Purification and characterization of prostacyclin receptor in mastocytoma cells

肥大细胞瘤细胞中前列环素受体的纯化和表征

基本信息

批准号：
03671048
负责人：
NEGISHI Manabu
金额：
$ 1.28万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

项目摘要

The prostacyclin (PGI_2) receptor of mouse mastocytoma P-815 cells was characterized by photoaffinity labeling with the stable PGI_2 analogue [15-^3H_1]-19-(3-azidophenyl)-20-norisocarbacyclin ([^3H]APNIC) used as a potential photoaffinity probe for the receptor. [^3H]APNIC bound to the mastocytoma membrane with high affinity and in a saturable manner. Scatchard plot analysis indicated a single binding site with a Kd of 4.7 nM and a Bmax of 0.58 pmol/mg protein. The binding of [^3H]APNIC was dose dependently inhibited by APNIC and iloprost, another stable PGI_2 agonist, and to a much lessor extent by PGE_2. The binding of the radioligand showed sensitivity to the guanine nucleotide guanosins 5'-OMICRON-(3-thiotriphosphate) (GTPgammaS). Photolysis of [^3H]APNIC-prelabeled membranes resulted in incorporation of radiolabel into a protein of approximately 43 kDa. Photolabeling was inhibited by PGI_2 agonists and other prostaglandins with specificity for the PGI_2 receptor and was modulated by GTPgammaS. A protein of approximately 45 kDa was also labeled by [^3H]APNIC in the membrane of porcine platelets, membranes that are known to be abundant in PGI_2 receptors. These results demonstrated that [^3H]APNIC specifically labels a protein that may represent the PGI_2 receptor and that this radioprobe will be a useful reagent for further characterization and purification of the PGI_2 receptor.

小鼠肥大细胞瘤 P-815 细胞的前列环素 (PGI_2) 受体通过使用稳定的 PGI_2 类似物 [15-^3H_1]-19-(3-叠氮苯基)-20-降异碳环素 ([^3H]APNIC) 进行光亲和标记来表征。受体的潜在光亲和探针。 [^3H]APNIC 以高亲和力并以饱和方式与肥大细胞瘤膜结合。 Scatchard 图分析表明单个结合位点的 Kd 为 4.7 nM，Bmax 为 0.58 pmol/mg 蛋白质。 [^3H]APNIC的结合受到APNIC和伊洛前列素（另一种稳定的PGI_2激动剂）的剂量依赖性抑制，并且受到PGE_2的较小程度的抑制。放射性配体的结合显示出对鸟嘌呤核苷酸鸟苷 5'-OMICRON-(3-硫代三磷酸) (GTPgammaS) 的敏感性。 [^3H]APNIC 预标记膜的光解导致放射性标记掺入约 43 kDa 的蛋白质中。光标记受到 PGI_2 激动剂和其他对 PGI_2 受体具有特异性的前列腺素的抑制，并受到 GTPgammaS 的调节。猪血小板膜上的 [^3H]APNIC 也标记了大约 45 kDa 的蛋白质，已知这些膜富含 PGI_2 受体。这些结果表明，[^3H]APNIC 特异性标记了可能代表 PGI_2 受体的蛋白质，并且该放射性探针将成为进一步表征和纯化 PGI_2 受体的有用试剂。