| Folate-mediated one-carbon metabolism is critical for the synthesis of numerous cellular constituents including nucleic acids, choline, glycine, and methyl groups for various metabolites. In mammalian systems, the β-carbon of serine provides the majority of carbon units with tetrahydrofolate (THF) serving as the carrier of carbon units via a network of oxidation-reduction reactions.; Our project was undertaken to investigate the contribution of cytosol and mitochondria in one-carbon metabolism of Chinese hamster ovary (CHO) cell lines, focusing on the one-carbon transfer from serine and formate to thymine and purine synthesis. The cells were incubated with serine or formate labeled with stable isotopes, then DNA was extracted from each cells, and the DNA was hydrolyzed to bases, and the bases were derivatized and the derivatized samples were applied to GC/MS for measurement.; The result from various experiments in this study revealed clearly the significance of mitochondrial serine hydroxy methyl transferase in one-carbon metabolism, i.e. in wild type CHO, two third of one carbon in thymine synthesis was shown to be originated from mSHMT. Formate was shown to be an important source of one-carbon in glycine-auxotrophic cells, Gly A, Gly B and Gly C, especially at a lower folate concentration (10 nM). The methyl flux toward methionine synthesis was measured and methionine de novo synthesis (met-DNS) was calculated and characterized for MR15 and Mat I cells, It showed an evident rise of met-DNS in MR15 and a reduction in Mat I. Met-DNS activity in wild type CHO was greatly stimulated when cells were provided with lower than 20 uM methionine. A series of competition studies provided useful information regarding the validity of labeled metabolites as internal standards for the future experiments.; This study provides a unique insight into the quantitative aspects of one carbon metabolism, which have not been accessible via other means. We also validate our results by quantitatively confirming previously deduced findings. |
