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

Journal Article · Mon Jul 01 00:00:00 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), Vol. 40:1; ISSN 0026-895X
• Country of Publication: United States
• Language: English

Related Subjects

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