Larionova N I, Balabushevitch N G, Zhatikov P A
School of Chemistry, Lomonosov Moscow State University, Moscow, 119899, Russia.
Biochemistry (Mosc). 1998 Sep;63(9):1078-82.
Binding of human leukocyte elastase (HLE) to human plasma fibronectin (Fn) was determined by its competition with an oligopeptide chromogenic substrate. Kinetic curves of accumulation of the substrate hydrolysis product in the presence of Fn were specific for slow-binding one-step inhibitors of the enzyme. Values of rate constants for HLE association with Fn and for the dissociation of the complex were, respectively, 2.2.10(3) M(-1).sec(-1) and 1.4.10(-3) sec(-1) at pH 7.5 and 25 degreesC. The dissociation constant of the HLE--Fn complex determined independently by titration of the enzyme with the protein substrate and neglecting the hydrolysis was 3.9.10(-7) M. The resulting values were suggested to describe a high-affinity site in the Fn molecule which was subjected to the primary attack by HLE. The soybean Bowman--Birk protease inhibitor (BBI) efficiently inhibited the HLE-induced degradation of Fn under conditions of both inhibitor preincubation with the protease and its addition into the reaction mixture of the protease with Fn.
通过人白细胞弹性蛋白酶（HLE）与一种寡肽发色底物的竞争来测定其与人血浆纤连蛋白（Fn）的结合。在存在Fn的情况下，底物水解产物积累的动力学曲线对于该酶的慢结合一步抑制剂具有特异性。在pH 7.5和25℃条件下，HLE与Fn结合的速率常数以及复合物解离的速率常数分别为2.2×10³ M⁻¹·sec⁻¹和1.4×10⁻³ sec⁻¹。通过用蛋白质底物滴定酶并忽略水解独立测定的HLE - Fn复合物的解离常数为3.9×10⁻⁷ M。这些结果表明，Fn分子中存在一个高亲和力位点，该位点是HLE的主要攻击目标。大豆鲍曼 - 伯克蛋白酶抑制剂（BBI）在抑制剂与蛋白酶预孵育以及将其添加到蛋白酶与Fn的反应混合物这两种条件下，均能有效抑制HLE诱导的Fn降解。
