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 cellular pharmacology of methotrexate was evaluated in freshly isolated rabbit hepatocytes in suspension with an analysis of drug metabolism by high-performance liquid chromatography. After exposure of hepatocytes at a cytocrit of 5% to 5 microM [3H]-methotrexate, intracellular 7-hydroxymethotrexate appears rapidly within the cell; within 15 sec, the level of 7-hydroxymethotrexate exceeds the level of intracellular methotrexate, although the latter has not achieved the dihydrofolate reductase binding capacity. Within 20 min, virtually all methotrexate is hydroxylated. There is minimal formation of methotrexate polyglutamyl derivatives even after exposure of cells to very high levels of methotrexate, and 7-hydroxymethotrexate polyglutamates do not accumulate in the cell at all after incubation with [3H]-7-hydroxymethotrexate. Because of the rapidity of the hydroxylation of methotrexate, transport of this agent could not be characterized. However, some aspects of the transport properties of 7-hydroxymethotrexate could be studied since the catabolite is neither bound nor metabolized in this system. Net 7-hydroxymethotrexate transport was reduced by the addition of 5-formyltetrahydrofolate. As observed for 4-aminoantifolate transport in other cell systems, net 7-hydroxymethotrexate transport was markedly stimulated by sodium azide, an inhibitor of energy metabolism. The data suggest that hydroxylation of methotrexate proceeds at a rate at least comparable to the rate of association of the drug with dihydrofolate reductase and that transport of methotrexate into rabbit hepatocytes is slow relative to the rate of catabolism to the 7-hydroxy derivative. Rabbit hepatocytes may be a useful model for exploring methotrexate catabolism at the cellular level and may provide insights into the interaction between methotrexate and/or other 4-aminoantifolates and the human liver.
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PMID:Characteristics of the formation and membrane transport of 7-hydroxymethotrexate in freshly isolated rabbit hepatocytes. 257 71

We have generated several mammalian cell lines that stably express high levels of intact glucocorticoid receptor. These cells were created by cotransfecting a glucocorticoid-dependent dihydrofolate reductase (DHFR) gene into DHFR-deficient Chinese hamster ovary (CHO) cells together with a plasmid directing the expression of human glucocorticoid receptor. Using this approach, transfection frequencies indicate that the inclusion of glucocorticoid receptor cDNA increased the efficiency of DHFR transformation greater than 10-fold over nonreceptor control DNA. When a stably cotransfected line (designated MG/hGR) was subjected to short term growth in cytotoxic concentrations of the antifolate methotrexate, these cells strongly resisted growth inhibition when dexamethasone was present in the medium. This effect was steroid specific and was inhibited by the glucocorticoid antagonist RU38486. In an effort to exploit the methotrexate-induced coamplification properties of the DHFR gene as a means of creating cell lines having increased levels of glucocorticoid receptor, MG/hGR cells were chronically exposed to a relatively low concentration of methotrexate (50 nM). After this treatment a resistant line was isolated (MG/hGR/MTX50) that displayed complete dependence on exogenous glucocorticoid for growth. To investigate the molecular basis for the enhanced ability of MG/hGR/MTX50 cells to resist the cytotoxic effects of methotrexate in the presence of dexamethasone, glucocorticoid receptor protein in these cells was characterized and compared to parental CHO cells and methotrexate sensitive MG/hGR cells. Affinity labeling with [3H]dexamethasone mesylate and Western blot analysis with antiglucocorticoid receptor antiserum revealed that nontransfected CHO cells have virtually undetectable levels of glucocorticoid receptor protein whereas cotransfected MG/hGR cells contain at least 3 times more intact monomeric receptor protein of Mr 94,000. Correspondingly, analysis of receptor protein in MG/hGR/MTX50 cells indicated that these cells contain 8 to 10 times more glucocorticoid receptor than nontransfected CHO cells. Scatchard analysis of steroid binding curves revealed that these increases correspond to 6,600, 22,000 and 63,000 dexamethasone binding sites per cell for nontransfected CHO cells, cotransfected MG/hGR cells, and MG/hGR/MTX50 cells, respectively. Sedimentation profiles of native receptor in transfected and methotrexate-resistant cells further support the progressive increase in receptor content and demonstrate that glucocorticoid receptor exists in cotransfected cels as an oligomeric complex under hypotonic conditions (9S complex in the presence of 20 mM sodium molybdate, 7S in the absence of molybdate), which dissociates to a monomeric 4S species in the presence of 0.4 M KCl. These physicochemical properties are indistinguishable from those observed for the endogenous hamster glucocorticoid receptor and suggest that stably transfected human glucocort
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PMID:Stable overproduction of intact glucocorticoid receptors in mammalian cells using a selectable glucocorticoid responsive dihydrofolate reductase gene. 260 55

Dihydrofolate reductase from Candida albicans was purified 31,000-fold and characterized. In addition, the C. albicans dihydrofolate reductase gene was cloned into a plasmid vector and expressed in Escherichia coli, and the enzyme was purified from this source. Both preparations showed a single protein-staining band with a molecular weight of about 25,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzymes were stable and had an isoelectric point of pH 7.1 on gel isoelectric focusing. Kinetic characterization showed that the enzymes from each source had similar turnover numbers (about 11,000 min-1) and Km values for NADPH and dihydrofolate of 3-4 microM. Like other eukaryotic dihydrofolate reductases, the C. albicans enzyme exhibited weak binding affinity for the antibacterial agent trimethoprim (Ki = 4 microM), but further characterization showed that the inhibitor binding profile of the yeast and mammalian enzymes differed. Methotrexate was a tight binding inhibitor of human but not C. albicans dihydrofolate reductase; the latter had a relatively high methotrexate Ki of 150 pM. The yeast and vertebrate enzymes also differed in their interactions with KCl and urea. These two agents activate vertebrate dihydrofolate reductases but inhibited the C. albicans enzyme. The sequence of the first 36 amino-terminal amino acids of the yeast enzyme was also determined. This portion of the C. albicans enzyme was more similar to human than to E. coli dihydrofolate reductases (50% and 30% identity, respectively). Some key amino acid residues in the C. albicans sequence, such as E-30 (human enzyme numbering), were "vertebrate-like" whereas others, such as I-31, were not. These results indicate that there are physical and kinetic differences between the eukaryotic mammalian and yeast enzymes.
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PMID:Characterization of Candida albicans dihydrofolate reductase. 264 98

The purpose of this study was to characterize the transport properties of trimetrexate in WI-L2 human lymphoblastoid cells and determine the mode of resistance that had developed in a subline, WI-L2/TMQ, that was grown in increasing concentrations of trimetrexate. WI-L2/TMQ cells were 62-fold resistant to trimetrexate and 68- and 96-fold cross-resistant to the other lipophilic antifolates metoprine and piritrexim (BW 301U). No cross-resistance was observed with vincristine or doxorubicin, and sensitivity was not increased with 5 micrograms/ml of verapamil, indicating that it was not a typical multidrug resistance phenotype. WI-L2/TMQ exhibited a 2-fold increase in dihydrofolate reductase; however, this did not contribute significantly to the observed resistance, since these cells retained full sensitivity to methotrexate. Nor were there any kinetic alterations in dihydrofolate reductase toward trimetrexate or differences in the levels of thymidylate synthase. The major difference between the sensitive and resistant cell line was a 50% decrease in the influx rate of WI-L2/TMQ cells which produced a corresponding decrease in cellular trimetrexate at the steady state. No difference in efflux rates was detected nor were there any differences in intracellular water or metabolism of trimetrexate. Additional characterization of trimetrexate transport in WI-L2 showed that influx was nonsaturable up to 5 mM extracellular trimetrexate, relatively insensitive to sodium azide, and exhibited a Q10 of 2.7. Influx was, however, inhibited in a dose-dependent manner by concentrations of p-chloromercuribenzylsulfonate above 10 microM. Efflux studies revealed a large nonexchangeable fraction of trimetrexate that was well above the dihydrofolate reductase binding capacity and varied depending on the initial level of cell-associated drug. The intracellular exchangeable trimetrexate concentration at the steady state was always several-fold higher than the extracellular concentration. Retention of trimetrexate appeared to be coupled to some component of energy metabolism, since the presence of sodium azide stimulated this process by 2- to 3-fold. The data suggest that trimetrexate enters cells by passive diffusion but then is distributed and concentrated within the cell through more complex mechanisms which may involve energy coupling, compartmentation, or binding to macromolecules or organelles, although some type of carrier-mediated process cannot be ruled out.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of trimetrexate transport in human lymphoblastoid cells and development of impaired influx as a mechanism of resistance to lipophilic antifolates. 297 70

A procaryotic high-level expression vector for human dihydrofolate reductase has been constructed and the protein characterized as a first step toward structure-function studies of this enzyme. A vector bearing the tac promoter, four synthetic oligodeoxynucleotides, and a restriction fragment from the dihydrofolate reductase cDNA were ligated in a manner which optimized the transcriptional and translational frequency of the enzyme mRNA. The reductase, comprising ca. 17% of the total soluble protein in the host bacteria, was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and characterized by amino acid composition, partial amino acid sequence, and steady-state kinetic analysis. This expression vector has been used as a template for double-stranded plasmid DNA site-specific mutagenesis. Functional studies on a Cys-6----Ser-6 mutant enzyme support the contention that Cys-6 is obligatory for organomercurial activation of human dihydrofolate reductase. The Ser-6 mutant enzyme was not activated to any extent following a 24-h incubation with p-(hydroxymercuri)benzoate and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), whereas the kcat for Cys-6 reductase increased 2-fold under identical conditions. The specific activities of the Cys-6 and Ser-6 enzymes were virtually identical as determined by methotrexate titration as were the Km values for both dihydrofolate and NADPH. The Ser-6 mutant showed a decreased temperature stability and was more sensitive to inactivation by alpha-chymotrypsin when compared to the wild-type enzyme. These results suggest that the Ser-6 mutant reductase is conformationally altered relative to the Cys-6 native enzyme.
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PMID:Expression and site-directed mutagenesis of human dihydrofolate reductase. 304 47

Lactobacillus casei cells contain a 25 kDa, membrane-associated, folate-binding protein (fbp), which is a component of the folate transport system. Polyclonal antibody to fbp (anti-fbp) has been prepared, and conditions have been established for detection and quantitation of the protein. Anti-fbp did not block [3H]folate transport or binding in L. casei cells. As judged by Western blots, the antibody reacted only with fbp on sodium dodecyl sulfate electrophoretograms of Triton X-100 extracts of L. casei membranes. Anti-fbp showed no cross-reactivity with L. casei dihydrofolate reductase, L. casei 5,10-methenyltetrahydrofolate synthetase, L1210 dihydrofolate reductase, rat liver dihydrofolate reductase, or L1210 folate-binding protein. Enzyme-linked immunosorbent assay measurements indicated the presence of an fbp in membranes of Lactobacillus salivarius and two transport-defective sublines of L. casei. Anti-fbp was used to demonstrate selective extraction, with n-butanol, of fbp from a mixture of Triton-solubilized L. casei membrane proteins; repression of fbp in membranes of L. casei cells grown on high levels of folate; and localization of fbp by electron microscopy, using anti-fbp in conjunction with goat anti-rabbit IgG gold conjugate, in L. casei membranes.
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PMID:Antibody for detection and quantitation of membrane-associated folate-binding protein from Lactobacillus casei. 310 28

We report here for the first time reconstitution and secretion of functionally active antibody in non-lymphoid cells. Expression vectors for the light and the heavy chain of a monoclonal antibody directed against creatine kinase (EC 2.7.3.2) were introduced into COS and CHO Chinese hamster ovary dhfr- cells. Introduction of the expression vectors separately gave rise to immuno-reactive material in the culture supernatants, but only cotransfection of the expression plasmids resulted in secretion of protein with immuno-reactivity against antibodies directed against mouse heavy and light chains as well as specific antigen-binding affinity, as determined by enzyme-linked immunosorbent assay. Secreted kappa and gamma chains from reconstituted antibody were characterized by immunoadsorption and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In COS cells, reconstituted antibody was transiently secreted; cotransfection of kappa and gamma chain expression plasmids with a dihydrofolate reductase (DHFR)-expression plasmid into CHO dhfr- cells gave rise to stable transformants secreting functionally active antibody.
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PMID:Reconstitution of functionally active antibody directed against creatine kinase from separately expressed heavy and light chains in non-lymphoid cells. 311 10

Analogues of methotrexate (MTX) with strong alkylating activity were prepared by replacing the L-glutamate side chain with N omega-haloacetyl derivatives of L-lysine and L-ornithine. Haloacetylation was accomplished in 30-40% yield by reaction of the preformed L-lysine and L-ornithine analogues of MTX with p-nitrophenyl bromoacetate or chloroacetate in aqueous sodium bicarbonate at room temperature. All four haloacetamides were potent inhibitors in spectrophotometric assays measuring noncovalent binding to purified dihydrofolate reductase (DHFR) from L1210 cells. In experiments designed to measure time-dependent inactivation of DHFR from L1210 cells and Candida albicans, the N epsilon-(bromoacetyl)-L-lysine and N delta-(bromoacetyl)-L-ornithine analogues gave results consistent with covalent binding, whereas N epsilon- and N delta-chloroacetyl analogues did not. The N delta-(bromoacetyl)-L-ornithine analogue appeared to be the more reactive one toward both enzymes. Amino acid analysis of acid hydrolysates of the L1210 enzyme following incubation with the bromoacetamides failed to demonstrate the presence of a carboxymethylated residue, suggesting that alkylation had perhaps formed an acid-labile bond. In growth inhibition assays with L1210 cultured murine leukemia cells, the four haloacetamides were all more potent than their nonacylated precursors but less potent than MTX. The greater than 40,000-fold MTX-resistant mutant cell line L1210/R81 was only partly cross-resistant to the haloacetamides. An analogue of MTX with acivicin replacing glutamate was a potent inhibitor of DHFR from chicken liver and L1210 cells but was 200 times less potent than MTX against L1210 cells in culture.
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PMID:Methotrexate analogues. 30. Dihydrofolate reductase inhibition and in vitro tumor cell growth inhibition by N epsilon-(haloacetyl)-L-lysine and N delta-(haloacetyl)-L-ornithine analogues and an acivicin analogue of methotrexate. 311 97

A radioiodinated photoaffinity analogue of methotrexate, N alpha-(4-amino-4-deoxy-10-methyl-pteroyl)-N epsilon-(4-azidosalicylyl)-L- lysine (APA-ASA-Lys), was recently used to identify the plasma membrane derived binding protein involved in the transport of this folate antagonist into murine L1210 cells [Price, E. M., & Freisheim, J. H. (1987) Biochemistry 26, 4757-4763]. The labeled protein has an apparent molecular weight of 46K-48K when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but no such labeling occurs in a methotrexate transport-defective cell line (L1210/R81). Labeling of the total cytosolic protein from disrupted cells, followed by electrophoresis and autoradiography, showed, among other proteins, a 21K band, corresponding to dihydrofolate reductase (DHFR), in both the parent and R81 cells and a 38K band only in the parent cells. However, when whole cells were UV irradiated at various times at 37 degrees C following addition of radiolabeled APA-ASA-Lys, the 38K protein and DHFR were the only cytosolic proteins labeled in the parent cells, while the intact R81 cells showed no labeled cytosolic protein, since the photoprobe is not transported. Further, when the parent cells were treated with a pulse of radiolabeled photoprobe, followed by UV irradiation at different times at 37 degrees C, the probe appeared sequentially on the 48K membrane protein and both the 38K cytosolic protein and dihydrofolate reductase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of the methotrexate transport pathway in murine L1210 leukemia cells: involvement of a membrane receptor and a cytosolic protein. 320 16

A photoaffinity analogue of methotrexate, APA-[125I]ASA-Lys, specifically binds to dihydrofolate reductase and covalently modifies the enzyme following irradiation. An excess of methotrexate blocks incorporation of the photoprobe. Following cyanogen bromide digestion of the radiolabeled enzyme and high-pressure liquid chromatographic separation of the generated peptides, a majority of the label was centered around residues 63-65 (Lys-Asn-Arg), part of the inhibitor binding domain. This photoprobe is also transported into murine L1210 cells in a temperature-dependent, sulfhydryl reagent inhibitable manner with a Vmax similar to that for methotrexate. Ultraviolet irradiation at 4 degrees C of a cell suspension that had been incubated with the radiolabeled photoprobe resulted in the covalent modification of a 46-48 Kd protein. This can be demonstrated when the plasma membranes from the labeled cells are analyzed via sodium dodecylsulfate-polyacrylamide gel electrophoresis and autoradiography. Labeling of this protein occurs half-maximally at a reagent concentration that correlates with the Kt for transport of the iodinated compound. Protection against labeling of this protein by increasing amounts of methotrexate parallels the concentration dependence of inhibition of photoprobe uptake by methotrexate. In addition, no labeling occurs when a cell line that has a defective methotrexate transport system is similarly treated. Evidence that, in the absence of irradiation and at 37 degrees C, the iodinated probe is actually internalized is demonstrated by the labeling of two soluble proteins (Mr = 38 Kd and 21 Kd) derived from the cell homogenate supernatant.
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PMID:Photoaffinity analogues of methotrexate as folate antagonist binding probes. 325 Feb 27


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