Fujii K, Nagasaki T, Huennekens F M
J Biol Chem. 1981 Oct 25;256(20):10329-34.
L1210 mouse leukemia cells were made cobalamin-deficient by propagation in a medium from which cyanocobalamin was omitted and fetal bovine serum (containing protein-bound cobalamins) was replaced by bovine serum albumin. These cobalamin-deficient cells exhibited a normal replication time of 12 h, provided that the medium contained excess folate or 5-formyltetrahydrofolate. The cells responded poorly, however, to 5-methyltetrahydrofolate unless exogenous cobalamin was added. A cobalamin dependency was also observed when low levels of folate or 5-formyltetrahydrofolate were used. With 5-methyltetrahydrofolate, optimal stimulation of growth was observed with free and transcobalamin-II-bound cobalamin at 4,000 pM and 2 pM, respectively. Under cobalamin-replete conditions, cells contained 2,000 to 4,000 molecules of cobalamin/cell, and in the deficient state, this value declined to less than 10 molecules/cell; optimal replication on 5-methyltetrahydrofolate required approximately 180 molecules/cell. Cobalamin-deficient cells cultured in the absence of folate reached an arrested state from which limited replication could be induced by the addition of aquacobalamin; normal replication was induced by aquacobalamin plus 5-methyltetrahydrofolate. Results of this investigation are interpreted in terms of the requirement for tetrahydrofolate in cell replication and the production of this compound from folate and 5-formyltetrahydrofolate (via cobalamin-independent pathways) and from 5-methyltetrahydrofolate (via the cobalamin-dependent methionine synthetase).
通过在不含氰钴胺素的培养基中传代培养，并将胎牛血清（含有与蛋白质结合的钴胺素）替换为牛血清白蛋白，使L1210小鼠白血病细胞缺乏钴胺素。只要培养基中含有过量的叶酸或5-甲酰四氢叶酸，这些缺乏钴胺素的细胞就表现出正常的12小时复制时间。然而，除非添加外源性钴胺素，否则细胞对5-甲基四氢叶酸的反应较差。当使用低水平的叶酸或5-甲酰四氢叶酸时，也观察到了对钴胺素的依赖性。对于5-甲基四氢叶酸，分别在游离钴胺素和与转钴胺素II结合的钴胺素浓度为4000 pM和2 pM时观察到最佳的生长刺激。在钴胺素充足的条件下，细胞每个细胞含有2000至4000个钴胺素分子，而在缺乏状态下，该值降至每个细胞少于10个分子；在5-甲基四氢叶酸上的最佳复制需要每个细胞约180个分子。在没有叶酸的情况下培养的缺乏钴胺素的细胞达到停滞状态，通过添加水钴胺素可以诱导有限的复制；水钴胺素加5-甲基四氢叶酸可诱导正常复制。本研究结果根据细胞复制中对四氢叶酸的需求以及从叶酸和5-甲酰四氢叶酸（通过不依赖钴胺素的途径）和从5-甲基四氢叶酸（通过依赖钴胺素的甲硫氨酸合成酶）产生该化合物来解释。
