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)

Four cell lines, SK-N-SH, SK-N-MC, SK-N-BE(2), and IMR-32, established in vitro from tumor tissue of patients with neuroblastoma were analyzed by trypsin-Giemsa banding methods. In two of the lines a large, abnormally staining chromosome region was observed. This "homogeneously staining region" (HSR) was considerably longer than any of the bands present in normal human cells and, as revealed by both G- and Q-banding, stained with an intermediate intensity. It was located on chromosomes No 6, 10, 17, or 19 of the SK-N-BE(2) cell line and on chromosome No 1 of the IMR-32 line. In concurrent studies, long HSR's were also observed in Chinese hamster sublines that had been exposed to and had developed high levels of resistance to methotrexate or methasquin and high levels of activity of target enzyme dihydrofolate reductase. For several sublines with the highest levels of enzyme activity, approximately 2% of the total cell protein was dihydrofolate reductase. Of 13 independently derived sublines with acquired resistance to antifolate, only those 7 with greater than 100-fold increases in enzyme activity consistently exhibited HSR's. These regions comprised 2-5% of the total length of the chromosome complement and were specifically localized, as demonstrated by G-banding. Analysis of chromosome replication patterns of the HSR in human neuroblastoma and in drug-resistant Chinese hamster cells by tritiated thymidine radioautography indicated that the long, abnormally staining region replicated relatively rapidly and synchronously and terminated replication before the midpoint of the S phase. The HSR thus appeared to represent a novel chromosome abnormality that may be present in cells with specialized functions. Drug-resistant Chinese hamster cells were characterized by overproduction of target enzyme, whereas human neuroblastoma cells had phenotypes of normal neuronal cells. Whether the HSR is transcriptionally active was not elucidated.
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PMID:A novel chromosome abnormality in human neuroblastoma and antifolate-resistant Chinese hamster cell lives in culture. 6 55

The complete amino acid sequence of dihydrofolate reductase from an amethopterin-resistant strain of Lactobacillus casei has been determined by sequence analysis of peptides produced by cleavage with cyanogen bromide, trypsin, staphylococcal protease, and myxobacter protease. Comparison of this sequence with those of reductases from other bacterial sources shows that the enzymes are homologous. The Lactobacillus casei reductase sequences shows a 29% sequence identity with that of the Escherichia coli enzyme and a 34% identity with the sequence of the enzyme from Streptococcus faecium. The NH2-terminal 68 residues of the L. casei reductase show a 54% sequence identity with that of the enzyme from S. faecium.
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PMID:Dihydrofolate reductase from amethopterin-resistant Lactobacillus casei. Sequences of the cyanogen bromide peptides and complete sequences of the enzyme. 9 27

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 major form of dihydrofolate reductase from a methotrexate-resistant mutant (strain A) of Streptococcus faecium var. Durans has been purified on a large scale. Amino acid analysis of this form of the enzyme (isoenzyme 2) reveals an absence of cystine or cysteine, and sedimentation studies indicate a molecular weight of 20,800. The NH2-terminal sequence was determined by Edman degradation of the intact protein and the COOH terminus by selective tritiation and by carboxypeptidase treatment. After the action of trypsin on the citraconylated protein, seven of the expected nine peptides were purified from the digest, and after cyanogen bromide treatment of the unmodified protein, all seven of the anticipated peptides were isolated. The amino acid composition of all of these peptides has been established as well as their complete or partial sequences. From the results it was possible to order these peptides within the sequence and to establish the sequence of the NH2-terminal 60 residues and the COOH-terminal 11 residues.
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PMID:The structure of the mutant dihydrofolate reductase from Streptococcus faecium. Partial sequence and order of the limited tryptic and cyanogen bromide peptides. 115 Jun 47

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 new computer program is described, which positions small molecules into clefts of protein structures (e.g. an active site of an enzyme) in such a way that hydrogen bonds can be formed with the enzyme and hydrophobic pockets are filled with hydrophobic groups. The program works in three steps. First it calculates interaction sites, which are discrete positions in space suitable to form hydrogen bonds or to fill a hydrophobic pocket. The interaction sites are derived from distributions of nonbonded contacts generated by a search through the Cambridge Structural Database. An alternative route to generate the interaction sites is the use of rules. The second step is the fit of molecular fragments onto the interaction sites. Currently we use a library of 600 fragments for the fitting. The final step in the present program is the connection of some or all of the fitted fragments to a single molecule. This is done by bridge fragments. Applications are presented for the crystal packing of benzoic acid and the enzymes dihydrofolate reductase and trypsin.
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PMID:The computer program LUDI: a new method for the de novo design of enzyme inhibitors. 158 40

The amino acid sequence of mouse dihydrofolate reductase was permuted circularly at the level of the gene. By transposing the 3'-terminal half of the coding sequence to its 5' terminus, the naturally adjacent amino and carboxyl termini of the native protein were fused, and one of the flexible peptide loops at the protein surface was cleaved. The steady-state kinetic constants, the dissociation constants of folate analogues, and the degree of activation by both mercurials and salt as well as the resistance toward digestion by trypsin were almost indistinguishable from those of a recombinant wild-type protein. Judged by these criteria, the circularly permuted variant has the same active site and overall structure as the wild-type enzyme. The only significant difference was the lower stability toward guanidinium chloride and the lower solubility of the circularly permuted variant. This behavior may be due to moving a mononucleotide binding fold from the interior of the sequence to the carboxyl terminus. Thus, dihydrofolate reductase requires neither the natural termini nor the cleaved loop for stability, for the conformational changes that accompany catalysis as well as the binding of inhibitors, and for the folding process.
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PMID:A fully active variant of dihydrofolate reductase with a circularly permuted sequence. 173 18

We characterized a highly purified preparation of the chromosomally encoded dihydrofolate reductase (DHFR) from a trimethoprim-susceptible (Tmp8; strain MAP) and two trimethoprim-resistant (TmpR) strains (MAP/47 and MAP/42) of Haemophilus influenzae. The enzymes were purified between 650- and 3000-fold by gel-filtration and dye-ligand chromatography. The apparent molecular mass of the three proteins was 18400 Da by PAGE under denaturing and nondenaturing conditions. Total enzyme activity was greater in all fractions from the TmpR strains compared with the Tmp8 isolate. The three enzymes had a similar Km for dihydrofolate (7, 9 and 5 microM) and NADPH (2, 5 and 6 microM). However, the Tmp IC50 (the concentration necessary for 50% inhibition of DHFR activity) for the Tmp8 strain MAP was 0.001 microM, whereas DHFR from the TmpR strains MAP/47 and MAP/42 had values of 0.1 microM and 0.3 microM respectively. The methotrexate IC50 of the MAP/42 DHFR was 0.06 microM in comparison with the enzyme from MAP (0.008 microM) and MAP/47 (0.007 microM). Isoelectric focusing indicated that the DHFR from MAP/42 had a different isoelectric point (pI 7.6) compared with the enzymes from MAP and MAP/47 (pI 7.3). Peptide mapping after digestion with trypsin revealed one major peptide fragment (7.9 kDa) in the DHFR of MAP and MAP/47 and three major tryptic fragments (7.9, 9.6 and 12.5 kDa) in DHFR from MAP/42. We conclude that trimethoprim resistance in H. influenzae results from overproduction of structurally altered DHFR(s).
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PMID:Trimethoprim resistance in Haemophilus influenzae is due to altered dihydrofolate reductase(s). 201 95

Molecular recognition is achieved through the complementarity of molecular surface structures and energetics with, most commonly, associated minor conformational changes. This complementarity can take many forms: charge-charge interaction, hydrogen bonding, van der Waals' interaction, and the size and shape of surfaces. We describe a method that exploits these features to predict the sites of interactions between two cognate molecules given their three-dimensional structures. We have developed a "cube representation" of molecular surface and volume which enables us not only to design a simple algorithm for a six-dimensional search but also to allow implicitly the effects of the conformational changes caused by complex formation. The present molecular docking procedure may be divided into two stages. The first is the selection of a population of complexes by geometric "soft docking", in which surface structures of two interacting molecules are matched with each other, allowing minor conformational changes implicitly, on the basis of complementarity in size and shape, close packing, and the absence of steric hindrance. The second is a screening process to identify a subpopulation with many favorable energetic interactions between the buried surface areas. Once the size of the subpopulation is small, one may further screen to find the correct complex based on other criteria or constraints obtained from biochemical, genetic, and theoretical studies, including visual inspection. We have tested the present method in two ways. First is a control test in which we docked the components of a molecular complex of known crystal structure available in the Protein Data Bank (PDB). Two molecular complexes were used: (1) a ternary complex of dihydrofolate reductase, NADPH and methotrexate (3DFR in PDB) and (2) a binary complex of trypsin and trypsin inhibitor (2PTC in PDB). The components of each complex were taken apart at an arbitrary relative orientation and then docked together again. The results show that the geometric docking alone is sufficient to determine the correct docking solutions in these ideal cases, and that the cube representation of the molecules does not degrade the docking process in the search for the correct solution. The second is the more realistic experiment in which we docked the crystal structures of uncomplexed molecules and then compared the structures of docked complexes with the crystal structures of the corresponding complexes. This is to test the capability of our method in accommodating the effects of the conformational changes in the binding sites of the molecules in docking.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:"Soft docking": matching of molecular surface cubes. 202 63

We have studied the specificity requirements for processing of the human insulin proreceptor by successively replacing each basic amino acid in the tetrabasic cleavage site with alanine. These mutated receptor cDNAs have then been overexpressed in Chinese hamster ovary cells, using vectors containing the mouse dihydrofolate reductase gene to amplify the transfected cDNAs in the presence of increasing concentrations of methotrexate. High levels of expression, ranging up to 6 x 10(7) receptors/cell were achieved in these experiments. Replacement of the P1 arginine with alanine led to the complete suppression of processing, as occurs also in a naturally occurring serine mutation at this site (Yoshimasa, Y., Seino, S., Whittaker, J., Kakehi, T., Kosaki, A., Kuzuya, H., Imura, H., Bell, G. I., and Steiner, D. F. (1988) Science 240, 783-787). A small amount of cleavage at alternative sites was detected. Replacement of the P2 arginine or P3 lysine with alanine did not in either case affect conversion to mature alpha and beta subunits, while replacement of the P4 arginine significantly inhibited processing. The binding isotherms for the processed versions of the receptor were comparable to previously published normal values. The unprocessed proreceptor bound insulin normally but was autophosphorylated less efficiently than processed versions of the receptor expressed in the same cells. These results suggest that a single processing protease with trypsin-like specificity may be involved in processing both insulin and insulin-like growth factor-I receptor precursors as well as a variety of viral envelope glycoprotein precursors.
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PMID:Effects of amino acid replacements within the tetrabasic cleavage site on the processing of the human insulin receptor precursor expressed in Chinese hamster ovary cells. 221 23


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