Properties of phosphodiesterase from Vipera lebetina venom immobilized on Agarose

Preparations of phosphodiesterase from Vipera lebetina venom immobilized on agarose were obtained. The kinetic properties for the hydrolyses of various substrates of soluble and immobilized phosphodiesterase, e. g. effects of pH, temperature, substrate concentrations, etc., were compared. The values of Km, V and activation energy for the substrate hydrolyses were determined.     

Populational differences in venom composition in the snake Vipera lebetina turanica]

Studies have been made on the toxicity, enzymic activity and fractional composition (polyacrylamide gel electrophoresis) of the venom obtained from various populations of the snake Vipera lebetina turanica. Animals were captured in different regions of the Mid-Asian part of the USSR. Electrophoretic studies reveal differences in fractional composition, mobility and density of proteins in the venoms studied. However, no significant variations were found in the enzymic activity and toxicity of the venoms.     

Vipera lebetina venom contains two disintegrins inhibiting laminin-binding beta1 integrins

To explain the myotoxic effects of snake venoms, we searched for inhibitors of alpha7beta1 integrin, the major laminin-binding integrin in skeletal muscle. We discovered two inhibitors in the venom of Vipera lebetina. One of them, lebein-1 (known as lebein), has already been proposed to be a disintegrin because of its RGD-containing primary sequence. The other, lebein-2, is a novel protein that also interacts firmly with alpha3beta1, alpha6beta1, and alpha7beta1 integrins, but not with the collagen-binding alpha1beta1 and alpha2beta1 integrins. Ligand binding of laminin-recognizing beta1 integrins was efficiently blocked by both lebein-1 and lebein-2. In cell attachment assays, lebein-1 and lebein-2 inhibited myoblast attachment not only to laminin, but also to fibronectin. However, neither lebein-1 nor lebein-2 interacted with alpha7beta1 integrin in an RGD-dependent manner, similar to the interaction of the laminin with alpha7beta1 integrin. Identical divalent cation dependence of integrin binding to laminin and to either of the two inhibitors and their mutually exclusive binding suggest that both lebein-1 and lebein-2 interact with the ligand-binding site of laminin-binding beta1 integrins by mimicking the yet unknown integrin-binding structure of laminins. Like lebein-1, lebein-2 is a soluble heterodimeric disintegrin of low molecular mass. Together with membrane-bound ADAM-2 and ADAM-9, the two inhibitors seem to form a small group of disintegrins that can bind to laminin-binding beta1 integrins. Because of their inhibitory capability both in vitro and in vivo, lebein-1 and lebein-2 may be valuable tools in influencing laminin-induced, integrin-mediated cell functions such as cell anchorage, migration, and mechanical force transduction on laminin-rich basement membranes.     

Factor X activator from Vipera lebetina venom is synthesized from different genes

Vipera lebetina venom contains specific coagulant Factor X activator (VLFXA) that cleaves the Arg52-Ile53 bond in the heavy chain of human factor X. VLFXA is a glycoprotein that is composed of a heavy chain (HC) and two light chains (LC) linked by disulfide bonds. The complete amino acid sequences of the three chains of the factor X activator from V. lebetina snake venom are deduced from the nucleotide sequences of cDNAs encoding these chains. The full-length cDNA (2347 bp) sequence of the HC encodes an open reading frame (ORF) of 612 amino acids that includes signal peptide, propeptide and mature metalloproteinase with disintegrin-like and cysteine-rich domains. The light chain LC1 contains 123 and LC2 135 amino acid residues. Both light chains belong to the class of C-type lectin-like proteins. The N-termini of VLFXA chains and inner sequences of peptide fragments detected by liquid chromatography-electrospray ionization tandem mass spectrometry (LC MS/MS) from protein sequence are 100% identical to the sequences deduced from the cDNA. The molecular masses of tryptic fragments of VLFXA chains analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) also confirm the protein sequences deduced from the cDNAs. These are the first cloned factor X activator heavy and light chains. We demonstrate that the heavy and light chains are synthesized from different genes.     

Purification and properties of a proteinase from Vipera lebetina (snake) venom

A proteinase from the venom of Vipera lebetima was purified by chromatography on Sephadex G-100 and CM-cellulose. The purified proteinase was homogeneous on SDS-polyacrylamide gel electrophoresis and consisted of a single chain with molecular weight of 37 000±1500. The isoelectric point of the proteinase was over 10. The enzyme was active on casein but not on esters and amides of arginine. It split the oxidized insulin B-chain at the peptide bonds of Tyr¹⁶-Leu¹⁷, Phe²⁴-Phe²⁵ and Phe²⁵-Tyr²⁶, and glucagon at the bonds Tyr¹⁰-Ser¹¹, Leu¹⁴-Asp¹⁵ and Leu²⁶-Met²⁷. The enzyme was inhibited by DFP and PMSF, and partially by soybean trypsin inhibitor, but not with EDTA.     

A permanent magnetic field alters the phospholipase activity in the snake venom from Vipera lebetina

It was found that at the exposure of Vipera lebetina snakes (during 10 days for 30 min daily) to SMF (0.15 T1) the specific activity of venom phospholipase A1, A2 and phosphodiesterase C increased by 20.6 +/- 2.8; 31.7 +/- 3.2 and 32.7 +/- 1.3% correspondingly. The above mentioned changes of venom enzyme activity were accompanied with the decrease of its total protein amount by 31.6 +/- 2.2%. It could be supposed that the described changes are able to cause significant changes in the total metabolic activity of cells and the organism as a whole.     

A new tyrosine-specific chymotrypsin-like and angiotensin-degrading serine proteinase from Vipera lebetina snake venom

Vipera lebetina venom contains different metallo- and serine proteinases that affect coagulation and fibrin(ogen)olysis. A novel serine proteinase from V. Lebetina venom having ChymoTrypsin Like Proteolytic activity (VLCTLP) was purified to homogeneity from the venom using Sephadex G-100sf, DEAE-cellulose, heparin-agarose and FPLC on Superdex 75 chromatographies. VLCTLP is a glycosylated serine proteinase with a molecular mass of 41926 Da. It reacts with N-acetyl-l-tyrosine ethyl ester (ATEE) but not with Suc-Ala-Ala-Pro-Phe-pNA or Suc-Ala-Ala-Pro-Leu-pNA. The complete amino acid sequence of the VLCTLP is deduced from the nucleotide sequence of the cDNA encoding this protein. The full-length cDNA sequence of the VLCTLP encodes open reading frame of 257 amino acid residues that includes a putative signal peptide of 18 amino acids, a proposed activation peptide of six amino acid residues and serine proteinase of 233 amino acid residues. VLCTLP belongs to the S1 (chymotrypsin) subfamily of proteases. The multiple alignment of its deduced amino acid sequence showed structural similarity with other serine proteases from snake venoms. The protease weakly hydrolyses azocasein, Aα-chain and more slowly Bβ-chain of fibrinogen. VLCTLP does not cleave fibrin and has no gelatinolytic activity. Specificity studies against peptide substrates (angiotensin I and II, oxidized insulin B-chain, glucagon, fibrinogen fragments etc.) showed that VLCTLP catalysed the cleavage of peptide bonds after tyrosine residues. VLCTLP is the only purified and characterized serine proteinase from snake venoms that catalyses ATEE hydrolysis. We detected ATEE-hydrolysing activities also in 9 different Viperidae and Crotalidae venoms.     

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.     

Factor X activator from Vipera lebetina snake venom,molecular characterization and substrate specificity

Our studies of the venom from the Levantine viper Vipera lebetina have demonstrated the existence of both coagulants and anticoagulants of the hemostatic system in the same venom. We showed that V. lebetina venom contains factor X activator (VLFXA) and factor V activator, fibrinolytic enzymes. VLFXA was separated by gel filtration on Sephadex G-100 superfine and ion exchange chromatography on CM-cellulose and on TSK-DEAE (for HPLC) columns. VLFXA is a glycoprotein composed of a heavy chain (57.5 kDa) and two light chains (17.4 kDa and 14.5 kDa) linked by disulfide bonds. VLFXA has multiple molecular forms distinguished by their isoelectric points. The differences in their pI values may be caused by dissimilarities in the respective charged carbohydrate content or in the primary sequence of amino acids. We synthesized 6–9 amino acid residues containing peptides according to physiological cleavage regions of human factor X and human factor IX. The peptides (Asn-Asn-Leu-Thr-Arg-Ile-Val-Gly-Gly – factor X fragment, and Asn-Asp-Phe-Thr-Arg-Val-Val-Gly-Gly – factor IX fragment) were used as substrates for direct assay of VLFXA. Cleavage products of peptide hydrolysis and the molecular masses of cleavage products of human factor X were determined by MALDI-TOF MS. The MALDI-TOF MS was highly efficient for the recovery and identification of peptides released by VLFXA hydrolysis. We can conclude that VLFXA cleaves the Arg⁵²-Ile⁵³ bond in the heavy chain of human factor X and the Arg²²⁶-Val²²⁷ bond in human factor IX precursor. VLFXA could not activate prothrombin nor had any effect on fibrinogen, and it had no arginine esterase activity toward benzoylarginine ethyl ester.     

Purification and characterization of a growth factor-like which increases capillary permeability from Vipera lebetina venom

We have investigated the effect of Vipera lebetina venom on capillary permeability and isolated an increasing capillary permeability protein (ICPP) which is devoid of arginine ester hydrolase and phospholipase A2 activities. This protein was purified with a yield of about 0.2% by fast protein liquid chromatography (FPLC) using successively Superose 12, Mono Q, and Mono S columns and by high-pressure liquid chromatography (HPLC) on a C8 reverse-phase column. The purified protein migrated on SDS-PAGE as a band of about 27 kDa under nonreducing conditions and as a band of about 16 kDa under reducing conditions. Chromatography on a C8 column of reduced and alkylated protein yielded a single peak suggesting that this protein is homodimeric. This protein was refractory to Edman degradation chemistry. We used successfully a chemical unblocking involving the incubation of the protein with HCl in anhydrous methanol. The N-terminal amino acid sequence clearly shows considerable similarity to that of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF).     

The stimulation of the delayed hypersensitivity reaction by the venom of the blunt-nosed viper Vipera lebetina and by its liposomal form]

Native poison of middleasia viper Vipera lebetina has been included into liposomes in order to get an effective preparation for immunotherapy. The preparation reduces the risk of complications taking place during the use of traditional methods. The immunomodulating action of the liposomal form of viper poison has been investigated on the model of reaction of hypersensitivity of delayed type. Together with the stimulation of functional activity T-cells, the absence of suppressive and toxic actions while using the liposomal preparation of poison has been discovered. It is suggested that the observed stimulating effect in connection with immune reactions is due to synergism of action of components of poison liposomal form and activation of T-cells and macrophage, taking part in immune response to the antigenic stimulus.