Multiple forms of L1210 dihydrofolate reductase differing in affinity for methotrexate.

  title={Multiple forms of L1210 dihydrofolate reductase differing in affinity for methotrexate.},
  author={T. Duffy and S. Beckman and F. M. Huennekens},
  journal={Biochemical and biophysical research communications},
  volume={119 1},
Dihydrofolate reductase, purified from a Methotrexate-resistant subline (R6) of L1210 mouse leukemia cells, consists of two forms (designated 1 and 2) differing in affinity for the drug. Form 1 is more sensitive to inhibition by Methotrexate. Form 2 is more heat-labile, but it can be stabilized by bovine serum albumin or NADPH. Isoelectric focusing resolves 1 and 2; pI values are 7.4 and 8.2. Forms 1 and 2 comprise about 10 and 90% of the total protein, but 1 has at least a 2-fold higher… Expand
11 Citations
L1210 dihydrofolate reductase: activation and enhancement of methotrexate sensitivity.
Activating agents appear to induce a more open (and labile) conformation of the enzyme, which leads to increased affinity for MTX accompanied, in some instances, by increased catalytic activity. Expand
Polymorphism of dihydrofolate reductase from a methotrexate-resistant subline of L1210 cells.
These conclusions are consistent with the well-known ability of eukaryotic dihydrofolate reductases to exhibit increased catalytic activity when treated with salts, chaotropes, or cysteine-modifying agents. Expand
Evidence for kinetic and immunologic heterogeneity of dihydrofolate reductase in L1210 leukemia cells.
Two species of DHFR were identified in wild-type L1210 murine leukemia cells by analysis of the kinetics of the binding of MTX and dissociation of the MTX-enzyme complex at pH 5.0 and pH 7.2. The twoExpand
A new form of dihydrofolate reductase in cancer cells.
It is suggested that the presence of a low affinity form of the enzyme in certain cancer cells may be one of the underlying causes of resistance to MIX therapy in these cells. Expand
Altered dihydrofolate reductase in pyrimethamine-resistant Plasmodium falciparum.
  • R. D. Walter
  • Biology, Medicine
  • Molecular and biochemical parasitology
  • 1986
Results presented here indicate that pyrimethamine resistance of P. falciparum depends on a modified dihydrofolate reductase, which shows less affinity for pyrimEThamine and dihydROfolate. Expand
Heterogeneity of methotrexate binding in human colon tumor cells.
Scatchard analysis of the [3H]-methotrexate binding data by a colon tumor sample suggests that the two binders of methotrexate may be the two forms of dihydrofolate reductase having different affinities for this anticancer drug. Expand
Pyrimethamin-resistantPlasmodium falciparum lack cross-resistance to methotrexate and 2,4-diamino-5-(substituted benzyl) pyrimidines
The efficiency of these newly designed 2,4-diamino-5-(substituted benzyl) pyrimidines was confirmed by their strong inhibitory effect on plasmodial dihydrofolate reductase as well as by in vitro screening against drug-sensitive and-resistant strains of P. falciparum. Expand
Biochemistry of Methotrexate: Teaching an Old Drug New Tricks
Information about the drug-binding sites, along with computer graphic modeling, then provides guidance for the chemical synthesis of compounds tailored to fit with a high degree of specificity and affinity. Expand
Drug Resistance: New Approaches to Treatment
Mechanisms by which malignant cells may become resistant to chemotherapeutic agents are reviewed, with emphasis on methotrexate resistance. At least four mechanisms of resistance have been describedExpand
Mechanisms of drug resistance in human leukemia.
Five-year disease-free survival rates are only 50% and 10%–15%, respectively, in these diseases, almost certainly due to the development of drug resistance even to the combination chemotherapy programs utilized to treat these diseases. Expand


Properties of a methotrexate-insensitive variant of dihydrofolate reductase derived from methotrexate-resistant L5178Y cells.
The presence of two distinct forms of dihydrofolate reductase in methotrexate-resistant L5178Y lymphoma cells is established and their properties have been compared. Expand
Inhibition of dihydrofolate reductase by methotrexate: a new look at an old problem.
Kinetic analyses indicate that the enzyme can exist in multiple forms varying in their Km values for NADPH and Ki values for MTX, and interaction of the enzyme with ligands appears to induce the formation of certain of these forms. Expand
Evidence for two molecular species of dihydrofolate reductase in amethopterin resistant and sensitive cells of the mouse leukemia L4946.
Evidence has been obtained for the formation of a new molecular species of the enzyme dihydrofolate reductase in an amethopterin-resistant strain of the mouse lymphocytic leukemia L4946. The evidenceExpand
Effect of a single amino acid substitution on Escherichia coli dihydrofolate reductase catalysis and ligand binding.
The two isozymes of dihydrofolate reductase (Forms 1 and 2) from, a Trimethoprim-resistant strain of Escherichia coli (RT500) were separated and purified to homogeneity using a simple procedure basedExpand
Methotrexate-resistant Chinese hamster ovary cells contain a dihydrofolate reductase with an altered affinity for methotrexate.
This study has shown that clonal isolates of Chinese hamster ovary cells that were resistant to the cytotoxic action of methotrexate and contained a dihydrofolate reductase that was less sensitive to inhibition by the drug than wild-type enzyme. Expand
Acquired methotrexate resistance in lymphoblasts resulting from altered kinetic properties of dihydrofoltate reductase.
Increasing the selective pressure by raising the methotrexate concentration in the medium above 1·6 × 10 −6 M, led to selection against the mutant, and cells with normal type dihydrofolate reductase again dominated the populalation. Expand
Isolation and characterization of dihydrofolic acid reductase from methotrexate-sensitive and -resistant human cell lines.
The available evidence indicates that the increased dihydrofolic acid reductase content of the two-resistant cell lines is at least 200-fold higher than that of the methotrexate-sensitive HeLa BU-25 cell line, presumably due to a selective dihydraulic acid reduction gene amplification, as previously reported for other cell lines of rodent origin. Expand
Properties of an altered dihydrofolate reductase encoded by amplified genes in cultured mouse fibroblasts.
Studies with various folic acid analogs suggest that modifications involving the para-aminobenzoyl moiety of the inhibitor molecules are associated with the most dramatic differential binding between the altered and wild type dihydrofolate reductases. Expand
Substrate and inhibitor complexes of dihydrofolate reductase from amethopterin-resistant L1210 cells.
Abstract Dihydrofolate reductase (EC, purified to homogeneity from an amethopterin-resistant subline (R6) of cultured L1210 murine leukemia cells, has been used to study enzyme-substrate andExpand
Interconversion of the multiple forms of dihydrofolate reductase from amethopterin-resistant Lactobacillus casei.
Abstract Dihydrofolate reductase from amethopterin-resistant Lactobacillus casei can be separated into two principal forms (I and II) by electrophoresis on polyacrylamide or by chromatography onExpand