Solubilization of a guanine nucleotide-sensitive form of the P2Y-purinergic receptor.
P2Y-Purinergic receptors were solubilized from turkey erythrocyte plasma membranes with the nonionic detergent digitonin. Adenosine 5'-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'-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'-O-(4-benzoyl)benzoyl adenosine 5'-[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.
Jeffs
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RA
Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill 27599.
Cooper
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CL
Harden
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TK
D000225:Adenine / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D004072:Digitonin / Q000737:chemistry; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D006150:Guanine Nucleotides / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D011983:Receptors, Purinergic / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D013462:Sulfur Radioisotopes; D013873:Thionucleotides / Q000378:metabolism; D014422:Turkeys
D006150:Guanine Nucleotides; D011983:Receptors, Purinergic; D013462:Sulfur Radioisotopes; D013873:Thionucleotides; C018444:adenosine 5'-O-(1-thiodiphosphate); D000244:Adenosine Diphosphate; D019204:GTP-Binding Proteins; D000225:Adenine; D004072:Digitonin