Lam H C, Takahashi K, Ghatei M A, Kanse S M, Polak J M, Bloom S R
Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London, England.
Eur J Biochem. 1990 Nov 13;193(3):725-9. doi: 10.1111/j.1432-1033.1990.tb19392.x.
Pituitary-adenylate-cyclase-activating polypeptide (PACAP) is a novel 38-amino-acid neuropeptide isolated from ovine hypothalamic tissues based on its activity of stimulating adenylate cyclase of rat pituitary cells. Binding sites for PACAP were studied in rat tissue membranes using a 27-amino-acid N-terminal derivative of PACAP [PACAP(1-27)] labelled with 125I. Particularly high specific binding sites of 125I-PACAP(1-27) were noted in the hypothalamus, brain stem, cerebellum and lung. Specific binding sites are also present in the pituitary gland, but at a lower concentration, and mainly in the anterior lobe. Very low concentration of 125I-PACAP(1-27)-binding sites were found in the colon, aorta and kidney membranes and no binding sites were detected in the pancreas and testis. Maximal binding of 125I-PACAP(1-27) was observed at pH 7.4. Interaction of 125I-PACAP(1-27) with its binding site was rapid, specific and saturable as well as time, pH and temperature dependent. PACAP(1-27) is more potent than PACAP in displacing the binding of 125I-PACAP(1-27) with brain membranes [concentration that inhibits 50% of the binding (IC50) = 7.45 +/- 1.52 nM and 11.45 +/- 3.65 nM, respectively; mean +/- SEM, n = 4] and lung membranes (IC50 = 4.41 +/- 0.87 nM and 10.68 +/- 3.09 nM, respectively). Vasoactive intestinal peptide displaced the binding of 125I-PACAP(1-27) in lung membrane (IC50 = 16.88 +/- 5.14 nM) but not in brain membranes. The equilibrium binding of 125I-PACAP(1-27) at 4 degrees C was characterized by a single class of binding site for the brain membrane with a dissociation constant (Kd) of 2.46 +/- 0.53 nM and a maximal binding capacity (Bmax) of 8.44 +/- 3.13 pmol/mg protein, but there were two classes of binding site for lung membranes with Kd of 1.02 +/- 0.51 nM and 5.19 +/- 0.99 nM, and Bmax of 2.84 +/- 0.72 pmol/mg protein and 9.13 +/- 1.89 pmol/mg protein, respectively. These findings suggest that subtypes of PACAP-binding sites exist and PACAP may have a physiological role in the hypothalamus/pituitary axis as well as in other regions of the brain and lung.
垂体腺苷酸环化酶激活多肽（PACAP）是一种新的由38个氨基酸组成的神经肽，它是根据其刺激大鼠垂体细胞腺苷酸环化酶的活性从绵羊下丘脑组织中分离出来的。利用用125I标记的PACAP的27个氨基酸的N末端衍生物[PACAP(1 - 27)]，在大鼠组织膜中研究了PACAP的结合位点。在丘脑、脑干、小脑和肺中发现了特别高的125I - PACAP(1 - 27)特异性结合位点。垂体中也存在特异性结合位点，但浓度较低，且主要存在于前叶。在结肠、主动脉和肾膜中发现125I - PACAP(1 - 27)结合位点的浓度非常低，而在胰腺和睾丸中未检测到结合位点。在pH 7.4时观察到125I - PACAP(1 - 27)的最大结合。125I - PACAP(1 - 27)与其结合位点的相互作用迅速、特异且可饱和，并且依赖于时间、pH和温度。在取代125I - PACAP(1 - 27)与脑膜的结合方面，PACAP(1 - 27)比PACAP更有效[抑制50%结合的浓度（IC50）分别为7.45±1.52 nM和11.45±3.65 nM；平均值±标准误，n = 4]，在取代与肺膜的结合方面也是如此（IC50分别为4.41±0.87 nM和10.68±3.09 nM）。血管活性肠肽可取代125I - PACAP(1 - 27)与肺膜的结合（IC50 = 16.88±5.14 nM），但不能取代与脑膜的结合。在4℃时，125I - PACAP(1 - 27)与脑膜的平衡结合以一类结合位点为特征，解离常数（Kd）为2.46±0.53 nM，最大结合容量（Bmax）为8.44±3.13 pmol/mg蛋白，但对于肺膜有两类结合位点，Kd分别为1.02±0.51 nM和5.19±0.99 nM，Bmax分别为2.84±0.72 pmol/mg蛋白和9.13±1.89 pmol/mg蛋白。这些发现表明存在PACAP结合位点的亚型，并且PACAP可能在下丘脑/垂体轴以及脑和肺的其他区域中具有生理作用。
