Selective solubilization by melittin of glycophorin A and acetylcholinesterase from human erythrocyte ghosts.

Melittin, the main basic and hydrophobic peptide of bee venom, has been used for solubilizing membrane components of the human erythrocyte ghost. Up to 1.0 mM, it does not extract any phospholipid. Between 0.1 and 1.0 mM, it solubilizes partially glycophorin A and acetylcholinesterase. When the membrane is first degraded by phospholipase A2, the solubilization of both proteins by melittin is total, and 48% of the phospholipids are removed, mainly as lysoproducts, whereas phospholipase A2, by itself, has no solubilizing properties. In its melittin-solubilized state, acetylcholinesterase is in a dimeric form and displays a slow time-dependent irreversible inactivation. Triton X-100 at 1.0% (v/v) interrupts the inactivation. We suggest that melittin binds to the hydrophobic site of acetylcholinesterase which anchors it in the lipid bilayer.

Receptor-like activity of a monoclonal anti-idiotypic antibody against an anti-acetylcholine receptor antibody.

A monoclonal anti-idiotypic antibody against an anti-acetylcholine receptor antibody from a patient with myasthenia gravis was shown to bind the cholinergic ligand alpha-bungarotoxin. This binding could be inhibited by other cholinergic ligands, both antagonists and agonists. The anti-idiotype was also able to elicit the production of anti-receptor antibodies in mice. Thus, the anti-idiotype had functional properties similar to those of the original antigen, the acetylcholine receptor.

Lack of cross-reactivity of a monoclonal antibody against the neurotransmitter site of Torpedo nicotinic acetylcholine receptor with muscle receptors from several sources.

A monoclonal antibody raised against Torpedo nicotinic acetylcholine receptor has been used to study the neurotransmitter binding site of the acetylcholine receptor from several sources. When tested on Torpedo receptor, this monoclonal antibody inhibited binding of alpha-bungarotoxin to 50% of the available sites. The failure to completely inhibit binding of the toxin is attributed to the orientation of the determinant for the monoclonal antibody on the receptor molecule. This monoclonal antibody bound very poorly to the acetylcholine receptor from muscle tissues. This suggests either a modification or a reduced accessibility of the determinant to the monoclonal antibody.

Encephalopathy, peripheral neuropathy, dysautonomia, myasthenia gravis, malignant thymoma, and antiacetylcholine receptor antibodies in the CSF.

A 54-year-old man suffered from multiple neurologic disturbances (polyneuropathy, encephalopathy, dysautonomia) associated with myasthenia gravis and malignant thymoma. No morphological signs of inflammation were present in the brain and peripheral nerves. Antiacetylcholine receptor antibodies were present in the brain and peripheral nerves. Antiacetylcholine receptor antibodies were present in serum and in cerebrospinal fluid. The association of thymoma, myasthenia gravis, multiple neurologic syndromes, and antiacetylcholine receptor antibodies in serum and cerebrospinal fluid has not been reported as yet. We suggest that this clinical picture is related to a generalized cholinergic dysfunction.

Reconstitution of pure acetylcholine receptor in phospholipid vesicles and comparison with receptor-rich membranes by the use of a potentiometric dye.

Acetylcholine receptor, isolated in Triton X-100 on a cobra alpha-neurotoxin affinity column was incorporated into unilamellar phospholipid vesicles by a detergent depletion method using Amberlite XAD-2. Vesicles of an average diameter of 25 nm were formed, as verified by freeze-fracture electron microscopy and gel filtration. 85 to 95% of the alpha-bungarotoxin binding sites of the reconstituted acetylcholine receptor were oriented towards the outside of the vesicles. In the reconstituted receptor one molecule of residual Triton X-100 per 2.5 alpha-bungarotoxin binding sites on the receptor molecule could be assessed. The reconstituted protein was not accessible to papain digestion, whereas the pure acetylcholine receptor, solubilized by Triton X-100 was split into smaller polypeptides under the same condition. Reconstituted acetylcholine receptor and receptor-rich membranes did not exhibit the same behavior as measured by use of a potentiometric dye. This is interpreted as an irreversible alteration of at least 95% of the receptors purified in the presence of Triton X-100. Furthermore, it could be shown that the fluorescence intensity changes induced by carbamylcholine in receptor-rich membranes did not reflect ion fluxes, but conformational changes of the protein or a displacement of the dye from the protein.

Evidence for sialic acid on the nicotinic acetylcholine receptor from Torpedo marmorata.

The nicotinic acetylcholine receptor from Torpedo marmorata was treated with neuraminidase. Direct determination of sialic acid released gave about 1 mole sialic acid per mole receptor. Lectin binding studies of the sugars accessible on the receptor molecule were performed after sialic acid hydrolysis. They indicated that the terminal sialic acid is linked to a galactose residue. The present findings confirm the presence of one terminal sialic acid residue per receptor molecule.

Specific binding to isolated acetylcholine receptor of a synthetic peptide duplicating the sequence of the presumed active center of a lethal toxin from snake venom.

To verify the existence of a lethal "active center" in snake venom neurotoxins and to assess its delineation within their polypeptide sequences, a tritriacontapeptide matching residues 16-48 of the natural "major" toxin of Naja naja philippinensis (Hauert, J., Maire, M., Sussmann, A., and Bargetzi, J. P. (1974) Int. J. Pept. Protein Research 6, 201-222) has been synthesized by solid-phase technology (Juillerat, M. A., and Bargetzi, J. P. (1980), results presented at the 16th European Peptide Symposium, Helsingor, Denmark, September, (1980). After deblocking, cyclization by reoxidation, and purification, one of the resulting peptides exhibiting the correct chemical and physical characteristics was found to be highly "active" in binding isolated, purified, and standardized acetylcholine receptor protein. A new assay procedure had been developed using 3H-labeled alpha-bungarotoxin as nonreversible back-titrant. It has the advantage of measuring only specific binding of an unknown ligand competing for the same receptor protein. The observed KD was 2.2 x 10(-7) M, a value attesting to a higher affinity than acetylcholine itself, 2.5 x 10(-6) M, as well as curare and the small organic cholinergic ligands, albeit somewhat lower than the affinity of the parent native toxin, as expected from differences in molecular size.

Inactivation and solubilization of opiate receptors by phospholipases A2.

(1) As previously shown, stereospecific binding of opiates to membrane bound receptors is inhibited by treatment with small amounts of phospholipase A2 from Vipera russelli. This effect is quantified and compared with the enzymes from the venoms of Naja Naja siamensis, Apis Mellifica and from porcine pancreas. All enzymes are equally effective. The inhibition is due to partial phospholipid hydrolysis leading to inactivation of membrane-bound receptor. (2) Bee venom phospholipase A2 together with the synergistically acting peptide, melittin, causes receptor solubilization up to 80% of preformed receptor-ligand complex can be solubilized in this manner. (3) Lysophosphatidylcholine, a product of phospholipid hydrolysis, solubilizes performed receptor-ligand complex to a similar extent. Several other detergents were tested for their ability to solubilize receptor-ligand complex. Digitonin appears to be most effective in solubilizing such a complex.

Monoclonal anti-torpedo receptor antibodies used to study antibody heterogeneity in myasthenic sera.

Monoclonal antibodies against Torpedo acetylcholine receptor were used to define the binding regions of cross-reacting, anti-receptor antibodies from sera of myasthenic patients. Cross-reacting antibodies were directed mainly against the toxin-binding region of the receptor and a region remote from the acetylcholine-binding site. Few patients had antibodies against the acetylcholine-binding site region. The monoclonal antibodies provide probes for defining the heterogeneity of anti-receptor antibodies in myasthenic sera.

Binding properties and subclass distribution of anti-acetylcholine receptor antibodies in myasthenia gravis.

Acetylcholine receptor antibodies were studied in the serum of 21 myasthenic patients. In 18 cases antibodies directed against sites other than the toxin binding were present whereas in 10 cases only there was a measurable inhibition of the ligand binding site. These 10 sera were from the 6 patients in stage IIB, III and IV and from 4 of the 12 patients in stage IIA. Antibodies against both non-toxin and ligand binding sites were measured in IgG subclasses. Most of the antibodies of the first type belonged to either subclass 1 or 3. They were, however, never absent from subclasses 2 and 4. Antibodies of the second type were not found in subclasses 2 and 4 except in one case. In 3 cases they were present exclusively in subclass 3. In 3 patients there was no correlation between the subclass distribution of the antibodies for the different binding sites.

Characterization of iodinated derivatives of alpha-bungarotoxin.

The iodination of alpha-bungarotoxin and the reactivity of iodinated derivatives towards nicotinic acetylcholine receptor are described. 125I2- and 125I-alpha-bungarotoxin can be resolved, but the latter was not separated from unreacted alpha-bungarotoxin. A study of the reactivities of the various forms of the toxin towards nicotinic acetylcholine receptor indicated that di-iodination had modified its reactivity. The 125I2-form bound with a slower rate constant than alpha-bungarotoxin to the receptor. 125I-alpha-bungarotoxin showed no modification of reactivity towards the receptor. Apart from the A280, two methods for calibrating 125I-alpha-bungarotoxin are described. They may be employed in the presence of other proteins. The first of these is an immunological assay using the complex formed between toxin and antitoxin antibodies. The second is a dilution assay, where competition between iodinated and noniodinated toxins for binding sites on nicotinic acetylcholine receptor is exploited.

Purification and chemical characterization of melittin and acetylated derivatives.

Melittin, the main basic and hydrophobic peptide of bee venom, displays marked detergent-like properties. At high peptide concentration, and depending on salt and pH, it forms a tetramer. This is prevented by using urea. A purification procedure in presence of 4.0 M urea was developed to prepare melittin in its monomeric form, free of other venom constituents such as N alpha-formyl melittin, degradation products of peptides and phospholipase A2. NH2-residues on the melittin molecule were modified by reaction with acetic anhydride to alter the asymmetrical charge distribution supposed to confer detergent-like properties to the molecule. This gave rise to di- and mono acetyl derivatives which could be used, once isolated, to study further the melittin structure-activity relationship.

A form of acute experimental autoimmune myasthenia gravis in mice occurring in absence of detectable circulating anti-acetylcholine receptor antibodies. Acute experimental myasthenia in mice.

A form of acute experimental autoimmune myasthenia gravis (EAMG) appears within 10 days after immunization of mice with rat acetylcholine receptor (AcChR) in complete adjuvant. This acute phase of EAMG differs from the chronic form reported earlier in the absence of detectable circulating anti-AcChR antibodies and by electrophysiologic signs of neuromuscular blockade which are not reversed by edrophonium injection. This form of acute EAMG occurs similarly in animals whose humoral response has been markedly reduced by pretreatment with cyclophosphamide. These data indicate that the pathogenesis of this acute form might differ from that of chronic EAMG and that it could involve mechanisms of cell-mediated immunity.