华广虻肠道溶纤活性蛋白的纯化和性质(英文)

经过 75 %饱和度硫酸铵沉淀、SephadexG 75凝胶过滤层析、Lys Sepharose 4B亲和层析和电泳制备洗脱 ,从华广虻 (TabanusamaenusWalker)腹部组织匀浆液中分离纯化出分子量约为 6 7KD的溶纤活性蛋白TAFP。经纤维蛋白平板测定表明 ,TAFP不仅具有纤溶酶作用 ,还具有激活纤溶酶原的作用 ;通过三肽生色底物测定发现 ,TAFP能分解纤溶酶原激活剂的生色底物—ChromozymUK及S 2 2 88。还能水解胰蛋白酶专一底物Bz Phe Val Arg NA及CBZ Gly Pro Arg NA ,表明TAFP具有类胰蛋白酶活性 ,专一水解精氨酸形成的酰胺键 (或肽键 )。TAFP无胰凝乳蛋白酶活性     

链霉菌C-3662产生的纤溶活性蛋白酶的纯化与理化性质

 从链霉菌 C- 3662发酵上清液中 ,通过硫酸铵沉淀 ,CM- Sepharose Fast Flow和 Phenyl-Sepharose Fast Flow等层析色谱 ,分离纯化得到了具有纤溶活性的蛋白酶 CGW- 3,反向 HPLC鉴定纯度为 90 % ;每立升发酵上清液可得到 8mg纯品 ,活性回收率 46% ,CGW- 3为一单肽链蛋白 ,分子量 2 2 72 1 ,对丝氨酸蛋白酶抑制剂 PMSF敏感 ,对 EDTA不敏感 ;其 N端 1 5个氨基酸的顺序为 VVGGTRAAQGEFPFM,与微生物来源的胰蛋白酶类丝氨酸蛋白酶有较高的同源性 . CGW- 3的等电点 p I9.0 ,纤溶活性的最适 p H为 7.5～ 8.0 ,对温度比较敏感 .CGW- 3不仅具有直接降解纤维蛋白作用 ,而且能够激活纤溶酶原     

黑蚂蚁纤溶活性蛋白的分离纯化及其性质研究

通过硫酸铵分段盐析、DEAE-52离子交换和Sephacryl S-100分子筛层析从黑蚂蚁(Polyrhachis vicina Roger)中纯化到一种纤溶活性蛋白,再通过SDS-PAGE凝胶电泳测定其分子质量,Braford法测定蛋白浓度,蒽酮硫酸法测定其含糖量,纤维蛋白平板法测定其酶活性。并研究温度、p H改变及不同金属离子对其活性的影响。结果显示,分离得到的活性蛋白具有纤溶活性,其分子质量约为25 k D,蛋白浓度为2.804 1 mg/m L,糖含量为8.171 4%,酶活力为67.455 2 U/g。研究表明,该活性蛋白最适温度为45℃,最适p H为3.0,p H在4.0~8.0基本稳定,Na+、K+对其酶活性影响较小,Mg2+对该酶纤溶活性有强烈的激活作用,而Zn2+、Ba2+、Mn2+、Ca2+等对此酶活力有没明显的抑制作用。     

纤溶酶抑制剂textilinin-1在毕氏酵母的表达及应用

目的研究真核表达的textilinin-1蛋白纯化品对纤溶酶抑制作用。方法根据textilinin-1的天然氨基酸序列,按照毕氏酵母偏好密码子进行优化,合成textilinin-1基因,重组到pPICZα载体上,并转化至Pichia p.X-33菌种中,实现高效分泌表达,并进行重组textilinin-1活力单位的定义及小鼠断尾试验。结果通过高密度发酵及两步柱层析纯化,最终从10L发酵液中得到6g纯度达97.0%以上的重组textilinin-1。活性研究表明,重组textilinin-1对纤溶酶的活性具有抑制作用,并对tPA所致的小鼠出血倾向有一定的抑制作用。结论重组textilinin-1可抑制纤溶酶活性,并对纤溶性出血小鼠模型有止血作用,具有开发为止血药的潜力。     

蝮蛇抗栓酶3号对心血管病高凝状态的影响

应用蝮蛇抗栓酶３号（以下简称Ｓｖａｔｅ—３）治疗心血管病高凝状态２４例,剂量１．０ｕ稀释后静滴,ｑｄ,３周为一疗程。治疗后患者全血粘度、血浆粘度、血小板聚集率、组织纤溶酶原激活物抑制剂（ＴＰＩ）、血栓素Ｂ２（ＴＸＢ２）、组织纤维连结蛋白（ＦＮ）均有显著下降,而组织纤溶酶原激活剂（ＴＰＡ）、６－酮前列腺素Ｆα（６－Ｋｅｔｏ－ＰＧＦα）明显升高。提示,该药治疗心血管病高凝状态有效。     

响应面法优化解淀粉芽孢杆菌发酵产豆豉纤溶酶的工艺条件

纤溶酶是指能专一降解纤维蛋白凝胶的蛋白水解酶,可用于治疗脑梗死、高凝血状态及血栓性脉管炎等外周血管疾病。解淀粉芽孢杆菌JXNUWX-1是一株从豆豉中分离得到的高产纤溶酶菌株,并保存于中国典型培养物保藏中心(CCTCC No.M2014638)。本研究利用单因素试验和响应曲面法对JXNUWX-1菌株产纤溶酶的发酵条件进行了优化。在单因素试验的基础上通过Design-Expert软件对玉米淀粉、牛肉粉、初始p H这三个较显著的因素进行响应面试验设计,最终获得最佳发酵条件为:玉米淀粉浓度为2.91%,牛肉粉的浓度为1.76%,最适p H为7.47,在此条件下,酶活力为346.62 IU/m L,较优化前纤溶酶活力提高了8.9倍。本研究为解淀粉芽孢杆菌JXNUWX-1大规模生产纤溶酶提供理论依据。     

单环刺螠纤溶酶UFE-Ⅰ的性质和溶栓活性

单环刺螠纤溶酶UFE-Ⅰ的最适反应温度为45 ℃;最适反应pH为7.0;Mg2+、Mn2+和Fe2+是该纤溶酶的强激活剂;Fe3+、Cu2+、Ag+、Hg+和Pb2+对该纤溶酶具有一定的抑制作用;SBTI和PMSF,完全抑制UFE-Ⅰ,说明该酶为丝氨酸蛋白酶;糜蛋白酶抑制剂部分抑制UFE-Ⅰ,亮抑酶肽、抑蛋白酶肽、苯甲脒较弱的抑制UFE-Ⅰ.与蚓激酶类似,UFE-Ⅰ不仅具有直接的纤溶活力更具有纤溶酶原激活活力(84.0%),另外分别将蚓激酶原料与该酶加入到家兔血栓块中,37 ℃孵育3 h,结果表明该酶具有比蚓激酶更为强大的溶栓能力.     

一株产纤溶酶凝结芽孢杆菌的筛选及其生物学特性

为了筛选性能良好的产纤溶酶菌株,本研究通过脱脂乳平板及纤维蛋白平板双层筛选从传统发酵食品豆豉中,分离到1株具有溶解纤维蛋白能力的凝结芽孢杆菌(Bacillus coagulans) HQ-1。分析其发酵上清液中纤溶酶的纤溶能力、组成特征和作用方式,并研究菌株HQ-1生物学特性。结果表明:菌株HQ-1发酵上清液中的纤溶酶活力为383.1 U/mL;Native-PAGE实验表明HQ-1发酵上清液中能溶解纤维蛋白的纤溶酶组分仅有一种,并且通过激活纤溶酶原实现HQ-1降解纤维蛋白。HQ-1生物学特性实验表明:菌株HQ-1对阿莫西林等11种常见抗生素敏感;对沙门氏菌、大肠杆菌和金黄色葡萄球菌的抑制作用显著。菌株HQ-1产纤溶酶活性及稳定性较好,具有潜在开发价值,本研究结果为产纤溶酶微生物资源的开发提供理论依据。     

三七皂苷Rg1对tPA和PAI-1活性的调节作用

探讨三七皂苷Rg1对组织型纤溶酶原激活物(tPA)和纤溶酶原激活物抑制物(PAI-1)活性的调节作用。运用发色底物方法测定三七皂苷Rg1在体外和静脉注射对家兔血浆纤溶酶原激活物(tPA)和血浆或血小板释放的纤溶酶原激活物抑制物(PAI-1)水平的影响。结果表明,三七皂苷Rg1在体外呈浓度依赖性明显抑制血浆PAI-1活性,同时提高血浆tPA活性;30和60 mg/kg的三七皂苷Rg1静脉注射显著抑制血浆PAI-1活性,提高血浆tPA活性,同时降低凝血酶激活的血小板所释放的PAI-1水平。本实验提示三七皂苷Rg1能抑制PAI-1活性,同时升高tPA活性可能是其抗血栓作用的分子机制之一。     

根霉12#发酵产生纤溶酶的酶学性质

溶栓疗法是血栓性疾病安全有效的治疗手段,开发新型纤溶酶具有实际应用意义.分离自南方小酒药的根霉12豆粕和麸皮为原料可产生纤溶酶.已采用盐析,疏水层析、离子交换层析和凝胶层析方法对纤溶酶分离提纯.提纯的纤溶酶比活力2143u/mg(尿激酶单位),有直接溶解血栓和激活纤溶酶原的双重溶栓作用,降解纤维蛋白α、β和γ肽链速度快;最适作用温度45℃,适宜作用pH范围6.8～8.8;等电聚焦方法测定该酶等电点8.5±0.1;只分解生色底物N-Succinvl-Ala-Ala-Pro-Phe-pNA,其米氏常数Km为O.23mmol/L,酶转换数Kcat为16.36 s-1;Molish实验和甲苯胺蓝实验均证明该酶为糖蛋白,地衣酚-硫酸法测得该酶含糖量4.70%;EDTA、PMSF、PCMB对该纤溶酶有抑制作用,说明活性中心含有巯基、金属和丝氨酸;N端12个氨基酸序列为NH2-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly,与其它生物来源的纤溶酶相比较没有同源性.根霉12#产生的纤溶酶为新型纤溶酶,有希望开发成溶栓药物.     

CHARACTERIZATION AND PURIFICATION OF GUT FIBRINOLYTIC PROTEASE FROM TABANUS AMAENUS

Abstract After ammonium sulphate precipitation, Sephadex G-75 gel filtration, Lys-Sepharose 4B affinity chromatography and elution from electrophoresis, the fibrinolytic protease (TAFP) was isolated and purified from the extract of T. amaenus Walker gut. It appeared a single band corresponding to molecular weight of approximately 67kD on SDS-PAGE and an probably pI of 7.2 on IEF. On fibrin plate and plasminogen-free fibrin plate (heated at 85°C for 30 minutes to eliminate plasminogen), TAFP showed same fibrinolytic activity. The result might indicate that TAFP is a fibrinolytic enzyme degrading fibrin, as well as a plasminogen activator degrading fibrin via activating plasminogen. The result of chromogenic substrates indicated that TAFP possesses trypsin-like activity specifically degrading argininyl amide bond or peptide bond, but has no chymotrypsin activity. TAFP was almost inhibited powerfully by antipain, PMSF, soybean trypsin inhibitor and soybean Bowman-Birk inhibitor. However, leupeptin, antitrypsin and TLCK was more powerful effective inhibitors of TAFP. Optimal reaction pH of TAFP was 7.5, and it was stable in 5.5–7.0 of pH range.     

华广虻(Tabanusamaenus Walker)溶纤活性蛋白的纯化及生物活性分析

经过 ７５％ 饱和度硫酸铵沉淀、 Ｓｅｐｈａｄｅｘ Ｇ ７５ 凝胶过滤层析、 Ｌｙｓ Ｓｅｐｈａｒｏｓｅ ４ Ｂ 亲和层析和电泳制备洗脱,从华广虻（ Ｔａｂａｎｕｓ ａｍ ａｅｎｕｓ Ｗ ａｌｋｅｒ）腹部组织匀浆液中分离纯化出分子量约为 ６７ｋ Ｄ 的溶纤活性蛋白 Ｔ Ａ Ｆ Ｐ经纤维蛋白平板测定表明, Ｔ Ａ Ｆ Ｐ 只具有纤溶酶作用,不具有激活纤溶酶原的作用;但 Ｔ Ａ Ｆ Ｐ 能分解纤溶酶原激活剂的生色底物—— Ｃｈｒｏｍ ｏｚｙｍ Ｕ Ｋ 及 Ｓ ２２８８还能水解胰蛋白酶专一底物 Ｂｚ Ｐｈｅ Ｖａｌ Ａｒｇ Ｎ Ａ 及 Ｃ Ｂ Ｚ Ｇｌｙ Ｐｒｏ Ａｒｇ Ｎ Ａ,表明 Ｔ Ａ Ｆ Ｐ具有类胰蛋白酶活性,专一水解精氨酸形成的酰胺键（或肽键） Ｔ Ａ Ｆ Ｐ无胰凝乳蛋白酶活性      

Magnetic urokinase: targeting of urokinase to fibrin clot

A plasminogen activator of human origin, urokinase, was endowed with magnetic property. The magnetic urokinase was composed of magnetite, polyethylene glycol derivative and urokinase, and dispersed in saline. Its particle size of magnetite was approximately 30-60 nm. It was selectively delivered to fibrin clot by magnetic force in continuously circulating plasma and exerted fibrinolytic activity without degrading fibrinogen.     

蛇毒纤溶酶Alfimeprase在大肠杆菌中的可溶表达和纯化

Alfimeprase是Fibrolase的突变体,是一种蛇毒纤溶酶,有纤溶活性而无出血性。根据Alfimeprase的氨基酸序列和大肠杆菌密码子偏爱性,利用PCR的方法合成Alfimeprase DNA序列,分别融合在NusA和MBP的C端,与分子伴侣FkpA在大肠杆菌Origami B(DE3)中共表达,融合蛋白NusA/Alfimeprase以部分可溶的形式存在,可溶部分占上清总蛋白的25%左右,通过镍柱亲合层析纯化和肠激酶切割得到具有纤溶活性的重组蛋白Alfimeprase。本研究是首次报道在大肠杆菌中可溶表达Alfimeprase,为以后深入研究其功能及应用奠定了基础。     

Activation of plasminogen by pro-urokinase. I. Mechanism

The mechanism of the activation of plasminogen by recombinant pro-urokinase (Rec-pro-UK), obtained by expression of the human pro-urokinase gene in Escherichia coli, was investigated in purified systems. In mixtures of Rec-pro-UK and plasminogen, both active urokinase and plasmin are quickly generated. Addition of plasmin inhibitors (aprotinin or alpha 2-antiplasmin) abolishes the conversion of Rec-pro-UK to urokinase but not the activation of plasminogen to plasmin, suggesting that Rec-pro-UK activates plasminogen directly. Human plasma competitively inhibits the activation of plasminogen by pro-urokinase with a Ki of 0.2% (v/v). This explains the relative stability of Rec-pro-UK in plasma and the lack of activation of the plasma fibrinolytic system in the absence of fibrin. The competitive inhibition by plasma is abolished by the addition of CNBr-digested fibrinogen although Rec-pro-UK has no specific affinity for fibrin. These findings suggest that the fibrin specificity of the activation of plasminogen by pro-urokinase is due to neutralization by fibrin of the competitive inhibition exerted by plasma and not to fibrin-enhanced activation of plasminogen.     

Fibrinogen and fibrinogen-fibrin degradation products in experimental African trypanosomiasis

Fibrinogen and fibrinogen/fibrin degradation products in experimental African trypanosomiasis. International Journal for Parasitology4: 143–151. Studies have been carried out on some components of the fibrinolytic system of rabbits infected with two strains of Trypanosoma (Trypanozoon) brucei. Significant increases in fibrinogen/ fibrin degradation products (FDP) occur with a peak of activity 14–21 days after infection. Plasma fibrinogen concentrations increase during the infection. Measurement of plasminogen concentrations and experimental inhibition of plasmin by drugs suggests that plasminogen is being activated during the infection giving rise to FDP production. Administration of the trypanocidal drug Mel B to infected rabbits did not cause a rapid change in FDP levels. The results suggest an increased synthesis of fibrinogen. This, together with the increased amount of FDP, may be indicative of an increased fibrinolytic activity suggesting the formation of microthrombi in the circulation.     

Functional effects of asparagine-linked oligosaccharide on natural and variant human tissue-type plasminogen activator

The role of Asn-linked oligosaccharide in the functional properties of both human tissue-type plasminogen activator (t-PA) and a genetic variant of t-PA was studied. Nonglycosylated and glycosylated wild-type t-PA were produced in mammalian cells which express recombinant t-PA. These proteins were compared in fibrin binding and 125I-labeled fibrin clot lysis assays, using purified components. The nonglycosylated form showed higher fibrin binding, as well as higher fibrinolytic potency than the glycosylated form. Subsequently, prevention of glycosylation of a t-PA variant which lacked the finger and epidermal growth factor domains (delta FE), was carried out in an attempt to enhance its fibrinolytic activity. Glycosylation was prevented by changing Asn to Gln; at Asn-117 to produce delta FE1X t-PA, and at Asn-117, -184, and -448 to produce delta FE3X t-PA. All variants were similar to wild-type t-PA in their catalytic dependence on fibrinogen fragments, fibrinolytic activity in fibrin autography analysis, and plasminogen activator activity. In a clot lysis assay, using citrated human plasma, the fibrinolytic potency of the variants were comparable to that of wild-type t-PA at activator concentrations of 17-51 nM (approximately 1-3 micrograms/ml). At 0.5-5.1 nM (approximately 0.03-0.3 micrograms/ml), however, the variant proteins had lower fibrinolytic potency than wild-type t-PA. Fifty percent lysis in 1.5 h for wild-type, delta FE, delta FE1X, and delta FE3X t-PA, required 2.5, 10, 7.5, and 5.5 nM t-PA, respectively. The fibrinogenolytic activity in human plasma was measured for wild-type, delta FE, delta FE1X, and delta FE3X t-PA, and showed significant fibrinogen depletion after 3 h of incubation at 51 nM, decreasing to 11, 11, 50, and 72% of basal levels, respectively. These data indicate that partial or total nonglycosylated t-PA variants have a higher fibrinolytic versus fibrinogenolytic ratio than their fully glycosylated counterparts.     

A rapid and strong increase of plasminogen activator induced by experimental anaphylaxis in rabbits.

Anaphylactic shock was induced in rabbits by injecting bovine serum albumin (BSA) as an antigen. Measurements of the enzyme activities in the fibrinolytic system confirmed that a rapid and strong increase of plasminogen activator (PA) was induced during anaphylaxis. The euglobulin fibrinolytic activity (EFA) as estimated by the plasminogen-rich fibrin plate method rose significantly, peaking at 15 min after the BSA injection (when the arterial pressure was minimum). However, EFA was not detected by the plasminogen-poor fibrin plate method. The tissue-type PA (t-PA) activity using the natural substrate plasminogen increased significantly with a peak at 15 min. The amidolytic activity also simultaneously increased significantly using the t-PA substrate, H-D-Ile-Pro-Arg-pNA. The plasminogen activator inhibitor (PAI) activity remained at baseline levels until 30 min, but rose fourfold at 90 min. The main plasma fibrinolytic enzyme which increased in anaphylaxis was proved by zymography to be t-PA with a molecular weight (MW) of 69,000.     

Effects of tissue-type plasminogen activator from a culture of calf kidney cells on hemostasis and fibrinolysis in experimental nephritis

The plasminogen activator 960 IU/mg protein activity isolated from cultured fluid of the calf kidney cells was introduced to albino rats (180-200 g) with experimental Heynmann nephritis every day during 4 days. Nephritis caused activation of haemostasis and inhibition of fibrinolysis in the blood. There was increased excretion of the fibrin, fibrinogen degradation products in urine as a results of the local fibrin deposition in diseased kidneys. The fibrinolytic activity of the cortical zone of kidney was markedly decreased. The plasminogen activator, infused to experimental animals, resulted in normalization of the altered indexes.     

Construction and characterization of a recombinant chimeric plasminogen activator consisting of a fibrin peptide and a low molecular mass single-chain urokinase

A recombinant chimeric plasminogen activator (f beta/scuPA-32k), with a fibrin beta-chain peptide (comprising Gly15 through Arg 42) linked to the N-terminal of a low molecular mass (32 kDa) single-chain urokinase (scuPA-32k, comprising Leu144 through Leu 411) via a 50 amino acid linker sequence, was produced by expression the corresponding chimeric cDNA in Escherichia coli cells. After refolding in vitro, the chimeric protein was purified to homogeneity by zinc chelate-Sepharose chromatography, Sephacryl S200 chromatography and benzamidine-Sepharose chromatography in sequence. The apparent molecular mass was 36 kDa shown by SDS-PAGE analysis. The special activity was 87,000 IU/mg detected by fibrin plate determination. F beta/scuPA-32k could directly activate plasminogen following Michaelis-Menten kinetics with K(m) = 0.52 microM and k(2) = 0.0024 s(-1). Mediated by plasmin, the single-chain molecule could be converted to the active two-chain molecule. The chimeric protein had 3.3 times higher fibrin affinity than scuPA-32k in the fibrin concentration of 3.2 mg/mL, while the chimeric protein inhibited the fibrin clotting and platelet aggregation. F beta/scuPA-32k showed a higher thrombolytic potency in vitro plasma clot lysis than scuPA-32k and depleted less fibrinogen in plasma. These results showed that the chimeric protein had not only higher fibrinolytic activity but also anti-thrombus activity. Further evaluation of the thrombolytic potential in appropriate animal models is required.