Eaton D L, Baker J B
J Cell Physiol. 1983 Nov;117(2):175-82. doi: 10.1002/jcp.1041170207.
Four criteria were used to examine serum-free conditioned cell culture medium for protease nexin (PN):(1) formation of SDS-stable approximately 77 K Da complexes between a medium component and [125I]thrombin; (2) acceleration by heparin of the rate of formation of these complexes; (3) cellular binding of these complexes; and (4) inhibition by heparin of the cellular binding of complexes. Listed in order of decreasing PN production, PN was detected in media conditioned by the following cell types: human foreskin fibroblasts (0.18 micrograms/10(6) cells), rat embryo heart muscle cells (0.13 micrograms/10(6) cells), mouse myotubes (0.1 micrograms/10(6) cells), monkey kidney epithelial cells, human fibrosarcoma cells, human lung fibroblasts, simian virus 40 (SV-40)-transformed human fibroblasts, human epidermoid carcinoma cells, bovine aortic endothelial cells (only after phorbol ester treatment), and mouse myoblasts. No PN was found in medium conditioned by mouse 3T3 cells, SV40 virus-transformed 3T3 cells, human lymphoblasts, or mouse leukemia cells. Eleven of the cell types examined for secretion of PN were also examined for the presence of cytoplasmic thrombin-binding factors. Lysates from all of these cell types contained a factor that formed approximately 60-65 K Da sodium dodecyl sulfate (SDS)-stable complexes with [125I] thrombin. This MW is significantly lower than that of [125I] thrombin-PN complexes, indicating that the factor is distinct from PN. Nevertheless, PN and the cytoplasmic factor share similarities. Production of both PN (by HF cells and WI-26 cells) and the cytoplasmic factor (by HF cells and 3T3 cells) are stimulated by epidermal growth factor and phorbol myristate acetate. Also, both PN and the cytoplasmic factor complex trypsin, plasmin, urokinase, and thrombin, but not pancreatic elastase. Because a number of the cells that produce PN or the cytoplasmic serine protease-binding factor are known to produce plasminogen activators, both PN and the cytoplasmic factor could regulate plasminogen activator activity.
采用四项标准检测无血清条件细胞培养基中的蛋白酶连接蛋白（PN）：（1）培养基成分与[125I]凝血酶之间形成SDS稳定的约77 kDa复合物；（2）肝素加速这些复合物的形成速率；（3）这些复合物的细胞结合；（4）肝素抑制复合物的细胞结合。按PN产生量递减顺序列出，在以下细胞类型条件培养基中检测到PN：人包皮成纤维细胞（0.18微克/10^6细胞）、大鼠胚胎心肌细胞（0.13微克/10^6细胞）、小鼠肌管（0.1微克/10^6细胞）、猴肾上皮细胞、人纤维肉瘤细胞、人肺成纤维细胞、猿猴病毒40（SV - 40）转化的人成纤维细胞、人表皮癌细胞、牛主动脉内皮细胞（仅在佛波酯处理后）和小鼠成肌细胞。在小鼠3T3细胞、SV40病毒转化的3T3细胞、人淋巴母细胞或小鼠白血病细胞条件培养基中未发现PN。对11种检测PN分泌的细胞类型也检测了细胞质凝血酶结合因子的存在。所有这些细胞类型的裂解物都含有一种因子，该因子与[125I]凝血酶形成约60 - 65 kDa十二烷基硫酸钠（SDS）稳定的复合物。该分子量明显低于[125I]凝血酶 - PN复合物，表明该因子与PN不同。然而，PN和细胞质因子有相似之处。表皮生长因子和佛波醇肉豆蔻酸酯乙酸盐可刺激PN（由HF细胞和WI - 26细胞产生）和细胞质因子（由HF细胞和3T3细胞产生）的产生。此外，PN和细胞质因子都能与胰蛋白酶、纤溶酶、尿激酶和凝血酶结合，但不与胰弹性蛋白酶结合。由于已知许多产生PN或细胞质丝氨酸蛋白酶结合因子的细胞会产生纤溶酶原激活剂，因此PN和细胞质因子都可能调节纤溶酶原激活剂的活性。
