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

Thesis/Dissertation · Thu Jan 01 00:00:00 EST 1987

OSTI ID:6281535

Price, E M

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
• Resource Relation: Other Information: Thesis (Ph. D.)
• Country of Publication: United States
• Language: English

Related Subjects

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