Digestive properties of the venom of the Australian Coastal Taipan, Oxyuranus scutellatus (Peters, 1867).

The digestive properties of Australian elapid snake venoms have not been studied to any great extent. To address this, the in vitro digestive properties of Oxyuranus scutellatus (Australian Coastal Taipan) venom were investigated in a simulation of the in vivo conditions using the parameters reported for the stomach of snakes and representative prey for this species. The amount of soluble protein released was measured over time using a bicinchoninic acid (BCA) assay. Dismembered mouse hindlegs were injected intramuscularly with 0.1 ml O. scutellatus venom (concentration 10 mg/ml) and maintained in a micro-anaerobic, acidic environment (pH approximately 1.2-1.7) at 25 degrees C. The bathing liquid was sampled every 24 h for 7 days, and assayed for soluble protein. Statistical analysis revealed that O. scutellatus venom increased the rate at which proteins were released when compared to a negative control suggesting the potential importance of envenomation in the digestion of whole prey.     

BJ-48, a novel thrombin-like enzyme from the Bothrops jararacussu venom with high selectivity for Arg over Lys in P1: Role of N-glycosylation in thermostability and active site accessibility.

BJ-48, a serine protease from the venom of Bothrops jararacussu, was purified to homogeneity using affinity chromatography on p-aminobenzamidine-agarose followed by HPLC gel filtration. BJ-48 presented 52kDa by SDS-PAGE analysis and 48,036Da by electron spray mass spectrometry. The enzyme was shown to be highly glycosylated with 42% of N-linked carbohydrates composed of Fuc(1):GalN(4):GlcN(5):Gal(1):Man(2) and a high content of sialic acid residues (8-12%). BJ-48 had optimal esterase activity at pH 7.5 and displayed maximum catalytic rate at 50 degrees C. Its hydrolytic activity was strongly inhibited by aprotinin and dithiothreitol while N-tosyl-l-phenylalanine chloromethyl ketone, 6-aminocaproic acid, E-64 and soybean trypsin inhibitor (SBTI) were ineffective. The kinetics of BJ-48 with chromogenic substrates revealed an unprecedented selectivity (10(4)-fold) for Arg over Lys in P1. BJ-48 proved to be a thrombin-like enzyme (TLE) with a specific fibrinogen-clotting activity of 73.4NIH units/mg. The TLE rapidly digested human fibrinogen Bbeta chain, but the Aalpha chain was cleaved specifically to release fibrinopeptide A with k(cat)/K(m)=2.1muM(-1)s(-1). The TLE showed no activity toward other thrombin substrates like protein C, protease-activated receptor-1 or inhibitors such as hirudin and antithrombin. A non-denaturing procedure using PNGase F and neuraminidase followed by hydrophobic interaction chromatography was employed to obtain active BJ-48 forms with variable carbohydrate content. Compared to the native enzyme, total or partially deglycosylated BJ-48 forms presented up to 2-fold reduction in their specific activities upon heating at 55/65 degrees C or treatment with SBTI. These results point out a role for BJ-48 glycosylation in thermostability and controlling the access of some canonical protein inhibitors to the active site.     

Expression, purification and molecular modeling of recombinant fibrinogenase [IV], a metalloproteinase from Deinakistrodon acutus venom.

A novel metalloproteinase, recombinant fibrinogenase IV (rFIV(a)), was expressed and purified from Deinakistrodon acutus venom. It was a single chain protein with an apparent molecular weight 27 kDa and an isoeletric point of pH 7.1. RFIV(a) cleaved preferentially the Aalpha-chain and also cleaved Bbeta, gamma-chains of fibrinogen when the incubation time was prolonged. The proteolytic activity was inhibited by EDTA, l-cysteine, and DTT, indicating rFIV(a) was a metalloproteinase requiring disulfide bonds for its activity. It kept above 85% of the initial activity from pH 4.5-11, showed an equal maximum activity at the temperature range from 30 to 50 degrees C, and was inactivated by Zn2+, Cu2+ and Cd2+. Homology modeling of rFIV(a) showed that two highly conserved disulfide bonds (Cys159-Cys164 and Cys117-Cys197) was maintained from its structure, and it exhibited the characteristic conserved motif H142E143XXH146XXGXXH152, whose three histidine residues were involved in binding of the catalytically essential zinc ion. This work demonstrates the expression, purification and characterization of recombinant fibrinogenase IV, which belongs to class P-I metalloproteinase from D. acutus venom.     

蛇毒Ⅱ类磷脂酶A2功能分化的分析研究

目的：研究蛇毒Ⅱ类磷脂酶A2(PLA2)中D49 PLA2和K49 PLA2的功能分化及其功能分化决定位点的鉴定。方法：运用序列比较分析，进化树构建和DIVERGE v1．04软件计算研究D49 PLA2和K49 PLA2的功能分化情况及其分化位点。结果：序列比较分析，进化树构建和DIVERGE v1．04软件计算结果表明蛇毒Ⅱ类PLA2中D49 PLA2和K49 PLA2的确发生了功能分化，对于K49 PLA2来说，1S，7K，11Q，E12，R34，T56，N88，L92，E108，K116，K128可能为功能分化决定位点。对于D49 PLA2，L2，G33，G35，F46和Y118可能为功能分化决定位点。结论：我们首次通过序列比较分析，进化树构建和DIVERGE v1．04软件计算鉴定出蛇毒Ⅱ类PLA2中D49 PLA2和K49 PLA2可能的功能分化位点，为今后通过基因重组和定点突变方法研究蛇毒Ⅱ类PLA2结构功能关系提供了线索。     

蛇毒溶栓素对实验动物血小板功能和环腺苷酸含量的影响

本文报道蛇毒溶栓素对实验动物血小板功能和血浆环腺苷酸含量的影响.(1)对血小板数的影响:给家犬以4 u/kg量静注1小时后,循环血的血小板数由药前130.44×10~9/L±50.02×10~9/L下降至60.20×10~9/L±20.50×10~9/L(P<0.05).(2)对血小板聚集功能的影响:猕猴和家兔分别按4 u/kg、8 u/kg量静注1小时后,对ADP诱导的血小板聚集率分别山药前的62.32±22.05%和43.66±10.60%下降至1.32±0.49%和4.95±1.96%;抑制率分别为97.50±1.75%和88.34±4.31%,与药前组相比均有非常显著的差异(P<0.001).(3)对血小板cAMP含量的影响:猕猴和家兔以蛋白竞争结合法测定血小板内cAMP的含量,药后1小时的含量65.43±31.35pmole/mg,32.25±15.43pmo1e/mg蛋白质,与药前12.02±0.95pmole/mg,4.25±3.13pmole/mg蛋白质相比有明显升高(P<0.001).     

Platelets as targets of snake venom metalloproteinases

For centuries snake venoms have been known to interfere with haemostasis and this is now known basically due either to toxins activating/inhibiting clotting factors, having effects on blood vessels or interfering with platelet function. In this short review, the interaction of one major group of toxins, the snake venom metalloproteinases, with platelets is considered. This is relevant for understanding the mechanism of haemorrhage induced by these toxins.     

蝮蛇抗栓酶对骨内高压降压作用实验研究

本实验随机将家兔一侧后肢于膝关节伸直位夹板外固定５周，制成固定侧胫骨上端骨内高压模型。对其全身和局部血液流变学指标进行测定，发现全血粘度，血浆粘度增大，血沉增快，血浆纤维蛋白原含量明显增高。对上述模型应用蝮蛇抗栓酶（ｓｖａｔｅ）连续治疗２１天，骨内压降低，血液流变学各项指标亦降低。本实验结果显示，蝮蛇抗栓酶通过改善骨内高压状态下的异常血液流变学状态可使骨内高压降低，从而认为血液流变学疗法可能成为临床治疗骨内高压症的一种新手段，而蝮蛇抗栓酶可能为其中高效药物之一。     

抗蝮蛇毒磷酸二酯酶单克隆抗体的制备及鉴定

从蝮蛇毒中提取磷酸二酯酶,用此酶免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞Fo融合,以间接ELISA检测杂交瘤细胞培养上清液和小鼠腹水中的特异性抗体,其效价分别为1∶128和1∶51 200.抗原阻断试验结果表明,此抗体对蛇毒磷酸二酯酶具有特异性.该杂交瘤细胞株定为G_8,该株单抗属鼠IgG_(2a)亚型,经体外持续培养6个月,其分泌抗体性能稳定.     

蝮蛇毒磷酸二酯酶抗原的制备及性质

为研究蛇类毒腺细胞分化过程中磷酸二酯酶的作用,需制备特异性和敏感性较高的该酶单克隆抗体用于免疫组化研究.本文所介绍为浙江蝮蛇蛇毒中磷酸二酯酶抗原的提取纯化过程,为制备单克隆抗体提供条件.作者采用离子交换技术和分子筛过滤法相结合的方法提取抗原,具有步骤少、分离效率高的特点.所得抗原经聚丙烯酰胺凝胶圆盘电泳呈单一条带,测定分子量为20900.     

圆斑蝰蛇毒中性磷脂酶A2对不同种类血管条作用的比较

本文比较了福建圆斑蝰蛇毒中性磷脂酶Ａ２（ＰＬＡ２－３）对人胃网膜动脉条、兔和大鼠主动脉条的作用，表面ＰＬＡ２－３对处于静息状态和预收缩状态的人网膜动脉能诱发松弛反应，而对大鼠和兔主动脉诱发剂量依赖性收缩反应，表明该ＰＬＡ２的血管作用具有血管种类差异性。ＰＬＡ２－３对大鼠主动脉的收缩反应是内皮细胞非依赖性的，去除内皮细胞能增强ＰＬＡ２－３所诱发的收缩反应；ＰＬＡ２－３所诱发的大鼠主动脉条收缩反应可能