Kinetics of the formation of thrombin-thrombospondin complexes: involvement of a 77-kDa intermediate.
Kinetics of the formation of thrombin-thrombospondin complexes: involvement of a 77-kDa intermediate.
复制标题
凝血酶-凝血反应蛋白复合物形成的动力学：77-kDa 中间体的参与。
DOI：
10.1016/0003-9861(88)90158-0
复制
发表时间：
1988
影响因子：
3.9
通讯作者：
Detwiler,TC
中科院分区：
文献类型：
--
作者：
Browne,PC;Miller,JJ;Detwiler,TC
凝血酶与活化血小板分泌的蛋白质形成十二烷基硫酸钠稳定的77和>450 kDa复合物。通过向A23187活化的血小板的上清液中加入125 I-凝血酶来研究这些复合物形成的动力学。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析复合物，无论是否有二硫键的还原。当在非还原性凝胶上分析时，77-kDa复合物在约3分钟时达到最大值，然后随着>450-kDa复合物的增加而下降。在还原性凝胶上（其上没有>450-kDa复合物），77-kDa复合物接近非还原性凝胶上>450-kDa复合物的水平。对于77-kDa复合物，形成的半衰期小于1分钟，而对于>450-kDa复合物，形成的半衰期约为15分钟。这些时间进程表明，77-kDa复合物作为必需前体掺入到>450-kDa复合物中。竞争性抑制剂或非竞争性抑制剂的凝血酶抑制复合物的形成，这两种复合物的形成的pH依赖性是类似的pH依赖于凝血酶的催化活性。Ca ~（2+）抑制>450-kDa复合物的形成，但不抑制77-kDa复合物的形成。一个模型中，凝血酶和分泌的蛋白质形成一个77-kDa的复杂的过程中，涉及凝血酶的活性位点。然后通过与血小板反应蛋白的巯基-二硫键交换将77-kDa复合物掺入>450-kDa复合物中，这是一个被Ca 2+抑制的过程。>450-kDa复合物中的凝血酶没有催化活性。
Thrombin forms sodium dodecyl sulfate stable complexes of 77 and >450 kDa with proteins secreted by activated platelets. The kinetics of formation of these complexes were investigated by addition of125I-thrombin to the supernatant solution of A23187-activated platelets. Complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis either with or without reduction of disulfide bonds. When analyzed on nonreduced gels, the 77-kDa complex reached a maximum at about 3 min and then declined as the >450-kDa complex increased. On reduced gels (on which there was no >450-kDa complex) the 77-kDa complex approached the level of the >450-kDa complex on nonreduced gels. The half-time of formation was less than 1 min for the 77-kDa complex and about 15 min for the >450-kDa complex. These time courses suggested that the 77-kDa complex was incorporated into the >450-kDa complex as an essential precursor. Formation of complexes was inhibited by a competitive inhibitor or a noncompetitive inhibitor of thrombin, and the pH dependence of formation of both complexes was similar to the pH dependence for catalytic activity of thrombin. Ca2+inhibited formation of the >450-kDa complex but not of the 77-kDa complex. A model is presented in which thrombin and a secreted protein form a 77-kDa complex by a process that involves the active site of thrombin. The 77-kDa complex is then incorporated into a >450-kDa complex by thiol-disulfide exchange with thrombospondin, a process that is inhibited by Ca2+. Thrombin in the >450-kDa complex had no catalytic activity.