Sui G C, Wiman B
Department of Clinical Chemistry and Blood Coagulation, Karolinska Hospital, Karolinska Institute, S-171 76 Stockholm, Sweden.
Biochem J. 1998 Apr 15;331 ( Pt 2)(Pt 2):409-15. doi: 10.1042/bj3310409.
Thirteen amino acid substitutions have been introduced within the stretch Phe113 to Asp138 in the plasminogen activator inhibitor 1 (PAI-1) molecule by site-directed mutagenesis. The different proteins and wild-type (wt) PAI-1 have been overexpressed in Escherichia coli and purified by chromatography on heparin-Sepharose and on anhydrotrypsin-agarose. The PAI-1 variants have been characterized by their reactivity with tissue plasminogen activator (tPA), interactions with vitronectin or heparin, and stability. Most PAI-1 variants, except for Asp125-->Lys, Phe126-->Ser and Arg133-->Asp, displayed a high spontaneous inhibitory activity towards tPA, which did not change greatly on reactivation with 4 M guanidinium chloride, followed by dialysis at pH 5.5. The variants Asp125-->Lys and Arg133-->Asp became much more active after reactivation and they were also more rapidly transformed to inactive forms (t12 22-31 min) at physiological pH and temperature than the other variants. However, in the presence of vitronectin they were both almost equally stable (t12 2.3 h) as wtPAI-1 (t12 3.0 h). The mutant Glu130-->Lys showed an increased stability, both in the absence and in the presence of vitronectin compared with wtPAI-1. Nevertheless a similar affinity between all the active PAI-1 variants and vitronectin was observed. Further, all mutants, including the three mutants with low activity, were to a large extent adsorbed on anhydrotrypsin-agarose and were eluted in a similar fashion. In accordance with these data, the three variants with a low activity were all to a large extent cleaved as a result of their reaction with tPA, suggesting that they occurred predominantly in the substrate conformation. Our results do not support the presence of a binding site for vitronectin in this part of the molecule, but rather that it might be involved in controlling the active PAI-1 to substrate transition. Partly, this region of the PAI-1 molecule (Arg115 to Arg118) seems also to be involved in the binding of heparin to PAI-1.
通过定点诱变，在纤溶酶原激活物抑制剂1（PAI - 1）分子中位于Phe113至Asp138片段内引入了13个氨基酸替换。不同的蛋白质和野生型（wt）PAI - 1已在大肠杆菌中过表达，并通过肝素 - 琼脂糖和脱水胰蛋白酶 - 琼脂糖柱层析进行纯化。PAI - 1变体已通过它们与组织纤溶酶原激活物（tPA）的反应性、与玻连蛋白或肝素的相互作用以及稳定性来进行表征。除了Asp125→Lys、Phe126→Ser和Arg133→Asp之外，大多数PAI - 1变体对tPA表现出高自发抑制活性，在用4M氯化胍重新激活后，接着在pH 5.5下透析，其活性变化不大。变体Asp125→Lys和Arg133→Asp在重新激活后变得更加活跃，并且在生理pH和温度下，它们比其他变体更快地转变为无活性形式（半衰期22 - 31分钟）。然而，在存在玻连蛋白的情况下，它们与wtPAI - 1（半衰期3.0小时）几乎同样稳定（半衰期2.3小时）。与wtPAI - 1相比，突变体Glu130→Lys在不存在和存在玻连蛋白的情况下都表现出稳定性增加。然而，观察到所有活性PAI - 1变体与玻连蛋白之间具有相似的亲和力。此外，所有突变体，包括三个低活性突变体，在很大程度上都吸附在脱水胰蛋白酶 - 琼脂糖上，并以类似的方式洗脱。根据这些数据，三个低活性变体在与tPA反应后在很大程度上都被切割，这表明它们主要以底物构象存在。我们的结果不支持在该分子的这一部分存在玻连蛋白结合位点，而是表明它可能参与控制活性PAI - 1向底物的转变。部分地，PAI - 1分子的这一区域（Arg115至Arg118）似乎也参与肝素与PAI - 1的结合。
