Related Experiment Videos
Identification of aminopeptidase activity in the secretory granules of mouse mast cells
W E Serafin1, U A Guidry, E T Dayton
1Department of Medicine, Harvard Medical School, Boston, MA.
Abstract:
Sonicates of mouse bone marrow-derived mast cells (BMMC) differentiated in vitro and of mouse serosal mast cells differentiated in vivo contained small but approximately equal amounts of aminopeptidase activity, as determined by cleavage of leucine-beta-naphthylamide and resolution of the reaction products by reverse-phase high-performance liquid chromatography. Aminopeptidase activity was exocytosed from antigen-activated, IgE-sensitized BMMC in proportion to the secretory granule enzyme beta-hexosaminidase, thereby localizing approximately 60% of the total cell-associated aminopeptidase activity to the secretory granules of the mast cells. A prominent secretory granule location for aminopeptidase was confirmed by activity measurement in subcellular fractions of disrupted BMMC. The secretory granule aminopeptidase had a pH optimum of 6.0-8.0 and a Km of 0.36 +/- 0.06 mM (mean +/- SD; n = 3) for leucine-beta-naphthylamide. When various amino acid beta-naphthylamides were used as substrates, the preference of the secretory granule enzyme was Ala greater than Leu greater than Phe much greater than Arg much greater than Asp = Tyr. Most of the aminopeptidase activity that was exocytosed from calcium ionophore-activated BMMC was bound to 35S-labeled proteoglycans in complexes of greater than 1 x 10(7) kDa as defined by exclusion during Sepharose CL-2B gel-filtration chromatography. We postulate that the amino-peptidase in the mast cell protease/proteoglycan complexes allows the removal of N-terminal amino acids from peptides that are generated by the action of mast cell endopeptidases.
Insights
Mast cells contain aminopeptidase activity primarily within their secretory granules. This enzyme is exocytosed with other granule contents and may process peptides generated by mast cell endopeptidases.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Mast cells are key players in allergic responses and immune modulation.
- Secretory granules of mast cells contain a rich array of enzymes, including proteases and proteoglycans.
- The precise enzymatic composition and function of mast cell secretory granules are still under investigation.
Purpose of the Study:
- To investigate the presence, localization, and characteristics of aminopeptidase activity in mast cells.
- To determine the role of aminopeptidase in mast cell degranulation and its association with other granule components.
Main Methods:
- Aminopeptidase activity was measured in sonicates of bone marrow-derived mast cells (BMMC) and serosal mast cells using leucine-beta-naphthylamide as a substrate.
- Activity was quantified using reverse-phase high-performance liquid chromatography.
- Subcellular fractions were analyzed to confirm enzyme localization.
- Enzyme kinetics (pH optimum, Km) and substrate specificity were determined.
- Exocytosed activity was analyzed for association with proteoglycans using gel-filtration chromatography.
Main Results:
- Both in vitro and in vivo differentiated mast cells exhibited comparable aminopeptidase activity.
- Approximately 60% of total cell-associated aminopeptidase activity was localized to secretory granules.
- The secretory granule aminopeptidase displayed a pH optimum of 6.0-8.0 and a Km of 0.36 mM for leucine-beta-naphthylamide.
- The enzyme showed a substrate preference for Ala > Leu > Phe.
- Exocytosed aminopeptidase activity was found in large complexes (>1 x 10(7) kDa) bound to proteoglycans.
Conclusions:
- Aminopeptidase is a significant component of mast cell secretory granules.
- The enzyme is released during degranulation and associates with proteoglycans.
- This association suggests a role for aminopeptidase in processing peptides generated by mast cell proteases within these complexes.