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 Journal Article
| Authors: | Chiao, J. H.; Yang, C. H.; Roy, K.; Pain, J.; Sirotnak, F. M. |
| Article Title: | 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 |
| Abstract: | 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 from transporter overproducing L1210/R83 cells. These cells were labeled with the N-hydroxysuccinimide ester of [3H]aminopterin (AMT), the isolated plasma membrane alkaline washed to remove nonintegral membrane proteins, detergent-solubilized, 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 (L1210/R83 ≫ L1210/0) a 46-kDa protein during SDS-polyacrylamide gel electrophoresis of plasma membrane from L1210/R83 and L1210 cells and in L1210/R83 cells after trichloroacetic acid precipitation. In contrast to that reported for human tumor cells, glycosidase treatment of RFT revealed 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 tumors. Blotting of plasma membrane from methotrexate resistant, transport defective L1210 cell variants exhibited wild-type levels of a less electrophoretically mobile RFT or greater levels of the same 46-kDa RFT which could not be affinity labeled with N-hydroxysuccinimide-[3H]AMT. The same antibody differentially blotted a 83-kDa plasma membrane protein from human HL-60 and CCRF-CEM cells with different levels of reduced folate transport and affinity labeling of RFT, verifying the conserved nature of this protein consistent with earlier functional studies. |
| Keywords: | leukemia; carrier protein; human cell; nonhuman; animal cell; mouse; animals; mice; tumor cells, cultured; monoclonal antibody; blotting, western; species specificity; protein purification; carrier proteins; cell membrane; protein transport; tumor cell; immunoblotting; ligands; folic acid; antibodies; cross reaction; aminopterin; polyacrylamide gel electrophoresis; electrophoresis, polyacrylamide gel; receptors, cell surface; tritium; oligosaccharide; immunoaffinity chromatography; antigen-antibody reactions; glycoside hydrolases; rabbits; chromatography, affinity; protein glycosylation; cross reactions; trichloroacetic acid; leukemia l1210; aminopterin derivative; humans; human; priority journal; article |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 270 |
| Issue: | 50 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 1995-12-15 |
| Start Page: | 29698 |
| End Page: | 29704 |
| Language: | English |
| PUBMED: | 8530358 |
| PROVIDER: | scopus |
| DOI: | 10.1074/jbc.270.50.29698 |
| DOI/URL: | |
| Notes: | Article -- Export Date: 28 August 2018 -- Source: Scopus |
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