Proopiocortin-converting enzyme activity in bovine neurosecretory granules.
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.
D000324:Adrenocorticotropic Hormone / Q000276:immunology; D000818:Animals; D002417:Cattle; D002458:Cell Fractionation; D003594:Cytoplasmic Granules / Q000201:enzymology*; D010450:Endopeptidases / Q000378:metabolism*; D004723:Endorphins / Q000276:immunology; D006863:Hydrogen-Ion Concentration; D007163:Immunosorbent Techniques; D009490:Neurosecretory Systems / Q000648:ultrastructure*; D010902:Pituitary Gland / Q000648:ultrastructure*; D010908:Pituitary Hormones, Anterior / Q000378:metabolism; D011333:Pro-Opiomelanocortin; D043484:Proprotein Convertases; D011480:Protease Inhibitors; D011498:Protein Precursors / Q000378:metabolism; D013379:Substrate Specificity; D014982:Xenopus laevis; D001615:beta-Endorphin
D004723:Endorphins; D010908:Pituitary Hormones, Anterior; D011480:Protease Inhibitors; D011498:Protein Precursors; D001615:beta-Endorphin; D011333:Pro-Opiomelanocortin; D000324:Adrenocorticotropic Hormone; D010450:Endopeptidases; D043484:Proprotein Convertases