Biochemical and Biophysical Research Communications
Purification of a phospholipase A2 from Lonomia obliqua caterpillar bristle extract
Section snippets
Materials and methods
Crude extract. Lonomia obliqua caterpillars were anesthetized with CO2 and bristles were removed and maintained on ice. Phosphate-buffered saline (PBS), pH 7.4, at 4 °C was added to achieve a 10% final extract solution. Bristles were homogenized by shaking and then centrifuged to obtain a suspension [21] which was stored at −70 °C. Protein concentration of bristle extracts was determined colorimetrically [22], using bovine serum albumin as a standard.
PLA2activity. PLA2 activity was determined
Results
A dose–response curve was observed for PLA2 activity in crude L. obliqua bristle extract, using soybean lecithin as substrate (Fig. 1A). This activity was augmented prominently by increasing CaCl2 concentration in medium (Fig. 1B). PLA2 activity in crude L. obliqua caterpillar bristle extract was inhibited 72% by 0.1 mM pBpb, 40% by 0.2 mM Na2–EDTA, and 67% by 2.0 mM Na2–EDTA (Fig. 2).
PLA2 was purified by three chromatographic steps (Fig. 3). PLA2 activity was detected in the first peak obtained
Discussion
Secreted PLA2s are important for digestion and immobilization of prey, and these enzymes are responsible for some of the physiological disturbances observed in humans following bee, wasp, spider, and snake envenomations [14], [16], [30], [31], [32]. PLA2 activity has been reported in the crude Euproctis caterpillar bristle extract [33] and, more recently, in the crude caterpillar bristle extract of L. obliqua[12]. In the present study, a secreted PLA2 was purified to homogeneity from the crude
Acknowledgments
This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP, Brazil (Proc. 00/11432-9 and 01/07643-7).
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2021, Toxicology LettersCitation Excerpt :As mentioned above, an important mechanism that may underlie venom-induced kidney effects is hemoglobin and heme release. Hematuria is a common clinical feature observed in envenomed victims (Malaque et al., 2006), and L. obliqua venom has a phospholipase A2 toxin that is able to specifically target erythrocyte membrane-releasing free hemoglobin (Seibert et al., 2006). In experimental models, there has been a drop in red blood cell counts between 2 h and 6 h after venom injection, a corresponding increase in plasma hemoglobin levels at the same time, and an increase in plasma haptoglobin and reticulocyte numbers between 24 h and 48 h after envenomation (Seibert et al., 2004; Berger et al., 2013, 2019).
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2014, Protein Expression and PurificationCitation Excerpt :The hydrolysis of phospholipids takes place in the monolayer, but without a suitable method to remove free fatty acids from the monolayer to the subphase (e.g. by means of soluble ciclodextrins), the characteristic decrease in the surface pressure indicating phospholipid hydrolysis, cannot be registered [84]. The optimum pH = 8.0 for phospholipase activity of the StI–StII mixture (Fig. 8) is in agreement with the values reported for other secreted phospholipases, generally located in the 7–9 range [15,19,21,22,25,44,71,85]. Interestingly, the permeabilizing ability of StI in lipid vesicles shows an optimum pH in the range 8–9 and a marked decrease at pH 10 and 11, probably due to the effect of pH on protein structure [29].
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2013, ToxiconCitation Excerpt :The case report of hemolysis-related AKI also indicated that treatment with ALS did not reduce hemoglobin levels to normal values and the patient did not completely recover renal function until 1 month after the accident, despite improvements in coagulation tests (Malaque et al., 2006). Until now, the main component with high in vitro hemolytic activity isolated from this venom was the phospholipase A2 enzyme, although the presence of proteolytic enzymes that act specifically on the membrane glycoproteins of erythrocytes cannot be ruled out (Seibert et al., 2006, 2010). Since myotoxins are commonly described in several snake, spider and bee venoms, the presence of myotoxic activity in L. obliqua was investigated using specific biochemical markers, in vitro experiments and histological analyses.