Studies on PGE_1/PGI_2 receptors in platelets from patients with thrombotic disorders

血栓性疾病患者血小板中PGE_1/PGI_2受体的研究

• 批准号: 63571113
• 负责人: ANDO Yasuhiko
• 金额: $ 0.9万
• 依托单位: Tokai 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: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1989
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-63571113/
• 关键词: PGE_1; PGI_2 receptor; platelet membrane proteins; thrombotic disorders; adenylate cyclase; cAMP; PGI_2レセプタ-; adenylafe cyclase; cAMP; PGE_1; PGI_2レセプター

The purpose of this report is to study PGE_1/PGI_2 receptors in platelets from patients with thrombotic disorders.PGE_1/PGI_2 receptor proteins in the platelet membranes were purified according to the method of Dutta-Roy. Briefly, PGE_1/PGI_2 receptor proteins were solubilized by Triton X-100 in Tris-HCl buffer. pH 7.4 containing PMSF. The soluble membrane proteins were chromatographed on a DEAE-cellulofine column and the receptor activity of each fraction was assayed by microfiber technique. The active fractions eluted at 0.7m KCl were pooled. concentrated and further fractionated by gel filtration on Sephadex G 200. The eluates containing [^3H] PGE_1 binding activity emerged in a single peak. The active fractions were concentrated and the homogeneity of the purified receptor proteins were tested by SDS polyacrylamide gel electrophoresis by Laemmli's method. When the gels were stained with Coomassie Brilliant Blue. one main band with the molecular weight of 190 kd and a few additional bands with smaller molecular weights were observed. Substitution of Mono Q with FPLC System for DEAE cellulofine failed to purify the receptor protein to homogeneity. In the next experiments we are planning to produce mouse monoclonal antibody against the 190 kd receptor protein, and to quantify the amounts of the receptor protein in platelets from patients with thrombotic disorders by the use of this antibody.

本报告的目的是研究血栓性疾病患者血小板中PGE_1/PGI_2受体。采用Dutta-Roy法纯化血小板膜上的PGE_1/PGI_2受体蛋白。简单地说，PGE_1/PGI_2受体蛋白被Triton X-100溶解在Tris-HCl缓冲液中。pH 7.4含PMSF。在deae -纤维素纤维柱上对可溶性膜蛋白进行层析，并采用超纤维技术测定各组分的受体活性。以0.7m KCl洗脱的活性组分池化。在Sephadex g200上进行浓缩和进一步的凝胶过滤。含有[^3H] PGE_1结合活性的洗脱液以单峰形式出现。对活性组分进行浓缩，用SDS聚丙烯酰胺凝胶电泳（Laemmli’s法）检测纯化后受体蛋白的均匀性。当凝胶用考马斯亮蓝染色时。发现了一条分子量为190 kd的主条带和一些分子量较小的附加条带。用FPLC系统代替DEAE纤维素不能使受体蛋白纯化到均匀。在接下来的实验中，我们计划生产针对190kd受体蛋白的小鼠单克隆抗体，并通过使用该抗体来量化血栓性疾病患者血小板中受体蛋白的含量。