REGULATION OF PROTEASE NEXIN 1 ACTIVITY AND SECRETION

我们的目标是阐明调节性丝氨酸蛋白酶 在细胞表面和细胞表面附近受三种蛋白酶连接素(Pn-1， Pn-2和Pn-3)。PNS是一种蛋白质 由多种细胞合成和释放的蛋白水解酶抑制剂 培养的细胞。它们与某些蛋白水解酶形成共价复合体。 细胞外环境；复合体与细胞结合，并 迅速内化和降解。使用纯化的PN-1和七种调节剂 它失活的蛋白水解酶，我们将测量二级缔合率 常量，为我们的研究提供一个量化的基础。然后, 基于我们的发现，固定成纤维细胞的表面加速了 PN-1和凝血酶之间的反应，我们将确定反应是否 Pn-1和其他蛋白酶之间的作用也同样加速。其中一些 PN-1与凝血酶反应的加速似乎 涉及细胞表面/细胞外基质糖胺多聚糖。我们会 通过用糖胺聚糖裂解酶处理成纤维细胞，修复 细胞，并检测这对细胞能力的影响 加快反应速度。在免费研究中，我们将检查 纯化的糖胺多聚糖在无细胞条件下对细胞周期的影响 Pn-1与蛋白水解酶的反应。大约50%的加速度 PN-1和凝血酶反应似乎涉及MR=150,000细胞 凝血酶的表面结合部位。我们会研究这方面的基础，并 确定是否存在其他六个细胞表面的结合部位 可能同样参与其控制的调节蛋白水解酶。 对于我们最近纯化到同质的Pn-2，我们将筛选 丝氨酸蛋白酶，它有效地灭活，测量二阶 将速率常数与它们联系起来，开始对Pn-2的研究 Pn-1的上述研究路线。最后，我们将从 以成纤维细胞为条件的无血清培养液。

Our goal is to elucidate mechanisms by which regulatory serine proteases are controlled at and near the cell surface by three protease nexins (PN-1, PN-2 and PN-3) which we identified in earlier studies. The PNs are protein protease inhibitors that are synthesized and released by a variety of cultured cells. They form covalent complexes with certain proteases in the extracellular environment; the complexes bind back to the cells and are rapidly internalized and degraded. With purified PN-1 and seven regulatory proteases it inactivates, we will measure second order association rate constants to provide a quantitative foundation for our studies. Then, based on our finding that the surface of fixed fibroblasts accelerates the reaction between PN-1 and thrombin, we will determine if the reactions between PN-1 and the other proteases are similarly accelerated. Some of the acceleration of the reaction between PN-1 and thrombin appears to involve cell surface/extracellular matrix glycosaminoglycans. We will check this by treating fibroblasts with glycosaminoglycan lyases, fixing the cells, and examining the effect of this on the ability of the cells to accelerate the reactions. In complimentary studies we will examine the effects of purified glycosaminoglycans, in the absence of cells, on the reactions between PN-1 and the proteases. About 50% of the acceleration of the PN-1 and thrombin reactions appears to involve the Mr=150,000 cell surface binding sites for thrombin. We will study the basis of this and determine if there are cell surface binding sites for the six other regulatory proteases that might similarly participate in their control. With PN-2 that we recently purified to homogeneity, we will screen for serine proteases it effectively inactivates, measure second order association rate constants with them, and begin studies on PN-2 along the lines of the above studies for PN-1. Finally, we will purify PN-3 from serum-free culture medium conditioned by fibroblasts.