Zimmerman M, Quigley J P, Ashe B, Dorn C, Goldfarb R, Troll W
Proc Natl Acad Sci U S A. 1978 Feb;75(2):750-3. doi: 10.1073/pnas.75.2.750.
A direct rate assay for plasminogen activator has been developed using a synthetic fluorogenic peptide substrate, 7-(N-Cbz-glycylglycylargininamido)-4-methylcoumarin trifluoroacetate. The assay correlates well with the standard 125I-labeled fibrin plate assay using highly purified urokinase, culture fluids from WI-38, Chinese hamster vary or HeLa cells, or Rous sarcoma virus-transformed chick fibroblasts as the source of plasminogen activator. The assay is sensitive, rapid, and linear throughout a wide range of enzyme concentrations. With this substrate it is possible to determine inhibitor profiles for the various plasminogen activators, independently of the interfering potential of plasmin. All of the enzymes tested are inhibited by leupeptin and antipain but not by the related aldehydes, elastatinal and chymostatin. The macromolecular inhibitors soybean trypsin inhibitor and trasylol have little or no effect on the plasminogen activators tested. This substrate should be useful for the study of the effect of various agents on functional changes in cells secreting this enzyme and also should allow kinetic measurements of potential inhibitors.
已开发出一种用于纤溶酶原激活剂的直接速率测定法,使用合成的荧光肽底物7-(N-苄氧羰基-甘氨酰甘氨酰精氨酰胺基)-4-甲基香豆素三氟乙酸盐。该测定法与使用高度纯化的尿激酶、WI-38细胞培养液、中国仓鼠卵巢细胞或HeLa细胞、或劳氏肉瘤病毒转化的鸡成纤维细胞作为纤溶酶原激活剂来源的标准125I标记纤维蛋白平板测定法相关性良好。该测定法灵敏、快速,在很宽的酶浓度范围内呈线性。使用这种底物,可以独立于纤溶酶的干扰潜力来确定各种纤溶酶原激活剂的抑制剂谱。所测试的所有酶都被亮抑酶肽和抑肽酶抑制,但不被相关的醛类、弹性蛋白酶抑制剂和抑糜酶素抑制。大分子抑制剂大豆胰蛋白酶抑制剂和抑肽酶对所测试的纤溶酶原激活剂几乎没有影响。这种底物对于研究各种试剂对分泌这种酶的细胞功能变化的影响应该是有用的,并且还应该允许对潜在抑制剂进行动力学测量。