Mammalian fibroblasts lacking mitochondrial NAD+-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase are glycine auxotrophs
Mammalian fibroblasts lacking mitochondrial NAD+-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase are glycine auxotrophs
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DOI：
10.1074/jbc.m301718200
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发表时间：
2003-05-23
影响因子：
4.8
通讯作者：
MacKenzie, RE
中科院分区：
文献类型：
--
作者：
Patel, H;Di Pietro, E;MacKenzie, RE
从NAD依赖性线粒体亚甲基四氢叶酸脱氢酶环化水解酶（NMDMC）敲除小鼠的胚胎中建立的原代成纤维细胞自发永生化或用SV 40大T抗原转化。细胞的Mitotracker Red CMXRos染色表明存在具有膜电位的完整线粒体。nmdmc（（-/-））细胞是甘氨酸营养缺陷型的，表明NMDMC是这些细胞系的线粒体中正常表达的唯一亚甲基四氢叶酸脱氢酶。在含有透析血清的完全培养基上，无效突变体而非野生型细胞的生长被添加的甲酸盐或次黄嘌呤刺激约2倍。放射性标记实验表明，nmdmc（（-/-））细胞从甲酸盐相对于丝氨酸掺入DNA的放射性增强3- 10倍。在nmdmc（（-/-））细胞中，线粒体产生的一碳单元被完全阻断，细胞质中叶酸途径本身不足以实现最佳嘌呤合成。结果表明NMDMC在支持嘌呤生物合成中的代谢作用。尽管突变细胞系中存在这些代谢缺陷，但当给予妊娠母鼠富含甘氨酸的饮食或每日注射甲酸钠时，开始于E13.5死亡的nmdmc（（-/-））胚胎的表型并未得到改善。
Primary fibroblasts established from embryos of NAD-dependent mitochondrial methylenetetrahydrofolate dehydrogenase-cyclohydrolase (NMDMC) knockout mice were spontaneously immortalized or transformed with SV40 Large T antigen. Mitotracker Red CMXRos staining of the cells indicates the presence of intact mitochondria with a membrane potential. The nmdmc((-/-)) cells are auxotrophic for glycine, demonstrating that NMDMC is the only methylenetetrahydrofolate dehydrogenase normally expressed in the mitochondria of these cell lines. Growth of null mutant but not wild type cells on complete medium with dialyzed serum is stimulated about 2-fold by added formate or hypoxanthine. Radiolabeling experiments demonstrated a 3-10x enhanced incorporation of radioactivity into DNA from formate relative to serine by nmdmc((-/-)) cells. The generation of one-carbon units by mitochondria in nmdmc((-/-)) cells is completely blocked, and the cytoplasmic folate pathways alone are insufficient for optimal purine synthesis. The results demonstrate a metabolic role for NMDMC in supporting purine biosynthesis. Despite the recognition of these metabolic defects in the mutant cell lines, the phenotype of nmdmc((-/-)) embryos that begin to die at E13.5 is not improved when pregnant dams are given a glycine-rich diet or daily injections of sodium formate.