Gabizon R, Schuldiner S
J Biol Chem. 1985 Mar 10;260(5):3001-5.
We have partially purified the amine transporter from bovine adrenal chromaffin granules in a single step utilizing affinity chromatography. A 5-hydroxytryptamine moiety has been coupled to a Sepharose 4B matrix in a position ortho to the hydroxyl group. When membranes solubilized with sodium cholate are chromatographed on the above matrix a 45,000 Mr polypeptide is highly enriched. The enrichment is dependent on the presence of the proper ligand on the matrix and is inhibited if the column is previously equilibrated with a soluble ligand. Enrichment of the above polypeptide is accompanied by an increase in the specific activity of the transporter as measured by its labeling by 4-azido-3-nitrophenylazo(5-hydroxytryptamine). The ability of reserpine, a competitive inhibitor of binding and transport, to inhibit labeling of the purified transporter correlates well with its known kinetic constants in the native membranes. The polypeptide purified is identical to the one previously identified as the putative transporter based on specific labeling by a photoaffinity label (Gabizon, R., Yetinzon, T., and Schuldiner, S. (1982) J. Biol. Chem. 257, 15145-15150). The results clearly support the contention that the 45,000 Mr peptide is the amine transporter or one of its subunits.
我们利用亲和色谱一步法部分纯化了牛肾上腺嗜铬颗粒中的胺转运体。5-羟色胺部分已连接到琼脂糖4B基质上羟基的邻位。当用胆酸钠溶解的膜在上述基质上进行色谱分析时，一种45000道尔顿的多肽高度富集。这种富集取决于基质上合适配体的存在，如果柱子事先用可溶性配体平衡，则会受到抑制。上述多肽的富集伴随着转运体比活性的增加，这是通过其被4-叠氮基-3-硝基苯偶氮（5-羟色胺）标记来测定的。利血平作为结合和转运的竞争性抑制剂，抑制纯化转运体标记的能力与其在天然膜中的已知动力学常数密切相关。纯化的多肽与先前基于光亲和标记的特异性标记被鉴定为假定转运体的多肽相同（加比松，R.，耶廷宗，T.，和舒尔迪纳，S.（1982年）《生物化学杂志》257，15145 - 15150）。结果清楚地支持了45000道尔顿的肽是胺转运体或其亚基之一的论点。
