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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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A fluorogenic method for measuring enteropeptidase activity: spectral shift in the emission of GD4K-conjugated 7-amino-4-methylcoumarin

  • Choi, Mal-Gi (Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology) ;
  • Lee, Eung-Yeong (Department of Biomedical Science, Daegu University) ;
  • Chung, Hye-Shin (Department of Biotechnology, Hannam University) ;
  • Jang, Sei-Heon (Department of Biomedical Science, Daegu University) ;
  • Lee, Chang-Woo (Department of Biomedical Science, Daegu University)
  • Received : 2011.04.13
  • Accepted : 2011.05.03
  • Published : 2011.07.31
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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 ($D_4K$). The assay for enteropeptidase has utilized $GD_4K$-conjugated 2-naphthylamine ($GD_4K$-NA) as a fluorogenic probe over the last 30 years. However, no other $D_4K$-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 $GD_4K$-conjugated 7-amino-4-methylcoumarin ($GD_4K$-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 $GD_4K$-AMC, whereas it has a $K_M$ of 0.5 to 0.6 mM and a $k_{cat}$ of 25 $sec^{-1}$ for $GD_4K$-NA. The optimum pH of $GD_4K$-AMC hydrolysis was pH 8.0. Our data indicate that $GD_4K$-AMC is more suitable as a substrate for enteropeptidase than $GD_4K$-NA.

Keywords

References

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