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)

Dihydrofolate reductase has been purified 40-fold to apparent homogeneity from a trimethoprim-resistant strain of Escherichia coli (RT 500) using a procedure that includes methotrexate affinity column chromatography. Determinations of the molecular weight of the enzyme based on its amino acid composition, sedimentation velocity, and sodium dodecyl sulfate gel electrophoresis gave values of 17680, 17470 and 18300, respectively. An aggregated form of the enzyme with a low specific activity can be separated from the monomer by gel filtration; treatment of the aggregate with mercaptoethanol or dithiothreitol results in an increase in enzymic activity and a regeneration of the monomer. Also, multiple molecular forms of the monomer have been detected by polyacrylamide gel electrophoresis. The unresolved enzyme exhibits two pH optima (pH 4.5 and pH 7.0) with dihydrofolate as a substrate. Highest activities are observed in buffers containing large organic cations. In 100 mM imidazolium chloride (pH 7), the specific activity is 47 mumol of dihydrofolate reduced per min per mg at 30 degrees. Folic acid also serves as a substrate with a single pH optimum of pH 4.5. At this pH the Km for folate is 16 muM, and the Vmax is 1/1000 of the rate observed with dihydrofolate as the substrate. Monovalent cations (Na+, K+, Rb+, and Cs+) inhibit dihydrofolate reductase; at a given ionic strength the degree of inhibition is a function of the ionic radius of the cation. Divalent cations are more potent inhibitors; the I50 of BaCl2 is 250 muM, as compared to 125 mM for KCl. Anions neither inhibit nor activate the enzyme.
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PMID:Purification and properties of Escherichia coli dihydrofolate reductase. 0 46

Dihydrofolate reductase plays a dual role in bacteriophage T4, first, as an enzyme of thymidylate metabolism, and second, as a protein component of the tail baseplate. Antibody to the purified enzyme has been used to study its synthesis and intracellular turnover. The antibody specifically precipitates one protein from T4D-infected cell extracts. This has been identified as dihydrofolate reductase, although the polypeptide molecular weight (22,000) is lower than that earlier determined for this enzyme. The protein comigrates on gels with pY, a genetically undefined protein component of the baseplate. However, it is not pY, for pY is synthesized late in infection, whereas virtually no dihydrofolate reductase synthesis occurs later than 10 min after infection at 37 degrees C. Dihydrofolate reductase, once formed, is neither degraded nor converted to proteins of higher or lower molecular weight. Thus, it is probably incorporated into virions at the same molecular weight as that of the soluble enzyme. 125I-radiolabeled antibody binds to the wedge substructure of the baseplate, and this binding is blocked by preincubation with purified T4 dihydrofolate reductase. Thus, the enzyme protein seems to be a component of the wedge.
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PMID:Bacteriophage T4-coded dihydrofolate reductase: synthesis, turnover, and location of the virion protein. 11 11

Dihydrofolate reductase from the wild type and aminopterin-resistant mutants of Diplococcus pneumoniae has been compared. Specific activity, optimum pH, Km, thermal stability, and inhibition by aminopterin are identical for both strains. Aminopterin resistance for such mutants is, therefore, not due to an alteration of the dihydrofolate reductase.
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PMID:Dihydrofolate reductases from the wild type and aminopterin-resistant mutants of Diplococcus pneumoniae. 23 45

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

Dihydrofolate reductase, specified by the type II plasmid of a trimethoprim-resistant Escherichia coli, was purified 40-fold to homogeneity using a combination of gel filtration, DEAE-Sephacel chromatography, and hydrophobic chromatography. The final product shows a single protein band on polyacrylamide gel electrophoresis and has a specific activity of 1.0 unit/mg. The molecular weight of the purified enzyme is 36,000 as determined both by gel filtration and Ferguson analysis of polyacrylamide gel electrophoresis. In contrast, a single polypeptide with a molecular weight of 8,500 was observed on sodium dodecyl sulfate-gel electrophoresis. These experiments suggest that, unlike any bacteria or vertebrate dihydrofolate reductase previously examined, the type II R plasmid reductase is a tetramer composed of four identical subunits. A partial amino acid sequence determination shows no heterogeneity of the subunits and also no clear homology with any reductase sequence previously reported.
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PMID:R plasmid dihydrofolate reductase with subunit structure. 37 28

Dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) from an amethopterin-resistant strain of Lactobacillus casei was inactivated by 2,4-pentanedione. The inactivation appears to be due to the specific interaction of 2,4-pentanedione with lysyl residues. Inactivation is concomitant with with the modification of three lysyl residues. Both NADPH and dihydrofolate protect the enzyme against inactivation, suggesting that the critical residue(s) lies at or near their binding sites. Unlike native dihydrofolate reductase, 2,4-pentanedione-modified enzyme does not form binary complexes with either NADPH, dihydrofolate or amethopterin which are stable to gel filtration. Treatment of the modified enzyme with nucleophilic reagents such as hydroxylamine, failed to promote reactivation of the enzyme. Reactivation was achieved following gel filtration at pH 6.0 and was found to be dependent on the degree to which the enzyme was inactivated.
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PMID:Modification of lysyl residues of dihydrofolate reductase with 2,4-pentanedione. 48 85

Dihydrofolate reductase from Escherichia coli exists as two species, which show large differences in their affinities for trimethoprim and for pyrimethamine. The two species are present in approximately equal proportions. Each possesses one binding site per mol with dissociation constants (KD) of 14 and 1400 nM, respectively, for the binding of trimethoprim in the binary complex, and of 5 and 47 nM for the pyrimethamine binary complex. In the formation of the ternary complex with NADPH, trimethoprim bound to dihydrofolate reductase as if all the enzyme existed as a single species with a KD for trimethoprim of 1.9 nM. Formation of the trimethoprim NADPH ternary complex thus involves strong cooperative effects, and interconversion of the two species. The binding of pyrimethamine in the ternary complex was indistinguishable from its binding in the binary complex, showing neither the cooperative effects, nor the interconversion of the two species observed with trimethoprim. The species with a low KD for trimethoprim and pyrimethamine could be isolated by selective proteolysis. It was quite stable, but could be converted to a mixture of the original species via formation of the ternary complex with trimethoprim, as predicted from the binding data. The results are interpreted in terms of a model in which the two species can be interconverted only via the formation of a common ternary complex, which can be formed after the binding of trimethoprim or dihydrofolate, but not pyrimethamine. The model is shown to be consistent with all data from the measurements of both binding and inhibition by both ligands.
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PMID:Interconvertible forms of Escherichia coli dihydrofolate reductase with different affinities for analogs of dihydrofolate. 79 43

Triazinate (TZT), a potent inhibitor of dihydrofolate reductase, was selected for detailed investigation to determine its mechanism of selective action as well as its metabolic fate in mice, rats, dogs, and monkeys. The serum disappearance of TZT in normal and tumor-bearing mice was similar, with a rapid tissue equilibration phase and a slower elimination phase. Serum disappearance in normal and tumor-bearing rats was 1.5 to 2.2 hr. Serum disappearance in dogs and monkeys was similar, with half-lives of 3 to 4 and 2 to 4 hr, respectively. Urinary excretion of TZT at 24 hr was only 5 to 6% of the injected dose in mice and rats; in contrast, the dogs excreted 60% of the injected dose in 8 hr. TZT accumulated to comparable degrees in the organs of rats and mice, with progressively lesser concentrations in liver, kidney, spleen, and brain. Dihydrofolate reductase activity became almost undectectable in all tissues studied within 15 min after drug adminsitration. An important difference in drug accumulation was in the ascites cells of tumor-bearing animals: in mice, the drug level was consistently lower in the L1210 cells than in the ascites fluid; in contrast, by 30 min after treatment with TZT the drug level in Walker 256 cells was 10-fold higher than the level in the ascites fluid. No evidence for drug metabolism was found in extracts of urine, feces, or organ tissues from either mice or rats. TZT and two related triazines were studied for their ability to accumulate in the cerbrospinal fluid of dogs after i.v. administration. TZT achieved a cerebrospinal fluid level of approximately 15% of the serum concentration at 1 hr; in contrast, the other two triazines reached maximum cerebrospinal fluid values of 1% at 1 hr.
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PMID:Pharmacology of a new triazine antifolate in mice, rats, dogs, and monkeys. 80 54

Dihydrofolate reductase was isolated from hamster cells sensitive to methotrexate and from a methotrexate-resistant subline which has an elevated enzyme level. The enzymes were compared by peptide map analysis, and no differences in primary structure could be detected. The rates of enzyme degradation and synthesis were determined in both cell lines by a novel approach based on the enzyme specific radioactivity (called radioaffinity labeling). Degradation of reductase was minimal in both cell lines, whereas the rates of synthesis were directly proportional to the steady state concentrations of enzyme. Thus the resistant cells synthesized reductase at a rate which was 140 times faster than that in sensitive cells. Therefore the high concentration of dihydrofolate reductase in the methotrexate-resistant cells is probably the result of an alteration of a cellular component which control the synthesis of the enzyme.
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PMID:Altered regulation of the rate of synthesis of dihydrofolate reductase in methotrexate-resistant hamster cells. 94 98

Dihydrofolate reductase activity of 0.2 nmole of dihydrofolate reduced/min/mg protein was detected in crude extracts of unsporulated oocysts of Eimeria tenella. The enzyme was purified by a combination of affinity and ion-exchange chromatography. Its molecular weight was estimated as 240,000 daltons. An anticoccidial drug pyrimethamine is a potent inhibitor of the activity of E. tenella dihydrofolate reductase (Ki = 3 nM), but it is less effective an inhibitor of dihydrofolate reductase from chicken liver. This difference may explain the in vivo therapeutic action of pyrimethamine against Coccidia.
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PMID:Dihydrofolate reductase from Eimeria tenella: rationalization of chemotherapeutic efficacy of pyrimethamine. 105 72


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