We have found that granule protease granzyme A can be inactivated by pretreatment of intact CTL with the covalent inhibitor PMSF. Because this reagent reacts with the active site serine in a pH dependent reaction, efficient inactivation also requires the presence of agents which raise the pH of acidic intracellular compartments. Thus these secretory granules have a low pH interior, as has been found in most other cells. Interestingly, inactivation of granzyme A in intact CTL does not significantly inhibit the lytic efficiency of the CTL, implying that this protease activity may not be required for cytotoxicity. However, target DNA breakdown is greatly reduced when the CTL has been pretreated with PMSF to inactivate granule proteases. We have found that cytotoxicity mediated by purified granules from CTL or NK tumors is accompanied by target DNA breakdown, and that this appears to require both cytolysin and another granule component. We have studied this other granule component(s) by assaying DNA release from detergent-isolated nuclei. In CTL granules this activity is dramatically inhibited by low concentrations of PMSF and DFP, which are specifics inhibitors of serine proteases. This reinforces the above results suggesting a role for serine proteases in target cell DNA breakdown. We have developed new quantitative methods for measurement of secretion from lymphocytes using fluorescent dyes such as quinacrine which localize in the acidic interior of the granules.
