Kane M A, Aguayo S M, Portanova L B, Ross S E, Holley M, Kelley K, Miller Y E
Medical Oncology Section, Denver Veterans Affairs Medical Center, Colorado 80220.
J Biol Chem. 1991 May 25;266(15):9486-93.
Purification of the gastrin-releasing peptide (GRP) or bombesin receptor has proved elusive in part due to technical difficulties. In the present studies, the problem of oxidized radioligand was avoided by the use of 125I-GRP, which was verified to be not oxidized by high performance liquid chromatography. Specific 125I-GRP binding (at 0 degrees C) to intact human small cell lung carcinoma NCI-H345 cells which had been subjected to a dilute acid wash was 6 fmol/10(6) cells. Inhibition of GRP degradation by human H345 cell membranes through the use of phenanthroline or phosphoramidon permitted the development of binding assays for the GRP receptor in detergent-solubilized crude membrane preparations. The solubilized GRP receptor exhibited saturable, high affinity (KD = 1.3 nM), temperature-dependent specific binding averaging 402 +/- 65 fmol/mg protein (mean +/- S.E. for eight separate membrane preparations with 125I-GRP concentration = 3 nM), with a Bmax = 434 fmol/mg protein using a gel filtration binding assay. That the GRP receptor had been solubilized was demonstrated by its failure to pellet when centrifuged at 100,000 x g for 60 min, its passage through a 0.22-micron filter without loss of binding activity, and its elution in the void volume of a Sephadex G-50 gel filtration column, but within the inclusion volume of a Sephacryl S-200 column (Ve/V0 = 1.1). Isolation of the GRP receptor from human H345 cell-solubilized membranes was achieved by ligand affinity chromatography. A unique 70-kDa band on silver-stained reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis was reproducibly eluted from GRP14-27 affinity columns by an acidic high salt buffer, but binding activity was denatured by these conditions. The protein nature of the GRP receptor was demonstrated by its sensitivity to proteases after isolation. In addition, two unique bands of 65 and 70 kDa were eluted from the GRP14-27 affinity column with GRP14-27 in neutral buffer, and this eluate possessed specific 125I-GRP binding with a stoichiometry of approximately 1:1. Thus, reported here is the isolation of a functional membrane-associated, saturable, high affinity GRP receptor with temperature-dependent binding from the solubilized membranes of human H345 cells.
胃泌素释放肽（GRP）或蛙皮素受体的纯化工作因技术难题而进展不顺。在本研究中，通过使用125I-GRP避免了氧化放射性配体的问题，经高效液相色谱验证其未被氧化。对经稀酸洗处理的完整人小细胞肺癌NCI-H345细胞，在0℃下125I-GRP的特异性结合量为6 fmol/10(6)个细胞。通过使用菲咯啉或磷酰胺抑制人H345细胞膜对GRP的降解，得以开发用于测定去污剂增溶的粗膜制剂中GRP受体的结合试验。增溶的GRP受体表现出可饱和的、高亲和力（KD = 1.3 nM）、温度依赖性的特异性结合，平均为402±65 fmol/mg蛋白质（八个单独膜制剂的平均值±标准误，125I-GRP浓度 = 3 nM），使用凝胶过滤结合试验时Bmax = 434 fmol/mg蛋白质。GRP受体已被增溶可通过以下几点证明：在100,000×g离心60分钟后其未沉淀、通过0.22微米滤膜后结合活性未丧失、在Sephadex G-50凝胶过滤柱的空体积中洗脱，但在Sephacryl S-200柱的包容体积内洗脱（Ve/V0 = 1.1）。通过配体亲和色谱从人H345细胞增溶膜中分离出GRP受体。在还原型十二烷基硫酸钠-聚丙烯酰胺凝胶电泳银染上一条独特的70 kDa条带可被酸性高盐缓冲液从GRP14-27亲和柱上重复洗脱下来，但这些条件会使结合活性变性。GRP受体的蛋白质性质在分离后通过其对蛋白酶的敏感性得以证明。此外，在中性缓冲液中用GRP14-27从GRP14-27亲和柱上洗脱下来两条独特的65 kDa和70 kDa条带，该洗脱液具有特异性125I-GRP结合，化学计量比约为1:1。因此，本文报道了从人H345细胞的增溶膜中分离出一种具有功能的膜相关、可饱和、高亲和力且具有温度依赖性结合的GRP受体。
