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 uptake of methotrexate by KB cells was observed to be dependent on time, temperature, and concentration of extracellular methotrexate. The Kd for methotrexate surface binding to KB cells was approximately 200 nM. Following exposure of KB cells to trace quantities of [3H]methotrexate for periods ranging from 6 min to 24 h, the cellular methotrexate was progressively formed into methotrexate polyglutamates and was bound to dihydrofolate reductase as well as to a particulate folate binding protein. To further study the mechanism of methotrexate uptake in KB cells, the N-hydroxysuccinimide ester of methotrexate was used to covalently label the surface of KB cells and to inhibit transport of methotrexate. The N-hydroxysuccinimide ester of methotrexate was bound to a species of protein with an apparent molecular weight of 160,000 in 1% (v/v) Triton X-100 that bound folic acid and was specifically precipitated by antiserum raised against the previously purified high-affinity folate binding protein (the folate receptor) from human KB cells. In addition, trypsin was utilized to remove surface-accessible covalently bound methotrexate. The amount of covalently bound methotrexate that could be released by trypsin initially decreased on incubation at 37 degrees C, suggesting that the methotrexate and binding protein were internalized. However, with time, trypsin could again release the covalently bound methotrexate, suggesting that the binding protein cycles from the external cell surface to the inside of the cell and out again.
...
PMID:Role of the membrane-associated folate binding protein (folate receptor) in methotrexate transport by human KB cells. 255 22

The HSITE program proposed in the previous paper was written to define putative ligand-point regions that could be found at protein surfaces. These regions would represent positions for hydrogen-bonding acceptor and donor atoms. In this paper the prediction of the location of these regions is compared with: (1) the position of the oxygen atoms of water molecules on the hydrated proteins myoglobin and plastocyanin; and (2) the position of hydrogen-bonded atoms in methotrexate and NADPH co-crystallized with dihydrofolate reductase, and in amidinophenyl-pyruvate co-crystallized with trypsin. The prediction of ligand-point regions is in agreement with the surveys of experimental data for water-molecule positions in protein crystals and with the positions of hydrogen-bonding atoms found in co-crystallized ligands.
...
PMID:Automated site-directed drug design: the prediction and observation of ligand point positions at hydrogen-bonding regions on protein surfaces. 256 76

In this paper the spacer skeleton concept is used to produce molecular graphs of putative ligands for binding sites. The skeletons are transformed into molecular templates within the constraints of the accessible surface of the ligand-binding site. A distance-matrix method is used to compare ligand points with vertices of the spacer skeleton through a permutation of all possible correspondences. A tolerance parameter is used to screen for poor matches. As a result, a small number of matched vertices and ligand points are produced. These are fitted into the site by a constrained optimization routine using an analytical function. Ligand points fall within the site and are optimally positioned adjacent to the corresponding site points; other vertices of the spacer skeleton lying beneath the accessible surface of the site are clipped off. A molecular template is thereby formed with its vertices linked to the ligand points. The final step is to verify that the bonding integrity of the skeleton remains. The computational methods outlined in this paper have been tested at two binding sites: the pteridine binding site in dihydrofolate reductase and the amidinophenylpyruvate site of trypsin. Molecular graphs for both sites were generated automatically; they showed strong similarity to those of the natural ligands.
...
PMID:Automated site-directed drug design: the formation of molecular templates in primary structure generation. 256 78

Nuclei rapidly purified from yeast Saccharomyces cerevisiae using a cytochalasin B enucleation procedure are substantially free of cell wall, secretory vesicle, plasma membrane vacuolar, and cytoplasmic and mitochondrial contamination. Nuclei obtained in this manner in high yield retain transport properties comparable to nuclei in situ. An in vitro nuclear import assay system has been developed using isolated nuclei and radiolabeled proteins prepared by a coupled in vitro transcription/translation system. Both wild-type SV40 large T-antigen and nucleoplasmin are imported into isolated yeast nuclei, whereas a missense cytoplasmic mutant of the SV40 large T-antigen (Lys128----Thr) and cytoplasmic dihydrofolate reductase are not imported. Association and import of these proteins in a time- and signal-dependent manner resulted in their protection from trypsin that was tethered to agarose beads. Greater than 70% of the labeled protein harboring a karyophilic signal was imported in a reaction that could be blocked by prior treatment of nuclei with trypsin-agarose. Nuclear accumulation of SV40 large T-antigen and nucleoplasmin was unidirectional, ATP and Ca2+ dependent, and was not inhibited by a vast excess of exogenous nonnuclear competitor protein. This system provides an important new tool in combination with powerful yeast genetic methods for analysis of the mechanism and the apparatus for transport at the nuclear envelope.
...
PMID:In vitro translocation through the yeast nuclear envelope. Signal-dependent transport requires ATP and calcium. 268 Nov 87

Hybrid genes were constructed. One, ompA153-dfr, encoded the precursor of the 325 residue Escherichia coli outer membrane protein OmpA up to residue 153 which was fused to the complete 186-residue dihydrofolate reductase of the mouse. The other, ompA219-lacZ, coded for the same precursor up to residue 219 which was fused to 1017 COOH-terminal residues of the 1023-residue subunit of the beta-galactosidase of E. coli. Full expression of the ompA153-dfr gene caused accumulation of its precursor and of that of the chromosomally encoded OmpA protein. When the amount of product was reduced, no pro-OmpA and very little pro-hybrid protein accumulated. The precursor was processed and the mature protein was fully accessible to trypsin in permeabilized cells. Expression of the ompA219-lacZ gene led to the presence of the hybrid protein at only 20-30% of the amount expected. About 20% of it appeared to be incorporated in the outer membrane. All of the hybrid was quantitatively accessible to trypsin in permeabilized cells. When the hybrid gene was overexpressed, the protein was found associated with the plasma membrane in the cytosol. It is concluded that both beta-galactosidase and dihydrofolate reductase could quantitatively traverse the plasma membrane, provided the amounts synthesized were sufficiently small.
...
PMID:Dihydrofolate reductase (mouse) and beta-galactosidase (Escherichia coli) can be translocated across the plasma membrane of E. coli. 314 14

Most mitochondrial proteins are encoded in the nucleus and synthesized in the cytoplasm as larger precursors containing NH2-terminal 'leader' peptides. To test whether a leader peptide is sufficient to direct mitochondrial import, we fused the cloned nucleotide sequence encoding the leader peptide of the mitochondrial matrix enzyme ornithine transcarbamylase (OTC) with the sequence encoding the cytosolic enzyme dihydrofolate reductase (DHFR). The fused sequence, joined with SV40 regulatory elements, was introduced along with a selectable marker into a mutant CHO cell line devoid of endogenous DHFR. In stable transformants, the predicted 26-K chimeric precursor protein and two additional proteins, 22 K and 20 K, were detected by immunoprecipitation with anti-DHFR antiserum. In the presence of rhodamine 6G, an inhibitor of mitochondrial import, only the chimeric precursor was detected. Immunofluorescent staining of stably transformed cells with anti-DHFR antiserum produced a pattern characteristic of mitochondrial localization of immunoreactive material. When the chimeric precursor was synthesized in a cell-free system and incubated post-translationally with isolated rat liver mitochondria, it was imported and converted to a major product of 20 K that associated with mitochondria and was resistant to proteolytic digestion by externally added trypsin. Thus, both in intact cells and in vitro, a leader sequence is sufficient to direct the post-translational import of a chimeric precursor protein by mitochondria.
...
PMID:A leader peptide is sufficient to direct mitochondrial import of a chimeric protein. 389 25

The amino acid sequence of the NADP+-dependent enzyme ovine 6-phosphogluconate dehydrogenase has been determined by conventional direct protein sequence analysis of peptides resulting from digestion of the protein with trypsin and chemical cleavages with cyanogen bromide, hydroxylamine, and iodosobenzoic acid. The polypeptide contains 466 amino acids and its NH2 terminus is acetylated. The Candida utilis enzyme is inactivated by reaction of pyridoxal phosphate with two lysine residues (Minchiotti, L., Ronchi, S., and Rippa, M. (1981) Biochim. Biophys. Acta 657, 232-242). These residues are conserved in the ovine enzyme. In contrast to NAD+ dehydrogenases which have weakly related sequences and spatially related folds in their nucleotide-binding sites, no significant sequence homologies were detected between 6-phosphogluconate dehydrogenase and any of three other NADP+-requiring enzymes, glutamate dehydrogenase, p-hydroxybenzoate hydroxylase, and dihydrofolate reductase. This is in accord with structural data that show no spatial relationship between NADP+-binding sites in these enzymes.
...
PMID:Amino acid sequence of ovine 6-phosphogluconate dehydrogenase. 668 25

The complete covalent structure of dihydrofolate reductase from chicken liver is described. The S-carboxymethylated protein was subjected to cleavage by cyanogen bromide which produced five fragments. Fragment CB2 contained an internal homoserine residue which was not cleaved by cyanogen bromide. Sequences and ordering of the cyanogen bromide fragments were established by means of automated sequencer analyses of the fragments and from smaller peptides generated by proteolysis with trypsin and staphylococcal protease. The covalent structure of the single polypeptide chain comprises 189 residues of molecular weight 21,651. The chicken liver enzyme is homologous to that from L1210 cells and shows regions of homology to dihydrofolate reductases from Streptococcus faecium, Escherichia coli, and Lactobacillus casei. These homologous regions in the chicken liver enzyme are primarily related to conserved amino acid residues implicated in the binding of NADPH and methotrexate by bacterial dihydrofolate reductases.
...
PMID:Primary structure of chicken liver dihydrofolate reductase. 676 36

Chicken liver dihydrofolate reductase is rapidly and stoichiometrically inactivated by a substituted 4,6-diaminodihydrotriazine containing a terminal benzenesulfonylfluoride (DTBSF). The substrate dihydrofolate largely prevents the enzyme inhibition by DTBSF, whereas NADPH had no effect, indicating that the inhibitor is bound at or near the folate site. Using radiolabeled inhibitor between 1.0 and 1.2 mol was incorporated/mol of enzyme (Mr = 21,651), following treatment with 8 M urea at 75 degrees C. Digestion of the maleylated, radiolabeled inhibitor-enzyme complex with trypsin and subsequent gel filtration on Sephadex G-50 SF yielded a single major peak of radioactivity. The covalently modified limited tryptic peptide was subsequently purified to homogeneity using high performance liquid chromatography. The radiolabeled tryptic peptide had the following sequence: Asn-Glu-Tyr (DTBS)-Lys-Tyr-Phe-Gln-Arg (residues 29-36). Automated Edman degradation of this peptide revealed that the radioactivity derived from the inhibitor was released at Step 3, identifying tyrosine-31 as the specific site of covalent attachment of the affinity label.
...
PMID:Affinity labeling of chicken liver dihydrofolate reductase by a substituted 4,6-diaminodihydrotriazine bearing a terminal sulfonyl fluoride. 702 56

The primary structure of dihydrofolate reductase from bovine liver has been established by Edman degradation of the intact carboxymethylated protein and of peptides obtained from the protein by the action of cyanogen bromide, trypsin, and the protease from Staphylococcus aureus, respectively. Since separation of some of the peptide mixtures by classical methods proved impossible, new systems were developed for the use of high-performance liquid chromatography to separate such mixtures. Some of the cleavage procedures used to obtain peptides gave atypical results at certain peptide bonds. The results are discussed in terms of the residues involved in these unexpectedly resistant or sensitive bonds. The sequence of the bovine liver enzyme is compared with those published for the enzyme from other sources, and known or probable functions of invariant residues are described. Sequences of vertebrate and bacterial reductases are compared and contrasted, and a possible role is considered for the residues which are invariant in bacterial reductases, but different in vertebrate reductases, in determining the selective inhibitory action of trimethoprim on bacterial reductases.
...
PMID:Structure of dihydrofolate reductase: primary sequence of the bovine liver enzyme. 711 69


<< Previous 1 2 3 4 Next >>

---