Rumberger B G, Barrueco J R, Sirotnak F M
Memorial Sloan Kettering Cancer Center, New York, New York 10021.
Cancer Res. 1990 Aug 1;50(15):4639-43.
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.
从鼠L1210白血病细胞、肉瘤180细胞以及小鼠小肠腔上皮的增殖部分（叶酸类似物毒性限制部位）中部分纯化得到的叶酰聚谷氨酸合成酶（FPGS），对其利用各种4-氨基叶酸作为底物的能力进行了检测。对于肿瘤来源的FPGS，氨蝶呤总体上是最优选的底物，其表观Km值最低，Vmax最高。其他类似物和叶酸的Vmax值低近2倍。叶酸的Km值比氨蝶呤高3倍。氨蝶呤在N-10位的烷基化（甲氨蝶呤）或碳氮取代（10-脱氮氨蝶呤）使Km增加3至6倍，而在C10位的烷基化（10-乙基-10-脱氮氨蝶呤）使Km恢复到与氨蝶呤接近相等的水平。对于来源于增殖性肠上皮的FPGS，氨蝶呤也是优选的底物，但Vmax值（由粗制无细胞提取物得出）比肿瘤细胞FPGS低6倍。其他4-氨基叶酸类似物和叶酸的Vmax值（由部分纯化的FPGS得出）与氨蝶呤相比相似（甲氨蝶呤）或低2倍（10-乙基-10-脱氮氨蝶呤）和5倍（叶酸）。氨蝶呤得出的Km值与肿瘤细胞FPGS得出的Km值相似。叶酸的Km值高2倍，氨蝶呤在N-10位的烷基化（甲氨蝶呤）或碳氮取代（10-脱氮氨蝶呤），无论有无烷基化（分别为甲氨蝶呤、10-乙基-10-脱氮氨蝶呤和10-脱氮氨蝶呤的27倍、90倍和大于100倍），都会显著增加Km。在其他研究中发现，与甲氨蝶呤二谷氨酸相比，氨蝶呤二谷氨酸（氨蝶呤 +G1）对于所有三种来源的FPGS而言都是相对较差的底物，这在每种情况下所测得的Km值和Vmax值方面均如此。通过对与母体化合物反应过程中产物形成的高压液相色谱分析，证实了从L1210细胞得到的FPGS的研究结果。本文讨论了这些结果对于这些类似物相对毒性和治疗活性问题的意义。
