Purification and further characterization of enteropeptidase from porcine duodenum

Masashi Matsushima, Masao Ichinose, Naohisa Yahagi, Shinko Tsukada-Kato, Kazumasa Miki, Masao Omata, Yong Tae Kim, Hisashi Ito, Takayuki Takahashi, Yasuko Sakurai, Yuichi Tsuchiya, Senarath B P Athauda, Hideshi Inoue, Kenji Takahashi

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Enteropeptidase [EC 3.4.21.9] is a membrane-bound serine endopeptidase present in the duodenum that converts trypsinogen to trypsin. We previously cloned the cDNA of the porcine enzyme and deduced its entire amino acid sequence. In the present study, we purified the porcine enzyme approximately 2200-fold in a 12% yield from a duodenal mucosal extract to apparent homogeneity by an improved procedure comprising four steps of chromatography including benzamidine-Sepharose affinity chromatography. Lectin blotting analysis suggested that the enzyme is glycosylated mainly with N-linked carbohydrate chains of the tri- and/ or tetraantennary complex type. The H and L chains of the enzyme were separated into two major bands upon SDS-PAGE under reducing conditions, suggesting that the enzyme mainly comprises two isoforms, a higher molecular weight form and a lower molecular weight form. The enzyme was also separated by lectin affinity chromatography into two major fractions, named isoforms I and II, which corresponded to the higher and lower molecular weight forms, respectively. These two isoforms appeared to be different only in the carbohydrate moiety, having essentially the same enzymatic properties. The enzyme was optimally active at pH 8.0 toward Gly-Asp-Asp-Asp-Asp-Lys-β-naphthylamide, and was inhibited strongly by various serine proteinase inhibitors. Furthermore, it was also strongly inhibited by E-64 [L-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane], a cysteine proteinase inhibitor. Substrate specificity studies involving various synthetic peptides indicated that acidic residues at the P2, P3, and/or P4 positions are especially favorable for maximal activity, but are not absolutely necessary, at least in the cases of peptide substrates.

Original languageEnglish
Pages (from-to)947-951
Number of pages5
JournalJournal of Biochemistry
Volume125
Issue number5
Publication statusPublished - 1999
Externally publishedYes

Fingerprint

Enteropeptidase
Duodenum
Purification
Swine
Enzymes
Affinity chromatography
Protein Isoforms
Molecular Weight
Molecular weight
Affinity Chromatography
Lectins
Carbohydrates
Trypsinogen
Cysteine Proteinase Inhibitors
Agarose Chromatography
Peptides
Serine Proteinase Inhibitors
Substrates
Substrate Specificity
Chromatography

Keywords

  • Enteropeptidase
  • Lectin blotting
  • Porcine duodenum
  • Purification
  • Substrate specificity

ASJC Scopus subject areas

  • Biochemistry

Cite this

Matsushima, M., Ichinose, M., Yahagi, N., Tsukada-Kato, S., Miki, K., Omata, M., ... Takahashi, K. (1999). Purification and further characterization of enteropeptidase from porcine duodenum. Journal of Biochemistry, 125(5), 947-951.

Purification and further characterization of enteropeptidase from porcine duodenum. / Matsushima, Masashi; Ichinose, Masao; Yahagi, Naohisa; Tsukada-Kato, Shinko; Miki, Kazumasa; Omata, Masao; Kim, Yong Tae; Ito, Hisashi; Takahashi, Takayuki; Sakurai, Yasuko; Tsuchiya, Yuichi; Athauda, Senarath B P; Inoue, Hideshi; Takahashi, Kenji.

In: Journal of Biochemistry, Vol. 125, No. 5, 1999, p. 947-951.

Research output: Contribution to journalArticle

Matsushima, M, Ichinose, M, Yahagi, N, Tsukada-Kato, S, Miki, K, Omata, M, Kim, YT, Ito, H, Takahashi, T, Sakurai, Y, Tsuchiya, Y, Athauda, SBP, Inoue, H & Takahashi, K 1999, 'Purification and further characterization of enteropeptidase from porcine duodenum', Journal of Biochemistry, vol. 125, no. 5, pp. 947-951.
Matsushima M, Ichinose M, Yahagi N, Tsukada-Kato S, Miki K, Omata M et al. Purification and further characterization of enteropeptidase from porcine duodenum. Journal of Biochemistry. 1999;125(5):947-951.
Matsushima, Masashi ; Ichinose, Masao ; Yahagi, Naohisa ; Tsukada-Kato, Shinko ; Miki, Kazumasa ; Omata, Masao ; Kim, Yong Tae ; Ito, Hisashi ; Takahashi, Takayuki ; Sakurai, Yasuko ; Tsuchiya, Yuichi ; Athauda, Senarath B P ; Inoue, Hideshi ; Takahashi, Kenji. / Purification and further characterization of enteropeptidase from porcine duodenum. In: Journal of Biochemistry. 1999 ; Vol. 125, No. 5. pp. 947-951.
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abstract = "Enteropeptidase [EC 3.4.21.9] is a membrane-bound serine endopeptidase present in the duodenum that converts trypsinogen to trypsin. We previously cloned the cDNA of the porcine enzyme and deduced its entire amino acid sequence. In the present study, we purified the porcine enzyme approximately 2200-fold in a 12{\%} yield from a duodenal mucosal extract to apparent homogeneity by an improved procedure comprising four steps of chromatography including benzamidine-Sepharose affinity chromatography. Lectin blotting analysis suggested that the enzyme is glycosylated mainly with N-linked carbohydrate chains of the tri- and/ or tetraantennary complex type. The H and L chains of the enzyme were separated into two major bands upon SDS-PAGE under reducing conditions, suggesting that the enzyme mainly comprises two isoforms, a higher molecular weight form and a lower molecular weight form. The enzyme was also separated by lectin affinity chromatography into two major fractions, named isoforms I and II, which corresponded to the higher and lower molecular weight forms, respectively. These two isoforms appeared to be different only in the carbohydrate moiety, having essentially the same enzymatic properties. The enzyme was optimally active at pH 8.0 toward Gly-Asp-Asp-Asp-Asp-Lys-β-naphthylamide, and was inhibited strongly by various serine proteinase inhibitors. Furthermore, it was also strongly inhibited by E-64 [L-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane], a cysteine proteinase inhibitor. Substrate specificity studies involving various synthetic peptides indicated that acidic residues at the P2, P3, and/or P4 positions are especially favorable for maximal activity, but are not absolutely necessary, at least in the cases of peptide substrates.",
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AU - Miki, Kazumasa

AU - Omata, Masao

AU - Kim, Yong Tae

AU - Ito, Hisashi

AU - Takahashi, Takayuki

AU - Sakurai, Yasuko

AU - Tsuchiya, Yuichi

AU - Athauda, Senarath B P

AU - Inoue, Hideshi

AU - Takahashi, Kenji

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