Thibault G, Grove K L, Deschepper C F
Cell Biology of Hypertension Laboratory, Institut de Recherches Cliniques de Montréal, Québec, Canada.
Mol Pharmacol. 1995 Dec;48(6):1046-53.
Tyr(O)CNP is an analogue of C-type natriuretic peptide (CNP) with a tyrosine residue added to the NH2 terminus to allow its iodination. In the present study, the suitability of iodinated Tyr(O)CNP as a ligand was tested, and its potency was compared with that of other natural rat natriuretic peptides or structural analogues by radioligand binding experiments. Binding studies were performed on membranes of COS-1 cells transfected with expression plasmids for either rat natriuretic peptide receptor (NPR)-A, rat NPR-B, or bovine NPR-C. 125I-ANP(99-126) was used as a ligand to assess the binding characteristics of NPR-A and -C, and 125I-Tyr(O)CNP was used to study NPR-B. Binding associated to membranes of nontransfected COS cells was always < 3% of the total binding observed in membranes from cells transfected with receptor expression plasmids. Receptor densities in transfected cells ranged from 500 to 2500 fmol/mg of protein. High performance liquid chromatography and ionspray mass spectrometry analyses revealed that the reagents used in the course of iodination (lactoperoxidase, chloramine T, or N-chloromorpholine altered the structure of Tyr(O)CNP, most likely by changing the thiol of the Met17 residue into a sulfoxide. To further evaluate the usefulness of forms of iodinated Tyr(O)CNP on the cGMP responses in cells transfected with NPR-B. In conclusion, the suitability iodinated forms of Tyr(O)CNP as radioligands, we performed iodination of the peptide with cold iodine (Na-127I-). After purification by high performance liquid chromatography, three different modified peptides (i.e. Tyr(O)Met(O)17CNP, 127I-Tyr(O)Met(O)17CNP, and 127I2-Tyr(O)Met(O)17CNP) were recovered, and they were compared with CNP-22, Tyr(O)CNP, ANP(99-126), BNP-32, and des[Gin18, Ser19, Gly20, Leu21, Gly22]ANP(4-23) NH2 (c-ANP) for their ability to bind to transfected receptors. The binding affinity of Tyr(O)CNP for NPR-A and -B receptors is similar to that of CNP. However, oxidation of the Met17 residue into methionine sulfoxide reduces the affinity of the compound for NPR-B by > 10-fold, whereas the addition of one or two iodines did not further reduce its affinity. Similar results were obtained on evaluation of the ability of the oxidized form of monoiodinated Tyr(O)CNP on the cGMP responses in cells transfected with NPR-B. In conclusion, the suitability of iodinated forms of Tyr(O)CNP as radioligands for binding studies on rat NPR-B is not optimal, and the results of studies using such compounds for the detection, identification, and quantification of this receptor should be interpreted with caution.
酪氨酰（O）环磷酸鸟苷是C型利钠肽（CNP）的类似物，在其氨基末端添加了一个酪氨酸残基以便于碘化。在本研究中，测试了碘化酪氨酰（O）环磷酸鸟苷作为配体的适用性，并通过放射性配体结合实验将其效力与其他天然大鼠利钠肽或结构类似物进行比较。结合研究在转染了大鼠利钠肽受体（NPR）-A、大鼠NPR-B或牛NPR-C表达质粒的COS-1细胞膜上进行。使用125I-心房钠尿肽（99-126）作为配体来评估NPR-A和-C的结合特性，使用125I-酪氨酰（O）环磷酸鸟苷来研究NPR-B。与未转染的COS细胞膜相关的结合始终小于转染了受体表达质粒的细胞膜中观察到的总结合的3%。转染细胞中的受体密度范围为500至2500 fmol/mg蛋白质。高效液相色谱和离子喷雾质谱分析表明，碘化过程中使用的试剂（乳过氧化物酶、氯胺T或N-氯吗啉）改变了酪氨酰（O）环磷酸鸟苷的结构，很可能是通过将Met17残基的硫醇转变为亚砜。为了进一步评估碘化酪氨酰（O）环磷酸鸟苷形式对转染了NPR-B的细胞中环鸟苷酸反应的有用性。总之，为了评估酪氨酰（O）环磷酸鸟苷碘化形式作为放射性配体的适用性，我们用冷碘（Na-127I-）对该肽进行碘化。通过高效液相色谱纯化后，回收了三种不同的修饰肽（即酪氨酰（O）甲硫氨酸（O）17环磷酸鸟苷、127I-酪氨酰（O）甲硫氨酸（O）17环磷酸鸟苷和127I2-酪氨酰（O）甲硫氨酸（O）17环磷酸鸟苷），并将它们与CNP-22、酪氨酰（O）环磷酸鸟苷、心房钠尿肽（99-126）、脑钠肽-32和去[甘氨酸18、丝氨酸19甘氨酸20、亮氨酸21、甘氨酸22]心房钠尿肽（4-23）NH2（c-心房钠尿肽）比较它们与转染受体结合的能力。酪氨酰（O）环磷酸鸟苷对NPR-A和-B受体的结合亲和力与CNP相似。然而，将Met17残基氧化为甲硫氨酸亚砜会使该化合物对NPR-B的亲和力降低10倍以上，而添加一个或两个碘并未进一步降低其亲和力。在用转染了NPR-B的细胞评估单碘化酪氨酰（O）环磷酸鸟苷氧化形式对环鸟苷酸反应能力时也得到了类似结果。总之，碘化酪氨酰（O）环磷酸鸟苷形式作为大鼠NPR-B结合研究的放射性配体的适用性并非最佳，使用此类化合物进行该受体检测、鉴定和定量研究的结果应谨慎解释。
