Substrate specificity of the chymotrypsin-like protease in secretory granules isolated from rat mast cells.
Substrate specificity of the chymotrypsin-like protease in secretory granules isolated from rat mast cells.
复制标题
从大鼠肥大细胞分离的分泌颗粒中胰凝乳蛋白酶样蛋白酶的底物特异性。
DOI：
10.1073/pnas.84.2.364
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发表时间：
1987
影响因子：
11.1
通讯作者：
Woodbury,RG
中科院分区：
文献类型：
--
作者：
LeTrong,H;Neurath,H;Woodbury,RG
大鼠肥大细胞蛋白水解酶I(RMCP I)是一种定位于肥大细胞分泌颗粒中的类糜蛋白酶样丝氨酸蛋白酶，用几种已知氨基酸序列的多肽和蛋白质底物，比较了它们的底物特异性。虽然这两种酶的总体特性相似，但观察到了细微但显著的差异。RMCP I比胰凝乳酶更容易水解由Leu-Xaa或两个疏水残基组成的多肽键--例如Phe-Phe。此外，由胰凝乳酶催化的血管紧张素I的水解，而不是由RMCP I催化的，导致了作为中间产物的血管紧张素II的产生。与溶解的酶相比，不溶性颗粒中的RMCP I活性在4℃、pH 8.0或pH 7.2的合适缓冲液中至少2个月是完全稳定的，与分离的肥大细胞颗粒相关的羧肽酶A活性可被10 mM邻菲咯啉完全抑制。颗粒结合的RMCP I能迅速降解小于脱脂肌红蛋白(17,199 Da)的多肽，而脱脂蛋白和其他较大的蛋白质则不能被水解。相反，游离酶很容易对较大的蛋白质进行水解。抑制游离RMCP I活性的正常大鼠血清和α1-抗胰蛋白酶均不能有效地抑制颗粒结合的RMCP I。结果表明，在离子强度和pH生理条件下，颗粒结合的RMCP I不能从分离的分泌颗粒释放到溶液中，颗粒结构限制了可被该酶水解的蛋白质的大小。
The substrate specificity of rat mast cell protease I (RMCP I), a chymotrypsin-like serine protease localized in the secretory granules of mast cells, was compared to that of bovine alpha-chymotrypsin by using several peptide and protein substrates of known amino acid sequences. Although the overall specificities of the two proteases appeared similar, subtle but significant differences were observed. RMCP I was more prone than chymotrypsin to hydrolyze peptide bonds consisting of Leu-Xaa or two hydrophobic residues--e.g., Phe-Phe. Additionally, the hydrolysis of angiotensin I catalyzed by chymotrypsin, but not by RMCP I, resulted in the generation of angiotensin II as an intermediate product. In contrast to the solubilized enzyme, the RMCP I activity within the insoluble granules was completely stable for at least 2 months in suitable buffers at pH 8.0 or pH 7.2, at 4 degrees C. Carboxypeptidase A activity associated with isolated mast cell granules was completely inhibited by 10 mM o-phenanthroline. Polypeptides smaller than apomyoglobin (17,199 Da) were rapidly hydrolyzed by granule-bound RMCP I, whereas apomyoglobin and other larger proteins were not hydrolyzed. In contrast, the free protease readily hydrolyzed the larger proteins. Neither normal rat serum nor alpha 1-antitrypsin, both of which inhibited the activity of free RMCP I, was effective in inhibiting granule-associated RMCP I. The results indicate that granule-bound RMCP I is not released into solution from isolated secretory granules under physiological conditions of ionic strength and pH and that the granule structure limits the size of proteins that can be hydrolyzed by the protease.