Functional reconstitution of the bovine brain GABA sub A receptor from solubilized components

Journal Article · Tue Mar 21 00:00:00 EST 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00432a030· OSTI ID:5547159

Dunn, S M.J.; Martin, C R; Agey, M W; Miyazaki, R ^[1]

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1. Univ. of Iowa, Iowa City (USA)

The GABA{sub A}/benzodiazepine receptor has been solubilized from membrane preparations of bovine cerebral cortex and has been reconstituted, in a functionally active form, into phospholipid vesicles. In preliminary experiments, the receptor was labeled with the photoactive benzodiazepine ({sup 3}H)flunitrazepam prior to solubilization. A peptide of apparent molecular weight 53,500 was specifically labeled by this method, and this was used as a marker for the receptor during the reconstitution procedures. The labeled protein was solubilized with approximately 40% efficiency by 1% {beta}-octyl glucoside. Reconstitution was achieved by mixing the solubilized proteins with a 4:1 mixture of soybean asolectin and bovine brain phospholipids, followed by chromatography on Sephadex G-50-80 to remove detergent. The incorporation of the GABA{sub A} receptor into membrane vesicles has been verified by sucrose gradient centrifugation in which the ({sup 3}H)-flunitrazepam-labeled peptide comigrated with ({sup 14}C)phosphatidylcholine used as a lipid marker. Vesicles prepared without labeled markers retained the ability to bind both ({sup 3}H)flunitrazepam and the GABA analogue ({sup 3}H)muscimol. A novel fluorescence technique has been used to measure chloride transport mediated by the GABA{sub A} receptor in reconstituted vesicles. Flux was also blocked by pretreatment with the competitive GABA{sub A} receptor blocker bicuculline or with the noncompetitive GABA{sub A} receptor antagonist picrotoxin.

• OSTI ID: 5547159
• Journal Information: Biochemistry; (USA), Vol. 28:6; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
MEMBRANE TRANSPORT
BIOCHEMICAL REACTION KINETICS
RECEPTORS
MOLECULAR STRUCTURE
AMINOBUTYRIC ACID
BRAIN
CARBON 14 COMPOUNDS
CATTLE
CELL MEMBRANES
CROSS-LINKING
ELECTROPHORESIS
FLUORESCENCE
MOLECULAR WEIGHT
PEPTIDES
PHOSPHOLIPIDS
SODIUM CHLORIDES
TRITIUM COMPOUNDS
ALKALI METAL COMPOUNDS
AMINO ACIDS
ANIMALS
AUTONOMIC NERVOUS SYSTEM AGENTS
BODY
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CENTRAL NERVOUS SYSTEM
CHEMICAL REACTIONS
CHLORIDES
CHLORINE COMPOUNDS
DOMESTIC ANIMALS
DRUGS
ESTERS
HALIDES
HALOGEN COMPOUNDS
HYDROGEN COMPOUNDS
KINETICS
LABELLED COMPOUNDS
LIPIDS
LUMINESCENCE
MAMMALS
MEMBRANE PROTEINS
MEMBRANES
NERVOUS SYSTEM
NEUROREGULATORS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
ORGANS
POLYMERIZATION
PROTEINS
REACTION KINETICS
RUMINANTS
SODIUM COMPOUNDS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques