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 (less than 0.5 nM), but down-regulated after transfer in standard high folate (2 microM) medium. The binding capacity of L1210B cells for [3H]folic acid and [3H]-methotrexate was identical (5-11 pmol/10(6) 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/10(7) 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 degrees 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.
|Number of pages||5|
|Publication status||Published - 15 Apr 1989|