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菜花烙铁头蛇毒中一种具有激肽释放酶与纤维蛋白原溶酶活性的Jerdonase 总被引:1,自引:1,他引:0
通过DEAESephadexA 5 0阴离子交换、超细SephadexG 10 0分子筛和反相高效液相C4 色谱层析 ,从菜花烙铁头蛇毒冻干粉中纯化出一种具有激肽释放酶活性和α纤维蛋白原溶酶活性的丝氨酸蛋白酶 ,命名为Jerdonase。在 12 .5 %胶浓度的SDS还原电泳条件下 ,该酶分子量大约为 5 5kD ,在非还原电泳条件下 ,分子量大约为 5 3kD。此酶是一种糖蛋白 ,含有约 35 .8%的中性糖。它的N末端氨基酸序列为IIGGDEENINEHPFLVALYDA ,其序列和蛇毒中其他丝氨酸蛋白酶具有非常高的序列相似性。Jerdonase能够催化BAEE、S 2 2 38和S 2 30 2的水解 ,其水解活性可被PMSF抑制 ,但是EDTA对此没有影响。Jerdonase能优先水解人纤维蛋白原的Aα链 ,同时伴随有微弱的Bβ链水解活性。另外 ,此酶能够水解牛低分子量的激肽原 ,释放舒缓激肽。总之 ,所有的结果表明Jerdonase是一个具有多功能活性的蛇毒丝氨酸蛋白酶 相似文献
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人组织激肽释放酶基因家族由KLK1-KLK15构成,编码一组丝氨酸蛋白酶。研究发现KLK基因家族涉及癌细胞的多种生物学功能,且其表达受类固醇激素的调节。人组织激肽释放酶4是丝氨酸蛋白酶家族的一个成员,在多种激素依赖性肿瘤如卵巢癌、前列腺癌、乳腺癌、子宫内膜癌中高表达,且表达量受雌激素、孕激素、雄激素不同程度的调节。近年来很多文献报道人组织激肽释放酶4涉及癌细胞的增殖、上皮间质转化及细胞外基质的降解等过程,可能促进了肿瘤的发生、发展,且与激素依赖性肿瘤的预后不良有关。这些研究显示人组织激肽释放酶4与激素依赖性肿瘤关系密切,是其潜在的肿瘤标记物和治疗靶点,随着研究的进一步深入,有望应用于激素依赖性肿瘤的早期诊断、病程监测和治疗。 相似文献
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蛇毒,特别是蝰亚科和腹亚科蛇毒,含有大量丝氨酸蛋白酶。这是自然界长期时化过程中演化出的一种既和哺乳动物蛋白酶相似,又存在相当程度的结构与功能分化的生物大分子,通过激活、灭活和转变机体中的凝血因子而广泛作用于血液凝固系统。它们的一级序列和胰蛋白酶-激肽释放酶同源。它们具有相同的活性中心构造和酶催化机制,但活性中心外可变区序列的差异造成了它们底物专一性差异,进一步体现为生物学和药理学功能的差异。它们和 相似文献
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组织型激肽释放酶1(kallikrein1,KLK1)和激肽释放酶相关肽酶(kallikrein-related peptidase 2~15,KLK2~15)是一类丝氨酸蛋白酶,具有广泛的生物学活性。在中枢神经系统中,它们不但在脑的生长、发育和学习记忆等方面起重要作用,同时也在多种脑部疾病中起重要作用,如帕金森病、痴呆、多发性硬化、肿瘤等,并在这些疾病的诊断、治疗和预后方面显示出潜在的应用价值。 相似文献
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本文报道烙铁头(Trimeresurusmucrosquamatus)蛇毒纤维蛋白原溶酶(TMVFg),眼镜王蛇(Ophiophagushannah)蛇毒纤维蛋白原溶酶(ohS1),竹叶青(Trimeresurusstejnegeri)蛇毒专一纤溶酶原激活剂(sv-pA)对5种小分子多肽底物的底物专一性,及这些蛇毒丝氨酸蛋白酶对各种凝血因子(第X因子、凝血酶原、纤溶酶原、蛋白C)的作用,并和其它蛇毒丝氨酸蛋白酶如矛头蝮(Bothropsatrox)蛇毒凝血酶样酶(Batroxobin)、铜头蝮(Agkistrodoncontortrixcontortrix)蛇毒蛋白C激活剂ACC-C、蝰蛇(Viperarusselli)毒第Ⅴ因子激活剂RVV-V进行比较研究。通过酶标偶联免疫反应研究了抗sv-PA抗体与各种丝氨酸蛋白酶的免疫交叉反应,并对蛇毒丝氨酸蛋白酶及相应功能的哺乳动物蛋白酶进行了序列比较分析。从底物专一性多样性及已知序列结构分化上对这一类蛇毒丝氨酸蛋白酶的结构与功能进行了探讨和研究。 相似文献
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浙江蝮蛇(Agkistrodon halys Pallas)蛇毒激肽释放酶Ⅰ的分离纯化及其性质的研究 总被引:1,自引:0,他引:1
浙江产蝮蛇(Agkistrodon halys Pallas)蛇毒中含有激肽释放酶,不需活化即可水解激肽原,释放激肽,并具有较弱的精氨酸酯酶的活力。粗毒经DEAE纤维素(DE-22,DE-52)和Sephadex G-75分离纯化后,可得到两个激肽释放酶的组分:Ⅰ与Ⅱ,二者电泳行为与酶活力有所不同,激肽释放酶Ⅰ在聚丙烯酰胺凝胶电泳中呈一条带,而组分Ⅱ中还杂有少量组分Ⅰ。激肽释放酶Ⅰ为一糖蛋白,含糖量20.3%,约由221个氨基酸残基组成,凝胶过滤和SDS电泳测定其分子量分别为31,000和30,000。此酶具有严格的底物专一性,能作用于激肽释放肽的专一底物Z-Phe-Arg-MCA及Bz-Pro-Phe-Arg-PA,不作用于一般蛋白质底物酪蛋白,对TAME的水解速度仅是对BAEE的14%。以BAEE为底物时,其最适pH为8~9,K_m值为2.85×10~(-4)M。本文测定了不同pH和不同温度下酶的稳定性,pH低于5或大于9,温度在40℃以上,酶活力迅速下降。其精氨酸酯酶及激肽释放酶的活力均能被丝氨酸蛋白酶抑制剂PMSF和DFP所抑制,两者呈平行关系,但都不被胰蛋白酶的专一抑制剂TLCK所抑制,慈菇抑制剂与大豆(Kunitz)抑制剂对此酶有部分抑制作用。经磷酸纤维素等阳离子交换树脂层析或交联的慈菇抑制剂Sepharose-4B亲合层析也能提纯激肽释放酶Ⅰ,但提纯后精氨酸酯酶活力下降,激肽释放活力几乎全部丧失。经圆二色光谱测定表明,酶的构象已发生改变。 相似文献
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本文报道烙铁头蛇毒纤维蛋白原溶酶,眼镜王蛇蛇毒纤维蛋白原溶酶,竹叶青蛇毒专一纤溶酶原激活剂对5种小分子多肽底物的底物专一性,及这些蛇毒丝氨酸蛋白酶对各种凝血因子(第X因子、凝血酶原、纤溶酶原、蛋白C)的作用,并和其它蛇毒丝氨酸蛋白酶如矛头蝮蛇毒凝血酶样酶、铜头蝮蛇毒蛋白C激活剂ACC-C、蝰蛇毒第V因子激活剂RVV-V进行比较研究。通过酶标偶联免疫反应研究了抗sv-PA抗体与各种丝氨酸蛋白酶的免疫 相似文献
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The extracellular proteinase complex of the microscopic fungus Aspergillus ochraceus 513 was isolated, purified, and separated by affinity chromatography on bacillichin-silochrom and subsequent column chromatography on DEAE-Toyopearl 650 M. The extracellular enzyme of the protein C activator type had a molecular mass of 36.5 kDa and activity close to that of the Agkistrodon snake venom protein C activator. The fibrinolytic and anticoagulant activities of the enzyme were investigated. 相似文献
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The extracellular proteinase complex of the microscopic fungus Aspergillus ochraceus513 was isolated, purified, and resolved by affinity chromatography on bacillichin-silochrom and subsequent column chromatography on DEAE-Toyopearl 650M. The extracellular enzyme of the protein C activator type had a molecular mass of 36.5 kDa and activity close to that of the Agkistrodonsnake venom protein C activator. The fibrinolytic and anticoagulant activities of the enzyme were investigated. 相似文献
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The sequence of two overlapping cDNA clones for the zinc metalloproteinase hemorrhagic toxin e (also known as atrolysin e, EC 3.4.24.44) from the venom gland of Crotalus atrox, the Western diamondback rattlesnake, is presented. The assembled cDNA sequence is 1975 nucleotides in length and encodes an open reading frame of 478 amino acids. The mature hemorrhagic toxin e protein as isolated from the crude venom has a molecular weight of approximately 24,000 and thus represents the processed product of this open reading frame. From the deduced amino acid sequence, it can be hypothesized that the enzyme is translated with a signal sequence of 18 amino acids, an amino-terminal propeptide of 169 amino acids, a central hemorrhagic proteinase domain of 202 amino acids, and a carboxy-terminal sequence of 89 amino acids. The propeptide has a short region similar to the region involved in the activation of matrix metalloproteinase zymogens. The proteinase domain is similar to other snake venom metalloproteinases, with over 57% identity to the low molecular weight proteinases HR2a and H2-proteinase from the Habu snake Trimeresurus flavoviridis. The carboxy-terminal region, which is not observed in the mature protein, strongly resembles the protein sequence immediately following the proteinase domain of HR1B (a high molecular weight hemorrhagic proteinase from the venom of T. flavoviridis) and the members of a different family of snake venom polypeptides known for their platelet aggregation inhibitory activity, the disintegrins. The cDNA sequence bears striking similarity to a previously reported sequence for a disintegrin cDNA. This report is evidence that this subfamily of venom metalloproteinases is synthesized in a proenzyme form which must be proteolytically activated.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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Henriques ES Fonseca N Ramos MJ 《Protein science : a publication of the Protein Society》2004,13(9):2355-2369
Pit viper venoms contain a number of serine proteinases that exhibit one or more thrombin-like activities on fibrinogen and platelets, this being the case for the kinin-releasing and fibrinogen-clotting KN-BJ from the venom of Bothrops jararaca. A three-dimensional structural model of the KN-BJ2 serine proteinase was built by homology modeling using the snake venom plasminogen activator TSV-PA as a major template and porcine kallikrein as additional structural support. A set of intrinsic buried waters was included in the model and its behavior under dynamic conditions was molecular dynamics simulated, revealing a most interesting similarity pattern to kallikrein. The benzamidine-based thrombin inhibitors alpha-NAPAP, 3-TAPAP, and 4-TAPAP were docked into the refined model, allowing for a more insightful functional characterization of the enzyme and a better understanding of the reported comparatively low affinity of KN-BJ2 toward those inhibitors. 相似文献
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Isolation and biochemical characterization of a fibrinolytic proteinase from Bothrops leucurus (white-tailed jararaca) snake venom 总被引:2,自引:0,他引:2
Bello CA Hermogenes AL Magalhaes A Veiga SS Gremski LH Richardson M Sanchez EF 《Biochimie》2006,88(2):189-200
In investigations aimed at characterizing snake venom clot-dissolving enzymes, we have purified a fibrinolytic proteinase from the venom of Bothrops leucurus (white-tailed jararaca). The proteinase was purified to homogeneity by a combination of molecular sieve chromatography on Sephacryl S-200 and ion-exchange chromatography on CM Sepharose. The enzyme called leucurolysin-a (leuc-a), is a 23 kDa metalloendopeptidase since it is inhibited by EDTA. PMSF, a specific serine proteinase inhibitor had no effect on leuc-a activity. The amino acid sequence was established by Edman degradation of overlapping peptides generated by a variety of selective cleavage procedures. Leuc-a is related in amino acid sequence to reprolysins. The protein is composed of 200 amino acid residues in a single polypeptide chain, possessing a blocked NH2-terminus and containing no carbohydrate. The proteinase showed proteolytic activity on dimethylcasein and on fibrin (specific activity=21.6 units/mg and 17.5 units/microg, respectively; crude venom=8.0 units/mg and 9.5 units/microg). Leuc-a degrades fibrin and fibrinogen by hydrolysis of the alpha chains. Moreover, the enzyme was capable of cleaving plasma fibronectin but not the basement membrane protein laminin. Leuc-a cleaved the Ala14-Leu15 and Tyr16-Leu17 bonds in oxidized insulin B chain. The pH optimum of the proteolysis of dimethylcasein by leuc-a was about pH 7.0. Antibody raised in rabbit against the purified enzyme reacted with leuc-a and with the crude venom of B. leucurus. In vitro studies revealed that leuc-a dissolves clots made either from purified fibrinogen or from whole blood, and unlike some other venom fibrinolytic metallopeptidases, leuc-a is devoid of hemorrhagic activity when injected (up to 100 microg) subcutaneously into mice. 相似文献
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C W Ward 《Biochimica et biophysica acta》1975,384(1):215-227
The metal chelator-sensitive proteinase activity from the larvae of the webbing clothes moth, Tineola bisselliella, was fractionated into two components by chromatography on DEAE-cellulose and the properties of the major fraction investigated. The approximate molecular weight obtained by gel filtration was 24 000. The pH optimum of 9.4 and the high stability between pH 9.0 and 11.5 are consistent with the alkaline conditions known to be present in the larval mid-gut. The enzyme also showed a second region of high stability around pH 2.3. The cleavage specificity against S-carboxy-methyl A and B chains of insulin was quite different to that of the metal chelator-sensitive proteinases from snake venoms and microorganisms. 10 bonds in the A-chain and 8 bonds in the B-chain were cleaved and the tentative rules governing the specificity limitations of this metal-chelator-sensitive proteinase are discussed. 相似文献
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Proteinase inhibitors and dendrotoxins. Sequence classification, structural prediction and structure/activity 总被引:3,自引:0,他引:3
M J Dufton 《European journal of biochemistry》1985,153(3):647-654
The amino acid sequences of four presynaptically active toxins from mamba snake venom (termed 'dendrotoxins') were compared systematically with homologous sequences of members of the proteinase inhibitor family (Kunitz). A comparison based on the complete sequences revealed that relatively few amino acid changes are necessary to abolish antiprotease activity and convert a proteinase inhibitor into a dendrotoxin. When comparison centred only on the sequence segments known to comprise the antiprotease site of bovine pancreatic trypsin inhibitor, the dendrotoxins were clearly classified apart from all the known inhibitors. Since the mode of action of the bovine pancreatic trypsin/kallikrein inhibitor involves beta sheet formation with the enzyme, predictions were obtained for this secondary structure in the region of the 'antiprotease site' throughout the homologues. Again, the dendrotoxins were clearly distinguished from the inhibitors. Structure/activity analyses, based on the crystal structures of inhibitor/enzyme complexes, suggest that unlike proteinase inhibitors, dendrotoxins might specifically co-ordinate the active-site 'catalytic' histidine residues of serine proteases. Although the significance of this remains to be studied, the presynaptic target is expected to involve an as yet uncharacterised member of the serine protease family. 相似文献
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Felicori LF Souza CT Velarde DT Magalhaes A Almeida AP Figueiredo S Richardson M Diniz CR Sanchez EF 《Protein expression and purification》2003,30(1):32-42
A kallikrein-like proteinase of Lachesis muta muta (bushmaster) venom, designated LV-Ka, was purified by gel filtration and anion exchange chromatographies. Physicochemical studies indicated that the purified enzyme is a 33 kDa monomeric glycoprotein, the Mr of which fell to 28 kDa after deglycosylation with PNGase F. Approximately 77% of the protein sequence was determined by sequencing the various fragments derived from digestions with endoproteases. The partial sequence obtained suggests that LV-Ka is of a similar size to other serine proteinases (i.e., approximately 234 amino acid residues). Sequence studies on the NH2-terminal region of the protein indicate that LV-Ka shares a high degree of sequence homology with the kallikrein-like enzymes EI and EII from Crotalus atrox, with crotalase from Crotalus adamanteus and significant homology with other serine proteinases from snake venoms and vertebrate serum enzymes. LV-Ka showed kallikrein-like activity, releasing bradikinin from kininogen as evidenced by guinea pig bioassay. In addition, intravenous injection of the proteinase (0.8 microg/g) was shown to lower blood pressure in experimental rats. In vitro, the isolated proteinase was shown to have neither fibrin(ogeno)lytic activity nor coagulant effect. LV-Ka was active upon the kallikrein substrates S-2266 and S-2302 (specific activity=13.0 and 31.5 U/mg, respectively; crude venom=0.25 and 6.0 U/mg) but had no proteolytic effect on dimethylcasein and insulin B chain. Its enzymatic activity was inhibited by NPGB and PMSF, indicating that the enzyme is a serine proteinase. Interestingly, one of the other reactions catalyzed by plasma kallikrein, the activation of plasminogen was one of the activities exhibited by LV-Ka. 相似文献
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Birrell GW Earl S Masci PP de Jersey J Wallis TP Gorman JJ Lavin MF 《Molecular & cellular proteomics : MCP》2006,5(2):379-389
Venom from the Australian elapid Pseudonaja textilis (Common or Eastern Brown snake), is the second most toxic snake venom known and is the most common cause of death from snake bite in Australia. This venom is known to contain a prothrombin activator complex, serine proteinase inhibitors, various phospholipase A2s, and pre- and postsynaptic neurotoxins. In this study, we performed a proteomic identification of the venom using two-dimensional gel electrophoresis, mass spectrometry, and de novo peptide sequencing. We identified most of the venom proteins including proteins previously not known to be present in the venom. In addition, we used immunoblotting and post-translational modification-specific enzyme stains and antibodies that reveal the complexity and regional diversity of the venom. Modifications observed include phosphorylation, gamma-carboxylation, and glycosylation. Glycoproteins were further characterized by enzymatic deglycosylation and by lectin binding specificity. The venom contains an abundance of glycoproteins with N-linked sugars that include glucose/mannose, N-acetylgalactosamine, N-acetylglucosamine, and sialic acids. Additionally there are multiple isoforms of mammalian coagulation factors that comprise a significant proportion of the venom. Indeed two of the identified proteins, a procoagulant and a plasmin inhibitor, are currently in development as human therapeutic agents. 相似文献