Biotin derivatives of methotrexate and folate. Synthesis and utilization for affinity purification of two membrane-associated folate transporters from L1210 cells.
Biotin derivatives of methotrexate and folate (2-(biotinamido)ethyl-1,3'-dithiopropionyldiaminopentyl methotrexate and/or folate), in which carboxyl groups of the functional components are joined by a disulfide-containing spacer, have been synthesized, purified by DEAE-Trisacryl chromatography, and characterized by high pressure liquid chromatography and mass spectrometry. These bifunctional, dissociable probes were utilized for the single-step purification to homogeneity of two folate transport proteins (43 and 39 kDa) from L1210 cells. Treatment of the 39-kDa protein with peptide N-glycosidase F produced a smaller component (32 kDa); the 43-kDa protein, conversely, was unchanged by this procedure. When the 39-kDa transporter in intact cells was labeled with a fluorescein derivative of folate and then treated with phosphoinositol-specific phospholipase C, complete loss of fluorescence was observed. Alternatively, there was no change in fluorescence when the 43-kDa transporter was labeled with a fluorescein derivative of methotrexate and treated with the enzyme. These results indicate that the 43-kDa transporter is a nonglycosylated, integral membrane protein, whereas the 39-kDa counterpart is heavily glycosylated and anchored exofacially to the membrane by a glycosylphosphatidylinositol component.
Fan
J
J
Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolla, California 92037.
Vitols
K S
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Huennekens
F M
FM
D000818:Animals; D001692:Biological Transport; D001710:Biotin / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000378:metabolism*; D007939:Leukemia L1210; D008727:Methotrexate / Q000378:metabolism*; D051379:Mice; D011956:Receptors, Cell Surface; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D011956:Receptors, Cell Surface; D001710:Biotin; D005492:Folic Acid; D008727:Methotrexate