A fluorogenic method for measuring enteropeptidase activity: spectral shift in the emission of GD₄K-conjugated 7-amino-4-methylcoumarin
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 (D₄K). The assay for enteropeptidase has utilized GD₄K-conjugated 2-naphthylamine (G...
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Published in | BMB reports Vol. 44; no. 7; pp. 458 - 461 |
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Main Authors | , , , , |
Format | Journal Article |
Language | Korean |
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생화학분자생물학회
30.07.2011
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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 (D₄K). The assay for enteropeptidase has utilized GD₄K-conjugated 2-naphthylamine (GD₄K-NA) as a fluorogenic probe over the last 30 years. However, no other D₄K- 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 GD₄K-conjugated 7-amino-4-methylcoumarin (GD₄K-AMC) as a fluorogenic method to measure enteropeptidase activity. The kinetic analysis revealed that enteropeptidase has a KM of 0.025 mM and a kcat of 65 sec-1 for GD₄K-AMC, whereas it has a KM of 0.5 to 0.6 mM and a kcat of 25 sec-1 for GD₄K-NA. The optimum pH of GD₄K-AMC hydrolysis was pH 8.0. Our data indicate that GD₄K-AMC is more suitable as a substrate for enteropeptidase than GD₄K-NA. |
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AbstractList | 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 (D₄K). The assay for enteropeptidase has utilized GD₄K-conjugated 2-naphthylamine (GD₄K-NA) as a fluorogenic probe over the last 30 years. However, no other D₄K- 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 GD₄K-conjugated 7-amino-4-methylcoumarin (GD₄K-AMC) as a fluorogenic method to measure enteropeptidase activity. The kinetic analysis revealed that enteropeptidase has a KM of 0.025 mM and a kcat of 65 sec-1 for GD₄K-AMC, whereas it has a KM of 0.5 to 0.6 mM and a kcat of 25 sec-1 for GD₄K-NA. The optimum pH of GD₄K-AMC hydrolysis was pH 8.0. Our data indicate that GD₄K-AMC is more suitable as a substrate for enteropeptidase than GD₄K-NA. |
Author | Hye Shin Chung Eung Yeong Lee Chang Woo Lee Mal Gi Choi Sei Heon Jang |
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SubjectTerms | 2-naphthylamine 7-amino-4-methylcoumarin Enteropeptidase Fluorogenic substrate |
Title | A fluorogenic method for measuring enteropeptidase activity: spectral shift in the emission of GD₄K-conjugated 7-amino-4-methylcoumarin |
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