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J. Biol. Chem., Vol. 266, Issue 23, 14862-14865, Aug, 1991

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

J Fan, KS Vitols and FM Huennekens
Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolla, California 92037.

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.
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