Isolation and biochemical and functional characterization of perforin 1 from cytolytic T-cell granules.

Abstract

The Ca2 -dependent cytolytic activity of isolated T-lymphocyte granules was purified to apparent homogeneity by high-salt extraction, gel filtration, and ion-exchange chromatography. The lytic activity resided in a 72-to 75-kDa protein of cytolytic granules. Incubation of the isolated protein

Impaired nonrestricted cytolytic activity in systemic lupus erythematosus

“…Third, we took advantage of the disparity between Fas-based and perforin-based killing in sensitivity to chelation of extracellular calcium. Perforin-mediated pore formation and ensuing cytolytic activity are strictly calcium-dependent (20)(21)(22)(23). That is, the effector phase of perforin-based cytotoxicity is highly calcium-dependent.…”

“…TO determine whether a defect in perforin expression could be implicated in the SLE defect, cultured PBMC were stained at various time points for intracellular perforin. Since perforin is associated with cytoplasmic granules (20,23,33,34), the cells were also stained in parallel for expression of the TIA-1 molecule, a 15-kd cytoplasmic granule-associated protein (13). At day 0, discrete antiperforin-and anti-TIA-1-staining populations were readily discernible in both normal and SLE cells (Figure 3).…”

The Involvement of Macrophages and Lymphocytes in the Apoptosis of Enterocytes.

“…Perforin, one of the predominant cytotoxic substances contained in the granules of LGLs, is a bioactive peptide which forms in its target cell nonspecific ion channels through which markers of intracellular components can readily pass (HENKART et al, 1984;PODACK et al, 1985). The formation of these ion channels effectively induces the cell death of target cells.…”

“…LGLs correspond to a heterogenous cell population including NK cells, cytotoxic T lymphocytes (CTLs) and lymphokine-activated killer (LAK) cells (KANEDA, 1989). Cytoplasmic granules of the lymphocytes contain cytotoxic substances including perforin (PODACK et al, 1985), a family of serine esterases (MUNGER et al, 1988;HAYES et al, 1989;ZUNINO et al, 1990), and TIA-1 (TIAN et al, 1991) as effector molecules which kill target cells.…”

Properties of a purified pore-forming protein (perforin 1) isolated from H-2-restricted cytotoxic T cell granules.

“…In method II, granules were extracted with 1 M NaKHPO4 buffer containing 1 mM EDTA, 10 mM benzamidine, and 1 mM PMSF, followed by sieving through Sephacryl-$300 column and ion-exchange through Mono Q column (Pharmacia Fine Chemicals, Uppsala, Sweden), essentially as described previously (15,18). The purified protein was used directly in functional assays or further purified through a TSK G3000 column, as described above.…”