title: A new method for simultaneous measurements of mast cell proteases in human vascular tissue.
abstract: 1. Human mast cells contain carboxypeptidase A, chymase and tryptase. In the present study, in order to analyse the mast cell proteases simultaneously, we investigated a method for the measurement of carboxypeptidase A, chymase and tryptase in human vascular tissues. 2. Human vascular tissues were homogenized in 10 mmol/L phosphate buffer containing 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 or 1.8 mol/L KCl and 0.1% non-idet P-40 and samples were then extracted. Because carboxypeptidase A and chymase convert angiotensin (Ang)I to Ang-(1-9) and AngII, respectively, the extracts were incubated with AngI in the presence of an angiotensin-converting enzyme (ACE) inhibitor. The extract prepared in buffer with over 0.8 mol/L KCl converted AngI to Ang-(1-9) and AngII. Formation of Ang-(1-9) and AngII plateaued in extracts with 1.0 and 1.2 mol/L KCl, respectively. 3. The formation of Ang-(1-9) and AngII in the extract with 1.2 mol/L KCl was inhibited by inhibitors of carboxypeptidase A and chymase, respectively, suggesting that Ang-(1-9) and AngII were generated from AngI by carboxypeptidase A and chymase, respectively. 4. Using a specific tryptase substrate, tryptase activity was detected in extract in buffer with over 0.8 mol/L KCl and reached a plateau at concentrations of KCl over 1.0 mol/L. 5. These findings show that the maximum activity of carboxypeptidase A, chymase and tryptase was detected in extracts of human homogenized vascular tissues in buffer at 1.2 mol/L KCl. The present study demonstrates a new method for the simultaneous measurement of proteases derived from mast cells in humans.
authors.affiliation: Department of Pharmacology, Osaka Medical College, Takatsuki, Japan. pha010@art.osaka-med.ac.jp
authors.forename: S
authors.initials: S
authors.lastname: Takai
authors.forename: M
authors.initials: M
authors.lastname: Sakaguchi
authors.forename: D
authors.initials: D
authors.lastname: Jin
authors.forename: M
authors.initials: M
authors.lastname: Miyazaki
mesh_terms: D000803:Angiotensin I / Q000378:metabolism; D000804:Angiotensin II / Q000096:biosynthesis; D001158:Arteries / Q000201:enzymology*; D002268:Carboxypeptidases / Q000378:metabolism; D043422:Carboxypeptidases A; D002851:Chromatography, High Pressure Liquid; D053818:Chymases; D004730:Endothelium, Vascular / Q000201:enzymology; D005260:Female; D006801:Humans; D008297:Male; D008407:Mast Cells / Q000201:enzymology*; D008875:Middle Aged; D009131:Muscle, Smooth, Vascular / Q000201:enzymology; D010446:Peptide Fragments / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*; D014020:Tissue Extracts / Q000032:analysis; D053802:Tryptases
chemical_list: D010446:Peptide Fragments; D014020:Tissue Extracts; D000804:Angiotensin II; D000803:Angiotensin I; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D012697:Serine Endopeptidases; C083294:chymase 2; D053818:Chymases; D053802:Tryptases