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.
Molineaux
C J
CJ
Department of Pharmacology, Mount Sinai School of Medicine, CUNY, NY 10029.
Yu
B
B
Ayala
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002421:Caudate Nucleus / Q000201:enzymology; D005917:Globus Pallidus / Q000201:enzymology; D007031:Hypothalamus / Q000201:enzymology; D008297:Male; D008666:Metalloendopeptidases / Q000032:analysis* / Q000378:metabolism; D008969:Molecular Sequence Data; D015260:Neprilysin / Q000032:analysis* / Q000378:metabolism; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013378:Substantia Nigra / Q000201:enzymology; D014018:Tissue Distribution
D009842:Oligopeptides; C069165:glutaryl-phenylalanyl-alanyl-alanyl-phenylalanine 4-methoxynaphthylamide; D008666:Metalloendopeptidases; D015260:Neprilysin; C022510:thimet oligopeptidase