Photoaffinity labeling of beef liver monoamine oxidase-B by 4-fluoro-3-nitrophenyl azide.
4-Fluoro-3-nitrophenyl azide (FNPA) competitively inhibited beef liver monoamine oxidase-B (MAO-B) in the dark (Ki = 2.8 microM). Upon irradiation in the presence of FNPA, a concentration-dependent photoinactivation of MAO-B was observed. The kinetic analysis showed that the photoinactivation of MAO-B resulted in a decrease in Vmax but no change in Km. This result suggests that an irreversible linkage may be formed between the enzyme and the photolyzed FNPA. When [3H]FNPA was photoirradiated with the purified MAO-B, a single radioactive band associated with MAO-B was observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The photo-dependent incorporation could be protected by phenylethylamine, the substrate for MAO-B, in a concentration-dependent manner. Complete tryptic-chymotryptic digestion of [3H]FNPA-labeled MAO-B resulted in three radioactive peaks on Sephadex G-25 column chromatography. With the same digestion and separation procedures, only one major radioactive peak was observed for the [3H]pargyline-labeled MAO-B, and its elution volume was different from that of [3H]FNPA-labeled peptides. These results suggest that, upon photolysis, FNPA may incorporate into a region in the active site of MAO-B which may be different from the pargyline binding site--the FAD prosthetic group of the enzyme.
D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D002850:Chromatography, Gel; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008995:Monoamine Oxidase / Q000378:metabolism*; D008996:Monoamine Oxidase Inhibitors / Q000494:pharmacology; D010293:Pargyline / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010782:Photolysis
D000345:Affinity Labels; D001386:Azides; D008996:Monoamine Oxidase Inhibitors; D010446:Peptide Fragments; D005182:Flavin-Adenine Dinucleotide; C007753:4-fluoro-3-nitrophenyl azide; D010293:Pargyline; D008995:Monoamine Oxidase