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Differing Specificities for 4-Aminofolate Analogues of Folylpolyglutamyl Synthetase from Tumors and Proliferative Intestinal Epithelium of the Mouse with Significance for Selective Antitumor Action¹

Memorial Sloan Kettering Cancer Center, New York, New York 10021

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 K_m and highest V_max. The other analogues and folic acid exhibited nearly 2-fold lower V_max. Folic acid exhibited a 3-fold higher K_m than aminopterin. Alkylation of aminopterin (methotrexate) or carbon for nitrogen substitution (10-deazaaminopterin) at N-10 increased K_m 3- to 6-fold, while alkylation at C¹⁰ (10-ethyl-10-deazaaminopterin) restored K_m to near equivalency with aminopterin. For FPGS derived from proliferative intestinal epithelium, aminopterin was also the preferred substrate, but the value for V_max (derived with crude cell-free extract) was 6-fold lower than for tumor cell FPGS. Values for V_max (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 K_m 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 K_m (27-, 90-, and >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 K_m and V_max 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.

¹ Supported in part by Grants CA08748, CA18856, and CA22764 from the National Cancer Institute and the Elsa U. Pardee Foundation.

² To whom requests for reprints should be addressed, at Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021.

Received 12/11/89. Revised 4/ 6/90.

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