Launay J M, Geoffroy C, Mutel V, Buckle M, Cesura A, Alouf J E, Da Prada M
Claude Bernard Neurochimie des Communications Cellulaires, Hôpital Saint-Louis, Paris, France.
J Biol Chem. 1992 Jun 5;267(16):11344-51.
A 68-kDa glycoprotein bearing the biological activity of the plasma membrane serotonin (5-hydroxytryptamine, 5-HT) transporter has been purified from human blood platelets, a classical cell model for the study of 5-HT uptake. After treatment of the whole platelet population or its plasma membrane fraction by sulfhydryl-dependent bacterial protein toxins or by digitonin, purification was reproducibly obtained by a one-step affinity chromatography using two different columns with 5-HT or 6-fluorotryptamine as ligands and elution by 5-HT or Na(+)-free buffer. The purified fraction migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band with an apparent molecular mass of 68 kDa and exhibited an apparent isoelectric point of 5.6-6.2. Two sialic acid residues were detected in the purified material. The purified glycoprotein bound the 5-HT uptake blocker [3H]paroxetine with a Kd (0.25 nM) similar to the one observed for intact human platelets. It also bound [3H] 5-HT but neither [3H]hydroxytetrabenazine nor [3H] ouabain, the respective markers of the granular monoamine transporter and of the Na+,K(+)-ATPase associated to the plasma membrane 5-HT transporter. 5-HT derivatives and 5-HT uptake inhibitors exhibited similar Ki values for 5-HT uptake and paroxetine binding in intact human platelets and in the purified glycoprotein. Under laser UV irradiation, 40% of this purified glycoprotein could be labeled by either [3H]paroxetine or [3H]cyanoimipramine. No labeling was detected with either [3H] gamma-aminobutyric acid or [3H]GBR 12783, the respective markers of gamma-aminobutyric acid and dopamine carriers. The purified 68-kDa protein is therefore likely to correspond at least to the binding domain of the 5-HT transporter located at the human platelet plasma membrane.
一种具有质膜血清素（5-羟色胺，5-HT）转运体生物活性的68 kDa糖蛋白已从人血小板中纯化出来，人血小板是研究5-HT摄取的经典细胞模型。在用巯基依赖性细菌蛋白毒素或洋地黄皂苷处理整个血小板群体或其质膜部分后，通过一步亲和色谱法可重复地获得纯化产物，该方法使用两根不同的以5-HT或6-氟色胺为配体的柱子，并通过5-HT或无钠缓冲液洗脱。纯化后的组分在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上迁移为一条表观分子量为68 kDa的单带，表观等电点为5.6 - 6.2。在纯化产物中检测到两个唾液酸残基。纯化后的糖蛋白与5-HT摄取阻滞剂[3H]帕罗西汀结合，其解离常数（Kd）（0.25 nM）与完整人血小板中观察到的相似。它也结合[3H] 5-HT，但不结合[3H]羟丁苯嗪和[3H]哇巴因，它们分别是颗粒单胺转运体和与质膜5-HT转运体相关的Na +,K(+)-ATP酶的标志物。5-HT衍生物和5-HT摄取抑制剂在完整人血小板和纯化后的糖蛋白中对5-HT摄取和帕罗西汀结合表现出相似的抑制常数（Ki）值。在激光紫外线照射下，这种纯化后的糖蛋白的40%可被[3H]帕罗西汀或[3H]氰咪帕明标记。用[3H]γ-氨基丁酸或[多巴胺载体的标志物[3H]GBR 12783均未检测到标记。因此，纯化后的68 kDa蛋白可能至少对应于人血小板质膜上5-HT转运体的结合结构域。
