PRGDWR-尿激酶原双功能嵌合分子的性质研究

doi:10.3321/j.issn:1000-3282.2001.02.018

CORC > 北京大学 > 生命科学学院

	PRGDWR-尿激酶原双功能嵌合分子的性质研究; Characterization of Bifunctional Chimeric Molecule of PRGDWR Containing Pro-Urokinase
	党昕 ; 杨晶鑫 ; 茹强 ; 茹炳根
刊名	生物化学与生物物理进展
	2001
关键词	PRGDWR片段尿激酶原溶栓活性血小板聚集 PRGDWR peptide single chain urokinase-type plasminoge n activator thrombolytic ability platelet aggregation
DOI	10.3321/j.issn:1000-3282.2001.02.018
英文摘要	为赋予单链尿激酶型纤溶酶原激活剂(single chain urokinase-type plasminogen activator, scu-PA, 尿激酶原)以抗血小板聚集的功能，在scu-PA的kringle区118 位Gly与119位Leu之间插入PRGDWR序列（insert mutant B，InB）.利用甲醇酵母（ Pichia pastoris）进行分泌表达，经金属离子螯合亲和层析与S强阳离子交换层析，得到纯蛋白.实验测定InB对人工合成底物S-2444的酰胺解活性为5 900 IU/mg，动力学常数为：KInBm,S-2444=56.8 μmolL-1，kInBca t, S-2444=0.33 s-1；水解天然底物plasminogen的动力学常数为：KInB m,plg=0.397 μmolL-1，kInBcat,plg=0.0164 s-1 .InB激活plasminogen的反应在有fibrin存在条件下InB的活性为无fibrin条件下的46.3 ％.该突变体在体外激活plasminogen的活性与同一系统表达的野生型scu-PA基本相同.该突变体表现出较强的抗血小板聚集活性，IC50＝12.7 μmolL-1，而野生型scu-PA无此功能.实验表明scu-PA的K区插入突变体InB是一种极具潜力的双功能溶栓分子.; In order to obtain the bifunctional chimeric molecule of single-chain urokinase-type plasminogen activator (scu-PA) which can inhibit platelet aggregation, PRGDWR peptide was inserted into the site between Gly 118 and Leu119 (called insertion mutant B, InB). The recombinant gene of InB was expressed by Pichia pastoris. The secreted protein was purified by metal chelate affinity and strong cation exchange chromatography. The amidolytic ability of mutant InB is 5 900 IU/mg, the kinetic constants is: KInB m,plg=56.8 μmolL-1，kInBcat,plg=0.33 s- 1. The kinetic constants of plasminogen activation reaction is: KInB m,plg=0.397 μmolL-1，kInBcat,plg=0.0164 s-1. Fibrin inhibit the catalytiv ability of InB during plasminogen activatio n, the influence factor is 0.463(means InB remain 46.3% of the catalytic abili ty when fibrin was involved in the reaction system). The mutant not only has alm ost the same catalytic ability as wild type scu-PA, but also has strong ability of anti-platelet aggregation(compared with scu-PA), IC50 of InB is 12.7 μmolL-1.; SCI(E); 中文核心期刊要目总览(PKU); 中国科学引文数据库(CSCD); 1; 2; 203-209; 28
语种	中文
内容类型	期刊论文
源URL	[http://ir.pku.edu.cn/handle/20.500.11897/45894]
专题	生命科学学院
推荐引用方式 GB/T 7714	党昕,杨晶鑫,茹强,等. PRGDWR-尿激酶原双功能嵌合分子的性质研究, Characterization of Bifunctional Chimeric Molecule of PRGDWR Containing Pro-Urokinase[J]. 生物化学与生物物理进展,2001.
APA	党昕,杨晶鑫,茹强,&茹炳根.(2001).PRGDWR-尿激酶原双功能嵌合分子的性质研究.生物化学与生物物理进展.
MLA	党昕,et al."PRGDWR-尿激酶原双功能嵌合分子的性质研究".生物化学与生物物理进展 (2001).