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)

The development of methotrexate (MTX) resistance in cultured cells results in increased levels of the drug's target enzyme dihydrofolate reductase (DHFR). Stepwise-selected MTX-resistant sublines originating from an MTX-sensitive human-mouse hybrid expressed elevated DHFR levels and human-DHFR specific gene sequence amplification. By high resolution two-dimensional polyacrylamide gradient electrophoresis, human DHFR was shown to be selectively overproduced in VB2a-100 MTX-resistant cells whereas mouse DHFR protein "spots" present in MTX-sensitive parental hybrid were absent in these cells exhibiting 100 microM MTX resistance. These findings and those in a parallel study indicate that concurrent with overproduction of human DHFR and amplification DHFR sequences in VB2a-100, a loss of mouse-specific DHFR gene sequences occurred.
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PMID:Selective overproduction of human dihydrofolate reductase in a methotrexate-resistant human-mouse somatic cell hybrid. 406 46

Dihydrofolate reductase is markedly hyperproduced in strains of Diplococcus pneumoniae which bear any one of a unique group of sense to sense mutations (ame(r)) in the corresponding structural gene. Increased enzyme levels mediated by the ame(r) mutations are apparently the result of increased rates of de novo synthesis. The basis for these effects could be transcriptional, translational or could involve an increase in messenger RNA stability. Data revealing no difference in stability of the related mRNA in a variety of mutants and the wild-type strain appear to eliminate the last possibility. Other data support the idea of an effect on transcription. This includes the extreme sensitivity of ame(r) mutation expression following genetic transformation, to inhibition by actinomycin D and rifampacin, and the presence in one extremely high level mutant (ame(r)-3 with 120 times the wild-type enzyme content) of increased amounts of mRNA. The data are most compatible with the idea of a regulatory function for the dihydrofolate reductase protein in this organism.
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PMID:Hyperproduction of dihydrofolate reductase in Diplococcus pneumoniae after mutation in the structural gene. Evidence for an effect at the level of transcription. 414 6

Dihydrofolate reductase (EC 1.5.1.3; 5,6,7,8-tetrahydrofolate:NADP(+) oxidoreductase) from antifolate-resistant Lactobacillus casei has been isolated in pure form and examined in solution by high resolution proton magnetic resonance spectroscopy. The 220 MHz proton magnetic resonance spectrum of this small enzyme (about 15,000 daltons) consists of several distinct resonance peaks that provide a sensitive nonperturbing probe of its conformational state. Comparison of catalytically active enzyme with preparations denatured in 6 M urea demonstrates dramatically the overall contribution of secondary and tertiary structure to its proton magnetic resonance spectra. More subtle differences existing among several catalytically active enzyme forms may also be readily differentiated by proton magnetic resonance spectroscopy, e.g., the spectra of the ligand-free enzyme and forms containing stoichiometric amounts of tightly bound folate and dihydrofolate, each obtained separately by affinity chromatography, are easily identified. Addition of ligands to these spectroscopically distinct forms may induce changes in their proton magnetic resonance spectra. For example, addition of equimolar dihydrofolate to the apoenzyme converts its relatively featureless aromatic proton magnetic resonance spectrum to one indistinguishable from that of the original enzyme-dihydrofolate binary complex obtained chromatographically. Interaction of the pyridine nucleotide coenzymes NADP(+) or NADPH or of the antifolate Methotrexate with apoenzyme induces additional distinct spectral changes. Enzyme-NADPH and enzyme-Methotrexate binary complexes, which have different aromatic region proton magnetic resonance spectra, are converted to ternary complexes having quite similar spectra by addition of Methotrexate and NADPH, respectively, thus suggesting that an ordered addition of ligands is not required.
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PMID:Conformational changes induced in dihydrofolate reductase by folates, pyridine nucleotide coenzymes, and methotrexate. 415 40

The protein component of the T-even bacteriophage coat which binds the phage-specific dihydropteroyl polyglutamate has been identified as the phage-induced dihydrofolate reductase. Dihydrofolate reductase activity has been found in highly purified preparations of T-even phage ghosts and phage substructures after partial denaturation. The highest specific enzymatic activity was found in purified tail plate preparations, and it was concluded that this enzyme was a structural component of the phage tail plate. Phage viability was directly correlated with the enzymological properties of the phage tail plate dihydrofolate reductase. All reactions catalyzed by this enzyme which changed the oxidation state of the phage dihydrofolate also inactivated the phage. Properties of two T4D dihydrofolate reductase-negative mutants, wh1 and wh11, have been examined. Various lines of evidence support the view that the product of the wh locus of the phage genome is normally incorporated into the phage tail structure. The effects of various dihydrofolate reductase inhibitors on phage assembly in in vitro complementation experiments with various extracts of conditional lethal T4D mutants have been examined. These inhibitors were found to specifically block complementation when added to extracts which did not contain preformed tail plates. If tail plates were present, inhibitors such as aminopterin, did not affect further phage assembly. This specific inhibition of tail plate formation in vitro confirms the analytical and genetic evidence that this phage-induced "early" enzyme is a component of the phage coat.
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PMID:Bacteriophage tail components. II. Dihydrofolate reductase in T4D bacteriophage. 439 96

1. Dihydrofolate reductase was purified from Lactobacillus casei MTX/R, and studied on affinity columns containing folic acid and methotrexate. Two forms of the enzyme were interconverted by incubation with substrates. 2. Affinity columns were prepared from agarose activated with cyanogen bromide and coupled with 1,6-diaminohexane. Stable folate derivatives were covalently attached by using a carbodi-imide condensation. 3. Columns containing folic acid retarded but did not retain the enzyme. 4. Methotrexate at pH 6.0 was particularly effective for retention of the enzyme. 5. There is selective loss of one form of the enzyme during affinity chromatography in the absence of added NADPH. This loss is due to conversion into a single enzyme form on the column. 6. NADPH has a dual effect in stabilizing the enzyme and in sensitizing it to inactivation by methotrexate, particularly in the presence of glycine. 7. Protein with affinity for methotrexate, but without dihydrofolate reductase activity, may also be eluted from the columns. 8. In a single-step procedure the enzyme was purified nearly 4000-fold from mammalian skin.
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PMID:Affinity chromatography of dihydrofolate reductase. 439 20

Dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) was purified from Escherichia coli strains that carried derivatives of the multicopy recombinant plasmid, pJFM8. The results of enzyme kinetic and two-dimensional gel electrophoresis experiments showed that the cloned enzyme is indistinguishable from the chromosomal enzyme. Therefore it can be concluded that these strains are ideal for use as a source of enzyme for further studies on the biochemistry and regulation of this important enzyme. The plasmid derivatives were constructed by recloning experiments that utilized several restriction endonucleases. From the analysis both of these plasmids and the purified dihydrofolate reductase enzymes it was possible to deduce the location and orientation of the dihydrofolate reductase structural gene on the parent plasmid, pJFM8.
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PMID:Characterization of the cloned Escherichia coli dihydrofolate reductase. 626 32

The Escherichia coli strain carrying pTP 6-10 which was constructed in our previous work (Iwakura, M., et al. (1983) J. Biochem. 93, 927-930) produces more than 400-fold dihydrofolate reductase as compared with the strain without the plasmid. Dihydrofolate reductase was highly purified from the cell-free extract of the plasmid strain simply by two steps; ammonium sulfate fractionation and ion-exchange chromatography. By 10-fold purification, the enzyme was essentially homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The restriction map of pTP 6-10 was also determined and the plasmid was shown to have an Ava I, an EcoR I, a Pst I, a Pvu I, and a Pvu II site. Our results indicate that the plasmid strain is suitable as a source of the enzyme and that plasmid pTP 6-10 is promising as a versatile plasmid vector for efficiently yielding the product of the cloned gene.
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PMID:Purification of dihydrofolate reductase amplified in Escherichia coli K-12. 631 90

Dihydrofolate reductase specified by plasmid R483 from a trimethoprim-resistant strain of Escherichia coli has been purified 2,000-fold to homogeneity using dye-ligand chromatography, gel filtration, and polyacrylamide gel electrophoresis. The protein migrated as a single band on nondenaturing polyacrylamide gel electrophoresis and had a specific activity of 250 mumol/mg min(-1). The molecular weight was estimated to be 32,000 by gel filtration and 39,000 by Ferguson analysis of polyacrylamide gel electrophoresis. When subjected to electrophoresis in the presence of sodium dodecyl sulfate, the protein migrated as a single 19,000-molecular weight species, a fact that suggests that the native enzyme is a dimer of similar or identical subunits. Antibody specific for R483-encoded dihydrofolate reductase did not cross-react with dihydrofolate reductase encoded by plasmid R67, T4 phage, E. coli RT500, or mouse L1210 leukemia cells. The amino acid sequence of the first 34 NH2-terminal residues suggests that the R483 plasmid dihydrofolate reductase is more closely related to the chromosomal dihydrofolate reductase than is the enzyme coded by plasmid R67.
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PMID:R plasmid dihydrofolate reductase with a dimeric subunit structure. 635 Feb 98

Dihydrofolate reductase has been purified from a trimethoprim-resistant strain of Neisseria gonorrhoeae. The enzyme showed a single component on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 18,000) and on isoelectric focusing in 5 M urea (pI = 6.8). Although gel electrophoresis under nondenaturing conditions resolved the preparation into two enzymatically active proteins (called form 1 and form 2), they were not genetically determined isozymes. Both had a similar dihydrofolate Km (2 microM), NADPH Km (10 microM), and trimethoprim Ki (20 nM), and form 2 (the slower migrating species) was shown to be generated from form 1 by the electrophoresis conditions. The complete covalent structure of the enzyme has also been determined. It is a single polypeptide composed of 162 residues and containing 4 cysteines. The gonococcal dihydrofolate reductase shares a 35% homology with the chicken liver enzyme and a 40% homology with the Escherichia coli enzyme. Most of these identities are residues that have been implicated in the binding of NADPH and methotrexate to the E. coli and Lactobacillus casei reductases.
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PMID:Characterization and amino acid sequence of Neisseria gonorrhoeae dihydrofolate reductase. 643 41

Plasmid DNA containing the human beta-interferon (IFN-beta) gene and mouse dihydrofolate reductase cDNA was transfected into dihydrofolate reductase-negative Chinese hamster ovary cells. Dihydrofolate reductase-positive transformants were obtained, and cells containing amplified copies of mouse dihydrofolate reductase were selected by exposure to increasing methotrexate concentrations. These cells were found to express high levels of human IFN-beta after polyriboinosinic acid-polyribocytidylic acid superinduction or NDV infection; this was a result of coamplification of the IFN-beta gene. Levels of expression of 1 U/cell per day were achieved on superinduction, giving corresponding titers of up to 10(10) U/liter medium in culture supernatants. Constitutive production of IFN-beta rates of about 0.5% of superinduced rates was observed; cells producing these levels of IFN-beta had acquired resistance to cytotoxic antiviral effects of IFN-beta. Two forms of human IFN-beta were produced; a major glycosylated 23,000-dalton form and an unglycosylated 18,500-dalton form. The latter had greatly reduced antiviral activity. IFN-beta production was very sensitive to cellular growth rate; the highest levels were produced by density-arrested cultures. Regulation of IFN-beta production by polyriboinosinic acid-polyribocytidylic acid or by cell density effects required the presence of DNA sequences 5' to the IFN-beta-coding sequences; replacement of these sequences with the simian virus 40 early promoter resulted in uninducible, density-independent production of IFN-beta.
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PMID:Inducible expression of amplified human beta interferon genes in CHO cells. 670 May 82


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