Isolation and functional characterization of the heavy and light chains of human tissue-type plasminogen activator.
Isolation and functional characterization of the heavy and light chains of human tissue-type plasminogen activator.
复制标题
人组织型纤溶酶原激活剂重链和轻链的分离和功能表征。
DOI：
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发表时间：
1986
影响因子：
4.8
通讯作者：
E. Groeneveld
中科院分区：
文献类型：
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作者：
D. Rijken;E. Groeneveld
双链组织型纤溶酶原激活剂（t-PA）由二硫键连接的重链（Mr = 38，000）和轻链（Mr = 31，000）组成，用2-巯基乙醇还原，然后在低蛋白浓度下空气再氧化并羧酰胺甲基化。通过锌螯合物-琼脂糖分离两条链，发现其选择性结合轻链。轻链对三肽底物H-D-异亮氨酰-L-脯氨酰-L-精氨酸对硝基苯胺（S-2288）具有完全活性，对纤溶酶原具有部分活性。纤溶酶原激活剂活性的轻链，在相反的双链t-PA，不刺激纤维蛋白或纤维蛋白原片段。放射性标记链的纤维蛋白-琼脂糖色谱显示，只有重链与纤维蛋白结合。这些结果表明，t-PA中含有活性位点的轻链需要重链来刺激纤维蛋白的纤溶酶原激活剂活性。
Two-chain tissue-type plasminogen activator (t-PA), which consists of a heavy chain (Mr congruent to 38,000) and a light chain (Mr congruent to 31,000) connected by a disulfide bridge, was reduced with 2-mercaptoethanol and then air-reoxidized at a low protein concentration and carboxamidomethylated. The two chains were separated by means of zinc chelate-agarose, which was found to bind the light chain selectively. The light chain was fully active on the tripeptide substrate H-D-isoleucyl-L-prolyl-L-arginine p-nitroanilide (S-2288) and partially active on plasminogen. The plasminogen activator activity of the light chain was, in contrast to that of two-chain t-PA, not stimulated by fibrin or fibrinogen fragments. Fibrin-agarose chromatography of radiolabeled chains showed that only the heavy chain bound to fibrin. These results indicate that the active site-containing light chain in t-PA needs the heavy chain for fibrin stimulation of its plasminogen activator activity.