Singh P, Draviam E, Guo Y S, Kurosky A
Department of Surgery, University of Texas Medical Branch, Galveston 77550.
Am J Physiol. 1990 May;258(5 Pt 1):G803-9. doi: 10.1152/ajpgi.1990.258.5.G803.
A biologically active, chemically defined, radioactive ligand was used for characterizing bombesin (BBS) receptors on rat pancreatic acinar cancer cells (AR42J). [Tyr4]BBS, iodinated with enzymobeads and fractionated by high-performance liquid chromatography, was monitored for biological activity as evidenced by gastrin release from perfused isolated rat stomach. The monoiodinated peptide peak was greater than 95% biologically active, with a specific activity of greater than 2,000 disintegrations.min-1.fmol-1. The maximum number of BBS receptors per cell were measured at 30 degrees C after 20-25 min of incubation; binding was submaximum at temperatures lower or higher than 30 degrees C. A single class of high-affinity binding sites (Kd = 1.77 +/- 0.21 nM) was identified for BBS on AR42J cells and nonspecific binding was less than 20-30% at all points. A total of 1.47 +/- 0.14 x 10(5) specific BBS binding sites per cell were measured that were specific for BBS and gastrin-releasing peptide (GRP) analogues. Iodinated GRP-(1-27) was cross-linked to BBS receptors on AR42J cells using several bifunctional cross-linking reagents followed by polyacrylamide gel electrophoresis of the solubilized receptor complex under reducing and nonreducing conditions. A densitometric analysis of the autoradiographs demonstrated the presence of an approximately 80- to 85-kDa molecular form of the receptor as a major component under both reducing and nonreducing conditions. These results indicated that the receptor molecule is a single subunit without multiple chains covalently attached by disulfide bonding.
一种具有生物活性、化学性质明确的放射性配体被用于表征大鼠胰腺腺癌细胞（AR42J）上的蛙皮素（BBS）受体。用酶珠碘化并通过高效液相色谱分离的[酪氨酸4]BBS，通过灌注分离的大鼠胃中胃泌素释放来监测其生物活性。单碘化肽峰的生物活性大于95%，比活性大于2000次衰变·分钟-1·飞摩尔-1。在30℃孵育20 - 25分钟后测量每个细胞的BBS受体最大数量；在低于或高于30℃的温度下结合未达到最大值。在AR42J细胞上鉴定出一类高亲和力结合位点（Kd = 1.77 +/- 0.21 nM），在所有点非特异性结合均小于20 - 30%。测量得出每个细胞共有1.47 +/- 0.14 x 10(5)个对BBS和胃泌素释放肽（GRP）类似物具有特异性的BBS特异性结合位点。使用几种双功能交联剂将碘化的GRP-(1 - 27)与AR42J细胞上的BBS受体交联，然后在还原和非还原条件下对溶解的受体复合物进行聚丙烯酰胺凝胶电泳。对放射自显影片的光密度分析表明，在还原和非还原条件下，受体的主要成分是一种分子量约为80 - 85 kDa的分子形式。这些结果表明，受体分子是一个单亚基，没有通过二硫键共价连接的多条链。
