Folylpolyglutamyl
synthetase (FPGS), partially purified from murine
L1210 leukemia and
Sarcoma 180 cells and the proliferative fraction of
luminal epithelium from mouse small intestine (the site of limiting toxicity to
folate analogues), was examined for its ability to utilize various 4-aminofolates as substrates. For
tumor-derived FPGS,
aminopterin was the most preferred substrate overall, exhibiting the lowest value for apparent Km and highest Vmax. The other analogues and
folic acid exhibited nearly 2-fold lower Vmax.
Folic acid exhibited a 3-fold higher Km than
aminopterin. Alkylation of
aminopterin (
methotrexate) or
carbon for
nitrogen substitution (10-deazaaminopterin) at N-10 increased Km 3- to 6-fold, while alkylation at C10 (10-ethyl-10-deazaaminopterin) restored Km to near equivalency with
aminopterin. For FPGS derived from proliferative intestinal epithelium,
aminopterin was also the preferred substrate, but the value for Vmax (derived with crude cell-free extract) was 6-fold lower than for
tumor cell FPGS. Values for Vmax (derived with partially purified FPGS) for the other 4-aminofolate analogues and
folic acid were similar (
methotrexate) or 2-fold (10-ethyl-10-deazaaminopterin) and 5-fold (
folic acid) lower than for
aminopterin. The value for Km derived with
aminopterin was similar to that derived for either
tumor cell FPGS. The value for
folic acid was 2-fold higher, and alkylation of
aminopterin (
methotrexate) or
carbon to
nitrogen substitution (10-deazaaminopterin) at N-10 with (10-ethyl-10-deazaaminopterin) or without alkylation markedly increased Km (27-, 90-, and greater than 100-fold, respectively, for
methotrexate, 10-ethyl-10-deazaaminopterin, and 10-deazaaminopterin). In other studies, it was found that the diglutamate of
aminopterin (
aminopterin +G1) was a relatively poor substrate for FPGS derived from all three sources compared with
methotrexate diglutamate, both in respect to values for Km and Vmax that were measured in each case. Findings with FPGS derived from L1210 cells were confirmed by high-pressure liquid chromatography analysis of product formation during the reaction with the parent compounds. The significance of the results presented here to the question of relative toxicity and therapeutic activity of these analogues is discussed.