Lomas D A, Elliott P R, Chang W S, Wardell M R, Carrell R W
Department of Haematology, University of Cambridge, United Kingdom.
J Biol Chem. 1995 Mar 10;270(10):5282-8. doi: 10.1074/jbc.270.10.5282.
Members of the serine proteinase inhibitor or serpin superfamily have a common molecular architecture based on a dominant five-membered A beta-pleated sheet and a mobile reactive center loop. The reactive center loop has been shown to adopt a range of conformations from the three turn alpha-helix of ovalbumin to the cleaved or latent inhibitor in which the reactive center loop is fully inserted into the A sheet of the molecule. While the cleaved state can be achieved in all inhibitory serpins only plasminogen activator inhibitor-1 and, more recently, antithrombin have been shown to adopt the latent conformation. We show here that the archetypal serpin, alpha 1-antitrypsin, can also be induced to adopt the latent conformation by heating at high temperatures in 0.7 M citrate for 12 h. The resulting species elutes at a lower sodium chloride concentration on an anion-exchange column and has a more cathodal electrophoretic mobility on non-denaturing polyacrylamide gel electrophoresis and isoelectric focusing than native M antitrypsin. Latent antitrypsin is inactive as an inhibitor of bovine alpha-chymotrypsin, is stable to unfolding with 8 M urea, and is more resistant to heat-induced loop-sheet polymerization than native but less resistant than cleaved antitrypsin. The reactive center loop of latent antitrypsin is inaccessible to proteolytic cleavage, and its occupancy of the A sheet prevents the molecule accepting an exogenous reactive center loop peptide. The activity of latent antitrypsin may be increased from < 1% to approximately 35% by refolding from 6 M guanidinium chloride.
丝氨酸蛋白酶抑制剂（serpin）超家族的成员具有基于一个主要的五元Aβ折叠片层和一个可移动的反应中心环的共同分子结构。反应中心环已被证明能呈现一系列构象，从卵清蛋白的三转角α螺旋到反应中心环完全插入分子A片层的裂解或潜伏抑制剂构象。虽然在所有抑制性丝氨酸蛋白酶抑制剂中都能实现裂解状态，但只有纤溶酶原激活物抑制剂-1以及最近发现的抗凝血酶已被证明能呈现潜伏构象。我们在此表明，原型丝氨酸蛋白酶抑制剂α1-抗胰蛋白酶，在0.7M柠檬酸盐中高温加热12小时也可被诱导呈现潜伏构象。所得产物在阴离子交换柱上以较低的氯化钠浓度洗脱，并且在非变性聚丙烯酰胺凝胶电泳和等电聚焦中比天然M抗胰蛋白酶具有更向阴极的电泳迁移率。潜伏抗胰蛋白酶作为牛α-糜蛋白酶的抑制剂无活性，对8M尿素变性稳定，并且比天然抗胰蛋白酶更耐热诱导的环-片层聚合，但比裂解抗胰蛋白酶的抗性小。潜伏抗胰蛋白酶的反应中心环不能被蛋白酶切割，并且其对A片层的占据阻止分子接受外源性反应中心环肽。通过从6M氯化胍中重折叠，潜伏抗胰蛋白酶的活性可从<1%提高到约35%。
