Bányai L, Patthy L
J Biol Chem. 1984 May 25;259(10):6466-71.
Incubation of human Glu-plasminogen with 1,5-difluoro-2,4-dinitrobenzene leads to the specific intra-molecular cross-linking of the kringle 1+2+3 region and the light (B) chain region of plasminogen. This cross-link was found to prevent the conformational change which is induced in Glu-plasminogen by lysine analogues or by proteolytic removal of the NH2-terminal peptide. Our results suggest that the cross-link freezes the closed conformation of Glu-plasminogen, and it seems likely that the transition to the loose conformer requires separation of the kringle 1+2+3 region from the light (B) chain portion. The change in the relative position of these regions during the conformational change in plasminogen is also indicated by our observation that the rate of formation of the intramolecular cross-link is significantly decreased when transition to the loose conformer is induced either by saturation of the lysine-binding sites or by conversion to Lys-plasminogen. Cross-linked Glu-plasminogen is slowly activated by urokinase and melanoma tissue plasminogen activator, but in contrast with uncross-linked Glu-plasminogen conversion to Lys-plasminogen or saturation of lysine-binding sites with ligand does not increase the rate of activation because the cross-link prevents transition to the loose conformer which is susceptible to activation. The fibrin affinity of cross-linked Glu-plasminogen is practically identical with that of Glu-plasminogen. As in the case of uncross-linked Glu-plasminogen, removal of the NH2-terminal peptide causes a marked increase in fibrin affinity although the resulting cross-linked Lys-plasminogen is fixed in the closed conformation. This result suggests that the NH2-terminal peptide inhibits binding of plasminogen to fibrin by direct interaction with the fibrin-binding site, and the conformational change that normally accompanies its removal is not a prerequisite of strong binding.
人谷氨酸纤溶酶原与1,5 - 二氟 - 2,4 - 二硝基苯温育会导致纤溶酶原kringle 1+2+3区域与轻链（B）区域发生特异性分子内交联。发现这种交联可防止赖氨酸类似物或通过蛋白水解去除NH2 - 末端肽在谷氨酸纤溶酶原中诱导的构象变化。我们的结果表明，这种交联使谷氨酸纤溶酶原的封闭构象冻结，并且向松散构象异构体的转变似乎需要kringle 1+2+3区域与轻链（B）部分分离。我们还观察到，当通过赖氨酸结合位点饱和或转化为赖氨酸纤溶酶原诱导向松散构象异构体转变时，分子内交联的形成速率显著降低，这也表明了纤溶酶原构象变化过程中这些区域相对位置的改变。交联的谷氨酸纤溶酶原可被尿激酶和黑色素瘤组织纤溶酶原激活剂缓慢激活，但与未交联的谷氨酸纤溶酶原不同，转化为赖氨酸纤溶酶原或用配体使赖氨酸结合位点饱和并不会增加激活速率，因为交联阻止了向易被激活的松散构象异构体的转变。交联的谷氨酸纤溶酶原对纤维蛋白的亲和力与谷氨酸纤溶酶原几乎相同。与未交联的谷氨酸纤溶酶原情况一样，去除NH2 - 末端肽会导致纤维蛋白亲和力显著增加，尽管生成的交联赖氨酸纤溶酶原固定在封闭构象中。这一结果表明，NH2 - 末端肽通过与纤维蛋白结合位点直接相互作用抑制纤溶酶原与纤维蛋白的结合，并且其去除通常伴随的构象变化不是强结合的先决条件。
