Jeffs R A, Cooper C L, Harden T K
Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill 27599.
Mol Pharmacol. 1991 Jul;40(1):85-92.
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.
使用非离子去污剂洋地黄皂苷从火鸡红细胞质膜中溶解P2Y嘌呤能受体。在平衡结合试验中，腺苷5'-O-(2-[35S]硫代二磷酸)（[35S]ADPβS）标记了单一群体的可溶性高亲和力位点（Kd = 12.9 nM；Bmax = 4.5 pmol/mg蛋白质）；腺嘌呤核苷酸类似物竞争性抑制[35S]ADPβS结合，其效力排序与P2Y嘌呤能受体一致。鸟嘌呤核苷酸对溶解的P2Y嘌呤能受体的放射性配体结合具有非竞争性抑制作用，其效力排序与在纯化的火鸡红细胞质膜中观察到的鸟嘌呤核苷酸介导的对[35S]ADPβS结合的抑制效力顺序一致。鸟苷5'-O-(3-硫代三磷酸)（GTPγS）使[35S]ADPβS从溶解受体上解离的速率常数增加了2.3倍。在溶解和Superose 12凝胶过滤色谱之前，质膜P2Y嘌呤能受体用[35S]ADPβS标记或用光亲和探针3'-O-(4-苯甲酰基)苯甲酰腺苷5'-[α-32P]三磷酸（[α-32P]BzATP）共价标记。[35S]ADPβS或[α-32P]BzATP标记的物质以单一放射性峰洗脱，表观Mr大于或等于300,000。在重新色谱之前，将表观Mr大于或等于300,000的蛋白质物种与GTPγS孵育，导致[35S]ADPβS对蛋白质的标记丢失，并且共价[α-32P]BzATP标记的物种的表观大小转移到单一放射性峰，表观Mr约为70,000。这些结果表明，即使在没有预结合激动剂的情况下，P2Y嘌呤能受体 - 鸟嘌呤核苷酸调节蛋白复合物对用洋地黄皂苷溶解膜也具有稳定性。
