Characterization of the thyroid Na+/I- symporter with an anti-COOH terminus antibody
Characterization of the thyroid Na+/I- symporter with an anti-COOH terminus antibody
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DOI：
10.1073/pnas.94.11.5568
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发表时间：
1997-05-27
影响因子：
11.1
通讯作者：
Carrasco, N
中科院分区：
文献类型：
--
作者：
Levy, O;Dai, G;Carrasco, N
Na+/I-同向转运蛋白（NIS）是一种质膜蛋白，在甲状腺中催化活性I-转运，这是甲状腺激素生物生成的第一步。最近，我们在实验室克隆了编码NIS的cDNA，并建立了二级结构模型，表明NIS是一种内源性膜蛋白(618个氨基酸；在这里，我们报道了一种位点导向的多克隆抗COOH末端NIS抗体（Ab）的产生，该抗体与大鼠甲状腺细胞系（FRTL-5）的膜部分中存在的约87 kDa的多肽发生免疫反应，模型预测的COOH末端的细胞质侧位置通过间接免疫荧光实验在渗透的FRTL-5细胞中使用抗COOH末端NIS Ab进行了证实。用肽基n -聚糖酶F处理FRTL-5细胞、非洲蟾卵母细胞和表达NIS的COS细胞的膜组分后，将大约87 kda的多肽转化为大约50 kda的多肽，其相对分子量与大肠杆菌中表达的NIS相同。Anti-NIS Ab免疫沉淀NIS前体分子（约56 kDa）和成熟分子（约87 kDa）。此外，体内循环促甲状腺激素水平与NIS表达之间存在直接相关性。
The Na+/I- symporter (NIS) is the plasma membrane protein that catalyzes active I- transport in the thyroid, the first step in thyroid hormone biogenesis. The cDNA encoding NIS was recently cloned in our laboratory and a secondary structure model proposed, suggesting that NIS is an intrinsic membrane protein (618 amino acids; approximate to 65.2 kDa predicted molecular mass) with 12 putative transmembrane domains, Here we report the generation of a site-directed polyclonal anti COOH terminus NIS antibody (Ab) that immunoreacts with a approximate to 87 kDa-polypeptide present in membrane fractions from a rat thyroid cell line (FRTL-5), The model-predicted cytosolic-side location of the COOH terminus was confirmed by indirect immunofluorescence experiments using anti-COOH terminus NIS Ab in permeabilized FRTL-5 cells, Immunoreactivity was competitively blocked by the presence of excess synthetic peptide, Treatment of membrane fractions from FRTL-5 cells, Xenopus laevis oocytes, and COS cells expressing NIS with peptidyl N-glycanase F converted the approximate to 87 kDa-polypeptide into a approximate to 50 kDa-species, the same relative molecular weight exhibited by NIS expressed in E. coli. Anti-NIS Ab immunoprecipitated both the NIS precursor molecule (approximate to 56 kDa) and the mature approximate to 87 kDa form, Furthermore, a direct correlation between circulating levels of thyroid-stimulating hormone and NIS expression in vivo was demonstrated.