Liposomes as targets for granule cytolysin from cytotoxic large granular lymphocyte tumors.

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Abstract
Purified cytoplasmic granules from rat large granular lymphocyte tumors having natural killer activity and/or antibody-dependent cell-mediated cytotoxicity induced a rapid, dose-dependent release of the water-soluble marker carboxyfluorescein from liposomes made of phosphatidylcholine. A solubilized, partially purified cytolytic preparation termed cytolysin from these granules showed identical properties. Marker release induced by granules or the cytolysin was strongly dependent on the presence of Ca2+ at a concentration of 0.1 mM or higher in the medium; Ca2+ could be replaced by higher concentration of Sr2+ but not by Ba2+ or by Mg2+. These properties strikingly parallel the lytic effects that granules and granule cytolysin exert on cells. Marker release from liposomes was stopped instantaneously when an excess of EGTA [ethylene glycol bis(.beta.-aminoethyl ether)N,N,N'',N''-tetracetic acid] was added to the medium. The remaining carboxyfluorescein inside the liposomes was present at the original internal concentration, indicating that marker release was all-or-none from individual liposomes. Liposomes comprised of lipid in the solid phase released marker more slowly than did comparable liposomes containing fluid-phase lipids. Variation of the lipid headgroup had only minor effects on the cytolysin-induced marker release. EM of liposomes exposed to cytolysin in the presence of Ca2+ showed cylindrical structures of 15-nm diameter inserted into the membrane concomitant with the penetration of negative stain into the liposome. These properties of large granular lymphocyte granule cytolysin strongly suggest that it operates through a mechanism similar to the membrane attack of complement.

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