Gils A, Vleugels N, Tobback K, Knockaert I, Declerck P J
Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, E. Van Evenstraat 4, B-3000 Leuven, Belgium.
Biochim Biophys Acta. 1998 Sep 8;1387(1-2):291-7. doi: 10.1016/s0167-4838(98)00139-3.
The serpin plasminogen activator inhibitor 1 (PAI-1) can occur, in vitro, in both an inhibitory and a non-inhibitory but cleavable substrate form. In the present study, we have evaluated the effect of replacing the P13 to P10 region of PAI-1 (Val-Ala-Ser-Ser), with the P13 to P10 region of either the non-inhibitory serpin ovalbumin (Glu-Val-Val-Gly; PAI-1-ovalbumin) or the inhibitory serpin antithrombin III (Glu-Ala-Ala-Ala; PAI-1-antithrombin III). In addition, we have replaced Val at position P13 with Glu (PAI-1-P13 (Val-->Glu)). Wild-type (wt) PAI-1 revealed specific activities of 80+/-9% (mean+/-S.D., n=4) of the theoretical maximum value towards t-PA. PAI-1-ovalbumin, PAI-1-antithrombin III and PAI-1-P13 (Val-->Glu) revealed specific activities of 86+/-15%, 77+/-11%, and 100+/-30% respectively, towards t-PA and similar inhibitory properties towards u-PA. Surprisingly, upon inactivation at 37 degreesC, the active conformation of the PAI-1 mutants converted partly into a substrate conformation (i.e. 52+/-5.2%, 55+/-8.2% and 46+/-4.6% for PAI-1-ovalbumin, PAI-1-antithrombin III and PAI-1-P13 (Val-->Glu), respectively) and partly into a latent conformation. This is in contrast to active wtPAI-1 which, as expected, is converted to the latent conformation (i.e. 86+/-1.0%). In conclusion, even though replacement of the P13 to P10 region of PAI-1 by the corresponding region of a non-inhibitory serpin or of an inhibitory serpin, does not directly affect its inhibitory properties, the nature of the amino acids in this region and of P13 in particular, contributes to its conformational transitions.
丝氨酸蛋白酶抑制剂纤溶酶原激活物抑制剂1（PAI-1）在体外可呈现抑制性形式和非抑制性但可裂解的底物形式。在本研究中，我们评估了用非抑制性丝氨酸蛋白酶抑制剂卵清蛋白（Glu-Val-Val-Gly；PAI-1-卵清蛋白）或抑制性丝氨酸蛋白酶抑制剂抗凝血酶III（Glu-Ala-Ala-Ala；PAI-1-抗凝血酶III）的P13至P10区域替换PAI-1的P13至P10区域（Val-Ala-Ser-Ser）的效果。此外，我们还用Glu替换了P13位的Val（PAI-1-P13（Val→Glu））。野生型（wt）PAI-1对组织型纤溶酶原激活物（t-PA）的比活性显示为理论最大值的80±9%（平均值±标准差，n = 4）。PAI-1-卵清蛋白、PAI-1-抗凝血酶III和PAI-1-P13（Val→Glu）对t-PA的比活性分别为86±15%、77±11%和100±30%，对尿激酶型纤溶酶原激活物（u-PA）具有相似的抑制特性。令人惊讶的是，在37℃失活时，PAI-1突变体的活性构象部分转变为底物构象（即PAI-1-卵清蛋白、PAI-1-抗凝血酶III和PAI-1-P13（Val→Glu）分别为52±5.2%、55±8.2%和46±4.6%），部分转变为潜伏构象。这与活性wtPAI-1相反，如预期的那样，wtPAI-1转变为潜伏构象（即86±1.0%）。总之，尽管用非抑制性丝氨酸蛋白酶抑制剂或抑制性丝氨酸蛋白酶抑制剂的相应区域替换PAI-1的P13至P10区域不会直接影响其抑制特性，但该区域中氨基酸的性质，特别是P13的氨基酸性质，有助于其构象转变。
