The high-affinity sulfonylurea receptor: distribution, glycosylation, purification, and immunoprecipitation of two forms from endocrine and neuroendocrine cell lines.
The high-affinity sulfonylurea receptor: distribution, glycosylation, purification, and immunoprecipitation of two forms from endocrine and neuroendocrine cell lines.
复制标题
高亲和力磺酰脲受体：内分泌和神经内分泌细胞系两种形式的分布、糖基化、纯化和免疫沉淀。
DOI：
10.1021/bi960777y
复制
发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Aguilar-Bryan,L
中科院分区：
文献类型：
--
作者：
Nelson,DA;Bryan,J;Wechsler,S;Clement4th,JP;Aguilar-Bryan,L
磺酰脲类受体是ATP结合盒超家族的新成员，是ATP敏感性钾通道的组成部分。该蛋白质对于调节胰腺β细胞的胰岛素分泌至关重要，并且受体中的突变与家族性高胰岛素血症有关，家族性高胰岛素血症是一种以尽管存在严重低血糖但胰岛素释放不受调节为特征的疾病。磺酰脲受体存在于许多内分泌和神经内分泌细胞系的膜中，包括HIT-T15、RINm 5 f、αTC-6、AtT-20和GH 3细胞。RINm 5 f和αTC-6细胞中存在两种形式的受体，表观SDS凝胶分子量为140和150 kDa。这两种形式对格列本脲（一种抗糖尿病磺酰脲）的碘化衍生物具有同样高的亲和力，KD <3 nM。该受体是一种糖蛋白;用衣霉素处理RINm 5 f或αTC-6细胞可将140和150 kDa物质还原为单个约137 kDa蛋白。140和150 kDa的受体结合差异刀豆球蛋白A和麦胚凝集素，凝集素亲和层析是理想的受体纯化的初始阶段。在凝集素亲和层析后，可以应用与纯化140 kDa受体相同的方法来纯化150 kDa形式。通过Ni-琼脂糖色谱法纯化具有组氨酸标记的羧基末端的瞬时表达的受体，并且该变体用于证明140 kDa多肽是全长的。针对受体氨基末端的抗肽抗体和针对核苷酸结合折叠的抗体免疫沉淀两种受体形式。结果表明140和150 kDa受体是同一多肽链的差异糖基化形式。
The high-affinity sulfonylurea receptor, a novel member of the ATP-binding cassette superfamily, is one component of the ATP-sensitive K+channel. The protein is critical for regulation of insulin secretion from pancreatic β-cells, and mutations in the receptor have been linked to familial hyperinsulinemia, a disorder characterized by unregulated insulin release despite severe hypoglycemia. The sulfonylurea receptor is present in membranes from a number of endocrine and neuroendocrine cell lines, including HIT-Tl5, RINm5f, αTC-6, AtT-20, and GH3cells. Two forms of the receptor are present in RINm5f and αTC-6 cells, with apparent SDS gel molecular masses of 140 and 150 kDa. The two forms have equally high affinity,KD≈ 3 nM, for an iodinated derivative of glyburide, an anti-diabetic sulfonylurea. The receptor is a glycoprotein; treatment of RINm5f or αTC-6 cells with tunicamycin reduces the 140 and 150 kDa species to a single ∼137 kDa protein. The 140 and 150 kDa receptors bind differentially to concanavalin A and wheat germ agglutinin, and lectin-affinity chromatography is ideal for the initial stages of receptor purification. After lectin-affinity chromatography, the same methods can be applied for purifying the 150 kDa form as for the 140 kDa receptor. A transiently expressed receptor with a histidine-tagged carboxy-terminus was purified by Ni-agarose chromatography, and this variant was used to demonstrate that the 140 kDa polypeptide is full length. Anti-peptide antibodies directed against the amino-terminus of the receptor and antibodies against the nucleotide binding folds immunoprecipitate both receptor forms. The results indicate the 140 and 150 kDa receptors are differentially glycosylated forms of the same polypeptide chain.