol. Chem. 242, 109-113). Similar activities were detected in Crithidia fasciculata, Crithidia oncopelti, the blood forms of Trypanosoma brucei, Trypansoma congolense and Trypanosoma lewisi and the blood, intracellular and culture forms of Trypanosoma cruzi, suggesting that all species synthesize at least some thymidylate de novo. The properties of the activities in C. fasciculata and the three forms of T. cruzi were compared with those of the isofunctional bacterial and mammalian enzymes. The trypanosotamid enzyme was inhibited by Mg2+, was much more sensitive to mercaptoethanol, had higher apparent Km values for substrate (dUMP) and cofactor (tetrahydrofolate), had a higher apparent molecular weight and was markedly more sensitive to inhibition by suramin. It is, therefore a possible target for chemotherapeutic attack, either on its own or in combination with a dihydrofolate reductase inhibitor. No evidence was obtained for the regulation of the trypanosomatid enzyme, either by its product, dTMP, or by dTDP or dTTp. This result agrees with previous studies which suggested that in trypanosomatids, the level of dTMP was regulated, at least in part, by a catabolic pathway consisting of a thymidylate phosphatase and a thymidine phosphorylase which degraded the excess of dTMP to thymine.
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PMID:Presence and properties of thymidylate synthase in trypanosomatids. 1 96

A DIRECT APPROACH IS DESCRIBED TO THE QUESTION: Are enzymes of DNA precursor synthesis organized into a supramolecular structure? This approach involved sedimentation analysis of several T4 phage-coded early enzyme activities in crude lysates of infected Escherichia coli. One-third to one-half of several activities tested-dCMP hydroxymethylase, dTMP synthetase, deoxynucleoside 5'-monophosphate kinase, deoxyuridine triphosphatase, and probably dCMP deaminase, but not dihydrofolate reductase or DNA polymerase-sedimented much more rapidly than expected from molecular weight. About 5% of the host cell nucleoside diphosphate kinase, known to participate in T4 DNA precursor synthesis, cosedimented with these activities. To show that this rapidly sedimenting material represents an organized enzyme complex rather than a nonspecific aggregate, we studied the kinetics of formation of dTTP with dUMP as the initial substrate. This three-step reaction sequence reached its maximal rate within a few seconds when catalyzed by enzymes in the aggregate, whereas an equivalent mixture of uncomplexed enzymes required nearly 20 min before dTTP synthesis reached its maximal rate. The effect of aggregation is evidently to decrease the volume into which intermediates are free to diffuse. Because there is reason to believe that intracellular concentration gradients of DNA precursors exist, the properties of this enzyme aggregate in vitro may help to explain how such gradients are maintained.
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PMID:Enzyme associations in T4 phage DNA precursor synthesis. 19 73

We have constructed a dihydrofolate reductase mutant (dfr1) of Saccharomyces cerevisiae. The mutant has auxotrophic growth requirements for the C1 metabolites dTMP, adenine, histidine and methionine, similar to those of wild-type (wt) strains grown in the presence of methotrexate (MTX). However, unlike wt strains treated with MTX, the growth requirements of the dfr1 mutant are not satisfied by exogenous 5-formyltetrahydrofolic acid (FA; folinic acid) in complex (YEPD) medium. This result is surprising, as yeast cells treated with MTX are expected to be phenocopies of dfr1 mutants. The inability of the mutants to metabolize FA suggests that the DFR1 gene product may have a role in folate metabolism in addition to its well-characterized function in the reduction of dihydrofolate. From dfr1 strains, we have isolated secondary mutants whose growth can be supported by FA in YEPD medium. This FA-utilizing phenotype is attributable to recessive mutations which we have designated fou. In addition to their inability to metabolize FA, the dfr1 strains are unable to grow on medium containing the non-fermentable carbon source glycerol, suggesting that the DFR1 gene product is also required for mitochondrial function. In order to overcome this lack of respiratory activity in the dfr1 mutants, we isolated strains containing a dominant mutation, DIR, which allows growth on glycerol in the presence of antifolate drugs. When crossed into dfr1 strains, the DIR mutation conferred respiratory competence. These strains should be useful in a variety of studies on the genetics and biochemistry of folate metabolism in this simple eukaryote.
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PMID:The phenotype of a dihydrofolate reductase mutant of Saccharomyces cerevisiae. 142 91

1-Acyl- and 1,2-diacyl-1,2,4-triazolidine-3,5-diones were found to be potent cytotoxic agents in murine and human cancer cell lines, e.g. L1210, P388, Tmolt3, colon adenocarcinoma, Hela cells and glioma. In vivo activity was demonstrated at 8 mg/kg/day against Ehrlich ascites carcinoma growth. In L1210 cells, 1-acetyl-4-phenyl-1,2,4-triazolidine-3,5-dione, 41, reduced DNA synthesis significantly with moderate reduction in RNA synthesis. Enzyme sites in L1210 cells which were markedly affected were m- and r-RNA polymerase, PRPP amidotransferase, IMP dehydrogenase, dihydrofolate reductase, thymidine, TMP and TDP kinases. Kinetic studies suggest the inhibition of rate limiting enzymes in the purine pathway by 41 was responsible for its cytotoxicity. Acute toxicity studies in mice indicated 41 was safe for therapeutic use at 20, 50, and 100 mg/ky/day.
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PMID:Antineoplastic activities and cytotoxicity of 1-acyl and 1,2-diacyl-1,2,4-triazolidine-3,5-diones in murine and human tissue culture cells. 144 91

MTX cytotoxicity is not fully explained by its well-known inhibition of dihydrofolate reductase activity which leads to a decrease in the dTMP synthase reaction, since TdR kinase which converts TdR to dTMP could readily circumvent MTX action through this salvage activity. TdR kinase is of particular significance, since in various types of carcinoma cells its activity is orders of magnitude higher than that of dTMP synthase. To throw light on this problem, we tested the hypothesis that the impact of MTX treatment might in fact involve an inhibition or decrease in TdR kinase activity. Injection in rat of MTX (i.p.) decreased TdR kinase activity in a time- and dose-dependent fashion in liver (t1/2 = 46 h; IC50 = 95 mg/kg), bone marrow (t1/2 = 10 h; IC50 = 5 mg/kg) and rapidly growing transplantable hepatoma 3924A (t1/2 = 56 h; IC50 = 5 mg/kg). Injection in rat of cycloheximide (15 mg/kg, i.p.), an inhibitor of protein biosynthesis, rapidly decreased TdR kinase activity in the hepatoma (t1/2 = 3.6 h); activities of other purine and pyrimidine synthetic enzymes, dTMP synthase, IMP dehydrogenase, GMP reductase and GMP synthase, declined at a markedly slower rate (t1/2 = 11, 11.6, 12 and 22 h, respectively). MTX, by curtailing purine and pyrimidine biosynthesis, limits product of TdR kinase which is more sensitive to unopposed protein degradation than other enzymes of nucleic acid biosynthesis. TdR kinase is a newly discovered target of MTX treatment.
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PMID:Methotrexate decreases thymidine kinase activity. 152 Mar 43

Since eucaryotic cell-derived thymidine or thymidine nucleotides are not incorporated into Chlamydia trachomatis DNA, we hypothesized that C. trachomatis must obtain dTTP for DNA synthesis by converting dUMP to dTMP. In most cells, this reaction is catalyzed by thymidylate synthase (TS) and requires 5,10-methylenetetrahydrofolate as a cofactor. We used C. trachomatis serovar L2 and a mutant CHO K1 cell line with a genetic deficiency in folate metabolism as a host for chlamydial growth. This cell line lacks a functional dihydrofolate reductase (DHFR) gene and, as a result, is unable to carry out de novo synthesis of dTTP. C. trachomatis inclusions form normally when DHFR- cells are starved for thymidine 24 h prior to and during the course of infection. When [6-3H]uridine is used as a precursor to label C. trachomatis-infected CHO DHFR- cells, radiolabel is readily incorporated into chlamydia-specific DNA. When DNA from [6-3H]uridine-labelled infected cultures is acid hydrolyzed and subjected to high-performance liquid chromatography analysis, radiolabel is detected in thymine and cytosine nucleobases. By using the DHFR- cell line as a host and [5-3H]uridine as a precursor, we could monitor intracellular C. trachomatis TS activity simply by following the formation of tritiated water. There is a good correlation between in situ TS activity and DNA synthesis activity during the chlamydial growth cycle. In addition, both C. trachomatis-specific DNA synthesis and 3H2O release are inhibited by exogenously added 5-fluorouridine but not by 5-fluorodeoxyuridine. Finally, we demonstrated in vitro TS activity in crude extracts prepared from highly purified C. trachomatis reticulate bodies. The activity is dependent on the presence of methylenetetrahydrofolic acid and can be inhibited with 5-fluoro-dUMP. Taken together, these results indicate that C. trachomatis contains a TS for the synthesis of dTMP.
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PMID:Biochemical evidence for the existence of thymidylate synthase in the obligate intracellular parasite Chlamydia trachomatis. 193 73

(Deoxy)thymidylate (dTMP) kinase is an enzyme which phosphorylates dTMP to dTDP in the presence of ATP and magnesium. This enzyme is important in cellular DNA synthesis because the synthesis of dTTP, either via the de novo pathway or through the exogenous supply of thymidine, requires the activity of this enzyme. It has been suggested that the activities of the enzymes involved in DNA precursor biosynthesis, such as thymidine kinase, thymidylate synthase, thymidylate kinase, and dihydrofolate reductase, are subjected to cell cycle regulation. Here we describe the cloning of a human dTMP kinase cDNA by functional complementation of a yeast dTMP kinase temperature-sensitive mutant at the non-permissive temperature. The nucleotide sequence of the cloned human cDNA is predicted to encode a 24 KD protein that shows considerable homology with the yeast and vaccinia virus dTMP kinase enzymes. The human enzyme activity has been investigated by expressing it in yeast. In this work, we demonstrate that the cloned human cDNA, when expressed in yeast, produces dTMP kinase activity.
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PMID:Molecular cloning and expression of the human deoxythymidylate kinase gene in yeast. 201 65

The pharmacologic responses of normal and malignant epidermal cells to chemotherapeutic agents were examined in a system which consists of a serum-free "defined" medium. The effects of methotrexate (MTX), fluorodeoxyuridine (FUDR), and hydroxyurea upon cell growth, DNA synthesis, thymidylate synthase activity, and dihydrofolate reductase (DHFR) activity were compared in normal human epidermal keratinocytes (NHEK), newborn epidermal cells (NEC), human squamous cell carcinoma (SCC-25), and a methotrexate-resistant human squamous cell carcinoma (SCC-15R). Normal keratinocytes were found to be 10(3) to 10(4) times less sensitive to the effects of MTX and FUDR than the malignant cells with respect to growth and DNA synthesis. The differential sensitivity to MTX and FUDR was not due to differences in growth media, increased target enzyme activity, e.g., DHFR and thymidylate synthase, or impaired transport of these drugs. The results indicate that the mechanism for the increased sensitivity of the squamous cell carcinoma to MTX and FUDR must involve a process which is distal to the de novo synthesis of dTMP.
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PMID:A comparison of the effects of antitumor agents upon normal human epidermal keratinocytes and human squamous cell carcinoma. 213 84

Trimethoprim-resistance genes of Shigella dysenteriae 1 strains, isolated from a different location of six different countries of Asia over a 5-year period were characterized by using three different dihydrofolate reductase (DHFR) gene probes. The trimethoprim-resistant (TMPR) strains hybridized only with the type I DHFR gene probe by colony hybridization. None of the strains hybridized with types II and III DHFR gene probes. Southern blot experiments using plasmid DNA extracted from these resistant strains indicated that the type I DHFR genes were either on a 20 MDa plasmid or might be located on the chromosome. None of the other plasmids present in S. dysenteriae 1 strains hybridized with the probe. This indicates that the TMP resistance in these S. dysenteriae 1 strains are mediated by type I DHFR enzyme, and there may be transposition of this type I DHFR gene occurs between the 20 MDa plasmid and the chromosome in this serotype of shigella.
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PMID:Trimethoprim resistance gene in Shigella dysenteriae 1 isolates obtained from widely scattered locations of Asia. 218 27

Metabolic inhibitors which act in the process of pyrimidine salvage influenced on the uracil incorporation into nucleic acids of Toxoplasma. Inhibitors of dihydrofolate reductase, pyrimethamine and methotrexate, and inhibitors of thymidylate synthase, fluoro-uridine, fluoro-dUMP and fluoro-uracil, diminished isotopic uracil uptake in dose-dependent manners. Azauridine which suppresses de novo pyrimidine biosynthesis did not affect the salvage even in a relatively high dose. These results suggested that the activation of uracil salvage should be closely related with the function of TMP biosynthetic enzymes. The pattern of thymidine uptake had no differences between control HL-60 cells and Toxoplasma infected cells, which did not reflect the specific proliferation of Toxoplasma. It can be exploited to characterize the effects of various compounds related with the proliferation of Toxoplasma, especially its DNA synthesis.
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PMID:Effects of pyrimidine salvage inhibitors on uracil incorporation of Toxoplasma gondii. 227 4


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