Isolation and Characterization of the Multiple Forms of Dihydrofolate Reductase from Methotrexate-resistant Hamster Cells

Hänggi, Urs J.; Littlefield, John W.

doi:10.1016/s0021-9258(19)42894-9

Cited by 45 publications

(4 citation statements)

References 25 publications

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“…nificantly different from the chromosomal enzyme. However, in other cases, such as with the dihydrofolate reductase from hamster kidney cells (Hanggi and Littlefield, 1974), Diplococcus pneumoniae (Sirotnak, 1973), and bovine liver (Kaufman and Kamerer, 1976;Baumann and Wilson, 1975), the occurrence of polymorphism is well documented, while the cause of the multiplicity is not completely understood.…”

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confidence: 99%

Escherichia coli dihydrofolate reductase: isolation and characterization of two isozymes

Baccanari¹,

Averett²,

Briggs³

et al. 1977

Biochemistry

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A combination of affinity column chromatography and preparative gel electrophoresis has been used to purify to homogeneity the two isozymes of dihydrofolate reductase from a trimethoprim-resistant strain of Escherichia coli B (RT 500). These enzyme forms are noninterconvertible and are present in crude cell lysates, but other electrophoretic species can be generated durng purification if sulfhydryl-protecting agents, such as dithiothreitol, are not present. The two isozymes, numbered form 1 and form 2 with respect to their decreasing electrophoretic mobilities, have similar molecular weights (18 500), molecular radii (21 A), and apparent Km values for reduced nico inamide adenin- dinucleotide (NADH) and NADH phosphate (NADPH). Both forms contain 2 mol of sulfhydryl/mol of enzyme which can be oxidized to intramolecular disulfide bonds. However, forms 1 and 2 differ physically in their electrophoretic mobility and isoelectric point and kinetically in their pH-activity profile, specific activity, Km for dihydrofolate, and their affinity toward a number of inhibitors.

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confidence: 99%

Escherichia coli dihydrofolate reductase: isolation and characterization of two isozymes

Baccanari¹,

Averett²,

Briggs³

et al. 1977

Biochemistry

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“…The presence of more than one dihydrofolate reductase component appears to be a common property of both proka-ryotic (Gundersen et al, 1972;Poe et al, 1972) and eukaryotic systems (Hanggi & Littlefield, 1974; Kaufman & Kemerer, 1977). Such multiple forms have been readily identified and separated by electrophoresis or chromatography, and the ratio of the forms appears to remain constant during purification procedures (Kaufman, 1971).…”

Section: Discussionmentioning

confidence: 99%

“…In microorganisms, two physiological controlled forms coexist inside the cell: the free enzyme and the cofactor-complexed enzyme (Dunlap et al, 1971;Gundersen et al, 1972;Poe et al, 1972;Harding et al, 1970). In eukaryotic systems, similar ligand-enzyme complex mechanisms may be involved, but at least in the case of the baby hamster kidney cell system, two basic enzyme forms, with similar amino acid sequences that can bind to ligands, are present (Hanggi & Littlefield, 1974). These two forms appear to remain in a constant ratio in wild-type cells and cells that greatly overproduce the enzyme (Hanggi & Littlefield, 1974).…”

Section: Discussionmentioning

confidence: 99%

“…In eukaryotic systems, similar ligand-enzyme complex mechanisms may be involved, but at least in the case of the baby hamster kidney cell system, two basic enzyme forms, with similar amino acid sequences that can bind to ligands, are present (Hanggi & Littlefield, 1974). These two forms appear to remain in a constant ratio in wild-type cells and cells that greatly overproduce the enzyme (Hanggi & Littlefield, 1974). In the CHO system at present, it is not clear what forms the basis for the two forms observed after isoelectric focusing.…”

Section: Discussionmentioning

confidence: 99%

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Methotrexate-resistant Chinese hamster ovary cells contain a dihydrofolate reductase with an altered affinity for methotrexate

1980

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Previous reports [Flintoff, W. F., Davidson, S. V., & Siminovitch, L. (1976) Somatic Cell Genet. 2, 245--261; Gupta, R. S., Flintoff, W. F., & Siminovitch, L. (1977) Can. J. Biochem. 55, 445--452] described a series 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. In this study, binding of labeled methotrexate to the reductase--NADPH complex and separation of free and bound drug by filtration through Sephadex G--25 have been used to demonstrate that clonal isolates of these resistant cells contain a dihydrofolate reductase varying between 2.5- and 6-fold lower in affinity for the drug than the wild-type enzyme. The apparent dissociation constant for the wild-type enzyme is 0.5 x 10(-9) M. Using two-dimensional polyacrylamide gel electrophoresis, 11 independently selected resistant isolates have been shown to contain a reductase with a similar overall net charge as the wild-type enzyme. Reductase purified from either wild-type or resistant cells contains two components after isoelectric focusing in polyacrylamide gels. The major component represents about 90% of the total protein and has a pI of about 8.0. The minor component representing about 10% of the reductase protein has a pI between 7.2 and 7.6.

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N‐terminal amino acid sequence analysis of dihydrofolate reductase from methotrexate‐resistant mosquito cells

Johnston¹,

Fallon²

1987

Arch Insect Biochem Physiol

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Methotrexate-resistant mosquito (Aedes albopictus) cell mutants were used as starting material for the purification of dihydrofolate reductase. Initial fractionation of a crude cell lysate by ammonium sulfate precipitation showed an enrichment of enzyme activity in the 60-100% pellet. Purification of this ammonium sulfate fraction on a Sephadex G-75 column prior to affinity chromatography was required for maximal yield of purified protein. A protein with dihydrofolate reductase activity and an M, of 24,000 was eluted from a methotrexate-agarose column with 0.5 M Tris-HCI, pH 8.5, containing 2 m M folic acid. A 21-fold increase in specific activity relative to that in the crude cell lysate was obtained, and amino acid sequence analysis confirmed that dihydrofolate reductase from the affinity column migrated as an electrophoretically pure band on SDS polyacrylamide gels. The initial 20 amino acid residues of the mosquito dihydrofolate reductase were identified by microsequencing and compared to those of murine, human, and bacterial d i hyd rofolate reductase molecu les.

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Isolation and Characterization of the Multiple Forms of Dihydrofolate Reductase from Methotrexate-resistant Hamster Cells

Cited by 45 publications

References 25 publications

Escherichia coli dihydrofolate reductase: isolation and characterization of two isozymes

Escherichia coli dihydrofolate reductase: isolation and characterization of two isozymes

Methotrexate-resistant Chinese hamster ovary cells contain a dihydrofolate reductase with an altered affinity for methotrexate

N‐terminal amino acid sequence analysis of dihydrofolate reductase from methotrexate‐resistant mosquito cells

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