A microassay for mammalian folylpolyglutamate synthetase

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Abstract

A new assay for the enzyme folylpoly-γ-glutamate synthetase (FPGS) that offers significant advantages over other published procedures has been developed. This assay is based on the addition of high specific activity [3H]glutamic acid to (6-S)-tetrahydrofolate followed by trapping of the labeled tetrahydropteroyldiglutamate product as a covalently bound macromolecular complex by the addition of formaldehyde, fluorodeoxyuridylate, and pure bacterial thymidylate synthase. This complex is then separated from excess labeled glutamic acid by centrifugal elution of a 1-ml Sephadex G-50 column. The assay was found to be useful for the measurement of FPGS on small tissue samples and is amenable with the assay of FPGS in cell sonicates. Typically, blank values of 100–200 cpm are seen with a signal normally more than 10 times higher. Analysis of 20–30 samples can be accomplished in less than 90 min. As a result, this assay has proven useful for detection of enzyme in elution fractions from chromatographic columns.

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Cited by (16)

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    2007, Leukemia Research
    Citation 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 Chemistry
    Citation 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.

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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.

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