Purification and reconstitution of serotonin receptors from bovine brain

Journal Article · Fri Apr 01 00:00:00 EST 1988 · Proceedings of the National Academy of Sciences of the United States of America; (USA)

DOI:https://doi.org/10.1073/pnas.85.7.2378· OSTI ID:6828881

Gallaher, T K; Wang, H H ^[1]

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1. Univ. of California, Santa Cruz (USA)

An affinity-chromatography column was used to isolate and purify 5-hydroxytryptamine (serotonin, 5-HT) receptors from bovine brain frontal cortex. The affinity ligand lysergic acid ethylamidoethylbromide was synthesized and coupled to an agarose matrix via a thioether bond. Receptors in the crude cortical membrane fragments were solubilized using 3-((3-cholamidopropyl)-dimethylammonio)-1-propanesulfonate (CHAPS) affinity purified, and reconstituted into lipid vesicles. ({sup 3}H)5-HT binding analysis indicates a single class of high-affinity binding site that was reconstituted. 5-Methoxytryptamine, a competitor for high-affinity serotonin sites, inhibited this binding and showed a K{sub i} of 27.4 nM. Ketanserin, a high-affinity ligand for 5-HT{sub 2} type receptors, was ineffective in displacing ({sup 3}H)5-HT binding at concentrations up to 4 {mu}M indicating a 5-HT{sub 1} receptor as the primary receptor type isolated. The average specific activity of 359 pmol/mg in the reconstituted fractions is an enrichment of 1,062-fold over crude membrane fragments. Sodium dodecylsulfate electrophoresis showed the presence of four proteins in the reconstituted vesicles with approximate relative M{sub r} values of 63,000, 70,000, 81,000, and 94,000.

• OSTI ID: 6828881
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (USA), Vol. 85:7; ISSN 0027-8424
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
NEUROREGULATORS
BIOCHEMICAL REACTION KINETICS
RECEPTORS
CROSS-LINKING
SEROTONIN
BRAIN
CATTLE
DOSE-RESPONSE RELATIONSHIPS
ELECTROPHORESIS
PURIFICATION
TRITIUM COMPOUNDS
TRYPTAMINES
AMINES
ANIMALS
AROMATICS
AUTONOMIC NERVOUS SYSTEM AGENTS
AZAARENES
AZOLES
BODY
CENTRAL NERVOUS SYSTEM
CHEMICAL REACTIONS
DOMESTIC ANIMALS
DRUGS
HETEROCYCLIC COMPOUNDS
HYDROGEN COMPOUNDS
HYDROXY COMPOUNDS
INDOLES
KINETICS
MAMMALS
MEMBRANE PROTEINS
NERVOUS SYSTEM
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANS
POLYMERIZATION
PROTEINS
PYRROLES
RADIOPROTECTIVE SUBSTANCES
REACTION KINETICS
RUMINANTS
SYMPATHOMIMETICS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques