title: Plant metacaspase activation and activity.
abstract: Metacaspases are essential for cell death regulation in plants. Further understanding of biochemistry of metacaspases and their molecular function in plant biology requires a set of robust methods for detection of metacaspase activation and quantitative analysis of corresponding proteolytic activity. Here we describe methods for purification of recombinant metacaspases, measurement of enzymatic activity of recombinant and endogenous metacaspases in vitro and in cell lysates, respectively, and finally detection of metacaspase activation in vivo. Additionally, an in vitro metacaspase protein substrate cleavage assay based on the cell-free production of substrate protein followed by proteolysis with recombinant metacaspase is presented. These methods have been originally developed for type II metacaspases from Arabidopsis and Norway spruce (Picea abies), but they can be used as templates for type I metacaspases, as well as for type II metacaspases from other species.
authors.affiliation: Department of Plant Biology, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, Uppsala, Sweden.
authors.forename: Elena A
authors.initials: EA
authors.lastname: Minina
authors.forename: Simon
authors.initials: S
authors.lastname: Stael
authors.forename: Frank
authors.initials: F
authors.lastname: Van Breusegem
authors.forename: Peter V
authors.initials: PV
authors.lastname: Bozhkov
mesh_terms: D017360:Arabidopsis / Q000201:enzymology*; D020169:Caspases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D016923:Cell Death / Q000235:genetics; D008967:Molecular Biology / Q000379:methods*; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D020169:Caspases