Petersen L C, Brender J, Suenson E
Biochem J. 1985 Jan 1;225(1):149-58. doi: 10.1042/bj2250149.
The kinetics of plasminogen activation catalysed by urokinase and tissue-type plasminogen activator were investigated. Kinetic measurements are performed by means of a specific chromogenic peptide substrate for plasmin, D-valyl-L-leucyl-L-lysine 4-nitroanilide. Two methods are proposed for the analysis of the resulting progress curve of nitroaniline formation in terms of zymogen-activation kinetics: a graphical transformation of the parabolic curve and transformation of the curve for nitroaniline production into a linear progress curve by the addition of a specific inhibitor of plasmin, bovine pancreatic trypsin inhibitor. The two methods give similar results, suggesting that the reaction between activator and plasminogen is a simple second-order reaction at least at plasminogen concentrations up to about 10 microM. The kinetics of both Glu1-plasminogen (residues 1-790) and Lys77-plasminogen (residues 77-790) activation were investigated. The results confirm previous observations showing that trans-4-(aminomethyl)cyclohexane-1-carboxylic acid at relatively low concentrations enhances the activation rate of Glu1-plasminogen but not that of Lys77-plasminogen. At higher concentrations both Glu1- and Lys77-plasminogen activation are inhibited. The concentration interval for the inhibition of urokinase-catalysed reactions is shown to be very different from that of the tissue-plasminogen activator system. Evidence is presented indicating that binding to the active site of urokinase (KD = 2.0 mM) is responsible for the inhibition of the urokinase system, binding to the active site of tissue-plasminogen activator is approx. 100-fold weaker, and inhibition of the tissue-plasminogen activator system, when monitored by plasmin activity, is mainly due to plasmin inhibition. Poly-D-lysine (Mr 160 000) causes a marked enhancement of plasminogen activation catalysed by tissue-plasminogen activator but not by urokinase. Bell-shaped curves of enhancement as a function of the logarithm of poly-D-lysine concentration are obtained for both Glu1- and Lys77-plasminogen activation, with a maximal effect at about 10 mg/litre. The enhancement of Glu1-plasminogen activation exerted by trans-4-(aminomethyl)cyclohexane-1-carboxylic acid is additive to that of poly-D-lysine, whereas poly-D-lysine-induced enhancement of Lys77-plasminogen activation is abolished by trans-4-(aminomethyl)cyclohexane-1-carboxylic acid. Analogies are drawn up between the effector functions of poly-D-lysine and fibrin on the catalytic activity of tissue-plasminogen activator.
研究了尿激酶和组织型纤溶酶原激活剂催化的纤溶酶原激活动力学。动力学测量通过纤溶酶的特异性显色肽底物D-缬氨酰-L-亮氨酰-L-赖氨酸4-硝基苯胺进行。提出了两种根据酶原激活动力学分析所得硝基苯胺生成进程曲线的方法：抛物线曲线的图形转换以及通过添加纤溶酶的特异性抑制剂牛胰蛋白酶抑制剂将硝基苯胺生成曲线转换为线性进程曲线。这两种方法给出了相似的结果，表明激活剂与纤溶酶原之间的反应至少在纤溶酶原浓度高达约10微摩尔时是简单的二级反应。研究了Glu1-纤溶酶原（第1-790位残基）和Lys77-纤溶酶原（第77-790位残基）的激活动力学。结果证实了先前的观察结果，即相对低浓度的反式-4-（氨甲基）环己烷-1-羧酸可提高Glu1-纤溶酶原的激活速率，但不能提高Lys77-纤溶酶原的激活速率。在较高浓度下，Glu1-和Lys77-纤溶酶原的激活均受到抑制。结果表明，抑制尿激酶催化反应的浓度区间与组织纤溶酶原激活剂系统的浓度区间非常不同。有证据表明，与尿激酶活性位点的结合（KD = 2.0毫摩尔）是尿激酶系统受到抑制的原因，与组织型纤溶酶原激活剂活性位点的结合约弱100倍，并且当通过纤溶酶活性监测时，组织型纤溶酶原激活剂系统的抑制主要是由于纤溶酶的抑制。聚-D-赖氨酸（Mr 160 000）可显著增强组织型纤溶酶原激活剂催化的纤溶酶原激活，但不能增强尿激酶催化的纤溶酶原激活。对于Glu1-和Lys77-纤溶酶原的激活，均获得了作为聚-D-赖氨酸浓度对数函数的增强的钟形曲线，在约10毫克/升时具有最大效应。反式-4-（氨甲基）环己烷-1-羧酸对Glu1-纤溶酶原激活的增强作用与聚-D-赖氨酸的增强作用相加，而反式-4-（氨甲基）环己烷-1-羧酸可消除聚-D-赖氨酸诱导的Lys77-纤溶酶原激活的增强作用。对聚-D-赖氨酸和纤维蛋白对组织型纤溶酶原激活剂催化活性的效应功能进行了类比。
