Strandberg L, Karolin J, Johansson L B, Fa M, Aleshkov S, Ny T
Department of Medical Biochemistry, University of Umeå, Sweden.
Thromb Res. 1994 Nov 1;76(3):253-67. doi: 10.1016/0049-3848(94)90197-x.
To investigate structural-functional aspects of plasminogen activator inhibitor 1 (PAI-1) we have taken advantage of the lack of cysteines in the PAI-1 molecule and replaced Ser344 (P3) and Asn329 (P18) with cysteine residues, thereby creating unique attachment sites for extrinsic fluorescent probes. After expression in E. coli and purification to homogeneity, both of the mutant proteins were found to have similar biochemical characteristics as wild type PAI-1 (wtPAI-1). Following labelling with 4-chloro-7-nitrobenzofurazan (NBD) and 2-(4'-iodoacetamido-anilino)naphtalene-6-sulfonic acid (IAANS) the mutant inhibitors showed similar inhibitory activities and heat stability as wtPAI-1. The purified complex between uPA and NBD-labelled P3cys mutant was found to be extremely stable, suggesting that no slow cleavage or reversible reaction occurs in complexes that have been properly formed. The rate of labelling of both mutants was decreased when the mutants were in the latent form indicating that these cysteine residues may be less accessible in the latent configuration. The PAI-1 mutants labelled with both NBD and IAANS could convert from the active to the latent form, but P3cys labelled with the larger IAANS chromophore showed a two fold decrease in the rate of conversion to latency, suggesting that a large chromophore in the P3 position may interfere with the active to latent conversion. The fluorescence spectra of the two NBD labelled mutants were similar, but the intensity was three times higher for the P3cys mutant than for P18cys. No significant spectral changes could be seen when the P3cys mutant was transferred to latency. In contrast, the P18cys mutant showed a major change in the excitation spectra characteristic of migration of the NBD chromophore from a thiol to an amine. Complex formation with uPA had no effect on the fluorescence spectrum of P18cys-NBD while the spectrum of P3cys-NBD revealed changes consistent with a restriction of the mobility of NBD probe in the uPA-PAI-1 complex.
为了研究纤溶酶原激活物抑制剂1（PAI-1）的结构功能方面，我们利用了PAI-1分子中缺乏半胱氨酸这一特性，将丝氨酸344（P3）和天冬酰胺329（P18）替换为半胱氨酸残基，从而为外在荧光探针创造了独特的连接位点。在大肠杆菌中表达并纯化至同质后，发现两种突变蛋白具有与野生型PAI-1（wtPAI-1）相似的生化特性。用4-氯-7-硝基苯并呋咱（NBD）和2-（4'-碘乙酰氨基苯胺基）萘-6-磺酸（IAANS）标记后，突变抑制剂显示出与wtPAI-1相似的抑制活性和热稳定性。发现尿激酶型纤溶酶原激活物（uPA）与NBD标记的P3cys突变体之间纯化的复合物极其稳定，这表明在正确形成的复合物中不会发生缓慢裂解或可逆反应。当突变体处于潜伏形式时，两种突变体的标记速率均降低，这表明这些半胱氨酸残基在潜伏构象中可能较难接近。用NBD和IAANS标记的PAI-1突变体可以从活性形式转变为潜伏形式，但用较大的IAANS发色团标记的P3cys在转变为潜伏状态的速率上降低了两倍，这表明P3位置的大发色团可能会干扰活性向潜伏的转变。两种NBD标记的突变体的荧光光谱相似，但P3cys突变体的强度比P18cys高3倍。当P3cys突变体转变为潜伏状态时，未观察到明显的光谱变化。相反，P18cys突变体在激发光谱上有重大变化，其特征是NBD发色团从硫醇迁移到胺。与uPA形成复合物对P18cys-NBD的荧光光谱没有影响，而P3cys-NBD的光谱显示出与uPA-PAI-1复合物中NBD探针迁移率受限一致的变化。
