Colás B, Valencia A M, Prieto J C, Arilla E
Department of Biochemistry and Molecular Biology, Medical School, University of Alcalá, Madrid, Spain.
Mol Cell Endocrinol. 1992 Oct;88(1-3):111-7. doi: 10.1016/0303-7207(92)90015-x.
Somatostatin (SS) receptors in membranes from ovine retinas were examined using 125I-Tyr11-SS as a ligand. Receptor binding was rapid, specific, saturable, reversible and dependent on temperature and membrane concentration. Conditions of apparent equilibrium were obtained at 25 degrees C after a 45 min incubation in the presence of about 0.25 mg membrane protein/ml. Native SS competitively inhibited the binding of 125I-Tyr11-SS in the range of 0.01-10 nM, and half-maximal inhibition was observed at 0.2 nM SS. Scatchard analysis of these data suggested the existence of a single population of SS receptors with a dissociation constant of 0.23 +/- 0.03 nM and a maximum binding capacity of 84 +/- 6 fmol/mg protein. The binding of 125I-Tyr11-SS was inhibited by various synthetic SS analogs in a dose-dependent manner whereas peptides unrelated to SS did not show practically any effect even at concentrations as high as 10(-6) M. SS receptor occupancy appears to be coupled to inhibition of adenylate cyclase activity by a guanine nucleotide-binding regulatory protein, as suggested by the facts that: (a) SS noncompetitively inhibited the stimulatory effect of vasoactive intestinal peptide (VIP) (3 x 10(-7) M) on membrane adenylate cyclase activity but it did not alter basal enzyme activity; and (b) the addition of guanosine 5'-triphosphate (GTP) (10(-5) M) decreased the specific binding of 125I-Tyr11-SS to 26.6% of the control value due to a decrease in SS receptor affinity. The present results support the hypothesis that SS may contribute to the physiological regulation of the functions of the retina.
使用125I-Tyr11-SS作为配体，对绵羊视网膜膜中的生长抑素（SS）受体进行了检测。受体结合迅速、具有特异性、可饱和、可逆且依赖于温度和膜浓度。在约0.25mg膜蛋白/ml存在的情况下，于25℃孵育45分钟后可达到表观平衡状态。天然SS在0.01-10nM范围内竞争性抑制125I-Tyr11-SS的结合，在0.2nM SS时观察到半数最大抑制。对这些数据进行Scatchard分析表明存在单一群体的SS受体，其解离常数为0.23±0.03nM，最大结合容量为84±6fmol/mg蛋白。125I-Tyr11-SS的结合受到各种合成SS类似物的剂量依赖性抑制，而与SS无关的肽即使在高达10(-6)M的浓度下也几乎没有任何作用。如以下事实所示，SS受体占据似乎通过鸟嘌呤核苷酸结合调节蛋白与腺苷酸环化酶活性的抑制相偶联：（a）SS非竞争性抑制血管活性肠肽（VIP）（3×10(-7)M）对膜腺苷酸环化酶活性的刺激作用，但不改变基础酶活性；（b）添加鸟苷5'-三磷酸（GTP）（10(-5)M）由于SS受体亲和力降低，使125I-Tyr11-SS的特异性结合降低至对照值的26.6%。目前的结果支持SS可能有助于视网膜功能生理调节的假说。
