Biosynthesis of Biopterin by Chinese Hamster Ovary (CHO K1) Cell Culture

By use of a Crithidia bioassay, the production of a 6-(Lerythrodihydroxypropyl)pterin (biopterin)-like substance was observed during the cultivation of Chinese hamster ovary cell line (CHO Kl) and mouse neuroblastoma cell line (C-1300).Further studies were carried out with the CHO Kl cells using various suspected precursors.In a cell culture [2J4C]folic acid was converted to 2-amino-4-hydroxypteridine (pterin), 6-hydroxymethylpterin, and 6-carboxypterin, but not to biopterin.When [2J4C]guanine was added to the culture, radioactive biopterin and pterin were obtained.However, when [2J4C]guanine was replaced by [8J%]guanine, little or no radioactivity was incorporated into biopterin and other pterins, indicating that the purine undergoes ring opening and elimination of carbon atom 8.When the cells were cultured in a bioptkrin-free medium with [2J4C]guanine, the specific radioactivities of the isolated biopterin and GMP from RNA were in close agreement.A similar experiment with [UJ4C]guanosine showed that only about 10% of the label found in the GMP of RNA appeared in the ribosyl moiety.Therefore, using this approach the possible involvement of the ribosyl moiety of GMP in biopterin biosynthesis could not be unequivocally determined.In addition, the results suggested that the radioactivity found in the other nucleotides of RNA was distributed among the ribose moiety.When [6-14C]glucase was used most of the radioactivity found in biopterin was localized in the side chain and in carbon atom 5 of the ribose moiety of GMP in RNA.The results indicate that guanosine or one of its phosphorylated derivatives is the precursor of biopterin and that the side chain is of ribose origin.The addition of 6-lactyl-7,Sdihydropterin (sepiapterin) to a cell culture with [2-'4Clguanine increased the amount of biopterin isolated and prevented the incorporation of radioactivity into the biopterin.This result indicates the possible intermediary role of sepiapterin.Based upon these observations, the metabolic relationship of pterins in CHO cell culture is proposed.