Speijer H, Govers-Riemslag J W, Zwaal R F, Rosing J
J Biol Chem. 1986 Oct 5;261(28):13258-67.
The prothrombin activator from the venom of Oxyuranus scutellatus (Taipan snake) was purified by gel filtration on Sephadex G-200 and ion-exchange chromatography on QAE-Sephadex. The activator is a large protein with a molecular weight of approximately 300,000, which is composed of subunits of Mr 110,000 and 80,000 and two disulfide-linked polypeptides of Mr 30,000. One or both of these Mr 30,000 subunits contain the active site. The venom activator readily converts Factor Xa-specific chromogenic substrates and is also able to activate prothrombin (Km = 166 microM, Vmax = 2.5 mumol of prothrombin activated per min/mg of venom). Gel electrophoretic analysis of prothrombin activation indicates that the venom activator randomly cleaves the Arg274-Thr275 and Arg323-Ile324 bonds of prothrombin since both thrombin and meizothrombin are formed as reaction products. Venom-catalyzed prothrombin activation is not affected by bovine Factor Va but is greatly stimulated by phospholipids plus Ca2+ ions. This stimulatory effect is explained by a decrease of the Km for prothrombin. In the presence of 50 microM phospholipid vesicles (25% phosphatidylserine/75% phosphatidylcholine; mole/mole), the Km is 0.34 microM and the Vmax is 7.1 mumol of prothrombin activated per min/mg of venom. The purified venom activator contains gamma-carboxyglutamic acid residues which presumably function in the interaction between the venom activator and phospholipids. Treatment of the activator with 0.8 M NaSCN strongly reduces its ability to activate prothrombin but has no effect on its amidolytic activity. The prothrombin-converting activity of the NaSCN-treated activator can be restored with bovine Factor Va. During prolonged gradient gel electrophoresis, the Mr 300,000 activator dissociates into smaller subunits. This causes a loss of the prothrombin-converting activity, while the amidolytic activity is recovered in a protein with an apparent molecular weight of 57,000. This protein can, however, rapidly activate prothrombin in the presence of Factor Va or in the presence of a protein component of Mr 220,000 that also migrates on the gel. These results suggest that the prothrombin activator from the O. scutellatus venom is a multimeric protein complex consisting of a Factor Xa-like enzyme and a Factor Va-like cofactor.
通过在葡聚糖凝胶G - 200上进行凝胶过滤以及在QAE - 葡聚糖凝胶上进行离子交换色谱法，从盾尖吻蛇（太攀蛇）毒液中纯化出凝血酶原激活剂。该激活剂是一种大分子蛋白质，分子量约为300,000，由分子量为110,000和80,000的亚基以及两条分子量为30,000的二硫键连接的多肽组成。这些分子量为30,000的亚基中的一个或两个含有活性位点。毒液激活剂能轻易地转化因子Xa特异性显色底物，并且也能够激活凝血酶原（Km = 166微摩尔，Vmax =每分钟每毫克毒液激活2.5微摩尔凝血酶原）。对凝血酶原激活的凝胶电泳分析表明，毒液激活剂随机切割凝血酶原的Arg274 - Thr275和Arg323 - Ile324键，因为凝血酶和中凝血酶都是反应产物。毒液催化的凝血酶原激活不受牛因子Va的影响，但受到磷脂加Ca2 +离子的极大刺激。这种刺激作用可以通过凝血酶原Km值的降低来解释。在存在50微摩尔磷脂囊泡（25％磷脂酰丝氨酸/ 75％磷脂酰胆碱；摩尔/摩尔）的情况下，Km为0.34微摩尔，Vmax为每分钟每毫克毒液激活7.1微摩尔凝血酶原。纯化的毒液激活剂含有γ - 羧基谷氨酸残基，推测其在毒液激活剂与磷脂之间的相互作用中起作用。用0.8 M NaSCN处理激活剂会强烈降低其激活凝血酶原的能力，但对其酰胺水解活性没有影响。经NaSCN处理的激活剂的凝血酶原转化活性可以用牛因子Va恢复。在长时间的梯度凝胶电泳过程中，分子量为300,000的激活剂会解离成较小的亚基。这导致凝血酶原转化活性丧失，而酰胺水解活性在一种表观分子量为57,000的蛋白质中恢复。然而，这种蛋白质在存在因子Va或存在一种分子量为220,000且也在凝胶上迁移的蛋白质成分的情况下，能够快速激活凝血酶原。这些结果表明，来自盾尖吻蛇毒液的凝血酶原激活剂是一种多聚体蛋白质复合物，由一种因子Xa样酶和一种因子Va样辅因子组成。
