PT  - JOURNAL ARTICLE
AU  - A Beaumont
AU  - J F Hernandez
AU  - P Chaillet
AU  - P Crine
AU  - B P Roques
TI  - Irreversible photolabeling of active site of neutral endopeptidase-24.11 "enkephalinase" by azidothiorphan and [14C]-azidothiorphan.
DP  - 1987 Nov 01
TA  - Molecular Pharmacology
PG  - 594--599
VI  - 32
IP  - 5
4099  - http://molpharm.aspetjournals.org/content/32/5/594.short
4100  - http://molpharm.aspetjournals.org/content/32/5/594.full
SO  - Mol Pharmacol1987 Nov 01; 32
AB  - 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.