We have isolated variants of L1210 cells (L1210B) expressing, in addition to the “classical” high affinity/low capacity system for reduced folate uptake, high levels of a membrane-associated folate binding protein. This folate binding protein was expressed in L1210 cells grown at low physiological folate levels (<0.5 nm), but down-regulated after transfer in standard high folate (2 µm) medium.

The binding capacity of L1210B cells for [3H]folic acid and [3H]-methotrexate was identical (5–11 pmol/106 cells) but affinities were different. The affinities relative to folic acid were 0.5 for 5-methyltetrahydrofolate, 0.25 for 5-formyltetrahydrofolate, 0.08 for 10-ethyl-10-deazaaminopterin, and 0.05 for methotrexate, respectively.

L1210B cells exposed to low extracellular concentrations of [3H]folic acid (25 nm) accumulated 15 pmol [3H]folic acid/107 cells over a 5-h period. [3H]Folic acid accumulation by wild-type L1210 cells could not be demonstrated under these conditions.

The folate binding protein in L1210B cells could be specifically and covalently labeled at 4°C with a N-hydroxysuccinimide ester of [3H]-methotrexate or [3H]folic acid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of detergent-solubilized membrane proteins showed a major labeled band with Mr 42,000–44,000.

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This study was supported by Grant UUKC 8516 of The Netherlands Cancer Foundation.

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