Ligand binding to the membrane-bound acetylcholine receptor from Torpedo marmorata: a complete mathematical analysis

Biochemistry ◽  
1992 ◽  
Vol 31 (29) ◽  
pp. 6728-6738 ◽  
Author(s):  
Heino Prinz ◽  
Alfred Maelicke
Keyword(s):  
Ligand Binding ◽  
Acetylcholine Receptor ◽  
Mathematical Analysis ◽  
Torpedo Marmorata ◽  
Membrane Bound

scholarly journals Oligomeric forms of the membrane-bound acetylcholine receptor disclosed upon extraction of the M(r) 43,000 nonreceptor peptide

1982 ◽  
Vol 92 (1) ◽  
pp. 60-68 ◽  
Author(s):  
FJ Barrantes
Keyword(s):  
Acetylcholine Receptor ◽  
Receptor Protein ◽  
Antigenic Determinants ◽  
Torpedo Marmorata ◽  
Carbon Supports ◽  
Nitrobenzoic Acid ◽  
Particle Aggregates ◽  
Membrane Bound ◽  
Monospecific Antibodies ◽  
Oligomeric Forms

Oligomeric forms of the acetylcholine receptor are directly visualized by electron microscopy in receptor-rich membranes from torpedo marmorata. The receptor structures are quantitatively correlated with the molecular species so far identified only after detergent solubilization, and further related to the polypeptide composition of the membranes and changes thereof. The structural identification is made possibly by the increased fragility of the membranes after extraction of nonreceptor peptides and their subsequent disruption upon drying onto hydrophilic carbon supports. Receptor particles in native membranes depleted of nonreceptor peptides appear as single units of 7-8 nm, and double and multiple aggregates thereof. Particle doublets having a main-axis diameter of 19 +/- 3 nm predominate in these membranes. Linear aggregates of particles similar to those observed in rotary replicas of quick-frozen fresh electrolytes (Heuser, J.E. and S. R. Salpeter. 1979, J. Cell Biol. 82: 150-173) are also present in the alkaline-extracted membranes. Chemical modifications of the thiol groups shift the distribution of structural species. Dithiothreitol reduction, which renders almost exclusively the 9S, monomeric receptor form, results in the observation of the 7-8 nm particle in isolated form. The proportion of doublets increases in membranes alkylated with N-ethylmaleimide. Treatment with 5,5'-dithiobis-(nitrobenzoic acid) increases the proportion of higher oligomeric species, and particle aggregates (n=oligo) predominate. The nonreceptor v-peptide (doublet of M(r) 43,000) appears to play a role in the receptor monomer-polymer equilibria. Receptor protein and v-peptide co-aggregate upon reduction and reoxidation of native membranes. In membranes protected ab initio with N- ethylmaleimide, only the receptor appears to self-aggregate. The v-peptide cannot be extracted from these alkylated membranes, though it is easily released from normal, subsequently alkylated or reduced membranes. A stabilization of the dimeric species by the nonreceptor v-peptide is suggested by these experiments. Monospecific antibodies against the v-peptide are used in conjunction with rhodamine- labeled anti-bodies in an indirect immunoflourescence assay to map the vectorial exposure of the v-peptide. When intact membranes, v-peptide depleted and "holey" native membranes (treated with 0.3 percent saponin) are compared, maximal labeling is obtained with the latter type of membranes, suggesting a predominantly cytoplasmic exposure of the antigenic determinants of the v-peptide in the membrane. The influence of the v-peptide in the thiol-dependent interconversions of the receptor protein and the putative topography of the peptide are analyzed in the light of the present results.

scholarly journals A combined ELISA-immunoelectron microscopic approach for topological mapping of membrane protein epitopes: application to the nicotinic acetylcholine receptor.

1994 ◽  
Vol 42 (3) ◽  
pp. 315-327 ◽  
Author(s):  
M Schmutz ◽  
D Kling ◽  
S Tzartos ◽  
A Brisson
Keyword(s):  
Membrane Proteins ◽  
Acetylcholine Receptor ◽  
Epitope Mapping ◽  
Lipid Membrane ◽  
Torpedo Marmorata ◽  
Microscopic Approach ◽  
Electron Microscopic ◽  
Experimental Conditions ◽  
General Application ◽  
Membrane Bound

Identification of epitope localization on either side of the lipid membrane by immunoelectron microscopy constitutes an intrinsic powerful method of structure determination for membrane proteins. We have developed a method allowing measurement and observation, under almost identical experimental conditions, of the binding of monoclonal antibodies (MAb) to membrane-bound acetylcholine receptor from Torpedo marmorata electric tissue. This method, based on ELISA and electron microscopy of negatively stained specimens, was developed with MAb of known epitope specificity. With native membrane fragments, we found that MAb bound to extracellular epitopes in a stoichiometric manner, whereas almost no binding was detected for intracellular epitopes. The treatment based on tissue homogenization in the presence of Zn2+ ions and sucrose resulted in the formation of large, stable openings, rendering accessible about 25% of intracellular epitopes. Electron microscopic observations showed a clear distinction between antibody binding to either intracellular or extracellular epitopes, both with native and Zn(2+)-treated membranes. In addition, the binding of one antibody directed against an extracellular epitope was strikingly dependent on the packing density of acetylcholine receptor molecules, thus enabling us to further distinguish between two levels of accessibility for extracellular epitopes. The method presented here is of general application for studies of epitope mapping in membrane proteins.

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