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纤溶酶原激活物抑制因子-1突变体E350G,E351K的构建及性质研究
引用本文:隋广超,张曼,胡美浩. 纤溶酶原激活物抑制因子-1突变体E350G,E351K的构建及性质研究[J]. 中国生物化学与分子生物学报, 1996, 12(5): 516-520
作者姓名:隋广超  张曼  胡美浩
作者单位:北京大学生命科学学院
摘    要:利用PCR扩增和合成突变引物的方法,将PAL-1的Glu350和Glu351分别突变为Gly和Lys,在大肠杆菌中表达并分离纯化突变体PAL-1(E350G,E351K),用盐酸胍激活并以ELISA法确定它与野生型rPAI-1的相对含量。通过对u-PA,t-PA抑制的动力学研究表明,突变体与野生型rPAI-1相比,对u-PA和t-PA的抑制活性都有明显下降,由活性态向潜伏态转变的半寿期也由0.83h缩短为0.57h。

关 键 词:PAI-1  突变体  u-PA  t-PA  抑制活性  
收稿时间:1996-10-20

Construction and Characterization of Plasminogen Activator Inhibitor Mutant (E350G , E351K)
Sui Guang-Chao,Zhang Man,Hu Mei-Hao. Construction and Characterization of Plasminogen Activator Inhibitor Mutant (E350G , E351K)[J]. Chinese Journal of Biochemistry and Molecular Biology, 1996, 12(5): 516-520
Authors:Sui Guang-Chao  Zhang Man  Hu Mei-Hao
Affiliation:(College of Life Sciences. Peking University, Beijing 100871
Abstract:Plasminogen activator inhibitor-1 (PAI-1 ) is a-physiological inhibitor of urine-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA).Using polymerase chain reaction (PCR) amplification and a synthesized mutation primer,a PAI-1 mutant, PAI-1 (E350G, E351K), was generated. The PAI-1 mutant expressed in E. coli was separated, followed by the activation with Guanidine HCI,and then the relative amounts of the mutant and wild type PAI-1 were determined by double-antibody ELISA. The kinetic studies showed that the second order rate constants , K_1, of wild type PAI-1 ,was 3. 38± 0. 20 towards u-PA and 3. 24± 0. 26 towards t-PA, while the K1 values of the mutant was 2. 84 ± 0. 12 towards u-PA and 2. 24± 0. 45 towards t-PA. It was also found that the half life of the mutant,for conversion from active form to latent form ,changed from 0. 83± 0. 11 h of wild type PAI-1 to 0. 57 ± 0. 04 h.The results suggested that the simultaneous mutations of Glu350 and Glu351might influence the inhibitory activities of PAI-1 towards u-PA and t-PA,as well as shorten the half life of its active form.
Keywords:PAI-1   Mutant   u-PA   t-PA   Inhibitory activity
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