MEMBRANE DAMAGE BY IMMUNE MECHANISMS

大鼠大颗粒淋巴细胞瘤细胞质颗粒的纯化 已经研究了NK活性，以确定它们在细胞毒性和 这些淋巴细胞的其他功能。 除了主要的溶解性 一种被称为溶细胞素的蛋白质，我们发现它具有一系列蛋白酶活性 存在于纯化的致密颗粒中。 其中两个，显示出强大的 对胰蛋白酶硫酯底物（称为BLT）的活性， 纯化至均一，并且是主要的颗粒蛋白。 之一 这些酶是两个大约30 kd蛋白质的二硫键连接的二聚体， 链，而另一个由约27 kd的单链组成。 这些酶不容易水解被蛋白质酶切割的蛋白质底物。 胰蛋白酶，但也可作用于几种合成肽 在P1位点具有精氨酸的对硝基苯胺底物。 抑制剂研究 表明两种酶都是丝氨酸蛋白酶，最适pH约为8。 一组抑制剂对两种细胞的活性抑制模式 酶是不同的，表明这两种酶不共享一个 简单的单体-二聚体关系。 除了这两 已纯化的BLT水解酶，颗粒提取物 通过凝胶过滤分离显示出另外的抗 胰蛋白酶对硝基苯胺底物和2-3个活性峰， 胰凝乳蛋白酶对硝基苯胺底物。 虽然生理作用 这些丝氨酸蛋白酶仍不清楚，每两个纯化的 BLT水解酶可以协同增强有核细胞的裂解， 用纯化的颗粒溶细胞素处理细胞。 颗粒蛋白多糖的研究 已经通过在体内标记生长的LGL肿瘤进行了研究， 35S-S04 发现该标记定位在两种致密的 （溶细胞素阳性）和轻（无溶细胞活性）颗粒级分 Percoll分级匀浆;动力学研究表明， 颗粒部分是致密颗粒部分的前体。 西方 用兔抗溶细胞素抗体进行Percoll梯度印迹显示， 溶细胞素蛋白存在于轻颗粒部分中， 缺乏可察觉的活动。

The cytoplasmic granules purified from rat large granular lymphocyte tumors with NK activity have been studied to determine their role in cytotoxic and other functions of these lymphocytes. In addition to the major lytic protein termed cytolysin, we have found a series of protease activities present in the purified dense granules. Two of these, which show potent activity against the trypsin thioester substrate known as BLT, have been purified to homogeneity and are among the major granule proteins. One of these enzymes is a disulfide linked dimer of two roughly 30kd protein chains, while the other is comprised of a single chain of about 27kd. These enzymes do not readily hydrolyze protein substrates cleaved by trypsin, but can be shown to act on several synthetic peptide p-nitroanilide substrates with arginine at the P1 site. Inhibitor studies show that both enzymes are serine proteases with a pH optimum of about 8. The pattern of activity inhibition by a panel of inhibitors on the two enzymes is distinct, indicating that these two enzymes do not share a simple monomer-dimer relationship. In addition to these two BLT-hydrolyzing enzymes which have been purified, granule extracts separated by gel filtration show an additional peak of activity against tryptic p-nitroanilide substrates, and 2-3 peaks of activity against chymotryptic p-nitroanilide substrates. Although the physiological roles of these serine proteases are still unclear, each of the two purified BLT-hydrolysing enzymes can synergistically enhance the lysis of nucleated cells by the purified granule cytolysin. Studies of granule proteoglycans have been undertaken by labeling the growing LGL tumors in vivo with 35S-S04. It was found that this label is localized in both dense (cytolysin positive) and light (lytically inactive) granule fractions of Percoll fractionated homogenates; kinetic studies suggest that the light granule fraction is a precursor of the dense granule fraction. Western blots of Percoll gradients with rabbit anti-cytolysin antibody show that the cytolysin protein is present in the light granule fraction in spite of the lack of dectable activity.