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)

A method for the high-voltage electrophoresis of dihydrofolate reductase from Escherichia coli W 3110 is described. Dihydrofolate reductase catalyses the reduction of dihydrofolic acid to tetrahydrofolic acid. By addition of a tetrazolium salt, tetrahydrofolic acid reacts by formation of a violet water insoluble formazane which is an indicator for the enzyme. Besides several unspecific bands, two isoenzymes of the dihydrofolate reductase from Escherichia coli W 3110 are found which are specificially inhibited by the folate antagonists methotrexate and trimethoprime in a concentration of 0, 1muM, 1 muM respectively.
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PMID:[High-voltage electrophoresis of dihydrofolate reductase from Escherichia coli W 3110 (author's transl)]. 32 Jun 38

Porcine liver dihydrofolate reductase has been purified 18,000-fold to homogeneity. The properties of the purified enzyme were compared to those of dihydrofolate reductase from L1210 cells, the only mammalian reductase for which complete amino acid sequence data are available. The enzymes are very similar when compared on the basis of mechanism and kinetic constants, molecular weights, isoelectric points, and stimulation by salt. A comparison of the amino acid sequences of both enzymes shows an overall identity of 89%. Thus, the similarities seen in inhibitor-binding profiles of mammalian enzymes reflect the close relationship of these enzymes at the molecular level.
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PMID:Porcine liver dihydrofolate reductase. Purification, properties, and amino acid sequence. 50 Jun 53

The crystal structure of the methotrexate-gamma-tetrazole (MTXT)-NADPH ternary complex with recombinant human dihydrofolate reductase (DHFR) has been determined and refined to R = 15.9% for 7003 data from 10.0 to 2.3 A resolution for the R3 lattice. Interpretation of difference Fourier electron density maps revealed that the cofactor NADPH is bound in an extended conformation, and the closest contact between cofactor and inhibitor is 3.1 A, between N(5) of the MTXT pteridine ring and the nicotinamide C(4) which transfers a hydride during the enzyme-catalyzed reaction. As in other DHFR complexes, MTXT is interpreted as protonated at N(1) by Glu-30, and the 2-amino group is hydrogen bonded to a structurally conserved water which also interacts with Glu-30 and Thr-136. The 4-amino group of MTXT hydrogen bonds to the carbonyl of Ile-7 and the phenolic hydroxyl of Tyr-121, and the alpha-carboxylate forms a salt bridge with the conserved Arg-70. In this structure, the amide carbonyl forms two hydrogen bonds with Asn-64 and a water molecule, whereas the gamma-tetrazole ring does not interact directly with the enzyme. The largest changes in the secondary structure on formation of the ternary complex involve the fold of a flexible loop near residues 40-46, and to a lesser extent the helical region near residues 102-109 and the beta-sheet regions near residues 71-75 and 157-159.
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PMID:Crystal structure determination at 2.3 A of recombinant human dihydrofolate reductase ternary complex with NADPH and methotrexate-gamma-tetrazole. 128 40

Two forms of a 6-methyladenine mRNA methyltransferase have been partially purified using a T7 transcript coding for mouse dihydrofolate reductase as an RNA substrate. Both enzyme forms modify internal adenine residues within the RNA substrate. The enzymes were purified 357- and 37-fold respectively from nuclear salt extracts prepared from HeLa cells using DEAE-cellulose and phosphocellulose chromatography. The activity of the first form of the enzyme eluted from DEAE-cellulose (major form) was at least 3-fold greater than that of the second (minor form). H.p.l.c. analysis of the hydrolysed, methylated mRNA substrates demonstrated that both forms of the enzyme produced only 6-methyladenine. The two forms of the enzyme differed in their RNA substrate specificity as well as in the dependence for a 5' cap structure. The 6-methyladenine mRNA methyltransferase activity was found to be elevated in HeLa nuclei as compared with nuclear extracts from rat kidney and brain. Enzymic activity could not be detected in nuclei from either normal rat liver or regenerating rat liver. In the case of the HeLa cell, activity could only be detected in nuclear extracts, with a small amount in the ribosomal fraction. Other HeLa subcellular fractions were void of activity.
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PMID:Partial purification of a 6-methyladenine mRNA methyltransferase which modifies internal adenine residues. 144 68

Dihydrofolate reductase (DHFR, EC 1.5.1.3) is an enzyme involved in the metabolism of nucleic acids; it is also an important target for folate antagonists such as methotrexate (MTX). The distribution and expression of DHFR both in human HeLa BU-25 cell line and in methotrexate-resistant (MTX-R) variant, deriving from the human VA2-B cell line (having the DHFR gene amplified) was studied by tetrazolium salt method and by flow cytometric analysis. The immunohistochemical labelling of DHFR was achieved by using the streptavidinbiotinilated complex technique. DHFR activity was low in the human HeLa BU-25 cell line, while it was very high in the MTX-R cell line; the activity level increased with the increasing concentration of the MTX. The results obtained with cytochemical and immunohistochemical technique were compared. These findings showed that the hyperproduction of DHFR is strictly related with the cells having the DHFR gene amplified. Since MTX resistance is a common finding in the cells of patients with acute leukaemia, these studies may be extended to tumour-bearing patients at onset and following chemotherapy with methotrexate.
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PMID:Immunohistochemistry of dihydrofolate reductase in methotrexate-sensitive and -resistant human cell lines by flow cytometry: a comparison with the cytochemical tetrazolium salt method. 152 71

Methylbenzoprim (MBP) is a potent inhibitor of dihydrofolate reductase, which is more selective for mammalian than bacterial enzymes. Crystal-structure studies on the free base of MBP, with two independent molecules, and the ethanesulfonate salt, have demonstrated three significantly different conformations for MBP. With the MOPAC optimized MBP cation as starting point, the COSMIC energy was monitored as torsion angles were changed in 5 degrees increments. The barrier to rotation about C(5)-C(11) can create two slowly interconverting rotamers, in agreement with NMR studies. Two conformations of the cation that fit the human DHFR structure from the Brookhaven Protein Data Bank have been found. They differ chiefly by a half-turn about C(5)-C(11), positioning the nitro group on opposite sides but allowing the central and benzylic rings to find hydrophobic surroundings. The central ring is close enough to the predicted position of the cofactor NADPH to make competition likely. Kinetic studies with rat liver DHFR show that MBP is an inhibitor that competes with NADPH as well as dihydrofolate.
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PMID:Structural studies on bio-active compounds. 20. Molecular modeling and crystallographic studies on methylbenzoprim, a potent inhibitor of dihydrofolate reductase. 161 54

The amino acid sequence of mouse dihydrofolate reductase was permuted circularly at the level of the gene. By transposing the 3'-terminal half of the coding sequence to its 5' terminus, the naturally adjacent amino and carboxyl termini of the native protein were fused, and one of the flexible peptide loops at the protein surface was cleaved. The steady-state kinetic constants, the dissociation constants of folate analogues, and the degree of activation by both mercurials and salt as well as the resistance toward digestion by trypsin were almost indistinguishable from those of a recombinant wild-type protein. Judged by these criteria, the circularly permuted variant has the same active site and overall structure as the wild-type enzyme. The only significant difference was the lower stability toward guanidinium chloride and the lower solubility of the circularly permuted variant. This behavior may be due to moving a mononucleotide binding fold from the interior of the sequence to the carboxyl terminus. Thus, dihydrofolate reductase requires neither the natural termini nor the cleaved loop for stability, for the conformational changes that accompany catalysis as well as the binding of inhibitors, and for the folding process.
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PMID:A fully active variant of dihydrofolate reductase with a circularly permuted sequence. 173 18

We describe the characterization of human lymphoblastoid cell lines with acquired resistance (greater than 20,000-fold) to a novel folate-based thymidylate synthase (TS) (EC 2.1.1.45) inhibitor, C2-desamino-C2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI198583). This acquired resistance was associated with a 64-fold amplification of the TS gene, a similar elevation in the corresponding mRNA, and an approximately 200-fold increase in both TS activity and TS protein. This amplification was maintained when the cells were grown in the absence of the selective agent, ICI198583, for 340 generations. TS isolated from one of the resistant cell lines, W1-L2:C1, displayed inhibition kinetic parameters similar to those of TS isolated from the parent W1-L2 cell line. It thus appears unlikely that resistance is due to an altered TS enzyme having a lower affinity for ICI198583. The resistant cell line, W1-L2:C1, was cross-resistant to other folate-based TS inhibitors but was as sensitive as the parent cell line, W1-L2, to 5-fluorodeoxyuridine. The W1-L2:C1 cell line was collaterally sensitive to the classical dihydrofolate reductase (EC 1.5.1.3) inhibitor methotrexate as well as to the lipophilic dihydrofolate reductase inhibitors metoprine and 2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyl]quinazolin e glucuronic acid salt (also called trimetrexate). When the W1-L2 and W1-L2:C1 cell lines were exposed to 1 microM ICI198583 for 24 h they accumulated the same concentration of total cellular ICI198583 polyglutamates despite the fact that the latter cell line accumulated a 300-fold greater concentration of ICI198583 monoglutamate. As polyglutamates, the tetra- and pentaglutamate forms predominated in the W1-L2 cell line, whereas the diglutamate form predominated in the W1-L2:C1 cell line, with few higher polyglutamates being detected. The lack of tri- and higher polyglutamates of ICI198583 (i.e., the more active species) in the W1-L2:C1 cell line may also contribute to the observed resistance. These findings may have important implications in light of the rapid onset of resistance to antifolates in the clinic.
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PMID:Human lymphoblastoid cells with acquired resistance to C2-desamino-C2-methyl-N10-propargyl-5,8-dideazafolic acid: a novel folate-based thymidylate synthase inhibitor. 173 72

Dihydrofolate reductase (DHFR, EC 1.5.1.3) is an important enzyme involved in DNA metabolism. In this connection the cell cycle modulation of DHFR levels in HeLa S3 and HL 60 cell lines was investigated by flow cytometric analysis. A concentration of 4 micrograms/ml of aphidicolin was employed to synchronize the cell lines. DHFR was cytochemically detected by using tetrazolium salt and immunofluorescence techniques; DNA content was evaluated by means of propidium iodide staining. At 0, 2, 4, 6, 8, 10, 12 hrs. after the removal of the drug we observed a low DHFR level in G0-G1 phase, followed by an increase during late S and G2/M phases. The variations of this enzyme may represent, under well defined conditions, a marker of cycling cells.
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PMID:Modulation of cytochemically detected dihydrofolate reductase during cell cycle. 180 92

A sequence of chromatographic procedures is described for the isolation of three consecutive enzymes of the folate pathway in Escherichia coli: hydroxymethyldihydropteridine pyrophosphokinase (E.C. 2.7.6.3) (I), 7,8-dihydropteroate synthase (E.C. 2.5.1.15) (II) and 7,8-dihydrofolate reductase, (E.C. 1.5.1.3) (III). Starting with the crude extract, ion-exchange chromatography on a DEAE-Sepharose CL-6B column with a salt gradient completely separated I, II and III. I and II were further purified by hydrophobic-interaction chromatography on Phenyl-Sepharose CL-4B, followed by size-exclusion chromatography on Ultrogel AcA 54. For III only size-exclusion chromatography was used. The overall enrichment factors, on the basis of protein, were 13,700-fold for I, 280-fold for II and 500-fold for III. Bacterial batches of more than 500 g were handled.
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PMID:Improved methods for the purification of enzymes of the folate pathway in Escherichia coli. I. Chromatographic methods. 196 13


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