Beck-Sickinger A G, Wieland H A, Brunner J
Departement Pharmazie, Zurich, Switzerland.
J Recept Signal Transduct Res. 1995 Jan-Mar;15(1-4):473-85. doi: 10.3109/10799899509045234.
Five photoactive analogues of porcine neuropeptide Y (NPY), a 36 amino acid hormone of the pancreatic polypeptide family, have been synthesized by solid phase peptide synthesis method, Fmoc/tBu strategy and carefully characterized. The analogues contain the photoactivatable amino acid 4'-(3-trifluoromethyl)-3H-diazirine-3-yl-phenyl-alanine ((Tmd)Phe) individually at different positions (1, 20, 21, 27 or 36) instead of tyrosine in the wildtype sequence. Affinity to membranes prepared from SMS-KAN cells, which stably express the Y2 receptor has been investigated by measuring the displacement of 125I-Bolton Hunter-NPY. After incubation of the membranes with different concentrations of the crosslinker and subsequent photolysis, the specific binding of 125I-Bolton Hunter-NPY at those membranes was tested in order to quantify the crosslinking efficiency. Whereas [(Tmd)Phe20] NPY, [(Tmd)Phe21] NPY and [(Tmd)Phe27] NPY revealed highest affinity to the Y2 receptor, crosslinking was most efficient when Tyr36 was replaced by (Tmd)Phe. This is in good agreement with the previously suggested C-terminal binding site of neuropeptide Y.
猪神经肽Y（NPY）是一种由36个氨基酸组成的胰多肽家族激素，通过固相肽合成法、Fmoc/tBu策略合成了五种光活性类似物，并对其进行了详细表征。这些类似物在野生型序列的不同位置（1、20、21、27或36）分别含有光活化氨基酸4'-（3-三氟甲基）-3H-重氮丙啶-3-基-苯丙氨酸（（Tmd）Phe），取代了酪氨酸。通过测量125I-博尔顿·亨特-NPY的置换，研究了它们对稳定表达Y2受体的SMS-KAN细胞制备的膜的亲和力。在用不同浓度的交联剂孵育膜并随后进行光解后，测试了125I-博尔顿·亨特-NPY在这些膜上的特异性结合，以量化交联效率。虽然[（Tmd）Phe20]NPY、[（Tmd）Phe21]NPY和[（Tmd）Phe27]NPY对Y2受体显示出最高亲和力，但当Tyr36被（Tmd）Phe取代时，交联效率最高。这与先前提出的神经肽Y的C末端结合位点非常一致。
