Roche P A, Pizzo S V
Biochemistry. 1987 Jan 27;26(2):486-91. doi: 10.1021/bi00376a021.
When human alpha 2-macroglobulin (alpha 2M) binds proteinases, it undergoes subunit cleavage. Binding of small proteinases such as trypsin results in proteolysis of each of the four subunits of the inhibitor. By contrast, previous studies suggest that reaction of plasmin with alpha 2M results in cleavage of only two or three of the inhibitor subunits. In this paper, we demonstrate that the extent of subunit cleavage of alpha 2M is a function of plasmin concentration. When alpha 2M was incubated with a 2.5-fold excess of plasmin, half of the subunits were cleaved; however, at a 20-fold enzyme to inhibitor ratio, greater than 90% of the subunits were cleaved with no additional plasmin binding. This increased cleavage was catalyzed by free rather than bound plasmin. It is concluded that this "nonproductive" subunit cleavage is dependent upon the molar ratio of proteinase to inhibitor. The consequence of complete subunit cleavage on receptor recognition of alpha 2M-plasmin (alpha 2M-Pm) complexes was studied. Preparations of alpha 2M-Pm with only two cleaved subunits bound to the murine macrophage receptor with a Kd of 0.4 nM and 60 fmol of bound complex/mg of cell protein. When preparations of alpha 2-M-Pm with four cleaved subunits were studied, the Kd was unaltered but ligand binding increased to 140 fmol/mg of cell protein. The receptor binding behavior of the latter preparation is equivalent to that observed when alpha 2M is treated with small proteinases such as trypsin. This study suggests that receptor recognition site exposure is not complete in the alpha 2M-Pm complex with half of the subunits cleaved. Proteolytic cleavage of the remaining subunits of the inhibitor results in a further conformational change exposing the remaining receptor recognition sites.
当人α2-巨球蛋白（α2M）与蛋白酶结合时，它会发生亚基裂解。诸如胰蛋白酶等小蛋白酶的结合会导致抑制剂的四个亚基各自发生蛋白水解。相比之下，先前的研究表明纤溶酶与α2M反应仅导致抑制剂的两到三个亚基裂解。在本文中，我们证明α2M亚基裂解的程度是纤溶酶浓度的函数。当α2M与2.5倍过量的纤溶酶一起孵育时，一半的亚基被裂解；然而，在酶与抑制剂的比例为20倍时，超过90%的亚基被裂解，且没有额外的纤溶酶结合。这种增加的裂解是由游离而非结合的纤溶酶催化的。得出的结论是，这种“无活性”的亚基裂解取决于蛋白酶与抑制剂的摩尔比。研究了α2M-纤溶酶（α2M-Pm）复合物亚基完全裂解对受体识别的影响。仅含有两个裂解亚基的α2M-Pm制剂与小鼠巨噬细胞受体结合，解离常数（Kd）为0.4 nM，每毫克细胞蛋白结合60 fmol的复合物。当研究含有四个裂解亚基的α2-M-Pm制剂时，Kd未改变，但配体结合增加到每毫克细胞蛋白140 fmol。后一种制剂的受体结合行为与用胰蛋白酶等小蛋白酶处理α2M时观察到的行为相当。这项研究表明，在α2M-Pm复合物中，当一半的亚基被裂解时，受体识别位点并未完全暴露。抑制剂其余亚基的蛋白水解裂解会导致进一步的构象变化，从而暴露其余的受体识别位点。
