Isolation and characterization of hemorrhagic metalloproteinase from Vipera berus berus (common viper) venom

1. Hemorrhagic metalloproteinase (HMP) was isolated by gel filtration on a Sephadex G-100 (superfine) and affinity chromatography on agarose HPS-7. 2. Hemorrhagic metalloproteinase is a glycoprotein with mol. wt 56.3 kDa. It contains 1 zinc atom per molecule of protein. 3. Hemorrhagic metalloproteinase hydrolyzes casein, fibrinogen and splits the insulin B chain at positions Ala14-Leu15, Tyr16-Leu17, His10-Leu11. It digests A alpha chain of fibrinogen.     

Isolation and characterization of nerve growth factor from Vipera berus berus (common viper) venom

Nerve growth factor from Vipera berus berus venom was purified by gel filtration on Sephadex G-100 (superfine), ion-exchange-chromatography on DEAE-Sephadex A-50 and chromatofocusing on PBE 118. The Vipera berus berus venom NGF consists of multiple molecular forms with pls in the interval 9.1-9.7. All isoforms have identical mol. wts approximately 35,000 +/- 3000 (in gel filtration) and 17,000 +/- 2000, 15,000 +/- 2000 (by SDS electrophoresis with beta-mercaptethanol). V. berus berus venom NGF reacted with monoclonal antibodies against Viper lebetina NGF and caused differentiation of pheochromocytoma PC12 cells.     

Isolation, structural, and functional characterization of an apoptosis-inducing L-amino acid oxidase from leaf-nosed viper (Eristocophis macmahoni) snake venom

The enzyme L-amino acid oxidase (LAO) from the leaf-nosed viper (Eristocophis macmahoni) snake venom was purified to homogeneity in a single step using high performance liquid chromatography on a Nucleosil 7C18 reverse phase column. The molecular mass of the purified enzyme was 58734.0 Da, as determined by matrix-assisted laser desorption/ionization mass spectrometry. The N-terminal amino acid sequence (ADDKNPLEEAFREADYEVFLEIAKNGL) and the chemical composition of the purified LNV-LAO shows close structural homology with other L-amino acid oxidases isolated from different snake venoms. The secondary structural contents analysis of LAO, established by means of circular dichroism, revealed ca. 49% alpha-helix, 19% beta-sheet, 10% beta-turn, and 22% random coil structure. The purified LNV-LAO not only retained its specific enzymatic activity (73.46 U/mg), determined against L-leucine as a substrate, but also exhibited potent haemolytic (1-10 microg/ml), edema- (MED 4.8 microg/ml) and human platelet aggregation-inducing (ED50 33 microg/ml) properties. Unlike other haemorrhagic snake venom L-amino acid oxidases, the LNV-LAO does not produce haemorrhage. In addition to these local effects, the purified LNV-LAO showed apoptosis-inducing activity in the MM6 cell culture assay. After 18 h treatment with 25-100 microg/ml of LAO, the typical DNA fragmentation pattern of apoptotic cells was observed by means of fluorescent microscopy and agarose gel electrophoresis.     

Isolation and characterization of an unusual form of L-amino acid oxidase from King cobra (Ophiophagus hannah) venom

The L-amino acid oxidase (EC 1. 4. 3. 2) from King cobra (Ophiophagus hannah) venom was purified to electrophoretic homogeneity. The molecular weight of the enzyme was determined to be 140000 when examined by gel filtration and 68000 by SDS-polyacrylamide gel electrophoresis. The enzyme had an isoelectric point of 4.5 and an intravenous LD50 of 5 micrograms/g in mice. It is a glycoprotein and contains two moles of FAD per mole of enzyme. The enzyme exhibited unusual thermal stability and unlike most other venom L-amino acid oxidases, it was stable in alkaline solution and was not inactivated by freezing.     

Purification and characterization of an organ specific haemorrhagic toxin from Vipera russelli russelli (Russell's viper) venom

A haemorrhagic toxin (VRR-12) from Vipera russelli russelli (Russell's viper) venom has been purified by ion-exchange chromatography on CM-Sephadex C-50 followed by size-exclusion HPLC to electrophoretically homogeneous state. It is a 12 kDa single polypeptide having 1 mole of Zn+2 ion. This toxin induces intense intestinal haemorrhage and to a lesser extent skeletal muscle haemorrhage in mice. It does not show detectable proteolytic and esterolytic activity with selected substrates under specified conditions, haemolytic and phospholipase activity. When VRR-12, preincubated with bivalent antiserum against Saw-scaled and Russell's viper venom or EDTA was injected, haemorrhagic activity was not reduced, on the other hand preincubation with phenylmethyl sulphonyl fluoride reduced the activity markedly. Biodistribution studies with 125I VRR-12 show that haemorrhagic manifestation by this toxin is not a direct function of the fraction of the totally administered toxin distributed to that tissue.     

Molecular characterization of L-amino acid oxidase from Agkistrodon halys blomhoffii with special reference to platelet aggregation

L-Amino acid oxidase (LAO, EC 1.4.3.2) is widely distributed in snake venom, and induces apoptosis in vascular endothelial cells, causing prolonged bleeding from vessel walls at bite sites. The effect of snake venom LAOs on platelet function is controversial. Further, we have little information on their structural characterization. We purified M (mamushi)-LAO, a single-chain glycoprotein with a molecular mass of 60 kDa and a pI of 4.9, from Agkistrodon halys blomhoffii (Japanese mamushi) venom, and determined the N-terminal and several internal amino acid sequences of this enzyme. Molecular cloning based on these data was conducted to elucidate its full-length cDNA structure (2192 nucleotides), which includes a putative 18 amino acid residue signal peptide and a 504 residue mature subunit. The predicted M-LAO translation product shares 87.3% identity with that of Crotalus adamanteus (Southeastern diamondback rattlesnake) LAO. M-LAO, up to a final concentration of 2.6 microM, inhibited both agonist- and shear stress-induced platelet aggregation (SIPA) dose-dependently. In agonist-induced platelet aggregation, M-LAO predominantly inhibited the second aggregation, but with a marginal inhibition of the first. In SIPA, the inhibition was more dramatic under low-shear stress than high-shear stress, and was enhanced by the presence of L-leucine, a substrate of this enzyme. Catalase, a H2O2 scavenger, totally quenched such enhancement. These results suggest that M-LAO inhibits the interaction between activated platelet integrin alphaIIb/beta3 and fibrinogen through the continuous generation of H2O2, and may contribute to prolonged bleeding from the vessels at snake bite sites.     

Biochemical and functional characterization of an L-amino acid oxidase isolated from Bothrops pirajai snake venom

In this work we describe the isolation of a new l-amino acid oxidase (LAAO) referred to as BpirLAAO-I from Bothrops pirajai snake venom, which was highly purified using a combination of molecular exclusion, affinity, and hydrophobic chromatography steps. BpirLAAO-I homodimeric acid glycoprotein (approximate Mr and pI of 130,000 and 4.9, respectively) displays high specificity toward hydrophobic/aromatic amino acids, while deglycosylation does not alter its enzymatic activity. The N-terminal LAAO sequence of its first 49 amino acids presented a high similarity between a amino acid sequence with other LAAOs from: Bothrops spp., Crotalus spp., Calloselasma rhodostoma, Agkistrodon spp., Trimeresurus spp., Pseudechis australis, Oxyuranus scutellatus, and Notechis scutatus. BpirLAAO-I induces time-dependent platelet aggregation, mouse paw edema, cytotoxic activity against Escherichia coli, Pseudomonas aeruginosa, Leishmania sp., and tumor cells, and also a typical fago (M13mp18) DNA fragmentation. Platelet aggregation, leishmanicidal and antitumoral activities were reduced by catalase. Thus, BpirLAAO-I is a multifunctional protein with promising biotechnological and medical applications.     

Cloning, characterization and mutagenesis of Russell's viper venom L-amino acid oxidase: Insights into its catalytic mechanism

To investigate the structure-function relationships and geographic variations of L-amino acid oxidase (LAAO) from Daboia venoms, a single LAAO (designated as DrLAO) was purified from eastern Indian Daboia russelii venom and characterized. The purified DrLAO showed subunit molecular mass of 60-64kDa; its N-terminal sequence (1-20) was identical to those of several true viper LAAOs. Its preferred substrates were hydrophobic l-amino acids and the kinetic specificities were ordered as follows: Phe, Tyr, Met, Leu, and Trp. Enzyme assay and Western blotting showed that the venom LAAO contents of D. russelii were higher than those of Daboia siamensis. DrLAO dose-dependently inhibited ADP- and collagen-induced platelet aggregation with IC(50) values of 0.27 and 0.82μM, respectively. Apparently, DrLAO may synergize with other venom components to prolong and enhance bleeding symptoms after Daboia envenoming. The full sequence of DrLAO was deduced from its cDNA sequence and then confirmed by peptide mass fingerprinting. Molecular phylogenetic analysis revealed that SV-LAAO family members could be differentiated not only by snake taxonomy but also by the variations at position 223, and they divided into H223, S223, N223, and D223 subclasses. We have further prepared recombinant DrLAO and mutants by the Pichia expression system. Mutagenic analyses of DrLAO His223 revealed that this residue bound substrates instead of serving as an essential base in the catalytic steps. Our results suggest a direct hydride transfer from substrate to FAD as the mechanism for SV-LAAOs.     

Characterization of two platelet aggregation inhibitor-like polypeptides from viper venom

Two polypeptides, eristocophins I and II, have been characterized from leaf-nosed viper (Eristocophis macmahoni) venom. They contain 10 half-Cys residues of a total of 61/62 residues, have 72% residue identity, and exhibit similarities to platelet aggregation inhibitors and segments of adhesive proteins. Eristocophin I contains the sequence Arg-Gly-Asp, known to inhibit fibrinogen interaction with the platelet receptor. Eristocophin II has Met instead of Arg in this sequence, and an adjacent Trp-Asn-Asp segment. The latter is also typical of adhesive proteins, thus linking two potentially functional segments in one molecule. Exchanges are maximal in these segments, suggesting that the polypeptides exhibit functional divergence with isoform differences in important regions.     

Purification,characterization and biological activities of phospholipase a from russell's viper (Vipera russelli) venom

• 1.1. The major phospholipase A has been purified to electrophoretic homogeneity from the venom of Vipera russelli (Russell's viper).
• 2.2. The molecular weight of the purified enzyme was estimated to be 31,000 by Sephadex G-75 gel filtration chromatography and 29,000 by SDS-polyacrylamide gel electrophoresis. The enzyme exhibited an apparent K_m value of 2.3 × 10⁻² M.
• 3.3. The phospholipase A showed edema forming, indirect hemolytic and myonecrotic activities but not hemorrhagic activity.

     

Isolation and characterization of nerve growth factor from Vipera lebetina (snake) venom

Nerve growth factor from Vipera lebetina venom was purified by gel filtration and ion exchange chromatography steps. The NGF preparation obtained is a glycoprotein with weak arginine esterase activity. It hydrolyzes benzoylarginine ethyl ester (BAEE). Vipera lebetina NGF consists of multiple forms of protein with pI in the interval 9-10.5. All isoforms have identical molecular weights of 32,500.     

Two L-amino acid oxidase isoenzymes from Russell's viper (Daboia russelli russelli) venom with different mechanisms of inhibition by substrate analogs

Two isoforms, L(1) and L(2), of L-amino acid oxidase have been isolated from Russell's viper venom by Sephadex G-100 gel filtration followed by CM-Sephadex C-50 ion exchange chromatography. The enzymes, with different isoelectric points, are monomers of 60-63 kDa as observed from size exclusion HPLC and SDS/PAGE. Partial N-terminal amino acid sequencing of L(1) and L(2) showed significant homology with other snake venom L-amino acid oxidases. Both the enzymes exhibit marked substrate preference for hydrophobic amino acids, maximum catalytic efficiency being observed with L-Phe. Inhibition of L(1) and L(2) by the substrate analogs N-acetyltryptophan and N-acetyl-L-tryptophan amide has been followed. The initial uncompetitive inhibition of L(1) followed by mixed inhibition at higher concentrations suggested the existence of two different inhibitor-binding sites distinct from the substrate-binding site. In the case of L(2), initial linear competitive inhibition followed by mixed inhibition suggested the existence of two nonoverlapping inhibitor-binding sites, one of which is the substrate-binding site. An inhibition kinetic study with O-aminobenzoic acid, a mimicking substrate with amino, carboxylate and hydrophobic parts, indicated the presence of three and two binding sites in L(1) and L(2), respectively, including one at the substrate-binding site. An inhibitor cross-competition kinetic study indicated mutually excluding binding between N-acetyltryptophan, N-acetyl-L-tryptophan amide and O-aminobenzoic acid in both the isoforms, except at the substrate-binding site of L(1). Binding of substrate analogs with different electrostatic and hydrophobic properties provides useful insights into the environment of the catalytic sites. Furthermore, it predicts the minimum structural requirement for a ligand to enter and anchor at the respective functional sites of LAAO that may facilitate the design of suicidal inhibitors.     

Primary culture of epithelial secretory cells from venom gland of the common adder Vipera berus

A primary culture of epithelial secretory cells from the venom gland of Vipera berus was obtained. The cells adhered to collagen 1 and to a mixture of adhesion proteins (Matrigel), proliferated and retained the features of differentiation. Electron microscopy demonstrated the presence of all ultrastructures typical of these cells in vivo, a full complex of intercellular junctions, and cellular membrane polarity. The immunohistochemistry confirmed the capacity of secretory cells to synthesize venom in culture. We have studied the role of carbochole, an agonist of M-cholinoreceptor, in the initiation of the secretory cycle in cells in vitro. We propose that M-cholinoreceptors may play an important role in the initiation of the secretory cycle in vivo.     

蛇毒L-氨基酸氧化酶酶学性质及对血小板功能的影响

L-氨基酸氧化酶(EC1·4·3·2)是一种黄素类蛋白酶,其酶活性为立体特异性催化L-氨基酸的氧化脱氨,生成α-酮酸,同时产生氨和过氧化氢。酶广泛存在于多种生物体种类中。蛇毒L-氨基酸氧化酶(LAAO)是该类酶中研究最为深入的一族,因其易于纯化而成为研究L-氨基酸氧化酶的酶学、结构生物学和药物学的有趣客体。近年来,越来越多的具有不同分子量、不同底物选择性的蛇毒LAAO被分离纯化,国内外文献越来越多的报道了蛇毒LAAO的特性,如准确的分子量、底物特性、与血小板的相互作用、诱发出血和凋亡。本文就蛇毒LAAO的性质及其对血小板功能的…     

beta-RTX. A receptor-active protein from Russell's viper (Vipera russelli russelli) venom

A single chain polypeptide, termed beta-RTX, with an apparent Mr = 9600 has been isolated from the venom of Vipera russelli russelli. It was purified by cation exchange chromatography, followed by preparative isoelectric focusing and chromatofocusing. Purity was confirmed by gel filtration, high performance liquid chromatography, gel electrophoresis, and analytical ultracentrifugation. Amino acid analysis revealed the presence of eight half-cystines, one being located at the NH2 terminus, which are linked to form four intramolecular disulfide bridges. Chromatofocusing revealed some microheterogeneity yielding three isoforms with pI values of 9.3, 9.37, and 9.48, respectively. In its native configuration, beta-RTX was not susceptible to tryptic degradation but was readily digested after reduction and alkylation. beta-RTX possesses weak phospholipase A2 activity and competes with the binding of monoamines and opiate ligands to their respective receptors. No binding to histamine, gamma-aminobutyric acid, benzodiazepine, or muscarinic receptors was observed. In vivo, whereas 100 micrograms/kg intravenous beta-RTX seemed to be without apparent effects in the rat, 10 ng/kg beta-RTX injected intracerebroventricularly caused marked sedation, with full recovery within 3 h.     

Isolation and structural characterization of a cytotoxic L-amino acid oxidase from Agkistrodon contortrix laticinctus snake venom: preliminary crystallographic data.

We have purified a cytotoxic L-amino acid oxidase (LAO) from Agkistrodon contortrix laticinctus snake venom by means of Superdex-200 gel filtration, followed by phenyl-Sepharose CL-4B chromatography. The purified enzyme (ACL LAO) is a dimer on gel filtration, with a M(r) of 60,000 for the monomer as estimated by SDS-PAGE. LAO activity was tested against 15 amino acids, but only 9 were oxidized by the enzyme, suggesting that it presents some degree of specificity. ACL LAO has apoptosis-inducing activity in an HL-60 cell culture assay. After 24 h treatment with 25 micrograms/ml of ACL LAO, the typical DNA fragmentation pattern of apoptotic cells was observed on agarose gel electrophoresis. NMR analysis showed the presence of a flavin mononucleotide prosthetic group. To solve its 3-D structure, crystals of the purified protein were grown in 0.1 M Tris-HCl, pH 8.5, and 2 M (NH(4))(2)SO(4). Diffraction data collected to 3.5 A showed that the protein crystallized in the tetragonal system, with unit cell a = b = 103.22 A, c = 183.45 A. This is the first report of preliminary crystallization data for a snake venom L-amino acid oxidase.