An improved fractionation and fast screening method for the identification of new and selective neurotoxins

Debont, Tom; Daenens, Paul; Tytgat, Jan

doi:10.1016/0168-0102(95)00982-5

Cited by 13 publications

(17 citation statements)

References 12 publications

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“…Both FPLC and RP-HPLC have been used for isolation and purification of specific peptides and proteins from the venoms of different scorpion species [17,[35][36][37]. Separation procedures leading to the identification of neurotoxins almost always include gel filtration chromatography combined with ion-exchange or RP-HPLC [27].…”

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confidence: 99%

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Rapid profiling of crude scorpion venom using liquid chromatography and its relevance to species identification

Asmari

Khan

Manthiri

2012

Acta Chromatographica

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Summary. We present the comprehensive chromatographic profiles of three scorpion species, Androctonus crassicauda, Androctonus bicolor, and Leiurus quinquestriatus, commonly inhabited to Middle East regions. Their venoms were milked by electrical stimulation, diluted with distilled water, properly mixed and centrifuged to separate the mucus from venom. The clear supernatant was filtered and the protein concentration was determined. Pre-diluted venoms were chromatographed on FPLC (fast protein liquid chromatography) and RP-HPLC (reversed-phase high-performance liquid chromatography) and the fractions were collected for molecular weight determination. Both techniques have resulted clearly distinguishable chromatographic patterns that can be used for identification of scorpion species and having a quick indication of venom toxicity.

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Section: Fig 2 (Continued)mentioning

confidence: 99%

“…The molecular weights of the scorpion venom fractions were estimated by gel filtration chromatography as described by Debont et al [27]. HiLoad 16/60 Superdex 200 Pg column was used with 0.05 M Tris-HCl buffer, containing 100 mM NaCl.…”

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Rapid profiling of crude scorpion venom using liquid chromatography and its relevance to species identification

Asmari

Khan

Manthiri

2012

Acta Chromatographica

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“…PBTx3 was isolated from the venom on the basis of its ability to inhibit the K + current through cloned voltage-dependent K + channels (Kv1) expressed in Xenopus oocytes. Separation procedures leading to the identification of this novel neurotoxin were performed by gel filtration and reversedphase HPLC, by using different types of columns, as described previously [28].…”

Section: Discussionmentioning

confidence: 99%

“…To identify the last C-terminal residue, a sample of peptide was also cleaved by cyanogen bromide. By this reaction, three fragments were produced (E 1 -M 4 , R 5 -M 28 and N 29 -R 37 ), separated by HPLC by using the same C 18 analytical column as described above, and then sequenced. The last amino acid (arginine) was elucidated.…”

Section: Sequence Determinationmentioning

confidence: 99%

“…The periplasmic extracts (400 mL) were loaded [24]), charybdotoxin-Lq-2 [10], Lqh 15-1 [25] and AgTx2 (agitoxin 2 [15]), were purified from Leiurus quinquestriatus var. Hebraeus; BmTx1-2 [26] was purified from Buthus martensi Karsch; HgTx2 (hongotoxin 2 [27]), and LbTx (limbatotoxin [34]), were purified from Centruroides limbatus; IbTx (iberiotoxin [28]), and TmTx (tamulotoxin [56]), were purified from Buthus tamulus; PBTx3 (parabutoxin 3, this study) was purified from Parabuthus transvaalicus. to an amylose affinity resin (1.5 · 23 cm column, Biolabs, NEB) at a flow rate of 1 mLAEmin )1 in column buffer containing 20 mM Tris/HCl, 200 mM NaCl (Merck Eurolabs, Belgium), and 1 mM Na 2 EDTA buffer, pH 7.4.…”

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Purification, characterization and biosynthesis of parabutoxin 3, a component of Parabuthus transvaalicus venom

et al. 2002

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A novel peptidyl inhibitor of voltage-gated K + channels, named parabutoxin 3 (PBTx3), has been purified to homogeneity from the venom of Parabuthus transvaalicus. This scorpion toxin contains 37 residues, has a mass of 4274 Da and displays 41% identity with charybdotoxin (ChTx, also called Ôa-KTx1.1Õ). PBTx3 is the tenth member (called Ôa-KTx1.10Õ) of subfamily 1 of K + channel-blocking peptides known thus far. Electrophysiological experiments using Xenopus laevis oocytes indicate that PBTx3 is an inhibitor of Kv1 channels (Kv1.1, Kv1.2, Kv1.3), but has no detectable effects on Kir-type and ERG-type channels. The dissociation constants (K d ) for Kv1.1, Kv1.2 and Kv1.3 channels are, respectively, 79 lM, 547 nM and 492 nM. A synthetic gene encoding a PBTx3 homologue was designed and expressed as a fusion protein with the maltose-binding protein (MBP) in Escherichia coli. The recombinant protein was purified from the bacterial periplasm compartment using an amylose affinity resin column, followed by a gel filtration purification step and cleavage by factor X a (fX a ) to release the recombinant toxin peptide (rPBTx3). After final purification and refolding, rPBTx3 was shown to be identical to the native PBTx3 with respect to HPLC retention time, mass spectrometric analysis and functional properties. The threedimensional structure of PBTx3 is proposed by homology modelling to contain a double-stranded antiparallel b sheet and a single a-helix, connected by three disulfide bridges. The scaffold of PBTx3 is homologous to most other a-KTx scorpion toxins.

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Antimicrobial and cytolytic peptides of venomous arthropods

Kuhn‐Nentwig

2003

Cellular and Molecular Life Sciences (CMLS)

185

131

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As a response to invading microorganisms, the innate immune system of arthropods has evolved a complex arrangement of constitutive and inducible antimicrobial peptides that immediately destroy a large variety of pathogens. At the same time, venomous arthropods have developed an additional offensive system in their venom glands to subdue their prey items. In this complex venom system, several enzymes, low-molecular-mass compounds, neurotoxins, antimicrobial and cytolytic peptides interact together, resulting in extremely rapid immobilization and/or killing of prey or aggressors. This review provides an overview of antimicrobial peptides identified in the hemolymph of venomous arthropods, and especially of cytolytic peptides in their venom. For these peptides a dual role is proposed: acting as antimicrobials as well as increasing the potency of the venom by influencing excitable cells.

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An improved fractionation and fast screening method for the identification of new and selective neurotoxins

Cited by 13 publications

References 12 publications

Rapid profiling of crude scorpion venom using liquid chromatography and its relevance to species identification

Rapid profiling of crude scorpion venom using liquid chromatography and its relevance to species identification

Purification, characterization and biosynthesis of parabutoxin 3, a component of Parabuthus transvaalicus venom

Antimicrobial and cytolytic peptides of venomous arthropods

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