蓝色染料配基亲和纯化眼镜蛇心脏毒素的研究

探索了蓝色染料（Cibacron Blue F3G-A）亲和分离中华眼镜蛇心脏毒素的可能性。采用环氧基活化法制备蓝色染料亲和介质,中性条件下提取眼镜蛇粗毒中的心脏毒素。Tricine系统SDS-PAGE多肽电泳和Lowry法蛋白定量分析纯化效果,发现蓝色染料琼脂糖一步纯化中华眼镜蛇心脏毒素的纯度达到84%,结合量为6.9mg/ml介质。这是首次利用小分子亲和配基纯化心脏毒素。与生物大分子配基相比,活性染料分子具有价格便宜,易于合成,性质稳定,不易降解和适合大规模生产等优点。     

Evidence showing an intermolecular interaction between KChIP proteins and Taiwan cobra cardiotoxins

Direct protein-protein interaction between Taiwan cobra cardiotoxin3 (CTX3) and potassium channel-interacting proteins (KChIPs) was investigated in the present study. It was found that KChIPs bound with CTX3, in which KChIP and CTX3 formed a 1:1 complex as evidenced by the results of chemical cross-linking. Pull-down assay revealed that the intact EF-hands 3 and 4 of KChIP1 were critical for CTX3-binding. Likewise, removal of EF-hands 3 and 4 distorted the ability of KChIP1 to bind with Kv4.2 N-terminal fragment (KvN) as well as fluorescent probe 8-anilinonaphthalene-1-sulfonate (ANS). In contrast to the interaction between KChIP1 and KvN, the binding of CTX3 to KChIP1 showed a Ca(2+)-independent manner. Fluorescence measurement revealed that CTX3 affected the binding of ANS to Ca(2+)-bound KChIP1, but not Ca(2+)-free KChIP1. Alternatively, KChIP1 simultaneously bound with KvN and CTX3, and the interaction between KChIP1 and KvN was enhanced by CTX3. In terms of the fact that KChIPs regulate the electrophysiological properties of Kv K(+) channel, the potentiality of CTX for this biomedical application could be considered.     

Cardiotoxin III-induced apoptosis is mediated by Ca2+-dependent caspase-12 activation in K562 cells

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. When K562 cells were treated with CTX III, cytosolic calcium concentration was rapidly and persistently increased. This CTX III-induced cell death was partially reversed by pretreatment with BAPTA/AM (20 microM), a chelator of intracellular Ca2+. Moreover, CTX III-induced apoptotic signals, such as caspase-12 and c-Jun N-terminal kinase (JNK) activation, were induced in a time-dependent manner and inhibited by BAPTA/AM. In contrast, the neutral protease micro-calpain, a key enzyme in endoplasmic reticulum (ER) stress-related apoptosis via caspase-12 activation, was unchanged during apoptosis. Taken together, our findings suggest CTX III-induced apoptosis is triggered by Ca2+ influx, then activated caspase-12 and JNK through micro-calpain-independent cascade, and consequently caused apoptosis.     

Heart and T-Lymphocyte Cell Surfaces Both Exhibit Positive Cooperativity in Binding a Membrane-Lytic Toxin

Cobra venom cytotoxins (CTX) have been shown to disrupt cells as different as immunocytes, skeletal myocytes, erythrocytes and tumor cells. Nevertheless, even subpopulations of tumor cells are differentially susceptible to CTX by an order of magnitude. In the present study, our objective was to compare CTX-specific binding with cytolytic potency for two disparate cell types in vitro. We investigated the lytic activity of cytotoxin-III from Naja naja atra (NNA, fraction D) using heart cells and human leukemic T-cells (CEM cells). For both cell types, 50% cytolysis, assessed by tetrazolium dye conversion, occurred with μm concentrations of toxin (EC₅₀= 2.2 μm). We examined the binding of radiolabeled CTX III to both heart cells and CEM cells and found the apparent dissociation constant (K _Dapp) to be 0.69 μm and 0.75 μm, for CEM and heart cells respectively. The B _max for the CEM cells was 1.0 fmoles/cell and that for heart cells was 5.2 fmoles/cell, both exhibiting positive cooperativity between the sites (Hill coefficients 1.4, T-cells; 1.6, heart). Relatively modest dissociation constants plus high numbers of binding sites per cell are consistent with a model of CTX binding to plasma membranes by interaction with phospholipids in the bilayer. Our results suggest that the lytic activity of this cytotoxin follows its binding to a population of sites on the cells in a cooperative fashion. Received: 8 May 1995/Revised: 17 November 1995     

The structural evolution of cobra venom cytotoxins

Summary In order to analyze the evolutionary behavior of the cobra venom cytotoxins, their probable tertiary structure was predicted using computer graphics. The 41 amino acid sequences known show that the major evolutionary changes have taken place in two particularly exposed areas of the molecular surface. In each area, neighboring residue positions seem to have evolved interdependently, but there is no obvious interdependence between the two areas. Indeed, the relative evolution of these two areas prompts a subdivision of the sequence set into four groups. According to the known cytotoxin circular dichroism spectra, one of these four groups could be characterized by a difference in molecular secondary structure. Sine the two variable areas have functional associations, it is suggested that their evolution may be governed by a target with several similar binding sites.     

A comparative in silico characterization of functional and physicochemical properties of 3FTx (three finger toxin) proteins from four venomous snakes

Snake venom is an abundant resource of diverse pharmacologically bioactive proteins and peptides and a good natural source of drug lead compounds and used as important research tools in the field of toxicology, pharmacology and neuroscience. Three finger toxins (3FTx) is an important super-family of snake venom proteins which has a conserved three finger like appearance in three dimensional structures. Members of 3FTx family show a wide array of pharmacological effects by targeting different receptors and ion channels with high specificity and many of them are being investigated as potential drug target. Therefore, with a vision to verdict a new edge and attempt we determined the amino acid compositional (%) profile, physiochemical properties, secondary structural and functional analysis and phylogenetic relationship of three finger toxins present in four different elapid snake species namely, Naja naja, Astrotia stokesii, Hydrophis cyanocintus and Pelamis platura using different bioinformatics tools. From the outcome of the current studies, it will be possible to know about a range of biological functions which are responsible mainly for the glowing amino acid composition profile of these proteins. Amino acid composition (%) profile although represents differential amount of different amino acid residues which encompasses a family precise model but all the protein sequence have a conserved amount of cysteine. The analysis of physicochemical properties can be used as a basic approach to contribute in developing rational drug through protein engineering and understanding different physiological function which will be beneficial for the welfare of human being.     

酶标抗原火箭免疫电泳法及其在蛇毒抗原成分含量测定中的应用

为解决目前蛇毒成份检测困难以及寻找一种可用于蛇伤快速鉴别诊断的方法,本研究建立了酶标抗原火箭免疫电泳法。本法结合了酶标法灵敏度高和火箭电泳操作简单的特点,对待测的同种抗原标记辣根过氧化物酶(酶标抗原),检测时把微量的酶标记抗原掺入被测样品中,在含多价抗血清的免疫电泳板进行电泳。最后用辣根过氧化物底物3.3′-二氨基联苯胺在板上直接显色,显色后观察到的免疫沉淀线高度与被测抗原含量呈正相关(r=+0.98)。采用本方法检测眼镜蛇毒细胞毒素和限镜王蛇毒 L-氨基酶氧化酶,细胞毒素的最低检测浓度为110ng/ml,氨基酸氧化酶为60ng/ml。比单向火箭免疫电泳法灵敏度高30倍。用同一原理的酶标记抗原融合电泳法监测L-氨基酸氧化酶的层析分离时,比酶活性法检测灵敏度高20倍。全部检测只需30分钟。初步观察商品眼镜蛇毒抗血清可对蛇毒15种成分产生免疫沉淀线,眼镜蛇的细胞毒与同科异科蛇毒的细胞毒素无交叉免疫反应。因此,采用本法可以确定不同蛇种蛇毒中所含的特异性抗原,获得蛇伤鉴别诊断的依据。     

The binding of snake venom cardiotoxins to heart cell membranes

Cobra venom cardiotoxins have the effect, inter alia, of causing systolic arrest of the heart. We have observed significant binding in vitro of ³⁵S-labelled cardiotoxins to mouse heart cell membranes. Part of the binding was saturable and could be displaced with homologous unlabelled cardiotoxins but not by neurotoxins or cardiotoxins inactivated by chemical modification. The specifically bound component represented more than 70% of total binding at saturation. Inclusion of Triton X-100 and NaCl in the phosphate-buffered incubation medium prevented nonspecific adsorption to centrifuge tube walls, and gave lower but more reproducible specific binding results, respectively. An apparent dissociation constant of 5·10^?7 M and a binding density of 500 pmol toxin/mg membrane protein were derived from the saturation isotherms.     

Lipid-protein interactions. A comparative study of the binding of cardiotoxins and neurotoxins to phospholipid vesicles

It has recently been shown that cardiotoxin II from Naja mossambica mossambica specifically interacts with negatively charged phospholipids (Dufourcq, J. and Faucon, J.F. (1978) Biochemistry 17, 1170–1176). In order to investigate whether or not short neurotoxins give rise to similar interactions, four techniques have been used, namely intrinsic fluorescence, fluorescence polarization of 1,6-diphenylhexatriene, turbidity measurements and release of 6-carboxyfluorescein trapped inside single shelled vesicles.Neurotoxin III from Naja mossambica mossambica and neurotoxin I from the venom of the scorpion Androctonus australis Hector, specifically interact with negatively charged phospholipids leading to changes in tryptophan fluorescence and to a decrease of the fluidity of the bilayer. Cardiotoxin II from the same snake venom gives similar results. On the other hand, it seems that either a very weak or no interaction at all occurs in the case of neurotoxin I from the same Naja venom.There are important differences in the behaviour of cardiotoxin and neurotoxins: (i) neurotoxins lead to only weak release of 6-carboxyfluorescein from lipid vesicles, whereas cardiotoxin II induces fast and quantitative escape of the dye and then a general breakdown of the vesicular structure; (ii) binding of neurotoxins can be easily reversed by 100–200 mM NaCl or less than 1 mM Ca²⁺ and so it is essentially electrostatic, whereas binding of cardiotoxin II seems to involve some hydrophobic contribution.The short neurotoxins and cardiotoxins from snake venom having a great homology in sequence, their differences on binding properties are discussed in terms of changes in a particular area of the sequence.