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

---

Query: EC:1.5.1.3 (dihydrofolate reductase)
5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The human ileum neurokinin NK2 receptor has been stably expressed in Chinese hamster ovary (CHO) cells using the dihydrofolate reductase (DHFR) expression system. Amplified cell populations expressing approximately 7 x 10(5) NK2 receptors/cell were selected in the presence of the DHFR inhibitor methotrexate. Cross-linking of [125I]NKA to NK2 receptor transfected cells revealed a specifically labeled protein of apparent molecular weight 64 kDa by SDS-polyacrylamide gel electrophoresis. This protein was deglycosylated by the enzymes N-glycosidase F and endoglycosydase F to a protein of apparent molecular weight of 39 kDa. The NK2 receptor was solubilized in an active form from CHO cell membranes using the zwitterionic detergent CHAPS. This method represents a valuable approach for the production of significant amounts of NK2 receptor protein from mammalian cells.
...
PMID:Biochemical characterization and solubilization of human NK2 receptor expressed in Chinese hamster ovary cells. 838 63

The Cryptococcus neoformans dihydrofolate reductase (DHFR) gene has been isolated from cDNA and genomic DNA libraries. The 690-base pair coding sequence codes for a 25,152-Da protein, which is the largest monofunctional DHFR yet reported. The gene contains two introns, and several putative regulatory sequences have been identified. The coding sequence was placed in a pUC-based expression vector, which expresses C. neoformans DHFR in Escherichia coli at a level of about 5% of the total soluble extract. The expressed DHFR was purified to homogeneity by methotrexate-Sepharose affinity chromatography, followed by anion exchange chromatography on Q-Sepharose. On SDS-polyacrylamide gel electrophoresis, the purified enzyme migrates as a single protein with apparent mass of 28 kDa. The molecular weight, as determined by electrospray mass spectral analysis, and the amino-terminal sequence are in accord with what was predicted from the DNA sequence. Steady state kinetic parameters, effects of pH, salts, and inhibition constants of several anti-folates have been determined.
...
PMID:Cloning, expression, and characterization of Cryptococcus neoformans dihydrofolate reductase. 847 32

The dihydrofolate reductase (DHFR) domain of the bifunctional dihydrofolate reductase-thymidylate synthase from Leishmania major has been subcloned and expressed as a soluble protein in Escherichia coli strain PA414 harboring plasmid pLMDHFR. Homogeneous L. major DHFR was obtained by chromatography on methotrexate-Sepharose followed by DE52. The purified enzyme migrated as a single 25-kDa protein on SDS-PAGE. The native molecular weight was determined to be 26 kDa, indicating that the isolated domain is a monomer. N-terminal sequence analysis revealed that serine, the second amino acid in the coding sequence, was the N-terminal amino acid of the protein. The enzyme showed a pH optimum similar to that of the bifunctional protein. For purified DHFR, the Km values were <1.0 microM for H2folate and <1.0 microM for NADPH. The kcat of the most active DHFR preparation was 5 s-1. The Km and kcat values were similar to those of the bifunctional enzyme.
...
PMID:Functional expression of the dihydrofolate reductase domain of Leishmania major dihydrofolate reductase-thymidylate synthase bifunctional protein. 881 31

The hamster polyomavirus (HaPV) is associated with spontaneously appearing skin epithelioma of the Syrian hamster Z3 strain. Virus particles prepared from the skin epithelioma cause lymphoma and leukemia when injected into newborn hamsters from a distinct Syrian hamster colony (HaP); in contrast to the skin epithelioma the hemopoietic tumors are virus free but accumulate viral DNA. To study the humoral immune response of HaPV-infected Z3 hamsters we produced recombinant HaPV proteins in Escherichia coli as beta-galactosidase-, TrpE- and dihydrofolate reductase-fusion proteins or as non-fused proteins. Recombinant plasmids carried segments of all putative early and late HaPV proteins. The recombinant proteins were detected in stained SDS polyacrylamide gels and in Western blots using monoclonal anti-TrpE and anti-beta-galactosidase antibodies and sera of HaPV-infected hamsters. Sera from HaPV-infected Z3 hamsters and crude lysates of all clones were applied to Western blots to characterize the humoral immune response in the animals. HaPV-specific antibodies were found to be directed against early protein segments translated from the first common exon and from the second unique exon of LT and MT, resp., as well as against the late proteins VP1 and VP2/3. The almost complete VP2 was recognized by all sera whereas VP1 was detected only by a half of the sera. Our data suggest the presence of at least 2 immunodominant regions in VP2, one in the C-terminal VP1 and at least 4 in early proteins.
...
PMID:Hamster polyomavirus-encoded proteins: gene cloning, heterologous expression and immunoreactivity. 888 64

R67 dihydrofolate reductase (DHFR) provides resistance to the antibacterial drug trimethoprim. This R-plasmid-encoded enzyme does not share any homology with chromosomal DHFR. A recent crystal structure of active, homotetrameric R67 DHFR (Narayana, N., Matthews, D. A., Howell, E. E., and Xuong, N.-H. (1995) Nat. Struct. Biol. 2, 1018-1025) indicates that a single active site pore traverses the length of the molecule. Since the center of the pore possesses exact 222 symmetry, site-directed mutagenesis of residues in the pore will produce four mutations/active site. To break this inevitable symmetry, four copies of the gene have been linked in frame to create an active monomer possessing the essential tertiary structure of native tetrameric R67 DHFR. The protein product, quadruple R67 DHFR, is 4 times the molecular mass of native R67 DHFR in SDS-polyacrylamide gel electrophoresis and is monomeric under nondenaturing conditions as measured by sedimentation equilibrium experiments. The catalytic activity of quadruple R67 DHFR is decreased only slightly when compared with native R67 DHFR. Folding of quadruple R67 DHFR is completely reversible at pH 5. However, at pH 8, folding is not fully reversible; this is likely due to a competition between productive intramolecular versus nonproductive intermolecular domain association. The production of a fully active, monomeric R67 DHFR variant will enable the design of more meaningful site-directed mutants where single substitutions per active site pore can be generated.
...
PMID:Redesigning the quaternary structure of R67 dihydrofolate reductase. Creation of an active monomer from a tetrameric protein by quadruplication of the gene. 891 Apr 13

We have cloned and expressed in Escherichia coli a 702-base pair gene coding for the dihydrofolate reductase (DHFR) domain of the bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Trypanosoma cruzi. The DHFR domain was purified to homogeneity by methotrexate-Sepharose chromatography followed by an anion-exchange chromatography step in a mono Q column, and displayed a single 27-kDa band on SDS-PAGE. Gel filtration showed that the catalytic domain was expressed as a monomer. Kinetic parameters were similar to those reported for the wild-type bifunctional enzyme with Km values of 0.75 microM for dihydrofolate and 16 microM for NADPH and a kcat value of 16.5 s-1. T. cruzi DHFR is poorly inhibited by trimethoprim and pyrimethamine and the inhibition constants were always lower for the bifunctional enzyme. The binding of methotrexate was characteristic of a class of inhibitors that form an initial complex which isomerizes slowly to a tighter complex and are referred to as 'slow, tight-binding' inhibitors. While the slow-binding step of inhibition was apparently unaffected in the individually expressed DHFR domain, the overall inhibition constant was two-fold higher as a consequence of the superior inhibition constant value obtained for the initial inhibitory complex.
...
PMID:Expression and characterization of the Trypanosoma cruzi dihydrofolate reductase domain. 892 5

Chinese hamster dihydrofolate reductase (ch-DHFR) was overexpressed in Escherichia coli DH5 alpha under the transcriptional control of PRPL promoters regulated by temperature-sensitive repressors. The desired recombinant product is soluble and constitutes about 30% of the total soluble proteins of the bacterial cell. With repeated cycles of freezing and thawing as a first step, the purification of the recombinant ch-DHFR to homogeneity requires only one further step, gel filtration on a Sephadex G-75 column with 85-90% enzyme recovery, two to three times higher than that obtained with the commonly used affinity chromatography on a methotrexate-Sepharose column. The purified enzyme migrates as a single protein band on SDS-polyacrylamide gel electrophoresis with approximate mass of 23 kDa, in accord with that calculated from the DNA sequence. The initiation methionine residue at the N-terminus of the enzyme is completely removed by E. coli methionine aminopeptidase as judged by amino-terminal analysis. The steady-state kinetic parameters, dissociation constants for binary complexes of dihydrofolate, NADPH, and methotrexate with ch-DHFR, and the inhibitor constant of methotrexate have also been determined. The enzyme is activated about 4-fold in 3 M urea and about 2.5-fold in 0.5 M guanidine hydrochloride.
...
PMID:Soluble expression in Escherichia coli, one-step purification, and characterization of Chinese hamster dihydrofolate reductase. 905 90

Dihydrofolate reductase is an essential bacterial enzyme necessary for the maintenance of intracellular folate pools in a biochemically active reduced state. In this report, the Mycobacterium avium folA gene was identified by functional genetic complementation, sequenced, and expressed for the first time. It has an open reading frame of 543 bp with a G + C content of 73%. The translated polypeptide sequence shows 58% identity to the consensus sequence of the conserved regions from eight other bacterial dihydrofolate reductases. Recombinant M. avium dihydrofolate reductase was expressed actively in Escherichia coli, and SDS-PAGE analysis revealed a 20 kDa species, agreeable with that predicted from the polypeptide sequence:
...
PMID:Identification and cloning of the Mycobacterium avium folA gene, required for dihydrofolate reductase activity. 936 62

Mucopolysaccharidosis type IIIA (MPS IIIA, Sanfilippo A syndrome) is a lysosomal storage disease that causes a profound neurological deterioration. The disorder is caused by a deficiency of the lysosomal enzyme sulphamidase which is a requisite for the degradation of heparan sulphate. To facilitate the development of enzyme-replacement strategies for MPS IIIA patients, we have constructed a high-level expression system for recombinant human sulphamidase in Chinese hamster ovary (CHO) cells. An expression construct containing a methotrexate-resistant dihydrofolate reductase (DHFR) gene allowed amplification of expression levels from less than 1 mg of sulphamidase per litre of culture medium to approx. 15 mg/l. Unlike many cell lines made by gene amplification in DHFR-deficient CHO cells, and utilizing the normal DHFR gene, these cell lines appeared to be stable in the absence of selective pressure. Recombinant human sulphamidase was purified from unamplified and amplified cell lines. The native enzyme was found to be a dimer of 115 kDa. Denaturing and reducing SDS/PAGE revealed a subunit size of 62 kDa. Kinetic analysis demonstrated that the recombinant enzyme had broadly similar kinetic characteristics to sulphamidase purified from liver. Recombinant human sulphamidase was able to correct the storage phenotype of MPS IIIA fibroblasts after endocytosis via the mannose-6-phosphate receptor.
...
PMID:Recombinant human sulphamidase: expression, amplification, purification and characterization. 940 87

Solving the crystallographic structure of the ring-shaped heptamer formed by protective antigen (PA), the B moiety of anthrax toxin, has focused attention on understanding how this oligomer mediates membrane translocation of the toxin's A moieties. We have developed an assay for translocation in which radiolabeled ligands are bound to proteolytically activated PA (PA63) at the surface of CHO or L6 cells, and translocation across the plasma membrane is induced by lowering the pH. The cells are then treated with Pronase E to degrade residual surface-bound material, and protected ligands are quantified after fractionation by SDS-PAGE. Translocation was most efficient (35%-50%) with LFN, the N-terminal PA binding domain of the anthrax lethal factor (LF). Intact LF, edema factor (EF), or fusion proteins containing LFN fused to certain heterologous proteins [the diphtheria toxin A chain (DTA) or dihydrofolate reductase (DHFR)] were less efficiently translocated (15%-20%); and LFN fusions to several other proteins were not translocated at all. LFN with different N-terminal residues was found to be degraded according to the N-end rule by the proteasome, and translocation of LFN fused to a mutant form of DHFR with a low affinity for methotrexate (MTX) protected cells from the effects of MTX. Both results are consistent with a cytosolic location of protected proteins. Evidence that a protein must unfold to be translocated was obtained in experiments showing that (i) translocation of LFNDTA was blocked by introduction of an artificial disulfide into the DTA moiety, and (ii) translocation of LFNDHFR and LFNDTA was blocked by their ligands (MTX and adenine, respectively). These results demonstrate that the acid-induced translocation by anthrax toxin closely resembles that of diphtheria toxin, despite the fact that these two toxins are unrelated and form pores by different mechanisms.
...
PMID:Characterization of membrane translocation by anthrax protective antigen. 984 79


<< Previous 1 2 3 4 5 Next >>

---