一种新的纤溶酶原激活反应动力学研究方法

 在溶栓研究中 ,纤溶酶原激活剂 (plasminogen activator,PA)激活纤溶酶原 (plasminogen,Plg)反应的动力学常数的测定占有重要地位 .在前人的研究中 ,虽然进行了这项实验 ,但是并未给出一个方便、快捷的测定方法 .所以建立一种更准确 ,更适合一般的实验室条件的 PA分子激活 Plg反应动力学常数的测定方法是必要的 .在对该反应进行数学分析的基础上 ,得到由可测量表示的纤溶酶 (plasmin,Plm)生成速度 (v(Plm) )的计算公式 ,由 v(Plm)及相应已知量可进一步推导出 Km、kcat的表达式 ,最终测得相应动力学常数 .用这种方法测定的由甲醇酵母 (pichia pastoris)表达的人单链尿激酶型纤溶酶原激活剂 (human single chain urokinase- PA,scu- PA)激活 Plg的反应的 Km=0 .648μmol·L-1,kcat=0 .0 62 6s-1,与文献报导相符 (Km=0 .4～ 1 .1 μmol· L-1,kcat=0 .0 2～ 0 .0 93) s-1,说明此方法是可靠的 .又因该法只需相应底物及酶标仪且为连续测定 ,所以十分简便 .     

单链尿激酶型纤溶酶原激活剂Kringle结构域催化功能探讨

 Kringle(K)结构域广泛存在于与凝血和纤溶相关的各种因子中 .虽然 K结构域在一级结构上是一种比较保守的结构域 (与其他结构域相比 ) ,但是 K结构域的功能在各种因子中的作用有很大的区别 .为探讨单链尿激酶型纤溶酶原激活剂 (scu- PA)的 K结构域功能 ,构建了在 scu- PA的 K结构域的 1 1 8位 Gly与 1 1 9位 Leu之间插入 PRGDWR序列的突变体 (称为 insert mutant B,In B) ,并测定了野生型 scu- PA与 In B的纤溶相关反应的动力学常数 . scu- PA与 In B水解 S- 2 4 4 4反应的 Km 值无明显变化 (分别为 60 .4与 56.8μmol·L-1) ,而 In B的 kcat值 (0 .33s-1)比 scu- PA的 kcat值 (7.31 s-1)降低很多 ;In B激活纤溶酶原反应的 Km 值 (0 .397μmol· L-1)比 scu- PA Km 值(0 .648μmol·L-1)降低 40 % ,但 kcat值 (0 .0 1 65s-1)比 scu- PA的 kcat值 (0 .0 62 6s-1)降低 74% .以上结果说明 :K结构域主要与反应活性相关 ,而与酶及底物的亲和性无关 .     

尿激酶型纤溶酶原激活剂的研究

尿激酶型纤溶酶原激活剂u-PA(urokinase-type plasminogen activator)属于丝氨酸蛋白酶类,能激活细胞外基质中丰富的纤溶酶原生成纤溶酶,从而催化细胞外基质降解,对纤溶和癌细胞侵染及扩散等一系列生理和病理过程中发生的胞外蛋白水解起重要调节作用。 人u-PA基因位于第10号染色体之上,表达产生一个约54kD的单链糖基化多肽——尿激酶原。尿激酶原经纤溶酶在其158位赖氨酸     

纤溶酶原激活物抑制因子—1蛋白分子的结构与功能

纤溶酶原激活物抑制因子－１（ＰＡＩ－１）是组织型纤溶酶原激活物（ｔ－ＰＡ）和尿型纤溶酶原激活物（ｕ－ＰＡ）的特异性抑制剂。对ＰＡＩ－１分子的结构与功能的认识,有助于了解ＰＡＩ－１作用的机理。本综述了近年来对ＰＡＩ－１蛋白分子结构与功能研究的进展,介绍了ＰＡＩ－１分子中一些区域的作用以及影响ＰＡＩ－１抑制活性的一些因子。     

凝血酶激活的单链尿激酶型纤溶酶原激活剂的构建、表达、纯化和性质研究

用Ｋｕｎｋｅｌ突变法,将单链尿激酶型纤溶酶原激活剂（ｓｃｕ－ＰＡ）ｃＤＮＡ基因中编码Ｐｒｏ１５５—Ｌｙｓ１５８的片段定点突变,并将此突变的ｓｃｕ－ＰＡ（ｔｓｃｕ－ＰＡ）的ｃＤＮＡ克隆到表达载体ｐＣＭ－β－ｎｅｏ中,与ｐＣＭ－ｄｈｆｒ共转染ＣＨＯ／ＤＨＦＲ－细胞．获得的稳定表达株在无血清培养基中２４ｈ的表达量为６２０ＩＵ／１０６细胞．经锌离子螯合Ｓｅｐｈａｒｏｓｅ亲和层析得到ｔｓｃｕ－ＰＡ纯品．ＳＤＳ－ＰＡＧＥ显示ｔｓｃｕ－ＰＡ分子量为５３ｋＤ左右,与预期的结果相符．ｔｓｃｕ－ＰＡ是由凝血酶激活而不是由纤溶酶激活,但激活后也能转变为双链分子（ｔｃｕ－ＰＡ）．ｔｓｃｕ－ＰＡ仍保持了ｓｃｕ－ＰＡ的血纤维蛋白亲和性．酶动力学研究表明,激活后的ｔｓｃｕ－ＰＡ水解Ｓ２４４４的Ｋｍ和Ｋｃａｔ值与高分子量尿激酶（ＨＵＫ）相似．体外溶栓实验结果表明,ｔｓｃｕ－ＰＡ可以选择性地溶解富含凝血酶的血凝块,对贫凝血酶的血凝块作用不大     

组织型纤溶酶原激活剂的纯化制备

简述了用于大规模生产组织型纤溶酶原激活剂(tPA)的重组动物细胞及其培养工艺。从重组tPA的大规模、快速纯化的角度考虑,对tPA的纯化制备方法进行了简要评述。     

抗PAI抑制作用的纤溶酶原激活剂突变体的活性研究

目的:重组表达抗PAI抑制作用的t-PA突变体,经诱导表达、复性、纯化后进行生物学活性和酶动力学分析。方法:构建pBV220-tpa重组表达质粒,经DNA测序确认后,转化至大肠杆菌DH5a,温控诱导表达,凝胶过滤法对包涵体蛋白进行初步纯化,复性后,过刺桐胰蛋白酶亲和层析柱纯化,酶动力学分析其活性。结果:测序证实,t-PA突变体的DNA序列正确,表达蛋白占总菌体蛋白的30%,经纯化后纯度达90%以上,比活性为7.0×108IU/mg,t-PA突变体与PAI-1反应后,其活性未受到抑制。t-PA突变体酶的米氏常数Km为0.5298,最大水解速度Vmax为0.0595。结论:经生物学活性测定,表达蛋白能够明显抵抗PAI的抑制作用,并具有良好的生物活性,该突变体有可能成为用量更少、疗效更佳的新型溶栓药物。     

组织纤溶酶原激活剂突变体（La—tPA）转基因鼠的建立

用羊β－乳球蛋白基因（ＢＬＧ）５’区５＊１０＾３ｂ（１０＾３ｂ,旧称ｋｂ）为调序列,构建了乳腺表达组织纤溶酶原激活剂突变体载体。对５４０枚小鼠受精卵进行显微注射,经ＰＣＲ和Ｓｏｕｔｈｅｒｎｂｌｏｔ检测,获得６只整合有人Ｌａ－ｔＰＡ的转基因小鼠,整合率为３２％。     

Fibrin Targeting of Echogenic Liposomes with Inactivated Tissue Plasminogen Activator

Fibrin-specific molecular targeting strategies are desirable for site-specific imaging and treatment of late stage atheroma, but fibrin-specific antibodies are difficult to produce and present immunogenicity problems. Tissue plasminogen activator (tPA) is an endogenous protein that has been shown to bind fibrin with high affinity and may circumvent antibody difficulties. Use of tPA-derived proteins or peptides, however, requires that the plasminogen-activating proteolytic activity be neutralized or removed. As an initial step in determining the feasibility of this targeting strategy, human recombinant tPA (Activase®) was irreversibly inhibited with D-phe-L-pro-L-arg-chloromethyl ketone (PPACK) and conjugated to intrinsically echogenic liposomes (ELIP) by a thioether coupling protocol. Fibrin-binding affinities were assessed with a novel two‐stage fibrin pad ELISA. We achieved 95–99% inactivation, while retaining both tPA fibrin-binding activities of K_D ～ 2 nM and 33 nM. Thermodynamic analysis of the PPACK-inactivated tPA (tPA(P)) revealed highly exothermic interactions, indicative of ionic associations, especially for the higher affinity. The conjugation efficiency of tPA(P) to ELIP was within the range of that previously achieved for IgG and exhibited satisfactory fibrin targeting, characterized by striking increases of enthalpy and entropy increments. Evidence for coupling of noncovalent association energetics with the phosphatidylethanolamine major phase transition, observed in previous IgG antibody conjugations, was also evident in this case, but the nature of the transduction mechanism was different. These results demonstrate that tPA-derived components lacking proteolytic activity can be employed as fibrin-targeting agents for delivery of therapeutic and diagnostic formulations.     

The potential improvement of thrombolytic therapy by targeting with bispecific monoclonal antibodies: Why they are used and how they are made

The generation of the proteolytic enzyme plasmin from its inactive precursor plasminogen, mediated by so called plasminogen activators, is the essential step in thrombolytic therapy. Plasmin is responsible for the degradation of the insoluble fibrin, the major component of a thrombus, to soluble fibrin degradation products. So far, the use of the more recently developed thrombolytic agents single-chain urokinase-type plasminogen activator (scu-PA) and tissue-type plasminogen activator (t-PA) were disappointing, mainly due to some of their negative propertiesin vivo, i.e., rapid inhibition and/or hepatic clearance. Besides some background information on the haemostatic balance; t-PA and scu-PA structure; and mechanisms of action, we here review some reported attempts to improve on these agents for thrombolytic therapy following various strategies. One of the more potential strategies, antibody-targeted thrombolytic therapy using bispecific monoclonal antibodies, is discussed somewhat more extensively, as are the several procedures that can befollowed for bispecific antibody preparation.     

PRGDWR-尿激酶原双功能嵌合分子的性质研究

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：K_m,S-2444^InB=56.8 μmol·L^-1,k_cat,S-2444^InB=0.33 s^-1;水解天然底物plasminogen的动力学常数为：K_m,plg^InB=0.397 μmol·L^-1,k_cat,plg^InB=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 μmol·L^-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.     

重组人组织型纤溶酶原激活剂(rht-PA)及其突变体的纯化

稳定高效表达重组人组织型纤溶酶原激活剂 (rht PA)的CHO细胞株和表达组合突变体的细胞株进行了 3L转瓶培养 .将培养上清分别进行了Lys Sepharose 4B亲和层析和Zn2 + Sepharose 4B层析两步纯化 ,rht PA纯度提高了 5 34倍 ,比活达 2 5× 10 5IU mg ,产率为 73% ;突变体纯度提高了1119倍 ,比活达 5 9× 10 5IU mg ,产率为 6 9% .纯化产物SDS PAGE分析显示 ,rht PA和突变体基本都呈单一条带 ,扫描分析均达到 98%以上纯度 .rht PA和突变体在纯化系统中的行为作对照分析发现 ,突变体的构建思想在Lys Sepharose 4B亲和层析过程中有充分体现 .这两步层析组合是很好的纯化t PA及其突变体的方法 ,尤其是Lys Sepharose 4B纯化突变体效果更好     

导向性纤溶酶原激活剂的研究

溶栓疗法是血栓治疗中的一种重要措施.研制具有高选择性的导向性纤溶酶原激活剂有着重大的理论意义和实用价值.采用血栓特异的单克隆抗体及其片段来介导溶栓剂已展示出较好的应用前景.双功能抗体以及同时具有抗栓,抗凝活性的小肽正逐渐拓宽人们有关导向分子研制的视野.所有这一切都将随着分子生物学技术的不断完善而付诸实现.     

人黑色素瘤细胞分泌的组织纤溶酶原激活剂(t-PA)的纯化

 本文报道了培养的人黑色素瘤细胞分泌的组织纤溶酶原激活剂(t-PA)的纯化方法。Bowes株人黑色素瘤细胞的分泌产物,经CM-Sephadex C--50层析,赖氨酸-Sepharose 4B,苯甲眯-sepharose 4B亲和层析后,即可得到纯化470倍的蛋白纯品。样品经聚丙烯酰胺凝胶电泳鉴定为均一单带,测得其分子量约为72kD。纯化的t-PA与尿激酶相比较,发现前者有更高亲和纤维蛋白的能力。     

Developmental Pattern of Plasminogen Activator Activity in Chick Brain Hemispheres

Plasminogen activators play key roles in several developmental events. In previous works we demonstrated the existence of typical developmental patterns of protease activity in the chick optic lobe and cerebellum. The aim of this work is to study the temporal pattern of development of plasminogen activator activity in the brain hemispheres. Plasminogen activator activity was assayed in soluble fractions derived by ultracentrifugation from Triton X-100 treated membrane fractions by using a radial fibrinolytic assay. Employing different inhibitors and anti-plasminogen activators antibodies we showed that developing brain hemispheres express only one type of enzyme which corresponds to the urokinase-type. Other results indicate that the protease activity displays a temporal pattern which completely differs from those of general parameters of development. This suggests that the plasminogen activator activity is developmentally regulated and could display specific functions during particular stages of development.     

人组织型纤溶酶原激活剂(t-PA)组合突变体FrGGl在CHO细胞中的高效表达

为了得到ｔＰＡ组合突变体ＦｒＧＧＩ在ＣＨＯ细胞中的高效表达,将表达质粒筛选基因启动子上游的增强子（ｅｎｈａｎｃｅｒ）去除,构建了ＦｒＧＧＩ真核表达质粒ｐＺＬＦｒＧＧＩ。酶切线性化后,采用大剂量ＤＮＡ电击介导法,转染ｄｈｆｒ基因缺陷型中国仓鼠卵巢细胞系（ＣＨＯｄｈｆｒ－）。氨甲喋呤（ＭＴＸ）筛选转染细胞,混合加压,挑选克隆,在１×１０－７ｍｏｌ／ＬＭＴＸ压力下,获得表达水平达１５００～２５００ＩＵ／１０６细胞·２４ｈ的细胞株。此细胞株表达水平稳定,形态良好,倍增时间约为３６ｈ,且有进一步提高表达水平的潜能,有望发展为工程细胞株     

Characterization of the Binding Sites for Plasminogen and Tissue-Type Plasminogen Activator in Cytokeratin 8 and Cytokeratin 18

Cytokeratin 8 (CK8) is an intermediate filament protein that penetrates to the external surfaces of breast cancer cells and is released from cells in the form of soluble heteropolymers. CK8 binds plasminogen and tissue-type plasminogen activator (t-PA) and accelerates plasminogen activation on cancer cell surfaces. The plasminogen-binding site is located at the C-terminus of CK8. In this study, we prepared GST-fusion proteins which contained either 174 amino acids from the C-terminus of CK8 (CK8f) or 134 amino acids from the C-terminus of CK18 (CK18f). A third GST-CK fusion protein was identical to CK8f except that the C-terminal lysine was mutated to glutamine (CK8f_K483Q). CK8f bound plasminogen; the K _D was 0.5 M. Binding was completely inhibited by ACA. CK8f_K483Q also bound plasminogen, albeit with decreased affinity (K _D 1.5 M). CK18f did not bind plasminogen at all. All three fusion proteins bound t-PA equivalently, providing the first evidence that CK18 may function as a t-PA receptor. t-PA and plasminogen cross-competed for binding to CK8f. Thus, t-PA and plasminogen cannot bind to the same CK8f monomer simultaneously. Nevertheless, CK8f still promoted plasminogen activation, probably reflecting the fact that CK8f was purified in dimeric or tetrameric form. These studies demonstrate that CK8 may promote plasminogen activation by t-PA only when present in an oligomerized state. CK18 may participate in the oligomer, together with CK8, based on its ability to bind t-PA.