Biochemical, Pharmacological, And Structural Characterization Of New Basic Pla2 Bbil-tx From Bothriopsis Bilineata Snake Venom

Bbil-TX, a PLA2, was purified from Bothriopsis bilineata snake venom after only one chromatographic step using RP-HPLC on μ7u-Bondapak C-18 column. A molecular mass of 14243.8 Da was confirmed by Q-Tof Ultima API ESI/MS (TOF MS mode) mass spectrometry. The partial protein sequence obtained was then submitted to BLASTp, with the search restricted to PLA2 from snakes and shows high identity values when compared to other PLA2s. PLA 2 activity was presented in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 25-37°C. Maximum PLA2 activity required Ca 2+ and in the presence of Cd2+, Zn2+, Mn 2+, and Mg2+ it was reduced in the presence or absence of Ca2+. Crotapotin from Crotalus durissus cascavella rattlesnake venom and antihemorrhagic factor DA2-II from Didelphis albiventris opossum sera under optimal conditions significantly inhibit the enzymatic activity. Bbil-TX induces myonecrosis in mice. The fraction does not show a significant cytotoxic activity in myotubes and myoblasts (C2C12). The inflammatory events induced in the serum of mice by Bbil-TX isolated from Bothriopsis bilineata snake venom were investigated. An increase in vascular permeability and in the levels of TNF-a, IL-6, and IL-1 was was induced. 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Cytotoxic Action In Myoblasts And Myotubes (c2c12) And Enzymatic Characterization Of A New Phospholipase A2 Isoform (bj-v) From Bothrops Jararacussu Venom

A new PLA2 Bj-V from Bothrops jararacussu (14039.49 Da determined by MALDI-TOF mass spectrometry) was isolated in only one chromatographic step by HPLC ion-exchange and its purity was confirmed by reverse phase. Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The N-terminal sequence (DLWQFGQMIL KETGKIPFPY YGAYGCYCGW GGRGGKPKDG TDRCCYVHD . . .) showed a high degree of homology with basic D49 PLA2 myotoxins from other Bothrops venoms. Bj V showed discrete sigmoidal enzymatic behavior, with maximal activity at pH 8.4 and 35-40°C. Full PLA2 activity required Ca 2+ (10 mM) and there was little catalytic activity in the presence of 1 mM Ca2+.The addition of Mn2+ or Mg2+ (10 mM) in the presence of low (1 mM) Ca2+ slightly increased the enzyme activity, whereas Zn2+ and Cu2+ (10 mM) diminished the activity. The substitution of Ca2+ for Mg2+ or Cu 2+ also reduced the enzymatic activity. 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Structural And Biological Characterization Of Two Crotamine Isoforms Iv-2 And Iv-3 Isolated From The Crotalus Durissus Cumanensis Venom (protein Journal (2007) 26, 8, (533-540) Doi: 10.1007/s10930-007-9094-z)

[No abstract available]283-419

Biological And Structural Characterization Of Crotoxin And New Isoform Of Crotoxin B Pla2 (f6a) From Crotalus Durissus Collilineatus Snake Venom

A new crotoxin B isoform PLA2 (F6a), from Crotalus durissus collilineatus was purified from by one step reverse phase HPLC chromatography using μ-Bondapack C-18 column analytic. The new crotoxin B isoform PLA 2 (F6a), complex crotoxin, the catalytic subunit crotoxin B isoform PLA2 (F6a) and two crotapotin isoforms (F3 and F4), were isolated from the venom of Crotalus durissus collilineatus. The crotapotins isoforms F3 and F4 had similar chemical properties, the two proteins different in their ability to inhibit of isoforms of PLA2 (F6 and F6a). The molecular masses estimated by MALDI-TOF mass spectrometry were: crotoxin B: 14,943.14 Da, crotapotin F3: 8,693.24 Da, and crotapotin F4: 9 314.56 Da. The new crotoxin B isoform PLA2 (F6a) contained 122 amino acid residues and a pI of 8.58. Its amino acid sequence presents high identity with those of other PLA2s, particularly in the calcium binding loop and active site helix 3. It also presents similarities in the C-terminal region with other myotoxic PLA2s. The new crotoxin B isoform PLA2 (F6a) contained 122 amino acid residues, with a primary structure of HLLQFNKMIK FETRRNAIPP YAFYGCYCGW GGRGRPKDAT DRCCFVHDCC YGKLAKCNTK WDFYRYSLKS GYITCGKGTW CEEQICECDR VAAECLRRSL STYRYGYMIY PDSRCRGPSE TC. A neuromuscular blocking activity was induced by crotoxin and new crotoxin B isoform PLA2 (F6a) in the isolated mouse phrenic nerve diaphragm and the biventer cervicis chick nerve-muscle preparation. Whole crotoxin was devoid of cytolytic activity upon myoblasts and myotubes in vitro, whereas new crotoxin B isoform PLA2 (F6a) was clearly cytotoxic to these cells. © 2006 Springer Science+Business Media, LLC.264221230Angulo, Y., Olamendi-Portugal, T., Alape-Girón, A., Possani, L.D., Lomonte, B., (2002) Int. J. Biochem., 34, pp. 1268-1278Araújo, F.A.A., Santalúcia, M., Cabral, R.F., (2003) Sarvier/FAPESP, São Paulo, pp. 6-12. , Cardoso, J. L. C., França, F. O. S., Fan, H. 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Isolation And Preliminary Enzymatic Characterization Of A Novel Pla2 From Crotalus Durissus Collilineatus Venom

A crotoxin homolog was purified from the Crotalus durissus collilineatus venom using molecular exclusion and reverse-phase HPLC. This crotoxin contained one PLA2 (Cdcolli III F6) and four crotapotin isoforms, whereas crotoxin from Crotalus durissus terrificus venom had three PLA2 isoforms and two crotapotin isoforms. SDS-PAGE showed that the C. d. collilineatus PLA2 and crotapotin had relative molecular mass of 15 and 9 kDa, respectively. Neither the PLA2 (Cdcolli III F6) nor the crotapotins (Cdcolli III F3 and F4) had any neurotoxicity in mouse phrenic nervediaphragm preparations when tested alone. However, when PLA2 and crotapotin were coincubated before testing, the neurotoxicity was restored to a level similar to test in the venom in native crotoxin. The two crotapotins (Cdcolli III F3 and F4) differed in their ability to inhibit PLA2 activity, perhaps because of variations in their affinities for this enzyme. Cdcolli III F6 showed allosteric enzymatic behavior, with maximal activity at pH 8.3 and 36°C. Full PLA2 activity required the presence of a low Ca2+ concentration and was inhibited by Cu2+ and Zn2+ and by Cu2+ and Mg2+ in the presence and absence of Ca2+, respectively. These results indicate that crotoxin from C. d. collineatus venom is very similar enzymatically to crotoxin from C. d. terrificus.213131136Azevedo-Marques, M.M., Cupo, P., Coimbra, T.M., Hering, S.E., Rossi, M.A., Laure, C.J., (1982) Toxicon, 23, pp. 631-636Beghini, D.G., Toyama, M.H., Hyslop, S., Sodek, L.C., Novello, J.C., Marangoni, S., (2000) J. Prot. Chem., 19, pp. 679-664Bon, C., Changeux, J.P., Jeng, T.W., Fraenkel-Conrat, H., (1979) Eur. J. Biochem., 99, pp. 471-481Bonfim, V.L., Toyama, M.H., Novello, J.C., Hyslop, S., Oliveira, C.R.B., Rodrigues-Simioni, L., Marangoni, S., (2001) J. Prot. Chem., 20, pp. 239-245Breithaupt, H., (1976) Toxicon, 14, pp. 221-233Cho, W., Kezdy, F.J., (1991) Methods Enzymol., 197, pp. 75-79Cupo, P., Azevedo-Marques, M.M., Hering, S.E., (1988) Trans. R. Soc. Trop. Med. Hyg., 82, pp. 924-929Faure, G., Bon, C., (1987) Toxicon, 25, pp. 229-234Faure, G., Bon, C., (1988) Biochemistry, 27, pp. 730-738Faure, G., Choumet, V., Bouchier, C., Camoin, L., Guillaume, J.L., Monegier, B., Vuilhorgne, M., Bon, C., (1994) Eur. J. Biochem., 223, pp. 161-164Gutiérrez, J.M., Lomonte, B., (1995) Toxicon, 33, pp. 1405-1424Habermann, E., Breithaupt, H., (1978) Toxicon, 16, pp. 19-30Hendon, R.A., Fraenkel-Conrat, H., (1971) Proc. Natl. Acad. Sci. USA, 68, pp. 1560-1563Holzer, M., Mackessy, S.P., (1996) Toxicon, 34, pp. 1149-1155Landucci, E.C.T., Condino-Neto, A., Perez, A.C., Hyslop, S., Corrado, A.P., Novello, J.C., Marangoni, S., De Nucci, G., (1994) Toxicon, 32, pp. 217-226Lennon, B.W., Kaiser, I., (1990) Comp. Biochem. Physiol. B., 97, pp. 695-699Pieterson, W.A., Volwerck, J.J., De Haas, G.H., (1974) Biochemistry, 13, pp. 1439-1445Rosenfeld, G., (1971) Venomous animals and their venoms, 2, pp. 345-362. , (Bücherl, W., and Buckley, E. E., eds), Academic Press, New YorkRubsamen, K., Breithaupt, H., Habermann, E., (1971) Naunyn-Schmiedeberg's Arch. Pharmacol., 270, pp. 274-288Schägger, H., Von Jagow, G., (1987) Anal. Biochem., 166, pp. 368-379Toyama, M.H., Mancuso, L.C., Giglio, J.R., Novello, J.C., Oliveira, B., Marangoni, S., (1995) Biochem. Mol. Biol. Int., 37, pp. 1047-1055Toyama, M.H., Carneiro, E.M., Marangoni, S., Barbosa, R.L., Corso, G., Boschero, A.C., (2000) Biochim. Biophys. Acta, 1474, pp. 56-60Tu, A.T., Passey, R.B., Toom, P.M., (1970) Arch. Biochem. Biophys., 140, pp. 96-10

Toxicity Of Phospholipases A2 D49 (6-1 And 6-2) And K49 (bj-vii) From Bothrops Jararacussu Venom

Purified phospholipase A2 (PLA2) enzymes from Bothrops jararacussu snake venom were examined to evaluate NIH 3T3 and COS7 fibroblast cytotoxicity, as well as muscle myotoxic and inflammatory activities. Separation of fractions Bj-VII (from BthTX-I; a Lys49 PLA2 homolog) and 6-1 and 6-2 (from BthTX-II; an Asp49 PLA2) from B. jararacussu snake venom by SDS-PAGE in tricine buffer in the absence and presence of dithiothreitol revealed a homodimer with an estimated molecular mass of ∼30 kDa (monomer mass ∼15 kDa). This finding indicates that these toxins form dimeric complexes-a previously reported tendency among PLA2s. These toxins were assayed for viability with the MTT assay, which is used to examine the effects of phospholipases on the mitochondrial viability of cells. The toxins were also assayed for cytolysis of the fibroblast cell lines NIH 3T3 and COS7 by quantification of lactate dehydrogenase released into the medium. The results indicate that the PLA2s 6-1, 6-2 and the Bj-VII PLA2 homolog studied here induce moderate footpad edema and local myotoxicity. Moreover, exposure to these phospholipases led to a reduction in fibroblast viability; at the 1 μM dose of PLA2 tested, a reduction of 50% in cell viability was observed. The present findings indicate that the inflammatory activity observed in envenomation may be correlated with the cytotoxicity observed in fibroblasts. © 2008 Springer Science+Business Media B.V.256523532Ahn, M.Y., Lee, B.M., Kim, Y.S., Characterization and cytotoxicity of L-amino acid oxidase from the venom of king cobra (Ophiophagus hannah) (1997) International Journal of Biochemistry and Cell Biology, 29 (6), pp. 911-919. , DOI 10.1016/S1357-2725(97)00024-1, PII S1357272597000241Andriao-Escarso, S.H., Soares, A.M., Rodrigues, V.M., Angulo, Y., Diaz, C., Lomonte, B., Gutierrez, J.M., Giglio, J.R., Myotoxic phospholipases A2 in Bothrops snake venoms: Effect of chemical modifications on the enzymatic and pharmacological properties of bothropstoxins from Bothrops jararacussu (2000) Biochimie, 82 (8), pp. 755-763. , DOI 10.1016/S0300-9084(00)01150-0Angulo, Y., Lomonte, B., Differential susceptibility of C2C12 myoblasts and myotubes to group II phospholipase A2 myotoxins from crotalid snake venoms (2005) Cell Biochemistry and Function, 23 (5), pp. 307-313. , DOI 10.1002/cbf.1208Arni, R.K., Ward, R.J., Phospholipase A2 - A structural review (1996) Toxicon, 34 (8), pp. 827-841. , DOI 10.1016/0041-0101(96)00036-0Bonfim, V.L., Ponce-Soto, L.A., Novello, J.C., Marangoni, S., Cytotoxic action in myoblasts and myotubes (C2C12) and enzymatic characterization of a new phospholipase A2 isoform (Bj-V) from Bothrops jararacussu venom (2006) Protein and Peptide Letters, 13 (7), pp. 707-713. , http://docstore.ingenta.com/cgi-bin/ds_deliver/1/u/d/ISIS/30400784.1/ben / ppl/2006/00000013/00000007/art00010/22524866E18A17B711525244689785D28A12EB6053. pdf?link=http://www.ingentaconnect.com/error/delivery&format=pdf, DOI 10.2174/092986606777790520Bonfim, V.L., Toyama, M.H., Novello, J.C., Hyslop, S., Oliveira, C.R.B., Rodrigues-Simioni, L., Marangoni, S., Isolation and enzymatic characterization of a basic phospholipase A 2 from Bothrops jararacussu snake venom (2001) Journal of Protein Chemistry, 20 (3), pp. 239-245. , DOI 10.1023/A:1010956126585Bonventre, J.V., Sapirstein, A., Group IV cytosolic phospholipase A2 (PLA2) function: Insights from the knockout mouse (2002) Adv Exp Med Biol, 507, pp. 25-31. , 12664560Brazil VO. 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Biochemical, Pharmacological And Structural Characterization Of Two Pla2 Isoforms Cdr-12 And Cdr-13 From Crotalus Durissus Ruruima Snake Venom

Cdr-12 and Cdr-13 isoforms of PLA2, a D49 protein, were purified from Crotalus durissus ruruima venom after one chromatographic step, reverse phase HPLC on μ-Bondapack C-18. The molecular mass by SDS-PAGE of Cdr-12 and Cdr-13 isoforms of PLA2 was 14333.49 Da and 14296.42 Da, respectively and confirmed by MALDI-TOF mass spectrometry .The amino acid composition showed that both isoforms Cdr-12 and Cdr-13 have a high content of Lys, Tyr, Gly, Arg, and 14 half-Cys residues, typical of a basic PLA2. The isoforms Cdr-12 and Cdr-13 had a sequence of amino acids of 122 amino acid residues, being Cdr-12: SLLQFNKMIK FETRKNAIPF YAFYGCYCGW GGQGRPKDAT DRCCIVHDCC YGKLAKCNTK WDFYRYSLRS GYFQCGKGTW CEQQICECDR VAAECLRRSL STYRYGYMIY PDSRCREPSE TC and pI value 8.37 and Cdr-13: SLVQFEKMIK EETGKNAVPF YAFYGCYCGW GGRGRPKDAT DRCCIVHDCC YEKLVKCNTK WDFYRYSLRS GYFQCGKGTW CEQQICECDR VAAECLRRSL STYRYGKMIY PDSRCREPSE TC with a pI value of 8.13 This sequence shows high identity values when compared to other D49 PLA2s isolated from venoms of crotalics snakes. Skeletal muscle preparations from the young chicken have been previously used in order to study the effects of toxins on neuromuscular transmission, providing an important opportunity to study the differentiated behavior of a toxin before more than one model, because it shows differences in its sensibilities. In mice, the PLA2 isoforms Cdr-12 and Cdr-13 induced myonecrosis and edema, upon intramuscular or subcutaneous injections, respectively. 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cDNA and deduced primary structure of basic phospholipase A2 with neurotoxic activity from the venom secretion of the Crotalus durissus collilineatus rattlesnake

To illustrate the construction of precursor complementary DNAs, we isolated mRNAs from whole venom samples. After reverse transcription polymerase chain reaction (RT-PCR), we amplified the cDNA coding for a neurotoxic protein, phospholipase A2 D49 (PLA2 D49), from the venom of Crotalus durissus collilineatus (Cdc PLA2). The cDNA encoding Cdc PLA2 from whole venom was sequenced. The deduced amino acid sequence of this cDNA has high overall sequence identity with the group II PLA2 protein family. Cdc PLA2 has 14 cysteine residues capable of forming seven disulfide bonds that characterize this group of PLA2 enzymes. Cdc PLA2 was isolated using conventional Sephadex G75 column chromatography and reverse-phase high performance liquid chromatography (RP-HPLC). The molecular mass was estimated using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. We tested the neuromuscular blocking activities on chick biventer cervicis neuromuscular tissue. Phylogenetic analysis of Cdc PLA2 showed the existence of two lines of N6-PLA2, denominated F24 and S24. Apparently, the sequences of the New World’s N6-F24-PLA2 are similar to those of the agkistrodotoxin from the Asian genus Gloydius. The sequences of N6-S24-PLA2 are similar to the sequence of trimucrotoxin from the genus Protobothrops, found in the Old World

Neutralisation Of The Pharmacological Activities Of Bothrops Alternatus Venom By Anti-pla2 Iggs

Basic phospholipases A2 (PLA2) are toxic and induce a wide spectrum of pharmacological effects, although the acidic enzyme types are not lethal or cause low lethality. Therefore, it is challenging to elucidate the mechanism of action of acidic phospholipases. This study used the acidic non-toxic Ba SpII RP4 PLA2 from Bothrops alternatus as an antigen to develop anti-PLA2 IgG antibodies in rabbits and used in vivo assays to examine the changes in crude venom when pre-incubated with these antibodies. Using Ouchterlony and western blot analyses on B. alternatus venom, we examined the specificity and sensitivity of phospholipase A2 recognition by the specific antibodies (anti-PLA2 IgG). Neutralisation assays using a non-toxic PLA2 antigen revealed unexpected results. The (indirect) haemolytic activity of whole venom was completely inhibited, and all catalytically active phospholipases A2 were blocked. Myotoxicity and lethality were reduced when the crude venom was pre-incubated with anti-PLA 2 immunoglobulins. CK levels in the skeletal muscle were significantly reduced at 6 h, and the muscular damage was more significant at this time-point compared to 3 and 12 h. When four times the LD50 was used (224 μg), half the animals treated with the venom-anti PLA2 IgG mixture survived after 48 h. All assays performed with the specific antibodies revealed that Ba SpII RP4 PLA2 had a synergistic effect on whole-venom toxicity. IgG antibodies against the venom of the Argentinean species B. alternatus represent a valuable tool for elucidation of the roles of acidic PLA2 that appear to have purely digestive roles and for further studies on immunotherapy and snake envenoming in affected areas in Argentina and Brazil. © 2014 Elsevier Ltd. All rights reserved.868995Acosta, O., (1999) Caracterización Bioquímica y Farmacológica Del Veneno de Bothrops Alternatus, , Facultad de Ciencias Veterinarias. 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