title: [3H]-(R)-N6-Phenylisopropyladenosine agonist binding to the solubilized A1 adenosine receptor from bovine epididymal spermatozoa.
abstract: The high-affinity agonist radioligand [3H]-(R)-N6-phenylisopropyladenosine ([3H]-R-PIA) was used to investigate agonist A1 adenosine receptors interactions in soluble preparations from bovine spermatozoa. The digitonin-solubilized receptor shows a high-affinity state with a Kd of 5.32+/-1.17 nM and a Bmax of 460+/-33 fmol/mg protein. The binding capacity, higher than that of the membrane-bound form, indicates that the soluble preparation is likely enriched with binding sites. In the presence of guanylyl-5'-imidodiphosphate (Gpp(NH)p), [3H]-R-PIA binds to the soluble receptor with a Kd of 7.97+/-1.44 nM and a Bmax of 400.8+/-27 fmol/mg protein. The radioligand rapidly dissociates with a K(-1) of 0.125 min(-1) although specific [3H]-R-PIA is still found in solubilized A1 receptor. The A1 agonist N6-cyclopentyladenosine differentiates two affinity states, whereas Gpp(NH)p shifts the agonist curve to the right and all the receptors are in the low-affinity state. In the presence of NaCl, the agonist still recognizes two affinity states with a lower affinity than that observed in the absence of NaCl. Analyses of sedimentation profiles show the existence of a population of A1 receptors tightly coupled to Gi, the pertussin toxin-sensitive component of the G protein family.
authors.affiliation: Dipartimento di Biologia Cellulare e Molecolare, Universitá di Perugia, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Minelli
authors.forename: C
authors.initials: C
authors.lastname: Allegrucci
authors.forename: I
authors.initials: I
authors.lastname: Mezzasoma
mesh_terms: D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D004822:Epididymis / Q000166:cytology; D005033:Ethylmaleimide / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000502:physiology; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D008024:Ligands; D008297:Male; D010660:Phenylisopropyladenosine / Q000378:metabolism*; D018047:Receptors, Purinergic P1 / Q000378:metabolism*; D015398:Signal Transduction; D013094:Spermatozoa / Q000378:metabolism*
chemical_list: D008024:Ligands; D018047:Receptors, Purinergic P1; D010660:Phenylisopropyladenosine; D006165:Guanylyl Imidodiphosphate; D019204:GTP-Binding Proteins; D005033:Ethylmaleimide