湖南永州尖吻蝮蛇蛇毒的蛋白组分及毒性分析

采用Nano–LC–ESI–MS/MS蛋白鉴定技术对湖南永州尖吻蝮蛇蛇毒蛋白组分进行质谱分析,应用常规体外试管法对尖吻蝮蛇蛇毒体外溶血值进行检测,采用改良寇式法对尖吻蝮蛇蛇毒LD_(50)进行测定。结果显示:尖吻蝮蛇蛇毒含有50种蛋白,蛋白相对分子质量集中在1.0×10~4～2.0×10~4(46.9%)、4.0×10~4～5.0×10~4(22.4%)、2.0×10~4～3.0×10~4(10.6%)和7.0×10~4～8.0×10~4(7.6%),其中代表性的高丰度蛋白有蛇毒金属蛋白酶A(11.7%)、蛇毒金属蛋白酶H1(9.8%)、蕲蛇类凝血酶–2(7.3%)、抗凝血酶A–A亚基(6.8%),低丰度蛋白(0.1%)有Ecto–5'–核苷酸酶、碱性磷脂酶A2 DAV–N6、生长分化因子11;蛇毒引起红细胞溶血的最低质量浓度为60.00μg/mL,尖吻蝮蛇蛇毒对KM小鼠的LD_(50)为7.1796mg/kg,攻毒小鼠出现呼吸急促、躁动不安、注射部位出现奇特瘙痒和大面积溃烂,至死亡。     

AsKC11, a Kunitz Peptide from Anemonia sulcata,Is a Novel Activator of G Protein-Coupled Inward-Rectifier Potassium Channels

(1) Background: G protein-coupled inward-rectifier potassium (GIRK) channels, especially neuronal GIRK1/2 channels, have been the focus of intense research interest for developing drugs against brain diseases. In this context, venom peptides that selectively activate GIRK channels can be seen as a new source for drug development. Here, we report on the identification and electrophysiological characterization of a novel activator of GIRK1/2 channels, AsKC11, found in the venom of the sea anemone Anemonia sulcata. (2) Methods: AsKC11 was purified from the sea anemone venom by reverse-phase chromatography and the sequence was identified by mass spectrometry. Using the two-electrode voltage-clamp technique, the activity of AsKC11 on GIRK1/2 channels was studied and its selectivity for other potassium channels was investigated. (3) Results: AsKC11, a Kunitz peptide found in the venom of A. sulcata, is the first peptide shown to directly activate neuronal GIRK1/2 channels independent from Gi/o protein activity, without affecting the inward-rectifier potassium channel (IRK1) and with only a minor effect on K_V1.6 channels. Thus, AsKC11 is a novel activator of GIRK channels resulting in larger K⁺ currents because of an increased chord conductance. (4) Conclusions: These discoveries provide new insights into a novel class of GIRK activators.     

意大利蜜蜂蜂毒透明质酸酶基因的克隆与序列分析

从意蜂毒腺抽提总RNA,用RT-PCR获得蜂毒透明质酸酶(AmHya)基因,其核苷酸序列全长为1 149 bp.将AmHya氨基酸序列与中蜂蜂毒透明质酸酶(AcHya)和其它5种透明质酸酶(Hya)氨基酸序列比较,结果显示,AmHya与 AcHya的同源性最高(91%),同时对7种Hya作了分子结构与分子进化分析.     

Protease Inhibitors from Marine Venomous Animals and Their Counterparts in Terrestrial Venomous Animals

The Kunitz-type protease inhibitors are the best-characterized family of serine protease inhibitors, probably due to their abundance in several organisms. These inhibitors consist of a chain of ~60 amino acid residues stabilized by three disulfide bridges, and was first observed in the bovine pancreatic trypsin inhibitor (BPTI)-like protease inhibitors, which strongly inhibit trypsin and chymotrypsin. In this review we present the protease inhibitors (PIs) described to date from marine venomous animals, such as from sea anemone extracts and Conus venom, as well as their counterparts in terrestrial venomous animals, such as snakes, scorpions, spiders, Anurans, and Hymenopterans. More emphasis was given to the Kunitz-type inhibitors, once they are found in all these organisms. Their biological sources, specificity against different proteases, and other molecular blanks (being also K⁺ channel blockers) are presented, followed by their molecular diversity. Whereas sea anemone, snakes and other venomous animals present mainly Kunitz-type inhibitors, PIs from Anurans present the major variety in structure length and number of Cys residues, with at least six distinguishable classes. A representative alignment of PIs from these venomous animals shows that, despite eventual differences in Cys assignment, the key-residues for the protease inhibitory activity in all of them occupy similar positions in primary sequence. The key-residues for the K⁺ channel blocking activity was also compared.     

苏木水提物抗蛇毒磷脂酶A2活性研究

中药苏木(Sappan Lignum)来源于植物苏木(Caesalpinia sappan L.)的干燥心材,具有止血 抗炎等功效。采用琼脂糖平板法研究不同浓度的苏木水提物（0.03g/ml,0.06g/ml,0.09g/ml, 0.12g/ml,0.15g/ml）对蛇毒sPLA2水解活性的抑制作用,测量透明圈和孔的直径,根据酶活标准曲 线计算其抑制率。结果表明0.15g/ml 苏木水提物对五步蛇毒sPLA2水解活性有抑制作用。     

白色霞水母刺细胞毒素抗烟草花叶病毒活性

为探索从水母毒素中获取抗植物病毒物质的应用前景,采用半叶枯斑法、整株法和漂浮叶圆片法,对白色霞水母Cyanea nozakii Kishinouye刺细胞毒素的抗烟草花叶病毒(Tobacco mosaic virus,TMV)活性进行了检测。结果表明,白色霞水母刺细胞毒素对TMV具有很强的直接钝化作用,枯斑抑制率随钝化时间的延长而增大。0.99 mg/m L毒素体外钝化TMV 30 min,枯斑抑制率达98.85%,将TMV与0.80 mg/m L毒素体外混合后立即接种,枯斑抑制率仍达89.57%;20~70℃之间,毒素对TMV的钝化作用随温度升高而降低,但又表现出一定的高温耐受性,70℃下处理30min,1.33 mg/m L毒素对枯斑的抑制率为61.73%。毒素对病毒初侵染有一定的预防作用,并可抑制TMV的增殖;1.93 mg/m L毒素处理接种TMV的叶圆片2 d后,对TMV的增殖抑制率为49.41%;但该毒素对TMV所致病害的治疗效果不明显。该毒素还具有较强的蛋白水解酶活性,可能与其抗TMV活性相关。表明白色霞水母刺细胞毒素抗TMV作用明显,其抗植物病毒活性值得深入研究。