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 alpha-(4-Amino-4-deoxy-10-methylpteroyl)-N epsilon-(4-azido-5- [125I]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 125I-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.
Price
E M
EM
Department of Biochemistry and Molecular Biology, University of Cincinnati College of Medicine, Ohio 45267-0522.
Smith
P L
PL
Klein
T E
TE
Freisheim
J H
JH
D000345:Affinity Labels / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000378:metabolism / Q000494:pharmacology*; D003488:Cyanogen Bromide; D005493:Folic Acid Antagonists; D007700:Kinetics; D007939:Leukemia L1210 / Q000201:enzymology*; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D008958:Models, Molecular; D010446:Peptide Fragments / Q000032:analysis; D010777:Photochemistry; D011485:Protein Binding; D011487:Protein Conformation
D000345:Affinity Labels; D001386:Azides; D005493:Folic Acid Antagonists; D010446:Peptide Fragments; C053282:N(alpha)-(4-amino-4-deoxy-10-methylpteroyl)-N(epsilon)-(4-azido-5-iodosalicylyl)lysine; D003488:Cyanogen Bromide; D008727:Methotrexate