Bovine tryptase, a mast cell trypsin-like protease, was isolated from liver capsula and from mast cells obtained from the same tissue. The purification procedure which leads to an increase in tryptase activity of 850 fold, involves high salt extraction, hydrophobic interaction chromatography on octyl-Sepharose and affinity chromatography on heparin-Sepharose. The enzyme is oligomeric, with an apparent Mr of 360000 +/- 40000 (as obtained by gel filtration in high salt). The constituent subunits with Mr 39000 and 41000 Da are both labeled with [H-3] diisopropyl fluorophosphate and cross-react with anti-rat tryptase immunoglobulins. Only a single N-terminal sequence was found, identical to that of human, dog and rat tryptases. Tripeptide fluorogenic substrates with basic residues in P1 and P2 positions are preferentially hydrolized by this enzyme, suggesting a possible processing role as proposed for other tryptases.Bovine tryptase activity is inhibited by NaCl and is insensitive to high molecular weight inhibitors, such as alpha-1 antitrypsin and soybean trypsin inhibitor, as for human and dog tryptases. However it is inhibited by low molecular weight serine protease inhibitors and, similarly to rat tryptase, by the bovine pancreatic trypsin inhibitor (BPTI or aprotinin), in a pH dependent fashion.

Fiorucci, L., Erba, F., Ascoli, F. (1992). Bovine tryptase: purification and characterization. BIOLOGICAL CHEMISTRY HOPPE-SEYLER, 373(7), 483-490 [10.1515/bchm3.1992.373.2.483].

Bovine tryptase: purification and characterization

Fiorucci, L;Erba, F;
1992

Abstract

Bovine tryptase, a mast cell trypsin-like protease, was isolated from liver capsula and from mast cells obtained from the same tissue. The purification procedure which leads to an increase in tryptase activity of 850 fold, involves high salt extraction, hydrophobic interaction chromatography on octyl-Sepharose and affinity chromatography on heparin-Sepharose. The enzyme is oligomeric, with an apparent Mr of 360000 +/- 40000 (as obtained by gel filtration in high salt). The constituent subunits with Mr 39000 and 41000 Da are both labeled with [H-3] diisopropyl fluorophosphate and cross-react with anti-rat tryptase immunoglobulins. Only a single N-terminal sequence was found, identical to that of human, dog and rat tryptases. Tripeptide fluorogenic substrates with basic residues in P1 and P2 positions are preferentially hydrolized by this enzyme, suggesting a possible processing role as proposed for other tryptases.Bovine tryptase activity is inhibited by NaCl and is insensitive to high molecular weight inhibitors, such as alpha-1 antitrypsin and soybean trypsin inhibitor, as for human and dog tryptases. However it is inhibited by low molecular weight serine protease inhibitors and, similarly to rat tryptase, by the bovine pancreatic trypsin inhibitor (BPTI or aprotinin), in a pH dependent fashion.
Pubblicato
Rilevanza internazionale
Articolo
Esperti anonimi
Settore BIO/10
English
Amino Acid Sequence
Animals
Autoradiography
Binding Sites
Blotting, Western
Cattle
Electrophoresis, Polyacrylamide Gel
Kinetics
Liver
Mast Cells
Molecular Sequence Data
Molecular Weight
Peptide Hydrolases
Protease Inhibitors
Fiorucci, L., Erba, F., Ascoli, F. (1992). Bovine tryptase: purification and characterization. BIOLOGICAL CHEMISTRY HOPPE-SEYLER, 373(7), 483-490 [10.1515/bchm3.1992.373.2.483].
Fiorucci, L; Erba, F; Ascoli, F
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2108/258264
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