4-Fluoro-3-nitrophenyl azide binding sites on purified beef liver monoamine oxidase B.
Previous work from this laboratory has shown that 4-fluoro-3-nitrophenyl azide (FNPA) is an effective photoaffinity labeling probe for MAO-B (Chen et al., Biochem. Pharmac.34, 781-785, 1985). The FNPA binding sites have been further studied by using [(3)H]FNPA. When [(3)H]FNPA was photolyzed with purified beef liver MAO, then subjected to tryptic and chymotryptic digestion, three radioactive peaks were observed after Sephadex G-25 column chromatography procedure. The extent of [(3)H]FNPA incorporation varied directly with [(3)H]FNPA concentration. They could be protected by the presence of the substrate (phenylethylamine) or inhibitors (pargyline and trans-phenylcyclopropylamine) of MAO-B during photolysis. These protections were concentration dependent. Furthermore, the decrease in [(3)H]FNPA labeling in the presence of inhibitors paralleled the decrease in MAO catalytic activity. These results suggest that the FNPA binding sites were related to the active site of MAO-B. Under the same conditions, the separation profiles of [(3)H]FNPA labeled and [(3)H]pargyline labeled tryptic-chymotryptic peptides after Sephadex G-25 column chromatography are distinctly different. This result suggests that FNPA labeling sites may be different from the pargyline binding site. Since pargyline binds to the prosthetic group(-FAD) of MAO, [(3)H]FNPA may label different domains of the active site. This probe may be useful for the characterization of the active site of MAO-B.
Hsu
M C
MC
Division of Biological Sciences, School of Pharmacy, University of Southern California, Los Angeles, Calif., U.S.A.
Chen
S
S
Shih
J C
JC