Purification of human fibroblast urokinase proenzyme and analysis of its regulation by proteases and protease nexin.
Purification of human fibroblast urokinase proenzyme and analysis of its regulation by proteases and protease nexin.
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发表时间：
1984-05
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通讯作者：
D. Eaton;R. W. Scott;J. Baker
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作者：
D. Eaton;R. W. Scott;J. Baker
最近我们提供的证据表明，正常人包皮成纤维细胞（HF 细胞）通过将其作为酶原分泌并通过分泌蛋白酶连接蛋白（尿激酶和某些其他丝氨酸蛋白酶的抑制剂）来限制分泌型尿激酶的活性（Scott, R.W.、Eaton, D. L. Duran, N. 和 Baker, J.B. (1983) J. Biol. Chem. 258， 4397-4403）。使用免疫亲和层析，我们现已纯化了 HF 细胞尿激酶原。它是一条 52 kDa 的多肽链，对纤溶酶原和低分子量底物均无活性。蛋白水解激活后，该物质（比活性为 3 X 10(4) 溶栓剂委员会单位/毫克）由两条二硫键桥接的 33-kDa 和 19-kDa 链组成，因此与尿液中发现的尿激酶的主要形式相似。 2 X 10(-10) M 的纤溶酶在 37 摄氏度下 30 分钟内可使酶原 (1 X 10(-9) M) 激活 50%。凝血酶和胰蛋白酶的效果是纤溶酶的二十分之一。活化的 HF 细胞 125I-尿激酶与纯化的蛋白酶连接蛋白或存在于 HF 细胞条件培养基中的蛋白酶连接蛋白形成十二烷基硫酸钠稳定复合物。纯化的蛋白酶连接蛋白抑制纯化的 HF 细胞尿激酶对纤溶酶原和低分子量底物的作用。蛋白酶连接蛋白和 HF 细胞尿激酶之间反应的关联速率常数约为 1.7 X 10(5) M-1 S-1。相反，蛋白酶连接蛋白与组织型纤溶酶原激活剂的单链和双链形式之间的反应的关联速率常数分别约为2 X 10(3) 和约3 X 10(4) M-1 S-1。抗蛋白酶连接蛋白抗体可增强与纤溶酶原一起孵育的心力衰竭细胞条件培养基的纤溶活性，这一发现支持了蛋白酶连接蛋白作为心力衰竭细胞尿激酶调节剂的重要性。
Recently we presented evidence that normal human foreskin fibroblasts (HF cells) limit the activity of secreted urokinase by secreting it as a proenzyme and by secreting protease nexin , an inhibitor of urokinase and certain other serine proteases (Scott, R.W., Eaton, D. L. Duran , N., and Baker, J.B. (1983) J. Biol. Chem. 258, 4397-4403). Using immunoaffinity chromatography we have now purified the HF cell urokinase proenzyme. It is a single 52-kDa polypeptide chain that is inactive toward both plasminogen and low molecular weight substrates. After proteolytic activation, this material (specific activity of 3 X 10(4) Committee on Thrombolytic Agents units/mg) is composed of two disulfide-bridged 33- and 19-kDa chains, and is thus similar to the predominant form of urokinase found in urine. Plasmin at 2 X 10(-10) M causes 50% activation of the proenzyme (1 X 10(-9) M) in 30 min at 37 degrees C. Thrombin and trypsin are one-twentieth as effective as plasmin. Activated HF cell 125I-urokinase forms sodium dodecyl sulfate stable complexes with purified protease nexin or protease nexin present in medium conditioned by HF cells. Purified protease nexin inhibits purified HF cell urokinase action on both plasminogen and low molecular weight substrates. The association rate constant for the reaction between protease nexin and HF cell urokinase is approximately 1.7 X 10(5) M-1 S-1. In contrast, the association rate constants for reactions between protease nexin and the one- and two-chain forms of tissue-type plasminogen activator are approximately 2 X 10(3) and approximately 3 X 10(4) M-1 S-1, respectively. The importance of protease nexin as a regulator of HF cell urokinase is supported by the finding that anti-protease nexin antibody potentiates the fibrinolytic activity of HF cell-conditioned medium incubated with plasminogen.