el Battari A, Martin J M, Luis J, Pouzol O, Secchi J, Marvaldi J, Pichon J
Institut de Chimie Biologique, Centre National de la Recherche Scientifique Unité Associée n.202, Université de Provence, Marseille, France.
J Biol Chem. 1988 Nov 25;263(33):17685-9.
The non-ionic detergent n-octyl-beta-D-glucopyranoside was used to solubilize the VIP (vasoactive intestinal peptide) receptor from human colonic adenocarcinoma cell line HT29-D4. The binding of monoiodinated 125I-VIP to the solubilized receptor was specific, time-dependent, and reversible. Scatchard analysis of data obtained from competitive displacement of monoiodinated 125I-VIP by native VIP suggested the presence of two classes of VIP binding sites with Kd values of 0.32 and 46.7 nM. The binding capacities of these two classes were 1.7 x 10(10) and 30.2 x 10(10) sites/mg of proteins, respectively. The solubilized receptor retained the specificity of the human VIP receptor towards the peptides of the VIP/secretin/glucagon family. The order of potency in inhibiting monoiodinated 125I-VIP binding was VIP (IC50 = 1.0 x 10(-9) M) much greater than peptide histidine methionine amide (IC50 = 10(-7) M) greater than growth hormone-releasing factor (IC50 = 3 x 10(-7) M) greater than secretin (IC50 greater than 10(-6) M); glucagon had no effect on VIP binding. The reducing agent dithiothreitol inhibited in a dose-dependent manner the binding of 125I-VIP. Covalent cross-linking experiments between the solubilized receptor and 125I-VIP showed that after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography two major and one minor polypeptides of Mr 67,000, 72,000, and 83,000 were specifically labeled. When analyzed by gel filtration, the n-octyl-beta-D-glucopyranoside-solubilized 125I-VIP-receptor complex was resolved into two major peaks with molecular mass in the range of 60-70 and 270-300 kDa. Thus, the soluble form of the VIP receptor was probably a multimeric complex in which disulfide bonds may play an important role to hold the receptor in an active configuration.
使用非离子去污剂正辛基-β-D-吡喃葡萄糖苷来溶解人结肠腺癌细胞系HT29-D4中的血管活性肠肽（VIP）受体。单碘化的125I-VIP与溶解后的受体的结合具有特异性、时间依赖性且可逆。对天然VIP竞争性取代单碘化125I-VIP所获得的数据进行Scatchard分析表明存在两类VIP结合位点，其解离常数（Kd）值分别为0.32和46.7 nM。这两类结合位点的结合容量分别为1.7×10(10)和30.2×10(10)个位点/毫克蛋白质。溶解后的受体保留了人VIP受体对VIP/促胰液素/胰高血糖素家族肽段的特异性。抑制单碘化125I-VIP结合的效力顺序为：VIP（半数抑制浓度[IC50]=1.0×10(-9) M）远大于肽组氨酸甲硫氨酸酰胺（IC50=10(-7) M）大于生长激素释放因子（IC50=3×10(-7) M）大于促胰液素（IC50大于10(-6) M）；胰高血糖素对VIP结合无影响。还原剂二硫苏糖醇以剂量依赖性方式抑制125I-VIP的结合。溶解后的受体与125I-VIP之间的共价交联实验表明，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和放射自显影后，分子量分别为67,000、72,000和83,000的两条主要多肽和一条次要多肽被特异性标记。通过凝胶过滤分析时，正辛基-β-D-吡喃葡萄糖苷溶解的125I-VIP-受体复合物被解析为两个主要峰，分子量范围为60 - 70 kDa和270 - 300 kDa。因此，VIP受体的可溶形式可能是一种多聚体复合物，其中二硫键可能在将受体保持在活性构象中起重要作用。
