Serafin W E, Guidry U A, Dayton E T, Kamada M M, Stevens R L, Austen K F
Department of Medicine, Harvard Medical School, Boston, MA.
Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):5984-8. doi: 10.1073/pnas.88.14.5984.
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.
通过亮氨酸-β-萘酰胺的裂解以及反相高效液相色谱法对反应产物的分离测定发现，体外分化的小鼠骨髓来源肥大细胞（BMMC）和体内分化的小鼠浆膜肥大细胞的超声裂解物含有少量但大致等量的氨肽酶活性。氨肽酶活性从抗原激活、IgE致敏的BMMC中以与分泌颗粒酶β-己糖胺酶成比例的方式胞吐，从而将约60%的总细胞相关氨肽酶活性定位于肥大细胞的分泌颗粒中。通过对破碎的BMMC亚细胞组分的活性测量，证实了氨肽酶在分泌颗粒中的显著定位。分泌颗粒氨肽酶的最适pH为6.0 - 8.0，对亮氨酸-β-萘酰胺的Km为0.36 +/- 0.06 mM（平均值 +/- 标准差；n = 3）。当使用各种氨基酸β-萘酰胺作为底物时，分泌颗粒酶的偏好顺序为丙氨酸＞亮氨酸＞苯丙氨酸＞＞精氨酸＞＞天冬氨酸 = 酪氨酸。从钙离子载体激活的BMMC中胞吐的大部分氨肽酶活性与大于1×10⁷ kDa的复合物中的35S标记蛋白聚糖结合，这是通过琼脂糖CL - 2B凝胶过滤色谱法中的排阻定义的。我们推测肥大细胞蛋白酶/蛋白聚糖复合物中的氨肽酶允许从肥大细胞内肽酶作用产生的肽中去除N端氨基酸。
