An attempt to identify amino groups of Naja naja siamensis neurotoxin that interact with acetylcholine receptor by a comparison of their reactivities in free and receptor-bound neurotoxin.
An attempt to identify amino groups of Naja naja siamensis neurotoxin that interact with acetylcholine receptor by a comparison of their reactivities in free and receptor bound neurotoxin. Toxicon 21, 219-229, 1983--Free Naja naja siamensis neurotoxin was acetylated with non-radioactive and acetylcholine receptor-bound neurotoxin with radioactive acetic anhydride. The toxins from the two experiments were combined and the monoacetyl derivatives isolated by chromatography on Bio-Rex 70. The yields were determined by spectrophotometry and scintillation counting. To localize the acetyl group, a radioactive monoacetyl toxin was oxidized with performic acid, digested with trypsin and a peptide with the radioactive acetyl group was isolated by gel filtration on Sephadex G-25 and high voltage paper electrophoresis. Amino acid analysis indicated from which part of the molecule the peptide was derived. In free toxin, Ac-Lys 23 and 49 account for 56% and 12%, respectively, of the monoacetyl derivatives, and in bound toxin for only 25% and 8%. Lys 49 is as reactive as Ile 1 in free toxin and 50-150% more reactive than Lys 69, 35 and 12, but it has the lowest reactivity in bound toxin, being only about half as reactive as any of these three residues. The large decrease in reactivity of Lys 23 and 49 indicates that they interact with the receptor. The proximity of the receptor makes them less accessible to acetic anhydride. The reactivities are compared to that of Lys 12, which in free toxin has the least reactive amino group. The yield of Ac-Lys 23 relative to that of Ac-Lys 12 drops from 12.4 to 1.5, or by 88%, Lys 49, 2.6 and 0.5 (81%); Ac-Ile 1, 2.6 and 1.1 (58%); Ac-Lys 69, 1.9 and 0.9 (53%); Ac-Lys 35, 1.8 and 1.0 (44%). The drop in reactivity relative to that of Lys 12 indicates a real decrease, provided that Lys 12 does not become more reactive in bound toxin. This is unlikely, since sequence homology shows that Lys 12 corresponds to Lys 15 of the neurotoxin oxiana II of Naja naja oxiana, a residue known to interact with the receptor. Sequence homology also supports the conclusion that the drop in the reactivity of Ile 1 has the same cause. The receptor-binding region of the siamensis toxin is rather large, containing the residue Lys 23 and 49, Ile 1 and probably also Lys 69 and 35.
D000107:Acetylation; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D003039:Cobra Neurotoxin Proteins / Q000032:analysis* / Q000378:metabolism; D004546:Elapid Venoms / Q000032:analysis*; D011950:Receptors, Cholinergic / Q000378:metabolism*
D000596:Amino Acids; D003039:Cobra Neurotoxin Proteins; D004546:Elapid Venoms; D011950:Receptors, Cholinergic