Purified human perforin efficiently binds to PAF.

Perforin was immunopurified from the perforin-positive YT2C2 NK-cell line using an appropriate binding gel (antiperforin 6.4-IgM, antiperforin mAb, coupled with Affi-Gel 10 gel). (A) Successful purification of perforin was controlled by a Western blot using the specific antiperforin antibody 2d4-perf. In lane C, for YT cell line extracts as positive control, the procedure revealed 2 bands at the molecular weight of 66 and 30-kd as recently reported by Uellner et al.24 Perforin was further purified by ultracentrifugation to obtain the active form of 66-kd (Uellner et al24), expressed in lane 1. No other band was detected in lanes 2, 3, 4, and 5 using, respectively, the perforin-negative Jurkat cell line lysate, the gel cross-linked to the mAb, the gel alone, or the gel with an irrelevant protein BSA instead of perforin. Active perforin was then exposed to [³H]PAF C18:0 during 30 minutes at 20°C and washed 5 times with the cold buffer as shown in “Materials and methods.” The resulting radioactivity was then measured for perforin. (B) In this assay, lane 1 shows the formation of perforin-[³H]PAF complexes expressed in disintegrations per minute. In contrast, no significant radioactivity was detected in lanes 2, 3, 4, and 5 using, respectively, the perforin-negative Jurkat cell line lysate, the gel cross-linked to the mAb, the gel alone, or the gel with an irrelevant protein BSA instead of perforin as already mentioned for Western blot analysis.