Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mercuration of 2'-deoxyuridine 5'-phosphate (dUMP) followed by alkylation with allylamine in the presence of K2PdCl4 afforded the 5-aminoallyl deoxynucleotide, which was isolated by sequential Dowex 50 H+ and DEAE-Sephadex chromatography. Further reaction of the product with the N-hydroxysuccinimide ester of methotrexate (MTX) in dry dimethyl sulfoxide gave an MTX-aminoallyl-dUMP covalent complex separable by DEAE-Sephadex chromatography. Reprecipitation with acid from basic solution offered further purification and the structure was confirmed by elemental analysis, NMR and absorbance spectra. The product was an inhibitor of rat liver dihydrofolate reductase (I50 approximately 250 nM, cf. MTX I50 approximately 60 nM) and Lactobacillus casei thymidylate synthase. With the latter enzyme, inhibition was competitive with both nucleotide and folate substrates (Ki = 2.6 and 3.5 microM, respectively) and partial enzyme-inhibitor binary complex could be detected by gel electrophoresis. Large fluorescence changes were observed on titration of the synthase with MTX-aminoallyl-dUMP and alterations in the UV difference spectra similar to those seen on titration of the enzyme with MTX were also noted. The compound was a poor growth inhibitor for cultured murine L1210 and human CCRF-CEM cell lines, which probably reflects low cellular uptake.
 ...
PMID:Methotrexate 5-aminoallyl-2'-deoxyuridine 5'-monophosphate: a potential bifunctional inhibitor of thymidylate synthase. 251 77

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

Properties of the methotrexate (MTX) transport carrier were examined in a stable single-step 16-fold MTX-resistant L1210 murine leukemia cell line with unchanged dihydrofolate reductase gene copy and thymidylate synthase and dihydrofolate reductase levels and activities. MTX influx was markedly depressed due to a decrease in Vmax without a change in Km. From this cell line a clonal variant with greater resistance to MTX was identified due solely to a further decrease in influx Vmax. Trans-stimulation of MTX influx by 5-formyltetrahydrofolate was induced in parental but not resistant cells. Analysis of specific MTX surface binding demonstrated a small increase in the number of carriers in the first- and second-step resistant lines. Affinity labeling of cells with an N-hydroxysuccinimide ester derivative of [3H]MTX demonstrated carriers with comparable molecular weights in the parent and second-step transport defective lines. In two partial revertants with increased MTX sensitivity isolated from the second-step resistant lines, MTX influx was increased but surface membrane-binding sites were unchanged suggesting that recovery of transport was due to normalization of carrier function rather than an increase in the number of carriers. These studies suggest that impaired MTX transport in these lines is not due to an alteration in the association of the transport carrier with its substrate at the cell surface. Rather, resistance may be due to an alteration in the mobility of the carrier possibly associated with a protein change in the carrier itself or the cell membrane that surrounds it.
 ...
PMID:Evidence for a functional defect in the translocation of the methotrexate transport carrier in a methotrexate-resistant murine L1210 leukemia cell line. 283 83