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.
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制备[3H]叶酸聚谷氨酸盐的改进方法：干酪乳杆菌生物合成和大肠杆菌叶酰聚谷氨酸合成酶酶法合成。
DOI：
10.1016/j.ab.2007.08.026
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发表时间：
2007
影响因子：
2.9
通讯作者：
Gregory3rd,JesseF
中科院分区：
文献类型：
--
作者：
Naponelli,Valeria;Hanson,AndrewD;Gregory3rd,JesseF
氚化形式的聚谷氨酰叶酸盐不是商业上可获得的，但在叶酸盐生物化学中经常需要用于实验用途。因此，从市售的单谷氨酰[3 H]叶酸盐制备聚谷氨酰[3 H]叶酸盐存在相当大的兴趣。然而，仍然需要完善已建立的酶和生物合成方法。为了满足这一需求，我们开发了将单谷氨酰[3 H]叶酸转化为各种聚谷氨酰形式的改进程序。在细菌合成中，干酪乳杆菌在叶酸干酪培养基中存在1 ng/ml（2.27nM）[3 H]叶酸的情况下生长。将洗涤的细胞重悬于含有10 mM 0-巯基乙醇的2%抗坏血酸钠中，并加热以释放叶酸。在叶酸结合蛋白亲和柱上纯化提取的[3 H]叶酸，然后将其施加到Sephadex G-10柱上以将洗脱的聚-叶酸与单谷氨酰叶酸物质分离。高效液相色谱与多通道电化学检测表明，细菌的合成主要产生聚谷氨酸[3 H]5-甲基四氢叶酸和[3 H]5-甲酰四氢叶酸（二至七谷氨酸）。替代方法包括由重组大肠杆菌叶酰聚谷氨酸合成酶催化的[3 H]叶酸的酶促聚谷氨酸化。这种酶促合成主要产生[3 H]叶酸底物的三-、四-和五谷氨酰物质。
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 1ng/ml (2.27nM) [3H]folic acid in Folic Acid Casei Medium. Washed cells were resuspended in 2% sodium ascorbate containing 10mM 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.