Schapira M, Scott C F, Colman R W
J Clin Invest. 1982 Feb;69(2):462-8. doi: 10.1172/jci110470.
Although Cl-inhibitor (Cl-INH) and alpha(2)-macroglobulin (alpha(2)M) have been reported as the major inhibitors of plasma kallikrein in normal plasma, there is little quantitative support for this conclusion. Thus, we studied the inactivation of purified kallikrein in normal plasma, as well as in plasma congenitally deficient in Cl-INH, or artificially depleted of alpha(2)M by chemical modification of the inhibitor with methylamine. Under pseudo-first-order conditions, the inactivation rate constant of kallikrein in normal plasma was 0.60 min(-1). This rate constant was reduced to 0.35, 0.30, and 0.06 min(-1), in plasma deficient respectively in Cl-INH, alpha(2)M, or both inhibitors. Thus Cl-INH (42%) and alpha(2)M (50%) were found to be the major inhibitors of kallikrein in normal plasma. Moreover all the other protease inhibitors present in normal plasma contributed only for 8% to the inactivation of the enzyme. To confirm these kinetic results, (125)I-kallikrein (M(r) 85,000) was completely inactivated by various plasma samples, and the resulting mixtures were analyzed by gel filtration on Sepharose 6B CL for the appearance of (125)I-kallikrein-inhibitor complexes. After inactivation by normal plasma, 52% of the active enzyme were found to form a complex (M(r) 370,000) with Cl-INH, while 48% formed a complex (M(r) 850,000) with alpha(2)M. After inactivation by Cl-INH-deficient plasma, >90% of the active (125)I-kallikrein was associated with alpha(2)M. A similar proportion of the label was associated with Cl-INH in plasma deficient in alpha(2)M. After inactivation by plasma deficient in both Cl-INH and alpha(2)M, (125)I-kallikrein was found to form a complex of M(r) 185,000. This latter complex, which may involve antithrombin III, alpha(1)-protease inhibitor, and/or alpha(1)-plasmin inhibitor, was not detectable in appreciable concentrations in the presence of either Cl-INH or alpha(2)M, even after the addition of heparin (2 U/ml). These observations demonstrate that Cl-INH and alpha(2)M are the only significant inhibitors of kallikrein in normal plasma confirming previous predictions based on experiments in purified systems. Moreover, in the absence of either Cl-INH or alpha(2)M, the inactivation of kallikrein becomes almost entirely dependent on the other major inhibitor.
虽然已有报道称C1酯酶抑制剂（C1-INH）和α2-巨球蛋白（α2M）是正常血浆中血浆激肽释放酶的主要抑制剂，但这一结论几乎没有定量支持。因此，我们研究了纯化的激肽释放酶在正常血浆、先天性缺乏C1-INH的血浆或通过用甲胺对抑制剂进行化学修饰而人工去除α2M的血浆中的失活情况。在准一级条件下，激肽释放酶在正常血浆中的失活速率常数为0.60 min-1。在分别缺乏C1-INH、α2M或两种抑制剂的血浆中，该速率常数分别降至0.35、0.30和0.06 min-1。因此，发现C1-INH（42%）和α2M（50%）是正常血浆中激肽释放酶的主要抑制剂。此外，正常血浆中存在的所有其他蛋白酶抑制剂对该酶失活的贡献仅为8%。为了证实这些动力学结果，用各种血浆样品使125I-激肽释放酶（相对分子质量85,000）完全失活，然后通过在Sepharose 6B CL上进行凝胶过滤分析所得混合物中125I-激肽释放酶-抑制剂复合物的出现情况。用正常血浆使其失活后，发现52%的活性酶与C1-INH形成复合物（相对分子质量370,000），而48%与α2M形成复合物（相对分子质量850,000）。用缺乏C1-INH的血浆使其失活后，>90%的活性125I-激肽释放酶与α2M结合。在缺乏α2M的血浆中，类似比例的标记物与C1-INH结合。用同时缺乏C1-INH和α2M的血浆使其失活后，发现125I-激肽释放酶形成相对分子质量为185,000的复合物。即使加入肝素（2 U/ml），在存在C1-INH或α2M的情况下，这种可能涉及抗凝血酶III、α1-蛋白酶抑制剂和/或α1-纤溶酶抑制剂的后一种复合物也无法以可观的浓度检测到。这些观察结果表明，C1-INH和α2M是正常血浆中激肽释放酶仅有的重要抑制剂，证实了基于纯化系统实验的先前预测。此外，在缺乏C1-INH或α2M的情况下，激肽释放酶的失活几乎完全依赖于另一种主要抑制剂。
