Olsson Magnus, Zhivotovsky Boris
Division of Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.
Methods Mol Biol. 2014;1133:155-73. doi: 10.1007/978-1-4939-0357-3_10.
The majority of caspases, cysteine-dependent aspartate-directed proteases, being in their activated state are involved in regulation of apoptosis by cleaving protein substrates harboring specific target motifs. Basically all biochemical and morphological changes in an apoptotic cell, including cell shrinkage, chromatin condensation, DNA fragmentation, and plasma membrane blebbing, are consequence of caspase-mediated proteolysis. Thus, uncovering activities of unique caspases are key determinants of the apoptotic process. This chapter describes a set of experimental protocols available for characterization, quantification and inhibition of caspase activities in mammalian cell cultures, including immunoblotting, usage of synthetic substrates, flow cytometry, and microscopic techniques.
大多数半胱天冬酶，即半胱氨酸依赖性天冬氨酸定向蛋白酶，处于激活状态时，通过切割含有特定靶基序的蛋白质底物参与细胞凋亡的调控。基本上，凋亡细胞中的所有生化和形态学变化，包括细胞萎缩、染色质浓缩、DNA片段化和质膜起泡，都是半胱天冬酶介导的蛋白水解的结果。因此，揭示独特半胱天冬酶的活性是凋亡过程的关键决定因素。本章描述了一组可用于表征、定量和抑制哺乳动物细胞培养中半胱天冬酶活性的实验方案，包括免疫印迹、合成底物的使用、流式细胞术和显微镜技术。
