ALPHA-BUNGAROTOXIN BINDS TO HUMAN ACETYLCHOLINE-RECEPTOR ALPHA-SUBUNIT PEPTIDE-185-199 IN SOLUTION AND SOLID-PHASE BUT NOT TO PEPTIDE-125-147 AND PEPTIDE-389-409

被引:34
作者
GRIESMANN, GE
MCCORMICK, DJ
DEAIZPURUA, HJ
LENNON, VA
机构
[1] MAYO CLIN & MAYO FDN, DEPT IMMUNOL,NEUROIMMUNOL LAB,ROOM 828, GUGGENHEIM BLDG, ROCHESTER, MN 55905 USA
[2] MAYO CLIN & MAYO FDN, DEPT NEUROL, ROCHESTER, MN 55905 USA
[3] MAYO CLIN & MAYO FDN, DEPT BIOCHEM & MOLEC BIOL, ROCHESTER, MN 55905 USA
关键词
Nicotinic acetylcholine receptor; Synthetic peptides; α‐Bungarotoxin;
D O I
10.1111/j.1471-4159.1990.tb01202.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Abstract: The nicotinic acetylcholine receptor (AChR) of human skeletal muscle has a reducible disulfide bond near the neurotransmitter binding site in each of its α‐subunits. By testing a panel of overlapping synthetic peptides encompassing the α‐subunit segment 177–208 (containing cysteines 192 and 193) we found that specific binding of 125I‐labelled α‐bungarotoxin (α‐BTx) was maximal in the region 185–199. Binding was inhibited by unlabelled α‐BTx d‐tubocurarine > atropine > carbamylcholine. Peptide 193–208 did not bind α‐BTx, whereas 177–192 retained 40% binding activity. Peptides corresponding to regions 125–147 (containing cysteines 128 and 142) and 389–409, or peptides unrelated to sequences of the AChR failed to bind α‐BTx. No peptide bound 125I‐α‐labelled parathyroid hormone. The apparent affinity (KD) of α‐BTx binding to immobilized peptides 181–199 and 185–199 was ∼25 μM and 80 μM, respectively, in comparison with α‐BTx binding to native Torpedo ACh receptor (apparent KD∼0.5 nM). In solution phase, both peptides effectively competed with solubilized native human AChR for binding of α‐BTx, and peptide 185–199 showed little evidence of dissociation after 24 h. Peptides that bound α‐BTx did so when sulfhydryls were reduced. Cysteine modification, by N‐ethylmaleimide or acetamidomethylation, abolished α‐BTx‐binding activity. The data implicate the region of cysteines 192 and 193 in the binding of neurotransmitter to the human receptor. Copyright © 1990, Wiley Blackwell. All rights reserved
引用
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页码:1541 / 1547
页数:7
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