Regulation of folate and one-carbon metabolism in mammalian cells. II. Effect of folylpoly-gamma-glutamate synthetase substrate specificity and level on folate metabolism and folylpoly-gamma-glutamate specificity of metabolic cycles of one-carbon metabolism.

The effect of folylpoly-gamma-glutamate synthetase (FPGS) levels on folate accumulation was investigated in Chinese hamster ovary cells expressing various levels of human and Escherichia coli FPGS activity. At low medium folate concentrations, folate accumulation was limited by influx and was independent of FPGS activity except in cells expressing extremely low levels of FPGS. Essentially all transported folate was metabolized to retained polyglutamate derivatives, the chain length of which varied with the level of FPGS activity. As medium folate concentration increased through the physiological to the pharmacological range, cellular folate accumulation became proportional to FPGS activity and the chain length of intracellular folates decreased. At high folate concentrations, competition between substrates for FPGS limited the extent of polyglutamylation and less than 5% of transported folate was retained by the cell. Pteroyltriglutamates functioned as effectively as the longer chain length polyglutamates normally found in mammalian cells in the metabolic cycles of de novo purine and thymidylate biosynthesis but were unable to support glycine and methionine synthesis. Transfectants expressing human FPGS and containing folates of glutamate chain length ranging from four to eight were equally effective at supporting glycine synthesis, and transfectants expressing higher levels of FPGS were able to grow in the absence of methionine. Growth in the absence of methionine required high (nonphysiological) intracellular folate levels and longer chain length polyglutamates.