Caspases: determination of their activities in apoptotic cells.
Caspases, a family of cysteine proteases, were identified as major regulators of apoptotic cell death. These enzymes are involved not only in initiation but also in an execution phase of apoptosis by cleaving more than 400 substrates. This cleavage mediates a majority of the typical biochemical and morphological changes in apoptotic cells, such as cell shrinkage, chromatin condensation, DNA fragmentation, and plasma membrane blebbing. In addition to their role in cell death, caspases fulfill various nonapoptotic functions in living cells. Thus, detection of caspase activation/activity is essential for the understanding of different biological processes. It can be used as a biochemical marker for apoptosis induced by diverse stimuli. This chapter describes a set of methods available for characterization and/or quantification of caspase activation, including immunoblotting, cleavage of synthetic substrates, affinity labeling, flow cytometry and different microscopic techniques. In addition, several methods discussing the attempts for in vivo analyzing of caspase activity are included. Advantages and disadvantages of each method are compared.
Vaculova
Alena
A
Institute of Environmental Medicine, Division of Toxicology, Karolinska Institute, Stockholm, Sweden.
Zhivotovsky
Boris
B
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D019169:Jurkat Cells; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D013379:Substrate Specificity
D061945:Caspase Inhibitors; D020169:Caspases