Distribution of endopeptidase-24.15 in rat brain nuclei using a novel fluorogenic substrate: comparison with endopeptidase-24.11.

A novel fluorogenic substrate for the neutral metalloendopeptidase-24.15 (E.C.3.4.24.15; EP-24.15) was synthesized which allowed continuous assay of the enzyme. The substrate, Glutaryl-Phe-Ala-Ala-Phe-4-methoxynaphthylamide (G-FAAF-4MN) is cleaved at the Phe-Ala bond by EP-24.15 (Km = 0.026 mM). The product, AAF-4MN is subsequently hydrolyzed to its constituent amino acids and the potent fluorophore 4MN by aminopeptidase M. This method has allowed the measurement of the specific activity EP-24.15 within microdissected nuclei of rat brain. The enzyme was found to have a relatively broad distribution within brain nuclei, and the activity ranged from 15-80 nmol 4MN/mg prot/h in all areas examined. The activity of EP-24.15 was relatively high in the medial and lateral pre-optic nuclei, where potential substrates include the dynorphin-like peptides and LHRH. The activity of EP-24.15 was compared with that of endopeptidase-24.11 (E.C.3.4.24.11, 'enkephalinase', EP-24.11), another peptide-cleaving metalloenzyme. EP-24.11 appeared to have a much more narrow distribution, with very high specific activity in basal ganglia as well as in the supraoptic and suprachiasmatic nuclei.