Purification and Preliminary Crystallographic Analysis of a New Lys49-PLA2 from B. Jararacussu

Santos, Marcelo Leite dos; Fagundes, Fábio H. R.; Teixeira, Bruno R. F.; Toyama, Marcos H.; Aparício, Ricardo

doi:10.3390/ijms9050736

Cited by 10 publications

(14 citation statements)

References 37 publications

(40 reference statements)

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“…They are responsible for multiple pharmacological effects, some of which are dependent on catalytic activity and others of which are not. The pharmacological or biological effects that do not depend on enzymatic activity are driven by other pharmacological regions or sites that include calcium-binding loop, beta-wing and C-terminal region [16,19,23,24]. The precise location or mapping of these pharmacological sites is not easy to find due to the fact that the residues involved in myotoxicity and neurotoxicity significantly overlap, suggesting that multiple biological effects observed in many snake venom PLA 2 s are a consequence of superposed structural determinants on the protein surface [24].…”

Section: Discussionmentioning

confidence: 99%

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Harpalycin 2 inhibits the enzymatic and platelet aggregation activities of PrTX-III, a D49 phospholipase A2 from Bothrops pirajai venom

Ximenes

Alves

Pereira

et al. 2012

BMC Complement Altern Med

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BackgroundHarpalycin 2 (HP-2) is an isoflavone isolated from the leaves of Harpalyce brasiliana Benth., a snakeroot found in northeast region of Brazil and used in folk medicine to treat snakebite. Its leaves are said to be anti-inflammatory. Secretory phospholipases A2 are important toxins found in snake venom and are structurally related to those found in inflammatory conditions in mammals, as in arthritis and atherosclerosis, and for this reason can be valuable tools for searching new anti-phospholipase A2 drugs.MethodsHP-2 and piratoxin-III (PrTX-III) were purified through chromatographic techniques. The effect of HP-2 in the enzymatic activity of PrTX-III was carried out using 4-nitro-3-octanoyloxy-benzoic acid as the substrate. PrTX-III induced platelet aggregation was inhibited by HP-2 when compared to aristolochic acid and p-bromophenacyl bromide (p-BPB). In an attempt to elucidate how HP-2 interacts with PrTX-III, mass spectrometry, circular dichroism and intrinsic fluorescence analysis were performed. Docking scores of the ligands (HP-2, aristolochic acid and p-BPB) using PrTX-III as target were also calculated.ResultsHP-2 inhibited the enzymatic activity of PrTX-III (IC50 11.34 ± 0.28 μg/mL) although it did not form a stable chemical complex in the active site, since mass spectrometry measurements showed no difference between native (13,837.34 Da) and HP-2 treated PrTX-III (13,856.12 Da). A structural analysis of PrTX-III after treatment with HP-2 showed a decrease in dimerization and a slight protein unfolding. In the platelet aggregation assay, HP-2 previously incubated with PrTX-III inhibited the aggregation when compared with untreated protein. PrTX-III chemical treated with aristolochic acid and p-BPB, two standard PLA2 inhibitors, showed low inhibitory effects when compared with the HP-2 treatment. Docking scores corroborated these results, showing higher affinity of HP-2 for the PrTX-III target (PDB code: 1GMZ) than aristolochic acid and p-BPB. HP-2 previous incubated with the platelets inhibits the aggregation induced by untreated PrTX-III as well as arachidonic acid.ConclusionHP-2 changes the structure of PrTX-III, inhibiting the enzymatic activity of this enzyme. In addition, PrTX-III platelet aggregant activity was inhibited by treatment with HP-2, p-BPB and aristolochic acid, and these results were corroborated by docking scores.

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Section: Discussionmentioning

confidence: 99%

“…The platelet aggregation activities were conducted as described by Oliveira et al [18] and dos Santos et al [19]. Venous blood was collected with informed consent from healthy volunteers who formally denied taking any medication in the previous 14 days.…”

Section: Methodsmentioning

confidence: 99%

Harpalycin 2 inhibits the enzymatic and platelet aggregation activities of PrTX-III, a D49 phospholipase A2 from Bothrops pirajai venom

Ximenes

Alves

Pereira

et al. 2012

BMC Complement Altern Med

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“…These effects are similar to those induced by the venom obtained from Crotalus species. It has been shown that convulxin, other class of C-type lectin from Crotalus durissus ssp., induced similar insulin secretion at both sub-(2.8 mM) and suprathreshold (16.7 mM) glucose concentrations (21).…”

Section: Discussionmentioning

confidence: 79%

“…For the first fractionation, whole venom was purified following the protocols described by dos Santos et al (21). In the second step, the whole venom (50 mg) was dissolved in 1 mL of calcium Trisbase saline (CTBS; Tris 20 mM, NaCl 150 mM, CaCl 2 5 mM, pH 7.5).…”

Section: Venommentioning

confidence: 99%

Purification and biological effects of a C-type lectin isolated from Bothrops moojeni

Barbosa¹,

Martins²,

Toyama³

et al. 2010

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

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Snake venom proteins from the C-type lectin family have very distinct biological activities despite their highly conserved primary structure, which is homologous to the carbohydrate recognition region of true C-type lectins. We purified a lectin-like protein (BmLec) from Bothrops moojeni venom and investigated its effect on platelet aggregation, insulin secretion, antibacterial activity, and isolated kidney cells. The BmLec was purified using two chromatographic steps: affinity chromatography and reverse phase high performance liquid chromatography (HPLC). BmLec showed a dose-dependent platelet aggregation and significantly decreased the bacterial growth rate in approximately 15%. During scanning electron microscopy, the profile of Xanthomonas axonopodis pv. passiflorae treated with lectin disclosed a high vesiculation and membrane rupture. BmLec induced a strong and significant increase in insulin secretion at 2.8 and 16.7 mM glucose concentrations, and this effect was seen in the presence of EGTA in both experiments. BmLec (10 µg/mL) increased the perfusion pressure, renal vascular resistance and urinary flow. The glomerular filtration rate and percentages of sodium, potassium and chloride tubular transport were reduced at 60 minutes of perfusion. Renal alterations caused by BmLec were completely inhibited by indomethacin in all evaluated parameters. In conclusion, the C-type lectin isolated from Bothrops moojeni affected platelet aggregation, insulin secretion, antibacterial activity and isolated kidney function

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“…Purification of sPLA2 from whole venom was performed as previously described (16). 10 mg of the crude venom were dissolved in 250 μl of loading buffer (0.05 M Tris-HCl, pH 8.0) and centrifuged at 4,500 g for 5 min.…”

Section: Purification Of Spla2mentioning

confidence: 99%

Analysis of Molecular Changes Induced By Mineral Trioxide Aggregate On sPLA2

et al. 2019

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The aim of this study was to analyze the effects of MTA on the structure and enzymatic activity of sPLA2 in order to provide subsidies for improvement in the formulation of the product. MTA powder was incubated for 60 min in the presence of sPLA2 and was analyzed by chromatography, electrospray mass (ESI-MS) and small-angle X-ray scattering (SAXS). It was find that the elution profile, retention time, and fragmentation of sPLA2 were altered after treatment with MTA. Calcium was the MTA component that most amplified the inflammatory signal. Significant interactions were found between MTA and sPLA2, which could aid in our understanding of the mechanisms of action of MTA during the inflammatory process and it may facilitate the structural modification of MTA, thereby improving its biological safety and consequently the rate of the treatment success.

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Purification and Preliminary Crystallographic Analysis of a New Lys49-PLA2 from B. Jararacussu

Cited by 10 publications

References 37 publications

Harpalycin 2 inhibits the enzymatic and platelet aggregation activities of PrTX-III, a D49 phospholipase A2 from Bothrops pirajai venom

Harpalycin 2 inhibits the enzymatic and platelet aggregation activities of PrTX-III, a D49 phospholipase A2 from Bothrops pirajai venom

Purification and biological effects of a C-type lectin isolated from Bothrops moojeni

Analysis of Molecular Changes Induced By Mineral Trioxide Aggregate On sPLA2

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