title: A new facile trinitrophenylated substrate for peptide alpha-amidation and its use to characterize PAM activity in chromaffin granules.
abstract: Carboxyl terminal alpha-amidation is a prevalent post translational modification in neuropeptide hormones, with amidation being essential for biological activity. We report a direct demonstration and characterization of peptidyl alpha-amidating monooxygenase (PAM) activity in chromaffin granules, secretory vesicles long known as loci for synthesis and storage of catecholamines but only recently recognized as processing and storage sites for neuropeptides. This finding, together with the recently recognized competence of dopamine-b-monooxygenase to carry out N-dealkylation, provides important information regarding the co-localization and co-secretion of multiple neuromodulators. In addition, we introduce a new substrate for both pituitary and chromaffin granule PAM--TNP-D-Tyr-Val-Gly. This substrate exhibits high turnover, and has the important advantage of allowing quantitative activity determinations using standard spectrophotometric techniques, thus facilitating mechanistic studies and inhibitor development.
authors.affiliation: School of Chemistry, Georgia Institute of Technology, Atlanta 30332.
authors.forename: A G
authors.initials: AG
authors.lastname: Katopodis
authors.forename: S W
authors.initials: SW
authors.lastname: May
mesh_terms: D000313:Adrenal Medulla / Q000201:enzymology*; D000818:Animals; D002417:Cattle; D002837:Chromaffin Granules / Q000201:enzymology*; D002838:Chromaffin System / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D006899:Mixed Function Oxygenases; D009097:Multienzyme Complexes; D009842:Oligopeptides / Q000378:metabolism*; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D009097:Multienzyme Complexes; D009842:Oligopeptides; C055092:2,4,6-trinitrophenyl-tyrosyl-valyl-glycine; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D000587:Oxidoreductases Acting on CH-NH Group Donors