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 (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) was partially purified from a cloned strain of pyrimethamine-sensitive Plasmodium chabaudi and a drug-resistant clone derived from it. A molecular weight of approximately 120000 was estimated by gel filtration for enzyme from both pyrimethamine-sensitive and resistant parasites. The specific activities of the crude enzyme at pH 7.4 were 2.7 +/- 0.8 and 1.4 +/- 0.6 nmol min-1 mg-1 protein for sensitive and resistant strains, respectively. Methotrexate titration (pH 7.4, 37 degrees C) indicated that the apparent turnover number of the enzyme from the sensitive parasites was 1229 +/- 322 mol min-1 mol-1 compared with 1238 +/- 179 mol min-1 mol-1 for the enzyme from the resistant parasites. There was therefore no significant difference in the amounts of the enzyme from both sources. The Km value for dihydrofolate (9.3 microM) of the enzyme from the drug-sensitive parasites at pH 7.4 was lower than that from the resistant parasites by a factor of approximately 4. The Km values for NADPH of the enzyme from both sources were similar. Inhibition by pyrimethamine of the enzyme from the sensitive parasites was competitive with dihydrofolate, with Ki of 0.26 nM. By contrast, noncompetitive inhibition was observed for the enzyme from the resistant parasites, with Kis of 50 nM and Kii of 33 nM. The enzyme from drug-sensitive and drug-resistant parasites had different activity profiles with respect to pH and temperature. Moreover, the former was more sensitive to heat denaturation than the latter. From these results, it was concluded that the major basis for drug resistance is not an increase in enzyme content, but a large decrease in drug binding with the structurally different enzyme.
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PMID:Kinetic and molecular properties of dihydrofolate reductase from pyrimethamine-sensitive and pyrimethamine-resistant Plasmodium chabaudi. 672 25

The protein-dependent retention of double-stranded DNA molecules on nitrocellulose filters has been used to show that pure dihydrofolate reductase from Lactobacillus casei has affinity for DNA. Dihydrofolate reductase will bind to end-labeled linear double-stranded DNA and to DNA in supercoiled form. Coenzymes and certain inhibitors do not affect the affinity of the protein to DNA, indicating that the DNA-binding region of the protein is distinct from the binding sites for these molecules. Comparison of the retention on filters by dihydrofolate reductase of two plasmid DNAs, differing only in a 3000-base pair insert containing the L. casei gene for dihydrofolate reductase, showed that in the presence of this DNA region lower concentrations of the protein were required to give significant retention; it is possible that a specific DNA-protein interaction underlies this effect. This presents the possibility of studying the interaction with DNA of a protein for which a crystal structure and considerable nuclear magnetic resonance data are already available.
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PMID:DNA binding by dihydrofolate reductase from Lactobacillus casei. 679 97

Dihydrofolate reductase isozyme 2 of Streptococcus faecium has been labeled with 13C in the C gamma position of tryptophan residues by growing the organism on a defined medium containing L-[gamma-13C]tryptophan (90% 13C). The 13C nuclear magnetic resonance (NMR) spectrum of the enzyme shows four well-resolved resonances which have nuclear Overhauser enhancements of 1.1-1.3. Values of T1 (spin-lattice relaxation time) and T2 (spin-spin relaxation time) are significantly less than predicted for an isotropically rotating, rigid sphere with no intermolecular dipole-dipole interactions. Three of the resonances have chemical shifts downfield from the 13C resonance of urea-denatured enzyme by amounts up to 1.43 ppm. The chemical shift of resonance 4 in the spectrum is 4.0 ppm upfield from Trp C gamma of urea-denatured enzyme. This large upfield shift is attributed to electric field effects generated by polar side chains. The two more upfield peaks both provide evidence that the corresponding tryptophan residues, WC and WD, each undergo chemical exchange between alternative microenvironments. In the case of WC, which gives a resonance with two components, exchange is slow (ve, exchange rate much less than 55 s-1), and the relative populations of the two stable states are in the ratio 2:3. WD is apparently in intermediate to fast exchange on the NMR time scale. With a two-state model, ve increases from approximately 90 to 150 s-1 as the temperature is increased from 5 to 25 degrees C. This increases in temperature is also accompanied by an increase in the fractional population of the minor downfield state(s), from about 0.062 at 5 degrees C to 0.24 at 25 degrees C. However, the data may also be interpreted as a temperature-dependent equilibrium between a continuum of many states. WD is tentatively identified with Trp-22 since comparison of the sequences of Lactobacillus casei dihydrofolate reductase and S. faecium dihydrofolate reductase and inspection of the crystal structure of the L. casei enzyme indicate that Trp-6, Trp-115, and Trp-160 are probably all involved in regions of beta sheet whereas Trp-22 is in a loop joining beta A to alpha B. Earlier crystallographic evidence for the Escherichia coli reductase suggests that in the methotrexate complex with this enzyme the corresponding loop has a good deal of flexibility. It is probable that in the uncomplexed S. faecium reductase the motion of this loop is the major mechanism for the exchange process involving Trp-22. The upfield chemical shift of resonance 4 is attributed to electric field effects on C gamma of Trp-22 produced by the carboxylate groups of Asp-27 and Asp-9. On the basis of the small difference between the chemical shift of resonance 3 and that of tryptophan C gamma in urea-denatured reductase, it is suggested that WC may be identified with Trp-6.
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PMID:Nuclear magnetic resonance study of dihydrofolate reductase labeled with [gamma-13C]tryptophan. 679 11

Dihydrofolate reductase from Lactobacillus casei was inactivated by reaction with tetranitromethane and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Loss of activity occurred with modification of four of the five tyrosine residues present in the enzyme. The presence of either substrate, NADPH or 7,8-dihydrofolate, as well as NADP and folate, provided extensive protection against inactivation, while NADH and tetrahydrofolate exhibited none. This protection from inactivation occurred on protection of two of the four susceptible tyrosines from modification. Nitration of the enzyme adversely affected its ability to bind substrates. Restoration of the pKa of the nitrated tyrosines by reduction of the nitro group to an amino group did not result in a regeneration of enzymatic activity. However, fluorotyrosine-containing enzyme, prepared by growing the bacterium in the presence of fluorotyrosine, exhibited specific activity identical to that of native enzyme over the pH range of 4.5-8. These results suggest that inactivation of dihydrofolate reductase by tyrosine modification occurs primarily due to a steric effect and that the active site tyrosines may participate in substrate binding.
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PMID:Modification of tyrosine residues in dihydrofolate reductase from Lactobacillus casei. 679 2

The role of histidine residues of dihydrofolate reductase from Lactobacillus casei was investigated with diethyl pyrocarbonate. This enzyme has no cysteine residues and differs in this respect from many nicotinamide nucleotide dehydrogenases, which have catalytically important sulfhydryl groups. X-ray studies of this enzyme have shown that histidine residues are involved in substrate binding but not in proton transfer [Matthews et al. (1978) J. Biol. Chem. 253, 6946]. Dihydrofolate reductase was inactivated by diethyl pyrocarbonate; the second-order rate constant for the reaction was 29 M-1 min-1 at 0 degrees C. The difference spectrum of native and diethyl pyrocarbonate inactivated enzyme had a maximum near 242 nm, which indicated a reaction with histidine residues. The absence of any spectral difference near 280 nm indicated that diethyl pyrocarbonate had not reacted with tyrosine residues. Dihydrofolate reductase lost all of its enzymatic activity after about six of the seven histidine residues had been modified. No catalytic activity was lost during an initial rapid reaction with about four histidine residues, but a subsequent slower reaction involving an additional one or two residues was associated with the loss of activity. The enzyme was protected from inactivation by either of the substrates NADPH or dihydrofolate. In fact, treatment with diethyl pyrocarbonate in the presence of either substrate, but particularly with NADPH, resulted in substantially greater activity than that found with untreated enzyme. Treatment with 1 M hydroxylamine partially restored activity to dihydrofolate reductase that had been inactivated by diethyl pyrocarbonate.
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PMID:Inactivation of dihydrofolate reductase from Lactobacillus casei by diethyl pyrocarbonate. 680 28

Infection of human cells by adenovirus results in multiple alterations of host gene expression. To examine the effects of viral infection on the expression of a single gene, a line of human cells was developed which is resistant to growth in methotrexate and which contains amplified RNA and protein specific for dihydrofolate reductase (DHFR). Cytogenetic evidence indicated the presence of amplified DNA. Adenovirus infection of these cells caused an induction and subsequent decline in the synthesis of DHFR protein. The maximum DHFR induction occurred 16 to 19 h after infection and reached a level 2.5-fold greater than that observed in uninfected cells. Induction of DHFR protein synthesis was accompanied by concomitant increases in the level of steady-state DHFR-specific cytoplasmic RNA. The relative rate of DHFR mRNA production (i.e., the appearance of DHFR-specific mRNA sequences in the cytoplasm) also increased 2.5-fold during induction. Later in infection, the relative rate of DHFR protein synthesis declined, reaching a level below that observed in uninfected cells. This decline was accompanied by a similar decline in the steady-state levels of DHFR RNA and in the relative rate of synthesis of DHFR mRNA. These data suggest that adenovirus infection controls DHFR gene expression by increasing and subsequently decreasing the relative rate at which DHFR-specific mRNA sequences appear in the cytoplasm and enter the pool of mRNA available for translation.
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PMID:Control of cellular gene expression during adenovirus infection: induction and shut-off of dihydrofolate reductase gene expression by adenovirus type 2. 686 43

The molecular weight of dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) from protozoa has been reported to be 5- to 10-fold larger than the isofunctional enzyme of most other organisms studied, based on gel filtration. This enzyme from the protozoal flagellate Crithidia fasciculata has been purified to homogeneity and found to be a bifunctional protein with thymidylate synthase (5,10-methylene tetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) activity. The purified protein, eluted from methotrexate-Sepharose columns by dihydrofolate, migrated as a single band on both nondenaturing and denaturing polyacrylamide gel electrophoresis. The monomer Mr is 56,700 +/- 200. The native Mr was calculated to be 107,000 from a sedimentation coefficient of 5.9 and Stokes radius of 4.4 nm. Dihydrofolate reductase and thymidylate synthase activities of the rodent malaria organism Plasmodium berghei also copurified on Sephadex G-200 and methotexate-Sepharose columns, suggesting that this unique bifunctional protein might occur throughout the Protozoa.
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PMID:Dihydrofolate reductase: thymidylate synthase, a bifunctional polypeptide from Crithidia fasciculata. 693 11

Crude X-methyl folate and two purified fractions (purified X-methyl folate and 9-methyl folate) were evaluated as possible folic acid antagonists. Antifolate activity was measured by the deoxyuridine suppression assay, direct measurement of dihydrofolate reductase and morphological changes characteristic of folate deficiency. Cytotoxicity was evaluated by cell growth curves. These drugs were studied in freshly obtained human normal evaluated by cell growth curves. These drugs were studied in freshly obtained human normal bone marrow and leukemia cells as well as in three established cell lines HL-60 (human promyelocytic leukemia), CEM (human T lymphocytes), and L1210 (murine lymphoid leukemia). Purified X-methyl folate and 9-methyl folate at concentrations up to 2X10(-5) M failed to induce cell toxicity or inhibit deoxyuridine suppression of 3H-thymidine into DNA. Dihydrofolate reductase was not inhibited by 9-methyl folate but was partially inhibited by purified X-methyl folate. Crude X-methyl folate (2X10(-1) M) is a weak folate antagonist as compared to methotrexate since it requires a 1,000-fold higher concentration to inhibit cell growth and dihydrofolate reductase activity. In addition, the crude X-methyl folate contains a folate-like fraction since it will correct the abnormal deoxyuridine suppression in folate-depleted cells. None of the drugs tested were able to induce myeloid differentiation in the HL-60 cells. The data suggest that 9-methyl folate would not be active as a folate antagonist antitumor agent and that purified and crude X-methyl folate in large concentrations will inhibit folate metabolism. Additional studies are needed to remove the folate-like activity in the crude material in order to further establish the antitumor effects of the crude X-methyl folate.
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PMID:Evaluation of X-methyl folate preparations as possible folic acid antagonists. 714 Apr 22

Our Chinese hamster ovary cells are extremely resistant to methotrexate (MTX) (100% survival after 500 microgram/ml for 13 hr). However, exposure to 43 degrees (but not 41 degrees or 42 degrees) for 1 hr sensitizes the cells to MTX so that a 50% cell kill in excess of that due to hyperthermia occurs. Treatment of cells at 43 degrees increases net MTX uptake by about 30% at 30 min but causes a substantial reduction after 1 hr. This negative effect is greater in cells continually heated at 43 degrees than in those exposed for only 1 hr. Treatment at 43 degrees for 1 hr also markedly increases efflux of MTX out of cells over the that 2 hr. Dihydrofolate reductase activity was found to decrease to about 50% of control values by 4 to 5 hr after exposure to 43 degrees. The biological half-life of dihydrofolate reductase in Chinese hamster ovary cells was determined to be about 4.5 hr, indicating that hyperthermia-induced cessation of protein synthesis may explain both the decrease in dihydrofolate reductase activity and the sensitization to MTX observed with heat exposure. In scheduling experiments, lethality due to exposure to 43 degrees for 1 hr in conjunction with MTX was maximum when 1-hr drug exposure began just at the end of heat treatment.
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PMID:Reversal of resistance to methotrexate by hyperthermia in Chinese hamster ovary cells. 728 91

Several reaction conditions of cell-free protein synthesis such as temperatures, buffers, tRNAs, and creatine phosphate were intensively investigated and optimized to prolong protein synthesis and make it more efficiently in a batch system. As a result of these modifications, the protein synthesis reaction continued for 10 h so that about 30 micrograms of dihydrofolate reductase (DHFR) protein derived from Escherichia coli was synthesized in 1 ml of reaction mixture. In this improved system, translational reactions of other mRNAs such as rabbit beta-globin, Xenopus beta-globin, and tobacco mosaic virus RNA also continued for about 10 h. In addition, protein synthesis directed by uncapped dhfr mRNA containing a viral cap-independent translation initiation-mediating sequence continued for 10 h, resulting in the synthesis of 18 micrograms of DHFR protein per milliliter of reaction mixture.
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PMID:A long-lived batch reaction system of cell-free protein synthesis. 779 34


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