Solubilization and molecular size determination of the P2x purinoceptor from rat vas deferens.
Membranes of the rat vas deferens were shown to contain a high density of binding sites for [3H] alpha, beta-methylene ATP ([3H] alpha, beta-MeATP), a ligand selective for the P2X purinoceptor. Analysis demonstrated two classes, of high affinity (Kd = 1.8 nM, Bmax (maximum density) = 9.3 pmol/mg of protein) and of low affinity (Kd = 34 nM, Bmax = 29 pmol/mg of protein). The high affinity [3H] alpha, beta-MeATP binding sites were successfully solubilized with 2% digitonin: the Kd was then 1.6 nM. Both the association and dissociation of the receptor-ligand complex were rapid (half-time for association = 6.5 min). The rank order of potency of purinergic ligands in displacing [3H] alpha, beta-MeATP binding from the solubilized preparation was in accord with the pharmacological criteria for P2X purinoceptors. The receptor-detergent complex was separated by sucrose gradient ultracentrifugation from the ATPase enzymes also present in the preparation. The sedimentation coefficient of the receptor-detergent complex was 12.1 S. It was shown that [3H] alpha, beta-MeATP can function as a photoaffinity labeling reagent upon exposure to ultraviolet light; in the rat vas deferens membranes, it thus became cross-linked in a specific manner to a polypeptide of apparent molecular mass = 62,000 daltons, proposed to be the ligand-binding subunit of the functional P2X purinoceptor.
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Molecular Neurobiology Unit, Medical Research Council Centre, Cambridge, United Kingdom.
Simon
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Burnstock
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Barnard
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EA
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D004072:Digitonin; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D011869:Radioligand Assay; D051381:Rats; D011919:Rats, Inbred Strains; D011983:Receptors, Purinergic / Q000302:isolation & purification* / Q000378:metabolism*; D014316:Tritium; D014649:Vas Deferens / Q000378:metabolism*
D000345:Affinity Labels; D011983:Receptors, Purinergic; D014316:Tritium; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D004072:Digitonin; C002630:alpha,beta-methyleneadenosine 5'-triphosphate