Tatsuno I, Gottschall P E, Arimura A
US-Japan Biomedical Research Laboratories, Tulane University Hebert Center, Belle Chasse, LA 70037.
Peptides. 1991 May-Jun;12(3):617-21. doi: 10.1016/0196-9781(91)90110-b.
Molecular identification of the binding sites for pituitary adenylate cyclase activating polypeptide (PACAP) and the effect of vasoactive intestinal peptide (VIP) on the specific binding sites for PACAP in rat cultured astrocyte membrane preparations were investigated. Affinity cross-linking of astrocyte membrane preparations with [125I]PACAP27 showed the presence of a 60 kDa radiolabeled ligand-receptor complex. The labeling of this band was completely abolished in the presence of 10(-8) M or higher concentrations of unlabeled PACAP27. The molecular weight of this binding protein was estimated to be 57 kDa assuming an equimolar interaction of ligand and receptor in the 60 kDa complex. The labeling of [125I]PACAP27 binding to this binding protein was partly reduced by the addition of 10(-6) M VIP, but not by 10(-8) M. In the binding assay, VIP displaced the specific binding of [125I]PACAP27 at 10(-7) M or a greater concentration. Displacement of [125I]PACAP27 binding by unlabeled PACAP27 was analyzed in the presence or absence of 10(-6) M VIP. VIP at 10(-6) M reduced the maximal binding capacity (Bmax) of the high affinity binding site for PACAP27 by about 50% but did not alter the Bmax of the low affinity binding site. The dissociation constants (Kd) for both the high and low affinity binding sites were unaltered. These results indicate that PACAP binds to a 57 kDa membrane protein with high affinity and that VIP, at much higher concentrations, binds to this same binding site, suggesting that VIP mimics the biological action of PACAP in astrocytes at high concentrations.
研究了垂体腺苷酸环化酶激活多肽（PACAP）结合位点的分子鉴定以及血管活性肠肽（VIP）对大鼠培养星形胶质细胞膜制剂中PACAP特异性结合位点的影响。用[125I]PACAP27对星形胶质细胞膜制剂进行亲和交联，结果显示存在一种60 kDa的放射性标记配体 - 受体复合物。在10^(-8) M或更高浓度的未标记PACAP27存在下，该条带的标记完全消失。假设在60 kDa复合物中配体和受体等摩尔相互作用，该结合蛋白的分子量估计为57 kDa。添加10^(-6) M VIP可部分降低[125I]PACAP27与该结合蛋白的结合标记，但10^(-8) M VIP则无此作用。在结合试验中，VIP在10^(-7) M或更高浓度时可取代[125I]PACAP27的特异性结合。在有或无10^(-6) M VIP存在的情况下，分析了未标记PACAP27对[125I]PACAP27结合的取代情况。10^(-6) M VIP使PACAP27高亲和力结合位点的最大结合容量（Bmax）降低约50%，但不改变低亲和力结合位点的Bmax。高亲和力和低亲和力结合位点的解离常数（Kd）均未改变。这些结果表明，PACAP以高亲和力结合到一种57 kDa的膜蛋白上，并且VIP在高得多的浓度下也结合到相同的结合位点，这表明VIP在高浓度下模拟了PACAP在星形胶质细胞中的生物学作用。
