Muscarinic acetylcholine receptors: location of the ligand binding site

Conference · Fri May 01 04:00:00 EDT 1987 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:5904179

Hulme, E; Wheatley, M; Curtis, C; Birdsall, N

The key to understanding the pharmacological specificity of muscarinic acetylcholine receptors (mAChR's) is the location within the receptor sequence of the amino acid residues responsible for ligand binding. To approach this problem, they have purified mAChR's from rat brain to homogeneity by sequential ion-exchange chromatography, affinity chromatography and molecular weight fractionation. Following labelling of the binding site with an alkylating affinity label, /sup 3/H-propylbenzilycholine mustard aziridinium ion (/sup 3/H-PrBCM), the mAChR was digested with a lysine-specific endoproteinase, and a ladder of peptides of increasing molecular weight, each containing the glycosylated N-terminus, isolated by chromatography on wheat-germ agglutinin sepharose. The pattern of labelling showed that a residue in the peptides containing transmembrane helices 2 and/or 3 of the mAChR was alkylated. The linkage was cleaved by 1 M hydroxylamine, showing that /sup 3/H-PrBCM was attached to an acidic residue, whose properties strongly suggested it to be embedded in a hydrophobic intramembrane region of the mAChR. Examination of the cloned sequence of the mAChR reveals several candidate residues, the most likely of which is homologous to an aspartic acid residue thought to protonate the retinal Schiff's base in the congeneric protein rhodopsin.

• Research Organization: National Institute for Medical Research, London, England
• OSTI ID: 5904179
• Report Number(s): CONF-870644-
• Conference Information: Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 46:6
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACETYLCHOLINE
AMINES
AMINO ACID SEQUENCE
AMMONIUM COMPOUNDS
ANIMALS
AUTONOMIC NERVOUS SYSTEM AGENTS
BIOCHEMICAL REACTION KINETICS
BODY
BRAIN
CENTRAL NERVOUS SYSTEM
CHROMATOGRAPHY
CONFIGURATION
DRUGS
ENZYMES
ESTERS
FRACTIONATION
HELICAL CONFIGURATION
HYDROLASES
ION EXCHANGE CHROMATOGRAPHY
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
LABELLING
LIGANDS
MAMMALS
MEMBRANE PROTEINS
MOLECULAR STRUCTURE
MOLECULAR WEIGHT
NERVOUS SYSTEM
NEUROREGULATORS
ORGANIC COMPOUNDS
ORGANS
PARASYMPATHOMIMETICS
PEPTIDE HYDROLASES
PROTEINS
QUATERNARY COMPOUNDS
RATS
REACTION KINETICS
RECEPTORS
RODENTS
SEPARATION PROCESSES
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VERTEBRATES