Photoaffinity analogues of methotrexate as folate antagonist binding probes.
A photoaffinity analogue of methotrexate, APA-[125I]ASA-Lys, specifically binds to dihydrofolate reductase and covalently modifies the enzyme following irradiation. An excess of methotrexate blocks incorporation of the photoprobe. Following cyanogen bromide digestion of the radiolabeled enzyme and high-pressure liquid chromatographic separation of the generated peptides, a majority of the label was centered around residues 63-65 (Lys-Asn-Arg), part of the inhibitor binding domain. This photoprobe is also transported into murine L1210 cells in a temperature-dependent, sulfhydryl reagent inhibitable manner with a Vmax similar to that for methotrexate. Ultraviolet irradiation at 4 degrees C of a cell suspension that had been incubated with the radiolabeled photoprobe resulted in the covalent modification of a 46-48 Kd protein. This can be demonstrated when the plasma membranes from the labeled cells are analyzed via sodium dodecylsulfate-polyacrylamide gel electrophoresis and autoradiography. Labeling of this protein occurs half-maximally at a reagent concentration that correlates with the Kt for transport of the iodinated compound. Protection against labeling of this protein by increasing amounts of methotrexate parallels the concentration dependence of inhibition of photoprobe uptake by methotrexate. In addition, no labeling occurs when a cell line that has a defective methotrexate transport system is similarly treated. Evidence that, in the absence of irradiation and at 37 degrees C, the iodinated probe is actually internalized is demonstrated by the labeling of two soluble proteins (Mr = 38 Kd and 21 Kd) derived from the cell homogenate supernatant.
Freisheim
J H
JH
Department of Biochemistry, Medical College of Ohio, Toledo 43699.
Ratnam
M
M
Smith
P L
PL
Delcamp
T J
TJ
Price
E M
EM
D000818:Animals; D001386:Azides / Q000493:pharmacokinetics; D001692:Biological Transport; D002352:Carrier Proteins / Q000032:analysis; D002460:Cell Line; D002462:Cell Membrane / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005493:Folic Acid Antagonists / Q000378:metabolism*; D007939:Leukemia L1210 / Q000378:metabolism; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000493:pharmacokinetics; D051379:Mice; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000378:metabolism; D014466:Ultraviolet Rays
D001386:Azides; D002352:Carrier Proteins; D005493:Folic Acid Antagonists; C053282:N(alpha)-(4-amino-4-deoxy-10-methylpteroyl)-N(epsilon)-(4-azido-5-iodosalicylyl)lysine; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate