Studies of dihydrofolate reductase and methotrexate transport utilizing the technique of photoaffinity labeling
Thesis/Dissertation
·
OSTI ID:6281535
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.
- Research Organization:
- Cincinnati Univ., OH (USA)
- OSTI ID:
- 6281535
- Country of Publication:
- United States
- Language:
- English
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Journal Article
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Characterization of the methotrexate transport pathway in murine L1210 leukemia cells: Involvement of a membrane receptor and a cytosolic protein
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Tue Oct 04 00:00:00 EDT 1988
· Biochemistry; (USA)
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OSTI ID:7004412
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
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· Biochemistry; (United States)
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Related Subjects
550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANTIMETABOLITES
BETA DECAY RADIOISOTOPES
DAYS LIVING RADIOISOTOPES
DRUGS
ELECTROMAGNETIC RADIATION
ELECTRON CAPTURE RADIOISOTOPES
ENZYME INHIBITORS
ENZYMES
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
LABELLING
MEMBRANE TRANSPORT
METHOTREXATE
NUCLEI
ODD-EVEN NUCLEI
OXIDOREDUCTASES
RADIATIONS
RADIOISOTOPES
TEMPERATURE DEPENDENCE
TRACER TECHNIQUES
ULTRAVIOLET RADIATION
59 BASIC BIOLOGICAL SCIENCES
ANTIMETABOLITES
BETA DECAY RADIOISOTOPES
DAYS LIVING RADIOISOTOPES
DRUGS
ELECTROMAGNETIC RADIATION
ELECTRON CAPTURE RADIOISOTOPES
ENZYME INHIBITORS
ENZYMES
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
LABELLING
MEMBRANE TRANSPORT
METHOTREXATE
NUCLEI
ODD-EVEN NUCLEI
OXIDOREDUCTASES
RADIATIONS
RADIOISOTOPES
TEMPERATURE DEPENDENCE
TRACER TECHNIQUES
ULTRAVIOLET RADIATION