Gils A, Declerck P J
Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit-Leuven, Belgium.
Thromb Haemost. 1998 Aug;80(2):286-91.
Plasminogen activator inhibitor-1 (PAI-1) is a unique member of the serpin superfamily because of its conformational and functional flexibility. In the present study, we have evaluated the influence of the non-ionic detergent Triton X-100 (TX-100) on the functional stability and conformational transitions of PAI-1. At 37 degrees C, TX-100 induced a concentration-dependent decrease of the functional half-life of PAI-1 resulting in half-lives of 177 +/- 54 min (mean +/- SD, n = 3), 19 +/- 2 min, 1.7 +/- 0.3 min and 0.53 +/- 0.03 min in the presence of 0.005, 0.010, 0.020 and 0.2% TX-100, respectively, compared to a half-life of 270 +/- 146 min in the absence of TX-100. Conformational analysis at various time points and at different temperatures (0 degrees C, 25 degrees C, 37 degrees C) revealed that this inactivation proceeds through the formation of a substrate-like intermediate followed by the formation of the latent form. Kinetic evaluation demonstrated that this conversion fits to two consecutive first-order transitions, i.e. active k1--> substrate k2--> latent. The k1 value was strongly dependent on the concentration of TX-100 (e.g. 0.002 +/- 0.0006 s(-1) and 0.029 +/- 0.003 s(-1) for 0.01% and 0.2% TX-100 at 37 degrees C) whereas the conversion of substrate to latent (k2) was virtually independent of the TX-100 concentration (i.e. 0.012 +/- 0.002 s(-1) and 0.011 +/- 0.001 s(-1) for 0.01 and 0.2% TX-100 at 37 degrees C). Experiments with a variety of other non-ionic amphiphilic compounds revealed that the amphiphilic character of the compound is, at least in part, responsible for the observed effects and strongly indicate that the currently reported mechanism of inactivation is of general importance for the conformational transitions in PAI-1. In conclusion, TX- 100 changes the initial conformation of PAI-1 resulting in altered functional properties. This observation allows us to develop a new model for the mechanism involved in the conformational flexibility of PAI-1 and may provide new insights for the development of strategies for interference with PAI-1 activity.
纤溶酶原激活物抑制剂-1（PAI-1）是丝氨酸蛋白酶抑制剂超家族中的一个独特成员，因其构象和功能具有灵活性。在本研究中，我们评估了非离子去污剂Triton X-100（TX-100）对PAI-1功能稳定性和构象转变的影响。在37℃时，TX-100诱导PAI-1的功能半衰期呈浓度依赖性降低，在存在0.005%、0.010%、0.020%和0.2%TX-100的情况下，半衰期分别为177±54分钟（平均值±标准差，n = 3）、19±2分钟、1.7±0.3分钟和0.53±0.03分钟，而在不存在TX-100时半衰期为270±146分钟。在不同时间点和不同温度（0℃、25℃、37℃）进行的构象分析表明，这种失活过程是通过形成底物样中间体，随后形成潜伏形式。动力学评估表明，这种转变符合两个连续的一级转变，即活性k1→底物k2→潜伏形式。k1值强烈依赖于TX-100的浓度（例如，在37℃时，0.01%和0.2%TX-100的k1值分别为0.002±0.0006 s-1和0.029±0.003 s-1），而底物向潜伏形式的转变（k2）实际上与TX-100浓度无关（即在37℃时，0.01%和0.2%TX-100的k2值分别为0.012±0.002 s-1和0.011±0.001 s-1）。用多种其他非离子两亲性化合物进行的实验表明，该化合物的两亲性至少部分地导致了所观察到的效应，并有力地表明，目前报道的失活机制对于PAI-1的构象转变具有普遍重要性。总之，TX-100改变了PAI-1的初始构象，导致其功能特性发生改变。这一观察结果使我们能够建立一个关于PAI-1构象灵活性所涉及机制的新模型，并可能为开发干扰PAI-1活性的策略提供新的见解。
