Moran R G, Werkheiser W C, Zakrzewski S F
J Biol Chem. 1976 Jun 25;251(12):3569-75.
Chromatographic procedures were developed which permit the separation of the monoglutamylfolic acid derivatives from each other and from their polyglutamyl congeners. Using these methods, the cellular folate derivatives in logarithmically growing L1210 cells were found to consist entirely of polyglutamyl derivatives. When ascorbate extracts of these cells were digested with hog kidney gamma-glutamylcarboxypeptidase and then chromatographed on DEAE-cellulose columns, the cellular folates eluted coincident with tetrahydrofolate, 5-formyltetrahydrofolate, 10-formyltetrahydrofolate, and 5-methyltetrahydrofolate, with minimal amounts associated with folate (approximately 0.8%) or dihydrofolate (approximately 0.4%). When these experiments were repeated on cells previously depleted of folates which were then grown on 0.4 muM pteroylglutamic acid (a concentration insufficient to maintain an optimal growth rate) more than 95% of the folates were again found in polyglutamyl form. These results imply that the polyglutamyl derivatives found are not storage forms of enzymatically active cofactor, but rather are the actual cofactor forms used by these mammalian cells.
已开发出多种色谱方法，可将单谷氨酰叶酸衍生物彼此分离，并与它们的多谷氨酰同类物分离。使用这些方法，发现对数生长期的L1210细胞中的细胞叶酸衍生物完全由多谷氨酰衍生物组成。当用猪肾γ-谷氨酰羧肽酶消化这些细胞的抗坏血酸提取物，然后在DEAE-纤维素柱上进行色谱分析时，细胞叶酸与四氢叶酸、5-甲酰基四氢叶酸、10-甲酰基四氢叶酸和5-甲基四氢叶酸同时洗脱，与叶酸（约0.8%）或二氢叶酸（约0.4%）相关的量极少。当对先前已耗尽叶酸、然后在0.4μM蝶酰谷氨酸（该浓度不足以维持最佳生长速率）上生长的细胞重复这些实验时，再次发现超过95%的叶酸以多谷氨酰形式存在。这些结果表明，所发现的多谷氨酰衍生物不是酶活性辅因子的储存形式，而是这些哺乳动物细胞实际使用的辅因子形式。
