Journal
ANALYTICAL BIOCHEMISTRY
Volume 371, Issue 2, Pages 127-134Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2007.08.026
Keywords
folate; polyglutamates; tritium; biosynthesis; enzymatic synthesis; folylpolyglutamate synthetase
Funding
- NIGMS NIH HHS [R01 GM071382, GM 071382] Funding Source: Medline
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Tritiated forms of polyglutamyl folates are not commercially available but are often needed for experimental uses in folate biochemistry. Thus, considerable interest exists in the preparation of polyglutamyl [H-3]folates from the commercial monoglutamyl [H-3]folates. However, refinement of established enzymatic and biological synthesis methods is still needed. To address this need we developed improved procedures for the conversion of monoglutamy] [H-3]folates to various polyglutamyl forms. In the bacterial synthesis, Lactobacillus casei was grown in the presence of I ng/ml (2.27 nM) [H-3]folic acid in Folic Acid Casei Medium. Washed cells were resuspended in 2% sodium ascorbate containing 10 mM P-mercaptoethanol and heated to release the folates. The extracted [H-3]folates were purified on a folate-binding protein affinity column and then applied to a Sephadex G-10 column to separate the eluted poly- from the monoglutamyl folate species. High performance liquid chromatography with multichannel electrochemical detection indicated that the bacterial synthesis yielded predominantly polyglutamates of [H-3]5-methyltetrahydrofolate and [H-3]5-formyltetrahydrofolate (di- through heptaglutamates). The alternative method consisted of enzymatic polyglutamylation of [H-3]folic acid catalyzed by recombinant Escherichia coli folylpolyglutamate synthetase. This enzymatic synthesis yielded predominantly tri-, tetra-, and pentaglutamyl species for the [H-3]folate substrate. (C) 2007 Elsevier Inc. All rights reserved.
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