REGULATION OF FOLATE AND ONE-CARBON METABOLISM IN MAMMALIAN-CELLS .2.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 fo late accumulation was investigated in Chinese hamster ovary cells expr essing various levels of human and Escherichia coli FPGS activity. At low medium folate concentrations, folate accumulation was limited by i nflux and was independent of FPGS activity except in cells expressing extremely low levels of FPGS. Essentially all transported folate was m etabolized to retained polyglutamate derivatives, the chain length of which varied with the level of FPGS activity. As medium folate concent ration increased through the physiological to the pharmacological rang e, cellular folate accumulation became proportional to FPGS activity a nd the chain length of intracellular folates decreased. At high folate concentrations, competition between substrates for FPGS limited the e xtent of polyglutamylation and less than 5% of transported folate was retained by the cell. Pteroyltriglutainates functioned as effectively as the longer chain length polyglutamates normally found in mammalian cells in the metabolic cycles of de novo purine and thymidylate biosyn thesis but were unable to support glycine and methionine synthesis. Tr ansfectants expressing human FPGS and containing folates of glutamate chain length ranging from four to eight were equally effective at supp orting glycine synthesis, and transfectants expressing higher levels o f FPGS were able to grow in the absence of methionine. Growth in the a bsence of methionine required high (nonphysiological) intracellular fo late levels and longer chain length polyglutamates.