The lymphocyte pore-forming protein perforin is associated with granules by a pH-dependent mechanism

Abstract

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.