Samson M, Osty J, Thibout H, Blondeau J P
Unité de Recherche sur la Glande Thyroïde et la Régulation Hormonale, Institut National de la Santé et de la Recherche Médicale, Le Kremlin-Bicêtre, France.
Eur J Endocrinol. 1996 May;134(5):660-8. doi: 10.1530/eje.0.1340660.
Triiodothyronine (T3) transport through the mammalian erythrocyte membrane is mediated by a transport system related to the aromatic amino acid transport system T. The T3-binding component of this transport system could be photolabeled with [125I]T3 as a 52-kD protein, and subsequently solubilized with non-ionic detergents. Upon purification by ion-exchange chromatography, the photolabeled 52-kD protein solubilized with octylglucoside (OG) resolved into several peaks, suggesting charge heterogeneity of labeled proteins. The saturable [125I]T3 binding to rat erythrocyte membranes was completely inhibited by non-ionic detergents at concentrations about 20 times lower than those that solubilized membrane. Therefore, detergent-free proteoliposomes were generated from the detergent-soluble extracts by treatment with a polystyrene adsorbent. Proteoliposomes prepared from OG-soluble extract contained the highest specific activity of T3 binding. The Kd of the T3 binding sites (4.5 nmol/l) and the competitive inhibition constant of tryptophan (120 mumol/l) were similar to those for native membranes. The photolabeling of the 52-kD protein in these proteoliposomes was prevented by tryptophan and T4, but not by leucine or the D-isomer of T3, corresponding to the transport specificity of system T. The 52-kD protein solubilized with OG from native membranes was partially purified by ion-exchange chromatography. The 52-kD protein was detected by photoaffinity labeling in the purified fraction only after addition of erythrocyte membrane phospholipids to generate proteoliposomes. This indicates that the association of 52-kD protein with phospholipids is critical for T3 binding.
三碘甲状腺原氨酸（T3）通过哺乳动物红细胞膜的转运是由一种与芳香族氨基酸转运系统T相关的转运系统介导的。该转运系统的T3结合成分可用[125I]T3光标记为一种52-kD蛋白，随后用非离子去污剂溶解。经离子交换色谱纯化后，用辛基葡糖苷（OG）溶解的光标记52-kD蛋白分离成几个峰，表明标记蛋白存在电荷异质性。非离子去污剂在浓度比溶解膜的浓度低约20倍时，就能完全抑制[125I]T3与大鼠红细胞膜的可饱和结合。因此，通过用聚苯乙烯吸附剂处理去污剂可溶提取物，制备了无去污剂的蛋白脂质体。由OG可溶提取物制备的蛋白脂质体具有最高的T3结合比活性。T3结合位点的解离常数（Kd）（4.5 nmol/l）和色氨酸的竞争抑制常数（120 μmol/l）与天然膜的相似。色氨酸和T4可阻止这些蛋白脂质体中52-kD蛋白的光标记，但亮氨酸或T3的D-异构体则不能，这与系统T的转运特异性一致。用OG从天然膜中溶解的52-kD蛋白经离子交换色谱部分纯化。只有在加入红细胞膜磷脂以生成蛋白脂质体后，才能在纯化组分中通过光亲和标记检测到52-kD蛋白。这表明52-kD蛋白与磷脂的结合对T3结合至关重要。
