| Autor: |
A S, Korotina, E V, Kriukova, E A, Azeeva, A F, Sheval'e, Iu N, Utkin, V I, Tsetlin |
| Rok vydání: |
2003 |
| Předmět: |
|
| Zdroj: |
Bioorganicheskaia khimiia. 29(4) |
| ISSN: |
0132-3423 |
| Popis: |
A sensitive nonradioactive method for detection of substances interacting with the neuronal nicotinic acetylcholine alpha 7-type receptor (AChR) was proposed. The method uses biotinylated alpha-cobratoxin (Bt-CTX) and is based on the ability of the N-terminal ligand-binding extracellular domain (LBED) of AChR to interact with alpha-cobratoxin (CTX) as does the whole receptor. LBED was produced by heterologic expression of a gene fragment of the alpha 7 subunit of AChR from the rat brain in Escherichia coli cells sorbed on wells of a 96-well plate and incubated with Bt-CTX. The specifically bound Bt-CTX was determined by staining with streptavidin-peroxidase complex. The ability of other compounds to interact with alpha 7-AChR was checked according to the degree with which they inhibit Bt-CTX binding to LBED. Nicotine, carbamylcholine, d-tubocurarin, anabaseine, conotoxin ImI, and neurotoxin II were used as model compounds. The sensitivity of this method was comparable with that of the radioligand method (up to 10 pmol). |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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