Gimpl G, Gerstberger R, Mauss U, Klotz K N, Lang R E
Department of Pharmacology, University of Heidelberg, Federal Republic of Germany.
J Biol Chem. 1990 Oct 25;265(30):18142-7.
Active neuropeptide Y receptors were solubilized from rabbit kidney membranes using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). In membrane fragments and soluble extracts neuropeptide Y binding was time dependent, saturable, reversible, and of high affinity. Scatchard analysis of equilibrium binding data indicated a single class of binding sites with respective KD and Bmax values of 0.09 nM and 530 fmol/mg of protein for the membrane-bound receptors and 0.10 nM and 1585 fmol/mg of protein for the soluble receptors. Neuropeptide Y binding was specifically inhibited by the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) in a concentration-dependent manner, with IC50 values of 28 and 0.14 microM for membrane-bound and soluble receptors, respectively, suggesting that neuropeptide Y receptors are functionally coupled to GTP-binding regulatory proteins. Cross-linking studies were performed with the heterobifunctional N-hydroxysuccinimidyl-4-azidobenzoate and the monofunctional neuropeptide Y derivative, azidobenzoyl and led to the identification of a 100 kDa peptide that should represent the covalently labeled neuropeptide Y receptor.
使用两性离子去污剂3-[(3-胆酰胺丙基)二甲基铵]-1-丙烷磺酸(CHAPS)从兔肾膜中溶解活性神经肽Y受体。在膜片段和可溶性提取物中，神经肽Y结合具有时间依赖性、饱和性、可逆性且亲和力高。对平衡结合数据进行Scatchard分析表明，对于膜结合受体，存在一类结合位点，其KD和Bmax值分别为0.09 nM和530 fmol/mg蛋白质；对于可溶性受体，KD和Bmax值分别为0.10 nM和1585 fmol/mg蛋白质。神经肽Y结合受到不可水解的GTP类似物鸟苷5'-O-(3-硫代三磷酸)的特异性抑制，且呈浓度依赖性，膜结合受体和可溶性受体的IC50值分别为28和0.14 microM，这表明神经肽Y受体在功能上与GTP结合调节蛋白偶联。使用异双功能N-羟基琥珀酰亚胺-4-叠氮苯甲酸酯和单功能神经肽Y衍生物叠氮苯甲酰进行交联研究，鉴定出一种100 kDa的肽，它应该代表共价标记的神经肽Y受体。
