Uptake and Metabolism of Methotrexate by Crithidia fasciculata

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The uptake and metabolism of methotrexate were examined in Crithidia fasciculata ATCC 12857. Whenthe cells grown in a mediumcontaining, biopterin were incubated with methotrexate, the uptake ofmethotrexate by the cells was completed within 1 min, and the amounts ofmethotrexate per 108 cells were maintained between 19 and 37 fmol/min throughout the remaining growth period. In contrast, the uptake offolate was linear for at least 20 min, and the uptake rate offolate by the cells varied from 31 to 247 fmol/min/108 cells during the growth. The folate uptake was inhibited by methotrexate and aminopterin. When C. fasciculata was cultivated in a medium containing methotrexate, the methotrexate taken up by the cells was metabolized to several compounds. Oneof the metabolites was isolated from the cells and the culture supernatant, using DEAE-cellulose and Sephadex G-10 column chromatographies, and identified as 4-amino-4-deoxy-10-methylpteroic acid (AMPte) on the basis of its behavior on thin-layer chromatography, and of its ultraviolet and infrared absorption spectra. An enzyme activity converting methotrexate to AMPtewas found in the cell-free extract of C. fasciculata.
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