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Effects of chymase on limited proteolysis and activation of protein kinase on the mechanism of degranulation from mast cells

食糜酶对有限蛋白水解和蛋白激酶激活对肥大细胞脱粒机制的影响

基本信息

批准号：
62570113
负责人：
HIROSHI Kido
金额：
$ 1.22万
依托单位：
The University of Tokushima, The Institute for Enzyme Research, Division of Enzyme Chemistry
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

Two types of serine proteases, such as chymase and tryptase, are localized in the grnules of mast cells. We found that antibody of chymase, substrate analogues and specific inhibitor of chymase inhibited histamine release from rat mast cells but that inhibitors of tryptase did not. In addition specific inhibitors of chymase and anti-chymase F(ab')_2 also inhibited protein phosphorylation by protein kinase C after bridging of IgE-receptor. From these results, We speculate that chumase activates protein kinse C on the process of degranulation. After degranulation, chymase retained on the surface of mast cells. In order to clarify which of the chymase in the granules or on the cell surface is involved in histamine release, we examined the intra-cellular localization of the inhibitores of both histamine release and chymase, such as antichymase f(ab')_2 and ^<125>I-Bowman Burk soybean inhibitor. After incubation of these inhibitors with mast cells at 37゜C, they first bound to plasma membrane of mast cells and then were incorporated into the granules time-dependently, resulting in the inhibition of histamine release after bridging of IgE receptor. When these inhibitors were incubated with mast cells at 4゜C, they bound to plasma membrane but did not incorporated into the granules and did not also inhibit histamine release. These results show that incorporation of these inhibitors into the granules, in which chymase is localized, is essential for inhibition of histamine release by these inhibitors.Studies on the mechanisms of activation of protein kinase C by chymase and of protein phosphorylation by the activated protein kinase C are now in progress.

肥大细胞的颗粒中存在两种丝氨酸蛋白酶，如类糜蛋白酶和类胰蛋白酶。我们发现糜酶抗体、底物类似物和特异性糜酶抑制剂均能抑制肥大细胞释放组胺，而类胰蛋白酶抑制剂则无此作用。此外，糜酶特异性抑制剂和抗糜酶F（ab '）_2也能抑制蛋白激酶C桥接IgE受体后的蛋白磷酸化。从这些结果，我们推测，chumase激活蛋白激酶C的脱粒过程。肥大细胞脱颗粒后，糜蛋白酶滞留在肥大细胞表面。为了阐明颗粒中或细胞表面的哪些糜酶参与组胺释放，我们检测了组胺释放和糜酶的两种媒介物，如抗糜酶f（ab '）_2和^ I-Bowman Burk大豆抑制剂的细胞内定位<125>。这些抑制剂与肥大细胞在37 ℃孵育后，首先与肥大细胞的质膜结合，然后时间依赖性地掺入颗粒中，从而抑制IgE受体桥接后的组胺释放。当这些抑制剂与肥大细胞在4 ℃下孵育时，它们与质膜结合，但不掺入颗粒中，也不抑制组胺释放。这些结果表明，这些抑制剂的掺入颗粒，其中糜酶定位，是必不可少的抑制组胺的释放。糜酶激活的蛋白激酶C和蛋白磷酸化的激活的蛋白激酶C的机制的研究正在进行中。