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Transport of Folate Compounds in L1210 Cells: Kinetic Evidence That Folate Influx Proceeds via the High-Affinity Transport System for 5-Methyltetrahydrofolate and Methotrexate¹

Division of Biochemistry, Department of Basic and Clinical Research, Research Institute of Scripps Clinic, La Jolla, California 92037

Folate is transported into L1210 mouse leukemia cells by the same system that mediates the uptake of methotrexate and reduced folate compounds. This conclusion is supported by the following observations: (a) methotrexate competitively inhibits the influx of folate and the K_i is comparable to the K_t for methotrexate influx; (b) the profile for inhibition of folate influx by methotrexate is monophasic and complete inhibition is achieved at high concentrations of the competitor; (c) folate inhibits the influx of methotrexate and the K_i is comparable to the K_t for folate influx; (d) the N-hydroxysuccinimide ester of methotrexate, a potent and specific irreversible inhibitor of the reduced folate system, also blocks the influx of folate; (e) folate and methotrexate influx are both inhibited by low concentrations of p-chloromercuriphenylsulfonate; and (f) folate influx fluctuates with the anionic composition of the medium in the same fashion as the influx of methotrexate.

Measurements of folate influx can be complicated by the fact that the ³H-labeled substrate is susceptible to decomposition and that labeled breakdown products at concentrations as low as 1% contribute appreciably to the observed uptake. Of these products, 6-hydroxymethylpterin appears to account for most of the extraneous uptake. Impurities can be eliminated by subjecting the [³H]folate to preparative thin-layer chromatography immediately prior to use.

¹ This work was supported by grants from the National Cancer Institute (CA 06522, CA 23970, and CA 32261) and the American Cancer Society (CH-31 and CH-229). Manuscript BCR 4024 from the Research Institute of Scripps Clinic.

² Present address: Laboratory of Molecular Biology, Medical Research Council Centre, University Medical School, Hills Road, Cambridge CB2 2QH, England.

Received 9/17/85. Revised 12/ 6/85. Accepted 12/27/85.