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

Journal Article · Tue Jul 28 04:00:00 EDT 1987 · Biochemistry; (United States)

OSTI ID:5934710

Price, E M; Smith, P L; Klein, T E; Freisheim, J H

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), Journal Name: Biochemistry; (United States) Vol. 26:15; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

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59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ALKALI METAL COMPOUNDS
AMINO ACID SEQUENCE
AMINO ACIDS
ANIMALS
ANTIMETABOLITES
AROMATICS
AZAARENES
BETA DECAY RADIOISOTOPES
BIOLOGICAL EFFECTS
BIOLOGICAL RADIATION EFFECTS
CARBOXYLIC ACIDS
CONFIGURATION INTERACTION
DAYS LIVING RADIOISOTOPES
DRUGS
ELECTROMAGNETIC RADIATION
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
ENZYME INHIBITORS
ENZYMES
FOLIC ACID
HALIDES
HALOGEN COMPOUNDS
HEMATINICS
HEMATOLOGIC AGENTS
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
INORGANIC PHOSPHORS
INTERMEDIATE MASS NUCLEI
IODIDES
IODINE 125
IODINE COMPOUNDS
IODINE ISOTOPES
ISOTOPES
LABELLED COMPOUNDS
LYSINE
MAMMALS
METHOTREXATE
MICE
MOLECULAR STRUCTURE
NEAR ULTRAVIOLET RADIATION
NUCLEI
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OXIDOREDUCTASES
PHOSPHORS
PTERIDINES
RADIATION EFFECTS
RADIATIONS
RADIOISOTOPES
RODENTS
SODIUM COMPOUNDS
SODIUM IODIDES
ULTRAVIOLET RADIATION
VERTEBRATES
VITAMIN B GROUP
VITAMINS