组织型纤溶酶原激活剂非催化链的结构决定因素，其调节与纤溶酶原激活剂抑制剂-1的结合速率。
Structural determinants of the noncatalytic chain of tissue-type plasminogen activator that modulate its association rate with plasminogen activator inhibitor-1.
作者信息
de Serrano V S, Castellino F J
机构信息
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.
出版信息
J Biol Chem. 1990 Jun 25;265(18):10473-8.
In order to identify the regions of recombinant (r) tissue plasminogen activator (tPA) that mediate its kinetically relevant interaction with r-plasminogen activator inhibitor-1 (rPAI-1), we have determined the second-order association rate (k1) constants of domain-altered variants of tPA with rPAI-1, at 10 degrees C. With two-chain, wild-type recombinant tPA (tcwt-rtPA), obtained by expression of the human cDNA for tPA in five different cell systems (viz. insect cells, human kidney 293 cells, Chinese hamster ovary cells, human melanoma cells, and mouse C127 cells), the average k1 was 1.45 x 10(7) M-1 s-1 (range, 1.34 10(7) M-1 s-1-1.68 x 10(7) M-1 s-1). Since this value was not significantly different for the different tcwt-rtPA preparations, it appears as though the nature of the glycosylation of tPA plays little role in its initial interaction with PAI-1. The k1 determined for tcwt-rtPA was slightly higher than that of 0.87 x 10(7) M-1 s-1, obtained for a similar inhibition of human urokinase by rPAI-1. The k1 value obtained for single-chain (sc) wt-rtPA was approximately 6-fold lower than that of the two-chain molecules, results consistent with previous conclusions on this matter. The k1 value for tcwt-rtPA was not influenced by the presence of epsilon-aminocaproic acid, suggesting that the lysine-binding site associated with the kringle 2 (K2) region of tPA does not modulate the rate of its initial interaction with rPAI-1. Removal of the K2 domain from tPA, by recombinant DNA technology, results in a protein, F-E-K1-P (tc-r delta K2-tPA), containing only the finger (F), growth factor (E), kringle 1 (K1), and serine protease (P) domains. This variant protein was more rapidly inhibited by rPAI-1 (k1 = 3.00 x 10(7) M-1 s-1) than its wild-type counterparts. Deletion of both the K1 and K2 domains resulted in a variant molecule, F-E-P (tc-r delta K1 delta K2-tPA), that was slightly more rapidly inhibited by rPAI-1 (k1 = 2.01 x 10(7) M-1 s-1).(ABSTRACT TRUNCATED AT 400 WORDS)
为了确定重组组织型纤溶酶原激活剂（r-tPA）中介导其与重组纤溶酶原激活剂抑制剂-1（rPAI-1）发生动力学相关相互作用的区域，我们测定了在10℃下tPA结构域改变变体与rPAI-1的二级缔合速率（k1）常数。通过在五种不同细胞系统（即昆虫细胞、人肾293细胞、中国仓鼠卵巢细胞、人黑色素瘤细胞和小鼠C127细胞）中表达人tPA cDNA获得的双链野生型重组tPA（tcwt-rtPA），其平均k1为1.45×10⁷ M⁻¹ s⁻¹（范围为1.34×10⁷ M⁻¹ s⁻¹至1.68×10⁷ M⁻¹ s⁻¹）。由于不同的tcwt-rtPA制剂的该值无显著差异，似乎tPA的糖基化性质在其与PAI-1的初始相互作用中作用不大。测定的tcwt-rtPA的k1略高于rPAI-1对人尿激酶类似抑制作用所获得的0.87×10⁷ M⁻¹ s⁻¹。单链（sc）wt-rtPA获得的k1值比双链分子低约6倍，结果与先前关于此事的结论一致。tcwt-rtPA的k1值不受ε-氨基己酸存在的影响，表明与tPA的kringle 2（K2）区域相关的赖氨酸结合位点不调节其与rPAI-1初始相互作用的速率。通过重组DNA技术从tPA中去除K2结构域，产生一种仅包含指状（F）、生长因子（E）、kringle 1（K1）和丝氨酸蛋白酶（P）结构域的蛋白质F-E-K1-P（tc-rδK2-tPA）。该变体蛋白比其野生型对应物被rPAI-1抑制得更快（k1 = 3.00×10⁷ M⁻¹ s⁻¹）。K1和K2结构域均缺失产生一种变体分子F-E-P（tc-rδK1δK2-tPA），其被rPAI-1抑制得稍快一些（k1 = 2.01×10⁷ M⁻¹ s⁻¹）。（摘要截短于400字）
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