Proopiocortin-converting enzyme activity in bovine neurosecretory granules

• Published in: Endocrinology (Philadelphia) Vol. 111; no. 5; p. 1607
• Main Authors: Chang, T L, Gainer, H, Russell, J T, Loh, Y P
• Format: Journal Article
• Language: English
• Published: United States 01-11-1982
• Subjects: Adrenocorticotropic Hormone - immunology; Animals; beta-Endorphin; Cattle; Cell Fractionation; Cytoplasmic Granules - enzymology; Endopeptidases - metabolism; Endorphins - immunology; Hydrogen-Ion Concentration; Immunosorbent Techniques; Neurosecretory Systems - ultrastructure; Pituitary Gland - ultrastructure; Pituitary Hormones, Anterior - metabolism; Pro-Opiomelanocortin; Proprotein Convertases; Protease Inhibitors; Protein Precursors - metabolism; Substrate Specificity; Xenopus laevis
• ISSN: 0013-7227
• DOI: 10.1210/endo-111-5-1607

Neurosecretory granules (NSGs) from neural lobes of bovine pituitary glands were isolated in a highly purified form by metrizamide-sucrose gradient centrifugation. The purified NSGs were lysed and centrifuged, and the supernatants were further fractionated by gel filtration on Sephadex G-75. Proopiocortin-converting enzyme activity was assayed by incubation of [3H]arginine- or [3H]phenylalanine-labeled toad proopiocortin with NSG supernatant fractions. The processed products were identified by immunoprecipitation with ACTH and beta-endorphin antisera, followed by acid-urea gel electrophoresis. The optimum pH for the enzyme-mediated conversion was around pH 5.0. Conversion of toad proopiocortin by NSG converting enzyme activity was inhibited by leupeptin, antipain, p-chloromercuribenzoate, and pepstatin A, but not by diisopropyl fluorophosphate, EDTA, or N-alpha-p-tosyl-L-lysine-chloromethyl ketone HCl. The results suggest that the proopiocortin-converting enzyme activity in bovine neurosecretory granules is due to an acid-thiol protease which may contain secondary hydrophobic binding sites that are involved in substrate recognition.