Photoaffinity analogues of methotrexate as folate antagonist binding probes. 1. Photoaffinity labeling of murine L1210 dihydrofolate reductase and amino acid sequence of the binding region
N/sup ..cap alpha../-(4-Amino-4-deoxy-10-methylpteroyl)-N/sup epsilon/-(4-azido-5-(/sup 125/I)iodosalicylyl)-L-lysine, a photoaffinity analogue of methotrexate, is only 2-fold less potent than methotrexate in the inhibition of murine L1210 dihydrofolate reductase. Irradiation of the enzyme in the presence of an equimolar concentration of the /sup 125/I-labeled analogue ultimately leads to an 8% incorporation of the photoprobe. A 100-fold molar excess of methotrexate essentially blocks this incorporation. Cyanogen bromide digestion of the labeled enzyme, followed by high-pressure liquid chromatography purification of the generated peptides, indicates that greater than 85% of the total radioactivity is incorporated into a single cyanogen bromide peptide. Sequence analysis revealed this peptide to be residues 53-111, with a majority of the radioactivity centered around residues 63-65 (Lys-Asn-Arg). These data demonstrate that the photoaffinity analogue specifically binds to dihydrofolate reductase and covalently modifies the enzyme following irradiation and is therefore a photolabeling agent useful for probing the inhibitor binding domain of the enzyme.
- Research Organization:
- Univ. of Cincinnati College of Medicine, OH
- OSTI ID:
- 5934710
- Journal Information:
- Biochemistry; (United States), Vol. 26:15
- Country of Publication:
- United States
- Language:
- English
Similar Records
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
Characterization of the methotrexate transport pathway in murine L1210 leukemia cells: Involvement of a membrane receptor and a cytosolic protein
Related Subjects
59 BASIC BIOLOGICAL SCIENCES
LABELLED COMPOUNDS
CONFIGURATION INTERACTION
METHOTREXATE
BIOLOGICAL RADIATION EFFECTS
OXIDOREDUCTASES
AMINO ACID SEQUENCE
ELECTROPHORESIS
ENZYME INHIBITORS
FOLIC ACID
IODINE 125
LYSINE
MICE
NEAR ULTRAVIOLET RADIATION
SODIUM IODIDES
ALKALI METAL COMPOUNDS
AMINO ACIDS
ANIMALS
ANTIMETABOLITES
AROMATICS
AZAARENES
BETA DECAY RADIOISOTOPES
BIOLOGICAL EFFECTS
CARBOXYLIC ACIDS
DAYS LIVING RADIOISOTOPES
DRUGS
ELECTROMAGNETIC RADIATION
ELECTRON CAPTURE RADIOISOTOPES
ENZYMES
HALIDES
HALOGEN COMPOUNDS
HEMATINICS
HEMATOLOGIC AGENTS
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
INORGANIC PHOSPHORS
INTERMEDIATE MASS NUCLEI
IODIDES
IODINE COMPOUNDS
IODINE ISOTOPES
ISOTOPES
MAMMALS
MOLECULAR STRUCTURE
NUCLEI
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHOSPHORS
PTERIDINES
RADIATION EFFECTS
RADIATIONS
RADIOISOTOPES
RODENTS
SODIUM COMPOUNDS
ULTRAVIOLET RADIATION
VERTEBRATES
VITAMIN B GROUP
VITAMINS
560120* - Radiation Effects on Biochemicals
Cells
& Tissue Culture
550201 - Biochemistry- Tracer Techniques