Extracellular release of lymphocyte cytolytic pore-forming protein (perforin) after ionophore stimulation.
The cytolytic pore-forming protein (PFP, perforin) of lymphocyte granules has recently been isolated and characterized. The lytic activities expressed by both the isolated granules and the purified PFP require the presence of Ca2+. Here, we report on the extracellular release of PFP after stimulation of lymphocytes with the Ca2+ ionophore A23187, which degranulates the cells. The secreted protein associates with lipid to form structural and functional channels and supramolecular complexes that partially resist dissociation by sodium dodecyl sulfate and reducing agents. Immunoblots of the released material reveal positive identification with antibodies specific for mouse PFP and human complement component C9, indicating cross-reactivity between these two molecules. By using these specific antibodies as immunoadsorbents, the lymphocyte PFP has been affinity purified from the supernatant of stimulated cells. The extracellular release of PFP is associated with simultaneous formation of functional ion-nonselective channels with conductances of 550-600 pS in 0.15 M NaCl, as measured in planar model bilayers. In the absence of extracellular Ca2+, 15% of the maximal release activity is observed. Ca2+ appears to be required to elicit both secretion by lymphocytes and the assembly of the released PFP into tubular polymers. Similar secretion of PFP may occur during cell killing by lymphocytes, resulting in its assembly on target membranes to form tubular transmembrane lesions.
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000502:physiology; D003600:Cytosol / Q000502:physiology; D003602:Cytotoxicity, Immunologic; D007473:Ion Channels; D008081:Liposomes; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D012636:Secretory Rate / Q000187:drug effects; D012995:Solubility; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
D007473:Ion Channels; D008081:Liposomes; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D052899:Pore Forming Cytotoxic Proteins; D054353:Perforin; D000001:Calcimycin; D002118:Calcium