Mapping the benzodiazepine photoaffinity-labelling site with sequence-specific gamma-aminobutyric acidA-receptor antibodies.
The gamma-aminobutyric acidA (GABAA) receptor purified from adult bovine cerebral cortex was photoaffinity-labelled with the agonist benzodiazepine [3H]flunitrazepam and the radioactivity shown to be coincident with a band with Mr 53,000 that was recognized by three anti-(GABAA receptor alpha 1 subunit sequence)-specific antibodies. Complete and limited CNBr cleavage of the purified photoaffinity-labelled receptor was carried out. The products of this reaction were analysed for radioactivity, for immunoreactivity with anti-[alpha 1-(1-15)-peptide], anti-[alpha 1-(324-341)-peptide] and anti-[alpha 1-(413-429)-peptide] polyclonal antibodies and for carbohydrate by biotinylated concanavalin A lectin overlay. Complete CNBr cleavage gave a radioactive peptide with Mr 10,000-12,000 that was not recognized by the above-mentioned specific antisera. By using the deduced amino acid sequence of the alpha 1 subunit [Schofield, Darlison, Fujita, Burt, Stephenson, Rodriguez, Rhee, Ramachandran, Reale, Glencorse, Seeburg & Barnard (1987) Nature (London) 328, 221-227], it is proposed that the site of the benzodiazepine-agonist photoaffinity-labelling reaction does not lie within the amino acid sequences alpha 1 1-58 and alpha 1 149-429.
Stephenson
F A
FA
Department of Pharmaceutical Chemistry, School of Pharmacy, London, U.K.
Duggan
M J
MJ
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001569:Benzodiazepines / Q000378:metabolism*; D001665:Binding Sites; D015153:Blotting, Western; D002417:Cattle; D003488:Cyanogen Bromide; D005445:Flunitrazepam / Q000378:metabolism; D007158:Immunologic Techniques; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D010777:Photochemistry; D011963:Receptors, GABA-A / Q000276:immunology / Q000378:metabolism*
D000345:Affinity Labels; D010446:Peptide Fragments; D011963:Receptors, GABA-A; D001569:Benzodiazepines; D005445:Flunitrazepam; D003488:Cyanogen Bromide