Purification, characterization, and fibrinogen cleavage sites of three fibrinolytic enzymes from the venom of Crotalus basiliscus basiliscus.
Purification, characterization, and fibrinogen cleavage sites of three fibrinolytic enzymes from the venom of Crotalus basiliscus basiliscus.
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响尾蛇毒液中三种纤溶酶的纯化、表征和纤维蛋白原裂解位点。
DOI：
10.1021/bi00134a003
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
MarklandJr,FS
中科院分区：
文献类型：
--
作者：
Retzios,AD;MarklandJr,FS
摘要：从墨西哥西海岸响尾蛇（Crotalusbasiliscus basiliscus）的毒液中纯化出三种不同的纤溶酶。基于高效液相色谱法的纯化包括以下步骤：（a）疏水相互作用色谱法;（B）羟基磷灰石色谱法;（c）阴离子交换色谱法。在疏水相互作用色谱法之后，检测到两个纤溶活性峰，Cbl 1和Cbl 2。在羟基磷灰石色谱法之后，使Cbd 2均匀。在羟基磷灰石层析后，显示CbdCl由两种组分组成，CbdCl。l和cbnl。2.两个人都是cbnl。l和cbnl。2用阴离子交换层析纯化至均一。SDS-聚丙烯酰胺凝胶电泳结果表明，Cbl. l和cbnl。2具有相似的分子量（约23500），而Cbf 2显示约22500的分子量。这些酶似乎没有被糖基化。通过高效反相色谱法分析的所有三种酶的胰蛋白酶活性表明，Cbfl. l和cbnl。2是密切相关的，并与Cbd 2不同。后者显示出更多的相似性，与Cbl。2比与cbabel。L.所有三种酶的特异性纤维蛋白溶解活性非常相似，但一般蛋白水解活性变化很大，其中Cbf 2显示出比Cbf 1高12倍的特异性蛋白水解活性。l和cbnl。2.当皮下注射（高达100 μ g）到小鼠体内时，这些酶都没有表现出出血活性。cbabel. l和cbnl。2对纤维蛋白原的作用同样针对A α链和B β链。针对纤维蛋白，α-链的降解速率显著高于β-链。Cbf 2主要表现出α-纤维蛋白原酶活性，β-链上的活性有限。已经鉴定了Aa-链上的几个纤维蛋白原切割位点：l和cbnl。2切割，
Revised Manuscript Received February 19, 1992 abstract: Three distinct fibrinolytic enzymes have been purified from the venom of Crotalusbasiliscus basiliscus (the Mexican westcoast rattlesnake). The high-performance liquid chromatography-based purification comprised the following steps:(a) hydrophobic interaction chromatography;(b) hydroxylapatite chromatography;(c) anion-exchange chromatography. Following hydrophobic interaction chromtography two fibrinolytic activity peaks were detected, Cbfibl and Cbfib2. Cbfib2 was rendered homogeneous following hydroxylapatite chromatography. Upon hydroxylapatite chromatography Cbfibl was shown to consist of two components, Cbfibl. l and Cbfibl. 2. Both Cbfibl. l and Cbfibl. 2 were purified to homogeneity using anion-exchange chromatography. SDS-polyacrylamide gel electrophoresis revealed that Cbfibl. l and Cbfibl. 2 had similar molecular weights (approximately 23 500), whereas Cbfib2 displayed a molecular weight of approximately 22500. The enzymes do not appear to be glycosylated. Tryptic digests of all three enzymes, analyzed by high-performance reverse-phase chromatography, suggest that Cbfibl. l and Cbfibl. 2 are closely related and different from Cbfib2. The latter displayed more similarity with Cbfibl. 2 than with Cbfibl. l. Specific fibrinolytic activity for all three enzymes was very similar, but general proteolytic activity varied substantially with Cbfib2 showing a 12-fold higher specific proteolyticactivity when compared to Cbfibl. l and Cbfibl. 2. None of these enzymes exhibited hemorrhagic activity when injected (up to 100 jug) subcutaneously into mice. Cbfibl. l and Cbfibl. 2 action against fibrinogen was directed equally against both the A a-and B^-chains. Against fibrin the rate of degradation of the a-chain was considerably higher than that of the/3-chain. Cbfib2 showed mainly a-fibrin (ogen) ase activity with limited activity on the (3-chain. Several fibrinogen cleavage sites on the Aa-chain have been identified: Cbfibl. l and Cbfibl. 2 cleave at