Roche P A, Pizzo S V
Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710.
Arch Biochem Biophys. 1988 Nov 15;267(1):285-93. doi: 10.1016/0003-9861(88)90034-3.
The structures of the two proteinase-binding sites in human alpha 2-macroglobulin (alpha 2M) were probed by treatment of alpha 2M with the serine proteinases thrombin and plasmin. Each proteinase forms an equimolar complex with alpha 2M (a binary alpha 2M-proteinase complex) which results in the activation and cleavage of two internal thiolester bonds in alpha 2M. Binary alpha 2M-proteinase complexes demonstrated an incomplete conformational change as determined by nondenaturing polyacrylamide gel electrophoresis and incomplete receptor recognition site exposure as determined by in vivo plasma elimination studies. Treatment of binary alpha 2M-proteinase complexes with CH3NH2, trypsin, or elastase resulted in cleavage of an additional one or two thiolester bonds in alpha 2M and complete receptor recognition site exposure, demonstrating that a limited conformational change had occurred. Treatment of the alpha 2M-thrombin complex with elastase resulted in the incorporation of approximately 0.5 mol proteinase/mol alpha 2M and completion of the conformational change in the complex. Similar treatment of the alpha 2M-plasmin complex resulted in the incorporation of less than 0.1 mol proteinase/mol alpha 2M. Unlike the alpha 2M-thrombin complex, the alpha 2M-plasmin complex did not undergo a complete conformational change following treatment with CH3NH2 or trypsin. Incubation of this complex with elastase resulted in proteolysis of the kringle 1-4 region of the alpha 2M-bound plasmin heavy chain, and following this treatment the alpha 2M-plasmin complex underwent a complete conformational change. The results of this investigation demonstrate that binary alpha 2M-proteinase complexes retain a relatively intact proteinase-binding site. In the case of the alpha 2M-plasmin complex, however, the heavy chain of alpha 2M-bound plasmin protrudes from the proteinase-binding site and prevents a complete conformational change in the complex despite additional thiolester bond cleavage.
通过用丝氨酸蛋白酶凝血酶和纤溶酶处理人α2-巨球蛋白（α2M），对其两个蛋白酶结合位点的结构进行了探究。每种蛋白酶与α2M形成等摩尔复合物（α2M-蛋白酶二元复合物），这导致α2M中两个内部硫酯键的激活和裂解。通过非变性聚丙烯酰胺凝胶电泳测定，α2M-蛋白酶二元复合物表现出不完全的构象变化；通过体内血浆清除研究测定，其受体识别位点暴露不完全。用甲胺、胰蛋白酶或弹性蛋白酶处理α2M-蛋白酶二元复合物，导致α2M中额外的一个或两个硫酯键裂解，并使受体识别位点完全暴露，表明发生了有限的构象变化。用弹性蛋白酶处理α2M-凝血酶复合物，导致每摩尔α2M掺入约0.5摩尔蛋白酶，并使复合物的构象变化完成。对α2M-纤溶酶复合物进行类似处理，导致每摩尔α2M掺入少于0.1摩尔蛋白酶。与α2M-凝血酶复合物不同，α2M-纤溶酶复合物在用甲胺或胰蛋白酶处理后未发生完全的构象变化。将该复合物与弹性蛋白酶一起孵育，导致α2M结合的纤溶酶重链的kringle 1-4区域发生蛋白水解，在此处理后，α2M-纤溶酶复合物发生了完全的构象变化。本研究结果表明，α2M-蛋白酶二元复合物保留了相对完整的蛋白酶结合位点。然而，在α2M-纤溶酶复合物的情况下，α2M结合的纤溶酶的重链从蛋白酶结合位点突出，尽管硫酯键进一步裂解，但仍阻止了复合物的完全构象变化。
