A microassay for mammalian folylpolyglutamate synthetase☆
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Cited by (16)
Molecular basis for decreased folylpoly-γ-glutamate synthetase expression in a methotrexate resistant CCRF-CEM mutant cell line
2007, Leukemia ResearchCitation Excerpt :Cell extracts from CCRF-CEM, CEM-p, and AUXB1 cells were prepared by sonication and protein concentrations determined using the Bio-Rad protein assay kit (BioRad). FPGS enzyme activity was determined at least twice using the FPGS microassay methodology described by Antonsson et al. [15]. Each experiment was performed with independently isolated cell extracts and in triplicate at each time point.
Molecular analysis of murine leukemia cell lines resistant to 5,10-dideazatetrahydrofolate identifies several amino acids critical to the function of folylpolyglutamate synthetase
2000, Journal of Biological ChemistryCitation Excerpt :Charcoal slurry (34) was added, the mixture was centrifuged in a Microfuge, and radioactivity in the supernatant was determined on a liquid scintillation spectrometer. FPGS activity was measured using a microprocedure previously described (39) in which cytosolic protein, prepared by a 110,000 ×g centrifugation step, was incubated with 10 μm (6S)-tetrahydrofolate in the presence of 5 mm ATP, 10 mm MgCl2, 30 mm KCl, and 1 mm [3H]glutamic acid in 200 mm Tris, pH 8.5, containing 36 mm2-mercaptoethanol. The product was isolated by Sephadex spin chromatography after conversion to a macromolecule in the presence of fluorodeoxyuridylate, pure Lactobacillus caseithymidylate synthase, and formaldehyde.
Cellular folates prevent polyglutamation of 5,10- dideazatetrahydrofolate: A novel mechanism of resistance to folate antimetabolites
1998, Journal of Biological ChemistryFolates and one-carbon metabolism in plants and fungi
1997, Phytochemistry
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Supported in part by Grant CA-27605 from the DHHS. R.G.M. is a Scholar of the Leukemia Society of America; this award was funded by the Scott Helping Hand Fund.
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Present address: Glaxo Institute for Molecular Biology, S.A., Route des Acacias 46, 1211 Geneva 24, Switzerland.