Abstract
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.
Highlights
Biotin derivatives of methotrexate and folate (2-(bio- tics, relatively little is known about the membrane-associated, tinamido)ethyl- 1,3’-dithiopropionyldiaminopentyl methotrexate and/or folate), in whichcarboxyl groups of the functional componentsare joined by a disulfidecontaining spacer, have been synthesized, purified by binding proteins that play key roles in the translocation process
Streptavidin-agarose beads wereemployed to collect the labeled proteins from detergentextracts of the no change in fluorescence when the 43-kDa trans- membranes, and the proteins were released by reduction of porter was labeled with a fluorescein derivative of the S-S bond in the biotin-SS-MTX or -folate
Two separate transport systems for folate compounds and methotrexate (MTX)’ have been described in L1210 mouse Materials-The following were obtained from commercial sources: NHSS-SS-biotin (Pierce Chemical Co.), streptavidin-agarose beads
Summary
Biotin derivatives of methotrexate and folate (2-(bio- tics, relatively little is known about the membrane-associated, tinamido)ethyl- 1,3’-dithiopropionyldiaminopentyl methotrexate and/or folate), in whichcarboxyl groups of the functional componentsare joined by a disulfidecontaining spacer, have been synthesized, purified by binding proteins that play key roles in the translocation process. When the 39-kDa transporter in intact cells was labeled with a fluorescein derivative of folate and treated with phosphoinositol-specific phospholipase C, complete loss of fluorescence was observed.
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