Isolation of an acidic phospholipase A2 from the venom of the snake Bothrops asper of Costa Rica: Biochemical and toxicological characterization☆ (original) (raw)
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Biochemical Journal, 1980
The water-soluble venom of Bothrops asper Garman (San Juan Evangelista, Veracruz, México) showed 15 polypeptide bands on polyacrylamide-gel electrophoresis. This material exhibited phospholipase, hyaluronidase, N-benzoyl-l-arginine ethyl hydrolase, N-benzoyl-l-tyrosine ethyl hydrolase and phosphodiesterase activity, but no alkaline phosphatase or acid phosphatase activity. Fractionation on Sephadex G-75 afforded seven protein fractions, which were apparently less toxic than the whole venom (LD50=4.3μg/g mouse wt.). Subsequent separation of the phospholipase-positive fraction (II) on DEAE-cellulose with potassium phosphate buffers (pH7.55) gave several fractions, two being phospholipase-positive (II.6 and II.8). These fractions were further purified on DEAE-cellulose columns with potassium phosphate buffers (pH8.6). Fraction II.8.4 was rechromatographed in the same DEAE-cellulose column, giving a pure protein designated phospholipase 1. The fraction II.6.3 was further separated by ge...
An aqueous endpoint assay of snake venom phospholipase A2
Toxicon, 1996
A, (PLA,), an enzyme found in most snake venoms, catalyzes the hydrolysis of phospholipids in biological membranes, and some have presynaptic neurotoxic activity. A synthetic substrate, 4-nitro-3-(octanoyloxy)benzoic acid, was synthesized and purified on a silica gel column using a published method. This substrate was used to develop an endpoint assay which is rapid and requires a minimum of equipment. This aqueous assay system allowed enzyme activity to be examined without the use of radioactive substrates or organic solvents, minimizing waste disposal concerns. Whole venoms, partially purified enzyme isolated from Crotalus mitchelli pyrrhus venom, tissue extracts and commercial preparations were employed as sources of PLA?. Results show that this method is a convenient and specific assay for PLA, from several sources and is particularly suited for assaying large numbers of fractions generated during purification procedures.
Toxicon: X, 2020
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Protein and Peptide …, 2009
A particular subgroup of toxins with phospholipase A 2 (PLA 2 ) structure, but devoid of this enzymatic activity, is commonly found in the venoms of snakes of the family Viperidae, and known as the PLA 2 homologues. Among these, the most frequent type presents a lysine residue at position 49 (Lys49), in substitution of the otherwise conserved aspartate (Asp49) of catalytically-active PLA 2 s. A brief and updated overview of these toxic PLA 2 homologues is presented, emphasizing their various biological activities, both in vivo and in vitro. The relevance of these bioactivities in relation to their possible adaptive roles for the snakes is discussed. Finally, experiments designed to assess the validity of such hypothetical roles are suggested, to stimulate future studies in this field.
An electrophoretic study on phospholipase A2 isoenzymes in the venoms of Central American crotaline snakes . Toxicon 30, 815-823, 1992 .-The number and isoelectric points of phospholipase A2 isoenzymes were studied in the venoms of 12 Central American crotaline snakes of the genera Bothrops, Crotalus, Lachesis and Agkistrodon . The study was carried out by using a methodology based on electrophoretic separation of venoms, transfer to nitrocellulose and detection of activity of the bands by an indirect hemolytic assay in agarose~rythrocyte~gg yolk gels . All venoms tested had indirect hemolytic activity, although they varied in the number and isoelectric point of their phospholipases A, . Most venoms had predominantly acidic isoenzymes, with the exception of Ä . bilineatus which had mainly basic isoenzymes and B. schlegelü which had both acidic and basic isoenzymes . Analysis of interindividual variability in B. riper venom demonstrated that two phospholipase A2 isoenzymes are present in some venoms but absent in others . Polyvalent antivenom was effective in neutralizing phospholipase A2 activity of the 12 venoms tested, when venoms and antivenom were incubated in the fluid phase. This work demonstrates a conspicuous interspecific variability in the number and isoelectric points of phospholipases A2 present in Central American crotaline snake venoms,
Biochimica Et Biophysica Acta-protein Structure and Molecular Enzymology, 1995
A neutral phospholipase A 2 (PLA 2) was separated from Pseudechis papuanus venom by a two-stage FPLC procedure of cation exchange and phenyl-Superose chromatography. It had a molecular mass of 15 kDa and a lower LDso value than a co-separated haemorrhagic fraction, indicating a higher lethal potency. In vitro tests confirmed the powerful inhibition of platelet aggregation by the PLA 2 and strong anticoagulant activity initially observed with whole venom. Ultrastructural studies showed that platelets lost their discoid shape and developed membranous projections with a general decrease in electron-density of the cytosol and disruption of the microfilaments following incubation with the enzyme. Amino acid sequence analysis of the N-terminus and some internal peptides demonstrated a high degree of homology with PLAzs from other Pseudechis venoms. Our results indicate that this fraction is the main agent responsible for the haemostatic disorders in envenomed patients.
Brazilian journal of medical and biological research = Revista brasileira de pesquisas médicas e biológicas / Sociedade Brasileira de Biofísica ... [et al.], 1993
Screening of the biochemical-pharmacological properties of the crude venom from the snake Lachesis muta indicated the presence of phospholipase A2 (PLA2; 5260 U/mg protein), procoagulant (2630 U/mg protein), platelet aggregating (43 U/mg protein) and caseinolytic activities (6670 U/mg protein). These activities were separated by filtration of the crude venom on Sephacryl S-200. The material containing PLA2 activity was further fractioned by DEAE-cellulose ion exchange chromatography into four active fractions (F-I to F-IV, containing 1.7, 1.2, 0.3, and 0.05% of the crude venom protein, respectively) by stepwise elution with buffers of increasing ionic strength. All fractions presented a molecular weight of approximately 15,000 and isoelectric points in the range pH 4.6-6.0. In addition to their indirect hemolytic activity, the partially purified fractions inhibited platelet aggregation induced either by collagen or thrombin. p-Bromophenacyl bromide-treated fractions lost both phosph...
Necrosis of muscular tissue is a serious consequence A new myotoxic phospholipase A 2 was isolated from of envenomation by snakebites (1). In envenomations the venom of the arboreal snake Bothriechis schlegelii caused by crotalid snakes of the genus Bothrops (Amer-(formerly Bothrops schlegelii) from Costa Rica, by ican lance-headed pit vipers), distributed from Mexico ion-exchange chromatography on CM-Sephadex. B. to Argentina, it has been shown that skeletal muscle schlegelii myotoxin I is a basic protein (pI ú 9.3) with necrosis develops mainly due to the action of basic toxa subunit molecular weight of 15 kDa, which migrates ins with group II phospholipase A 2 (PLA 2 ) 2 structure as a dimer in sodium dodecyl sulfate-polyacrylamide (reviewed in Ref. 2). In recent years, several species gel electrophoresis under nonreducing conditions. within the genus Bothrops have been reclassified into This myotoxin is recognized by antibodies generated the new genera Bothriechis, Bothriopsis, and Porthidagainst Bothrops asper myotoxin II (a lysine-49 phosium (3). The venoms of many of these species contain pholipase A 2 ), by both enzyme-immunoassay and gel basic PLA 2 myotoxins (4), which display sequence variimmunodiffusion, in the latter case with a pattern of ability (2, 5). Furthermore, several myotoxin variants partial identity. The toxin induces rapid myonecrosis or isoforms can exist in the venom of individual speciupon intramuscular injection in mice, as evidenced by mens, as shown in Bothrops asper (6). Given the subthe early increase in plasma creatine kinase activity stantial inter-and intraspecific diversity observed in and by direct intravital microscopic observation. B. this family of basic PLA 2 s, the biochemical and pharschlegelii myotoxin I also induces edema in the mouse macological characterization of new variants may add footpad assay and exerts lethal activity (LD 50 Ç2.5 mg/ valuable information for the understanding of their g) upon intravenous injection. The toxin has a low structure-function relationship.
Archives of biochemistry …, 1997
Necrosis of muscular tissue is a serious consequence A new myotoxic phospholipase A 2 was isolated from of envenomation by snakebites (1). In envenomations the venom of the arboreal snake Bothriechis schlegelii caused by crotalid snakes of the genus Bothrops (Amer-(formerly Bothrops schlegelii) from Costa Rica, by ican lance-headed pit vipers), distributed from Mexico ion-exchange chromatography on CM-Sephadex. B. to Argentina, it has been shown that skeletal muscle schlegelii myotoxin I is a basic protein (pI ú 9.3) with necrosis develops mainly due to the action of basic toxa subunit molecular weight of 15 kDa, which migrates ins with group II phospholipase A 2 (PLA 2 ) 2 structure as a dimer in sodium dodecyl sulfate-polyacrylamide (reviewed in Ref. 2). In recent years, several species gel electrophoresis under nonreducing conditions. within the genus Bothrops have been reclassified into This myotoxin is recognized by antibodies generated the new genera Bothriechis, Bothriopsis, and Porthidagainst Bothrops asper myotoxin II (a lysine-49 phosium (3). The venoms of many of these species contain pholipase A 2 ), by both enzyme-immunoassay and gel basic PLA 2 myotoxins (4), which display sequence variimmunodiffusion, in the latter case with a pattern of ability (2, 5). Furthermore, several myotoxin variants partial identity. The toxin induces rapid myonecrosis or isoforms can exist in the venom of individual speciupon intramuscular injection in mice, as evidenced by mens, as shown in Bothrops asper (6). Given the subthe early increase in plasma creatine kinase activity stantial inter-and intraspecific diversity observed in and by direct intravital microscopic observation. B. this family of basic PLA 2 s, the biochemical and pharschlegelii myotoxin I also induces edema in the mouse macological characterization of new variants may add footpad assay and exerts lethal activity (LD 50 Ç2.5 mg/ valuable information for the understanding of their g) upon intravenous injection. The toxin has a low structure-function relationship.