Amino acid sequence of kappa-flavitoxin: establishment of a new family of snake venom neurotoxins

The complete amino acid sequence of kappa-flavitoxin, a neurotoxin isolated from the venom of Bungarus flaviceps, has been determined by automated Edman analysis of the intact protein and of peptides derived from digests with trypsin and chymotrypsin. kappa-Flavitoxin consists of a single 66-residue polypeptide chain which is completely devoid of methionine. The amino acid sequence of kappa-flavitoxin demonstrates that although the toxin is related to the alpha-neurotoxin family, it displays a much higher degree of homology with kappa-bungarotoxin. The conserved structural features of the kappa-neurotoxins and their pharmacological profiles, which are distinct from those of all known alpha-neurotoxins, provide evidence for a new, structurally and functionally unique family of snake venom neurotoxins.     

kappa-Bungarotoxin. Self-association of a neuronal nicotinic receptor probe

kappa-Bungarotoxin is a postsynaptic neurotoxin purified from the venom of the elapid snake Bungarus multicinctus. The amino acid sequence of this basic polypeptide reveals a single chain containing 66 amino acids having a Mr of 7,313. kappa-Bungarotoxin is a potent antagonist of nicotinic cholinergic transmission in avian and murine autonomic ganglia, a characteristic which distinguishes the toxin from other postsynaptic neurotoxins isolated from snake venoms. The self-association of kappa-bungarotoxin has now been examined using molecular sizing columns, sedimentation velocity, and sedimentation equilibrium. The results demonstrate that, under physiological solvent conditions, kappa-bungarotoxin exists as a dimer (Mr = 14,000 +/- 3,000) of identical subunits. kappa-Bungarotoxin monomers are not observed at toxin concentrations typically used in electrophysiological experiments (0.5-22 micrograms/ml), indicating that the dimer may be physiologically active. Denaturation with sodium dodecyl sulfate or urea dissociates kappa-bungarotoxin dimers into monomers. Significant amounts of monomers are also produced under nondenaturing conditions of high ionic strength and high pH. However, complete reassociation of nondenatured monomers occurs following return to a physiological buffer. The unique pharmacological spectrum of kappa-bungarotoxin may be due in part to its strong tendency to self-associate.     

Crystallization of kappa-bungarotoxin: preliminary X-ray data obtained from the venom-derived protein.

kappa-Bungarotoxin is a 66 residue polypeptide found in the venom of the Taiwanese banded krait, Bungarus multicinctus. It binds tightly to neuronal nicotinic acetylcholine receptors and inhibits nerve transmission mediated by these postsynaptic receptors. It is related, by similarity in amino acid sequence, to alpha-bungarotoxin and other alpha-neurotoxins, but differs sharply in physiologic action. The alpha-neurotoxins inhibit nerve transmission in nicotinic acetylcholine receptors associated with vertebrate skeletal muscle and fish electric organs. The kappa-neurotoxins inhibit nerve transmission in neuronal nicotinic acetylcholine receptors such as those found in chick ciliary ganglia. The kappa-neurotoxins display a low level of interaction with receptors that are strongly affected by alpha-neurotoxins, but alpha-neurotoxins are completely without effect on receptors that are affected by kappa-bungarotoxin. The structural basis for this physiologic differentiation is not known. Crystals of kappa-bungarotoxin have now been obtained that diffract to at least 2.3 A. These crystals are hexagonal, space group P6, and have dimensions of a = b = 80.2 A, c = 39.6 A, and angles of alpha = beta = 90 degrees and gamma = 120 degrees. Each unit cell contains 12 molecules of the 66 residue protein or two molecules per asymmetric unit. Comparison of the structure of kappa-bungarotoxin, which will result from further diffraction analysis of these crystals, with the structures of the alpha-neurotoxins that have been determined may provide information on the structural basis of physiologic action in these acetylcholine receptor antagonists.     

kappa-Bungarotoxin: complete amino acid sequence of a neuronal nicotinic receptor probe

The complete amino acid sequence of kappa-bungarotoxin, a neurotoxin isolated from the venom of the banded krait Bungarus multicinctus, has been determined by automated Edman analyses of the intact protein and peptides derived from digests with trypsin and chymotrypsin. kappa-Bungarotoxin consists of a single polypeptide chain of 66 amino acids with a molecular weight of 7313. It contains 10 cysteinyl residues, presumably arranged in 5 disulfide bonds, and is completely devoid of methionine and tryptophan. The amino acid sequence of kappa-bungarotoxin shows greatest homology to the curaremimetic postsynaptic long neurotoxins of which alpha-bungarotoxin is also a member. However, there are some striking differences between kappa-bungarotoxin and other members of this group which may explain its unusual ability to block neuronal acetylcholine receptors.     

cDNA sequence analysis of a novel member of the three loop protein family from the Chinese continental banded krait.

The cDNA encoding a novel three loop protein was cloned from cellular RNA isolated from the venom gland of Bungarus multicinctus multicinctus by RT-PCR. The mature protein has 82 amino acid residues. It shared only 25-38% similarity with some cardiotoxins and did not have sequence similarity with neurotoxins, while its cDNA was about 70% similar to both the cDNAs encoding neurotoxins and the cDNAs encoding cardiotoxins.     

An ISFET-based immunosensor for the detection of beta-Bungarotoxin

An ion-sensitive field effect transistor (ISFET)-based immunosensor was developed to detect/quantitate beta-Bungarotoxin (beta-BuTx), a potent presynaptic neurotoxin from the venom of Bungarus multicinctus. A murine monoclonal antibody (mAb 15) specific to beta-BuTx was immobilized onto silicon nitride wafers after silanization and activation with glutaraldehyde. A chip based enzyme linked-immunosorbantassay (ELISA) was performed to ascertain antigen binding to the immobilized antibody. To develop an electrochemical immunosensing system for the detection/quantitation of beta-BuTx, an ISFET was used as a solid phase detector. MAb 15 was immobilized on the gate region of the ISFET. The antigen antibody reaction was monitored by the addition of urease conjugated rabbit anti-beta-BuTx antibodies. The sensor can detect toxin level as low as 15.6 ng/ml. The efficacy of the sensor for the determination of beta-BuTx from B. multicinctus venom was demonstrated in mouse model. Toxin concentration was highest at the site of injection (748.0+/-26 ng/ml) and moderate amount was found in the plasma (158.5+/-13 ng/ml).     

Isolation, toxicity and amino terminal sequences of three major neurotoxins in the venom of Malayan krait (Bungarus candidus) from Thailand

We isolated the most lethal toxins in the venom of the Malayan krait (Bungarus candidus), one of the medically most important snake species in southeast Asia. Three beta-BTx like basic neurotoxins, T1-1, T1-2, and T2, with PLA2 activity were isolated from pooled venom of eight B. candidus from southern Thailand by cation-exchange chromatography, followed by adsorption chromatography on hydroxylapatite and RP-HPLC, with 14-, 16-, and 4-fold increases in toxicity compared to crude venom. The LDs50 determined in mice weighing 18-20 g were 0.26, 0.22, and 0.84 micro g per mouse with i.v. injection. T1-1 and T1-2 possessed comparable lethal toxicities to those of beta1-BTx, the most toxic neurotoxin in B. multicinctus venom, and the major neurotoxin in B. flaviceps venom. The apparent molecular weights of the native toxins were approximately 25-25.5 kDa. They consist of two polypeptide chains with apparent molecular weights of 15.5-16.5 and 8-8.5 kDa, respectively. The amino terminal sequences of the two chains of each of the toxins determined by Edman degradation exhibited considerable similarity with those of the A-chains and B-chains of beta-BTxs in the venom of Bungarus multicinctus.     

An inhibitory domain of E12 transcription factor prevents DNA binding in E12 homodimers but not in E12 heterodimers.

The kappa E2 sequence binding proteins, E12 and E47, are generated by alternative splicing of the E2A gene, giving closely related basic and helix-loop-helix structures crucial for DNA binding and dimerization. Measurements of dimerization constants and binding strengths to the optimal DNA sequence (the kappa E2 site or its near relatives) showed that E47 homodimers and MyoD heterodimers with E12 or E47 dimerized and bound avidly, but E12 homodimerized efficiently and bound to DNA poorly; MyoD homodimerized poorly and bound strongly. An inhibitory domain N-terminal to the basic region of E12 prevents E12 homodimers but not E12/MyoD heterodimers from binding to DNA. Thus, E47 binds to DNA both as a heterodimer with MyoD and as a homodimer, while E12 and MyoD bind to DNA efficiently only as heterodimers.     

The complete amino-acid sequence of a basic phospholipase A2 in the venom of Bungarus fasciatus

A basic phospholipase A2 (PLA2) was isolated and purified from the venom of Bungarus fasciatus. Four kinds of enzymes, lysyl endopeptidase, endoproteinase Asp-N, endoproteinase Glu-C and trypsin, were employed to elucidate the complete primary structure by means of gas-phase sequencing. The amino-acid sequence reveals 118 amino-acid residues containing seven pairs of half-cystine. It has 78% and 61% structural identities with PLA2 from Bungarus multicinctus and Naja melanoleuca DE-II, respectively.     

基于动物学、临床医学跨界研究快速准确诊断1例不明蛇类咬伤的分析

目的 探讨动物学、临床医学跨界研究成果联合运用于快速准确诊断不明蛇类咬伤中的作用及效果.方法 通过回顾性分析1例不明蛇类咬伤患者的临床资料,从病史、动物学生活习性、咬痕鉴别、流行病学、临床表现、蛇类动物学分布6个方面进行剖析不明蛇类咬伤的诊断思路与方法.结果 该例蛇伤患者的病史特征以及诊断与鉴别诊断:(1)病史特征符合...     

缺抗蛇毒血清银环蛇伤的临床观察

目的探讨临床上缺乏抗银环蛇蛇毒血清治疗银环蛇咬伤的临床效果。方法将我院急诊科2011年3月~2013年10月收治的银环蛇咬伤患者9例与同期67例应用抗银环蛇毒血清治疗的病例进行比较,观察两组患者住院期间的临床变化与转归。结果经机械通气支持、胆碱酯酶抑制剂联合抗胆碱药物,以及肺部抗感染治疗,9例未经抗银环蛇毒血清治疗的患者均成活,但较使用抗银环蛇毒血清的患者住院时间延长、费用明显增加。结论重症银环蛇伤患者在缺乏抗银环蛇毒血清治疗时,应用机械呼吸支持,胆碱酯酶抑制剂联合抗胆碱药物,以及积极抗感染治疗也能救治患者生命。     

Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence

A DNA binding and dimerization motif, with apparent amphipathic helices (the HLH motif), has recently been identified in various proteins, including two that bind to immunoglobulin enhancers (E12 and E47). We show here that various HLH proteins can bind as apparent heterodimers to a single DNA motif and also, albeit usually more weakly, as apparent homodimers. The HLH domain can mediate heterodimer formation between either daughterless, E12, or E47 (Class A) and achaete-scute T3 or MyoD (Class B) to form proteins with high affinity for the kappa E2 site in the immunoglobulin kappa chain enhancer. The achaete-scute T3 and MyoD proteins do not form kappa E2-binding heterodimers together, and no active complex with N-myc was evident. The formation of a heterodimer between the daughterless and achaete-scute T3 products may explain the similar phenotypes of mutants at these two loci and the genetic interactions between them. A role of E12 and E47 in mammalian development, analogous to that of daughterless in Drosophila, is likely.     

The VLA protein family. Characterization of five distinct cell surface heterodimers each with a common 130,000 molecular weight beta subunit

The family of human cell surface heterodimers which includes VLA-1 and VLA-2 is now shown to include three additional heterodimers, here called VLA-3, VLA-4, and VLA-5. Each of these separate VLA structures is composed of a distinct alpha subunit (Mr 110,000-200,000 nonreduced) noncovalently associated with a common beta subunit (Mr 110,000 nonreduced). Chemical cross-linking experiments provided evidence that each VLA complex exists predominantly as a 1:1 alpha beta heterodimer. The VLA proteins are widely distributed, with one or more of the heterodimers present on nearly all cell types tested. Evidence for five distinct VLA alpha subunits was obtained from differences observed in antibody recognition, cell distribution patterns, two-dimensional gel analyses, and V8 protease cleavage patterns. On the other hand, the beta subunit present in each heterodimer was immunochemically and electrophoretically indistinguishable, and yielded identical V8 cleavage fragments. Immunoblotting experiments revealed that besides the Mr 110,000 beta normally seen, another beta protein was present that is smaller in size (Mr 90,000 nonreduced), altered or deficient in glycosylation, and not available for cell surface radiolabeling.     

Evolutionary and structural analyses of heterodimeric proteins composed of subunits with same fold

Heterodimeric proteins with homologous subunits of same fold are involved in various biological processes. The objective of this study is to understand the evolution of structural and functional features of such heterodimers. Using a non‐redundant dataset of 70 such heterodimers of known 3D structure and an independent dataset of 173 heterodimers from yeast, we note that the mean sequence identity between interacting homologous subunits is only 23–24% suggesting that, generally, highly diverged paralogues assemble to form such a heterodimer. We also note that the functional roles of interacting subunits/domains are generally quite different. This suggests that, though the interacting subunits/domains are homologous, the high evolutionary divergence characterize their high functional divergence which contributes to a gross function for the heterodimer considered as a whole. The inverse relationship between sequence identity and RMSD of interacting homologues in heterodimers is not followed. We also addressed the question of formation of homodimers of the subunits of heterodimers by generating models of fictitious homodimers on the basis of the 3D structures of the heterodimers. Interaction energies associated with these homodimers suggests that, in overwhelming majority of the cases, such homodimers are unlikely to be stable. Majority of the homologues of heterodimers of known structures form heterodimers (51.8%) and a small proportion (14.6%) form homodimers. Comparison of 3D structures of heterodimers with homologous homodimers suggests that interfacial nature of residues is not well conserved. In over 90% of the cases we note that the interacting subunits of heterodimers are co‐localized in the cell. Proteins 2015; 83:1766–1786. © 2015 Wiley Periodicals, Inc.     

Effect of β-Bungarotoxin on the Release of Endogenous Amino Acids from the Sensorimotor Cortex

Abstract: β-Bungarotoxin, a snake neurotoxin purified from the venom of Bungarus multicinctus , caused a significant increase in the in vivo release of glutamate from the superfused sensorimotor cortex of awake animals. A smaller effect on GABA release was observed, but no change was detected in the release of six other amino acids measured. The effects on glutamate and GABA release were entirely blocked by tetrodotoxin (1 μM) and were reversible when the cortical tissue was washed with saline.     

Alkaline phosphatase from adult rat femur

Four alkaline phosphatase forms from adult rat femur were distinguished on polyacrylamide gel electrophoresis: two soluble forms of Mr 165,000 and 110,000 in the water extract, and three membrane-bound forms of Mr 130,000, 110,000 and 100,000 extractable with deoxycholate. Alkaline phosphatase after SDS-treatment disintegrated into three kinds of monomers: of Mr 80,000, 65,000 and 50,000. The soluble fraction (extract I) contained subunits of Mr 80,000 and 55,000--whereas the pellet fraction (extract II), subunits of Mr 65,000 and 50,000. Since for native forms only three types of subunits were found it seems that, apart from homodimers, there are also some heterodimers composed of the Mr 65,000 and 50,000 subunits forming the native enzyme of Mr 110,000-115,000. Two denatured monomers: of Mr 80,000 and 50,000 may form two native homodimeric forms of Mr 165,000 and 100,000 while in the pellet two monomers: of Mr 65,000 and 50,000 may correspond to three native alkaline phosphatase forms: of Mr 130,000, 110,000-115,000 and 100,000. Probably the Mr 110,000-115,000 form is a heterodimer composed of subunits of Mr 65,000 and 50,000.     

Smooth muscle tropomyosin coiled-coil dimers. Subunit composition, assembly, and end-to-end interaction

Subunits of gizzard smooth muscle tropomyosin, dissociated by guanidinium chloride and reassociated by high salt dialysis, form a 1:1 mixture of the beta beta and gamma gamma homodimers (Graceffa, P. (1989) Biochemistry 28, 1282-1287). The homodimers have now been separated by anion-exchange chromatography and native gel electrophoresis, enabling us to show that the native protein is composed of more than 90% heterodimer. The in vitro equilibrium distribution of heterodimer and homodimers, at close to physiological temperature and ionic conditions, was calculated from thermal unfolding profiles of separated homodimers and heterodimer, as monitored by circular dichroism. The results, for an equal proportion of beta and gamma chains, indicate a predominant formation of heterodimer via chain dissociation and chain exchange, although the proportion of heterodimer was much less than the 90-100% found in the native protein. However, the proportion of heterodimer for actin-bound tropomyosin, determined by analyzing tropomyosin sedimented with actin, was greater than 90%, which may provide a model for assembly in vivo. The end-to-end interactions of the homodimers are about the same but are much less than that of the native heterodimer, as determined by viscometry. The greater end-to-end interaction of heterodimers may lead to stronger binding to actin compared to homodimers and thus would further shift the equilibrium between heterodimer and homodimers toward heterodimer and possibly account for the almost exclusive population of heterodimer in the presence of actin. The greater end-to-end interaction of the heterodimer may also provide a functional advantage for its preferred assembly. This study also shows that the two-step thermal unfolding of the homodimer mixture is due to the formation of heterodimer via an intermediate which is a new type of tropomyosin species which forms a gel in low salt. This tropomyosin is also present in small amounts in native tropomyosin preparations.