Higazi A, Cohen R L, Henkin J, Kniss D, Schwartz B S, Cines D B
Department of Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, Philadelphia 19104, USA.
J Biol Chem. 1995 Jul 21;270(29):17375-80. doi: 10.1074/jbc.270.29.17375.
Single-chain urokinase (scuPA), the unique form of urokinase secreted by cells, is converted to an active two-chain molecule through the cleavage of a single peptide bond by plasmin and other specific proteinases. Although scuPA may express limited enzymatic activity, its contribution to plasminogen activation on cell surfaces remains uncertain. Further, although it is well known that scuPA binds to a specific extracellular urokinase-type plasminogen activator receptor, the effect of this interaction on the enzymatic activity of scuPA has not been described. In the present paper we report that the binding of scuPA to cellular an to recombinant soluble urokinase-type plasminogen activator receptors (suPAR) increases its catalytic activity as measured by the cleavage of a urokinase-specific chromogenic substrate. suPAR increased the Vmax of scuPA 5-fold with little change in its Km. suPAR also stimulated the plasminogen activator activity of scuPA by decreasing its Km for Glu-plasminogen from 1.15 microM to 0.022 microM and by increasing the kcat of this reaction from 0.0015 to 0.022 s-1. Preincubation of scuPA with suPAR also enhances its susceptibility to inhibition by plasminogen activator inhibitor type 1, consistent with exposure of its catalytic site. The activity of scuPA bound to suPAR is not accompanied by cleavage of scuPA, which continues to migrate as a single band in SDS-polyacrylamide gel electrophoresis under reducing conditions. Moreover, suPAR increases the plasminogen activator activity of a plasmin-insensitive variant, scuPA (scuPA-Glu158), as well. Enhancement of scuPA activity by suPAR is both prevented and reversed by its aminoterminal fragment (amino acids 1-135), which competes for receptor binding, suggesting that continued binding to the receptor is required for expression of scuPA's enzymatic activity. Thus, our data suggest that scuPA may undergo a reversible transformation between a latent and an active state. The urokinase receptor may induce or stabilize scuPA in its active conformation, thereby contributing to the initiation of plasminogen activation on cell surfaces.
单链尿激酶（scuPA）是细胞分泌的尿激酶的独特形式，通过纤溶酶和其他特定蛋白酶切割单个肽键而转化为活性双链分子。尽管scuPA可能表现出有限的酶活性，但其对细胞表面纤溶酶原激活的贡献仍不确定。此外，尽管众所周知scuPA与特定的细胞外尿激酶型纤溶酶原激活剂受体结合，但这种相互作用对scuPA酶活性的影响尚未见报道。在本文中，我们报告scuPA与细胞型和重组可溶性尿激酶型纤溶酶原激活剂受体（suPAR）的结合增加了其催化活性，这通过尿激酶特异性显色底物的切割来测定。suPAR使scuPA的Vmax增加了5倍，而其Km变化很小。suPAR还通过将scuPA对Glu-纤溶酶原的Km从1.15 microM降低到0.022 microM，并将该反应的kcat从0.0015增加到0.022 s-1，刺激了scuPA的纤溶酶原激活剂活性。scuPA与suPAR的预孵育也增强了其对1型纤溶酶原激活剂抑制剂抑制的敏感性，这与催化位点的暴露一致。与suPAR结合的scuPA的活性并不伴随着scuPA的切割，在还原条件下，scuPA在SDS-聚丙烯酰胺凝胶电泳中继续作为单一条带迁移。此外，suPAR也增加了纤溶酶不敏感变体scuPA（scuPA-Glu158）的纤溶酶原激活剂活性。suPAR对scuPA活性的增强被其氨基末端片段（氨基酸1-135）阻止并逆转，该片段竞争受体结合，表明持续与受体结合是scuPA酶活性表达所必需的。因此，我们的数据表明scuPA可能在潜伏状态和活性状态之间经历可逆转变。尿激酶受体可能诱导或稳定scuPA处于其活性构象，从而有助于细胞表面纤溶酶原激活的起始。
