Photolabeling of membrane-bound Torpedo nicotinic acetylcholine receptor with the hydrophobic probe 3-trifluoromethyl-3-(m-(/sup 125/I)iodophenyl)diazirine
The hydrophobic, photoactivatable probe 3-trifluoromethyl-3-(m-(/sup 125/I)iodophenyl)diazirine ((/sup 125/I)TID) was used to label acetylcholine receptor rich membranes purified from Torpedo californica electric organ. All four subunits of the acetylcholine receptor (AChR) were found to incorporate label, with the ..gamma..-subunit incorporating approximately 4 times as much as each of the other subunits. Carbamylcholine, an agonist, and histrionicotoxin, a noncompetitive antagonist, both strongly inhibited labeling of all AChR subunits in a specific and dose-dependent manner. In contrast, the competitive antagonist ..cap alpha..-bungarotoxin and the noncompetitive antagonist phencyclidine had only modest effect on (/sup 125/I)TID labeling of the AChR. The regions of the AChR ..cap alpha..-subunit that incorporate (/sup 125/)TID were mapped by Staphylococcus aureus V8 protest digestion. The carbamylcholine-sensitive site of labeling was localized to a 20-kDa V8 cleavage fragment that begins at Ser-173 and is of sufficient length to contain the three hydrophobic regions M1, M2, and M3. A 10-kDa fragment beginning at Asn-339 and containing the hydrophobic region M4 also incorporated (/sup 125/I)TID but in a carbamylcholine-insensitive manner. Two further cleavage fragments, which together span about one-third of the ..cap alpha..-subunit amino terminus, incorporated no detectable (/sup 125/I)TID. The mapping results place constraints on suggested models of AChR subunit topology.
- Research Organization:
- Washington Univ. School of Medicine, St. Louis, MO (USA)
- OSTI ID:
- 6245151
- Journal Information:
- Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 27:24; ISSN BICHA
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
ACETYLCHOLINE
AMINES
AMINO ACIDS
AMMONIUM COMPOUNDS
ANIMALS
ANTIGENS
AUTONOMIC NERVOUS SYSTEM AGENTS
AZINES
BACTERIA
BETA DECAY RADIOISOTOPES
BIOCHEMISTRY
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL REACTIONS
CHEMISTRY
DAYS LIVING RADIOISOTOPES
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
ESTERS
HALOGENATION
HETEROCYCLIC COMPOUNDS
HYDROXY ACIDS
INTERMEDIATE MASS NUCLEI
INVERTEBRATES
IODINATION
IODINE 125
IODINE ISOTOPES
ISOTOPES
LABELLING
MATERIALS
MEMBRANE PROTEINS
MEMBRANES
MICROORGANISMS
MOLECULAR STRUCTURE
NEUROREGULATORS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PARASYMPATHOMIMETICS
PROTEINS
QUATERNARY COMPOUNDS
RADIOISOTOPES
RECEPTORS
SERINE
STAPHYLOCOCCUS
STOICHIOMETRY
TOXIC MATERIALS
TOXINS