title: Irreversible photolabeling of active site of neutral endopeptidase-24.11 "enkephalinase" by azidothiorphan and [14C]-azidothiorphan.
abstract: Azidothiorphan and its [14C]-labeled analogue have been developed as photoaffinity ligands for the active site of the neutral endopeptidase 24.11. In in vitro assays azidothiorphan inhibits the endopeptidase activity with a Ki of 0.75 nM. After ultraviolet irradiation the inhibitor binds irreversibly to the enzyme, and many factors suggest that the photolabeling occurs at the active site. The binding is accompanied by a loss of enzymatic activity, and the inclusion of the competitive inhibitor thiorphan protects the endopeptidase from this inactivation. In addition the binding of another competitive inhibitor [3H]N-[(R,S)-3-hydroxyaminocarbonyl-2-benzyl-1-oxopropyl]-glycine to the active site of endopeptidase-24.11 is inhibited after irradiation with azidothiorphan. Experiments with [14C]-azidothiorphan have shown that very little nonspecific binding of inhibitor to enzyme occurs and the the labeled probe remains bound under denaturing conditions. Azidothiorphan has also been found to produce a long-lasting naloxone-reversible analgesia after intracerebroventricular administration. The results show that azidothiorphan should prove useful both for structural studies and for investigations on the synthesis and turnover of the neutral endopeptidase-24.11.
mesh_terms: D000818:Animals; D001386:Azides / Q000037:antagonists & inhibitors / Q000138:chemical synthesis / Q000378:metabolism*; D001665:Binding Sites; D002250:Carbon Radioisotopes; D007202:Indicators and Reagents; D007668:Kidney / Q000201:enzymology; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors* / Q000528:radiation effects; D015260:Neprilysin; D011817:Rabbits; D015244:Thiorphan / Q000031:analogs & derivatives*; D014466:Ultraviolet Rays
chemical_list: D001386:Azides; D002250:Carbon Radioisotopes; D007202:Indicators and Reagents; C036451:azidothiorphan; D015244:Thiorphan; D008666:Metalloendopeptidases; D015260:Neprilysin