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)

A purification procedure is reported for obtaining bovine liver dihydrofolate reductase in high yield and amounts of 100-200 mg. A key step in the procedure is the use of an affinity gel prepared by coupling pteroyl-L-lysine to Sepharose. The purified reductase has a specific activity of about 100 units/mg and is homogeneous as judged by analytical ultracentrifugation, polyacrylamide gel electrophoresis, and titration with methotrexate. The products of the first step of Edman degradation indicated a minimum purity of 79%. The reductase has a molecular weight of about 21500 on the basis of amino acid composition and 22100 +/- 300 from equilibrium sedimentation. It is not inhibited by antiserum to the Streptococcus faecium reductase (isoenzyme 2). Unlike the reductase of many other vertebrate tissues, the bovine enzyme is inhibited by mercurials rather than activated and it has a single pH optimum at both low and high ionic strength. However, the position of the pH optimum is shifted and the activity increased by increasing ionic strength. Automatic Edman degradation has been used to determine 34 of the amino-terminal 37 amino acid residues. Considerable homology exists between this region and the corresponding regions of the reductase from S. faecium and from Escherichia coli. This strengthens the idea that this region contributes to the structure of the binding site for dihydrofolate.
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PMID:Bovine liver dihydrofolate reductase: purification and properties of the enzyme. 0 45

Methotrexate and [(3)H]methotrexate were conjugated through a carbodiimide-catalyzed reaction to a 70,000 molecular weight poly(L-lysine) in molar ratios of approximately 13 to 1. The cellular uptake of labeled conjugate was far in excess of the uptake of free drug in cells that were either proficient or deficient in methotrexate transport. The conjugate markedly inhibited the growth of PRO(-)3 Mtx(RII) 5-3 Chinese hamster ovary cells, which are known to be drug resistant by virtue of a deficient methotrexate transport. The cells, however, were not inhibited by the same concentrations of free poly(Lys) and free drug. The 100-fold difference in drug concentration needed to inhibit the mutant cells and their corresponding wild type was totally abolished by exposing the methotrexate-resistant cells to methotrexate-poly(Lys). That the drug is carried into the resistant cells as intact drug-poly(Lys) is evident also from the fact that the conjugate is rendered inactive by brief trypsinization in vitro. Because the conjugate fails to inhibit dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP(+) oxidoreductase; EC 1.5.1.3) in vitro, it must be concluded that the strong growth inhibitory effect of the conjugate is due to the intracellular hydrolysis of its polymeric backbone, followed by the release inside the cell of a pharmacologically active form of methotrexate. Our date show that in methotrexate-resistant cells the intracellular release of active drug after uptake of conjugate is of the same order of magnitude as the uptake of free drug by transport-proficient cells and, hence, that the drug resistance due to deficient transport can be totally overcome.
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PMID:Conjugation of methotrexate to poly(L-lysine) increases drug transport and overcomes drug resistance in cultured cells. 27 1

In vitro studies were made on four synthetic polymeric derivatives of the antitumor agent methotrexate (MTX): 1) divinylether-maleic anhydride-MTX (DIVEMA-MTX), 2) poly-L-lysine-MTX (PL-MTX), 3) polyethyleneimine-MTX (PEI-MTX), and 4) carboxymethyl cellulose-MTX (CMC-MTX). They were tested for their ability to inhibit tetrahydrofolate dehydrogenase (dihydrofolate reductase). Their growth inhibition of murine L5178Y leukemia cells was also studied. 1wo of these polymers, DIVEMA-MTX and PEI-MTX, had similar or only slightly reduced activity compared to equivalent concentrations of MTX, whereas PL-MTX and CMC-MTX had significantly higher (1--3 logs) minimal inhibitory concentrations.
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PMID:In vitro inhibitory effects of polymer-linked methotrexate derivatives on tetrahydrofolate dehydrogenase and murine L5178Y cells. 28 2

The determination of the amino acid sequence of the dihydrofolate reductase from Escherichia coli RT500 is described. The sequence, comprising 159 residues, has been derived from automatic sequencing of the intact protein in conjunction with manual sequencing of lysine-blocked tryptic peptides, Staphylococcus aureus protease peptides, and alpha-lytic protease peptides. Comparison of the sequence with that of the dihydrofolate reductase from a methotrexate-resistant strain of E. coli (MB1428) shows that 145 of the residues are identical. The distribution of the differences along the length of the molecule is discussed.
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PMID:The amino-acid sequence of the dihydrofolate reductase of a trimethoprim-resistant strain of Escherichia coli. 32 5

Nalpha-(pteroyltetra (gamma-glutamyl))-lysine Sepharose was synthesized and shown to be a stable high capacity affinity matrix capable of bringing about the purification of Lactobacillus casei thymidylate synthetase to maximum specific activity from crude extracts in high yield. Under conditions optimal for binding of thymidylate synthetase, dihydrofolate reductase was not bound.
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PMID:Nalpha-(pteroyltetra (gamma-glutamyl))-lysine as a ligand for the purification of thymidylate synthetase by affinity chromatography. 71 78

A series of Nepsilon-poly-alpha-glutamyl and Nepsilon-polylysyl derivatives of Nalpha-pteroyllysine and Nalpha-homopteroyllysine, analogues of the naturally occurring gamma-polyglutamyl forms of folate, was prepared and tested as substrates for dihydrofolate reductase and as substrates and inhibitors of thymidylate synthetase. Nalpha-Dihydropteroyl-Nepsilon-(tri-alpha-glutamyl)lysine was 1.8 times as active as Nalpha-dihydropteroyl glutamate (dihydrofolate) as a substrate for L1210 murine leukemia dihydrofolate reductase. N-alpha-Dihydropteroyl-Nepsilon-(di-alpha-lysyl)lysine was 1.2 times as active as dihydrofolate in spite of its strong positive charge. The most active compound tested, Nepsilon-(tert-butyloxycarbonyl)lysine, was 3.5 times as active as dihydrofolate. None of the enzymatically prepared Nalpha-tetrahydropteroyllysine derivatives tested was as active as Nalpha-tetrahydropteroyl glutamate (tetrahydrofolate) as a substrate for E. coli thymidylate synthetase. However, there was a progressive increase in activity with the addition of each alpha-glutamyl residue, the Nepsilon-(penta-alpha-glutamyl)lysine being 88% as active as tetrahydrofolate. Nalpha-Tetrahydropteroyl-Nepsilon-(di-alpha-lysyl)lysine was the most active thymidylate synthetase substrate of the polylysine derivatives, being 67% as active as tetrahydrofolate. Addition or deletion of lysyl residues resulted in diminished activity. It is noteworthy that substrate activity is retained in spite of the positively charged poly(amino acid) side chain. None of the enzymatically prepared tetrahydrohomopteroyl derivatives tested was as active as Nalpha-tetrahydrohomopteroyl glutamate (tetrahydrohomofolate) as an inhibitor of E. coli thymidylate synthetase.
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PMID:Polyglutamyl and polylysyl derivatives of the lysine analogues of folic acid and homofolic acid. 79 72

Naturally occurring oligoglutamate derivatives of folic acid in extracts of Escherichia coli have been isolated on the basis of their inhibitory actions toward thymidylate synthetase and dihydrofolate reductase. The inhibitor of thymidylate synthetase has been identified as N-5-formyl-H4pteroyloligoglutamate (approximately 5 amino acid residues). It is 150-fold more inhibitory than the monoglutamate. Synthetic N-5-formyl derivatives containing 3 to 6 glutamyl residues were prepared and found to be 67- to 200-fold more inhibitory than the monoglutamate. N-5-Formimino-H4pteroyltriglutamate is one-twentieth as inhibitory as the corresponding N-5-formyl derivative. The inhibitor of mouse leukemia dihydrofolate reductase has been identified as N-10-formylpteropentaglutamate. It is approximately 7 times as inhibitory as N-10-formylpteroylmonoglutamate. It is 4,400 times as inhibitory toward mouse leukemia dihydrofolate reductase compared with the enzyme from E. coli. Lysine analogs of N-5-formyl-H4folate containing alpha0glutamyl groups in peptide linkage to the epsilon-amino group of lysine were relatively poor inhibitors of thymidylate synthetase. The inhibitory action of folic acid oligoglutamates on E. coli thymidylate synthetase was subject to reversal with 0.4 M NaCl, an effect that was more marked with various pteroyloligoglutamates than with H4homopteroylmonoglutamate and N-5, N-8-deaza-N-10-methylpteroylmonoglutamate.
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PMID:Inhibition of thymidylate synthetase and dihydrofolate reductase by naturally occurring oligoglutamate derivatives of folic acid. 109 83

Secretion of fusion proteins composed of cytoplasmic protein dihydrofolate reductase (DHFR) and the Escherichia coli alpha-haemolysin (HlyA) C-terminal sequence was examined through the haemolysin secretion machinery of E. coli. DHFR of various lengths was combined with the HlyA C-terminal region, and both secretion and DHFR activity of the fusions were measured. The secretion was found to be inversely correlated with the intracellular DHFR activity. Moreover, when one amino acid (Ile155) in a beta-sheet of the DHFR C-terminal region was replaced with Lys, the enzymatically active DHFR fusion protein was secreted into the medium. We discuss the possibility of a relationship between folding and secretion of HlyA-fused protein in the HlyA secretion system.
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PMID:Secretion of genetically-engineered dihydrofolate reductase from Escherichia coli using an E. coli alpha-hemolysin membrane translocation system. 136 20

A pentapeptide which potently inhibits primary IgE antibody formation, Asp-Ser-Asp-Gly-Lys (DSDGK), has been efficiently produced with the aid of the dihydrofolate reductase (DHFR) handle [M. Iwakura, et al. (1992) J. Biochem. 111, 37-45]. The genes coding fused proteins comprising DHFR and multimeric forms of DSDGK, namely, DHFR-(DSDGK)3, DHFR-(DSDGK)14, and DHFR-(DSDGK)28, were constructed and expressed in Escherichia coli. The C-terminal peptides attached to DHFR did not affect the expression or the function of the DHFR handle, even when the length of the C-terminal peptide was as long as 160 amino acid residues. The fused proteins were easily purified by methotrexate affinity chromatography, one of the major advantages of the DHFR handle. The fused proteins were digested with trypsin and the monomeric peptide, DSDGK, was purified by HPLC. The yields of the peptide were estimated to be 11, 43, and 99 mg per 1 gram of the total cell proteins from E. coli cells producing DHFR-(DSDGK)3, DHFR-(DSDGK)14, and DHFR-(DSDGK)28, respectively.
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PMID:Efficient production of a small peptide by expression as a multimeric form fused with the dihydrofolate reductase affinity handle. 147 25

A fluorescein derivative of the lysine analogue of folic acid, N alpha-pteroyl-N epilson-(4'-fluoresceinthiocarbamoyl)-L-lysine (PLF), was synthesized as a probe for dihydrofolate reductase (DHFR) and a membrane folate binding protein (m-FBP). Excitation of PLF at 282 nm and at 497 nm gave a fluorescence emission maximum at 518 nm. Binding of PLF to human DHFR or human placental m-FBP results in approximately a 20-fold enhancement in the magnitude of the fluorescence emission, suggesting that the ligand interacts with a hydrophobic region on these proteins. Additional evidence suggests that an energy transfer may occur between the pteridine and the fluorescein moieties. PLF binds to the active site of human DHFR since methotrexate (MTX) competes stoichiometrically and the denatured enzyme in the presence of PLF did not exhibit fluorescent enhancement. The dissociation constant for the fluorescein derivative with respect to human DHFR is 115 nM as compared to 111 nM for folic acid. The Ki value for the competitive inhibition of human DHFR by the fluorescent analogue of folic acid is 2.0 microM compared to 0.48 microM for folic acid. PLF was reduced to N alpha-(7,8-dihydropteroyl)-N epilson-(4'-fluoresceinthiocarbamoyl)-L-lysine (H2PLF) and assayed by the enzymatic conversion to the tetrahydro derivative. The Km value for human DHFR for the dihydrofolate analogue is 2.0 microM. The KD value for H2PLF to human DHFR is 47 nM as compared to 44 nM for dihydrofolate. The KD values for both H2PLF and PLF indicate that the fluorescein moiety does not significantly affect folate binding in enzyme binary complexes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Synthesis and biological evaluation of a fluorescent analogue of folic acid. 190 73


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