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1.
蚯蚓体内一种纤溶酶原激活剂(e-PA)的分离纯化 总被引:21,自引:4,他引:21
为获得一种高效,低廉的溶栓药物,从赤子爱胜蚓(Eiseniafaetida)体内分离纯化出一种可体外激活纤溶酶原从而间接降解纤维蛋白的酶(e-PA).纯化过程包括:粗品的盐析,离子交换层析,凝胶过滤层析及疏水相互作用层析.该组份是由二个亚基通过疏水相互作用维系在一起的.通过凝胶过滤层析,可测得全酶的分子量为45000;SDS电泳显示大、小亚基的分子量分别是26000与18000;而质谱法测得的大、小亚基的分子量分别为24556.7与15546.6.对大小亚基进行了氨基酸组成分析,结果显示大亚基不含Lys而小亚基不含Cys.测定了大亚基N端25个氨基酸序列:VIGGTNASPGEIPWQLSQQRQSGSW.并与部分已知蛋白质序列进行了比较.e-PA在纤维蛋白平板上表现有三种不同的纤溶活性 相似文献
2.
蚯蚓体内一种纤溶酶原激活剂(e-PA)对BAEE的降解 总被引:2,自引:0,他引:2
以苯甲酰-L-精氨酸乙酯(benzoyl-L-arginineethylester,BAEE)为底物,研究了蚯蚓体内纤溶酶原激活剂(plasminogenactivatorfromEiseniafetida,e-PA)的酶学性质.酶促反应的最适pH为8.4,e-PA降解BAEE的Km为1.24±0.16×10-5mol/L,Kcat为13.80±4.02s-1.测定了构成e-PA的大,小亚基分别降解BAEE的Km和Kcat.结果表明,大亚基的Km与全酶的Km相差不多,但比小亚基小约10倍,即对底物的亲和力比小亚基强约一个数量级.大小亚基的Kcat比较接近,分别是全酶的1/6和1/3.研究了8种抑制剂对e-PA降解BAEE活性的影响,其中pepstatin和E-64(一种巯基抑制剂)对酶促反应有激活作用,TPCK,TL-CK,PMSF,chymostatin和leupeptin对其有不同程度的抑制作用,EDTA对e-PA的活性没有影响.对e-PA的BAEE活性和e-PA的纤溶活性之间作了比较. 相似文献
3.
蚯蚓体内一种纤溶酶原激活剂(e-PA)对ATEE的降解 总被引:3,自引:0,他引:3
赤子爱胜蚓(Eiseniafetida)体内的一种纤溶酶原激活剂(e-PA)能够降解人工合成底物N-乙酰-L-酪氨酸乙酯(ATEE),该降解反应的最适pH为8.5,而且在0.2mol/LNa2HPO4中的活性要强于在0.05mol/LTris-HCl(pH8.5)中.分别测定了e-PA的大小亚基及全酶在0.2mol/LNa2HPO4与0.05mol/LTris-HCl(pH8.5)两种体系中的Km和Kcat.结果表明,在0.2mol/LNa2HPO4中,全酶的ATEE活性远远高于大小亚基单独的ATEE活性,而在0.05mol/LTris-HCl(pH8.5)中则没有这种现象.从蛋白质结构的角度对这一结果作了解释.用不同抑制剂和e-PA作用,结果表明,pepstatin,E-64和EDTA对e-PA的ATEE活性都有不同程度的抑制,这一点与e-PA的BAEE活性不同. 相似文献
4.
蚯蚓纤溶酶原激活剂的分离纯化及其性质 总被引:5,自引:0,他引:5
为了从赤子爱胜蚓中获得主要表现为纤溶酶原激活活性的蛋白酶,采用盐析、离子交换层析、凝胶过滤层析和疏水吸附层析从蚯蚓组织匀浆中纯化出6种具有纤溶活性的酶组分(F1、F2、F3、F4、F5和F6).它们均为单一多肽链;表观分子量分别为28500、29500、26100、26300、14850和32800;经非还原型SDSPAGE和扫描仪扫描,纯度分别为100%、95.2%、96.5%、93.6%、98%和97.8%;等电点(pI)均不高于3;SDSPAGE后,用Schiff试剂染色显示F5为糖蛋白;纯化的6种酶于20℃~50℃保温1h,酶活力基本不变;F1和F2、F3和F4、F5和F6分别在pH4~10、4~11、7~12范围内稳定;水解BAEE试验及纤溶活性抑制试验表明,F6既是丝氨酸蛋白酶,又是含金属离子的蛋白酶,其它5种酶为胰蛋白酶样的丝氨酸蛋白酶;免疫双扩散试验结果初步表明,F1和F5以及它们和其它4种组分之间无共同的抗原决定簇;用纤维蛋白平板法及以ChromozymU和ChromozymPL为底物测定,除F1外,其余5种酶的纤溶酶原激活活性明显强于其直接纤溶活性. 相似文献
5.
尿激酶型纤溶酶原激活剂的研究 总被引:1,自引:0,他引:1
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 相似文献
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7.
蚯蚓纤溶酶原激活剂(e-PA)小亚基活性中心的酶学性质及CD光谱的研究 总被引:2,自引:0,他引:2
蚯蚓纤溶酶原激活剂(e-PA)具有多种底物特异性.对构成其的小亚基的活性中心的研究有利于阐述e-PA的结构与功能的关系.利用胰蛋白酶的特异性抑制剂TLCK(N-α-p-tosyl-L-lysinechloromethylketone)对小亚基进行抑制活性的研究,结果表明TLCK对酶促反应的抑制曲线与可逆抑制曲线相似,但在抑制剂存在下的最大反应速度与抑制剂不存在时的最大反应速度不同.结合小亚基在TLCK存在下的CD光谱变化,证明了小亚基分子表面存在两个活性位点,这两个活性位点对TLCK的敏感性不同,从而造成TLCK抑制行为的异常.推测不同的活性结构对应于不同的底物特异性. 相似文献
8.
重组纤溶酶原激活剂抑制剂-2基因在HT1080亚克隆中的表达 总被引:1,自引:0,他引:1
重组纤溶酶原激活剂抑制剂-2基因在HT1080亚克隆中的表达曹祥荣(南京师范大学生物学系,210024)关键词纤溶酶原激活剂,基因重组,纤溶酶原激活剂抑制剂活性表达目前认为纤溶酶系统是肿瘤细胞浸润和转移蛋白水解过程中重要的酶,纤溶酶能直接水解细胞外间... 相似文献
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10.
纤溶酶原活化物抑制剂 总被引:1,自引:0,他引:1
纤溶酶原活化物抑制剂(PAI)能专一性地抑制纤溶酶原活化物,在纤溶系统中起重要的调节作用。本文综述了四种PAI的来源、性质、基因结构、生理功能及与疾病的关系。 相似文献
11.
Jacqueline M. Cale Shih-Hon Li Mark Warnock Enming J. Su Paul R. North Karen L. Sanders Maria M. Puscau Cory D. Emal Daniel A. Lawrence 《The Journal of biological chemistry》2010,285(11):7892-7902
Plasminogen activator inhibitor type 1, (PAI-1) the primary inhibitor of the tissue-type (tPA) and urokinase-type (uPA) plasminogen activators, has been implicated in a wide range of pathological processes, making it an attractive target for pharmacologic inhibition. Currently available small-molecule inhibitors of PAI-1 bind with relatively low affinity and do not inactivate PAI-1 in the presence of its cofactor, vitronectin. To search for novel PAI-1 inhibitors with improved potencies and new mechanisms of action, we screened a library selected to provide a range of biological activities and structural diversity. Five potential PAI-1 inhibitors were identified, and all were polyphenolic compounds including two related, naturally occurring plant polyphenols that were structurally similar to compounds previously shown to provide cardiovascular benefit in vivo. Unique second generation compounds were synthesized and characterized, and several showed IC50 values for PAI-1 between 10 and 200 nm. This represents an enhanced potency of 10–1000-fold over previously reported PAI-1 inactivators. Inhibition of PAI-1 by these compounds was reversible, and their primary mechanism of action was to block the initial association of PAI-1 with a protease. Consistent with this mechanism and in contrast to previously described PAI-1 inactivators, these compounds inactivate PAI-1 in the presence of vitronectin. Two of the compounds showed efficacy in ex vivo plasma and one blocked PAI-1 activity in vivo in mice. These data describe a novel family of high affinity PAI-1-inactivating compounds with improved characteristics and in vivo efficacy, and suggest that the known cardiovascular benefits of dietary polyphenols may derive in part from their inactivation of PAI-1. 相似文献
12.
Mario Gonzalez-Gronow Rupa Ray Fang Wang Salvatore V. Pizzo 《The Journal of biological chemistry》2013,288(1):498-509
The voltage-dependent anion channel (VDAC), a major pore-forming protein in the outer membrane of mitochondria, is also found in the plasma membrane of a large number of cells where in addition to its role in regulating cellular ATP release and volume control it is important for maintaining redox homeostasis. Cell surface VDAC is a receptor for plasminogen kringle 5, which promotes partial closure of the channel. In this study, we demonstrate that VDAC binds tissue-type plasminogen activator (t-PA) on human neuroblastoma SK-N-SH cells. Binding of t-PA to VDAC induced a decrease in Km and an increase in the Vmax for activation of its substrate, plasminogen (Pg). This resulted in accelerated Pg activation when VDAC, t-PA, and Pg were bound together. VDAC is also a substrate for plasmin; hence, it mimics fibrin activity. Binding of t-PA to VDAC occurs between a t-PA fibronectin type I finger domain located between amino acids Ile5 and Asn37 and a VDAC region including amino acids 20GYGFG24. These VDAC residues correspond to a GXXXG repeat motif commonly found in amyloid β peptides that is necessary for aggregation when these peptides form fibrillar deposits on the cell surface. Furthermore, we also show that Pg kringle 5 is a substrate for the NADH-dependent reductase activity of VDAC. This ternary complex is an efficient proteolytic complex that may facilitate removal of amyloid β peptide deposits from the normal brain and cell debris from injured brain tissue. 相似文献
13.
为获得一种高效的溶栓药物。从赤子爱胜蚓(Eiseniafoelida)中分离纯化得到了一种纤溶酶组分。用Lowry法测定蛋白质浓度,SDSPAGE鉴定纯度为98%,表观相对分子质量(Mr)为14850,纤维蛋白平板法测定其总纤溶活性为65.51×103mm2/mg,直接纤溶活性为15.61×103mm2/mg,间接纤溶活性为26.34×103mm2/mg。水解BAEE的米氏常数(Km)为1.82×105mol/L。水解ChromozymPL的米氏常数(Km)3.98×105mol/L,水解ChromozymtPA的米氏常数(Km)5.55×105mol/L活性,N端氨基酸序列测定的结果为VIGGTNAIPGEFPYQ。结果表明该纤溶酶分子量较小,间接活性较高,适宜作为一种新型的溶栓药物。 相似文献
14.
Changes of ovarian tPA,uPA and PA inhibitor activities were examined in PMSG-and hCG-treatedimmature mice during periovulatory periods.The results show that 15% of the gonadotropin-treatedanimals ovulated 8 hrs after hCG administration,about 6-8 hrs earlier than in rat.It is also shownthat not only tPA activity,but also uPA activity,was regulated by gonadotropins in ovarianhomogenates and granulosa cells,and they reached maximum prior to ovulation.No measurableamount of PAI-1 activity could be detected in mouse granulosa cell conditioned medium andfollicular fluid,but considerable amount of α_2-antiplasmin,a specific inhibitor for plasmin,wasfound in follicular fluid.Cumulus-oocyte complexes contain mainly tPA.Since the ovulated eggsstill have high tPA activity,it is thought that the enzyme in the oocyte may play an important rolein implantation. 相似文献
15.
Previous studies from this laboratory have demonstrated that plasminogen and angiostatin bind to endothelial cell (EC) surface-associated
actin via their kringles in a specific manner. Heat shock proteins (hsps) like hsp 27 are constitutively expressed by vascular
ECs and regulate actin polymerization, cell growth, and migration. Since many hsps have also been found to be highly abundant
on cell surfaces and there is evidence that bacterial surface hsps may interact with human plasminogen, the purpose of this
study was to determine whether human plasminogen and angiostatin would interact with human hsps. ELISAs were developed in
our laboratory to assess these interactions. It was observed that plasminogen bound to hsps 27, 60, and 70. In all cases,
binding was inhibited (85–90%) by excess (50 mM) lysine indicating kringle involvement. Angiostatin predominantly bound to
hsp 27 and to hsp 70 in a concentration- and kringle-dependent manner. As observed previously for actin, there was concentration-dependent
inhibition of angiostatin’s interaction with hsp 27 by plasminogen. In addition, 30-fold molar excess actin inhibited (up
to 50%), the interaction of plasminogen with all hsps. However, 30-fold molar excess actin could only inhibit the interaction
of angiostatin with hsp 27 by 15–20%. Collectively, these data indicate that (i) while plasminogen interacts specifically
with hsp 27, 60, and 70, angiostatin interacts predominantly with hsp 27 and to some extent with hsp 70; (ii) plasminogen
only partially displaces angiostatin’s binding to hsp 27 and (iii) actin only partially displaces plasminogen/angiostatin
binding to hsps. It is conceivable therefore that surface-associated hsps could mediate the binding of these ligands to cells
like ECs. 相似文献
16.
Ola Fjellstr?m Johanna Deinum Tove Sj?gren Carina Johansson Stefan Geschwindner Viveca Nerme Anne Legnehed Jane McPheat Karolina Olsson Cristian Bodin Amalia Paunovic David Gustafsson 《The Journal of biological chemistry》2013,288(2):873-885
A novel class of small molecule inhibitors for plasminogen activator inhibitor type 1 (PAI-1), represented by AZ3976, was identified in a high throughput screening campaign. AZ3976 displayed an IC50 value of 26 μm in an enzymatic chromogenic assay. In a plasma clot lysis assay, the compound was active with an IC50 of 16 μm. Surprisingly, AZ3976 did not bind to active PAI-1 but bound to latent PAI-1 with a KD of 0.29 μm at 35 °C and a binding stoichiometry of 0.94, as measured by isothermal calorimetry. Reversible binding was confirmed by surface plasmon resonance direct binding experiments. The x-ray structure of AZ3976 in complex with latent PAI-1 was determined at 2.4 Å resolution. The inhibitor was bound in the flexible joint region with the entrance to the cavity located between α-helix D and β-strand 2A. A set of surface plasmon resonance experiments revealed that AZ3976 inhibited PAI-1 by enhancing the latency transition of active PAI-1. Because AZ3976 only had measurable affinity for latent PAI-1, we propose that its mechanism of inhibition is based on binding to a small fraction in equilibrium with active PAI-1, a latent-like prelatent form, from which latent PAI-1 is then generated more rapidly. This mode of action, with induced accelerated latency transition of active PAI-1 may, together with supporting x-ray data, provide improved opportunities for small molecule drug design in the hunt for therapeutically useful PAI-1 inhibitors. 相似文献
17.
用偶联有L-赖氨酸的Sepharose 4B亲和柱对猪血纤维蛋白溶酶原进行分离纯化,所得酶原在酸性及SDS-聚丙烯酰胺凝胶电泳中显示单一蛋白质区带,在碱性条件下电泳及等电聚焦电泳表现出较明显的不均一性。还原及非还原SDS-聚丙烯酰胺凝胶电泳测得酶原分子量为88kD,经尿激酶激活后,进行还原SDS-凝胶电泳,出现两条新的蛋白质区带,分子量分别为63kD和26kD。酶原含中性糖1.35%,N-末端为异亮氨酸。尿激酶激活后产生的血纤维蛋白溶酶以对甲苯磺酰-L-精氨酸甲酯为底物,测得Km为4.2m mol/L,V_(max)为13.5 IU。6-氨基已酸对酶活性有双重影响。此外,还观察了胰蛋白酶对猪血纤维蛋白溶酶原的激活。 相似文献
18.
用正常人胎肺细胞体外培养,从其培养液中分离纤溶酶原活化物(PA),通过CM-SephadexC-50层析,硫酸铵沉淀和Fibrin-Sepharose,Lysine-Sepharose亲和层析及SephadexG-50凝胶过滤等步骤,从10.5l条件培养液中分离纯化得到两种类型的纤溶酶原活化物,t-PA90μg,u-PA800μg.在还原条件下SDS-PAGE均显示单带,分子量t-PA为72kD,u-PA为54kD,纤溶比活分别为156000IU/mg蛋白和106000IU/mg蛋白. 相似文献
19.
Characterization of PauB, a Novel Broad-Spectrum Plasminogen Activator from Streptococcus uberis
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A bovine plasminogen activator of atypical molecular mass ( approximately 45 kDa) from Streptococcus uberis strain SK880 had been identified previously (L. B. Johnsen, K. Poulsen, M. Kilian, and T. E. Petersen. Infect. Immun. 67:1072-1078, 1999). The strain was isolated from a clinical case of bovine mastitis. The isolate was found not to secrete PauA, a bovine plasminogen activator expressed by the majority of S. uberis strains. Analysis of the locus normally occupied by pauA revealed an absence of the pauA open reading frame. However, an alternative open reading frame was identified within the same locus. Sequence analysis of the putative gene suggested limited but significant homology to other plasminogen activators. A candidate signal peptide sequence and cleavage site were also identified. Expression cloning of DNA encoding the predicted mature protein (lacking signal peptide) confirmed that the open reading frame encoded a plasminogen activator of the expected size, which we have named PauB. Both native and recombinant forms of PauB displayed an unexpectedly broad specificity profile for bovine, ovine, equine, caprine, porcine, rabbit, and human plasminogen. Clinical and nonclinical field isolates from nine United Kingdom sites were screened for the pauB gene and none were identified as carrying it. Similarly, clinical isolates from 20 Danish herds were all found to encode PauA and not PauB. Therefore, PauB represents a novel but rare bacterial plasminogen activator which displays very broad specificity. 相似文献