Affinity labeling of the folate-methotrexate transporter from Leishmania donovani

Joanne T. Beck, Buddy Ullman

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

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 μM 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 μM 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. Inhibitors of folate-methotrexate transport, including dihydrofolate, tetrahydrofolate, 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, and (p-aminobenzoyl)glutamate, blocked the incorporation of radiolabeled ligand into the 46 000-dalton polypeptide, while biopterin and pteroic acid, two pterins that did not block folate-methotrexate entry into wild-type Leishmania donovani, did not interfere with the labeling of the 46 000-dalton moiety. 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.

Original languageEnglish (US)
Pages (from-to)6931-6937
Number of pages7
JournalBiochemistry
Volume28
Issue number17
StatePublished - 1989

Fingerprint

Folic Acid Transporters
Leishmania donovani
Folic Acid
Methotrexate
Labeling
Ligands
Membranes
Carrier Proteins
Biopterin
Pterins
Derivatives
Peptides
Time and motion study
Proteins
Leucovorin
Dimethyl Sulfoxide
Electrophoresis
Complex Mixtures
Purification
Polyacrylamide Gel Electrophoresis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Affinity labeling of the folate-methotrexate transporter from Leishmania donovani. / Beck, Joanne T.; Ullman, Buddy.

In: Biochemistry, Vol. 28, No. 17, 1989, p. 6931-6937.

Research output: Contribution to journalArticle

Beck, JT & Ullman, B 1989, 'Affinity labeling of the folate-methotrexate transporter from Leishmania donovani', Biochemistry, vol. 28, no. 17, pp. 6931-6937.
Beck, Joanne T. ; Ullman, Buddy. / Affinity labeling of the folate-methotrexate transporter from Leishmania donovani. In: Biochemistry. 1989 ; Vol. 28, No. 17. pp. 6931-6937.
@article{17bb6e09424d4046933f8c4963fdf596,
title = "Affinity labeling of the folate-methotrexate transporter from Leishmania donovani",
abstract = "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 μM 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 μM 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. Inhibitors of folate-methotrexate transport, including dihydrofolate, tetrahydrofolate, 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, and (p-aminobenzoyl)glutamate, blocked the incorporation of radiolabeled ligand into the 46 000-dalton polypeptide, while biopterin and pteroic acid, two pterins that did not block folate-methotrexate entry into wild-type Leishmania donovani, did not interfere with the labeling of the 46 000-dalton moiety. 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.",
author = "Beck, {Joanne T.} and Buddy Ullman",
year = "1989",
language = "English (US)",
volume = "28",
pages = "6931--6937",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "17",

}

TY - JOUR

T1 - Affinity labeling of the folate-methotrexate transporter from Leishmania donovani

AU - Beck, Joanne T.

AU - Ullman, Buddy

PY - 1989

Y1 - 1989

N2 - 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 μM 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 μM 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. Inhibitors of folate-methotrexate transport, including dihydrofolate, tetrahydrofolate, 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, and (p-aminobenzoyl)glutamate, blocked the incorporation of radiolabeled ligand into the 46 000-dalton polypeptide, while biopterin and pteroic acid, two pterins that did not block folate-methotrexate entry into wild-type Leishmania donovani, did not interfere with the labeling of the 46 000-dalton moiety. 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.

AB - 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 μM 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 μM 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. Inhibitors of folate-methotrexate transport, including dihydrofolate, tetrahydrofolate, 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, and (p-aminobenzoyl)glutamate, blocked the incorporation of radiolabeled ligand into the 46 000-dalton polypeptide, while biopterin and pteroic acid, two pterins that did not block folate-methotrexate entry into wild-type Leishmania donovani, did not interfere with the labeling of the 46 000-dalton moiety. 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.

UR - http://www.scopus.com/inward/record.url?scp=0024453984&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0024453984&partnerID=8YFLogxK

M3 - Article

C2 - 2554960

AN - SCOPUS:0024453984

VL - 28

SP - 6931

EP - 6937

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 17

ER -