GrM is targeted by SPI-CI. (A) CMT93-RS is lysed by GrB cluster knock-out LAK cells. CMT93-RS was incubated for 5 hours with different E/T ratios of wild-type and GrB knock-out LAKs, and membranolysis was determined using chromium release. (B) Several chymotrypsin inhibitors inhibit lysis of CMT93-RS by GrB cluster knock-out LAK cells. Lysis assays were performed as described in panel A, and inhibitors used are as follows: for tryptase: BAEE (10 mM; Sigma) and leupeptin (0.1 mM; Sigma); for chymotrypsin: ATEE (10 mM; Sigma), TPCK (10 μM; Sigma), AEBSF (0.4 mM; Sigma), and GrM inhibitor z-Met^P-(OPh-4-Cl)₂ (0.1 mM; kind gift from Jim Powers); for calpain: E64D (40 μM; Sigma) and z-LLY-fmk (20 μM; Enzyme Systems Products, Livermore, CA); for Asp proteases: pepstatin A (25 μg/mL; Calbiochem, San Diego, CA); and for cathepsin B: z-FA-fmk (10 μM; Enzyme Systems Products) and Ca074-ME (25 μM, Calbiochem). None of the inhibitors displayed toxicity on the LAK cells as determined by trypan blue exclusion. (C) Met-ase activity in granule isolates of CTLs and LAK cells was determined using the preferred substrate Boc-Ala-Ala-Met-SBzl. Activity is depicted as absorbance at 405 nm. (D) In vitro association between purified GrB and in vitro–translated SPI-6 and purified GrM and in vitro–translated SPI-CI. Complexes are visualized on reducing SDS-PAGE. (E) Lysis of MFF and MFF/SPI-CI using purified perforin in combination with purified GrM. GrM was added to cells either with or without perforin, and cell death was determined using PI exclusion. One representative experiment of 3 is shown.