The mechanism of activation of bovine prothrombin by an activator isolated from Echis carinatus venon and characterization of the new active intermediates.
The mechanism of activation of bovine prothrombin by an activator isolated from Echis carinatus venon and characterization of the new active intermediates.
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从 Echis carinatus 毒液中分离出的激活剂激活牛凝血酶原的机制以及新活性中间体的表征。
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发表时间：
1976
期刊：
影响因子：
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通讯作者：
T. Suzuki
中科院分区：
文献类型：
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作者：
T. Morita;S. Iwanaga;T. Suzuki
从锯鳞蝰蛇（Echis carinatus，ECV）蛇毒中提取的一种激活剂，在磷酸二异丙酯（DEP）和苯甲脒（benzamidine）存在和不存在的情况下，均可激活牛凝血酶原。通过十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳监测活化过程，并对反应产物进行分离和化学表征。在不存在抑制剂的情况下，凝血酶原产生分子量为28，000和57，000的两个片段，其中前者是酶原的N-末端片段，后者是由单一多肽链组成的中间体1。中间体1随后转化为活性中间体，称为中间体ECV，而分子量不降低。这种新的中间体ECV几乎没有凝血活性，但具有很强的α-N-甲苯磺酰基-L-精氨酸甲酯（驯服）酯分解活性，并与水蛭素或抗凝血酶III结合，由两条分子量为35，000或27，000道尔顿的多肽链组成。前者为凝血酶B链，N端为Ile-Val-Glu-Gly，C端为丝氨酸;后者为N端为Ser-Gly-Gly，与凝血酶A链相连。在长时间孵育后，中间体ECV自动裂解产生一个14，000道尔顿的片段（内部片段），具有N-末端丝氨酸和凝血酶α-凝血酶[EC 3.4.21.5]，由A和B链组成。在抑制剂的存在下，中间体ECV和N-末端片段在活化混合物中积累。另一方面，当凝血酶原在水蛭素、抗凝血酶III或p ′-胍基苯甲酸对硝基苯基酯存在下被毒液激活剂激活时，它不产生任何片段，而是转化为具有分子量为51，000和34，000道尔顿的两条多肽链的衍生物，其中前者由N-末端片段、内部片段和凝血酶A链组成，后者为凝血酶B链。这种新的凝血酶原衍生物，命名为凝血酶原ECV，形成一个高分子量的复合物，与抗凝血酶III。该复合物即使在SDS存在下也不能解离。此外，凝血酶原ECV与对硝基苯基对胍基苯甲酸酯反应。根据上述结果，本文认为隆突刺蛇毒激活剂激活凝血酶原的机理是：蛇毒激活剂首先裂解酶原分子中连接凝血酶A、B链的Arg-Ile键，形成活性衍生物凝血酶原ECV。该活性衍生物自催化转化为中间体ECV，释放N-末端片段，活性中间体ECV产生α-凝血酶，释放内部片段。因此，只有沿着凝血酶原多肽链的单个肽键断裂与毒液激活剂的激活有关。
Bovine prothrombin was activated, in both the absence and presence of dissopropyphosphofluoridate (DEP) and benzamidine, by an activator which was highly purified from the venom of Echis carinatus (saw-scaled viper, ECV). The process of activation was monitored by sodium dodecysulfate (SDS)-polyacrylamide gel electrophoresis, and the reaction products were isolated and chemically characterized. In the absence of the inhibitors, prothrombin yielded two fragments with molecular weights of 28,000 and 57,000, of which the former was the N-terminal fragment of the zymogen and the latter was intermediate 1, consisting of a single polypeptide chain. Intermediate 1 was subsequently converted to an active intermediate, named intermediate ECV, without decrease of molecular weight. This new intermediate ECV, which showed little clotting activity but a strong alpha-N-tosyl-L-arginine methyl ester (TAME)-esterolytic activity and which bound with hirudin or antithrombin III, consisted of two polypeptide chains with molecular weights of 35,000 of 27,000 daltons. The former was indentified as the thrombin B chain with the N-terminal sequence Ile-Val-Glu-Gly and C-terminal serine, and the latter was a fragment with N-terminal Ser-Gly-Gly, linked to the thrombin A chain. On prolonged incubation, intermediate ECV autocaralytically yielded a fragment (inner fragment) of 14,000 daltons with N-terminal serine and the clotting enzyme alpha-thrombin [EC 3.4.21.5], which consists of A and B chains. In the presence of the inhibitors, intermediate ECV and the N-terminal fragment were accumulated in the activation mixture. On the other hand, when prothrombin was activated by the venom activator in the presence of hirudin, antithrombin III, or p-nitrophenyl p'-guanidinobenzoate, it did not yield any fragments but was converted to a derivative with two polypeptide chains having molecular weights of 51,000 and 34,000 daltons, of which the former consisted of N-terminal fragment, the inner fragment, and thrombin A chain, and the latter was thrombin B chain. This new prothrombin derivative, named prothrombin ECV, formed a high-molecular-weight complex, associating with antithrombin III. The complex was not dissociable even in the presence of SDS. Moreover, prothrombin ECV reacted with p-nitrophenyl p'-guanidinobenzoate. On the basis of the results described above, the mechanism of activaton of prothrombin by Echis carinatus venom activator can be summarized as follows: The venom activator first cleaves an Arg-Ile bond liniking thrombin A and B chains in the zymogen molecule, forming an active derivative, prothrombin ECV. This active derivative converts autocatalytically to intermediate ECV, liberating the N-terminal fragment, and active intermediate ECV generates alpha-thrombin, releasing the inner fragment. Thus, only a single peptide bond cleavage along the polypeptide chain of prothrombin is associated with activation by the venom activator...