Identification of aminopeptidase activity in the secretory granules of mouse mast cells.
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.
Serafin
W E
WE
Department of Medicine, Harvard Medical School, Boston, MA.
Guidry
U A
UA
Dayton
E T
ET
Kamada
M M
MM
Stevens
R L
RL
Austen
K F
KF
D000626:Aminopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002458:Cell Fractionation; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D003594:Cytoplasmic Granules / Q000201:enzymology*; D007700:Kinetics; D008407:Mast Cells / Q000201:enzymology*; D051379:Mice; D008807:Mice, Inbred BALB C; D013379:Substrate Specificity
D000626:Aminopeptidases