Affinity labeling of the folate-methotrexate transporter from Leishmania donovani.
An affinity labeling technique has been developed to identify the folate-methotrexate transporter of Leishmania donovani promastigotes using "activated" derivatives of the ligands. These "activated" derivatives were synthesized by incubating folate and methotrexate with a 10-fold excess of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) for 10 min at ambient temperature in dimethyl sulfoxide. Preincubation of intact cells with nonradioactive "activated" folate or methotrexate at a concentration of 40 microM inhibited the capacity of wild-type cells to transport submicromolar concentrations of unmodified ligand. When intact wild-type (DI700) Leishmania donovani or preparations of their membranes were incubated with a 0.4 microM concentration of either "activated" [3H]folate or "activated" [3H]methotrexate, the radiolabeled ligands were covalently incorporated into a polypeptide with a molecular weight of approximately 46,000, as demonstrated by SDS-polyacrylamide gel electrophoresis. No affinity labeling of a 46,000-dalton protein was observed when equimolar concentrations of "activated" radiolabeled ligands were incubated with intact cells or membranes prepared from a methotrexate-resistant mutant clone of Leishmania donovani, MTXA5, that is genetically defective in folate-methotrexate transport capability [Kaur, K., Coons, T., Emmett, K., & Ullman, B. (1988) J. Biol. Chem. 263, 7020-7028]. However, some labeling of a 46,000-dalton protein was observed when MTXA5 cells were incubated with higher concentrations of "activated" ligands. Time course studies indicated that maximal labeling of the 46,000-dalton protein occurred within 5-10 min of incubation of intact cells with "activated" ligand.(ABSTRACT TRUNCATED AT 250 WORDS)
Beck
J T
JT
Department of Biochemistry, Oregon Health Sciences University, Portland 97201.
Ullman
B
B
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000378:metabolism; D005493:Folic Acid Antagonists / Q000494:pharmacology; D007700:Kinetics; D007893:Leishmania donovani / Q000378:metabolism*; D008024:Ligands; D008727:Methotrexate / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism
D000345:Affinity Labels; D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D005493:Folic Acid Antagonists; D008024:Ligands; D011956:Receptors, Cell Surface; D005492:Folic Acid; D008727:Methotrexate