Horizontal SINE transfer between vertebrate classes.
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Animals↗
Biomedical subjects
F Gubensek
Publications and source records attributed to F Gubensek.
Human cathepsin B is a metastable enzyme stabilized by specific ionic interactions associated with the active site.
The effect of neutral or alkaline pH on cathepsin B activity and structure was investigated. An irreversible loss of activity, accompanied by large structural changes, was observed at pH > or = 7.0. The high activation energy of 183.5 kJ mol-1, calculated for the inactivation process, is in good agreement with structural changes observed by circular dichroism. Both the pH-induced inactivation and the pH-induced unfolding of cathepsin B were found to be first-order processes, exponentially increasing with increasing pH of the solution. The good agreement of the rate constants of inactivation and unfolding of the enzyme indicates an important structure-function relationship. Cathepsin B was also found to be destabilized both by increasing ionic strength and organic solvent content.
Binding Sites↗
Identification of the neuronal acceptor in bovine cortex for ammodytoxin C, a presynaptically neurotoxic phospholipase A2.
A specific, high-affinity binding site for ammodytoxin C in synaptic membranes from bovine cerebral cortex was detected and partially characterized. Equilibrium binding analysis revealed a single population of [125I]ammodytoxin C acceptors with the following binding parameters: Kd = 6.0 nM and Bmax = 5.7 pmol/mg membrane protein. Such binding was strongly inhibited by three ammodytoxins (A, B, and C) and by crotoxin B. Vipera berus berus phospholipase A2 was a weaker inhibitor; nontoxic phospholipase A2, ammodytin I2, and the myotoxic phospholipase A2 homologue, ammodytin L, both from Vipera ammodytes ammodytes venom, inhibited binding only at very high concentrations, whereas alpha-dendrotoxin, beta-bungarotoxin, and crotoxin A had no influence on the [125I]ammodytoxin C-specific binding. The ammodytoxin C neuronal binding site therefore overlaps, at least partially, with the neuronal acceptors for some of the related presynaptically neurotoxic phospholipases A2 (beta-neurotoxins). [125I]-Ammodytoxin C was covalently attached to its acceptor by chemical cross-linking. Subsequent SDS-PAGE analysis followed by autoradiography revealed saturably labeled membrane components with apparent M(r) values of 51,000 (weaker band) and 53,000-56,000 (stronger band). Pretreatment of synaptic membranes with Staphylococcus aureus V-8 proteinase and proteinase K, heat, or low pH decreased the [125I]ammodytoxin C-specific binding to various extents, but never abolished it completely. Membrane protein and certain phospholipids residing in its vicinity are therefore most likely involved in the binding of ammodytoxin C to bovine synaptic membranes.
Animals↗
Primary and secondary structure of a pore-forming toxin from the sea anemone, Actinia equina L., and its association with lipid vesicles.
The complete amino acid sequence of equinatoxin II, a potent pore-forming toxin with hemolytic, cytotoxic and cardiotoxic activity from the venom of the sea anemone, Actinia equina L., is reported. In addition, circular dicroism was used to estimate the secondary structure of this toxin either in the water-soluble or in the membrane-anchored form. Equinatoxin II when in water was found to contain about 29-33% of alpha-helical structure, 53-58% of beta-strand+beta-turn and 10-16% of random structure. Upon association with phospholipids, in particular with sphingomyelin, a rearrangement of the secondary structure occurs resulting in an increase of the alpha-helix content. An amphiphilic alpha-helical segment is predicted at the N-terminus, which shares structural homology with membrane active peptides like melittin and viral fusion peptides. In analogy to the behaviour of these peptides we propose that at least part of the alpha-helix content increase of equinatoxin II is due to the insertion of its N-terminus into the lipid bilayer. As in the case of melittin, association of 3-4 equinatoxin molecules is necessary to induce membrane permeabilisation.
Amino Acid Sequence↗
Presynaptically acting phospholipase A2 from Vipera ammodytes venom.
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Amino Acid Sequence↗
Antipeptide antibodies directed to the C-terminal part of ammodytoxin A react with the PLA2 subunit of crotoxin and neutralize its pharmacological activity.
Crotoxin and ammodytoxin A are snake venom neurotoxic phospholipases A2. Polyclonal antibodies against three synthetic peptides selected from the C-terminal part of the primary structure of ammodytoxin A were tested by ELISA for their interaction with crotoxin and its subunits, CA and CB. All three antipeptide antibodies reacted specifically with corresponding parts of ammodytoxin A and CB, either native or reduced. Conversely, polyclonal antibodies produced against ammodytoxin A and CB reacted strongly with all three peptides, suggesting that they constitute at least a part of natural epitopes in both proteins. All antipeptide antibodies reacted also with the corresponding peptides derived from CB by cyanogen bromide cleavage. The biological activity of the immune complexes was tested. No significant change in the enzymatic activity of CB, ammodytoxin A or crotoxin was observed with any of the three antipeptide antibodies. These antibodies were, however, able to protect mice against the lethal potency of CB and to prolong survival time of mice injected with crotoxin. These antipeptide antibodies were assayed in vitro for their protective effect against the action of CB or crotoxin on synaptosomes from Torpedo marmorata electric organ. They partly inhibited the acetylcholine release induced by both proteins. These results indicate that the C-terminal part of CB is likely to be involved in the pharmacological action of crotoxin.
Amino Acid Sequence↗
Molecular cloning of a putative homolog of proline/arginine-rich antibacterial peptides from porcine bone marrow.
Screening of a porcine bone marrow cDNA library with a PCR-derived probe from rabbit LPS-binding protein CAP18 led to the discovery of two closely related clones. The longer, full-length cDNA clone encodes a 228 amino acid residue protein similar to the family of antibacterial/LPS-binding cationic peptides. In contrast to other hitherto discovered precursors of Pro/Arg-rich peptides from this family, they have a novel, unique structure of the C-terminal region of 100 amino acid residues with a repeating sequence of ten residues (FPPPNXPGPR, where X = V or F). These precursors could represent a part of the antibacterial peptide repertoire of porcine bone marrow.
Amino Acid Sequence↗
Expression of fully active ammodytoxin A, a potent presynaptically neurotoxic phospholipase A2, in Escherichia coli.
A cDNA encoding the most presynaptically neurotoxic phospholipase A2, ammodytoxin A, from the venom of the long-nosed viper (Vipera ammodytes ammodytes) has been expressed in Escherichia coli. Ammodytoxin A was produced as a fusion protein with the 81 N-terminal residues of adenylate kinase followed by the tetrapeptide recognition site for factor Xa (IEGR) just preceding the first amino acid residue of the toxin. The fusion protein was expressed under the control of tac promoter without IPTG induction in the form of insoluble inclusion bodies. It was dissolved in guanidine hydrochloride, S-sulfonated and refolded in a reoxidation mixture including a reduced/oxidized glutathione redox couple. Ammodytoxin A was fully activated by limited hydrolysis with trypsin that preferentially cleaves the fusion protein at the factor Xa recognition site and purified by cation-exchange chromatography. The correct N-terminus was confirmed by protein sequencing. Recombinant ammodytoxin A has been proved to be indistinguishable from the native toxin in its enzymatic activity and toxicity.
Animals↗
Monoclonal antibodies to human stefin B and determination of their epitopes.
Monoclonal antibodies (MAbs) to human stefin B were developed and three of them were characterised. Stefin B was cleaved into four peptides which were later subfragmented to smaller peptides. Only two peptides, of 24 and 30 amino acids, could bind to MAbs. In one instance, two peptides that were not consecutive in the sequence were recognised by the same antibody, proving that the epitope was discontinuous. Location of the epitopes was further narrowed by measuring the binding of MAbs to the complex of stefin B with papain. A sandwich ELISA (enzyme-linked immunosorbent assay), which measures the concentration of free inhibitor, was developed. It confirms that two out of three MAbs bind to different sites of stefin B. On the basis of the crystal structure of complex of stefin B with papain, the surface, accessible to a sphere with a radius of 5 A which simulates the accessibility of variable regions of antibody was determined. From the difference between accessibilities of free stefin B and stefin B in complex, the epitope location was determined more accurately.
Amino Acid Sequence↗
Isolation, partial characterization and complete amino acid sequence of the toxic phospholipase A2 from the venom of the common viper, Vipera berus berus.
A basic, toxic phospholipase A2 was purified from the venom of Vipera berus berus (Vbb) by a single purification step, using hydrophobic chromatography. The primary structure of isolated protein was established from peptides generated by Gly-specific papaya proteinase IV, beta-trypsin, CNBr and mild acid hydrolysis. The enzyme consists of a single chain of 122 amino acid residues with 14 Cys in positions characteristic for the phospholipase A2 subgroup IIA. As far as we know, this is the first complete Vipera berus phospholipase A2 amino acid sequence reported.
Amino Acid Sequence↗
Improved purification of steroid 1:2-dehydrogenase from Nocardia opaca and partial characterization of its cloned gene sequence.
We have purified a steroid-inducible 1:2-dehydrogenase from Nocardia opaca. The final enzyme preparation was purified 120-fold with a recovery of 38%. The N-terminal amino acid sequence was determined to be: Met-Gln-Asp-Trp-Thr-Ser-Glu-(Cys)-Asp-Val-Leu-Val-Val-Gly-. From the genomic library of Nocardia opaca in the plasmid pUC19, a clone designated as pSTD23 containing a 0.9 kb KpnI-PstI fragment was found to hybridize with an oligonucleotide probe corresponding to the first six amino acids from the N-terminal of the purified protein. The nucleotide sequence of the upstream region and a part of the structural domain were determined. The sequence of the first 56 amino acids of the steroid 1:2-dehydrogenase from Nocardia opaca as deduced from its gene sequence showed a 58% homology with the corresponding gene from Pseudomonas testosteroni, and the conservative sequences in the FAD-binding domain were also determined.
Amino Acid Sequence↗
Snake-venom phospholipase A2 neurotoxins. Potentiation of a single-chain neurotoxin by the chaperon subunit of a two-component neurotoxin.
The venoms from Crotalinae and Viperinae snakes contain only two kinds of phospholipase A2 neurotoxins (beta-neurotoxins): single-chain beta-neurotoxins, such as agkistrodotoxin and ammodytoxin-A, and dimeric beta-neurotoxins, which, in the case of the best studied ones, crotoxin-like toxins, consist of the non-covalent association of a phospholipase A2 (CB) and a non-enzymatic chaperon (CA). Possible evolutionary relationships of these beta-neurotoxins have been investigated by analyzing whether CA could behave as a chaperon toward agkistrodotoxin and ammodytoxin, as it does in the crotoxin complex. CA increased the lethal potency of agkistrodotoxin and modified its pharmacological effect on Torpedo synaptosomes. Sedimentation experiments proved that CA can form an heterocomplex with agkistrodotoxin. Agkistrodotoxin prevented the binding to CA of an anti-CA mAb which recognizes an epitope at the zone of interaction between crotoxin subunits, suggesting the association of CA and agkistrodotoxin implicated the same zone. A 10-fold molar excess of CA over ammodytoxin modified the effect of ammodytoxin on acetylcholine release but did not increase the lethal potency of ammodytoxin. Sedimentation experiments showed CA and ammodytoxin can form an heterocomplex which is less stable than CA.agkistrodotoxin. Ammodytoxin A did not compete with the anti-CA mAb. These observations are in good agreement with the sequence similarities between CB and agkistrodotoxin (80%) and ammodytoxin A (60%).
Animals↗
A thrombin-like enzyme from bushmaster (Lachesis muta stenophyrs) venom.
The clotting enzyme (Stenoxobin), from the venom of Lachesis muta stenophyrs, was purified by gel chromatography on Bio-gel P-100 followed by agmatine CH-Sepharose-4B and FPLC on Mono Q column. By SDS polyacrylamide gel electrophoresis the mol. wt was found to be 37,000. The enzyme is a glycoprotein with 1.6 moles of sialic acid per mole of protein and has an average content of 7.0% of neutral carbohydrates. The clotting and esterolytic (BAEE) activities were 843 NIH units/mg and 60.1 +/- 1.2 OD225 ml/min/mg, respectively, and could not be inhibited by heparin or hirudin. Amino acid analysis revealed a low content of tryptophan and a high content of acid residues. Stenoxobin acts upon human fibrinogen by releasing consecutively fibrinopeptides A and B from the alpha- and beta-chains of fibrinogen.
Animals↗
Calcium ion independent membrane leakage induced by phospholipase-like myotoxins.
The two snake venom myotoxins ammodytin L and myotoxin II, purified respectively from Vipera ammodytes ammodytes and Bothrops asper, have phospholipase-like structures but lack an Asp-49 in the active site and are without normal phospholipase activity. The interaction of these proteins with different types of liposomes indicated that the myotoxins were able to provoke rapid and extensive release of the aqueous content of liposomes. Leakage was measured by two different methods: fluorescence dequenching of liposome-entrapped carboxyfluorescein and ESR measurement of intravesicular TEM-POcholine reduction by external ascorbate. The process was independent of Ca2+ and took place without any detectable phospholipid hydrolysis. Nonmyotoxic phospholipases tested under the same conditions were unable to induce liposome leakage, which could be detected only when Ca2+ was added to the medium and with the concomitant hydrolysis of phospholipids. The kinetics of Ca(2+)-dependent and Ca(2+)-independent leakage were completely different, indicating two different mechanisms of interaction with the lipid bilayer. Studies using diphenylhexatriene as a probe of lipid membrane organization indicated that the myotoxins gave rise to a profound perturbation of the arrangement of the lipid chains in the membrane interior, whereas interaction of Naja naja phospholipase A2 with the membrane surface did not affect lipid organization. On the basis of these results we suggest that a new type of cytolytic reaction mechanism is responsible for the effects of phospholipase-like myotoxins in vivo.
Calcium↗
Amino acid and cDNA sequences of a neutral phospholipase A2 from the long-nosed viper (Vipera ammodytes ammodytes) venom.
The amino acid sequence of a non-toxic phospholipase A2, ammodytin I2, from the venom of the long-nosed viper (Vipera ammodytes ammodytes) and its cDNA sequence have been determined. The protein sequence was elucidated by sequencing the peptides generated by CNBr cleavage, mild acid hydrolysis and tryptic digestion of maleylated and non-maleylated protein. Sequencing of the cDNA showed that the protein is synthesized as an 137-amino-acid-residue precursor molecule consisting of a 16-residue signal peptide, followed by a 121-residue mature enzyme. Ammodytin I2 cDNA shows 73% nucleotide and 59% amino acid identities in the mature protein region in comparison to that of ammodytoxin A, the most presynaptically neurotoxic phospholipase A2 from the long-nosed viper. Identities in the signal-peptide region are considerably higher, 96% and 100%, respectively.
Amino Acid Sequence↗
The role of tryptophan in structural and functional properties of equinatoxin II.
A pore-forming, cytolytic and lethal polypeptide, equinatoxin II, from the sea anemone Actinia equina, was subjected to oxidation with N-bromosuccinimide to study the role of five present tryptophan residues in structure-function relationships. In the folded toxin molecule, 1-2 tryptophan residues were readily susceptible to oxidation with N-bromosuccinimide, whereas modification of a single residue resulted in complete impairment of the toxin lethal and hemolytic activities as well as the ability of an oxidized toxin to precipitate with serum lipoproteins. CD and fluorescence spectra indicated a slight alteration of a toxin secondary structure following N-bromosuccinimide treatment. Incubation with sphingomyelin of the toxin prior to oxidation did not prevent subsequent modification with N-bromosuccinimide and loss of its activities, indicating that the modified tryptophan residue is not directly involved in toxin binding and insertion into lipid membranes. It was concluded that the modified tryptophan residue is essential for the structure of equinatoxin II.
Animals↗
Isolation and sequence analysis of the genomic DNA fragment encoding an aspartic proteinase inhibitor homologue from potato (Solanum tuberosum L.).
A genomic DNA clone encoding an aspartic proteinase inhibitor of potato was isolated from a lambda EMBL3 phage library using the aspartic proteinase inhibitor cDNA as a hybridization probe. The gene has all characteristic sequences normally found in eucaryotic genes. Typical CAAT and TATA box sequences were found in the 5'-upstream region. In this part are also two putative regulatory AGGA box sequences located. In the genomic sequence there are no intron sequences interrupting the coding region. An open reading frame of the gene encodes a precursor protein of 217 amino acids which shows high percent identity with the aspartic proteinase inhibitor cDNA.
Amino Acid Sequence↗
Characterization of aspartic proteinase inhibitors from potato at the gene, cDNA and protein levels.
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Amino Acid Sequence↗