Muscarinic acetylcholine receptors: location of the ligand binding site
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-; TRN: 87-037185
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 46:6; Conference: 78. annual meeting of the American Society of Biological Chemists conference, Philadelphia, PA, USA, 7 Jun 1987
- Country of Publication:
- United States
- Language:
- English
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ACETYLCHOLINE
RECEPTORS
AMINO ACID SEQUENCE
FRACTIONATION
LABELLING
BIOCHEMICAL REACTION KINETICS
BRAIN
HELICAL CONFIGURATION
ION EXCHANGE CHROMATOGRAPHY
LIGANDS
MOLECULAR WEIGHT
PEPTIDE HYDROLASES
RATS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
AMINES
AMMONIUM COMPOUNDS
ANIMALS
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CHROMATOGRAPHY
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KINETICS
LABELLED COMPOUNDS
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MEMBRANE PROTEINS
MOLECULAR STRUCTURE
NERVOUS SYSTEM
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ORGANS
PARASYMPATHOMIMETICS
PROTEINS
QUATERNARY COMPOUNDS
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550201* - Biochemistry- Tracer Techniques