Ray P, Ali S T
Biology Department, Walter Reed Army Institute of Research, Washington, D. C. 20307-5100, USA.
Drug Chem Toxicol. 1998 Aug;21(3):319-27. doi: 10.3109/01480549809002208.
An in vitro normal human epidermal keratinocytes (NHEK) model was used to study and to characterize the protease stimulated by the mustards 2-chloroethyl ethyl sulphide (CEES), 2-chloro-N-(2-chloroethyl)-N-methylethanamine hydrochloride (nitrogen mustard, HN2), and Bis-2-chloroethyl sulfide (sulfur mustard, HD). The results obtained by using a chromozym (TRY) peptide substrate protease assay showed the optimum mustard concentration and time for protease stimulation to be about 200 microM CEES, 100 microM HN2 or HD, and 16 hours. The mustard-stimulated protease was membrane-bound, and was inhibited by adding a Ca2+ chelator EGTA (2 mM), BAPTA AM (50 microM) or a serine protease inhibitor diisopropyl fluoro-phosphate DFP (1 mM), or a protein synthesis inhibitor cycloheximide (10 microM) in the extracellular medium. These results suggest that one of the mechanisms of mustard toxicity is via the stimulation of a trypsin/chymotrypsin like serine protease, which is dependent on Ca2+ and new protein synthesis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a mustard-stimulated approximately equal to 70-80 KDa protein band that was associated with protease activity which was inhibitable by EGTA, BAPTA, DFP or cycloheximide. This mustard-stimulated protein (protease) may serve as a diagnostic tool for mustard exposure as well as an assay for screening prospective antivesicant protease inhibitor drugs.
利用体外正常人表皮角质形成细胞（NHEK）模型研究并表征由芥子气2-氯乙基乙基硫醚（CEES）、2-氯-N-（2-氯乙基）-N-甲基乙胺盐酸盐（氮芥，HN2）和双-2-氯乙基硫醚（硫芥，HD）刺激产生的蛋白酶。使用色原底物（TRY）蛋白酶测定法获得的结果表明，蛋白酶刺激的最佳芥子气浓度和时间分别约为200 microM CEES、100 microM HN2或HD以及16小时。芥子气刺激产生的蛋白酶与膜结合，在细胞外培养基中添加Ca2+螯合剂乙二醇双四乙酸（EGTA，2 mM）、1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸四乙酰甲酯（BAPTA AM，50 microM）、丝氨酸蛋白酶抑制剂二异丙基氟磷酸酯（DFP，1 mM）或蛋白质合成抑制剂环己酰亚胺（10 microM）可抑制该蛋白酶。这些结果表明，芥子气毒性的机制之一是通过刺激一种胰蛋白酶/糜蛋白酶样丝氨酸蛋白酶，该蛋白酶依赖于Ca2+和新的蛋白质合成。十二烷基硫酸钠聚丙烯酰胺凝胶电泳显示，芥子气刺激产生的一条约70 - 80 kDa的蛋白条带与蛋白酶活性相关，该活性可被EGTA、BAPTA、DFP或环己酰亚胺抑制。这种芥子气刺激产生的蛋白质（蛋白酶）可作为芥子气暴露的诊断工具以及筛选潜在抗水疱蛋白酶抑制剂药物的检测方法。
