Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Large-scale purification and characterization of dihydrofolate reductase from a methotrexate-resistant strain of Lactobacillus casei.

ABSTRACT: Dihydrofolate reductase has been purified from a methotrexate-resistant strain of Lactobacillus casei NCB 6375. By careful attention to growth conditions, up to 2.5 g of enzyme is obtained from a 400 litre culture. The purification procedure, involving poly-ethyleneimine treatment, DEAE-cellulose chromatography and affinity chromatography on methotrexate-aminohexyl-Sepharose, operates on the gram scale, with overall yields of 50-60%. Elution of the affinity column by reverse (upward) flow was used, as it led to recovery of the enzyme in a much smaller volume. The enzyme obtained appears to be more than 98% pure, as judged by gel electrophoresis, isoelectric focusing, and gel filtration. It has a mol.wt. of approx. 17900 and a turnover number of 4s-1 (50mM-triethanolamine/400mM-KCl, pH 7.2, 25 degrees C) with dihydrofolate and NADPH as substrates. The turnover number for folate is 0.02s-1. Michaelis constants for a variety of substrates have been measured by using a new fluorimetric assay (0.36 muM-dihydrofolate; 0.78 muM-NADPH), and binding constants determined by using the quenching of protein fluorescence (dihydrofolate, 2.25 X 10(6)M-1; NADPH, greater than 10(8)M-1). The pH/activity profile shows a single maximum at pH 7.3; at this pH, marked activation by 0.5M-NaCl is observed.

SUBMITTER: Dann JG

PROVIDER: S-EPMC1163897 | biostudies-other | 1976 Sep

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

3H-n.m.r. studies of multiple conformations and dynamic processes in complexes of folate and methotrexate with Lactobacillus casei dihydrofolate reductase.

Project description:[7,3',5'-3H3]- and [7,9-3H3]-folic acid and [7,3',5'-3H3]methotrexate (MTX) have been prepared and 3H-n.m.r. spectra obtained for their complexes with Lactobacillus casei dihydrofolate reductase (DHFR). The 3H results confirm the presence of three pH-dependent different conformational forms in the complex DHFR.NADP+.folate. The folate benzoyl ring could be shown to be in essentially the same environment in the different forms, with the major differences being associated with the pterin ring. The appearance of a single resonance for the 3',5'-tritons showed that the benzoyl ring is flipping rapidly in all three forms. In contrast, the MTX complex was shown to exist as a single conformational state with the benzoyl ring flipping rate being too low to give a single averaged signal for the 3',5'-nuclei over the temperature range 283-313 K.

| S-EPMC1137341 | biostudies-other

Ultraviolet difference-spectroscopic studies of substrate and inhibitor binding to Lactobacillus casei dihydrofolate reductase.

Project description:The u.v. difference spectra generated when methotrexate, trimethoprim or folate bind to Lactobacillus casei dihydrofolate reductase were analysed. The difference spectrum producted by methotrexate binding is shown to consist of three components: (a) one closely resembling that observed on protonation of methotrexate, reflecting an increased degree of protonation on binding; (b) a pH-independent contribution corresponding to a 40 nm shift to longer wavelengths of a single absorption band of methotrexate: (c) a component arising from perturbation of tryptophan residue(s) of the enzyme. Quantitative analysis of the pH-dependence of component (a) shows that pK of methotrexate is increased from 5.35 to 8.55 (+/-0.10) on binding. In contrast, folate is not protonated when bound to the enzyme at neutral pH. At pH7.5, where methotrexate is bound 2000 times more tightly than folate, one-third of the difference in binding energy between the two compounds arises from the difference in chaarge stage. A similar analysis of the difference spectra generated on trimethoprim binding demonstrates that this compound, too, shows an increase in pK on binding but only from 7.22 to 7.90 (+/-0.10), suggesting that its 2,4-diaminopyrimidine ring does not bind to the enzyme in precisely the same way as the corresponding moiety of methotrexate.

| S-EPMC1183964 | biostudies-other

Circular-dichroism studies of ligand binding to dihydrofolate reductase from Lactobacillus casei MTX/R.

Project description:Circular-dichroism spectra (200--450 nm) were recorded for Lactobacillus casei MTX/R dihydrofolate reductase and its complexes with substrates, inhibitors and coenzymes. These spectra are compared with those reported by others for dihydrofolate reductase from other sources. The binding of NADP+ or NADPH is associated with the perturbation of one or more aromatic amino acid residues, and there is marked enhancement of the negative c.d. band at 340 nm arising from the dihydronicotinamide chromophore of NADPH. The substrates folate and dihydrofolate give rise to substantial extrinsic c.d. bands on binding, which show a number of specific differences between enzymes from different sources. The binary complexes between the enzyme and the inhibitors methotrexate or trimethoprim also show strong c.d. bands, and these are qualitatively very similar for all dihydrofolate reductases studied so far. The ternary complexes between enzyme, NADPH and trimethoprim or methotrexate are very different from the sum of the spectra of the binary complexes. Trimethoprim leads to the disappearance of the 340 nm c.d. band of bound NADPH, whereas in the methotrexate--NADPH--enzyme ternary complex a "couplet" c.d. spectrum is observed at long wavelengths. Analysis of this latter feature suggests that it arises from a direct interaction between the dihydronicotinamide and pteridine rings in the ternary complex.

| S-EPMC1186391 | biostudies-other

Efficient selection of genetically modified human T cells using methotrexate-resistant human dihydrofolate reductase.

Project description:Genetic modification of human T cells to express transgene-encoded polypeptides, such as tumor targeting chimeric antigen receptors, is an emerging therapeutic modality showing promise in clinical trials. The development of simple and efficient techniques for purifying transgene(+) T cells is needed to facilitate the derivation of cell products with uniform potency and purity. Unlike selection platforms that utilize physical methods (immunomagnetic or sorting) that are technically cumbersome and limited by the expense and availability of clinical-grade components, we focused on designing a selection system on the basis of the pharmaceutical drug methotrexate (MTX), a potent allosteric inhibitor of human dihydrofolate reductase (DHFR). Here, we describe the development of self inactivating (SIN) lentiviral vectors that direct the coordinated expression of a CD19-specific chimeric antigen receptor (CAR), the human EGFRt tracking/suicide construct, and a methotrexate-resistant human DHFR mutein (huDHFR(FS); L22F, F31S). Our results demonstrate that huDHFR(FS) expression renders lentivirally transduced primary human CD45RO(+)CD62L(+) central memory T cells resistant to lymphotoxic concentrations of MTX up to 0.1??M. Our modular complementary DNA (cDNA) design insures that selected MTX-resistant T cells co-express functionally relevant levels of the CD19-specific CAR and EGFRt. This selection system on the basis of huDHFR(FS) and MTX has considerable potential utility in the manufacturing of clinical-grade T cell products.

| S-EPMC4028078 | biostudies-literature

Isolation and characterization of a dihydrofolate reductase gene mutation in methotrexate-resistant Drosophila cells.

Project description:Stepwise increases in methotrexate (MTX) concentration over a 4-year period led to the selection of a highly drug-resistant (2 x 10(-4) M MTX) Drosophila cell line. Uptake experiments with [3H]MTX showed a slightly lower level of intracellular MTX in the resistant S3Mtx cells than in the susceptible S3 parental cell line. Southern blot analysis demonstrated that the gene for the MTX target, dihydrofolate reductase (DHFR), was not significantly amplified in the resistant line. To determine the molecular basis for resistance, the DHFR cDNA sequence was amplified by polymerase chain reaction from both the resistant and susceptible cells. Sequence comparison revealed a single T to A base change at nucleotide 89, which resulted in the substitution of Gln for Leu at residue 30 in S3Mtx cells. Expression and purification of the wild-type and mutant DHFR from E. coli cells showed that the S3Mtx enzyme had a reduced binding affinity for the antifolates, MTX and trimethoprim, with 15-fold higher K[d] and K[i] values than those from the wild-type enzyme. Molecular modeling confirmed that the replacement of the hydrophobic Leu by the more polar Gln was in the substrate binding site and thus would decrease the binding of MTX. These results suggest that the high level of MTX resistance in the selected cell line can be attributed to the mutation in the DHFR gene and also provides a model for pesticide resistance in insects.

| S-EPMC6148270 | biostudies-literature

Very Long-Term Treatment with a Lactobacillus Probiotic Preparation, Lactobacillus Casei Strain Shirota

Project description:Primary outcome(s): life and death

| 103768 | ecrin-mdr-crc

The role of large-scale motions in catalysis by dihydrofolate reductase.

Project description:Dihydrofolate reductase has long been used as a model system to study the coupling of protein motions to enzymatic hydride transfer. By studying environmental effects on hydride transfer in dihydrofolate reductase (DHFR) from the cold-adapted bacterium Moritella profunda (MpDHFR) and comparing the flexibility of this enzyme to that of DHFR from Escherichia coli (EcDHFR), we demonstrate that factors that affect large-scale (i.e., long-range, but not necessarily large amplitude) protein motions have no effect on the kinetic isotope effect on hydride transfer or its temperature dependence, although the rates of the catalyzed reaction are affected. Hydrogen/deuterium exchange studies by NMR-spectroscopy show that MpDHFR is a more flexible enzyme than EcDHFR. NMR experiments with EcDHFR in the presence of cosolvents suggest differences in the conformational ensemble of the enzyme. The fact that enzymes from different environmental niches and with different flexibilities display the same behavior of the kinetic isotope effect on hydride transfer strongly suggests that, while protein motions are important to generate the reaction ready conformation, an optimal conformation with the correct electrostatics and geometry for the reaction to occur, they do not influence the nature of the chemical step itself; large-scale motions do not couple directly to hydride transfer proper in DHFR.

| S-EPMC3590880 | biostudies-literature

In vivo selection of human embryonic stem cell-derived cells expressing methotrexate-resistant dihydrofolate reductase.

Project description:Human embryonic stem cells (hESCs) provide a novel source of hematopoietic and other cell populations suitable for gene therapy applications. Preclinical studies to evaluate engraftment of hESC-derived hematopoietic cells transplanted into immunodeficient mice demonstrate only limited repopulation. Expression of a drug-resistance gene, such as Tyr22-dihydrofolate reductase (Tyr22-DHFR), coupled to methotrexate (MTX) chemotherapy has the potential to selectively increase the engraftment of gene-modified, hESC-derived cells in mouse xenografts. Here, we describe the generation of Tyr22-DHFR-GFP-expressing hESCs that maintain pluripotency, produce teratomas and can differentiate into MTXr-hemato-endothelial cells. We demonstrate that MTX administered to nonobese diabetic/severe combined immunodeficient/IL-2Rgammac(null) (NSG) mice after injection of Tyr22-DHFR-hESC-derived cells significantly increases human CD34(+) and CD45(+) cell engraftment in the bone marrow (BM) and peripheral blood of transplanted MTX-treated mice. These results demonstrate that MTX treatment supports selective, long-term engraftment of Tyr22-DHFR cells in vivo, and provides a novel approach for combined human cell and gene therapy.

| S-EPMC2820606 | biostudies-literature

Metformin sensitises hepatocarcinoma cells to methotrexate by targeting dihydrofolate reductase.

Project description:Metformin, the first-line drug for type II diabetes, has recently been considered an anticancer agent. However, the molecular target and underlying mechanism of metformin's anti-cancer effects remain largely unclear. Herein, we report that metformin treatment increases the sensitivity of hepatocarcinoma cells to methotrexate (MTX) by suppressing the expression of the one-carbon metabolism enzyme DHFR. We show that the combination of metformin and MTX blocks nucleotide metabolism and thus effectively inhibits cell cycle progression and tumorigenesis. Mechanistically, metformin not only transcriptionally represses DHFR via E2F4 but also promotes lysosomal degradation of the DHFR protein. Notably, metformin dramatically increases the response of patient-derived hepatocarcinoma organoids to MTX without obvious toxicity to organoids derived from normal liver tissue. Taken together, our findings identify an important role for DHFR in the suppressive effects of metformin on therapeutic resistance, thus revealing a therapeutically targetable potential vulnerability in hepatocarcinoma.

| S-EPMC8487431 | biostudies-literature

Isolation and characterization of a variant dihydrofolate reductase cDNA from methotrexate-resistant murine L5178Y cells.

Project description:Dihydrofolate reductase (DHFR) cDNA sequences were isolated from a methotrexate-resistant mouse L5178Y cell line previously shown to contain methotrexate-resistant dihydrofolate reductase enzyme activity. Specifically-primed reverse transcription products were amplified using the polymerase chain reaction and then cloned into a mammalian expression plasmid. Candidate clones were identified by restriction analysis and then functionally tested by transfection into mouse 3T3 fibroblasts, selecting for methotrexate-resistant colonies. Sequence analysis of the cDNA clones demonstrated the substitution of tryptophan (TGG) in place of the wild-type phenylalanine (TTC) at codon 31. Sequencing of PCR-amplified genomic DNA extracted from the drug-resistant L5178Y cells confirmed the tryptophan codon at position 31. Transfection of mammalian tissue culture cells with expression plasmids containing the trp31 DHFR sequence resulted in substantial methotrexate-resistant colony formation. Recombinant trp31 DHFR enzyme activity expressed in stably-transfected Chinese hamster ovary cells was approximately 20-fold less sensitive to methotrexate inhibition than wild-type mouse DHFR enzyme activity. We conclude that the cloned Trp31 DHFR sequence encodes an enzyme substantially resistant to methotrexate which confers a drug-resistance phenotype to cells in which it is expressed.

| S-EPMC332765 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data