Petersen L C, Suenson E
Biochim Biophys Acta. 1986 Sep 4;883(2):313-25. doi: 10.1016/0304-4165(86)90324-7.
Trinitrobenzyl alkylation of poly(D-lysine) provides a novel powerful stimulator of tissue-type plasminogen activator. Its stimulatory effect on plasminogen activation is far greater than that of the original poly(D-lysine), and even surpasses that of fibrin. Its effect on plasmin-catalysed modification of both tissue-type plasminogen activator (t-PA) and native (Glu-1-) plasminogen are also investigated. Cleavage of one-chain t-PA to its two-chain form is monitored by measuring the increase in amidolytic activity which accompanies this transformation. Presupposing apparent first-order reaction kinetics, a theory is developed by which the rate constant, kcat/Km = 1.0 X 10(6) M-1 X s-1 of plasmin cleavage of one-chain t-PA can be calculated. Plasmin-catalysed transformation of 125I-labelled Glu-1- to Lys-77-plasminogen is quantified following separation by polyacrylamide gel electrophoresis at pH 3.2. A rate constant, kcat/Km = 4.4 X 10(3) M-1 X s-1 is obtained for the reaction between plasmin and Glu-1-plasminogen in the presence of 1 mM trans-4-(aminomethyl)cyclohexane-1-carboxylic acid. Both of the above plasmin-catalysed reactions are strongly enhanced by trinitrobenzoylated poly(D-lysine). The mechanism of action of this stimulator is elucidated by studying its binding to both activator and plasmin(ogen), and by direct comparison of the results with measurements of plasminogen activation kinetics in the presence of the stimulator. Binding studies are performed exploiting the observation that an insoluble yellow complex is formed between plasminogen and modified poly(D-lysine). Protein-polymer interactions are also studied with solubilised components in an aqueous two-phase partition system containing dextran and poly(ethylene glycol). The rate enhancement of plasminogen activation is found to be closely correlated to the association of plasminogen to the stimulator. It is proposed that the stimulator effects of this simple polymer on the enzymatic activities of both plasminogen activator and plasmin are brought about by association of the proteinase and its substrate to a common matrix. Similarities between the action of the artificial and the natural stimulator (fibrin) are stressed. These properties of trinitrobenzoylated poly(D-lysine) makes it useful as a model for the study of the regulatory mechanism of the fibrinolytic process at the molecular level.
聚（D - 赖氨酸）的三硝基苄基烷基化提供了一种新型的强力组织型纤溶酶原激活剂刺激物。它对纤溶酶原激活的刺激作用远大于原始的聚（D - 赖氨酸），甚至超过纤维蛋白。还研究了其对纤溶酶催化的组织型纤溶酶原激活剂（t - PA）和天然（Glu - 1 -）纤溶酶原修饰的影响。通过测量伴随这种转变的酰胺水解活性增加来监测单链t - PA裂解为其二链形式。假定为表观一级反应动力学，建立了一种理论，据此可以计算纤溶酶裂解单链t - PA的速率常数kcat/Km = 1.0×10⁶ M⁻¹×s⁻¹。在pH 3.2条件下通过聚丙烯酰胺凝胶电泳分离后，对125I标记的Glu - 1 - 向Lys - 77 - 纤溶酶原的纤溶酶催化转化进行定量。在1 mM反式 - 4 -（氨甲基）环己烷 - 1 - 羧酸存在下，纤溶酶与Glu - 1 - 纤溶酶原反应的速率常数kcat/Km = 4.4×10³ M⁻¹×s⁻¹。上述两种纤溶酶催化反应均被三硝基苯甲酰化的聚（D - 赖氨酸）强烈增强。通过研究其与激活剂和纤溶酶（原）的结合，并将结果与刺激剂存在下纤溶酶原激活动力学的测量结果直接比较，阐明了这种刺激物的作用机制。利用纤溶酶原与修饰的聚（D - 赖氨酸）之间形成不溶性黄色复合物这一观察结果进行结合研究。还在含有葡聚糖和聚（乙二醇）的水两相分配系统中用可溶成分研究蛋白质 - 聚合物相互作用。发现纤溶酶原激活的速率增强与纤溶酶原与刺激剂的缔合密切相关。提出这种简单聚合物对纤溶酶原激活剂和纤溶酶的酶活性的刺激作用是通过蛋白酶及其底物与共同基质的缔合实现的。强调了人工刺激物（三硝基苯甲酰化聚（D - 赖氨酸））与天然刺激物（纤维蛋白）作用之间的相似性。三硝基苯甲酰化聚（D - 赖氨酸）的这些特性使其成为在分子水平研究纤溶过程调节机制的有用模型。
