Isolation of a Ca-dependent erythrolytic protein (perforin) from cytotoxic T-lymphocytes

A Ca-dependent erythrolytic protein (perforin) was isolated from a cytotoxic T-cell line (CTLL2). Cellular extracts were fractionated on DEAE-cellulose and hydrophobic Phenyl-Sepharose columns. Lytic activity was tightly bound to the hydrophobic column and was eluted with 50% ethyleneglycol. The erythrolytic activity was dependent on the concentration of Ca2+ ions, and heparin accelerated the lysis of erythrocytes by perforin 10-fold, with a half maximal concentration of 12 ng/ml. The activity was strongly inhibited by micromolar concentrations of heavy metal ions, such as Zn2+ and Fe2+, and glycylarginine-methylcoumarinamide (Gly-Arg-MCA) in the presence of 100 ng/ml heparin.