Biotin derivatives of methotrexate and folate. Synthesis and utilization for affinity purification of two membrane-associated folate transporters from L1210 cells.

摘要: Biotin derivatives of methotrexate and folate (2-(biotinamido)ethyl-1,3'-dithiopropionyldiaminopentyl and/or folate), in which carboxyl groups the functional components are joined by a disulfide-containing spacer, have been synthesized, purified DEAE-Trisacryl chromatography, characterized high pressure liquid chromatography mass spectrometry. These bifunctional, dissociable probes were utilized for single-step purification to homogeneity two transport proteins (43 39 kDa) from L1210 cells. Treatment 39-kDa protein with peptide N-glycosidase F produced smaller component (32 kDa); 43-kDa protein, conversely, was unchanged this procedure. When transporter intact cells labeled fluorescein derivative then treated phosphoinositol-specific phospholipase C, complete loss fluorescence observed. Alternatively, there no change when enzyme. results indicate that is nonglycosylated, integral membrane whereas counterpart heavily glycosylated anchored exofacially glycosylphosphatidylinositol component.