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Expression of a Folate Binding Protein in L1210 Cells Grown in Low Folate Medium¹

Department of Internal Medicine, Oncology Unit [G. J., I. K., B. C. R., G. R. W., J. H. S.], and Laboratory of Medical Enzymology, Department of Haematology [G. R.], University Hospital Utrecht; and the Department of Otolaryngology [B. J. M. B.], Free University Hospital Amsterdam; The Netherlands

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 M_r 42,000–44,000.

¹ This study was supported by Grant UUKC 8516 of The Netherlands Cancer Foundation.

² To whom requests for reprints should be addressed, at Oncology Unit, Department of Internal Medicine, University Hospital Utrecht, Catharijnesingel 101, 3511 GV Utrecht, The Netherlands.

Received 2/23/88. Revised 12/ 1/88. Accepted 12/29/88.

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