Purified perforin induces target cell lysis but not DNA fragmentation.
Rapid and extensive target cell DNA fragmentation is a unique characteristic of CTL-mediated killing. We studied the role of the granule pore-forming protein (PFP/perforin/cytolysin) of CTL in mediating lysis and DNA fragmentation of target cells. Perforin was isolated from murine CTL by sequential application of perforin-enriched granule fractions to four chromatographic columns: DEAE-Sepharose, Q-Sepharose, Polyanion SI, and Superose 12. Purified perforin was eluted as a single band of 70 kD in SDS-PAGE. While purified perforin produced potent lysis of a variety of target cells tested, it did not induce any measurable amount of DNA fragmentation. In parallel experiments, intact CTL produced marked DNA fragmentation of the same target cell populations. Our results suggest that perforin alone is not responsible for the DNA fragmentation observed during CTL-mediated killing and that other, as yet unknown, mediators or mechanisms are likely to be involved in the induction of target cell nuclear damage.
Duke
R C
RC
Department of Microbiology and Immunology, University of Colorado School of Medicine, Denver 80262.
Persechini
P M
PM
Chang
S
S
Liu
C C
CC
Cohen
J J
JJ
Young
J D
JD
D000818:Animals; D003602:Cytotoxicity, Immunologic; D004249:DNA Damage; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D051379:Mice; D008970:Molecular Weight; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D013602:T-Lymphocytes, Cytotoxic / Q000502:physiology*
D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D052899:Pore Forming Cytotoxic Proteins; D054353:Perforin