PLASMINOGEN/ANGIOSTATIN CONVERTING ENZYME

血管抑素是一种有效的抑制某些固体转移生长的药物 肿瘤。它是由纤溶酶原蛋白分解而来的，由 纤溶酶原Kringle1-4结构域。尽管转化为蛋白质的酶 纤溶酶原到血管抑素在一些原发肿瘤中表达， 这种酶的特性，以及哪些肿瘤表达这种酶 未知。我们已经证明了PC-3和DU-145人前列腺 癌细胞表达一种或多种酶，这种酶是 血管抑素的生成，暂定为纤溶酶原- 血管抑素转换酶(PACE)。更多数据表明， 活性是一种丝氨酸蛋白酶。在初步试验中，几个 已经确定和表征了净化工具，这将使 在这个项目中对PACE进行提纯。其中包括活性红 120-琼脂糖凝胶(染料-配基层析)，Hi-Q-琼脂糖凝胶(阴离子交换 柱层析)和大豆胰酶抑制剂-琼脂糖(亲和力 层析)。PC-3细胞体外产生的血管抑素是 基于抑制内皮细胞增殖的生物活性和 迁移。血管抑素抗原已在人的血浆中被证实 荷瘤裸鼠PC-3。这项提议是利用 确定了净化工具，以完成净化的节奏 一种或多种酶。如果这种酶是新的，就会克隆出它的cdna。 将产生针对PACE的单抗，这将有助于 PACE免疫分析和免疫组织化学染色方法的建立。 PACE与纤溶酶原相互作用的体外动力学 以及PACE潜在的抗血管生成和抗转移作用 体内给药将被分析。

Angiostatin is a potent inhibitor of metastatic growth of some solid tumors. It is derived by proteolysis of plasminogen, and consists of the kringle 1-4 domain of plasminogen. Although the enzyme that converts plasminogen to angiostatin is expressed by some primary tumors, the identity of this enzyme, and which tumors express the enzyme remain unknown. We have demonstrated that the PC-3 and DU-145 human prostate cancer lines express the enzyme (or enzymes) necessary and sufficient for angiostatin generation, and this is tentatively designated as plasminogen- angiostatin converting enzyme (PACE). Additional data indicate that PACE activity is a serine protease. In preliminary experiments several purification tools have been identified and characterized which will allow for purification of PACE during this project. These include Reactive Red 120-Agarose (Dye-Ligand chromatography), Hi-Q-Sepharose (anion exchange chromatography) and soybean trypsin inhibitor-sepharose (affinity chromatography). The angiostatin generated in vitro by PC-3 cells is bioactive based on inhibition of endothelial cell proliferation and migration. Angiostatin antigen has been demonstrated in the plasma of athymic mice bearing PC-3 tumors. This proposal is to utilize the purification tools identified to complete the purification of the PACE enzyme or enzymes. If the enzyme is novel, the cDNA will be cloned. Monoclonal antibodies to PACE will be generated which will facilitate development of immunoassays and immunohistochemical staining for PACE. The kinetics of the interaction between PACE and plasminogen in vitro, as well as potential antiangiogenic and anti-metastatic effects of PACE administration in vivo will be analyzed.