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F Gubensek

Publications and source records attributed to F Gubensek.

At least 73 records · Page 4Linked to original sources
PubMed1991

The primary structure of ammodytin L, a myotoxic phospholipase A2 homologue from Vipera ammodytes venom.

A new myotoxic phospholipase A2 homologue, having a serine residue in position 49 instead of highly conserved aspartic acid, was found in the venom of Vipera ammodytes. The primary structure revealed additional mutations in the positions important for enzymatic activity. Tyr28 is exchanged for a histidine and Gly33 for asparagine. These changes render earlier-reported weak enzymatic activity unlikely. The role of this rather abundant venom fraction is apparently in myotoxicity, which was confirmed in the muscle-cell culture from neonatal rats. The muscle-cell culture proved to be a good tool to investigate the effects of various myotoxins on muscle cells.

Amino Acid Sequence↗
PubMed1991

Immunological studies of the toxic site in ammodytoxin A.

Two monoclonal antibodies against the native ammodytoxin A and four site-directed polyclonal antibodies against synthetic peptides derived from the primary structure of the toxin were prepared in order to estimate the localization of its toxic site. Some of the antibodies neutralized the lethal toxicity of the toxin, thus indicating an approximate position of the toxic or receptor binding site on the molecule that is different from those predicted by comparison with a number of known sequences.

Amino Acid Sequence↗
PubMed1991

Cloning and nucleotide sequence of a cDNA encoding ammodytoxin A, the most toxic phospholipase A2 from the venom of long-nosed viper (Vipera ammodytes).

A venom gland cDNA library was constructed in pUC9 and screened with a mixed oligonucleotide probe deduced from the unique Glu-4 to Ile-9 region of ammodytoxins. Twenty-one strongly positive clones were found by hybridization of about 5000 bacterial colonies, nine of them with the inserts encoding ammodytoxin A. The cDNA for ammodytoxin A encodes a 122 amino acid residue mature protein, preceded by a 16 residue signal peptide. Its complete nucleotide sequence shows 99% similarity to those of ammodytoxins B and C.

Amino Acid Sequence↗
PubMed1989

Primary structure of ammodytoxin C further reveals the toxic site of ammodytoxin.

The sequence of ammodytoxin C, a presynaptically toxic, basic phospholipase A2 of Vipera ammodytes ammodytes venom was determined. The toxin differs only in two amino acid residues from the most toxic isotoxin ammodytoxin A and is 18-times less lethal. Ammodytoxin B which is 30-times less lethal than ammodytoxin A differs from it only in three amino acid residues. From the three-dimensional model of ammodytoxin A, it can be seen that mutated regions of ammodytoxin B and ammodytoxin C are on the surface, and relatively distant from each other. The observed decrease in toxicity of ammodytoxin C could be a consequence of changed charge in position 128 where a Lys is exchanged for Glu. The resulting change in electrostatic properties of the molecule which influences the orientation of the molecule during the approach to the charged nerve-terminal membrane might be responsible for the observed decrease in toxicity.

Amino Acid Sequence↗
PubMed1989

Chemical modification of equinatoxin II, a lethal and cytolytic toxin from the sea anemone Actinia equina L.

The role of arginine and tyrosine in cytolytic properties of equinatoxin II, isolated from the sea anemone Actinia equina L., was studied by means of chemical modifications. The toxin was modified with 2,3 butanedione and tetranitromethane, respectively. The extent of modification and physico-chemical properties of the modified proteins were checked with amino acid analysis, isoelectric focusing and circular dichroic spectra. Extensive treatment of the toxin with 2,3 butanedione modified seven arginines and also two tyrosines, with resulting loss of hemolytic activity. Modification of two out of nine arginine residues resulted in a 25% loss of hemolytic activity, whereas nitration of three out of ten tyrosines decreased hemolytic activity by 95%. The nitrated toxin had at least a 30-fold higher i.v. LD50 than the native toxin. None of the modifications significantly affected the secondary structure of the toxin as revealed by the CD spectra. It is concluded that tyrosine residues are involved in both lethal and cytolytic activity, while the role of arginine residues is not evident because of the non-specific alteration of tyrosine residues with 2,3 butanedione.

Amino Acids↗
PubMed1989

Characterization of a lectin-like protein isolated from Lachesis muta snake venom.

A lectin-like protein was isolated from L. muta venom by gel filtration on BIO Gel P-100 followed by column Chromatography on DEAE-sephades A-50. The protein eluted at 0.4 M Nacl in 0.01 Tris pH 7.3 and exhibited agglutinin activity toward 0+ human erythrocytes. The protein is a dimer with Mr 28 kDa. Amino acid analysis revealed high content of tryptophan and acid recidues and low content of cysteine and methionine residues. No neutral carbohydrates and sialic acid were detected. Circular dichroic spectrum shows 78% of B structure and 1% of alpha structure. In vitro experiments with erythrocytes from rat, rabbit and dog revealed strong agglutination while red blood cells from mice, sheep and goat were not agglutinated. In vivo experiments using anesthetized rats, a sharp and prolonged fall in the blood pressure was observed at protein dose of 1.5 mg/kg. Double dose of protein caused the death of the animal.

Amino Acids↗
PubMed1987

Characterization of a metallo-proteinase from Bothrops asper (terciopelo) snake venom.

Metalloproteinase from the venom of Bothrops asper (proteinase G) is a glycoprotein with 1% neutral hexose and 3.5 moles of sialic acid per mole of protein. It hydrolyses a number of protein substrates such as casein, hemoglobin, gelatin and fibrinogen, whose alpha chain is degraded preferentially. The pH optimum of hydrolysis of casein is approximately 9.5. The protease is devoid of hemorraghic, esterolytic and amidolytic activities. The proteolytic activity of the enzyme increases by about 20% in the presence of 0.2 mM Ca2+ and Mg2+. Among the other ions tested, only Cd2+ and Fe2+ markedly decreased its activity. EDTA and cysteine are also strong inhibitors. In the presence of Ca2+ and EDTA, Zn2+ ions restored 50% of the activity. The amino acid composition shows fewer acidic residues than in related proteinases from other snake venoms.

Animals↗
PubMed1986

Amino-acid sequence of ammodytoxin B partially reveals the location of the site of toxicity of ammodytoxins.

The complete amino-acid sequence of ammodytoxin B, a presynaptically toxic phospholipase A2 isolated from Vipera ammodytes ammodytes venom, was determined by manual and automated protein sequencing. Ammodytoxin B (i.v. LD50 = 0.58 mg/kg for white mice) is 30-fold less toxic than ammodytoxin A, the most toxic phospholipase isolated from the same venom. The two proteins (each 122 residues long) differ in only 3 residues located in positions 115, 118 and 119 (numbering according to R. Renetseder et al. (1985) J. Biol. Chem. 260, 11627-11634) suggesting that an exposed hydrophobic residue in position 115 and a basic residue in position 118 may be responsible for the increased toxicity of ammodytoxin A and should form at least one part of the site of toxicity in ammodytoxins.

Amino Acid Sequence↗
PubMed1985

Ammodytoxin A, a highly lethal phospholipase A2 from Vipera ammodytes ammodytes venom.

The amino acid sequence of ammodytoxin A, the most toxic presynaptically active phospholipase A2 isolated from Vipera ammodytes ammodytes venom, was determined. The primary structure was deduced from peptides obtained by Staphylococcus aureus proteinase and trypsin digestion of reduced and carboxymethylated protein and from the automated Edman degradation of the N-terminal part of the non-reduced molecule. According to the sequence, the enzyme classifies to the subgroup IIA of the phospholipase A2 family of enzymes. The location of basic residues believed to be responsible for the toxic activity of presynaptically active phospholipases differs substantially from those in the highly toxic enzymes of other subgroups. Comparison of the sequence with sequences of other snake venom enzymes indicates that the toxic site(s) may not be the same in all subgroups of presynaptically active phospholipases.

Amino Acid Sequence↗
PubMed1984

Mode of neuromuscular blocking action of toxic phospholipases A2 from Vipera ammodytes venom.

The effects of toxic phospholipases A2 ( fraxtions "j", "k1" and "k2") isolated from the venom of Vipera ammodytes were studied on the chick biventer cervicis muscle and the mouse phrenic nerve-diaphragm preparations. In the chick muscle, all of these PLA2s caused neuromuscular (N-M) blockade without producing contracture or affecting the response of the muscle to acetylcholine. In the mouse diaphragm, these PLA2s inhibited completely the indirectly elicited contraction without affecting that evoked directly. The order of their N-M blocking potency is "k2" greater than "k1" greater than or equal to "j". In a low Ca2+ (0.5 mM) medium, they produced a triphasic change in the indirectly elicited contractions: an initial inhibition followed by an enhancement and then a progressive depression leading to complete N-M blockade. The frequency of miniature endplate potentials (m.e.p.p.s) in the mouse diaphragm first increased 2-3 fold and then gradually decreased after "k2" treatment, while the amplitude of m.e.p.p.s did not decrease even after the evoked release of transmitter failed. Giant potentials and bursts of m.e.p.p.s were frequently observed. The quantal content of e.p.p.s was first increased and then decreased gradually. The resting membrane potential was only slightly reduced at 30 micrograms per ml. The ultrastructure of motor nerve terminals in the "k2"-intoxicated mouse diaphragm showed an increase in omega-shaped indentation in the axolemma. The mitochondria in the nerve terminal were swollen and vacuolized. No structural changes were found in the muscle fibers, fibrocytes and myelinated axons in the diaphragm. It is concluded that the toxic PLA2s from Vipera ammodytes venom produce a N-M blockade by acting selectively on the presynaptic site.

Action Potentials↗
PubMed1983

The primary structure of Vipera ammodytes venom trypsin inhibitor I.

The primary structure of Vipera ammodytes venom trypsin inhibitor I consists of 61 amino acid residues [sequence in text]. The N-terminal group of the inhibitor is pyrrolidonecarboxylic acid. The sequential data were obtained by analysis of peptides isolated from tryptic and chymotryptic digests and by analysis of peptides derived from the hydrolysis of the aspartyl-prolyl bond of the carboxymethylated inhibitor. The primary structure of trypsin inhibitor I presented shows approximately 80% sequence homology with chymotrypsin inhibitor isolated from the venom of the same snake, and nearly 50% homology with bovine basic pancreatic trypsin inhibitor. It belongs to the Kunitz-pancreatic trypsin inhibitor family of inhibitors.

Amino Acid Sequence↗
PubMed1983

Serine proteinase inhibitors from Vipera ammodytes venom. Isolation and kinetic studies.

Three protein inhibitors of serine proteinases were isolated from the crude venom of the long-nosed viper Vipera ammodytes ammodytes by ion-exchange and gel chromatography. Two of them strongly inhibit trypsin (Ki = 3.4 X 10(-10) and 5.6 X 10(-10) M), while the third one primarily inhibits chymotrypsin (Ki = 4.3 X 10(-9) M). Their Mr values are close to 7000, and pI is 9.8 in both trypsin inhibitors and 10.0 in the chymotrypsin inhibitor. The N-terminal group in the former inhibitors is blocked; arginine is the N-terminal amino acid in the latter. Besides trypsin and alpha-chymotrypsin, the trypsin inhibitors also inhibit plasmin, human plasma kallikrein and porcine pancreatic kallikrein. The chymotrypsin inhibitor inhibits trypsin and human plasma kallikrein only weakly and does not inhibit plasmin and porcine pancreatic kallikrein. According to their properties, all three inhibitors belong to the Kunitz-pancreatic trypsin inhibitor family of inhibitors.

Amino Acids↗