scholarly journals Localization and synthesis of the acetylcholine-binding site in the α-chain of the Torpedo californica acetylcholine receptor

1984 ◽  
Vol 224 (3) ◽  
pp. 995-1000 ◽  
Author(s):  
D J McCormick ◽  
M Z Atassi
Keyword(s):  
Binding Site ◽  
Acetylcholine Receptor ◽  
Synthetic Peptide ◽  
Solid Phase ◽  
Essential Elements ◽  
Binding Activity ◽  
Binding Assays ◽  
Free Peptide ◽  
Loop Region ◽  
Alpha Bungarotoxin

The sequence of the alpha-chain of the acetylcholine receptor of T. californica has been determined by recent cloning studies. The integrity of the disulphide bond between Cys-128 and cys-142 has been shown to be important for the maintenance of the binding activity of the receptor, thus implicating the regions around the disulphide bridge in binding with acetylcholine. In the present work, a synthetic peptide containing this loop region (residues 125-147) was synthesized. Solid-phase radiometric binding assays demonstrated a high binding of 125I-labelled alpha-bungarotoxin to the synthetic peptide. It was further shown that the free peptide bound well to [3H]acetylcholine. Additional experiments demonstrated that pretreatment of peptide 125-147 with 2-mercaptoethanol destroyed its binding activity, clearly showing that the integrity of the disulphide structure was essential for binding. Unlabelled acetylcholine also inhibited the binding of labelled acetylcholine to the synthetic peptide. The region 125-147, therefore, contains essential elements of the acetylcholine binding site of the Torpedo receptor.

scholarly journals The short-neurotoxin-binding regions on the α-chain of human and Torpedo californica acetylcholine receptors

1991 ◽  
Vol 274 (3) ◽  
pp. 849-854 ◽  
Author(s):  
K H Ruan ◽  
B G Stiles ◽  
M Z Atassi
Keyword(s):  
Binding Site ◽  
Acetylcholine Receptors ◽  
Binding Activity ◽  
Lower Activity ◽  
Binding Region ◽  
Torpedo Californica ◽  
Binding Regions ◽  
Alpha Bungarotoxin ◽  
Erabutoxin B ◽  
Low Activity

The continuous regions for short-neurotoxin binding on the alpha-chains of Torpedo californica (electric ray) and human acetylcholine receptors (AChR) were localized by reaction of 125I-labelled cobrotoxin (Cot) and erabutoxin b (Eb) with synthetic overlapping peptides spanning the entire extracellular part of the respective alpha-chains. On Torpedo AChR, five Cot-binding regions were found to reside within peptides alpha 1-16, alpha 23-38/alpha 34-49 overlap, alpha 100-115, alpha 122-138 and alpha 194-210. The Eb-binding regions were localized within peptides alpha 23-38/alpha 34-49/alpha 45-60 overlap, alpha 100-115 and alpha 122-138. The main binding activity for both toxins resided within region alpha 122-138. In previous studies we had shown that the binding of long alpha-neurotoxins [alpha-bungarotoxin (Bgt) and cobratoxin (Cbt)] involved the same regions on Torpedo AChR as well as an additional region within residues alpha 182-198. Thus region alpha 182-198, which is the strongest binding region for long neurotoxins on Torpedo AChR, was not a binding region for short neurotoxins. On human AChR, peptide alpha 122-138 possessed the highest activity with both toxins, and lower activity was found in the overlap alpha 23-38/alpha 34-49/alpha 45-60 and in peptide alpha 194-210. In addition, peptides alpha 100-115 and alpha 56-71 showed strong and medium binding activities to Eb, but low activity to Cot, whereas peptide alpha 1-16 exhibited low binding to Cot and no binding to Eb. Comparison with previous studies indicated that, for human AChR, the binding regions of short and long neurotoxins were essentially the same. The finding that the region within residues alpha 122-138 of both human and Torpedo AChR possessed the highest binding activity with short neurotoxins indicated that this region constitutes a universal binding site for long and short neurotoxins on AChR from various species.

scholarly journals Interaction of nuclear proteins with a positive cis-acting element of rat embryonic myosin heavy-chain promoter: identification of a new transcriptional factor.

1989 ◽  
Vol 9 (5) ◽  
pp. 1839-1849 ◽  
Author(s):  
Y T Yu ◽  
B Nadal-Ginard
Keyword(s):  
Binding Site ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Hela Cells ◽  
Promoter Activity ◽  
Point Mutations ◽  
Binding Activity ◽  
Transcriptional Factor ◽  
Binding Assays ◽  
Cis Acting

A DNA fragment of the rat embryonic myosin heavy-chain promoter (MHCemb) has been found to specifically bind a nuclear factor (NFe) present in extracts prepared from mouse C2 myoblasts, myotubes, and HeLa cells. The nucleotide sequence of the binding site (BSe) has been identified as 5'-GTGTCAGTCA-3' and was located between -93 and -84. Transient expression studies on MHCemb promoter deletion constructs in C2 myoblasts and C2 myotubes suggested that NFe is a transcriptional factor. Deletion of the NFe-binding site resulted in four- to sixfold and twofold reduction of promoter activity in C2 myotubes and C2 myoblasts, respectively. Furthermore, point mutations at the BSe not only abolished the NFe-binding activity of the MHCemb promoter but also resulted in reduction of the promoter activity to levels similar to those of the deletion constructs in C2 myotubes, myoblasts, and Hela cells (four- to sixfold). Although BSe and the binding site of the recently identified transcriptional factors AP-1 and ATF share significant homology, the results from competition binding assays indicated that NFe is different from both AP-1 and ATF.

scholarly journals BiP forms stable complexes with unassembled subunits of the acetylcholine receptor in transfected COS cells and in C2 muscle cells

1992 ◽  
Vol 117 (4) ◽  
pp. 841-847 ◽  
Author(s):  
JR Forsayeth ◽  
Y Gu ◽  
ZW Hall
Keyword(s):  
Acetylcholine Receptor ◽  
Muscle Cells ◽  
Binding Activity ◽  
Alpha Subunit ◽  
Stable Complex ◽  
Cos Cells ◽  
Toxin Binding ◽  
Immunoglobulin Binding Protein ◽  
Alpha Bungarotoxin ◽  
Immunoglobulin Binding

We have investigated the role of the immunoglobulin-binding protein (BiP) in the folding and assembly of subunits of the acetylcholine receptor (AChR) in COS cells and in C2 muscle cells. Immunoprecipitation in COS cells showed that alpha, beta, and delta subunits are associated with BiP. In the case of the alpha subunit, which first folds to acquire toxin-binding activity and is then assembled with the other subunits to form the AChR, BiP was associated only with a form that is unassembled and does not bind alpha-bungarotoxin. Similar results were found in C2 cells. Although the alpha and beta subunits of the AChR are minor membrane proteins in C2 cells, they were prominent among the proteins immunoprecipitated by antibodies to BiP, suggesting that BiP could play a role in their maturation or folding. In pulse-chase experiments in C2 cells, however, labeled alpha subunit formed a stable complex with BiP that was first detected after most of the alpha subunit had acquired toxin-binding activity and whose amount continued to increase for several hours. These kinetics are not compatible with a role for the BiP complex in the folding or assembly pathway of the AChR, and suggest that BiP is associated with a misfolded form of the subunit that is slowly degraded.

Development of MS Binding Assays targeting the binding site of MB327 at the nicotinic acetylcholine receptor

2018 ◽  
Vol 293 ◽  
pp. 172-183 ◽  
Author(s):  
S. Sichler ◽  
G. Höfner ◽  
S. Rappenglück ◽  
T. Wein ◽  
K.V. Niessen ◽  
...  
Keyword(s):  
Binding Site ◽  
Acetylcholine Receptor ◽  
Nicotinic Acetylcholine Receptor ◽  
Binding Assays ◽  
Nicotinic Acetylcholine

scholarly journals Genetic variants of C2 muscle cells that are defective in synthesis of the alpha-subunit of the acetylcholine receptor.

1987 ◽  
Vol 105 (3) ◽  
pp. 1329-1336 ◽  
Author(s):  
R Black ◽  
D Goldman ◽  
S Hochschwender ◽  
J Lindstrom ◽  
Z W Hall
Keyword(s):  
Acetylcholine Receptor ◽  
Genetic Variants ◽  
Muscle Cells ◽  
Binding Activity ◽  
Alpha Subunit ◽  
Beta Subunits ◽  
Wild Type ◽  
S1 Nuclease ◽  
Toxin Binding ◽  
Alpha Bungarotoxin

We have analyzed two genetic variants of C2 muscle cells that have reduced levels of binding activity for alpha-bungarotoxin and have found that both synthesize only low levels of the alpha-subunit of the acetylcholine receptor. In both variants the uptake of 22Na in response to carbachol is diminished in proportion to the reduction in toxin-binding activity. In addition, the kinetic and sedimentation properties of the residual toxin-binding activity in both is indistinguishable from that seen in wild-type cells. Immunoblotting experiments on extracts of the variants using subunit-specific antibodies to alpha- and beta-subunits of the acetylcholine receptor demonstrated that the beta-subunit was present, but failed to detect alpha-subunit. In both variants, the amount of alpha-subunit accumulated after a 5-min period of labeling with [35S]methionine was reduced by over 90%, leading to the conclusion that the alpha-subunit is synthesized at greatly reduced rates. Northern blot and S1 nuclease analysis showed no differences between the alpha-subunit mRNA in wild-type and variant cells.

scholarly journals Mapping of the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor.

1986 ◽  
Vol 83 (9) ◽  
pp. 3008-3011 ◽  
Author(s):  
D. Neumann ◽  
D. Barchan ◽  
A. Safran ◽  
J. M. Gershoni ◽  
S. Fuchs
Keyword(s):  
Binding Site ◽  
Acetylcholine Receptor ◽  
Alpha Subunit ◽  
Alpha Bungarotoxin
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