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 accumulation of [3H]pyrimethamine by pyrimethamine-resistant (Pyrr) mutants of the Plasmodium falciparum strain FCR3 was examined by measuring the accumulation of drug in infected red blood cells. [3H]Pyrimethamine was stage specifically accumulated in trophozoites and schizont infected red blood cells. The mutant parasites accumulated drug as efficiently as FCR3. Pyrimethamine was associated with a high molecular weight protein that eluted from a Sephadex G200 column exactly as [3H]fluorodeoxyuridinemonophosphate (FdUMP) labeled parasite dihydrofolate reductase-thymidylate synthetase (DHFR-TS) enzyme. These results suggested that the pyrimethamine resistance was not associated with decreased drug permeability of the membrane. DHFR-TS-[3H]FdUMP enzyme complex of all the Pyrr mutants and FCR3 had a monomer of 70 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One highly resistant mutant, FCR3-D7, exhibited a 5-10 fold higher uptake of pyrimethamine and a proportionately higher amount of DHFR-TS protein than FCR3 but only a normal level of DHFR activity. The genomic DNA of FCR3-D7 was shown to contain at least twice as much DHFR-TS specific DNA than either FCR3-D8, another Pyrr mutant, or FCR3. Preliminary results suggested some of the DHFR-TS genetic material in FCR3-D7 is associated with a gene duplication.
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PMID:Pyrimethamine resistant Plasmodium falciparum: overproduction of dihydrofolate reductase by a gene duplication. 332 3

Arsenic is a well-established carcinogen in humans, but there is little evidence for its carcinogenicity in animals and it is inactive as an initiator or tumor promoter in two-stage models of carcinogenicity in mice. Two arsenic salts (sodium arsenite and sodium arsenate) induced a high frequency of methotrexate-resistant 3T6 cells, which were shown to have amplified copies of the dihydrofolate reductase gene. The ability of arsenic to induce gene amplification may relate to its carcinogenic effects in humans since amplification of oncogenes is observed in many human tumors. The inability of arsenic to induce gene mutations may relate to the negative results of arsenic in long-term animal studies and suggests that these experiments may not detect some environmental agents that act late in the carcinogenic process in humans.
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PMID:Induction of gene amplification by arsenic. 338 20

Recombinant type 1 transforming growth factor beta (TGF-beta) was expressed to high levels in CHO cells by using dihydrofolate reductase (dhfr) gene amplification. The expression plasmid was derived from the pSV2 vectors and contained, in tandem, the simian TGF-beta and mouse dhfr cDNAs. Transcription of both cDNAs was controlled by the simian virus 40 early promoter. Stepwise selection of transfected CHO cells in increasing concentrations of methotrexate yielded cell lines that expressed amplified TGF-beta nucleic acid sequences. The expression plasmid DNA was amplified greater than 35-fold in one of the methotrexate-selected transfectants. The major proteins secreted by these cells consisted of latent TGF-beta and TGF-beta precursor polypeptides, as judged by immunoblots by using site-specific anti-peptide antibodies derived from various regions of the TGF-beta precursor. Levels of recombinant TGF-beta protein secreted by these cells approached 30 micrograms/24 h per 10(7) cells and required prior acidification for optimal activity; nonacidified supernatants were approximately 1% as active as acidified material. Antibodies directed toward sequences present in the mature growth factor readily identified a proteolytically processed recombinant TGF-beta which, on sodium dodecyl sulfate-polyacrylamide gels, comigrated with highly purified natural TGF-beta. In addition to mature recombinant TGF-beta, site-specific antibodies demonstrated the existence of larger TGF-beta precursor polypeptides. The availability of biologically active recombinant type 1 TGF-beta and precursor forms should provide a means to examine the structure, function, and potential in vivo therapeutic use of this growth factor.
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PMID:Type 1 transforming growth factor beta: amplified expression and secretion of mature and precursor polypeptides in Chinese hamster ovary cells. 347 80

Factor IX has been expressed to high levels within a recombinant host cell and the biologically active fraction subsequently purified to homogeneity for characterization. The coding sequence for Factor IX was inserted into a mammalian cell expression vector and transfected into dihydrofolate reductase-deficient Chinese hamster ovary cells. The integrated DNA was amplified to a high copy number by selection for increasingly higher expression levels of the marker gene, dihydrofolate reductase, contained within a co-transfected plasmid. Cloned cell lines secreting over 100 micrograms/ml Factor IX antigen and up to 1.5 microgram/ml native Factor IX antigen have been obtained. Expression of biologically active Factor IX was dependent on the presence of vitamin K in the culture media. The gamma-carboxylated Factor IX was isolated from cell culture fluid by immunoaffinity chromatography using antibodies conformation-specific for the metal-stabilized conformer of Factor IX. This conformation is dependent upon metal ions and gamma-carboxyglutamic acid. Purified recombinant Factor IX migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an electrophoretic mobility equivalent to plasma-derived Factor IX. The purified recombinant Factor IX demonstrated Factor IX coagulant activity, measured in Factor IX-deficient plasma, of 35-75 units/mg. Amino acid analysis of the alkaline hydrolysate of recombinant Factor IX demonstrated an average of 6-7 mol of gamma-carboxyglutamic acid per mol of Factor IX. NH2-terminal sequence analysis of the first 17 residues revealed equivalent amino acid sequences for both purified recombinant and plasma-derived Factor IX. The results represent the first purification and characterization of a biologically active, gamma-carboxylated vitamin K-dependent protein expressed in a recombinant DNA system.
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PMID:Expression, purification, and characterization of recombinant gamma-carboxylated factor IX synthesized in Chinese hamster ovary cells. 373 88

Methotrexate(MTX)-resistant human promyelocytic-leukaemia cells (HL-60) derived from MTX-sensitive cells have a 20-fold increase in dihydrofolate reductase (DHFR) activity as compared with the sensitive cells. This increase is not associated with a concomitant increase in DHFR protein as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by immunological methods using mouse anti-DHFR antibody. The rate of DHFR synthesis is similar in both cell lines. Furthermore, both the sensitive and resistant cells have similar amounts of RNA hybridizing to a DHFR complementary-DNA probe, correlating well with the lack of increase in DHFR protein. DHFR-gene dosages were similar in both types of cells. We conclude that the 20-fold increase in DHFR activity present in these MTX-resistant cells is not due to the overproduction of DHFR but due to the expression of a more active form of the enzyme.
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PMID:Increased dihydrofolate reductase activity in methotrexate-resistant human promyelocytic-leukaemia (HL-60) cells. Lack of correlation between increased activity and overproduction. 385 28

Dihydrofolate reductase from a MTX-resistant human promyelocytic leukemia cell line (HL-60 R4-29) had previously been found to have a higher specific activity than the DHFR from the parent MTX-sensitive cell line, in the absence of enzyme overproduction (Dedhar et al, Biochem. J. 225, 609-617, 1985). The enzymes from these two cell lines have been purified to apparent homogeneity as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The dihydrofolate reductase from the sensitive cells has an apparent molecular weight of 42,000 daltons, whereas that from the resistant cells has an apparent molecular weight of 21,000 daltons. The dihydrofolate reductase activity from the resistant cells is characterized by marked heat instability and substantially higher Vmax and Km values for the dihydrofolic acid/NADPH combination. The enzyme from the resistant cells can be protected against heat inactivation by either dihydrofolic acid or NADPH, or both. A 50 fold higher concentration of methotrexate was required to totally inhibit the R4-29 dihydrofolate reductase activity as compared to the (S) activity when the enzymes were assayed using equivalent amounts of enzyme protein. These data show that the increased dihydrofolate reductase specific activity present in the (R4-29) cells is associated with an alteration in the structure of this enzyme.
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PMID:Methotrexate-resistant human promyelocytic leukemia (HL-60) cells express a dihydrofolate reductase with altered properties associated with increased enzyme activity. 386 Feb 3

Influx of [3H]methotrexate into freshly isolated hepatocytes in suspension is mediated by two routes, one with a high affinity (Km = 5.9 microM) and another with a low affinity for methotrexate. Both transport routes are equally sensitive to the sulfhydryl group inhibitor, p-chloromercuriphenylsulfonic acid, alterations in temperature, substitution of extracellular Na+ with choline, and inhibition by ouabain or azide. The high-affinity pathway for methotrexate shows specificity for the 4-amino group of the pteridine moiety as methotrexate and aminopterin similarly inhibit influx of the labeled drug. On the other hand, 100 microM concentrations of the naturally occurring folates, folic acid, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate, are not inhibitory to influx of 1 microM methotrexate. Once in the cell, methotrexate rapidly reaches molar equivalence with dihydrofolate reductase following which both exchangeable and nonexchangeable intracellular methotrexate accumulates. The exchangeable component reaches steady state within 0.5 hr while the nonexchangeable component increases for at least 1 hr. The nonexchangeable component represents both bound methotrexate and methotrexate polyglutamates. Polyglutamates of methotrexate are a trivial component of total 3H within the cell until about 15 min, but thereafter, their rate of accumulation is constant so that by 1 hr they represent approximately 30% of total intracellular 3H. At steady state, there is a transmembrane chemical gradient for exchangeable methotrexate of 2.4:1; this is 24 times greater than the chemical gradient predicted for equilibrium when the transcellular membrane potential is considered. These results indicate that there are multiple routes for methotrexate transport in the rat hepatocyte that appear to be, at least in part, distinct from the routes for folic acid and the tetrahydrofolate cofactors. The data suggest that transport is energy and Na+ dependent and that the transport carrier requires intact sulfhydryl groups. Net association of methotrexate with the cells is a complex process determined by transport and binding to multiple sites within the cell and metabolism to polyglutamate derivatives that are retained within the cell.
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PMID:Transport, binding, and polyglutamation of methotrexate in freshly isolated rat hepatocytes. 616 51

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


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