Van Leuven F, Cassiman J J, Van den Berghe H
Biochem J. 1982 Jan 1;201(1):119-28. doi: 10.1042/bj2010119.
The unique steric inhibition of endopeptidases by human alpha(2)M (alpha(2)-macroglobulin) and the inactivation of the latter by methylamine were examined in relation to each other. Progressive binding of trypsin by alpha(2)M was closely correlated with the loss of the methylamine-reactive sites in alpha(2)M: for each trypsin molecule bound, two such sites were inactivated. The results further showed that, even at low proteinase/alpha(2)M ratios, no unaccounted loss of trypsin-binding capacity occurred. As alpha(2)M is bivalent for trypsin binding and no trypsin bound to electrophoretic slow-form alpha(2)M was observed, this indicates that the two sites must react (bind trypsin) in rapid succession. Reaction of [(14)C]methylamine with alpha(2)M was biphasic in time; in the initial rapid phase complex-formation with trypsin caused a largely increased incorporation of methylamine. In the subsequent slow phase trypsin had no such effect. These results prompted further studies on the kinetics of methylamine inactivation of alpha(2)M with time of methylamine treatment. It was found that conformational change of alpha(2)M and decrease in trypsin binding (activity resistant to soya-bean trypsin inhibitor) showed different kinetics. The latter decreased rapidly, following pseudo-first-order kinetics. Conformational change was much slower and followed complex kinetics. On the other hand, binding of (125)I-labelled trypsin to alpha(2)M did follow the same kinetics as the conformational change. This discrepancy between total binding ((125)I radioactivity) and trypsin-inhibitor-resistant binding of trypsin indicated formation of anomalous complexes, in which trypsin could still be inhibited by soya-bean trypsin inhibitor. Further examination confirmed that these complexes were proteolytically active towards haemoglobin and bound (125)I-labelled soya-bean trypsin inhibitor to the active site of trypsin. The inhibition by soya-bean trypsin inhibitor was slowed down as compared with reaction with free trypsin. The results are discussed in relation to the subunit structure of alpha(2)M and to the mechanism of formation of the complex.
研究了人α2M（α2-巨球蛋白）对肽链内切酶的独特空间抑制作用以及甲胺对α2M的失活作用，并探讨了二者之间的相互关系。α2M与胰蛋白酶的逐步结合与α2M中甲胺反应性位点的丧失密切相关：每结合一个胰蛋白酶分子，就有两个这样的位点失活。结果还表明，即使在低蛋白酶/α2M比例下，胰蛋白酶结合能力也没有出现无法解释的损失。由于α2M对胰蛋白酶结合具有二价性，且未观察到胰蛋白酶与电泳慢型α2M结合，这表明这两个位点必须快速连续反应（结合胰蛋白酶）。[(14)C]甲胺与α2M的反应在时间上呈双相性；在初始快速阶段，与胰蛋白酶形成复合物导致甲胺掺入量大幅增加。在随后的缓慢阶段，胰蛋白酶没有这种作用。这些结果促使进一步研究甲胺处理时间对α2M失活动力学的影响。发现α2M的构象变化和胰蛋白酶结合能力的降低（对大豆胰蛋白酶抑制剂有抗性的活性）表现出不同的动力学。后者以伪一级动力学快速降低。构象变化要慢得多，遵循复杂的动力学。另一方面，(125)I标记的胰蛋白酶与α2M的结合确实与构象变化遵循相同的动力学。胰蛋白酶的总结合量（(125)I放射性）与对胰蛋白酶抑制剂有抗性的结合量之间的这种差异表明形成了异常复合物，其中胰蛋白酶仍可被大豆胰蛋白酶抑制剂抑制。进一步检查证实这些复合物对血红蛋白具有蛋白水解活性，并将(125)I标记的大豆胰蛋白酶抑制剂结合到胰蛋白酶的活性位点。与游离胰蛋白酶反应相比，大豆胰蛋白酶抑制剂的抑制作用减缓。结合α2M的亚基结构和复合物形成机制对结果进行了讨论。
