title: Bovine tryptase: purification and characterization.
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 M(r) of 360,000 +/- 40,000 (as obtained by gel filtration in high salt). The constituent subunits with M(r) 39,000 and 41,000 Da are both labeled with [3H] 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 hydrolyzed 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.
authors.affiliation: Department of Experimental Medicine and Biochemical Sciences, Tor Vergata University, Rome, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Fiorucci
authors.forename: F
authors.initials: F
authors.lastname: Erba
authors.forename: F
authors.initials: F
authors.lastname: Ascoli
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D001665:Binding Sites; D015153:Blotting, Western; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008407:Mast Cells / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000032:analysis* / Q000302:isolation & purification; D011480:Protease Inhibitors / Q000494:pharmacology
chemical_list: D011480:Protease Inhibitors; D010447:Peptide Hydrolases; C023073:tosylarginine methyl ester hydrolase