Shigeno C, Hiraki Y, Keutmann H T, Stern A M, Potts J T, Segre G V
Massachusetts General Hospital, Department of Medicine, Harvard Medical School, Boston, Massachusetts.
Anal Biochem. 1989 Jun;179(2):268-73. doi: 10.1016/0003-2697(89)90126-7.
Photoaffinity radiolabeling techniques have been widely used to characterize the properties of peptide hormone receptors. However, the identity of authentic receptors is often uncertain because many macromolecules are labeled. These ambiguities are due, in part, to the use of a heterogeneous mixture of photoreactive photoligands, many of which have no or low affinity for the relevant hormone receptor. In this report, we describe the synthesis, purification, and structural analysis of the photoreactive parathyroid hormone analog, [Nle8,Lys(N-epsilon-4-azido-2-nitrophenyl)13,Nle18,Tyr34]-bovine parathyroid hormone-(1-34)NH2. The sulfur-free, oxidation-resistant, synthetic analog of bovine parathyroid hormone (PTH), [Nle8,Nle18,Tyr34]bovine PTH-(1-34)NH2 (NlePTH), was reacted with 4-fluoro-3-nitrophenylazide under nonaqueous conditions to yield several derivatives which were separated by reverse-phase high-performance liquid chromatography and analyzed by amino acid compositional analysis, thin-layer chromatography, and ultraviolet and visible absorption spectroscopy. Among the NlePTH derivatives generated, one of the least hydrophobic was shown to retain the highest potency as assessed in the canine renal cortical membrane radioreceptor assay. Sequence analysis of this peptide, after it had been derivatized with 4-fluoro-3-nitro-[2,6-3H]phenylazide and purified to homogeneity, permitted us to determine that the structure of this analog is [Nle8,Lys(N-epsilon-4-azide-2-nitrophenyl)13,Nle18,Tyr34]bovine PTH-(1-34)NH2. We emphasize the importance of using photoreactive ligands which are purified and subjected to detailed chemical and biological analyses for characterizing the properties of parathyroid hormone receptors and receptors for other peptide hormones.
光亲和放射标记技术已被广泛用于表征肽类激素受体的特性。然而，由于许多大分子被标记，真正受体的身份往往不确定。这些模糊性部分归因于使用了光反应性光配体的异质混合物，其中许多对相关激素受体没有亲和力或亲和力很低。在本报告中，我们描述了光反应性甲状旁腺激素类似物[Nle8,Lys(N-ε-4-叠氮基-2-硝基苯基)13,Nle18,Tyr34]-牛甲状旁腺激素-(1-34)NH2的合成、纯化及结构分析。将无硫、抗氧化的牛甲状旁腺激素（PTH）合成类似物[Nle8,Nle18,Tyr34]牛PTH-(1-34)NH2（NlePTH）在非水条件下与4-氟-3-硝基苯叠氮反应，生成几种衍生物，通过反相高效液相色谱分离，并通过氨基酸组成分析、薄层色谱以及紫外和可见吸收光谱进行分析。在所生成的NlePTH衍生物中，经犬肾皮质膜放射受体测定评估，疏水性最低的一种显示保留了最高活性。用4-氟-3-硝基-[2,6-3H]苯叠氮对该肽进行衍生化并纯化至同质后进行序列分析，使我们能够确定该类似物的结构为[Nle8,Lys(N-ε-4-叠氮基-2-硝基苯基)13,Nle18,Tyr34]牛PTH-(1-34)NH2。我们强调使用经过纯化并进行详细化学和生物学分析的光反应性配体对于表征甲状旁腺激素受体及其他肽类激素受体特性的重要性。
