LIGAND-DIRECTED IMMUNOAFFINITY PURIFICATION AND PROPERTIES OF THE ONE-CARBON, REDUCED FOLATE TRANSPORTER - INTERSPECIES IMMUNO-CROSS-REACTIVITY AND EXPRESSION OF THE NATIVE TRANSPORTER IN MURINE AND HUMAN TUMOR-CELLS AND THEIR TRANSPORT-ALTERED VARIANTS

Almost complete purification (>95%) of the 46-kDa murine, one-carbon, reduced folate transporter (RFT) at a recovery of 20% was obtained by Ligand-directed immunoaffinity fractionation hom transporter overprodu cing L1210/R83 cells, These cells were labeled with the N-hydroxysucci nimide ester of [H-3]aminopterin (AMT), the isolated plasma membrane a lkaline washed to remove nonintegral membrane proteins, detergent-solu bilized, and RFT-separated on an anti-AMT antibody-protein G-Sepharose column followed by preparative SDS-polyacrylamide gel electrophoresis . Anti-RFT antibody, subsequently derived, differentially blotted (L12 10/R83 >> L1210/0) a 46-kDa protein during SDS-polyacrylamide gel elec trophoresis of plasma membrane from L1210/R83 and L1210 cells and in L 1210/R83 cells after trichloroacetic acid precipitation. In contrast t o that reported for human tumor cells, glycosidase treatment of RFT re vealed no common N- or O-linked core oligosaccharides associated with this protein. The same 46-kDa protein at different relative levels was revealed in a Western blot of plasma membrane from other murine tumor s. Blotting of plasma membrane from methotrexate resistant, transport defective L1210 cell variants exhibited wild-type levels of a less ele ctrophoretically mobile RFT or greater levels of the same 46-kDa RFT w hich could not be affinity labeled with N-hydroxysuccinimide-[H-3]AMT. The same antibody differentially blotted a 83-kDa plasma membrane pro tein from human HL-60 and CCRF-CEM cells with different levels of redu ced folate transport and affinity labeling of RFT, verifying the conse rved nature of this protein consistent with earlier functional studies .