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 在细胞表面和附近由三种蛋白酶连接蛋白（PN-1， PN-2和PN-3），我们在早期的研究中发现。 PN是蛋白质 由多种蛋白酶合成和释放的蛋白酶抑制剂 培养细胞 它们与某些蛋白酶形成共价复合物， 细胞外环境;复合物结合回细胞， 迅速内化和降解。 用纯化的PN-1和七种调节性的 蛋白酶失活，我们将测量二阶缔合速率 常数，为我们的研究提供定量基础。 然后， 基于我们的发现，固定的成纤维细胞表面加速了 PN-1和凝血酶之间的反应，我们将确定是否反应 PN-1和其他蛋白酶之间的相互作用也同样被加速。 一些 PN-1和凝血酶之间反应的加速似乎 涉及细胞表面/细胞外基质糖胺聚糖。 我们将 用糖胺聚糖裂解酶处理成纤维细胞， 细胞，并检查这对细胞能力的影响， 加速反应。 在补充研究中，我们将研究 在没有细胞的情况下，纯化的糖胺聚糖对 PN-1与蛋白酶之间的反应。 大约50%的加速度 PN-1和凝血酶反应似乎涉及Mr= 150，000的细胞 凝血酶的表面结合位点。 我们会研究这方面的基础， 确定是否有其他六个细胞表面结合位点 调节蛋白酶可能同样参与其控制。 用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，测量二级 结合速率常数，并开始研究PN-2沿着 上述研究的PN-1行。 最后，我们将从 成纤维细胞条件的无血清培养基。

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.