STRUCTURE AND FUNCTION OF CYTOTOXIC AND HELPER T LYMPHOCYTE GRANULES

我们发现颗粒蛋白水解酶颗粒酶A可以灭活 用共价抑制剂PMSF对完整的CTL进行预处理。 因为该试剂在pH值下与活性部位丝氨酸发生反应 依赖反应，有效的失活也需要 存在能提高酸性细胞内pH的试剂 车厢。因此，这些分泌颗粒的pH值较低。 内部，就像在大多数其他细胞中发现的那样。有趣的是， 完整CTL中颗粒酶A的失活并不显著 抑制CTL的裂解效率，意味着这 蛋白酶活性可能不是细胞毒性所必需的。然而， 靶DNA的破坏在CTL已经被 用PMSF处理以灭活颗粒蛋白水解酶。我们有 发现CTL或CTL纯化颗粒介导的细胞毒作用 NK肿瘤伴有靶向DNA的破坏，而这 似乎既需要溶细胞素，也需要另一种颗粒成分。 我们已经通过DNA分析研究了这个颗粒成分(S) 从洗涤剂分离的细胞核中释放。在CTL颗粒中 低浓度的PMSF显著抑制了活性。 和DFP，这是丝氨酸蛋白酶的特异性抑制剂。这 加强了上述结果，表明丝氨酸蛋白酶的作用 靶细胞DNA的分解。我们已经开发出了新的定量 淋巴细胞分泌物的测量方法 荧光染料，如奎纳克林，定位于酸性 颗粒的内部。

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.