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Title: Photoaffinity analogues of methotrexate as folate antagonist binding probes. 2. Transport studies, photoaffinity labeling, and identification of the membrane carrier protein for methotrexate from murine L1210 cells

Abstract

A membrane-derived component of the methotrexate/one-carbon-reduced folate transport system in murine L1210 cells has been identified by using a photoaffinity analogue of methotrexate. The compound, a radioiodinated 4-azidosalicylyl derivative of the lysine analogue of methotrexate, is transported into murine L1210 cells in a temperature-dependent, sulfhydryl reagent inhibitable manner with a K/sub t/ of 506 +/- 79 nM and a V/sub max/ of 17.9 +/- 4.2 pmol min/sup -1/ (mg of total cellular protein)/sup -1/. Uptake of the iodinated compound at 200 nM is inhibited by low amounts of methotrexate. The parent compounds of the iodinated photoprobe inhibit (/sup 3/H)methotrexate uptake, with the uniodinated 4-azidosalicylyl derivative exhibiting a K/sub i/ of 66 +/- 21 nM. UV irradiation, at 4 /sup 0/C, of a cell suspension that had been incubated with the probe results in the covalent modification of a 46K-48K protein. This can be demonstrated when the plasma membranes from the labeled cells are analyzed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Labeling of this protein occurs half-maximally at a reagent concentration that correlates with the K/sub t/ for transport of the iodinated compound. Protection against labeling of this protein by increasing amounts of methotrexate parallels the concentration dependence ofmore » inhibition of photoprobe uptake by methotrexate. Evidence that, in the absence of irradiation and at 37/sup 0/C, the iodinated probe is actually internalized is demonstrated by the labeling of two soluble proteins (M/sub r/ 38K and 21K) derived from the cell homogenate supernatant.« less

Authors:
;
Publication Date:
Research Org.:
Univ. of Cincinnati College of Medicine, OH
OSTI Identifier:
5964988
Resource Type:
Journal Article
Journal Name:
Biochemistry; (United States)
Additional Journal Information:
Journal Volume: 26:15
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; LABELLED COMPOUNDS; MEMBRANE TRANSPORT; METHOTREXATE; PROTEINS; BIOCHEMICAL REACTION KINETICS; ANIMAL CELLS; CELL MEMBRANES; FOLIC ACID; INHIBITION; IODINE 125; MICE; TRITIUM COMPOUNDS; AMINO ACIDS; ANIMALS; ANTIMETABOLITES; AROMATICS; AZAARENES; BETA DECAY RADIOISOTOPES; CARBOXYLIC ACIDS; CELL CONSTITUENTS; DAYS LIVING RADIOISOTOPES; DRUGS; ELECTRON CAPTURE RADIOISOTOPES; HEMATINICS; HEMATOLOGIC AGENTS; HETEROCYCLIC COMPOUNDS; HYDROXY COMPOUNDS; INTERMEDIATE MASS NUCLEI; IODINE ISOTOPES; ISOTOPES; KINETICS; MAMMALS; MEMBRANES; NUCLEI; ODD-EVEN NUCLEI; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; PTERIDINES; RADIOISOTOPES; REACTION KINETICS; RODENTS; VERTEBRATES; VITAMIN B GROUP; VITAMINS; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Price, E M, and Freisheim, J H. Photoaffinity analogues of methotrexate as folate antagonist binding probes. 2. Transport studies, photoaffinity labeling, and identification of the membrane carrier protein for methotrexate from murine L1210 cells. United States: N. p., 1987. Web.
Price, E M, & Freisheim, J H. Photoaffinity analogues of methotrexate as folate antagonist binding probes. 2. Transport studies, photoaffinity labeling, and identification of the membrane carrier protein for methotrexate from murine L1210 cells. United States.
Price, E M, and Freisheim, J H. 1987. "Photoaffinity analogues of methotrexate as folate antagonist binding probes. 2. Transport studies, photoaffinity labeling, and identification of the membrane carrier protein for methotrexate from murine L1210 cells". United States.
@article{osti_5964988,
title = {Photoaffinity analogues of methotrexate as folate antagonist binding probes. 2. Transport studies, photoaffinity labeling, and identification of the membrane carrier protein for methotrexate from murine L1210 cells},
author = {Price, E M and Freisheim, J H},
abstractNote = {A membrane-derived component of the methotrexate/one-carbon-reduced folate transport system in murine L1210 cells has been identified by using a photoaffinity analogue of methotrexate. The compound, a radioiodinated 4-azidosalicylyl derivative of the lysine analogue of methotrexate, is transported into murine L1210 cells in a temperature-dependent, sulfhydryl reagent inhibitable manner with a K/sub t/ of 506 +/- 79 nM and a V/sub max/ of 17.9 +/- 4.2 pmol min/sup -1/ (mg of total cellular protein)/sup -1/. Uptake of the iodinated compound at 200 nM is inhibited by low amounts of methotrexate. The parent compounds of the iodinated photoprobe inhibit (/sup 3/H)methotrexate uptake, with the uniodinated 4-azidosalicylyl derivative exhibiting a K/sub i/ of 66 +/- 21 nM. UV irradiation, at 4 /sup 0/C, of a cell suspension that had been incubated with the probe results in the covalent modification of a 46K-48K protein. This can be demonstrated when the plasma membranes from the labeled cells are analyzed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Labeling of this protein occurs half-maximally at a reagent concentration that correlates with the K/sub t/ for transport of the iodinated compound. Protection against labeling of this protein by increasing amounts of methotrexate parallels the concentration dependence of inhibition of photoprobe uptake by methotrexate. Evidence that, in the absence of irradiation and at 37/sup 0/C, the iodinated probe is actually internalized is demonstrated by the labeling of two soluble proteins (M/sub r/ 38K and 21K) derived from the cell homogenate supernatant.},
doi = {},
url = {https://www.osti.gov/biblio/5964988}, journal = {Biochemistry; (United States)},
number = ,
volume = 26:15,
place = {United States},
year = {Tue Jul 28 00:00:00 EDT 1987},
month = {Tue Jul 28 00:00:00 EDT 1987}
}