Stump D C, Thienpont M, Collen D
J Biol Chem. 1986 Sep 25;261(27):12759-66.
Urokinase-related proteins in human urine occur mainly as a 1:1 complex of urokinase with an inhibitor (Stump, D. C., Thienpont, M., and Collen, D. (1986) J. Biol. Chem. 261, 1267-1273). BALB/c mice were immunized with this urokinase-urokinase inhibitor complex and spleen cells fused with mouse myeloma cells, resulting in hybridomas producing monoclonal antibodies. Three antibodies reacting with the complex but not with urokinase were utilized to develop a sensitive (0.5 ng/ml) enzyme-linked immunosorbent assay for the urokinase inhibitor, which was used for monitoring its purification by chromatography on zinc chelate-Sepharose, concanavalin A-Sepharose, SP-Sephadex C-50, and Sephadex G-100. A homogenous glycoprotein of apparent Mr 50,000 was obtained with a yield of 40 micrograms/liter urine and a purification factor of 320. One mg of the purified protein inhibited 35,000 IU of urokinase within 30 min at 37 degrees C. This protein was immunologically related to both the purified urokinase-urokinase inhibitor complex and to the inhibitor portion dissociated from it by nucleophilic dissociation. It was immunologically distinct from all known protease inhibitors, including the endothelial cell-derived fast-acting inhibitor of tissue-type plasminogen activator, the placental inhibitor of urokinase and protease nexin. In electrophoresis the protein migrated with beta-mobility. Inhibition of urokinase occurred with a second order rate constant (k) of 8 X 10(3) M-1 s-1 in the absence and of 9 X 10(4) M-1 s-1 in the presence of 50 IU of heparin/ml. The urokinase inhibitor was inactive towards single-chain urokinase-type plasminogen activator and plasmin, but it inhibited two-chain tissue-type plasminogen activator with a k below 10(3) M-1 s-1 and thrombin with a k of 4 X 10(4) M-1 s-1 in the absence and 2 X 10(5) M-1 s-1 in the presence of heparin. The concentration of this urokinase inhibitor in plasma from normal subjects determined by immunoassay was 2 +/- 0.7 micrograms/ml (mean +/- S.D., n = 25). The protein purified from plasma by immunoabsorption had the same Mr, amino acid composition, and immunoreactivity as the urinary protein. Furthermore, when urokinase was added to plasma, time-dependent urokinase-urokinase inhibitor complex formation was observed at a rate similar to that observed for the inhibition of urokinase by the purified inhibitor from urine. This urokinase inhibitor, purified from human urine, most probably represents a new plasma protease inhibitor.
人尿中的尿激酶相关蛋白主要以尿激酶与一种抑制剂的 1:1 复合物形式存在（斯顿普，D.C.，廷蓬特，M.，和科伦，D.（1986 年）《生物化学杂志》261 卷，1267 - 1273 页）。用这种尿激酶 - 尿激酶抑制剂复合物免疫 BALB/c 小鼠，并将脾细胞与小鼠骨髓瘤细胞融合，产生了分泌单克隆抗体的杂交瘤。利用三种与该复合物反应但不与尿激酶反应的抗体，开发了一种灵敏的（0.5 纳克/毫升）尿激酶抑制剂酶联免疫吸附测定法，用于监测其通过锌螯合 - 琼脂糖凝胶、伴刀豆球蛋白 A - 琼脂糖凝胶、SP - 葡聚糖凝胶 C - 50 和葡聚糖凝胶 G - 100 柱层析进行的纯化过程。得到了一种表观分子量为 50,000 的均一糖蛋白，产率为 40 微克/升尿液，纯化因子为 320。1 毫克纯化蛋白在 37℃下 30 分钟内可抑制 35,000 国际单位的尿激酶。该蛋白在免疫上与纯化的尿激酶 - 尿激酶抑制剂复合物以及通过亲核解离从其中解离出的抑制剂部分均相关。它在免疫上与所有已知的蛋白酶抑制剂不同，包括内皮细胞衍生的组织型纤溶酶原激活剂的快速作用抑制剂、尿激酶的胎盘抑制剂和蛋白酶连接蛋白。在电泳中，该蛋白以β迁移率移动。在不存在肝素的情况下，尿激酶的抑制反应二级速率常数（k）为 8×10³ M⁻¹ s⁻¹，在存在 50 国际单位/毫升肝素的情况下为 9×10⁴ M⁻¹ s⁻¹。尿激酶抑制剂对单链尿激酶型纤溶酶原激活剂和纤溶酶无活性，但在不存在肝素时，它以低于 10³ M⁻¹ s⁻¹ 的 k 抑制双链组织型纤溶酶原激活剂，在存在肝素时以 4×10⁴ M⁻¹ s⁻¹ 的 k 抑制凝血酶，在存在肝素时以 2×10⁵ M⁻¹ s⁻¹ 的 k 抑制凝血酶。通过免疫测定法测定，正常受试者血浆中这种尿激酶抑制剂的浓度为 2±0.7 微克/毫升（平均值±标准差，n = 25）。通过免疫吸附从血浆中纯化的蛋白与尿蛋白具有相同的分子量、氨基酸组成和免疫反应性。此外，当向血浆中加入尿激酶时，观察到尿激酶 - 尿激酶抑制剂复合物随时间的形成速率与用从尿液中纯化的抑制剂抑制尿激酶时观察到的速率相似。这种从人尿中纯化的尿激酶抑制剂很可能代表一种新的血浆蛋白酶抑制剂。
