Biomedical subjects

H Rochat

Publications and source records attributed to H Rochat.

At least 181 records · Page 10Linked to original sources

PubMed1985

Characterization of ten proteins from the venom of the Moroccan scorpion Androctonus mauretanicus mauretanicus, six of which are toxic to the mouse.

When the venom of the scorpion Androctonus mauretanicus mauretanicus was submitted to purification procedures, ten proteins were obtained; six were lethal to mice and four were devoid of toxicity in the biological tests used. The ten molecules were characterized by their amino acid composition, and among them toxin V and polypeptide P2 by their amino acid sequences. Peptide P2 (35 amino acid residues), a structural homologue of the so called Buthus epeus short 'insectotoxins' I1 and I5, was inactive on fly larvae and absent from the 'manual venom' obtained by manual handling and excitation of the scorpions.

Amino Acid Sequence↗

PubMed1984

Amino acid sequence of toxin VII, a beta-toxin from the venom of the scorpion Tityus serrulatus.

The sequence of the 61 amino acids of toxin VII, a beta-toxin from the venom of the South American scorpion Tityus serrulatus, has been determined by automatic sequencing of the reduced and S-[14C] carboxymethylated protein and of tryptic peptides obtained before or after citraconylation of this protein. This toxin, the most active beta-toxin from this venom, is the first Tityus toxin to be fully sequenced. The results clearly show that toxin VII belongs to the structural group of scorpion toxins originating from Central and North America.

Amino Acid Sequence↗

PubMed1984

Classification of scorpion toxins according to amino acid composition and sequence.

Scorpion venom toxins were systematically classified according to amino acid composition, insertion/deletion events and sequence. The significance of each comparison method and its outcome is discussed in relation to known immunological and structural properties. A general classification of the toxins is proposed that accounts for both the immunological groupings and the differences in mode of action.

Amino Acid Sequence↗

PubMed1984

Purification of thirteen toxins active on mice from the venom of the North African scorpion Buthus occitanus tunetanus.

A combination of several equilibrium chromatographic steps permitted us to purify thirteen proteins from the venom of Buthus occitanus tunetanus using the mouse as the test animal for lethality measurement. These proteins have been characterized by their amino acid composition and, for seven of them, by their N-terminal or complete amino acid sequence. The structural data obtained correlate well with the pharmacological and antigenic properties of the neurotoxins.

Amino Acid Sequence↗

PubMed1984

Purification and amino acid sequence of toxin I" from the venom of the North African scorpion Androctonus australis Hector.

When a large quantity of the venom of the scorpion Androctonus australis Hector is submitted to a purification procedure it is possible to purify a new toxin, toxin I", which is lethal to mouse, and present in very low quantities. The yield is 0.04%. The amino acid sequence of this newly discovered toxin only differs from toxin I of the same scorpion by an extra arginine residue at the C-terminal end provided that the sequence of toxin I, at positions 34 and 35, is in agreement with newer information provided in this paper.

Amino Acid Sequence↗

PubMed1984

Immunochemistry of scorpion alpha-toxins: purification and characterization of two functionally independent IgG populations raised against toxin II of Androctonus australis Hector.

We report the isolation and characterization of two IgG populations specific to two synthetic peptides corresponding to two antigenic sites of toxin II of the North African scorpion Androctonus australis Hector. Firstly, thanks to the use of: (1) antigenic homology studies between toxin II of A. australis Hector and toxin III of Buthus occitanus tunetanus, (2) chemical modification of toxin II of A. australis Hector, and (3) prediction of the localization of the four major antigenic sites of scorpion alpha-toxins by the method developed by Hopp and Woods [Proc. natn. Acad. Sci. U.S.A. 78, 3824-3828 (1981)], we have established that the region around the disulfide bridge between cysteines 12 and 63 as well as the stretch of residues 50-59 probably each enclosed an antigenic site. Secondly, the synthetic replicates of these regions linked to Sepharose allowed us to isolate, by immunoaffinity chromatography, two IgG populations from the whole anti-toxin II of A. australis Hector IgGs. Finally, each of these two IgG populations was shown to be specific to one antigenic site as evidenced by the multideterminant effect on the slopes of binding curves developed by Berzofsky et al. [Biochemistry 15, 2113-2121 (1976)]. Furthermore, these two IgG populations were found to be functionally independent and this could be related to the fact that the two regions carrying the two antigenic sites are not close to each other in space and that there is neither steric hindrance nor cooperative effects between them. The association constant of these site-specific IgG populations was calculated and found to be equal to 1.18-5.14 X 10(9) l/mole for IgG anti-site 1 and 1.16-5.62 X 10(9) l/mole for IgG anti-site 2 respectively by Sips [J. chem. Phys. 16, 490-495 (1948)], Scatchard [Am. N.Y. Acad. Sci. 51, 660-772 (1949)] and Steward and Petty [Immunology 23, 881-887 (1972)] representations. The index of heterogeneity of 0.9 for anti-P1 and anti-P2 indicates the purification of essentially homogeneous affinity IgG populations.

Amino Acid Sequence↗

PubMed1984

Synthesis and immunological characterization of two peptides which are models for two of the four major antigenic sites of a scorpion toxin.

We have synthesized, by the solid phase procedure, then purified and chemically characterized two peptides. They mimic two regions of toxin II of the scorpion Androctonus australis Hector, one around disulfide bridge 12-63 and another at sequence 50-59. Each of these two regions was supposed to include an antigenic site. We have shown that the synthetic replicas of these regions are individually recognized by a part of IgGs raised against native toxin II. This is a strong argument for the involvement of these areas in the antigenicity of the toxin. Furthermore, region-specific IgGs purified by affinity chromatography on the two Sepharose linked peptides were able to bind 125I-labelled toxin II.

Amino Acid Sequence↗

PubMed1984

Interaction of scorpion toxins with the sodium channel.

Scorpion toxins are miniproteins that have been characterized with regard to their molecular properties including their pharmacological action. On these bases, toxins specific to mammals or to insects have been described and within the first category alpha- and beta-toxins identified. These toxins were chemically modified and used as molecular probes of the sodium channel. Thus, the interaction of the toxins with the pharmacological target could be followed and a better definition of this target could be obtained.

Animals↗

PubMed1983

Photoaffinity labeling of alpha- and beta- scorpion toxin receptors associated with rat brain sodium channel.

Azido nitrophenylaminoacetyl [125I]iodo derivative of toxin II from Centruroides suffusus suffusus, a beta-toxin, and azido nitrophenylaminoacetyl [125I]iodo derivative of toxin V from Leiurus quinquestriatus quinquestriatus, an alpha-toxin, have been covalently linked after binding to their receptor sites that are related to the voltage sensitive sodium channel present in rat brain synaptosomes. Both derivatives labeled two polypeptides of 253000 +/- 20000 and 35000 +/- 2000 mol. wt. Labeling was blocked for each derivative by a large excess of the corresponding native toxin but no cross inhibition was obtained. These results suggest that both alpha - and beta - scorpion toxin receptors are located on or near the same two membrane polypeptides which may be part of the voltage dependent sodium channel.

Affinity Labels↗

PubMed1983

Isolation and characterization of colipase from porcine and human pancreatic juice by immunoaffinity chromatography.

Pure colipase was prepared by immunoaffinity chromatography from porcine and human pancreatic juice. A single form of the porcine colipase was obtained, having the structural and biological properties of previously characterized porcine procolipase A. Two forms of activated colipase (N-terminal Gly) were isolated from human pancreatic juice by the same procedure. The existence of two forms of activated colipase might arise from rapid activation of a precursor form of human colipase during collection of the pancreatic juice.

Animals↗

PubMed1983

Are interactions with phospholipids responsible for pharmacological activities of cardiotoxins?

Cardiotoxins are small basic proteins (7 000 daltons) that are found in the venoms of Elapidae snakes. Although they are structurally close to alpha-neurotoxins present in the same secretions, their activity is related to their ability to interact with every cell membrane inducing, near micromolar concentration, the modification of its biological properties and/or physical structure. The mode of action of cardiotoxins, on a molecular level, is still under investigation. However, lipid-protein interactions are more and more involved in their binding to membrane and in their activities. Using new experimental data a better definition of phospholipid-cardiotoxin interaction is arrived at and a tentative molecular explanation of the pharmacological activities of these proteins is presented and discussed.

Amino Acid Sequence↗

PubMed1983

Covalent structure of toxins I and II from the scorpion Buthus occitanus tunetanus.

The amino acid sequences of neurotoxins I and II, which are active on mammals, purified from the venom of Buthus occitanus tunetanus have been determined using standard methods, including mainly automatic phenylisothiocyanate degradation of S-carboxymethylated derivatives of the two proteins and peptides derived by enzymatic hydrolyses. Both toxins are made of sixty-five amino acid residues cross-linked with four disulfide bridges. For toxin II, the complete covalent structure, including the positions of the four disulfide bridges was determined: the positions are similar to those previously found in toxin II of another scorpion from Africa, Androctonus australis Hector. This finding is in favor of a similar structure for all of the scorpion neurotoxins active on mammals.

Amino Acid Sequence↗

PubMed1983

Immunochemistry of scorpion alpha-toxins: antigenic homologies checked with radioimmunoassays (RIA).

Three toxins, i.e. toxins II and III of Androctonus australis Hector and toxin I of Buthus occitanus tunetanus, were labeled with 125I. High specific radioactivities were obtained (490-1100 Ci/mmole) that allowed the setting up of three radioimmunoassays. We were able to quantify the amount of toxin in 0.1 ml of 10(-9)-10(-10) M solutions and to test antigenic homologies between toxins belonging to different structural groups. Three possibilities exist: (1) no cross-reactivity when sequence difference is greater than 25%; (2) full cross-reactivity when this difference is lower than 25%; (3) partial cross-reactivity, which is interpreted as a loss of some common antigenic sites.

Amino Acid Sequence↗

PubMed1983

Immunochemistry of scorpion alpha-neurotoxins. Determination of the antigenic site number and isolation of a highly enriched antibody specific to a single antigenic site of toxin II of Androctonus australis Hector.

Scorpion neurotoxins active on mammals, i.e. alpha- and beta-toxins, have been divided into several groups according to structural and also immunological criteria. A study of the alpha-toxins has been performed in order to determine the number of antigenic sites; a minimum of four Fab fragments were found to bind simultaneously to toxins I and II of Androctonus australis Hector, and toxin I of Buthus occitanus tunetanus. Taking advantage of the loss of one common antigenic site between toxin II of Androctonus australis Hector and toxin III of Buthus occitanus tunetanus, a highly purified antibody population towards a single site of toxin II of Androctonus australis Hector was isolated and characterized. This result is discussed in terms of sequence homologies between the two proteins as the amino acid sequence of toxin III of Buthus occitanus tunetanus has been determined using standard procedures: the two proteins differ at three positions, two of which (positions 10 and 64) are in the vicinity of the disulfide bridge Cys 12-Cys 63, the third (position 51) is the only one to be conservative.

Amino Acid Sequence↗

PubMed1983

Alpha-scorpion neurotoxin derivatives suitable as potential markers of sodium channels. Preparation and characterization.

Modified scorpion neurotoxins, i.e. mono-biotinylated and mono-azido derivatives either on lysine 58 or lysine 60 have been characterized both at the structural level (sequence and circular dichroism) and by their pharmacological activity (toxicity to mice and ability to displace 125I-Androctonus australis Hector toxin II from its receptor sites. The results allowed us to pinpoint a region of the molecule including lysine residues 58 and 60 that is important for neurotoxin receptor interaction. Furthermore, as these derivatives retain, after 125I labeling, high binding capacities to synaptosomal membranes, they can be used as potential labels of the sodium channel either by covalent binding or using the avidin-biotin system.

Animals↗

PubMed1982

Two types of scorpion receptor sites, one related to the activation, the other to the inactivation of the action potential sodium channel.

The action of the neurotoxin in Buthinae scorpion venoms (Androctonus, Buthus or Leiurus genera) has been extensively studied. These proteins induce a prolongation of the action potential of nerves and muscles by slowing down inactivation of the sodium channel. Their affinity for their receptor site depends on membrane potential. In the present report we describe a toxin from a Centrurinae scorpion, Centruroides suffusus, which binds rat brain synaptosomes at a receptor site distinct from the Buthinae scorpion site independently of voltage. We name Androctonus-like toxins, alpha-scorpion toxins (alpha-ScTX), and Centruroides-like toxins, beta-scorpion toxins (beta-ScTX). We further report that beta-ScTX induces repetitive firing in frog myelinated nerve fibres by producing an abnormal sodium permeability. The beta-toxin binds specifically to rat brain synaptosomes (Kd = 3 nM) and induces an inhibition of the uptake and a stimulation of the release of GABA at concentrations which are in good agreement with the Kd value. These effects are blocked by tetrodotoxin. The binding site of beta -ScTX is distinct from those of other neurotoxins acting on the sodium channel like tetrodotoxin, alpha-ScTX and veratridine. The alpha-ScTX/beta-ScTX binding site capacities decreases as development of rat brain synaptosomes progresses ; at day 7 after birth, it is 1.1. and at day 39, 0.3.

Action Potentials↗

PubMed1982

Structure-function relationships for cardiotoxins interacting with phospholipids.

Four cardiotoxins (CTX I-IV) from Naja mossambica mossambica were compared for their ability to interact with phospholipid vesicles and their capacity to bind erythrocytes. It is concluded that the affinity of the toxins always increases in the order: I approximately equal to II less than III less than IV. The binding is specific for charged lipids even in lipid mixtures. Proteolytic attack of the free and lipid-bound cardiotoxin indicates that at least the first loop Leu1-Thr13 is at the lipid contact. Tryptic and synthetic peptides constitutive of this loop are shown to interact with lipids. Arg5 residue increases the affinity toward the bilayer. The Raman spectra of lipid-bound cardiotoxin indicate a secondary and tertiary structure mainly similar to that of the free toxin. On charged lipids cardiotoxins induce a decrease of the enthalpy and an increase of disorder without change in the transition temperature; at saturating amounts of toxin the transition is abolished. In binary mixtures of phosphatidylcholine and charged lipids the observed effects can be accounted by a phase separation induced by the toxin.

Amino Acid Sequence↗

PubMed1982

A possible orientation change of cardiotoxin molecule during its interaction with phospholipid monolayer.

The monomolecular film technique was used in order to study the specific interactions of 4 cardiotoxins from Elapidae snake venom Naja mossambica mosambica with different phospholipids. The interaction, at pH 7.5, of cardiotoxin (10(-7) M) with both neutral and negatively charged phospholipids occurs up to a very high critical surface pressure (pi = 45 dynes/cm with the latest). The apparent molecular area of cardiotoxin molecule, during its insertion into a negatively charged phospholipid film, presents only two characteristic values: 1400 A2 for pi less than 20 dynes/cm and 420 A2 for pi greater than 30 dynes/cm, the transition occurring in a very narrow range of surface pressure (25 +/- 5 dynes/cm). Thus, according to the surface pressure, the cardiotoxin may present two different orientations: "flat" or "edgewise" and the transition between both could account for lytic activity of cardiotoxin.

Cobra Cardiotoxin Proteins↗