Structure of peptide fragments of a cross-linked complex of [Lys(Abz){sup 26}]neurotoxin II from Naja naja oxiana with the nicotinic acetylcholine receptor from Torpedo californica
- M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Moscow (Russian Federation); and others
After irradiation of a complex of the nicotinic acetylcholine receptor (AChR) with iodinated [Lys(Abz){sup 26}]neurotoxin II, the labeled {delta}-subunit of AChR was isolated, and it was cleaved with the aid of LysC endoproteinase, the hydrolysate being separated by rfHPLC. In a mass-spectrometric analysis of the radioactive fraction, the peptide of the {delta}-subunit (M{sub r} 2593) was detected. By purification of the radioactive rfHPLC fraction with the aid of electrophoresis in tricine gel, three radioactive bands were obtained (M {approximately} 16, 10, and 8 kDa). Edman degradation gave for all of them the sequence of a fragment of the {delta}-subunit beginning from Phe{sup 148}. On further cleavage of the radioactive fraction within the gel by the action of AspN proteinase, followed by rfHPLC, the radioactive peak was eluted under conditions close to those for the elution of the single radioactive peptide 30-44 obtained by the successive cleavage of the [{sup 125}I] neurotoxin II by LysC/AspN proteinases. This result shows the presence of the corresponding neurotoxin fragment in the sample in which the above-mentioned sequence of the receptor was detected. Since no sequences of the neurotoxin were detected in the radioactive products of the cross-linkages in model experiments at the picomolar level, neurotoxin II and its fragments were investigated by Edman degradation at the picomole level and so was the influence of the p-azidobenzyl group and its photoactivation on the degradation. On the whole, the sequencing of neurotoxin II and its fragments containing photolabeled and iodinated residues took place with extremely low initial yields; a further fall in the yields was observed on the degradation of irradiated Lys{sup 26}-peptides. The results obtained explain the difficulties in the detection of the sequences of the neurotoxin in cross-linkage products available in amounts of only 10-20 pmole.
- OSTI ID:
- 114873
- Journal Information:
- Russian Journal of Bioorganic Chemistry, Vol. 20, Issue 10; Other Information: PBD: 10 Sep 1994; TN: Translated from Bioorganicheskaya Khimiya; 20: No. 10, 1047-1059(Oct 1994)
- Country of Publication:
- United States
- Language:
- English
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