GROWTH-DEPENDENT CHANGES OF FOLATE METABOLISM AND BIOSYNTHESIS IN CULTURED DAUCUS-CAROTA CELLS

Metabolism of folates has been studied in cultures of Daucus carota cells. With the addition to the culture medium of natural folates as folic acid, 5-HCO-H4PteGlu, 5-CH3-H4-PteGlu and pteroic acid the final yield was scarcely affected but folic acid stimulated the initial rate of growth. On the contrary, the growth rate was reduced by 5-HCO-H4-PteGlu. Growth was severely inhibited by folate analogues such aminopterin and methotrexate and by sulfanilamide, the inhibition of the latter being easily reversed by equimolar amount of folic acid. Microbiological assays of cell folates with Lactobacillus casei were performed at different stages of growth, covering the entire culture cycle. Most folates can be measured only after treatment of the extracts with conjugase (gamma-glutamylhydrolase, an enzyme removing glutamate from the glutamate chain of folates) thus indicating an high ratio between poly- and mono-plus oligo-glutamate forms. Radiochromatographic analyses of folates obtained by cells cultured in [H-3]folic acid show the presence, besides of tri- and penta-glutamates, of forms with very long chains, of unidentified length. A strong shortening of the glutamate chain length was obtained with aminopterin and methotrexate. The ratio poly/mono, as well as the cell amount of folates, varies during the culture cycle. Rate of folate accumulation reaches its maximum before that of culture growth and sharply decreases before the slowing down of cell divisions, suggesting that derepression or repression of folate biosynthetic pathways precedes the changes in the trend of cell replication.