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SYNTHESIS OF [3H]POLYGLUTAMYL FOLATES FOR MEASURING VACUOLAR FOLATE UPTAKE
Author(s) -
Naponelli Valeria,
Ziemak Michael J,
Hanson Andrew D,
Gregory Jesse F
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a346-b
Subject(s) - vacuole , chemistry , biochemistry , high performance liquid chromatography , tris , chromatography , centrifugation , in vitro , folic acid , medicine , cytoplasm
This work aimed to develop methods to prepare and purify tritiated polyglutamyl folate derivatives of high specific radioactivity. These are not commercially available. Recombinant folylpolyglutamate synthetase (FPGS) from E. coli was examined as a route for in vitro synthesis of radiolabeled polyglutamyl folates. FPGS catalyzes the conversion of folates to polyglutamate derivatives by the addition of glutamate units via γ‐linkage. The standard synthesis mixture contained 2 nmol of [3H]folic acid monoglutamate, 50 mM Tris‐HCl pH 8.6, 10 mM β‐mercaptoethanol, 10 mM MgCl2, 50 mM KCl, 5 mM ATP, 25 μg BSA, 20 mM sodium glutamate and 25 μg of E. coli FPGS in a final volume of 50 μl. After incubation for 24 h at 37°C, the reaction was stopped by boiling for 5 min and centrifugation. HPLC analysis showed that the products consisted of 40–45% folic acid triglutamate, 50–55% tetraglutamate and 3–5% pentaglutamate. This preparation will be suitable for use in assays for measurement of [3H]folate polyglutamate uptake by plant vacuoles or vacuolar vesicles. If polyglutamyl folate uptake is observed in intact vacuoles, assays will be modified to include HPLC separation of the [3H]folates taken up to test for progressive shortening of the polyglutamyl tail, as occurs in mammalian lysosomes. This research was supported by NIH Grant R01 GM071382