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Title: Studies of dihydrofolate reductase and methotrexate transport utilizing the technique of photoaffinity labeling

Abstract

The methotrexate (MTX) transport mechanism in murine L1210 leukemia cells appears to involve a temperature- and energy-dependent, carrier-mediated, saturable process. Although the intracellular target for MTX, dihydrofolate reductase (DHFR), has been well characterized, the protein(s) involved in MTX uptake have yet to be unequivocally identified. In order to characterize the MTX transport system of L1210 cells, photoaffinity analogues of the drug have been synthesized. They are the azidosalicylyl derivatives of lysine and ornithine analogues of MTX, and their iodinated counterparts. All of the compounds were potent inhibitors of DHFR. When the L1210 DHFR:APA-(/sup 125/I)ASA-Lys complex was irradiated with long-wave UV light, the photoaffinity analogue modified the enzyme with high efficiency. This reaction was specific as an excess of MTX prevented the covalent incorporation of the probe into DHFR. Cyanogen bromide digestion of the labeled enzyme, followed by sequence analysis of the singularly labeled peptide revealed that the enzyme was labeled in a region that encompasses Lys 63, Asn 64 and Arg 65 of native DHRF.

Authors:
Publication Date:
Research Org.:
Cincinnati Univ., OH (USA)
OSTI Identifier:
6281535
Resource Type:
Thesis/Dissertation
Resource Relation:
Other Information: Thesis (Ph. D.)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; METHOTREXATE; MEMBRANE TRANSPORT; OXIDOREDUCTASES; LABELLING; ENZYME INHIBITORS; IODINE 125; TEMPERATURE DEPENDENCE; TRACER TECHNIQUES; ULTRAVIOLET RADIATION; ANTIMETABOLITES; BETA DECAY RADIOISOTOPES; DAYS LIVING RADIOISOTOPES; DRUGS; ELECTROMAGNETIC RADIATION; ELECTRON CAPTURE RADIOISOTOPES; ENZYMES; INTERMEDIATE MASS NUCLEI; IODINE ISOTOPES; ISOTOPE APPLICATIONS; ISOTOPES; NUCLEI; ODD-EVEN NUCLEI; RADIATIONS; RADIOISOTOPES; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Price, E M. Studies of dihydrofolate reductase and methotrexate transport utilizing the technique of photoaffinity labeling. United States: N. p., 1987. Web.
Price, E M. Studies of dihydrofolate reductase and methotrexate transport utilizing the technique of photoaffinity labeling. United States.
Price, E M. Thu . "Studies of dihydrofolate reductase and methotrexate transport utilizing the technique of photoaffinity labeling". United States.
@article{osti_6281535,
title = {Studies of dihydrofolate reductase and methotrexate transport utilizing the technique of photoaffinity labeling},
author = {Price, E M},
abstractNote = {The methotrexate (MTX) transport mechanism in murine L1210 leukemia cells appears to involve a temperature- and energy-dependent, carrier-mediated, saturable process. Although the intracellular target for MTX, dihydrofolate reductase (DHFR), has been well characterized, the protein(s) involved in MTX uptake have yet to be unequivocally identified. In order to characterize the MTX transport system of L1210 cells, photoaffinity analogues of the drug have been synthesized. They are the azidosalicylyl derivatives of lysine and ornithine analogues of MTX, and their iodinated counterparts. All of the compounds were potent inhibitors of DHFR. When the L1210 DHFR:APA-(/sup 125/I)ASA-Lys complex was irradiated with long-wave UV light, the photoaffinity analogue modified the enzyme with high efficiency. This reaction was specific as an excess of MTX prevented the covalent incorporation of the probe into DHFR. Cyanogen bromide digestion of the labeled enzyme, followed by sequence analysis of the singularly labeled peptide revealed that the enzyme was labeled in a region that encompasses Lys 63, Asn 64 and Arg 65 of native DHRF.},
doi = {},
url = {https://www.osti.gov/biblio/6281535}, journal = {},
number = ,
volume = ,
place = {United States},
year = {1987},
month = {1}
}

Thesis/Dissertation:
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