Purification and partial characterization of two phospholipases A2 from Bothrops leucurus (white-tailed-jararaca) snake venom

Higuchi, Debora Ayame; Barbosa, Christiano M.V.; Bincoletto, Cláudia; Chagas, Jair Ribeiro; Magalhães, Ana C.; Richardson, Michael; Sanchez, Eládio Flores; Pesquero, João Bosco; Araújo, Ronaldo Carvalho; Pesquero, Jorge L.

doi:10.1016/j.biochi.2006.10.010

Cited by 51 publications

(43 citation statements)

References 53 publications

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“…The diversity of snake venom PLA 2 functions includes: neurotoxicity, cardiotoxicity, myotoxicity, edema, hypotension, hyperalgesia as well as activation and inhibition of platelet aggregation (12)(13)(14)(15)(16)(17)(18)(19). The diversity of biological and pharmacological functions of PLA 2 denotes that accelerated or positive Darwinian evolution has occurred and appears to confer a better fitness on the snake venom (10,20).…”

Section: Introductionmentioning

confidence: 99%

Cloning of a novel acidic phospholipase A2 from the venom gland of Crotalus durissus cascavella (Brazilian northeastern rattlesnake)

Guarnieri¹,

Melo²,

Melo³

et al. 2009

J. Venom. Anim. Toxins incl. Trop. Dis

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The phospholipase A2 superfamily encompasses 15 groups that are classified into: secreted PLA2 (sPLA2); cytosolic PLA2 (cPLA2); Ca2+-independent intracellular PLA2 (iPLA2); platelet-activating factor acetylhydrolase (PAF-AH); and lysosomal PLA2. Currently, approximately 700 PLA2 sequences are known, of which 200 are obtained from the venom gland of Crotalinae snakes. However, thus far, little information is available on cloning, purification and structural characterization of PLA2 from Crotalus durisssus cascavela venom gland. In the present work, we report the molecular cloning of a novel svPLA2 from C. d. cascavella (Cdc), a predominant rattlesnake subspecies in northeastern Brazil. The Cdc svPLA2 cDNA precursor is 689 nucleotides long and encodes a protein of 138 amino acid residues, with a calculated molecular mass of approximately 13,847 Da and an estimated isoelectric point of 5.14. Phylogenetic analysis of Crotalinae PLA2 reveals that Cdc PLA2 clustered with other acidic type IIA PLA2 homologues is also present in the venom of North American rattlesnakes. Hitherto, this study presents a novel PLA2 cDNA precursor from C. d. cascavella and data reported herein will be useful for further steps in svPLA2 purification and analysis

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Section: Introductionmentioning

confidence: 99%

Cloning of a novel acidic phospholipase A2 from the venom gland of Crotalus durissus cascavella (Brazilian northeastern rattlesnake)

Guarnieri¹,

Melo²,

Melo³

et al. 2009

J. Venom. Anim. Toxins incl. Trop. Dis

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“…Nevertheless, SDS-PAGE under non-reducing conditions showed Btae TX-I running as a monomer and possessing a single polypeptidic chain of ~14 kDa (13,889.89 Da) after reduction, what was confirmed by MALDI-TOF mass spectrometry. Such configuration of Btae TX-I is similar to other basic Asp49 PLA 2 s that also have monomeric structure like the PhTX-I from Porthidium hyoprora [28], blD-PLA 2 from Bothrops leucurus [29] and LmTX-I from Lachesis muta muta venoms [22]. The amino acid composition of the toxin revealed a high content of basic and hydrophobic residues, with 14 half-Cys, in agreement with the reported composition and primary structure of PLA 2 myotoxins isolated from Bothrops venoms [21,30,31].…”

Section: Discussionmentioning

confidence: 60%

Biochemical Characterization of a PLA2 Btae TX-I Isolated from Bothriopsis taeniata Snake Venom: A Pharmacological and Morphological Study

Romero-Vargas¹,

Rocha²,

Cruz‐Höfling

et al. 2014

J Clin Toxicol

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In this research a preliminary identification and biochemical and biological characterization of a PLA 2 (Btae TX-I) from the venom of a viperid snake, Bothriopsis taeniata (Speckled forest pit viper) were obtained. Btae TX-I was purified by two chromatographic steps, molecular exclusion chromatography followed by analytical chromatography reverse phase HPLC. Molecular mass behaved as a homogeneous single chain protein on SDS-PAGE, confirmed by MALDI-TOF spectrometry, indicating a molecular mass of 13889.98 Da. Tryptic peptides were determined in tandem mass spectrometry and showed similarity with other myotoxic PLA 2 s. Btae TX-I belongs to the Asp49 PLA 2 class, is enzymatically active in presence of a synthetic substrate and shows a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 35-45°C. PLA 2 activity in presence of Mn 2+ , Mg 2+ , Cd 2+ and Zn 2+ was reduced either in presence or absence of Ca 2+ , suggesting that the arrangement of the catalytic site presents an exclusive structure for Ca 2+ . Crotalic crotapotins from rattlesnake venom has significantly inhibited (p<0.05) the enzymatic activity of Btae TX-I. In ex vivo experiment, Btae TX-I caused partial blockade of the neuromuscular transmission in chick biventer cervicis preparations in a similar way to other Bothrops species. Btae TX-I also inhibited contractures in the upper concentration (50 µg) to exogenous KCl (20 mM). Histological analysis of the biventer cervicis incubated with Btae TX-I showed that just the highest Btae TX-I PLA 2 dose (50 µg) caused almost 27.4 ± 0.3% damaged fibers. The results give evidence that the main effect of type Asp49 Btae TX-I PLA 2 from Bothriopsis taeniata is at the post-synaptic site.

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“…Also, with the use of a multiple step procedure [38] successfully isolated two isoforms of PLA2s from B. leucurus venom. After a first molecular exclusion chromatography using Sephacryl S-200®, 7 fractions were obtained, from which the named "P6" showed to be composed by proteins with apparent molecular mass bellow 30 kDa, and a major fraction of approximately 14 kDa concentrated the phospholipase activity.…”

Section: Midca1mentioning

confidence: 99%

Purification of Phospholipases A2 from American Snake Venoms

Stábeli¹,

Simões-Silva²,

Kayano³

et al. 2012

Chromatography - The Most Versatile Method of Chemical Analysis

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Purification and partial characterization of two phospholipases A2 from Bothrops leucurus (white-tailed-jararaca) snake venom

Cited by 51 publications

References 53 publications

Cloning of a novel acidic phospholipase A2 from the venom gland of Crotalus durissus cascavella (Brazilian northeastern rattlesnake)

Cloning of a novel acidic phospholipase A2 from the venom gland of Crotalus durissus cascavella (Brazilian northeastern rattlesnake)

Biochemical Characterization of a PLA2 Btae TX-I Isolated from Bothriopsis taeniata Snake Venom: A Pharmacological and Morphological Study

Purification of Phospholipases A2 from American Snake Venoms

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