Binding of a curarimimetic toxin from cobra venom to the nicotinic acetylcholine receptor. Interactions of six biotinyltoxin derivatives with receptor and avidin.
We have reacted N-hydroxysuccinimidyl biotin with the principal curarimimetic toxin in Naja naja siamensis venom, biotinylating each of the five lysine residues and the N-terminal isoleucine. The six monobiotinyl-toxins were isolated by ion-exchange chromatography, and the residue modified in each was identified by peptide mapping and amino acid analysis. We evaluated the role of each lysine in the binding of toxin to the acetylcholine receptor by measuring the affinity of each biotinyltoxin for receptor and by determining which biotinyltoxins could bind receptor and avidin simultaneously. The effect of biotinylation of each residue decreased the affinity of toxin for receptor in the order Lys 23 greater than Lys 49 greater than Lys 35 greater than Lys 69 congruent to Lys 12 greater than Ile 1. Biotinyltoxin modified either at Lys 12 or at Lys 69 is effective in cross-linking avidin to receptor, while biotinyltoxin modified at Lys 49 can form a low-affinity avidin-biotinyltoxin-receptor complex. Taken together, these results help define the surface of toxin that binds to receptor.
Lobel
P
P
Kao
P N
PN
Birken
S
S
Karlin
A
A
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001360:Avidin / Q000378:metabolism*; D001710:Biotin / Q000031:analogs & derivatives* / Q000378:metabolism; D004546:Elapid Venoms / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine; D008958:Models, Molecular; D003473:Neuromuscular Nondepolarizing Agents / Q000378:metabolism*; D010047:Ovalbumin / Q000031:analogs & derivatives*; D011487:Protein Conformation; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*
D000596:Amino Acids; D004546:Elapid Venoms; D003473:Neuromuscular Nondepolarizing Agents; D011978:Receptors, Nicotinic; D001360:Avidin; D001710:Biotin; D010047:Ovalbumin; D008239:Lysine