Section 21
Chapter 20,880

Distribution of -bungarotoxin binding sites over residues 173-204 of the subunit of the acetylcholine receptor

Wilson, PT.; Hawrot, E.; Lentz, TL.

Molecular Pharmacology 345: 643-650


ISSN/ISBN: 0026-895X
Accession: 020879151

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The binding of .alpha.-bungarotoxin to several synthetic peptides comprising different segments of the region 173-204 of the .alpha. subunit of the Torpedo acetylcholine receptor was investigated to further localize the neurotoxin-binding site on the primary sequences. When tested in a solid phase microwell assay system, a 32-amino acid peptide corresponding to residues 173-204 (32-mer) bound 125I-.alpha.-bungarotoxin with the same affinity (4.2 .times. 10-8 M as determined from IC50 values) as the isolated .alpha. subunit (4.6 .times. 10-8 M). The relative affinities of other antagonists (.alpha.-cobratoxin, d-tubocurarine) maintained the same rank order in this assay system as has been demonstrated with the intact receptor. Agonists competed with binding of toxin at millimolar concentrations but lost all rank order of potency. These findings demonstrate that peptide 173-204 contains many of the antagonist-binding determinants present on denatured .alpha. subunit but has lost specificity of agonist binding. To further localize the toxin-binding site, .alpha.-bungartoxin binding to seven shorter peptides corresponding to portions of the 32-mer was investigated. 125I-.alpha.-Bungarotoxin bound to .alpha. subunit peptides 179-192, 181-198, 185-196, 186-196, and 193-204, but not to .alpha. subunit peptides 173-180 and 194-204. In a second assay, all of the peptides competed with binding of 125I-acetylcholine receptor to immobilized .alpha.-bungarotoxin. The apparent affinity was highest for the 173-204 32-mer (1.4 .times. 10-7 M) and lowest for peptides 173-180 and 194-204 (> 10-4 M). The affinity of the other peptides was intermediate (.apprx. 10-5 M) and about 100-fold less than that of the 32-mer. The affinity of .alpha.-bungarotoxin was 3.5 .times. 10-10 M, of isolated, native acetylcholine receptor, 3.2 .times. 10-9 M, and of isolated denatured subunit, 1.2 .times. 10-8 M, with this assay.

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