Characterization, application and potential uses of biotin-tagged inhibitors for lymphocyte serine proteases (granzymes).
Characterization, application and potential uses of biotin-tagged inhibitors for lymphocyte serine proteases (granzymes).
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淋巴细胞丝氨酸蛋白酶（颗粒酶）生物素标记抑制剂的表征、应用和潜在用途。
DOI：
10.1016/0161-5890(96)00025-9
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发表时间：
1996
影响因子：
3.6
通讯作者：
Hudig,D
中科院分区：
文献类型：
--
作者：
Winkler,U;Allison,NJ;Woodard,SL;Gault,RA;Ewoldt,GR;Kam,CM;Abuelyaman,A;Powers,JC;Hudig,D
细胞毒性淋巴细胞和自然杀伤细胞能够在几分钟内杀死它们的靶细胞。靶细胞的死亡发生在效应细胞释放胞质颗粒之后。这些颗粒含有成孔蛋白穿孔素和丝氨酸蛋白酶（颗粒酶）。迄今为止，已发现10个编码淋巴细胞颗粒酶的基因;其中只有4个已被纯化并表征其底物特异性。预测有几种具有常见的糜酶样特异性，其在颗粒提取物中发现。其他酶可能需要作为活性酶进行富集，然后才能对它们进行底物水解评价。由于底物水解检测的局限性，需要一种更灵敏的检测稀释颗粒的方法。我们报告了七种生物素（Bi）标记的异香豆素（IC）抑制剂对Asp-ase、糜酶、类胰蛋白酶和Met-ase颗粒酶的不同反应性。这些抑制剂在其3号位上含有不同的取代基：甲氧基（OMe）、乙氧基（OEt）、丙氧基（OPr）或2-苯基乙氧基（OEtPh）基团。OMe组赋予一般反应性，而OEtPh组赋予与糜酶颗粒酶的选择性反应性。在生物素标签和异香豆素环之间含有最长的氨基己酰基（Aca）间隔子的抑制剂介导最稳定的颗粒酶失活。这些抑制剂在阻断颗粒提取物对红细胞的溶解方面最有效。在蛋白质印迹实验中使用抑制剂，其中用抗生物素蛋白-酶复合物检测生物素。超过10种颗粒酶被抑制剂Bi-Aca-Aca-IC-OMe标记。抑制剂检测颗粒酶时，他们不容易检测到底物水解。
Cytotoxic lymphocytes and natural killer cells are able to kill their target cells in minutes. The death of the target cell occurs after the release of cytoplasmic granules from the effector cell. These granules contain the pore-forming protein perforin and serine proteases (granzymes). To date 10 genes encoding lymphocyte granzymes have been discovered; of these only four have been purified and characterized for their substrate specificity. Several are predicted to have a common chymase-like specificity which is found in the granule extracts. Others may need to be enriched as active enzymes before they can be evaluated for substrate hydrolysis. Due to the limitations of detection by substrate hydrolysis, a more sensitive method for the detection of dilute granules was needed. We report the differing reactivities of seven biotin (Bi)-tagged isocoumarin (IC) inhibitors for Asp-ase, chymase, tryptase and Met-ase granzymes. The inhibitors contained different substituents at their no. 3 position: methoxy (OMe), ethoxy (OEt), propoxy (OPr) or 2-phenylethoxy (OEtPh) groups. The OMe group conferred general reactivity, whereas the OEtPh group conferred selective reactivity with chymase granzymes. The inhibitors that contained the longest aminocaproyl (Aca) spacers between the biotin-tag and the isocoumarin ring mediated the most stable granzyme inactivation. These inhibitors were the most effective at blocking lysis of red blood cells by the granule extracts. The inhibitors were used in protein blotting experiments where the biotin was detected with an avidin-enzyme complex. Over 10 granzymes were labelled by the inhibitor Bi-Aca-Aca-IC-OMe. The inhibitors detected granzymes when they were not readily detected by substrate hydrolysis.
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作者：
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DOI：
10.1016/0304-4165(88)90095-5
发表时间：
1988
期刊：
Biochimica et biophysica acta
影响因子：
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作者：
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通讯作者：
Hudig,D