MEMBRANE DAMAGE BY IMMUNE MECHANISMS

从大鼠大颗粒淋巴细胞瘤中提纯的胞浆颗粒 已有研究确定其在细胞毒和细胞毒性中的作用 这些淋巴细胞的其他功能。除了主要的裂解 一种名为细胞溶素的蛋白质，我们发现了一系列的蛋白酶活性 存在于纯化的致密颗粒中。其中的两个，它们显示出强大的 对胰酶硫酯底物(BLT)的活性一直是 纯化至均一，是主要的颗粒蛋白之一。其中之一 这些酶是由两个约30kd蛋白质组成的二硫键连接的二聚体。 链，而另一个由约27kd的单链组成。 这些酶不容易水解被 胰酶，但可以作用于几种合成肽 对-硝基苯胺底物与精氨酸在P1位。抑制剂研究 结果表明，两种酶均为丝氨酸蛋白酶，最适pH约为8。 一组抑制剂对两种酶的活性抑制模式 酶是不同的，这表明这两种酶不共享 简单的单体-二聚体关系。除了这两个之外 纯化的BLT-水解酶、颗粒提取物 经凝胶过滤分离后显示出一个额外的抗病毒活性峰 胰酶对硝基苯胺底物和2-3个抗菌活性高峰 胰凝乳酶对硝基苯胺底物。虽然生理上的作用 这些丝氨酸蛋白酶中的每一种仍不清楚，分别是纯化的两种 BLT-水解酶能协同促进有核物质的裂解 用纯化的细胞溶血素颗粒培养细胞。颗粒状蛋白多糖的研究进展 已经通过标记体内生长的LGL肿瘤进行了研究 35S-S04。研究发现，该标记在致密的两种细胞中均有定位 (细胞溶血素阳性)和轻(裂解不活跃)颗粒组分 Percoll分级匀浆；动力学研究表明，光 颗粒组分是致密颗粒组分的前驱物质。西式 Percoll梯度与兔抗细胞溶血素抗体免疫印迹显示 溶细胞素蛋白存在于轻颗粒组分中，尽管 缺乏可分解的活动。

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.