title: Carboxypeptidase A in mouse mast cells. Identification, characterization, and use as a differentiation marker.
abstract: By using a conventional spectrophotometric assay with hippuryl-L-phenylalanine as the substrate, 10(6) BALB/c mouse serosal mast cells possessed 1.5 +/- 0.43 U (mean +/- SE, n = 5, range = 0.48 to 2.5) of carboxypeptidase A activity, while T cell factor-dependent, mouse bone marrow-derived mast cells (BMMC) had barely detectable levels of 0.01 +/- 0.001 U/10(6) cells (mean +/- SE, n = 3). In order to characterize the carboxypeptidase A present in the BMMC, a sensitive assay was developed that used angiotensin I as the substrate and reverse phase-high performance liquid chromatography to separate and quantify production of the cleavage product des-leu-angiotensin I. Using this assay, mouse BMMC carboxypeptidase A had a neutral to basic pH optimum and hydrolyzed angiotensin I with a Km of 0.78 mM. The antigen-induced net percent release of carboxypeptidase A from IgE-sensitized BMMC was proportional to that of the secretory granule component beta-hexosaminidase which indicates a secretory granule location for the exopeptidase. As defined by exclusion during Sepharose CL-2B chromatography, carboxypeptidase A was exocytosed as a greater than 1 X 10(7) m.w. complex bound to proteoglycans. Because BMMC cocultured with mouse skin-derived 3T3 fibroblasts are known to undergo an increase in histamine content and biosynthesis of 35S-labeled heparin proteoglycans, carboxypeptidase A activity was measured during BMMC/fibroblast coculture for 0 to 28 days. The carboxypeptidase A activity increased progressively during 28 days of co-culture from 0.004 +/- 0.002 U/10(6) starting BMMC (mean +/- SE, n = 3) to 0.36 +/- 0.10 U/10(6) co-cultured mast cells. These findings indicate that carboxypeptidase A, a neutral protease, is exocytosed from the secretory granules of mouse mast cells bound to proteoglycan and is increased during the in vitro differentiation of mouse BMMC from mucosal-like mast cells to serosal-like mast cells.
authors: Department of Medicine, Harvard Medical School, Boston, MA.
authors: W E
authors: WE
authors: Serafin
authors: E T
authors: ET
authors: Dayton
authors: P M
authors: PM
authors: Gravallese
authors: K F
authors: KF
authors: Austen
authors: R L
authors: RL
authors: Stevens
mesh_terms: D000803:Angiotensin I / Q000378:metabolism; D000818:Animals; D001854:Bone Marrow Cells; D002268:Carboxypeptidases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D043422:Carboxypeptidases A; D002454:Cell Differentiation; D002478:Cells, Cultured; D003594:Cytoplasmic Granules / Q000201:enzymology; D005089:Exocytosis; D005347:Fibroblasts; D006863:Hydrogen-Ion Concentration; D008407:Mast Cells / Q000166:cytology / Q000201:enzymology*; D051379:Mice; D008807:Mice, Inbred BALB C; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D000803:Angiotensin I; D002268:Carboxypeptidases; D043422:Carboxypeptidases A