The lymphocyte pore-forming protein perforin is associated with granules by a pH-dependent mechanism.
A pore-forming protein (PFP, perforin or cytolysin) has been found in the cytoplasmic granules of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Extraction of granules with high-salt buffers or by freezing-and-thawing results in the release of perforin, which occurs only when the buffer pH is above 7.0. While high-salt extraction and freezing-and-thawing of granules at low pH (below 7.0) do not result in perforin release, these treatments render granules susceptible to a subsequent incubation with low-salt buffers (pH 7-8) that then solubilizes perforin completely. Granules may thus have been made leaky by high-salt extraction or freezing-and-thawing that may occur regardless of the buffer pH, while dissociation of perforin from granules may be exquisitely pH-sensitive. Freezing-and-thawing intact CTL and NK cells in physiological buffers with pH in the range of 7-8 (but not below 7) also causes release of perforin activity to the cell supernatant, thus providing a simple procedure by which perforin activity can be quantitated in small cell samples. Our results suggest that during lymphocyte-mediated killing, the extracellularly released perforin may rapidly dissociate from granules as a result of pH change and, in the process, become cytolytically active.
Persechini
P M
PM
Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, NY 10021.
Liu
C C
CC
Jiang
S
S
Young
J D
JD
D002021:Buffers; D002460:Cell Line; D003594:Cytoplasmic Granules / Q000032:analysis*; D005615:Freezing; D006863:Hydrogen-Ion Concentration; D007694:Killer Cells, Natural / Q000378:metabolism; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000032:analysis* / Q000378:metabolism; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D012995:Solubility; D013602:T-Lymphocytes, Cytotoxic / Q000378:metabolism
D002021:Buffers; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D052899:Pore Forming Cytotoxic Proteins; D054353:Perforin