title: Improved methods for the preparation of [3H]folate polyglutamates: biosynthesis with Lactobacillus casei and enzymatic synthesis with Escherichia coli folylpolyglutamate synthetase.
abstract: 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 [(3)H]folates from the commercial monoglutamyl [(3)H]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 monoglutamyl [(3)H]folates to various polyglutamyl forms. In the bacterial synthesis, Lactobacillus casei was grown in the presence of 1 ng/ml (2.27 nM) [(3)H]folic acid in Folic Acid Casei Medium. Washed cells were resuspended in 2% sodium ascorbate containing 10mM beta-mercaptoethanol and heated to release the folates. The extracted [(3)H]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 [(3)H]5-methyltetrahydrofolate and [(3)H]5-formyltetrahydrofolate (di- through heptaglutamates). The alternative method consisted of enzymatic polyglutamylation of [(3)H]folic acid catalyzed by recombinant Escherichia coli folylpolyglutamate synthetase. This enzymatic synthesis yielded predominantly tri-, tetra-, and pentaglutamyl species for the [(3)H]folate substrate.
authors.affiliation: Department of Food Science and Human Nutrition, University of Florida, Gainesville, FL 32611, USA.
authors.forename: Valeria
authors.initials: V
authors.lastname: Naponelli
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Hanson
authors.forename: Jesse F
authors.initials: JF
authors.lastname: Gregory
mesh_terms: D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology*; D005492:Folic Acid / Q000378:metabolism; D007780:Lacticaseibacillus casei / Q000378:metabolism*; D010453:Peptide Synthases / Q000302:isolation & purification / Q000378:metabolism*; D011624:Pteroylpolyglutamic Acids / Q000032:analysis / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors; D014316:Tritium
chemical_list: D011624:Pteroylpolyglutamic Acids; D011994:Recombinant Proteins; D014316:Tritium; D005492:Folic Acid; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase