کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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1175722 | 961815 | 2007 | 8 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Improved methods for the preparation of [3H]folate polyglutamates: biosynthesis with Lactobacillus casei and enzymatic synthesis with Escherichia coli folylpolyglutamate synthetase Improved methods for the preparation of [3H]folate polyglutamates: biosynthesis with Lactobacillus casei and enzymatic synthesis with Escherichia coli folylpolyglutamate synthetase](/preview/png/1175722.png)
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 [3H]folates from the commercial monoglutamyl [3H]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 [3H]folates to various polyglutamyl forms. In the bacterial synthesis, Lactobacillus casei was grown in the presence of 1 ng/ml (2.27 nM) [3H]folic acid in Folic Acid Casei Medium. Washed cells were resuspended in 2% sodium ascorbate containing 10 mM β-mercaptoethanol and heated to release the folates. The extracted [3H]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 [3H]5-methyltetrahydrofolate and [3H]5-formyltetrahydrofolate (di- through heptaglutamates). The alternative method consisted of enzymatic polyglutamylation of [3H]folic acid catalyzed by recombinant Escherichia coli folylpolyglutamate synthetase. This enzymatic synthesis yielded predominantly tri-, tetra-, and pentaglutamyl species for the [3H]folate substrate.
Journal: Analytical Biochemistry - Volume 371, Issue 2, 15 December 2007, Pages 127–134