Hydrolysis of artificial substrates by enterokinase and trypsin and the development of a sensitive specific assay for enterokinase in serum

Grant, David A.; Hermon‐Taylor, John

doi:10.1016/0005-2744(79)90187-6

Cited by 45 publications

(26 citation statements)

References 28 publications

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“…Samples were removed at intervals, and the reaction was terminated either by adding sample loading buffer (2% SDS and 2% ␤-mercaptoethanol) for analysis by SDS-PAGE (15) and silver staining (16) or by adding ovomucoid to a final concentration of 50 nM for assay of activated enteropeptidase with GD 4 K-NA (17). The amino-terminal amino acid sequences of activated HL-BEK and L-BEK were determined after SDS-PAGE and electroblotting onto a polyvinylidene difluoride membrane as described previously (18).…”

Section: Materials-bovine Enteropeptidase (Bek)mentioning

confidence: 99%

“…Kinetic parameters for cleavage of the synthetic peptide substrate GD 4 K-NA were determined as described previously (17). Assays (60 l) contained 0 -1 mM GD 4 K-NA, 25 mM Tris-HCl, pH 8.4, and 10 mM CaCl 2 at 37°C.…”

Section: Materials-bovine Enteropeptidase (Bek)mentioning

confidence: 99%

“…E and q, activation of pro-HL-BEK; Ⅺ and f, activation of pro-L-BEK. 0.28 mM and k cat ϭ 28.3 s Ϫ1 (17). The kinetics of trypsinogen activation were analyzed under two conditions.…”

Section: Fig 4 Comparison Of Pro-hl-bek and Pro-l-bek Activation Bymentioning

confidence: 99%

“…At selected times, 10-l samples were removed and added to 3.5 l of 2 M HCl. Free ␤-naphthylamine concentration was determined as described previously (17).…”

Section: Materials-bovine Enteropeptidase (Bek)mentioning

confidence: 99%

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Bovine Proenteropeptidase Is Activated by Trypsin, and the Specificity of Enteropeptidase Depends on the Heavy Chain

Lu¹,

Yuan²,

Zheng

et al. 1997

Journal of Biological Chemistry

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Enteropeptidase, also known as enterokinase, initiates the activation of pancreatic hydrolases by cleaving and activating trypsinogen. Enteropeptidase is synthesized as a single-chain protein, whereas purified enteropeptidase contains a Ϸ47-kDa serine protease domain (light chain) and a disulfide-linked Ϸ120-kDa heavy chain. The heavy chain contains an amino-terminal membrane-spanning segment and several repeated structural motifs of unknown function. To study the role of heavy chain motifs in substrate recognition, secreted variants of recombinant bovine proenteropeptidase were constructed by replacing the transmembrane domain with a signal peptide. Secreted variants containing both the heavy chain (minus the transmembrane domain) and the catalytic light chain (pro-HL-BEK (where BEK is bovine enteropeptidase)) or only the catalytic domain (pro-L-BEK) were expressed in baby hamster kidney cells and purified. Single-chain pro-HL-BEK and pro-L-BEK were zymogens with extremely low catalytic activity, and both were activated readily by trypsin cleavage. Enteropeptidase, originally named enterokinase when it was discovered by Pavlov (1), is a membrane-bound serine protease of the duodenal mucosa that cleaves trypsinogen to generate active trypsin. In almost all vertebrate species, a short trypsinogen activation peptide is released that terminates with the sequence Asp-Asp-Asp-Asp-Lys (2). Following activation, trypsin cleaves and activates other zymogens in pancreatic secretions, including chymotrypsinogen, proelastase, procarboxypeptidases, and some prolipases. Thus, enteropeptidase initiates a simple two-step enzymatic cascade that activates digestive hydrolases within the lumen of the gut. The biological importance of this pathway is demonstrated by the severe intestinal malabsorption and diarrhea that is caused by congenital enteropeptidase deficiency (3, 4).Bovine enteropeptidase is synthesized as a single-chain precursor of 1035 amino acid residues (5) that appears to require proteolytic activation, suggesting that enteropeptidase may not be the "first" protease of the digestive hydrolase cascade. Active enteropeptidase has been cleaved after Arg-800 to produce a disulfide-linked heterodimer with an amino-terminal Ϸ120-kDa heavy chain and a Ϸ47-kDa light chain; ϳ40% of the apparent mass of these polypeptides is due to glycosylation (6, 7). The enteropeptidase heavy chain consists of an aminoterminal membrane-spanning domain, a mucin-like domain, two repeats found in complement serine proteases C1r and C1s, a MAM domain (so named for similar motifs first identified in the metalloprotease meprin, the Xenopus laevis neuronal protein A5, and protein-tyrosine phosphatase Mu), and a macrophage scavenger receptor cysteine-rich repeat (reviewed in Ref. 8). The light chain is a typical chymotrypsin-like serine protease. The activation cleavage site between the heavy and light chains has the sequence Val-Ser-Pro-Lys2Ile, which might be recognized by trypsin or other trypsin-like proteases. The identity of the endogenous...

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Section: Materials-bovine Enteropeptidase (Bek)mentioning

confidence: 99%

Section: Materials-bovine Enteropeptidase (Bek)mentioning

confidence: 99%

“…E and q, activation of pro-HL-BEK; Ⅺ and f, activation of pro-L-BEK. 0.28 mM and k cat ϭ 28.3 s Ϫ1 (17). The kinetics of trypsinogen activation were analyzed under two conditions.…”

Section: Fig 4 Comparison Of Pro-hl-bek and Pro-l-bek Activation Bymentioning

confidence: 99%

“…At selected times, 10-l samples were removed and added to 3.5 l of 2 M HCl. Free ␤-naphthylamine concentration was determined as described previously (17).…”

Section: Materials-bovine Enteropeptidase (Bek)mentioning

confidence: 99%

See 2 more Smart Citations

Bovine Proenteropeptidase Is Activated by Trypsin, and the Specificity of Enteropeptidase Depends on the Heavy Chain

Lu¹,

Yuan²,

Zheng

et al. 1997

Journal of Biological Chemistry

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“…We also examined the effect of calcium ions on the activity of enteropeptidase. Grant and Hermon-Talor showed that the KM of enteropeptidase for GD4K-NA decreased from 0.525 mM to 0.28 mM when calcium concentration was changed from 0.1 mM to 10 mM (12). However, as shown in Fig.…”

Section: Resultsmentioning

confidence: 84%

A fluorogenic method for measuring enteropeptidase activity: spectral shift in the emission of GD₄K-conjugated 7-amino-4-methylcoumarin

Choi

Lee²,

Chung³

et al. 2011

BMB Reports

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Fig. 1. Structure of 2-naphthylamine (A) and 7-amino-4-methylcoumarin (B).A fluorogenic method for measuring enteropeptidase activity: spectral shift in the emission of GD 4 K-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 (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. [BMB reports 2011; 44(7): 458-461]

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Enteropeptidase

Takahashi

2002

Wiley Encyclopedia of Molecular Medicine

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Hydrolysis of artificial substrates by enterokinase and trypsin and the development of a sensitive specific assay for enterokinase in serum

Cited by 45 publications

References 28 publications

Bovine Proenteropeptidase Is Activated by Trypsin, and the Specificity of Enteropeptidase Depends on the Heavy Chain

Bovine Proenteropeptidase Is Activated by Trypsin, and the Specificity of Enteropeptidase Depends on the Heavy Chain

A fluorogenic method for measuring enteropeptidase activity: spectral shift in the emission of GD₄K-conjugated 7-amino-4-methylcoumarin

Enteropeptidase

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Hydrolysis of artificial substrates by enterokinase and trypsin and the development of a sensitive specific assay for enterokinase in serum

Cited by 45 publications

References 28 publications

Bovine Proenteropeptidase Is Activated by Trypsin, and the Specificity of Enteropeptidase Depends on the Heavy Chain

Bovine Proenteropeptidase Is Activated by Trypsin, and the Specificity of Enteropeptidase Depends on the Heavy Chain

A fluorogenic method for measuring enteropeptidase activity: spectral shift in the emission of GD4K-conjugated 7-amino-4-methylcoumarin

Enteropeptidase

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A fluorogenic method for measuring enteropeptidase activity: spectral shift in the emission of GD₄K-conjugated 7-amino-4-methylcoumarin