Gottschall P E, Tatsuno I, Arimura A
U.S.-Japan Biomedical Research Laboratories, Tulane University, Belle Chasse, Louisiana 70037.
FASEB J. 1991 Feb;5(2):194-9. doi: 10.1096/fasebj.5.2.1848519.
The goal of these experiments was to identify and characterize binding sites in the rat hypothalamus for the peptide, pituitary adenylate cyclase activating polypeptide (PACAP). The 27 amino acid form of PACAP (PACAP27) was used as the radiolabeled ligand in these experiments. Binding of [125I]PACAP27 to hypothalamic membrane preparations was rapid, reversible on addition of unlabeled peptide, and at least partially regulated by GTP. Nonhydrolyzable GTP analogs, guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S), guanosine-5'-(2-thiodiphosphate) (GDP beta S), and guanylylimidophosphate (GppNHp) also displaced [125I]PACAP27 binding to hypothalamic membrane preparations in a dose-dependent manner. The order of potency for the three analogs was GTP gamma S greater than GDP beta S greater than GppNHp. Both forms of the peptide, PACAP27 and PACAP38, were highly potent in displacing bound [125I]PACAP27, whereas VIP or PACAP(1-23) were unable to displace binding at concentrations of up to 500 nM. Scatchard analysis of the PACAP27 and PACAP38 displacement curves revealed that the fit of both curves was consistent with a single class of high-affinity binding sites, although the site exhibited a greater affinity for PACAP38 compared with PACAP27 (PACAP27 Kd = 1452 +/- 59 pM; PACAP38 Kd = 175 +/- 13 pM; Bmax 23.2 +/- 1.1 pmol/mg protein). The possibility of the existence of a class of binding sites with extremely low affinity cannot be discounted. After covalent cross-linking of [125I]PACAP27 with its receptor, the molecular weights of the complexes were estimated by electrophoresis and autoradiography. A major band of 60 Kd was evident when membranes were incubated with VIP or PACAP(1-23). Previous incubation with unlabeled PACAP27 or PACAP38 eliminated visualization of this band. These results suggest that a specific, high-affinity binding site for PACAP27 is present in rat hypothalamus, and that this site shows a greater affinity for PACAP38 compared with PACAP27. The molecular weight of the peptide-receptor complex is 60,000 kDa, and therefore the receptor itself has an apparent molecular weight 57,000.
这些实验的目的是鉴定和表征大鼠下丘脑内肽类物质垂体腺苷酸环化酶激活多肽（PACAP）的结合位点。在这些实验中，使用了27个氨基酸形式的PACAP（PACAP27）作为放射性标记配体。[125I]PACAP27与下丘脑膜制剂的结合迅速，加入未标记的肽后可逆，并且至少部分受GTP调节。不可水解的GTP类似物，鸟苷-5'-O-（3-硫代三磷酸）（GTPγS）、鸟苷-5'-（2-硫代二磷酸）（GDPβS）和鸟苷酰亚胺磷酸（GppNHp）也以剂量依赖性方式取代[125I]PACAP27与下丘脑膜制剂的结合。这三种类似物的效力顺序为GTPγS大于GDPβS大于GppNHp。两种形式的肽，PACAP27和PACAP38，在取代结合的[125I]PACAP27方面都具有高效力，而VIP或PACAP(1-23)在浓度高达500 nM时无法取代结合。对PACAP27和PACAP38取代曲线的Scatchard分析表明，两条曲线的拟合都与一类高亲和力结合位点一致，尽管该位点对PACAP38的亲和力比对PACAP27更高（PACAP27 Kd = 1452 +/- 59 pM；PACAP38 Kd = 175 +/- （此处原文可能有误，推测为175 +/- 13 pM）13 pM；Bmax 23.2 +/- 1.1 pmol/mg蛋白）。不能排除存在一类亲和力极低的结合位点的可能性。在将[125I]PACAP27与其受体进行共价交联后，通过电泳和放射自显影估计复合物的分子量。当膜与VIP或PACAP(1-23)一起孵育时，明显出现一条60 Kd的主要条带。预先用未标记的PACAP27或PACAP38孵育可消除该条带的可见性。这些结果表明，大鼠下丘脑存在一个针对PACAP27的特异性高亲和力结合位点，并且该位点对PACAP38的亲和力比对PACAP27更高。肽-受体复合物的分子量为60,000 kDa，因此受体本身的表观分子量为57,000。
