Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: HUMANGGP:041777 (acetylcholine receptor)
 5,663 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Following reduction of the acetylcholine receptor in the electroplax with dithiothreitol, the quaternary ammonium compounds bromoacetylcholine bromide and the p-nitrophenyl ester of (p-carboxyphenyl) trimethylammonium iodide react near the active site probably with a sulfhydryl group. The covalently attached quaternary ammonium moieties additionally interact with the active site noncovalently to activate the receptor and cause depolarization of the cell.
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PMID:Acetylcholine receptor: covalent attachment of depolarizing groups at the active site. 578 18

1. The interaction of two specific ligands for the vertebrate nicotinic acetylcholine receptor were investigated on the solubilized form of a proposed acetylcholine receptor from the invertebrate Limulus polyphemus. 2. The affinity agent 4-(N-maleimodo)benzyltrimethylammonium iodide exhibited no effect on the binding of alpha-bungarotoxin to the Limulus receptor protein. 3. Torpedo acetylcholine receptor antibody neither inhibited alpha-bungarotoxin binding nor produced any alteration in the sedimentation profile of the Limulus receptor. 4. The lack of interaction of 4-(N-maleimido)benzyltrimethylammonium iodide and Torpedo acetylcholine receptor antibody with the Limulus acetylcholine receptor was interpreted to reflect significant difference between the molecular structures of this invertebrate receptor and the acetylcholine receptor of vertebrate.
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PMID:The use of specific ligands for the vertebrate acetylcholine receptor in the characterization of invertebrate acetylcholine receptors. 612 23

Antibody against acetylcholine receptor (AChR) of human skeletal muscle was measured using enzyme-linked immunosorbent assay and found in 23 (74%) of 31 Japanese patients with generalized myasthenia gravis. In 15 patients with generalized myasthenia gravis who had not undergone thymectomy and who were not receiving adrenocorticosteroids, the antibody was found in 13 (87%). Antibody was also found in 13 (54%) of 24 patients with myasthenia gravis against AChR fractions obtained from fetal calf thymus. Based on the subunit structures of the AChR protein, the double precipitation assay using iodine 125-alpha-bungarotoxin is also capable of detecting antibody against the toxin binding site, by cross reactivity. This is among the first reports of experiments in which enzyme-linked immunosorbent assay was used to measure the antibodies in human myasthenia gravis and provides evidence of anti-AChR antibody against antigens from fetal calf thymus.
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PMID:Enzyme-linked immunosorbent assay for antibody against the nicotinic acetylcholine receptor in human myasthenia gravis. 636 20

Proteolytic fragments of the alpha subunit of the acetylcholine receptor retain the ability to bind alpha-bungarotoxin following resolution by polyacrylamide gel electrophoresis and immobilization on protein transfers. The alpha subunit of the acetylcholine receptor of Torpedo electric organ was digested with four proteases: Staphylococcus aureus V-8 protease, papain, bromelain, and proteinase K. The proteolytic fragments resolved on 15% polyacrylamide gels were electrophoretically transferred onto positively charged nylon membrane filters. When incubated with 0.3 nM 125I-labeled alpha-bungarotoxin and autoradiographed, the transfers yielded patterns of labeled bands characteristic for each protease. The molecular masses of the fragments binding toxin ranged from 7 to 34 kDa, with major groupings in the 8-, 18-, and 28-kDa ranges. The apparent affinity of the fragments for alpha-bungarotoxin as determined from the IC50 value was 6.7 X 10(-8) M. The labeling of fragments with alpha-bungarotoxin could be inhibited by prior affinity alkylation of receptor-containing membranes with 4-(N-maleimido)-alpha-benzyltrimethylammonium iodide. These findings demonstrate that immobilized proteolytic fragments as small as 1/5 the size of the alpha subunit retain the structural characteristics necessary for binding alpha-bungarotoxin, although the toxin is bound to the fragments with lower affinity than to the native receptor. The effect of affinity ligand alkylation demonstrates that the alpha-bungarotoxin binding site detected on the proteolytic fragments is the same as the affinity-labeled acetylcholine binding site on the intact acetylcholine receptor.
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PMID:Binding of alpha-bungarotoxin to proteolytic fragments of the alpha subunit of Torpedo acetylcholine receptor analyzed by protein transfer on positively charged membrane filters. 637 17

The 13 amino acid toxic peptide from the marine snail Conus geographus, conotoxin GI, blocks the acetylcholine receptor at the neuromuscular junction. In this report, we describe a method for analyzing disulfide bonding in nanomole amounts of small cystine-rich peptides. The procedure involves partial reduction and a double-label alkylation of cysteine residues. Using this method, we show that the natural conotoxin GI has a (2-7, 3-13) disulfide configuration. The structure of conotoxin GI has been confirmed by chemical synthesis. The preparation and purification of molecularly homogeneous, iodinated derivatives of this toxin are also described. All derivatives, including the [diiodohistidine,diiodotyrosine]conotoxin GI, retained at least half of the biological activity of unmodified toxin. Since the tetraiodinated toxin, which is greater than 25% by weight iodine, retains considerable toxicity, unmodified histidine and tyrosine residues in conotoxin GI are not crucial for biological activity.
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PMID:Conotoxin GI: disulfide bridges, synthesis, and preparation of iodinated derivatives. 646 16

The direct binding of alpha-bungarotoxin to the alpha subunit of the acetylcholine receptor from Torpedo electric organ immobilized onto protein blots was demonstrated. Protein blots were prepared by electrophoretically transferring resolved acetylcholine receptor subunits from 10% polyacrylamide gels onto Zetabind, positively charged nylon membrane filters. Such blots, when incubated with 125I-labeled alpha-bungarotoxin, washed, and autoradiographed, gave rise to a single labeled band corresponding to the alpha subunit of the receptor. The labeling with alpha-bungarotoxin could be inhibited by pretreating the receptor-containing membranes with the affinity ligand 4-(N-maleimido)-alpha-benzyltrimethylammonium iodide. In addition, the association of toxin with the alpha subunit could be inhibited by d-tubocurarine (IC50 = 0.9 mM). Furthermore, removal of high-mannose oligosaccharide chains from the alpha subunit by treatment with endoglycosidase H did not interfere with the observed toxin binding. Thus it is demonstrated that isolated, immobilized alpha subunit of the Torpedo acetylcholine receptor can bind alpha-bungarotoxin. However, the observed binding of alpha-bungarotoxin to immobilized alpha subunit is reduced in affinity to 1/1,000 to 1/10,000 of that obtained with native receptor. The endoglycosidase H-susceptible oligosaccharide side chain(s) is not required for this interaction. Binding of alpha-bungarotoxin is to the physiologically relevant acetylcholine binding site as defined by affinity ligand alkylation.
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PMID:Binding of alpha-bungarotoxin to isolated alpha subunit of the acetylcholine receptor of Torpedo californica: quantitative analysis with protein blots. 657 69

The isolated subunits of the acetylocholine receptor from Torpedo californica were digested with proteolytic enzymes, and the resulting polypeptide fragments were analyzed by gel electrophoresis. We have identified those fragments which contain carbohydrate and those from the alpha subunit which are labelled with the acetylcholine binding site specific reagent [4-(N-maleimido)benzyl]tri[3H]methylammonium iodide. We have tested several monoclonal antibodies raised to the acetylcholine receptor from torpedo, some of which react with the denatured subunits [Tzartos, S.J., & Lindstrom, J.M. (1980) Proc. Natl. Acad. Sci. U.S.A.77, 755; Tzartos, S.J., & Lindstrom, J.M. (1981) in Monoclonal antibodies in Endocrine Research (Fellows, R., & Eisenbarth, G., Eds.) Raven Press (in press)]. The binding specificities of these antibodies to radioiodinated proteolytically generated fragments of the alpha subunit were determined by immunoprecipitation followed by gel electrophoresis. The antibodies tested fell into at least three main groups on the basis of their binding specificities. These antibodies were also tested for their capacity to bind to acetylcholine receptor solubilized in Triton X-100, sodium cholate, or sodium cholate supplemented with exogenous lipids. A monoclonal antibody raised to the denatured delta subunit, was tested for its ability to select radioiodinated proteolytic fragments of these subunits. These molecules provide probes for many sites on the acetylcholine receptor with affinities and specificities comparable to alpha-neurotoxins.
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PMID:Monoclonal antibodies as probes of acetylcholine receptor structure. 1. Peptide mapping. 678 26

Acetylcholine receptors from fish electric organ tissue and mammalian muscle were compared by peptide mapping. The alpha subunits from receptors of Torpedo californica and Electrophorus electricus electric organ tissue were digested with V8 protease and the resulting fragments separated on polyacrylamide gels and stained for protein or for carbohydrate. 125I-Labeled alpha subunits of acetylcholine receptors from Electrophorus electric organ tissue and bovine muscle were digested with V8 protease, and the resulting fragments were also separated on polyacrylamide gels. Intact receptors from both the fish electric organs and mammalian muscle were labeled with [4-(N-maleimido) benzyl]tri[3H]-methylammonium iodide which binds specifically to the acetylcholine binding site on alpha subunits, and the isolated alpha subunits were subjected to the same peptide mapping procedure. The fragment patterns produced were stained for protein and fluorographed to identify active site containing polypeptides. None of these peptide mapping approaches revealed extensive homologies between alpha subunits. Intact and V8 proteolyzed sodium dodecyl sulfate denatured receptors from Torpedo and Electrophorus electric organs and bovine muscle were electrophoretically transferred to diazophenyl thioether paper and probed with antisera to Torpedo receptor subunits and two monoclonal antibodies. Unique fragment patterns were obtained with each antisubunit serum. A fragment of the same size was derived from the beta subunit of each acetylcholine receptor and was shown to specifically bind the same monoclonal antibody in each case. These results indicate that only in the beta subunits from all of the species examined is a large sequence nearly identical. However, it is likely that corresponding receptor subunits from receptors of all of these species have extensive structural homologies.
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PMID:Structural similarities between acetylcholine receptors from fish electric organs and mammalian muscle. 713 15

Iodination of alpha-bungarotoxin (alpha BuTx) gives rise to two products, mono- and diiodo-alpha BuTx. A combination of enzymatic digestion and Edman degradation revealed that the iodinated derivatives contain label in residue Tyr 54 exclusively. This labeling pattern is reminiscent of the reactivity toward iodination of "short" neurotoxins. The binding properties of the two derivatives were compared. At 20 degrees C, in 10 mM sodium phosphate, pH 7.4, the association of mono-125I-alpha BuTx to detergent-solubilized Torpedo californica electroplax acetylcholine receptor is characterized by a rate constant of 5.2 X 10(6) M-1 s-1, which is indistinguishable from the on-rate constant of the native toxin. Introduction of the second iodine atom reduces the association rate constant to 1.7 X 10(6) M-1 s-1. The diiodo derivative was also found to bind more slowly than the monoiodinated toxin by factors of 2.6 and 2.5 to partially purified preparations of acetylcholine receptor from denervated chick muscle and of toxin receptor from chick brain, respectively. No difference in dissociation kinetics was observed; both derivatives bind irreversibly to electric tissue and muscle acetylcholine receptor and dissociate from the neuronal receptor with an off-rate constant of 5.9 X 10(-5) s-1. In muscle, two sets of binding sites are kinetically distinguishable as originally observed by Brockes and Hall (Brockes, J. P., and Hall, Z. W. (1975) Biochemistry 14, 2092-2099). Di-125I-alpha BuTx binds more slowly to each site than mono-125I-alpha BuTx by factors of 2.0 and 3.7, respectively.
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PMID:Primary structure and binding properties of iodinated derivatives of alpha-bungarotoxin. 744 May 35

We demonstrated previously that a phencyclidine-displaceable quinacrine binding site exists at the lipid-protein interface of the Torpedo acetylcholine receptor (AcChR) (Valenzuela, C. F., Kerr, J. A., and Johnson, D. A. (1992) J. Biol. Chem. 267, 8238-8244). In this manuscript, we assess (1) the transverse position of this site in the lipid bilayer by examining the ability of a series of paramagnetic n-doxyl stearates (n-SALs) and iodide to quench receptor-bound quinacrine and membrane-partitioned octadecyl rhodamine B (C18-Rho) fluorescence and (2) the stoichiometry of histrionicotoxin- or phencyclidine-displaceable quinacrine binding. Initial experiments established what fraction of the n-doxyl stearates partitioned into the membranes and that the n-doxyl stearates do not interfere with quinacrine binding to the receptor at the concentrations used in the quenching studies. The n-doxyl stearate quenching experiments indicated relatively small (< 2) differences between the n-doxyl stearates to quench receptor-bound quinacrine fluorescence, with a rank order of 7-SAL > or = 5-SAL > 12-SAL > 16-SAL. This contrasts with the n-doxyl stearate quenching of the membrane-partitioned C18-Rho which showed as much as an 8.6-fold difference between the various isomers with a rank order of quenching efficiencies of 5-SAL > 7-SAL > 12-SAL > or = 16-SAL. Iodide quenching measurements indicated significant solute accessibility to membrane-partitioned C18-Rho but not to receptor-bound quinacrine. The ratios of the bimolecular quenching rate constants for free to bound quinacrine and for free rhodamine B to membrane-partitioned C18-Rho were 53.4 and 6.6, respectively. Direct titration of quinacrine into suspensions of a high concentration of AcChR-associated membranes yielded an upper limit to the binding stoichiometry of 1.4 HTX- or PCP-displaceable quinacrine binding sites/AcChR functional units. The results suggest that there is a single phencyclidine- or histrionicotoxin-displaceable quinacrine binding site located at or somewhat below the level of the C5-C7 in the phospholipid acyl chains at the lipid-protein interface.
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PMID:Transverse localization of the quinacrine binding site on the Torpedo acetylcholine receptor. 845 5


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