Purification and identification of two structural variants of porcine tissue plasminogen activator by affinity adsorption on fibrin.
Purification and identification of two structural variants of porcine tissue plasminogen activator by affinity adsorption on fibrin.
复制标题
通过纤维蛋白亲和吸附纯化和鉴定猪组织纤溶酶原激活剂的两种结构变体。
DOI：
10.1016/0304-4165(82)90105-2
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发表时间：
1982
期刊：
影响因子：
--
通讯作者：
M. Rånby
中科院分区：
文献类型：
--
作者：
P. Wallén;N. Bergsdorf;M. Rånby
用亲和吸附法从脱脂血化的心脏组织中纯化了猪组织纤溶酶原激活物。用硫酸铵沉淀从酸性组织提取物中制备粗馏分。该组分的组织活化剂通过纤维蛋白吸附和KSCN洗脱分离。该程序还包括精氨酸- sepharose层析和两个凝胶过滤步骤。与国际尿激酶参考制剂相比，最终产物的比活性为250000 IU/mg（±16000）。活性硫酸铵沉淀物的产率约为28%。一个约。比活性增加了7000倍，其中大部分是在纤维蛋白步骤中实现的。天然组织纤溶酶原激活剂由单链分子组成，sds -聚丙烯酰胺凝胶电泳测定分子量为64000。在之前的报告中，有人声称激活剂是由两个二硫连接的多肽链组成的。这些结果是由于组织提取物中存在的蛋白水解活性引起的制备伪影。在纯化过程中引入蛋白酶抑制剂抑肽蛋白和6-氨基己酸，消除了蛋白酶污染物的影响，从而产生了单链活化剂。用纤溶酶处理将天然的单链组织激活剂转化为由两条大小相等的链（m32000）通过二硫键连接而成的变体。这种改性活化剂与对蛋白水解酶保护不足时获得的活化剂没有区别。通过测定h - d - val - gly - arg -p-硝基analide，纤溶酶的裂解使酶解活性增加了8倍。通过血块溶解测定的纤溶活性仅略有增加。讨论了卵裂的生理意义。
Porcine tissue plasminogen activator has been purified from delipidized heart tissue by affinity adsorption to fibrin. A crude fraction is prepared from an acid tissue extract by precipitation with ammonium sulphate. The tissue activator of this fraction is isolated by adsorption on fibrin and elution with KSCN. The procedure also includes chromatography on arginine-Sepharose and two gel-filtration steps. The final product has a specific activity of 250 000 IU/mg (±16 000) as compared to an international urokinase reference preparation. The yield calculated from the active ammonium sulphate precipitate is about 28%. An approx. 7 000-fold increase of specific activity is obtained, most of which is achieved in the fibrin step. The native tissue plasminogen activator consists of a single chain molecule with a molecular weight of 64 000 as measured by SDS-polyacrylamide gel electrophoresis. In a previous report, it was claimed that the activator is composed of two disulphide-connected polypeptide chains. These results were due to a preparation artefact, caused by proteolytic activity present in the tissue extracts. The introduction of the protease inhibitor aprotinin and 6-amino-hexanoic acid in the purification procedure has abolished the effect of the protease contaminant, leading to the production of a one-chain activator. Treatment with plasmin transforms the native, one-chain tissue activator into a variant composed of two chains of about equal size (M r 32 000) connected by disulphide bonding. This modified activator is indistinguishable from the one obtained at insufficient protection against proteolytic enzymes. The cleavage by plasmin causes about an 8-fold increase of amidolytic activity as measured on H-D-Val-Gly-Arg-p-nitroanalide. The fibrinolytic activity as measured by clot lysis is only slightly increased. The physiological significance of the cleavage is discussed.