Cytochemical analysis of acid phosphatase activity in the venom secretory cells of Bothrops jararaca (original) (raw)
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Toxicon, 1995
Localization and expression of phospholipases A2 in Trimeresurusflavoviridis (habu snake) venom gland. Toxicon 33, 1645-1652, 1995.--The localization and expression profiles of phospholipases A2 (PLA2 s) in Trimeresurus flavoviridis venom gland were studied by means of in situ hybridization and immunohistochemical techniques. Venom gland cells are tightly arrayed in a single layer along the inlet-like lumens in which venom proteins are stored, mRNAs for PLA2s were detected at the high level in cytoplasm. Using the immunohistochemical technique with polyclonal anti-Asp-49-PLA2 antibody, Asp-49-PLA2 and, possibly, its isozymes were detected in intracellular granules and in venom lumens. The intracellular granules containing PLA2 proteins appear to be transferred from the nucleus towards the outer membrane facing the lumen, and then to be secreted.
Toxicon, 2006
There is an increasing interest of obtaining venom by other ways than from extracting it from snakes captured in the wild. A readily available source of this venom will be useful for all pharmacological and biotechnological studies, as well as providing an improved avenue for treatments of snakebites. Here, we show that secretory cells of venom gland can be a good in vitro apparatus to produce venom. We have maintained and morphologically characterized the secretory cells of the Bothrops jararaca venom gland cultured up to 21 days. The isolated cells assemble into acini that growth in size up to 21st day, instead of adhering to the substrate. Bothropasin, a venom metalloprotease, was localized in secretory vesicles by immunoelectron microscopy and venom was also detected in culture medium in a concentration as high as 63 mg/ml. These data show that the acini formed in culture are functionally viable; they can produce and secrete venom.
Detection of alkaline phosphatase in venom by blotting methods
Toxicon, 1994
Venom alkaline phosphatase was detected using a blotting method following electrophoresis. The enzyme gave strong reactions in some venoms, but was absent in other venoms, some within the same species. The mol. wt of the enzyme is close to 100,000 and its pl is between 3.6 and 4.8. The enzyme was inactivated by EDTA and 2-mercaptcethanol, and lost activity by freezing and thawing. Endogenous venom alkaline phosphatase can interfere with alkaline phosphatase-based detection methods. Pre-screening for endogenous venom alkaline phosphatase is recommended prior to using alkaline phosphatasebased detection methods when studying snake venom.
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...
Activation of Bothrops jararaca snake venom gland and venom production: A proteomic approach
2013
Viperidae venom glands have a basal-central lumen where the venom produced by secretory cells is stored. We have shown that the protein composition of venom gland changes during the venom production cycle. Here, we analyzed the venom gland proteins during the venom production cycle by proteomic approach. We identified specific proteins in each stage of the cycle. Protein species from endoplasmic reticulum (PDI and GPR78) and cytoplasm (actin, vimentin, tropomyosin, proteasome subunit alpha type-1, thioredoxin, and 40S ribosomal protein) are more abundant in the activated stage, probably increasing the synthesis and secretion of toxins. We also showed for the first time that many toxins are present in the secretory cells during the quiescent stage. C-type lectin-like and serine proteinases were more abundant in the quiescent stage, and GPIb-BP and coagulation factor IX/X were present only in this stage. Metalloproteinases, L-amino acid oxidases, PLA 2 and snake venom metalloproteinase and PLA 2 inhibitors, and disintegrins were more abundant in the activated stage. Regarding metalloproteinases, the presence of peptides corresponding to the pro-domain was observed. These results allow us to better understand the mechanism of venom gland activation and venom production, contributing to studies about snake toxins and their diversity.
BioMed Research International, 2014
Abbreviations: PLA 2 : phospholipase A 2 ; BjussuCV: Bothrops jararacussu crude venom; BthTX-I: phospholipase A 2 Lys49 from B. jararacussu venom; BthTXII: phospholipase A 2 Asp49 from B. jararacussu venom; BPB: p-bromophenacyl bromide; anti-BjussuCV: antibodies against B. jararacussu crude venom; anti-BthTX-I: antibodies against B. jararacussu myotoxin BthTX-I; anti-BthTX II: antibodies against B. jararacussu myotoxin BthTX-II; anti-BjussuCV-BPB, antibodies against B. jararacussu BPB modified crude venom; anti-BthTX-I-BPB: antibodies against B. jararacussu myotoxin BPB modified BthTX-I; anti-BthTX-II-BPB: antibodies against B. jararacussu myotoxin BPB modified BthTX-II.
Journal of Protein Chemistry
A novel basic phospholipase A 2 (PLA 2) isoform was isolated from Bothrops jararacussu snake venom and partially characterized. The venom was fractionated by HPLC ion-exchange chromatography in ammonium bicarbonate buffer, followed by reverse-phase HPLC to yield the protein Bj IV. Tricine SDS-PAGE in the presence or absence of dithiothreitol showed that Bj IV had a molecular mass of 15 and 30 kDa, respectively. This enzyme was able to form multimeric complexes (30, 45, and 60 kDa). Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The N-terminal sequence (DLWSWGQMIQETGLLP-SYTTY. . .) showed a high degree of homology with basic D49 PLA 2 myotoxins from other Bothrops venoms. Bj IV had high PLA 2 activity and produced moderate myonecrosis in skeletal muscle, but showed no neuromuscular activity in mouse phrenic nerve-diaphragm preparations. Bj IV showed allosteric enzymatic behavior, with maximal activity at pH 8.2 and 35-45°C. Full PLA 2 activity required Ca 2ϩ but was inhibited by Cu 2ϩ and Zn 2ϩ , and by Cu 2ϩ and Mg 2ϩ in the presence and absence of Ca 2ϩ , respectively. Crotapotins from Crotalus durissus terrificus rattlesnake venom significantly inhibited the enzymatic activity of Bj IV. The latter observation suggested that the binding site for crotapotin in this PLA 2 was similar to that in the basic PLA 2 of the crotoxin complex from C. d. terrificus venom. The presence of crotapotin-like proteins capable of inhibiting the catalytic activity of D49 PLA 2 could partly explain the low PLA 2 activity of Bothrops venoms.
Purification and Characterization of a Phosphodiesterase from Bothrops alternatus Snake Venom
A phosphodiesterase was purified from the venom of the snake Bothrops alternatus by a combination of gel filtration and ion exchange chromatographies. In SDS-PAGE, the enzyme gave a single band with a molecular mass of 105 kDa, which was unaltered in the presence of b-mercaptoethanol, indicating that the protein contained no subunits. A single protein band was also observed in native PAGE. There were no contaminating 59-nucleotidase, alkaline phosphatase and protease activities. The enzyme was recognized by commercial bothropic antiserum and gave a single band in immunoblotting. The enzyme had a pH optimum in the range of 7.5–9.5 and the optimum temperature was 60°C, with activity being rapidly lost within 1 min at $70°C. The Km of the enzyme was 2.69 mM. PDE activity was potentiated by cobalt and, to a lesser extent, by calcium, whereas copper, manganese, zinc, EDTA, and b-mercaptoethanol were inhibitory. These properties show thatthis enzyme is very similar to that isolated from other snake venoms.