Biochemical and immunological studies of the Muscarinic acetylcholine receptor
Muscarinic acetylcholine receptors were solubilized from bovine brain membranes with 3(3-cholamidopropyl)dimethylammonio)propanesulfonate (CHAPS). A combination of 10 mM CHAPS and 1 M NaCl solubilized 15-40% of the specific receptor binding sites from these membranes. The solubilized receptors displayed high affinity binding of the muscarinic antagonist, (/sup 3/H)quinuclidinyl benzilate with a K/sub D/ = 300 pM. In addition, the solubilized and retained guanyl nucleotide regulation of agonist binding characteristic of membrane bound receptors. Gel filtration experiments showed that solubilized receptors from cortex and cerebellum had different elution profiles. Analysis by sucrose density gradient centrifugation showed that receptors in the lower molecular weight peak sedimented with a coefficient of 5S. Receptors in the larger molecular weight peak sedimented to the bottom of the gradient. Attempts to purify receptors by chromatography on propylbenzilycholine Sepharose were unsuccessful. The technique used to attach the ligand to the solid support, however, was used to synthesize a PrBCM-BSA conjugate and the conjugate used as an antigen in the production of anti-ligand antibodies. Two anti-PrBCM monoclonal antibodies were isolated that recognize muscarinic but not nicotinic cholinergic ligands. The abilities of the antibodies to recognize other muscarinic ligands indicated the antibodies recognized a portion of PrBCM involved in binding to the receptor. Construction of an antibody affinity resin resulted in the purification of this fragment a minimum of 170 fold.
- Research Organization:
- Washington Univ., Seattle (USA)
- OSTI ID:
- 6936157
- Resource Relation:
- Other Information: Thesis
- Country of Publication:
- United States
- Language:
- English
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ACETYLCHOLINE
BIOCHEMICAL REACTION KINETICS
RECEPTORS
IMMUNOLOGY
BRAIN
CATTLE
CELL MEMBRANES
CENTRIFUGATION
FILTRATION
ION EXCHANGE CHROMATOGRAPHY
TRITIUM COMPOUNDS
AMINES
AMMONIUM COMPOUNDS
ANIMALS
AUTONOMIC NERVOUS SYSTEM AGENTS
BODY
CELL CONSTITUENTS
CENTRAL NERVOUS SYSTEM
CHROMATOGRAPHY
DOMESTIC ANIMALS
DRUGS
ESTERS
KINETICS
LABELLED COMPOUNDS
MAMMALS
MEMBRANE PROTEINS
MEMBRANES
NERVOUS SYSTEM
NEUROREGULATORS
ORGANIC COMPOUNDS
ORGANS
PARASYMPATHOMIMETICS
PROTEINS
QUATERNARY COMPOUNDS
REACTION KINETICS
RUMINANTS
SEPARATION PROCESSES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques