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 location of T4D phage-induced dihydrofolate reductase (dfr) has been determined in intact and incomplete phage particles. It has been found that phage mutants inducing a temperature-sensitive dfr (dfrts) procude heat-labile phage particles. The structural dfr produced by these ts mutants was shown to assume different configurations depending on the temperature at which the phage is assembled. Morphogenesis of incomplete phage particles lacking the gene 11 protein on their baseplates was found to be inhibited by reagents binding to dfr, such as antibodies to dfr. Further, cofactor molecules for dfr, such as reduced nicotinamide adenine dinucleotide phosphate and reduced nicotinamide adenine dinucleotide, also inhibited the step in morphogenesis involving the addition of gene 11 product. On the other hand, inhibitors of dfr, such as adenosine dephosphoribose, stimulated the addition of the gene 11 protein. It has been concluded that the phage-induced dfr is a baseplate component which is partially covered by the gene 11 protein. The properties of phage particles produced after infection of the nonpermissive host with the one known T4D mutant containing a nonsense mutation in its dfr gene suggested that these progeny particles contained a partial polypeptide, which was large enough to serve as a structural element.
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PMID:Bacteriophage T4 baseplate components. II. Binding and location of bacteriophage-induced dihydrofolate reductase. 0 May 16

A central eight-stranded beta-pleated sheet is the main feature of the polypeptide backbone folding in dihydrofolate reductase. The innermost four strands and two bridging helices are geometrically similar to but are connected in a different way from those in the dinucleotide binding domains found in nicotinamide-adenine dinucleotide-linked dehydrogenases. Methotrexate is bound in a 15-angstrom-deep cavity with the pteridine ring buried in a primarily hydrophobic pocket, although a strong interaction occurs between the side chain of aspartic acid 27 and N(1), N(8), and the 2-amino group of methotrexate.
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PMID:Dihydrofolate reductase: x-ray structure of the binary complex with methotrexate. 1 20

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, 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

The amino acid sequence of a trimethoprim-resistant dihydrofolate reductase (EC 1.5.1.3) specified by the R-plasmid R67 is described. The sequence was deduced from automatic and manual sequence analysis of the intact protein, the fragments produced by cyanogen bromide cleavage, and peptides derived from the largest cyanogen bromide fragment by digestion with trypsin, Staphylococcus aureus V8 proteus, chymotrypsin, and Lysobacter enzymogenes alpha-lytic protease. The complete sequence comprises 78 residues in a single polypeptide chain of molecular weight 8444. No evidence of heterogeneity was obtained, indicating that all subunits of the native enzyme are identical. Comparison of the sequence with that of all known dihydrofolate reductases shows no significant sequence homology.
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PMID:The amino acid sequence of the trimethoprim-resistant dihydrofolate reductase specified in Escherichia coli by R-plasmid R67. 38 58

The amino acid sequence of beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) has been compared to itself and to other proteins. Two segments, each of about 380 amino acids, comprising the first three-fourths of the polypeptide chain, were found to be very similar to each other. It is concluded that they are homologous. The carboxyl-terminal fourth has a high percentage of amino acid identities with dihydrofolate reductase of Escherichia coli, suggesting these sequences also are homologous. A model for the origin of beta-galactosidase is presented. The overall similarity of beta-galactosidase to lac repressor does not appear to be significant.
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PMID:On the evolution of beta-galactosidase. 41 4

The determination of the amino acid sequence of the dihydrofolate reductase (EC 1.5.1.3) from cells of the mouse lymphoma L1210 is described. The protein was cleaved by cyanogen bromide to produce the six fragments CB1 (residues 1 to 14), CB2 (residues 15 to 52), CB3 (residues 53 to 111), CB4 (residues 115 to 125), CB5 (residues 126 to 139), and CB6 (residues 140 to 186). One of the fragments, CB2, contained an internal homoserine derived from a methionine which was not cleaved by cyanogen bromide. The amino acid sequences and order of the cyanogen bromide fragments were determined by a combination of automatic and manual sequence analyses of the fragments and small peptides from tryptic, thermolytic, and Staphylococcus aureus protease digestions. The complete sequence comprises 186 residues in a single polypeptide chain of molecular weight 21,458. Comparison of the sequence of the L1210 dihydrofolate reductase with the sequences of the enzymes from Streptococcus faecium, escherichia coli RT500, and Lactobacillus casei indicates that all enzymes show some homology, which is strongest in the regions forming the substrate binding cleft.
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PMID:The amino acid sequence of dihydrofolate reductase from the mouse lymphoma L1210. 76 74

A single polypeptide chain containing two dihydrofolate reductase (DHFR) sequences from Escherichia coli was constructed to determine if a repeat sequence fusion protein could be expressed in an active form. The possibility that intersequence interactions could play a significant role for this enzyme is suggested by the results of Hall and Frieden (1989, Proc. Natl Acad. Sci. USA, 86, 3060-3064) who observed a substantial decrease in the yield of active enzyme when folded in the presence of a large C-terminal fragment. The fusion protein [DHFR(Cys152Glu)--Ile--DHFR (Met1Gln)] was efficiently expressed in E. coli cells and has an activity which is twice that of the wild-type enzyme in the standard assay. The Michaelis constants of the fusion protein for the substrate, dihydrofolate and the cofactor, NADPH, are essentially unchanged from those of the wild-type protein. The urea-induced in vitro unfolding reaction of the fusion protein at low concentrations was found to be fully reversible and follow a three state model, suggesting that the two domains unfold independently. At higher protein concentrations the unfolding transition broadened and shifted to a higher urea concentration. Size-exclusion chromatography results are consistent with the formation of aggregates at the higher protein concentration, even in the absence of denaturant.
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PMID:Construction and characterization of a single polypeptide chain containing two enzymatically active dihydrofolate reductase domains. 128 60

The transit peptide of the lumenal 33-kDa oxygen-evolving polypeptide (OEE1) is capable of directing the import and targeting of the foreign protein dihydrofolate reductase (DHFR) to the thylakoid lumen. The import results from the first part of this study indicate that methotrexate cannot block the import or intraorganellar targeting of OEE1-DHFR in chloroplasts in contrast to that reported for the import of cytochrome oxidase subunit IV (COXIV)-DHFR in mitochondria. These results suggest that the fusion of the OEE1 transit sequence to DHFR affected the protein's methotrexate binding properties. We further examined and compared the transport characteristics of a number of carboxyl-terminal truncated native chloroplast precursors to determine whether carboxyl domains contribute to the import and intraorganellar targeting mechanism of these proteins. The plastid precursors chosen for this study are targeted to one of the following chloroplast compartments: the stroma, the thylakoid membrane, and the lumen. In most cases, removal of carboxyl domains had a dramatic effect on one or more stages of the translocation pathway, such as import, processing, and intraorganellar targeting. The effects of carboxyl deletions varied from precursor to precursor and were dependent on the extent of the deletion. These combined results suggest that carboxyl domains in the mature part of the proteins can influence the function of the transit peptide, and as a result play an important role in determining the import and targeting competence of chloroplast precursors.
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PMID:Carboxyl-terminal sequences can influence the in vitro import and intraorganellar targeting of chloroplast protein precursors. 132 Nov 29

A neu/erb B2 ligand growth factor (NEL-GF) was purified to homogeneity from bovine kidney by a procedure involving ammonium sulfate fractionation (35-70% saturation) followed by sequential column chromatography on DEAE-cellulose (DE52), Sulfadex (sulfated Sephadex G-50), heparin-Sepharose 4B, and Superdex 75 (fast protein liquid chromatography). NEL-GF was found to be a 25-kDa polypeptide according to the analysis by gel filtration on Superdex 75 and 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NEL-GF stimulated the tyrosine-specific autophosphorylation of the neu/erb B2 gene product purified by immunoabsorbent and tyrosine-specific phosphorylation of the neu/erb B2 gene product in intact dihydrofolate reductase (DHFR/G-8 cells (NIH 3T3 cells transfected with rat c-neu). NEL-GF also down-regulated the cell surface neu/erb B2 gene product in DHFR/G-8 cells. NEL-GF was mitogenic toward NIH 3T3 cells, DHFR/G-8 cells, A431 cells (human epidermoid carcinoma cells), and SK-BR-3 cells (human breast carcinoma cells) but inactive toward bovine aorta endothelial cells. NEL-GF was sensitive to 0.1% trifluoroacetic acid but resistant to 5% beta-mercaptoethanol and appeared to be distinct from a neu protein-specific activating factor (Davis, J. G., Hamuro, J., Shim, C. Y., Samanta, A., Greene, M. I., and Dobashi, K. (1991) Biochem. Biophys. Res. Commun. 179, 1536-1542) and a 30-kDa glycoprotein which competed with a monoclonal antibody for binding to the neu/erb B2 gene product (Lupu, R., Colomer, R., Zugmaier, G., Sarup, J., Shepard, M., Slamon, D., and Lippman, M. E. (1990) Science 249, 1552-1555).
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PMID:Purification and characterization of the neu/erb B2 ligand-growth factor from bovine kidney. 135 Jul 85


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