Enghild J J, Salvesen G, Thøgersen I B, Pizzo S V
Duke University Medical Center, Durham, North Carolina 27710.
J Biol Chem. 1989 Jul 5;264(19):11428-35.
The inhibitory capacity of the alpha-macroglobulins resides in their ability to entrap proteinase molecules and thereby hinder the access of high molecular weight substrates to the proteinase active site. This ability is thought to require at least two alpha-macroglobulin subunits, yet the monomeric alpha-macroglobulin rat alpha 1-inhibitor-3 (alpha 1I3) also inhibits proteinases. We have compared the inhibitory activity of alpha 1I3 with the tetrameric human homolog alpha 2-macroglobulin (alpha 2M), the best known alpha-macroglobulin, in order to determine whether these inhibitors share a common mechanism. alpha 1I3, like human alpha 2M, prevented a wide variety of proteinases from hydrolyzing a high molecular weight substrate but allowed hydrolysis of small substrates. In contrast to human alpha 2M, however, the binding and inhibition of proteinases was dependent on the ability of alpha 1I3 to form covalent cross-links to proteinase lysine residues. Low concentrations of proteinase caused a small amount of dimerization of alpha 1I3, but no difference in inhibition or receptor binding was detected between purified dimers or monomers. Kininogen domains of 22 and 64 kDa were allowed to react with alpha 1I3- or alpha 2M-bound papain to probe the accessibility of the active site of this proteinase. alpha 2M-bound papain was completely protected from reaction with these domains, whereas alpha 1I3-bound papain reacted with them but with affinities several times weaker than uncomplexed papain. Cathepsin G and papain antisera reacted very poorly with the enzymes when they were bound by alpha 1I3, but the protection provided by human alpha 2M was slightly better than the protection offered by the monomeric rat alpha 1I3. Our data indicate that the inhibitory unit of alpha 1I3 is a monomer and that this protein, like the multimeric alpha-macroglobulins, inhibits proteinases by steric hindrance. However, binding of proteinases by alpha 1I3 is dependent on covalent crosslinks, and bound proteinases are more accessible, and therefore less well inhibited, than when bound by the tetrameric homolog alpha 2M. Oligomerization of alpha-macroglobulin subunits during the evolution of this protein family has seemingly resulted in a more efficient inhibitor, and we speculate that alpha 1I3 is analogous to an evolutionary precursor of the tetrameric members of the family exemplified by human alpha 2M.
α-巨球蛋白的抑制能力在于其捕获蛋白酶分子的能力，从而阻碍高分子量底物接近蛋白酶活性位点。这种能力被认为至少需要两个α-巨球蛋白亚基，但单体α-巨球蛋白大鼠α1-抑制剂-3（α1I3）也能抑制蛋白酶。我们比较了α1I3与四聚体人类同源物α2-巨球蛋白（α2M）（最著名的α-巨球蛋白）的抑制活性，以确定这些抑制剂是否具有共同机制。α1I3与人类α2M一样，能阻止多种蛋白酶水解高分子量底物，但允许小底物水解。然而，与人类α2M不同的是，α1I3对蛋白酶的结合和抑制取决于其与蛋白酶赖氨酸残基形成共价交联的能力。低浓度的蛋白酶会导致α1I3少量二聚化，但纯化的二聚体或单体之间在抑制或受体结合方面未检测到差异。让22 kDa和64 kDa的激肽原结构域与α1I3或α2M结合的木瓜蛋白酶反应，以探测该蛋白酶活性位点的可及性。α2M结合的木瓜蛋白酶完全受到保护，不与这些结构域反应，而α1I3结合的木瓜蛋白酶则与它们反应，但亲和力比未结合的木瓜蛋白酶弱几倍。当组织蛋白酶G和木瓜蛋白酶与α1I3结合时，它们与抗血清的反应非常弱，但人类α2M提供的保护略优于单体大鼠α1I3提供的保护。我们的数据表明，α1I3的抑制单元是一个单体，并且这种蛋白质与多聚体α-巨球蛋白一样，通过空间位阻抑制蛋白酶。然而，α1I3与蛋白酶的结合取决于共价交联，并且与四聚体同源物α2M结合时相比，结合的蛋白酶更容易接近，因此抑制效果更差。在这个蛋白质家族的进化过程中，α-巨球蛋白亚基的寡聚化似乎产生了一种更有效的抑制剂，并且我们推测α1I3类似于以人类α2M为例的该家族四聚体成员的进化前体。
