Kane M A, Kelley K, Ross S E, Portanova L B
Medical Oncology Section, Denver Veterans Affairs Medical Center, CO 80220.
Peptides. 1991 Mar-Apr;12(2):207-13. doi: 10.1016/0196-9781(91)90001-6.
The presence of a putative GRP receptor on rat pancreatic particulate membranes was demonstrated by covalent cross-linking to 125I-gastrin releasing peptide (GRP), which revealed a radioactive band with Mr = 80-90 kDa on reduced SDS-PAGE. Fresh rat pancreatic membranes contained a GRP receptor which was solubilized with Triton X-100 as assessed by its failure to sediment at 100,000 x g for one hour and its ability to pass through a 0.22 mu filter. When 125I-GRP binding was studied using Sephadex G50 gel filtration chromatography to separate bound from unbound ligand, substantial amounts of 125I-GRP binding were observed in rat crude solubilized pancreatic membranes, but essentially no specific binding was observed until the crude solubilized membranes were fractionated by ammonium sulfate precipitation. Specific 125I-GRP binding was 500, 700 and 1400 fmol/mg protein, respectively, in the 0-25%, 25-50% and 50-80% saturated ammonium sulfate fractions (125I-GRP concentration = 1 nM). Specific binding was temperature dependent, saturable and of high affinity, (KD = 2.3 nM). A unique 70 kDa band was visualized by silver staining of the SDS-PAGE of eluates of GRP(14-27) affinity gel compared with eluates of control affinity gels incubated with the 25-50% (NH4)2SO4 fraction. The lower Mr than that observed with covalent cross-linking may represent the binding subunit of a larger receptor protein. This ligand-affinity isolated protein is thus a good candidate for the GRP receptor, or the binding subunit of it, from normal rat pancreas.
通过与125I-胃泌素释放肽（GRP）共价交联，证实了大鼠胰腺微粒体膜上存在一种假定的GRP受体，在还原型SDS-PAGE上显示出一条Mr = 80 - 90 kDa的放射性条带。新鲜大鼠胰腺膜含有一种GRP受体，用Triton X-100可将其溶解，这可通过其在100,000 x g离心1小时后未沉淀以及能够通过0.22μm滤膜来评估。当使用Sephadex G50凝胶过滤色谱法研究125I-GRP结合以分离结合态与游离态配体时，在大鼠粗制溶解胰腺膜中观察到大量的125I-GRP结合，但在粗制溶解膜经硫酸铵沉淀分级分离之前基本未观察到特异性结合。在0 - 25%、25 - 50%和50 - 80%饱和度的硫酸铵级分中（125I-GRP浓度 = 1 nM），特异性125I-GRP结合分别为500、700和1400 fmol/mg蛋白。特异性结合是温度依赖性的、可饱和的且具有高亲和力（KD = 2.3 nM）。与用25 - 50%（NH4）2SO4级分孵育的对照亲和凝胶洗脱物相比，GRP(14 - 27)亲和凝胶洗脱物的SDS-PAGE经银染后可见一条独特的70 kDa条带。比共价交联观察到的Mr更低可能代表更大受体蛋白的结合亚基。因此，这种通过配体亲和分离的蛋白是正常大鼠胰腺中GRP受体或其结合亚基的良好候选者。
