Solubilization of a guanine nucleotide-sensitive form of the P2Y-purinergic receptor

Journal Article · Mon Jul 01 04:00:00 EDT 1991 · Molecular Pharmacology; (United States)

OSTI ID:5451645

Jeffs, R A; Cooper, C L; Harden, T K ^[1]

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1. Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill (USA)

P2Y-Purinergic receptors were solubilized from turkey erythrocyte plasma membranes with the nonionic detergent digitonin. Adenosine 5{prime}-O-(2-(35S)thiodiphosphate) ((35S)ADP {beta} S) labeled a single population of soluble high affinity sites (Kd = 12.9 nM; Bmax = 4.5 pmol/mg of protein) in an equilibrium binding assay; adenine nucleotide analogs competitively inhibited (35S)ADP {beta} S binding with a rank order of potency consistent with that for P2Y-purinergic receptors. Radioligand binding to solubilized P2Y-purinergic receptors was noncompetitively inhibited by guanine nucleotides with a rank order of potency that was in agreement with the potency order observed for guanine nucleotide-mediated inhibition of (35S)ADP {beta} S binding in purified turkey erythrocyte plasma membranes. The rate constant for dissociation of (35S)ADP beta S from solubilized receptors was increased 2.3-fold by guanosine 5{prime}-O-(3-thiotriphosphate) (GTP {gamma} S). Plasma membrane P2Y-purinergic receptors were labeled with (35S)ADP {beta} S or covalently labeled with the photoaffinity probe 3{prime}-O-(4-benzoyl)benzoyl adenosine 5{prime}-({alpha}-32P)triphosphate (({alpha}-32P)BzATP) before solubilization and gel filtration chromatography on Superose 12. (35S)ADP {beta} S- or (alpha-32P)BzATP-labeled species eluted as a single peak of radioactivity of apparent Mr greater than or equal to 300,000. Incubation of the Mr greater than or equal to 300,000 protein species with GTP {gamma} S before rechromatography resulted in loss of labeling of proteins by (35S)ADP {beta} S and a shift in apparent size of the covalently ({alpha}-32P)BzATP-labeled species to a single peak of radioactivity of approximate Mr 70,000. These results suggest that a P2Y-purinergic receptor-guanine nucleotide regulatory protein complex is stable to membrane solubilization with digitonin, even in the absence of prebound agonist.

• OSTI ID: 5451645
• Journal Information: Molecular Pharmacology; (United States), Journal Name: Molecular Pharmacology; (United States) Vol. 40:1; ISSN 0026-895X; ISSN MOPMA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ADP
ANIMALS
AUTONOMIC NERVOUS SYSTEM AGENTS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL MATERIALS
BIRDS
BLOOD
BLOOD CELLS
BODY FLUIDS
CELL CONSTITUENTS
CELL MEMBRANES
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
DRUGS
ERYTHROCYTES
EVEN-ODD NUCLEI
FOWL
GEL PERMEATION CHROMATOGRAPHY
INHIBITION
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
MATERIALS
MEMBRANE PROTEINS
MEMBRANES
MOLECULAR WEIGHT
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
RECEPTORS
SEPARATION PROCESSES
SOLUBILITY
SULFUR 35
SULFUR ISOTOPES
SYMPATHOMIMETICS
TRACER TECHNIQUES
VERTEBRATES