Tayeh M A, Olson S T, Shore J D
Division of Biochemical Research, Henry Ford Hospital, Detroit, Michigan 48202.
J Biol Chem. 1994 Jun 10;269(23):16318-25.
We have studied the cleavage of human high molecular weight kininogen (HK) by plasma kallikrein in the absence and presence of the surfaces, dextran sulfate (DxSO4) and sulfatides. Using a combination of SDS-polyacrylamide gel electrophoresis, Western blotting with polyclonal antibodies that specifically recognize the COOH terminus of the bradykinin moiety, and high pressure liquid chromatography analyses of the cleavage reaction, we have identified two intermediates in the formation of bradykinin from intact kininogen and demonstrated that alternative cleavage pathways are followed in the absence and presence of surfaces. The COOH-terminal bradykinin cleavage occurred first both in the absence and presence of DxSO4, producing a 103-kDa HK intermediate consisting of disulfide-linked heavy and light chains that retained the kinin moiety. In the presence of DxSO4, this was followed exclusively by the NH2-terminal bradykinin cleavage and release of kinin with no apparent change in molecular mass. Subsequently, a slower cleavage of an 8-kDa peptide from the amino terminus of the HK light chain occurred to form a 95-kDa end product. In contrast to this sequential cleavage pattern, NH2-terminal bradykinin and light chain cleavages occurred randomly in the absence of DxSO4, resulting in the production of an additional 95-kDa intermediate that retained bradykinin but had lost the 8-kDa peptide from the HK light chain. Comparison of the relative rates of the three kallikrein cleavages in the absence and presence of DxSO4 indicated that the surface enhanced the rates of both bradykinin cleavages 2-4-fold, but inhibited the light chain cleavage rate approximately 10-fold, thereby accounting for the change from a partially random to a sequential cleavage pattern in the presence of the surface. Steady-state kinetic analysis revealed that DxSO4 enhanced the kcat/KM for bradykinin release by the rate-limiting NH2-terminal bradykinin cleavage by approximately 2-fold due exclusively to an increase in kcat. Sulfatides appeared to produce the same effects on the pattern of HK cleavages as DxSO4. Blocking of the nonactive site, i.e. exosite, interaction between kallikrein and HK with excess prekallikrein or a synthetic peptide containing the region of HK that interacts with the kallikrein exosite significantly reduced the rate of bradykinin release as well as HK cleavages detected by SDS-polyacrylamide gel electrophoresis either in the absence or presence of DxSO4, indicating that the exosite interaction facilitates bradykinin cleavage.
我们研究了在不存在和存在表面物质硫酸葡聚糖（DxSO4）和硫苷脂的情况下，血浆激肽释放酶对人高分子量激肽原（HK）的裂解作用。通过结合使用十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳、用特异性识别缓激肽部分COOH末端的多克隆抗体进行蛋白质印迹分析以及对裂解反应进行高压液相色谱分析，我们确定了完整激肽原形成缓激肽过程中的两种中间体，并证明在不存在和存在表面物质的情况下遵循不同的裂解途径。在不存在和存在DxSO4的情况下，COOH末端缓激肽裂解均首先发生，产生一种103 kDa的HK中间体，其由保留激肽部分的二硫键连接的重链和轻链组成。在存在DxSO4的情况下，随后仅发生NH2末端缓激肽裂解并释放激肽，分子量无明显变化。随后，从HK轻链的氨基末端缓慢裂解一个8 kDa的肽，形成95 kDa的终产物。与这种顺序裂解模式相反，在不存在DxSO4的情况下，NH2末端缓激肽和轻链裂解随机发生，导致产生另一种95 kDa的中间体，其保留缓激肽但已从HK轻链丢失8 kDa的肽。比较在不存在和存在DxSO4的情况下三种激肽释放酶裂解的相对速率表明，表面物质使两种缓激肽裂解速率提高了2 - 4倍，但使轻链裂解速率降低了约10倍，从而解释了在存在表面物质时从部分随机裂解模式到顺序裂解模式的变化。稳态动力学分析表明，DxSO4仅通过增加催化常数（kcat）使限速的NH2末端缓激肽裂解释放缓激肽的kcat/KM提高了约2倍。硫苷脂似乎对HK裂解模式产生与DxSO4相同的影响。用过量的前激肽释放酶或含有与激肽释放酶外位点相互作用的HK区域的合成肽阻断激肽释放酶与HK之间的非活性位点即外位点相互作用，显著降低了缓激肽释放速率以及在不存在或存在DxSO4的情况下通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳检测到的HK裂解速率，表明外位点相互作用促进缓激肽裂解。
