A human lung mast cell chymotrypsin-like enzyme. Identification and partial characterization.
A human lung mast cell chymotrypsin-like enzyme. Identification and partial characterization.
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一种人肺肥大细胞胰凝乳蛋白酶样酶。
DOI：
10.1172/jci112276
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Proud,D
中科院分区：
文献类型：
--
作者：
Wintroub,BU;Kaempfer,CE;Schechter,NM;Proud,D
我们使用了高效液相色谱法，检测血管紧张素I的phe8-his9键的凝乳胰蛋白酶裂解以产生血管紧张素II，以检查人肺肥大细胞是否存在凝乳胰蛋白酶活性。通过酶解分散、逆流洗脱和Percoll梯度离心纯化人肺肥大细胞，裂解或用山羊抗人IgE激发。在多个实验中，在10-99%的纯肥大细胞制剂的裂解物中检测到血管紧张素ii转化活性。对组胺净释放值和血管紧张素i转化活性的回归分析表明，这两个参数之间存在相关性，表明凝乳胰蛋白酶样酶是肥大细胞分泌颗粒的组成部分。10(-3) M苯基甲基磺酰氟完全抑制了乳糜蛋白酶的活性，而10(-3)M卡托普利对乳糜蛋白酶的活性没有抑制作用，活性的最适pH为7.5 ~ 9.5。凝胶过滤释放的物质分离了胰蛋白酶的活性，并证明其分子量约为30- 35000。肥大细胞酶，像人皮肤乳凝胰蛋白酶样蛋白酶，可以从白细胞组织蛋白酶G中区分出来，对牛胰胰蛋白酶抑制剂的抑制缺乏敏感性。因此，在人肺肥大细胞中存在一种具有有限凝乳胰蛋白酶特异性的酶。血管紧张素I酶的米切利斯常数为6.0 X 10(-5) M，与内皮细胞血管紧张素转换酶的米切利斯常数相似，与具有重要生理意义的反应一致。
We have used a high performance liquid chromatography assay, which detects chymotryptic cleavage of the phe8-his9 bond of angiotensin I to yield angiotensin II, in order to examine human lung mast cells for the presence of chymotryptic activity. Mast cells, purified from human lung by enzymatic dispersion, countercurrent elutriation, and Percoll gradient centrifugation, were lysed or challenged with goat anti-human IgE. In multiple experiments angiotensin II-converting activity was detected in lysates of 10-99% pure mast cell preparations. Regression analysis of net percent release values of histamine and the angiotensin I-converting activity from dose-response experiments demonstrated a correlation between the two parameters, indicating that the chymotrypsin-like enzyme is a constituent of the mast cell secretory granule. The chymotryptic activity was completely inhibited by 10(-3) M phenylmethylsulfonylfluoride but not by 10(-3) M Captopril, and the pH optimum of activity was 7.5-9.5. Gel filtration of released material separated the activity from tryptase and demonstrated an approximate molecular weight of 30-35,000. The mast cell enzyme, like a human skin chymotrypsin-like proteinase, can be distinguished from leukocyte cathepsin G by lack of susceptibility to inhibition by bovine pancreatic trypsin inhibitor. Thus, an enzyme with limited chymotryptic specificity is present in human lung mast cells. The Michaelis constant of the enzyme for angiotensin I of 6.0 X 10(-5) M is similar to that of endothelial cell angiotensin-converting enzyme and is consistent with a reaction of physiologic importance.