Effect of a polyhistidine terminal extension on eglin c stability.
Effect of a polyhistidine terminal extension on eglin c stability.
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多组氨酸末端延伸对eglin c 稳定性的影响。
DOI：
10.1006/abio.1998.2808
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Pielak,GJ
中科院分区：
文献类型：
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作者：
Waldner,JC;Lahr,SJ;Edgell,MH;Pielak,GJ
多聚组氨酸末端延伸对从韩国传统发酵食品Doen-Jang分离的芽孢杆菌菌株的Eglin c稳定性培养基的影响。反向纤维蛋白自显影（图2A）显示在纤维蛋白/琼脂指示凝胶上几乎看不到的两条宽带（图2B）。另一方面，几种具有纤溶活性的多肽在纤维蛋白酶谱上被检测为清晰的条带（图2B）。分子标记也可以用眼睛看到，但它们的照片效果不好。已经证明，从Doen-Jang分离的两种芽孢杆菌菌株中的每一种都分泌至少四种表观分子大小约为64、53、38和29 kDa的纤维蛋白溶解酶。蛋白酶的纯化和表征将在别处描述。尽管明胶和酪蛋白是纤溶酶的理想底物（7，8），但所有纤溶酶对底物的敏感性都不如纤溶酶。显然，纤维蛋白是纤溶酶的体内底物。我们利用这一事实，探讨了用纤维蛋白酶谱法测定纤溶酶样酶的可能性，纤维蛋白酶谱法是一种电泳技术，其中纤维蛋白在分离凝胶的浇铸过程中与丙烯酰胺共聚。我们已经证明，酶谱技术可以可靠地用于快速定性评价纤溶酶样酶，这有能力恢复酶活性的凝胶共聚与纤维蛋白后，除去SDS，与其他酶谱技术的灵敏度水平相当。
Effect of a Polyhistidine Terminal Extension on Eglin c Stability culture media of Bacillus strains isolated from Doen-Jang, the Korean traditional fermented food. The reverse fibrin autography (Fig. 2A) shows two broad bands barely visualized on the fibrin/agar indicator gel (Fig. 2B). On the other hand, several polypeptides possessing fibrinolytic activity are detected as sharp, clear bands on the fibrin zymogram (Fig. 2B). Molecular markers are also perceptible by eye; they do not photograph well, though. It has been demonstrated that at least four fibrinolytic enzymes with apparent molecular sizes of approximately 64, 53, 38, and 29 kDa are secreted from each of two Bacillus strains isolated from Doen-Jang. The purification and characterization of the proteases will be described elsewhere. Even though gelatin and casein are satisfactory substrates for plasmin (7, 8), all fibrinolytic enzymes are not sensitive for the substrates as much as plasmin is. Apparently fibrin is an in vivo substrate for plasmin. We took advantage of this fact to explore the possibility of assaying plasmin-like enzymes with fibrin zymography, an electrophoretic technique in which fibrin is copolymerized with the acrylamide during casting of the separating gel. We have demonstrated that the zymographic technique can be used reliably for the rapid qualitative evaluation of plasmin-like enzymes, which have the ability to restore enzymatic activity on the gel copolymerized with fibrin upon removal of the SDS, with a sensitivity level comparable to that of other zymographic techniques.