title: Detection of human and mouse granzyme B activity in cell extracts.
abstract: The serine protease granzyme B (GrB) is a key effector molecule in cell-mediated immunity, released by cytotoxic lymphocytes (CLs) to induce cell death in neoplastic or virus-infected cells. The ability to detect and measure GrB activity is important for understanding CLs. Unfortunately, such analyses are complicated by significant differences in the substrate specificities of human and mouse GrB, which is reflected by their different activities on commonly used peptide substrates. Here, we present methods for the detection of active human and mouse GrB in extracts from primary cells, and evaluate the sensitivity of the various substrates and inhibitors. Mouse splenocytes produce approximately 120-fold more GrB than similarly activated human cells, which allows the use of the hGrB substrate IETD-AFC to follow mouse GrB activity despite its unfavourable kinetic properties.
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, VIC, Australia.
authors.forename: Sarah Elizabeth
authors.initials: SE
authors.lastname: Stewart
authors.forename: Matthew Stephen James
authors.initials: MS
authors.lastname: Mangan
authors.forename: Phillip Ian
authors.initials: PI
authors.lastname: Bird
authors.forename: Dion
authors.initials: D
authors.lastname: Kaiserman
mesh_terms: D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000201:enzymology; D002457:Cell Extracts / Q000737:chemistry*; D004151:Dipeptides / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D057075:Enzyme Assays / Q000379:methods*; D053804:Granzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D051379:Mice; D011994:Recombinant Proteins / Q000378:metabolism; D013154:Spleen / Q000166:cytology / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D002457:Cell Extracts; D004151:Dipeptides; D011994:Recombinant Proteins; C028342:compound 20; D053804:Granzymes