Measuring elastase, proteinase 3 and cathepsin G activities at the surface of human neutrophils with fluorescence resonance energy transfer substrates
Measuring elastase, proteinase 3 and cathepsin G activities at the surface of human neutrophils with fluorescence resonance energy transfer substrates
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DOI：
10.1038/nprot.2008.63
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发表时间：
2008-01-01
期刊：
影响因子：
14.8
通讯作者：
Gauthier, Francis
中科院分区：
文献类型：
--
作者：
Korkmaz, Brice;Attucci, Sylvie;Gauthier, Francis
中性粒细胞丝氨酸蛋白酶(NSPs)、弹力酶、蛋白酶3和组织蛋白酶G是参与病原体破坏和炎症过程调控的多功能蛋白酶。分泌的一小部分NSP仍与外质膜结合，在那里它们保持酶活性。本方案描述了使用高灵敏度的Abz-多肽-EDDnp荧光共振能量转移(FRET)底物对中性粒细胞表面NSP活性的荧光光谱测量，该底物完全区分了三种人类NSP。我们描述了FRET底物的合成，中性粒细胞的纯化和处理，以及对静止和激活细胞的动力学实验。这些方法被用来测量低结合微板中膜结合或游离NSP的亚纳摩尔浓度，并定量测定咯痰和支气管肺泡灌洗等生物液中个别蛋白酶的活性。整个过程，包括中性粒细胞纯化和动力学测量，可以在4-5小时内完成，因为中性粒细胞的寿命不应该更长。使用这一方案将有助于确定单个NSP在炎症性疾病发展中的作用，并可能揭示这些蛋白酶是治疗抑制剂的靶点。
The neutrophil serine proteases (NSPs) elastase, proteinase 3 and cathepsin G are multifunctional proteases involved in pathogen destruction and the modulation of inflammatory processes. A fraction of secreted NSPs remains bound to the external plasma membrane, where they remain enzymatically active. This protocol describes the spectrofluorometric measurement of NSP activities on neutrophil surfaces using highly sensitive Abz-peptidyl-EDDnp fluorescence resonance energy transfer (FRET) substrates that fully discriminate between the three human NSPs. We describe FRET substrate synthesis, neutrophil purification and handling, and kinetic experiments on quiescent and activated cells. These are used to measure subnanomolar concentrations of membrane-bound or free NSPs in low-binding microplates and to quantify the activities of individual proteases in biological fluids like expectorations and bronchoalveolar lavages. The whole procedure, including neutrophil purification and kinetic measurements, can be done in 4-5 h and should not be longer because of the lifetime of neutrophils. Using this protocol will help identify the contributions of individual NSPs to the development of inflammatory diseases and may reveal these proteases to be targets for therapeutic inhibitors.