江浙蝮蛇(Agkistrodon halys Pallas)毒的抑菌作用及其抑菌组分L-氨基酸氧化酶的分离纯化

我们用平皿测活法研究了江浙蝮蛇毒对真菌和大肠杆菌生长的抑制作用，并对其抗细菌的组分进行柱层析分离纯化，得到组分单一的LAO不仅有抑菌作用还有L-氨基酸氧化酶的活性。纯化后LAO的比活力为808U／mg。蛇毒经酸性聚丙烯酰胺电泳后测抑菌活性发现有3个抑菌组分(Ⅰ、Ⅱ、Ⅲ)，LAO为抑菌组分Ⅱ。     

清栓酶（蝮蛇抗栓酶）的药理研究与临床应用进展

清栓酶（蝮蛇抗栓酶）的药理研究与临床应用进展中国蛇协蛇毒研究所邓禄延,覃公平,胡征林自本世纪八十年代起,蛇毒抗凝制剂在我国研制及应用于临床以来,相继有尖吻蝮蛇毒制剂去纤酶,长白山陆生白眉蝮蛇毒制剂清栓酶（蝮蛇抗栓酶）及江浙蝮蛇毒制剂江浙蝮蛇毒抗栓酶（...     

墨旱莲对4种蝮蛇毒引起的炎症和出血的影响

目的探讨墨旱莲提取液对短尾蝮蛇毒、蛇岛蝮蛇毒、白眉蝮蛇毒及尖吻蝮蛇毒所致的炎症和出血的影响。方法应用短尾蝮蛇毒、蛇岛蝮蛇毒、白眉蝮蛇毒及尖吻蝮蛇毒所致大鼠足跖肿胀的致炎模型,观察墨旱莲提取液对蛇毒所致大鼠足跖肿胀的影响。墨旱莲提取液分别与不同蛇毒混合,给小鼠腹部皮下注射,观察其对蛇毒引起的小鼠皮下出血的影响。结果墨旱莲提取液15g／kg连续2次灌胃给药,对短尾蝮蛇毒、蛇岛蝮蛇毒、白眉蝮蛇毒或尖吻蝮蛇毒所致大鼠足跖肿胀的急性炎症造模和短尾蝮蛇毒棉球肉芽肿的慢性炎症造模(20g／kg)均有明显的抑制作用,对这些蛇毒引起的小鼠皮下出血也能明显抑制。结论墨旱莲提取液对短尾蝮蛇毒、蛇岛蝮蛇毒、白眉蝮蛇毒及尖吻蝮蛇毒引起的炎症和出血均有明显的抑制作用。     

何谓“清栓酶”“AT—3”?

編辑同志: 近来我们发现蝮蛇抗栓酶的名目较多,有“清栓酶”,报纸上又见有“AT—3”,到底是不是同一样的产品? 田阳县医院邓有辉我国从蛇毒中提取有效成份制成抗血栓药物目前有三种,即用东北白眉蝮蛇毒制成“东北蝮蛇抗栓酶”江浙蝮蛇毒制成的,“江浙蝮蛇抗栓酶”和用五步蛇毒制成的“去纤酶”。     

眼镜王蛇毒柱层析分离及其组份的活性测定和L—氨基酸氧化酶的分离纯化

用ＣＭ—ＳｅｐｈａｄｅｘＣ—２５分离广西产眼镜王蛇毒,得到了１３个蛋白峰,测定了６种酶在这些峰的分布,其中７个峰有酸活性。并对含量高的Ｌ—氨基酸氧化酶进一步纯化,得到聚丙烯酰胺凝胶电泳纯一的Ｌ—氨基酸氧化酶     

钙离子对江浙蝮蛇毒酸性磷脂酶A2结构与功能的影响

钙离子在江浙蝮蛇毒酸性磷脂酶Ａ２中的作用是多方面的,它不仅能够引起酸性磷脂酶Ａ２在溶液中构象的变化,而且对该酶活性有较大的影响,Ｃａ＾２＋为酶活力所必需,当Ｃａ＾２＋浓度达到０．０６ｍｍｏｌ／Ｌ时,酶表现出很的活力;Ｃａ＾２＋浓度超过０．５ｍｍｏｌ／Ｌ时,催化反应出现一个明显的延滞期。化学修饰表明Ｈｉｓ４７在表现活力方面起重要作用,Ｃａ＾２＋的存在可降低其修饰反应的速度,提示这是由于Ｃａ＾２＋引起     

福建圆斑蝰蛇（Vipera russelli siamensis Smith）毒新的磷脂酶A_2的分离纯化及理化、药理性质的初步研究

本文应用离子交换柱层析和凝胶过滤技术从福建产圆斑蝰蛇泰国亚种蛇毒中纯化出一个新的ＰＬＡ,（ＰＬＡ２－３）。经聚丙烯酰胺凝胶电泳,ＳＤＳ－聚丙烯酰胺凝胶电泳和Ｏｕｃｈｔｅｒｌｏｎｙ双向免疫扩散证明为均一蛋白质。其理化及酶学性质如下：由约１２０个氨基酸残基组成（不包括半胱氨酸和色氨酸）;分子量为１４８００;等电点为７．８;ＰＬＡ２酶比活力用自动电位滴定法测定为２５５μｍｏｌ／ｍｉｎ·ｍｇ,用间接溶血法测定半数溶血量（ＨＵ５０）在家兔和大白鼠分别为０．０２５μｇ／ｍｌ和０．０５μｇ／ｍｌ;小鼠静脉注射的近似ＬＤ５０为３１５４ｍｇ／ｋｇ。与从该蛇毒中分离出的毒性和碱性ＰＬＡ,相比,具有ＰＬＡ。酶比活力高,毒性小的特点。此酶具有明显降压作用。抗血小板聚集效应和可逆性负性心肌收缩力作用,但未观察到心肌挛缩现象．     

蛇毒抗菌的比较性研究

目的 研究蛇毒粗毒的抗菌作用,并比较不同蛇毒对不同细菌的抑制效果.方法 采用抑菌环法测定蛇岛蝮、江浙蝮和短尾蝮蛇毒粗毒对金黄色葡萄球菌、枯草芽孢杆菌、大肠埃希菌、铜绿假单胞菌以及白色念珠菌的抗菌作用,比较不同蛇毒粗毒的抗菌效果;加入过氧化氢酶(catalase),考察蛇毒抗菌作用的变化.结果 (1)3种蛇毒粗毒对5种细菌均呈现不同程度的抑制作用.(2)3种蛇毒粗毒对5种细菌的抑制作用大小依次为:金黄色葡萄球菌＞白色念珠菌＞大肠埃希菌或铜绿假单胞菌＞枯草芽孢杆菌.(3)加入catalase后,蛇毒的抗菌作用明显减弱.结论 (1)3种蛇毒粗毒均具有一定的抗菌作用,并具有明显抑菌选择性.(2)3种蛇毒的抗菌效果不同,蛇岛蝮蛇抗菌作用最强.(3) catalase能显著降低蛇毒的抗菌活性,表明L-氨基酸氧化酶是蛇毒起抗菌作用的主要成分.     

谈清栓酶与东北白眉蝮蛇毒市场

近几年来,在防治各种心、脑、周围闭塞性血管病过程中,清栓酶(蝮蛇抗栓酶)显示出良好疗效,市场需求量较大。从而自86年起,生产厂家逐渐增多,目前国内已有32个厂家或单位生产此类药品。随着厂家增多,其原料东北白眉蝮蛇毒(粗原毒)也相应的畅销,售价高于江浙蝮蛇毒8～10倍。由于蛇毒差价原因,给某些商贩造成可乘之机,将江浙蝮蛇毒冒充东北白眉蝮蛇毒进行兕售。据了解不少厂家或单位直接以个体养蛇户或商贩手中购买粗原毒,造成原料不纯,直接影响清栓酶药品质量。江浙蝮蛇毒以外观上与东北白眉蝮蛇毒相似,但含神经毒、出血毒素均较高,一般分离制药残留较多,临床用药容易出现其反应,并且降低血小板作用明显。而东北白眉蝮蛇毒含神经毒极微,出血毒素极低,经一般分离制药即可以除去,临床用药安全,一般不降低血小板。根据调查分析,假冒原料有以下几个原因:     

钙离子对江浙蝮蛇毒酸性磷脂酶A_2结构与功能的影响

钙离子在江浙蝮蛇毒酸性磷脂酶Ａ_２中的作用是多方面的。它不仅能够引起酸性磷脂酶Ａ_２在溶液中构象的变化,而且对该酶活性有较大的影响。Ｃａ~（２＋）为酶活力所必需,当Ｃａ~（２＋）浓度达到０．０６ｍｍｏｌ／Ｌ时,酶表现出很高的活力;Ｃａ~（２＋）浓度超过０．５ｍｍｏｌ／Ｌ时,催化反应出现一个明显的延滞期。化学修饰表明Ｈｉｓ_（４７）在表现活力方面起重要作用,Ｃａ~（２＋）的存在可降低其修饰反应的速度,提示这是由于Ｃａ~（２＋）引起构象发生变化而造成的。     

Viper metalloproteinase (Agkistrodon halys pallas) with antimicrobial activity against multi-drug resistant human pathogens

Metalloproteinases are abundant enzymes in crotalidae and viperidae snake venoms. Snake venom metalloproteinases (SVMPs) comprise a family of zinc-dependent enzymes, which display many different biological activities. A 23.1 kDa protein was isolated from Agkistrodon halys (pallas, Chinese viper) snake venom. The toxin is a single chain polypeptide with a molecular weight of 23146.61 and an N-terminal sequence (MIQVLLVTICLAVFPYQGSSIILES) relatively similar to that of other metalloprotein-like proteases isolated from the snake venoms of the Viperidae family. The antibacterial effect of Agkistrodon halys metalloproteinase (AHM) on Burkholderia pseudomallei (strains TES and KHW), Escherichia coli, Enterobacter aerogenes, Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa (Gram-negative bacteria) and Staphylococcus aureus (Gram-positive bacterium) was studied at a concentration 120 microM. Interestingly, we found that the metalloproteinase exhibited antibacterial properties and was more active against S. aureus, P. vulgaris, P. mirabilis and multi-drug resistant B. pseudomallei (strain KHW) bacteria. AHM variants with high bacteriostatic activity (MIC 1.875-60 microM) also tended to be less cytotoxic against U-937 human monocytic cells up to 1 mM concentrations. These results suggest that this metalloprotein exerts its antimicrobial effect by altering membrane packing and inhibiting mechanosensitive targets.     

Antibacterial activity of snake, scorpion and bee venoms: a comparison with purified venom phospholipase A2 enzymes

AIMS: Venoms of snakes, scorpions, bees and purified venom phospholipase A(2) (PLA(2)) enzymes were examined to evaluate the antibacterial activity of purified venom enzymes as compared with that of the crude venoms. METHODS AND RESULTS: Thirty-four crude venoms, nine purified PLA(2)s and two L-amino acid oxidases (LAAO) were studied for antibacterial activity by disc-diffusion assay (100 microg ml(-1)). Several snake venoms (Daboia russelli russelli, Crotalus adamanteus, Naja sumatrana, Pseudechis guttata, Agkistrodon halys, Acanthophis praelongus and Daboia russelli siamensis) showed activity against two to four different pathogenic bacteria. Daboia russelli russelli and Pseudechis australis venoms exhibited the most potent activity against Staphylococcus aureus, while the rest showed only a moderate activity against one or more bacteria. The order of susceptibility of the bacteria against viperidae venoms was -S. aureus > Proteus mirabilis > Proteus vulgaris > Enterobacter aerogenes > Pseudomonas aeruginosa and Escherichia coli. The minimum inhibitory concentrations (MIC) against S. aureus was studied by dilution method (160-1.25 microg ml(-1)). A stronger effect was noted with the viperidae venoms (20 microg ml(-11)) as compared with elapidae venoms (40 microg ml(-1)). The MIC were comparable with those of the standard drugs (chloramphenicol, streptomycin and penicillin). CONCLUSION: The present findings indicate that viperidae (D. russelli russelli) and elapidae (P. australis) venoms have significant antibacterial effects against gram (+) and gram (-) bacteria, which may be the result of the primary antibacterial components of laao, and in particular, the PLA(2) enzymes. The results would be useful for further purification and characterization of antibacterial agents from snake venoms. SIGNIFICANCE AND IMPACT OF THE STUDY: The activity of LAAO and PLA(2) enzymes may be associated with the antibacterial activity of snake venoms.     

Cloning, sequence analysis and expression in E. coli of the cDNA of the thrombin-like enzyme (pallabin) from the venom of Agkistrodon halys pallas

The cDNA of the thrombin-like enzyme (pallabin) from the venom of Agkistrodon halys pallas was cloned and sequenced. The length of the cDNA is 923bp which includes 120bp of noncoding region and 780bp of coding region. Pallabin was synthesized as a prozymogen with 260 amino acids, which includes a signal peptide of 18 amino acids, a proposed propeptide of 6 amino acids and a matured peptide of 236 amino acids. Pallabin exhibits a strong amino acid similarity to the serine proteases isolated from other snake venoms. It contains 12 cysteins which form 6 disulfide bridges. Like other serine proteases, it also has three conserved catalytically active sites: His41, Asp86 and Ser182. To our knowledge, this study is the first report concerning the cDNA of a thrombin-like enzyme from Agkistrodon halys pallas. The cDNA was cloned into the expression plasmid pT7ZZa and expressed in E.coli. The recombinant pallabin immunologically reacted with its specific antibody.     

Inhibitory effects on phospholipase A2 and antivenin activity of melanin extracted from Thea sinensis Linn

Antivenin activity of melanin extracted from black tea (MEBT) was reported for the first time. The antagonistic effect of MEBT was evaluated for Agkistrodon contortrix laticinctus (broadbanded copperhead), Agkistrodon halys blomhoffii (Japanese mamushi), and Crotalus atrox (western diamondback rattlesnake) snake venoms administered i.p. to ICR mice. MEBT was injected i.p. immediately after the venom administration in dose of 3 mg per mouse in the same place of venom injection. MEBT demonstrated neutralization effect against all venoms tested. The greatest antivenin effect of MEBT was found against Japanese mamushi snake venom. In this case, half the mice died within 2.5 +/- 0.7 h after injection of 0.9 mg/kg of venom. An immediate injection of MEBT substantially reduced the toxic effect of venom and extended time at the 50% level of survival up to 52.3 +/- 2.3 h. The antivenin activity of MEBT is due to chelating of Ca++ and non-specific binding of phospholipase A2. The inhibitory effect of MEBT on phospholipase A2 assessed for different venoms was similar to that obtained with pure enzyme. Low toxicity of MEBT in combination with its antagonistic activity against different venoms may allow effective life-saving treatment against snakebites. Such application of MEBT is important when identification of the snake is impossible or if specific treatment is unavailable.     

蛇毒中抗癌组分对Hca-16局部肿瘤生长抑制的实验研究报告

本文报告从中华眼镜蛇毒及长白山白眉蝮蛇毒中分离出的抗癌组分蛇毒抗癌宁(SVAT)对Hca-16局部肿瘤生长抑制进行实验研究,结果证明蛇毒抗癌宁对白鼠的 Hca-16接种生长有一定抑制作用,与对照组相比差异显著。实验还发现蛇毒抗癌宁用量过大对肝脏有损害,用量适当可减轻损伤,并且损伤在一定范围内是可逆的.     

蛇毒的核磁共振氢谱研究

本文对不同产地的蝮蛇毒和眼镜蛇毒、五步蛇毒等十六个冻干样品,进行了核磁共振氢谱测试.列出了具有代表性的的氢谱图。从谱图中可看出:每种种属蛇毒均有其特征的核磁共振氢谱,此法在准分子水平上是鉴定蛇毒的一种有效而可靠的方法.     

用尖吻蝮蛇毒、蝗蛇毒及抗蛇毒血清处理小鼠S_(180)、EAC腹水癌的初步观察

本文报道了尖吻蝮蛇毒、蝮蛇毒及抗蛇毒血清能使接种S_(180)、EAC腹水癌的小鼠明显延长存活时间、降低接种率,但不能完全阻止痛细胞生长。体外具有较明显的导致,菡细胞肿胀、膜破裂,核纤维化,坏死等。从腹水酶活力测定及抗血清初步研究结果表明,癌细胞病变中产生的某些抗原物质可能与蛇毒中的酶和毒蛋白相近。因此注射蛇毒后可在体内产生相关抗体,中和癌细胞产生的毒素以达到治疗目的。     

蝮蛇抗栓酶与精制蝮蛇抗栓酶制剂的比较研究

本文作者对以东北长白山白眉蝮蛇毒为原料生产的两种酶制剂——蝮蛇抗栓酶与精制蝮蛇抗栓酶进行了比较研究。用 HPLC 柱层析粗毒得到15个蛋白峰,同时对两种酶制剂以 HPLC 层析用两根层析柱串联上样层析以提高其分辩率,得到两个图谱,蝮蛇抗栓酶有7个蛋白峰,而精制品有3～4个蛋白峰.同时以 SDS-PAGE 电泳图谱.蝮蛇抗栓酶呈现7～8条谱带,精制品有4～5条谱带,并与已知分子量的标准蛋白进行对比,结果表明两种酶制剂均非单一组分.蝮蛇抗栓酶谱带较多,精制品较纯.其分子量均在2～6万之间.蝮蛇抗栓酶几个组分有协同作用,每次剂量在0.25～0.50单位,即有明显疗效,而精制品临床用药剂量较大,每次0.75～1.0单位,多者达1.25单位(3～5支).     

The effect of calmodulin and chlorpromazine on the activity of phospholipases A2 from various sources

The effects of calmodulin and chlorpromazine on purified phospholipase A2 preparations from snake venoms: cobra (Naja naja oxiana), echis (Ehis multisquamatus) and Agkistrodon halys halys, as well as on phospholipases A2 from rat liver mitochondria and human platelets were studied. It was shown that within the concentration range of 1-5 microM calmodulin stimulates the phospholipase activity. Chlorpromazine inhibits the activity of these enzymes, the degree of inhibition being different for various phospholipases. Calmodulin was shown to interact with the phospholipases in the absence of exogenous Ca2+. The results obtained indicate that all phospholipases tested are calmodulin-dependent enzymes.     

The action of various venoms on Escherichia coli

The antibacterial activity of honeybee venom ( Apis mellifera ), three snake venoms ( Naja naja sputatrix, Vipera russellii and Crotalus adamanteus ) and the polypeptide melittin was investigated against Escherichia coli . Minimum inhibitory concentration values, cell lysis and alterations in cell permeability were determined and action against E. coli was in the order: A. mellifera venom > melittin > N. naja sputatrix venom ≫ V. russellii venom > C. adamanteus venom. Cellular damage by A. mellifera and N. naja sputatrix venoms was evident in electron micrographs.