Demoliou-Mason C, Epand R M
Biochemistry. 1982 Apr 27;21(9):1996-2004. doi: 10.1021/bi00538a004.
The photoreactive 125I-labeled glucagon-NAPS [125I-labeled 2-[2-nitro-4-azidophenyl)sulfenyl]-Trp25-glucagon] was used to label the glucagon receptor sites in rat liver plasma membranes. The proteins labeled were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with or without reduction with dithiothreitol. The photoaffinity peptide specifically labeled a number of bands with apparent molecular weights greater than 200000 and probably at least two protein bands in the molecular weight range 52000-70000. The relative amounts of radioactivity associated with these bands and their relative mobilities differed in samples from reduced and unreduced membranes. Their relative mobilities also differed with percent acrylamide cross-linking, suggesting a glycoprotein nature and the presence of intramolecular disulfide bonds. A nonspecifically labeled band with an apparent molecular weight of 27000-28000 also displayed a similar behavior. Photolabeling in the presence of 0.1 mM guanosine 5'-triphosphate (GTP) decreased the amount of radiolabeling of these bands, suggesting their involvement in the glucagon stimulation of adenylate cyclase. The photolabeled receptor in the membranes, solubilized with Lubrol-PX and fractionated on an Ultrogel AcA22 column, eluted with an apparent molecular weight of 200000-250000. Addition of GTP to the solubilized glucagon receptor of nonirradiated membranes caused complete dissociation of the complex. Gel electrophoresis of the partially purified radiolabeled receptor identified the same protein components observed in photolabeled membranes. These results indicate that the glucagon receptor is an oligomer probably composed of at least two different subunits that are linked together or greatly stabilized by disulfide bonds. They also show that 125I-labeled glucagon-NAPS can be used effectively to covalently label the putative glucagon receptor and thus aid in its further characterization.
光反应性125I标记的胰高血糖素-NAPS[125I标记的2-[2-硝基-4-叠氮基苯基）亚磺酰基]-Trp25-胰高血糖素]用于标记大鼠肝细胞膜中的胰高血糖素受体位点。对标记的蛋白质进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析，电泳过程中有的样品用二硫苏糖醇还原，有的则不还原。光亲和肽特异性标记了一些表观分子量大于200000的条带，可能还至少标记了两条分子量在52000-70000范围内的蛋白质条带。来自还原和未还原膜的样品中，与这些条带相关的放射性相对量及其相对迁移率有所不同。它们的相对迁移率也随丙烯酰胺交联百分比的不同而不同，这表明其具有糖蛋白性质且存在分子内二硫键。一条表观分子量为27000-28000的非特异性标记条带也表现出类似行为。在0.1 mM鸟苷5'-三磷酸（GTP）存在下进行光标记，会减少这些条带的放射性标记量，表明它们参与了胰高血糖素对腺苷酸环化酶的刺激作用。用Lubrol-PX溶解膜中的光标记受体，并在Ultrogel AcA22柱上进行分级分离，洗脱时表观分子量为200000-250000。向未辐照膜的溶解型胰高血糖素受体中添加GTP会导致复合物完全解离。对部分纯化的放射性标记受体进行凝胶电泳，鉴定出在光标记膜中观察到的相同蛋白质成分。这些结果表明，胰高血糖素受体是一种寡聚体，可能由至少两个不同的亚基组成，这些亚基通过二硫键连接在一起或高度稳定。它们还表明，125I标记的胰高血糖素-NAPS可有效用于共价标记假定的胰高血糖素受体，从而有助于对其进一步表征。
