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Query: HUMANGGP:041777 (acetylcholine receptor)
 5,663 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The acetylcholine receptor from denervated rat skeletal muscle was purified by affinity chromatography and, after reduction, was treated with the affinity alkylating agent 4-(N-maleimido)benzyltri[3H]methylammonium iodide. The receptor specifically incorporated approximately 1 mol of alkylating agent per mol of 125I-labeled alpha-bungarotoxin bound. Analysis of the labeled receptor by polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that two subunits were labeled; their apparent molecular weights were 45,000 and 49,000. These results suggest that the affinity reagent labels a second site for acetylcholine binding in the muscle receptor that is not labeled in receptors from Electrophorus or Torpedo.
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PMID:Affinity alkylation labels two subunits of the reduced acetylcholine receptor from mammalian muscle. 27 Jul 7

The frog sartorius motor endplate was treated with the specific disulfide bond reducing agent dithiothreitol and subsequently exposed to a covalently reacting compound (the nitrophenyl ester of p-carboxyphenyltrimethylammonium iodide, NPTMB) known to activate the dithiothreitol-reduced acetylcholine receptor in Electrophorus electroplax. NPTMB causes a maximum depolarization of about 35 mV when applied to the dithiothreitol-treated sartorious motor endplate. It is ineffective on postjunctional membrane prior to disulfide bond reduction and on extrajunctional regions, reduced or unreduced. High concentrations of a competitive antagonist such as (+)-tubocurarine prevent reaction between NPTMB and the reduced receptor and cause a repolarization of the membrane when applied to the already-depolarized preparation. We conclude that in frog muscle, as in electroplax, the attached activator bridges the acetylcholine binding site of the reduced receptor between a sulfhydryl group, to which it is covalently bound, and a negative subsite, with which it forms a reversible ionic band.
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PMID:Activation of the frog sartorius acetylcholine receptor by a covalently attached group. 31 27

We have purified the junctional acetylcholine receptor from normal rat skeletal muscle and compared its structure with that of the extrajunctional receptor from denervated muscle. The two receptors from leg muscle were distinguished by isoelectric focusing and by reaction with sera from patients with myasthenia gravis. The junctional form of the acetylcholine receptor was purified from normal leg muscle by affinity chromatography on concanavalin A/Sepharose and cobrotoxin/Sepharose followed by sucrose gradient centrifugation. Analysis of radioiodinated receptor by polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated that the subunit structure of the junctional receptor was similar to that previously determined for the extra-junctional form (Froehner, S. C., et al. (1977) J. Biol. Chem. 252, 8589-8596), with major polypeptides, whose apparent molecular weights in 9% polyacrylamide gels were 45 000 and 51 000. In addition, several minor polypeptides were found. When the two receptors were labeled with different isotopes of iodine and run together on a sodium dodecyl sulfate gel, the subunits of one receptor could not be resolved from those of the other. As seen earlier with the extrajunctional form, the affinity alkylating reagent [3H]MBTA labeled the 45 000- and 49 000-dalton polypeptides of the junctional receptor. Peptide mapping showed that the two MBTA binding subunits are structurally related, although they are unrelated to the other polypeptides, and that the 45 000- and 51 000-dalton polypeptides of the junctional receptor were indistinguishable from those of the extrajunctional receptor. In addition, peptide mapping of the four subunits of acetylcholine receptor isolated from Torpedo californica electric organ showed that these four polypeptides appear to be structurally unrelated.
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PMID:Subunit structure and peptide mapping of junctional and extrajunctional acetylcholine receptors from rat muscle. 46 80

The pharmacological specificity of the binding of 125I-labeled alpha-bungarotoxin to a 1% Emulphogene BC-720 extract of a rat brain particulate fraction has been investigated. The extract contains a component which possesses the binding characteristics of a nicotinic acetylcholine receptor protein. The crude soluble acetylcholine receptor protein was purified by affinity chromatography utilizing the alpha-neurotoxin of Naja naja siamensis as ligand and 1.0 M carbamylcholine chloride as eluant. A single, batch-wise, affinity chromatography procedure yields an average purification of 510-fold. When this purified material is treated a second time by affinity chromatography, purification as high as 12600-fold has been obtained. Binding of 125I-labeled alpha-bungarotoxin to this purified acetylcholine receptor protein is saturable with a Kd of 1 - 10(-8) M. Nicotine and acetylcholine iodide at concentrations of 10(-5) M inhibit 125I-labeled toxin-acetylcholine receptor protein complex formation by 41 and 61% respectively. At 10(-4) M, carbamylcholine chloride and (+)-tubocurarine chloride give respectively 52 and 82% inhibition. Eserine sulfate and atropine sulfate have no effect on complex formation at a concentration of 10(-4) M. These data support the isolation of a partially purified nicotinic acetylcholine receptor protein.
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PMID:Studies of nicotinic acetylcholine receptor protein from rat brain. II. Partial purification. 88 57

A high-affinity muscarinic receptor antagonist, 123IQNB (3-quinuclidinyl-4-iodobenzilate labeled with iodine 123), was used with single photon emission computed tomography to image muscarinic acetylcholine receptors in 14 patients with dementia and in 11 healthy controls. High-resolution single photon emission computed tomographic scanning was performed 21 hours after the intravenous administration of approximately 5 mCi of IQNB. In normal subjects, the images of retained ligand showed a consistent regional pattern that correlated with postmortem studies of the relative distribution of muscarinic receptors in the normal human brain, having high radioactivity counts in the basal ganglia, occipital cortex, and insular cortex, low counts in the thalamus, and virtually no counts in the cerebellum. Eight of 12 patients with a clinical diagnosis of Alzheimer's disease had obvious focal cortical defects in either frontal or posterior temporal cortex. Both patients with a clinical diagnosis of Pick's disease had obvious frontal and anterior temporal defects. A region of interest statistical analysis of relative regional activity revealed a significant reduction bilaterally in the posterior temporal cortex of the patients with Alzheimer's disease compared with controls. This study demonstrates the practicability of acetylcholine receptor imaging with 123IQNB and single photon emission computed tomography. The data suggest that focal abnormalities in muscarinic binding in vivo may characterize some patients with Alzheimer's disease and Pick's disease, but further studies are needed to address questions about partial volume artifacts and receptor quantification.
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PMID:The distribution of cerebral muscarinic acetylcholine receptors in vivo in patients with dementia. A controlled study with 123IQNB and single photon emission computed tomography. 195 90

Radioligand binding and ESR were used to study the association of a spin-labeled local anesthetic, 2-[N,N-dimethyl-N-(2,2,6,6-tetramethylpiperidinooxyl)] ethyl-4-hexyloxybenzoate iodide (C6SLMel), with acetylcholine receptor-enriched membranes from Torpedo californica. In the presence of carbamylcholine, we found that C6SLMel competitively inhibits [3H]phencyclidine binding with high affinity (KD = 8.7 X 10(-7) M for C6SLMel), whereas in the presence of alpha-toxin or in the absence of agonist, C6SLMel binds with lower affinity (KD = 2 X 10(-5) M). At concentrations lower than 1 X 10(-5) M, C6SLMel does not bind to the agonist site but enhances [3H]acetylcholine binding. These findings show that C6SLMel binds to the allosterically coupled noncompetitive inhibitor site which is regulated by agonist binding. In addition, C6SLMel preferentially associates with the desensitized receptor state known to exhibit high affinities for agonists and local anesthetics. ESR measurements of C6SLMel bound to receptor membranes in the absence of agonist show moderately immobilized spectra. Addition of carbamylcholine results in the appearance of a strongly immobilized component. Prior exposure to alpha-toxin blocks the carbamylcholine-induced, strongly immobilized component in the ESR spectrum. Furthermore, in the presence of carbamylcholine, back-titration of bound C6SLMel with phencyclidine decreases the highly immobilized component at concentrations consistent with the KD for phencyclidine. These findings indicate that C6SLMel detects conformational changes between the resting and desensitized acetylcholine receptor states that occur at the noncompetitive inhibitor binding site. The strongly mobilized component is not affected by ferricyanide addition, suggesting that the binding site is in a region not readily accessible to anion collision from the aqueous phase.
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PMID:Association of a spin-labeled local anesthetic with the allosterically coupled noncompetitive inhibitor site on the acetylcholine receptor. 301 81

The portions of the Torpedo californica nicotinic acetylcholine receptor (AChR) alpha-subunit that contribute to the allosteric antagonist-binding site and to the agonist-binding site have been localized by affinity labeling and proteolytic mapping. [3H]Meproadifen mustard was employed as an affinity label for the allosteric antagonist-binding site and [3H]tubocurare as a photoaffinity label for the agonist-binding site. Both labels were found in a 20-kDa proteolytic fragment generated from the AChR alpha-subunit by Staphylococcus aureus V8 protease. This 20-kDa peptide also contains the 3H-labeled 4-(N-maleimido)-alpha-benzyltrimethylammonium iodide-reactive site and binds 125I-alpha-bungarotoxin. N-terminal sequencing established that the 20-kDa fragment began at Ser-173 of the alpha-subunit. Fluorescein isothiocyanate-conjugated concanavalin A could be bound to the second of the two major V8 cleavage products, an 18-kDa peptide. This peptide was also sensitive to treatment with endo-beta-N-acetyl-glucosaminidase H, consistent with the presence of N-linked carbohydrate on this fragment. The N terminus of this peptide was found to be Val-46 of the alpha-subunit sequence. Experiments designed to map disulfide bonds within the AChR alpha-subunit indicate that no bonds exist between the 18-kDa fragment (containing Cys-128 and Cys-142) and the 20-kDa fragment (containing Cys-192, Cys-193, and Cys-222). These results establish that the 20-kDa fragment contributes to both the acetylcholine and the allosteric antagonist-binding sites, whereas there is no evidence that the 18-kDa fragment is part of either site.
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PMID:Location of ligand-binding sites on the nicotinic acetylcholine receptor alpha-subunit. 309 82

Neuronal acetylcholine receptors (AChRs), which bind nicotine with high affinity but do not bind alpha-bungarotoxin (alpha Bgt), have recently been immunoaffinity-purified from chicken (Whiting and Lindstrom, 1986a) and rat (Whiting and Lindstrom, 1987a) brain using monoclonal antibodies (mAbs). Here we report the characterization of nicotinic AChRs of bovine and human brain using as probes mAbs prepared to AChRs from rat brain. Both the human and bovine brain AChRs exhibit high affinity for L-nicotine (Ki = 16 nM for bovine AChR and Ki = 6.5 nM for human AChR) and other cholinergic agonists, relatively lower affinity for cholinergic antagonists, and do not bind alpha Bgt. These AChRs are affinity-labeled with bromoacetylcholine and 4-(N-maleimido)benzyltrimethylammonium iodide (MBTA) after reduction with dithiothreitol, indicating that amino acid residues homologous to cysteines 192 and 193 of alpha subunits of Torpedo electric organ AChRs are conserved. Immunoaffinity-purified bovine brain AChR consists of 2 types of subunit, Mr 50,600 and Mr 74,400. The Mr 74,400 subunit was affinity-labeled with 3H-MBTA, indicating that it contains the ACh binding site. Thus, mAbs have proven to be excellent probes for these proteins, and have been used to show that neuronal nicotinic AChRs of chickens, rats, and cattle are macromolecules approximately 10 S in size and composed of only 2 kinds of subunits: an ACh-binding subunit and a structural subunit.
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PMID:Characterization of bovine and human neuronal nicotinic acetylcholine receptors using monoclonal antibodies. 317 81

To obtain information on the disposition of alpha-toxin when bound to the acetylcholine receptor (AChR), we evaluated the accessibility of solutes to fluorescein isothiocyanate (FITC) conjugated to alpha-toxin (siamensis 3) at lysine 23 (FITC-toxin) by measuring the rate constants for iodide quenching of the fluorescence of fluorescein free in solution and FITC-toxin free in solution and bound to AChR. Relative to the free fluorescein, we observed a 55% reduction in the quenching rate constant for the unbound FITC-toxin and 80% reduction for the AChR-bound FITC-toxin. It is tempting to interpret a decrease in the quenching rate constant as due to an increase in the masking of the labeling fluorophore, which in our case would then be indicative of masking of fluorescein conjugated to the free toxin and masking of FITC-toxin, in the region of lysine 23, when bound to AChR. However, elementary considerations indicate that the quenching rate depends not only on geometrical masking factors but also on the translational and rotational mobilities of the labeled molecules as well as orientational constraints. To evaluate these effects we have established quantitative relations between the rate of fluorescence quenching, the degree of masking of fluorophore, translational and rotational rates, and orientational constraints of the labeled macromolecules, using recent formulations for the rate of reaction between asymmetric molecules (Shoup et al., 1981, Biophys. J., 36:619-714). These relations predict that the decrease in quenching constant observed for the labeled FITC-toxin as well as the AChR-bound FITC-toxin is largely due to differences in translational and rotational rates and orientational constraints and not to significant increases in geometrical masking. Our theoretical formulation shows that the quenching rate can be decreased by a factor of 2-5 merely by immobilizing a fluorophore on the surface of a large protein without any significant increase in geometrical masking.
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PMID:Solute accessibility to N epsilon-fluorescein isothiocyanate-lysine-23 cobra alpha-toxin bound to the acetylcholine receptor. A consideration of the effect of rotational diffusion and orientation constraints on fluorescence quenching. 393 57

Electroplax, single cells dissected from electric tissue of Electrophorus, are labeled in a two-step procedure: reduction by dithiothreitol followed by alkylation by the affinity label 4-(N-maleimido)-alpha-benzyltri-[methyl-(3)H]methylammonium iodide, either alone or in combination with [2,3-(14)C]N-ethylmaleimide. Electrophoresis in sodium dodecyl sulfate on polyacrylamide gel of an extract, prepared with this detergent, of single-labeled or of double-labeled cells results in a major peak of (3)H activity, with a mobility corresponding to a polypeptide of molecular weight 42,000. In addition, in the double-labeled samples, there is a unique peak in the ratio of (3)H to (14)C that is coincident with the (3)H peak. The electrophoretic patterns of extracts of cells in which affinity alkylation of the reduced receptor has been suppressed by dithiobischoline, an affinity oxidizing agent, by cobratoxin, an irreversible ligand, or by hexamethonium, a reversible ligand, show a considerably diminished peak of (3)H activity in the region of molecular weight 42,000. This is the predominant difference between the electrophoretic patterns of extracts of unprotected and of protected cells. Furthermore, extracts of cells protected with dithiobischoline before labeling with both tritiated affinity label and [(14)C]N-ethylmaleimide do not show the peak in the (3)H to (14)C ratio seen in the absence of protection. Thus, by several diverse criteria, the peak of (3)H activity corresponding to a molecular weight of 42,000 contains affinity-labeled acetylcholine receptor or receptor subunit.
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PMID:Affinity labeling of the acetylcholine receptor in the electroplax: electrophoretic separtion in sodium dodecyl sulfate. 450 31


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