title: A fluorogenic method for measuring enteropeptidase activity: spectral shift in the emission of GD4K-conjugated 7-amino-4-methylcoumarin.
abstract: Enteropeptidase is a serine protease secreted by the pancreas and converts inactive trypsinogen to active trypsin. Enteropeptidase cleaves the C-terminal end of the substrate recognition sequence Asp-Asp-Asp-Asp-Lys (D(4)K). The assay for enteropeptidase has utilized GD(4)K-conjugated 2-naphthylamine (GD(4)K-NA) as a fluorogenic probe over the last 30 years. However, no other D(4)K-conjugated fluorogenic substrates of enteropeptidase have been reported. Furthermore, naphthalene is known as carcinogenic to humans. In this study, we used shift in the emission spectrum of GD(4)K-conjugated 7-amino-4-methylcoumarin (GD(4)K-AMC) as a fluorogenic method to measure enteropeptidase activity. The kinetic analysis revealed that enteropeptidase has a K(M) of 0.025 mM and a k(cat) of 65 sec(-1) for GD(4)K-AMC, whereas it has a K(M) of 0.5 to 0.6 mM and a k(cat) of 25 sec(-1) for GD(4)K-NA. The optimum pH of GD(4)K-AMC hydrolysis was pH 8.0. Our data indicate that GD(4)K-AMC is more suitable as a substrate for enteropeptidase than GD(4)K-NA.
authors.affiliation: Department of Biomedical Science, Daegu University, Gyeongsan, Korea.
authors.forename: Mal-Gi
authors.initials: MG
authors.lastname: Choi
authors.forename: Eungyeong
authors.initials: E
authors.lastname: Lee
authors.forename: Hye-Shin
authors.initials: HS
authors.lastname: Chung
authors.forename: Sei-Heon
authors.initials: SH
authors.lastname: Jang
authors.forename: ChangWoo
authors.initials: C
authors.lastname: Lee
mesh_terms: D015081:2-Naphthylamine / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003374:Coumarins / Q000737:chemistry / Q000378:metabolism*; D004765:Enteropeptidase / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D013057:Spectrum Analysis / Q000379:methods*
chemical_list: D003374:Coumarins; D005456:Fluorescent Dyes; D010455:Peptides; D015081:2-Naphthylamine; D004765:Enteropeptidase; C028743:7-amino-4-methylcoumarin