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A Maelicke

Publications and source records attributed to A Maelicke.

At least 109 records · Page 6Linked to original sources
PubMed1986

A monoclonal antibody interfering with binding and response of the acetylcholine receptor.

Employing a monoclonal antibody raised against the receptor protein, we have probed the mechanism of ligand interaction of the nicotinic acetylcholine receptor from Torpedo marmorata. Antibody WF6 specifically binds to alpha-subunits of the receptor with a stoichiometry of one molecule per receptor monomer. At saturating concentrations, WF6 blocks half of the binding sites for acetylcholine, all of the binding sites for alpha-neurotoxins, and none of the binding sites for representative cholinergic antagonists (with the exception of alpha-toxins) at the receptor. In the presence of saturating concentrations of antibody WF6, acetylcholine (or its agonists) cannot induce T1+ influx into Torpedo membrane vesicles. Rapid oversaturation of the receptor by agonist also cannot overcome this blockade of channel gating. The observed competition patterns of WF6 and representative cholinergic ligands with the receptor are evidence for separate binding sites for groups of ligands and for a network of allosterically linked effector regions at the receptor. The blockade by saturating concentrations of WF6 of the agonist-induced channel gating supports the conclusion that two molecules of agonist are required to activate the receptor-integral ion channel.

Acetylcholine↗
PubMed1986

Equilibrium binding of cholinergic ligands to the membrane-bound acetylcholine receptor.

We have studied the binding equilibria of the membrane-bound acetylcholine receptor from Torpedo marmorata with representative cholinergic ligands by means of two fluorescence and a rapid centrifugation assay. Based on the established mechanism of acetylcholine binding to the receptor (Fels, G., Wolff, E. K., and Maelicke, A. (1982) Eur. J. Biochem. 127, 31-38), the obtained binding and competition data were analyzed assuming two classes of interacting sites for all ligands studied. The experimental data were consistent with this assumption and, based on the obtained KD values, suggest weak positively cooperative interactions of binding sites when occupied by agonists but independent (or negatively cooperative interacting) sites when occupied by antagonists. Based on the fluorescence binding assay employed, agonists and antagonists induce different conformational states of the liganded receptor. These states seem to be similar for all antagonists tested but differ for the different agonists tested. The existence of ligand-specific conformational states suggests a close link of these states with receptor function.

4-Chloro-7-nitrobenzofurazan↗
PubMed1984

Antibodies against preselected peptides to map functional sites on the acetylcholine receptor.

Rabbit immune sera and mouse monoclonal antibodies were raised against the synthetic peptide Tyr-Cys-Glu-Ile-Ile-Val matching in sequence residues 127-132 of the alpha-subunit of all nicotinic acetylcholine receptors sequenced so far. Representative cholinergic ligands did not interfere with the binding of these antibodies to the receptor from Torpedo marmorata, indicating that this sequence is not part of the binding sites for cholinergic ligands. The applicability of antigenic sites analysis to the mapping of functional sites on receptor proteins is discussed.

Animals↗
PubMed1984

Ligand-specific state transitions of the membrane-bound acetylcholine receptor.

We have developed a simple, direct and time-resolved method to monitor ligand-induced changes in agonist affinity of the membrane-bound acetylcholine receptor. The assay is based on the quenching of fluorescence of NBD-5-acylcholine observed upon binding of this cholinergic agonist to the receptor. Under conditions of partial saturation with the fluorescent agonist, agonists and local anesthetics but not antagonists can induce an increase in affinity of the receptor for NBD-5-acylcholine. The effect is not observed with receptor fully saturated with the fluorescent agonist. The half-life of the observed change in affinity is independent of the nature of the agonist or local anesthetic applied (t1/2 approximately 60 s at 22 degrees C). We conclude that the same state transition of the receptor can be induced by two groups of cholinergic ligands that are assumed to be non-competitive with each other and to have distinctly different modes of action. The time course of the transition is reminiscent of the slow process of desensitization observed in vivo.

4-Chloro-7-nitrobenzofurazan↗
PubMed1984

Antibodies directed against functional sites on the acetylcholine receptor from Torpedo marmorata.

Antibodies directed against functional sites on the acetylcholine receptor from Torpedo marmorata have been obtained by the following two procedures: (i) Our library of monoclonal antibodies raised against the whole receptor protein was screened for antibodies competing with cholinergic agonists, antagonists and local anesthetics for receptor binding, (ii) antibodies were raised against short peptides matching the sequence of predetermined sites on the receptor protein. In this way, a topographic map of the functional sites on the receptor surface can be constructed.

Acetylcholine↗
PubMed1983

Synthesis and properties of NBD-n-acylcholines, fluorescent analogs of acetylcholine.

We have synthesized a homologous series of fluorescent analogs of acetylcholine, N-7-(4-nitrobenzo-2-oxa-1,3-diazolyl)-omega-amino-n-alkanoic acid beta (N,N,N-trialkylammonium) ethylesters (NBD-n-acylcholines) and report here on their physiological and biochemical properties. All NBD-n-acylcholines trimethylated at the cholinergic nitrogen are agonists of acetylcholine at the frog neuromuscular junction. Their potencies in depolarizing frog muscle cells decrease with decreasing chain length. The affinities of binding to the purified receptor from Electrophorus electricus also decrease with decreasing chain length with a large drop in affinity for the derivatives n = 4 and n = 3. The rate constants of association to acetylcholine receptor and to acetylcholine esterase are of the order of 10(8) M-1 S-1 and do not vary significantly with the chain length of the NBD-n-acylcholines. In contrast, the dissociation rate constants decrease with increasing chain length. The quenching of fluorescence of NBD-n-acylcholines accompanying binding to purified receptor and esterase from E. electricus appears to be due to the formation of a hydrogen bond between the omega-amino group as donor and an unidentified acceptor group in a hydrophobic pocket of the protein. With their advantageous fluorescence properties, their simple pharmacology, and their clear structure-function relationships, these compounds are useful tools for the study of cholinergic mechanisms.

4-Chloro-7-nitrobenzofurazan↗
PubMed1983

Interaction of cholinergic ligands with the purified acetylcholine receptor protein. I. Equilibrium binding studies.

We have studied the binding equilibria of two fluorescent ligands and several nonfluorescent cholinergic ligands with the purified acetylcholine receptor from Electrophorus electricus. The assay was based on the specifically cholinergic and reversible quenching of fluorescence observed upon complex formation between the receptor protein and N-7-(4-nitrobenzo-2-oxa-1,3-diazole)-5-aminopentanoic acid beta-(N-trimethylammonium) ethyl ester. This way, a large body of accurate, true equilibrium data was obtained. We find 1) all ligands studied compete for the same number of binding sites at the receptor; 2) agonists compete for half of these sites with high affinity and for the other half of these sites with significantly lower affinity; 3) antagonists compete for all of these sites with one affinity; and 4) in the presence of disulfide reducing agents, the binding patterns of some agonists and antagonists are changed in accordance with the electrophysiological changes observed under the same conditions with Rana pipiens Sartorius muscle fibers. Our studies indicate that the mechanism of ligand recognition is still functional at equilibrium and is not subject to the presence of an intact membrane environment. Furthermore, the existence of two types of agonist sites at every receptor molecule excludes most of the presently discussed two-state models as the basis for a mechanism of receptor-ligand interaction. To explain sigmoidal dose-response curves, a two-site model is already sufficient.

4-Chloro-7-nitrobenzofurazan↗
PubMed1983

Interaction of cholinergic ligands with the purified acetylcholine receptor protein. II. Kinetic studies.

We have studied the kinetics of interaction of N-7-(4-nitrobenzo-2-oxa-1,3,diazole)-5-aminopentanoic acid beta-(N-trimethylammonium) ethyl ester with the purified acetylcholine receptor from Electrophorus electricus. By employing a laser stopped flow fluorimeter, the full course of the kinetics was observed. The initial analysis of data yielded the following results. 1) Both association and dissociation kinetics are composed of several overlapping reaction steps. 2) Dissociation of monoliganded receptor is biphasic. This indicates the existence of two forms in equilibrium of the monoliganded receptor. 3) Displacement of fluorescent ligand by competing ligand follows a more complicated pattern. In particular, the late stages of the dissociation kinetics are increasingly slowed down the higher the concentration of competing ligand. This indicates a strictly ordered mode of association and dissociation, i.e. first on-last off for cholinergic ligands and the receptor. These and the previous finding of two agonist binding sites at the receptor (Prinz, H., and Maelicke, A. (1983) J. Biol. Chem. 258, 10263-10272) were employed to develop minimal reaction schemes that can account for the experimental data. They require two sites but four different ligand-receptor complexes and a strictly ordered mode of binding. The reaction schemes were used to simultaneously fit whole sets of association and dissociation kinetics. The fits reinforced the conclusions drawn by the initial analysis and also provided the rate constants for all reaction steps considered in the schemes. The following additional results were obtained. 4) One form of the monoliganded and diliganded receptor are formed with very large rate constants (greater than 2 X 10(8) M-1 s-1); the other complexes are formed more slowly. 5) The rapidly formed diliganded receptor (AR-A) has a rather short half-life while the other form of diliganded receptor (AR*A) develops more slowly and dominates at equilibrium. The obtained reaction schemes and rate constants invite comparison with physiological data. For example, the properties of the two forms of diliganded receptor correlate with those of the active and desensitized state of the endplate as defined by electrophysiological studies.

4-Chloro-7-nitrobenzofurazan↗
PubMed1983

The sites of neurotoxicity in alpha-cobratoxin.

We have chemically modified groups of amino acids in the sequence of alpha-cobratoxin and have studied the derivatives as to their affinity of binding to the acetylcholine receptor protein from Torpedo marmorata. (i) The toxin derivatives which were fully modified at lysine (penta-epsilon-N,N-dimethyl lysine; penta-epsilon-N-acetyl lysine), arginine (penta-N7,N8-(1,2-dihydroxycyclohex-1,2-ylene arginine), and tyrosine (mononitrotyrosine) all had significant remaining toxicity and affinity of binding. (ii) The "extra" disulfide of alpha-cobratoxin was selectively reduced and alkylated. Depending on the charge, size, and hydrophobicity of the attached groups, derivatives were obtained that bound to the acetylcholine receptor with higher (di-S-carboxyamidomethyl), about equal (di-S-pyridylethyl), or lower (di-iodoacetaminoethylnaphthylamine-5-sulfonic acid) affinity than the unmodified toxin. (iii) A fully reduced and carbamidomethylated derivative of alpha-cobratoxin obtained by repeating the procedure for selective reduction six times still bound with appreciable affinity (KD approximately 3 X 10(-6) M) to the acetylcholine receptor. We conclude that neither a single positively charged residue nor tyrosine nor the integrity of the disulfides is absolutely essential for toxicity. Furthermore, the single tyrosine and the area around the extra disulfide do not participate in the binding to the receptor. Together with previous findings on this interaction, this suggests a multipoint attachment of toxin and receptor involving several locally separate structural elements of the toxin.

Acylation↗
PubMed1983

Organization of ligand binding sites at the acetylcholine receptor: a study with monoclonal antibodies.

We have studied 20 monoclonal antibodies directed against both the solubilized and the membrane-bound receptor from Torpedo marmorata. We find the following: (i) Six of the antibodies compete with cholinergic ligands for receptor binding and, hence, are directed against the ligand binding regions. (ii) Of these six antibodies, two cross-react with receptor from Electrophorus electricus, rat myotubes, and chicken sympathetic ganglia. These two antibodies therefore define a preserved structure within the ligand binding regions. The other four antibodies bind to structures not common between the receptor preparations tested. (iii) From competition binding studies using internally 3H-labeled antibodies, nine nonoverlapping antigenic regions were defined at the surface of the receptor. Three of these regions overlap with the ligand binding regions. Since two of these three regions do not overlap with each other, two structurally distinct ligand binding regions must exist at the receptor. (iv) From competition binding studies with representative cholinergic ligands, the antibodies directed against the ligand binding regions can be subdivided into three groups: one group competes with all ligands tested; the second group competes with all ligands except the bismethonium compounds; the third group competes with all ligands except the bismethonium compounds and tubocurarine. The results are summarized in a model of the organization of ligand binding sites at the receptor: There are two ligand binding regions differing in their antigenic properties. Furthermore, either there exists separate sites for distinct groups of ligands within each of these binding regions or some ligands produce conformational changes of the receptor that reversibly abolish some antigenic sites. In any case, the cholinergic ligands must interact with the receptor by more and/or other structural determinants than are provided by the structure of acetylcholine.

Acetylcholine↗
PubMed1983

Nylon tube linked acetylcholine receptor: a tool for affinity isolations and immunosorption.

We have covalently coupled acetylcholine receptor and other proteins to the inner surface of nylon tubes and employ these affinity tubes in binding assays and for chromatographic purposes. Here we describe two applications: (i) The concentration determination of toxins and antibodies directed against the acetylcholine receptor, and (ii) the isolation and chromatography of specific immunoglobulins.

Animals↗
PubMed1982

Equilibrium binding of acetylcholine to the membrane-bound acetylcholine receptor.

We have studied the binding of acetylcholine to membrane-bound acetylcholine receptor from Torpedo marmorata employing a highly accurate airfuge assay procedure. At equilibrium the receptor displays two classes of acetylcholine binding sites; these interact with only weak positive cooperativity. As a further difference to binding data deduced from electrophysiological dose/response curves, the equilibrium constants for the two classes of sites (Kd1 = 25 nM, Kd2 = 8 nM) are orders of magnitude lower than the concentration required for half-maximal response. Both the weaker-than-expected cooperativity of sites and the high binding affinities are likely to be due to desensitisation of the receptor during the period of incubation. The positively cooperative interaction of acetylcholine binding sites is only observed with membrane preparations obtained in the presence of appropriate chelating, sulfhydryl-blocking and active-serine-blocking agents. Aged membrane preparations loose the ability of site interactions while only small changes in the total number of binding sites are observed. In the absence of divalent ions, the affinity of binding of acetylcholine to the receptor is reduced. To assess the significance of the binding data obtained, several alternative reaction schemes for non-random binding to two sites at the receptor are considered. In addition, the effects of possible sources of experimental error on the shape of Scatchard plots are analysed.

Acetylcholine↗
PubMed1981

Affinity chromatography and immunosorption with acetylcholine receptor attached to nylon tubes.

Nylon-linked proteins were used for affinity trapping and chromatography. As representative examples purified acetylcholine receptor, alpha-cobratoxin and bovine serum albumin were coupled to the activated matrix to serve as biospecific ligands. In particular, acetylcholine receptor was coupled without significant loss of biochemical properties. The resulting affinity tubes bind receptor-specific ligands including immunoglobulins and thus can be used for affinity-chromatographic purposes and immunoassays.

Chromatography, Affinity↗
PubMed1981

Agonist-activated ionic channels in acetylcholine receptor reconstituted into planar lipid bilayers.

Planar lipid bilayers were formed with the mixed chain phospholipid 1-stearoyl-3-myristolglycero-2-phosphocholine. Acetylcholine receptor membrane fragments or the purified receptor protein was incorporated into these bilayers by fusing receptor-containing vesicles with the planar membranes a few degrees below the lipid phase transition temperature. Single-channel currents activated by nicotinic agonists in the reconstituted system resembled those observed in intact rat and frog muscle membrane as measured by the patch clamp technique. The observed channel characteristics did not depend on the degree of receptor purification. Thus, the receptor-enriched fragments and those depleted of nonreceptor peripheral peptides, the purified receptor monomer/dimer mixtures, and the isolated receptor monomer as defined by gel electrophoresis all shared similar electrochemical properties in the synthetic lipid bilayer. The agonist-activated ionic channel seems, therefore, to be contained within the receptor monomer.

Acetylcholine↗