Affinity labeling of the folate-methotrexate transporter from Leishmania donovani
- Oregon Health Sciences Univ., Portland (USA)
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. When intact wild-type (DI700) Leishmania donovani or preparations of their membranes were incubated with a 0.4 {mu}M concentration of either activated ({sup 3}H)folate or activated ({sup 3}H)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. 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. These studies provide biochemical evidence that the folate-methotrexate transporter of Leishmania donovani can be identified in crude extracts by an affinity labeling technique and serve as a prerequisite to further analysis of the transport protein by providing a vehicle for subsequent purification of this membrane carrier. Moreover, these investigations suggest that the affinity labeling technique using EDC-activated ligands may be exploitable to analyze other cell surface binding proteins in Leishmania donovani, as well as in other organisms.
- OSTI ID:
- 5353225
- Journal Information:
- Biochemistry; (USA), Vol. 28:17; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
FOLIC ACID
RECEPTORS
MEMBRANE PROTEINS
LABELLING
MEMBRANE TRANSPORT
BIOCHEMICAL REACTION KINETICS
METHOTREXATE
PROTEINS
ELECTROPHORESIS
LIGANDS
MEMBRANES
PARASITES
PROTOZOA
TRITIUM COMPOUNDS
AMINO ACIDS
ANIMALS
ANTIMETABOLITES
AROMATICS
AZAARENES
CARBOXYLIC ACIDS
DRUGS
HEMATINICS
HEMATOLOGIC AGENTS
HETEROCYCLIC COMPOUNDS
HYDROGEN COMPOUNDS
HYDROXY COMPOUNDS
INVERTEBRATES
KINETICS
MICROORGANISMS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PTERIDINES
REACTION KINETICS
VITAMIN B GROUP
VITAMINS
550201* - Biochemistry- Tracer Techniques