Ahmad S, Berezin I, Vincent J P, Daniel E E
Biochim Biophys Acta. 1987 Jan 26;896(2):224-38. doi: 10.1016/0005-2736(87)90183-0.
To study the binding of (Tyr3-125I)-labelled neurotensin to intestinal muscle, plasma membranes have been purified from dog intestinal circular smooth muscle. Purification was done by differential centrifugation followed by separation on a sucrose gradient. Electron microscopic study revealed that the dissected circular muscles used as the source of membranes were free of myenteric plexus and that the plasma membrane fraction obtained was free of any mitochondria or synaptosomes. The fraction used was obtained at the interface of 14%-33% sucrose density on the gradient and was 25-times enriched in the plasma membrane marker enzyme 5'-nucleotidase activity as compared to post-nuclear supernatant. This fraction contained negligible activity of mitochondrial membrane marker enzyme cytochrome c oxidase and low activity of a putative endoplasmic reticulum marker enzyme NADPH-cytochrome-c reductase. This membrane fraction contained a high density of neurotensin binding sites. This binding was studied by kinetic and by saturation approaches. Analysis of data from saturation binding studies by the computer programs (EBDA and LIGAND) suggested the presence of a two-site model (Kd1 = 0.118 nM, Kd2 = 3.18 nM, Bmax1 = 9.73 fmol/mg and Bmax2 = 129.8 fmol/mg). A part of specifically bound neurotensin was rapidly dissociated. No cooperativity between the two receptor types could be detected. A kinetic analysis of binding gave the Kd value equal to 0.107 nM. Carboxy terminal amino acid residues 8-13 were found to be essential for the binding activity and replacement of Tyr11 by tryptophan reduced the affinity of the peptide by 10 times in displacement studies. Binding was modulated by sodium ions and a guanine nucleotide Gpp[NH]p. MgCl2, CaCl2 and KCl were also found to reduce the specific binding. Evidence was found of a high specific binding to another membrane fraction poor in plasma membranes and rich in synaptosomes. We concluded that plasma membrane of canine intestinal circular muscle contains neurotensin receptors with recognition properties distinct from those obtained in previous studies of neurotensin binding sites in murine tissues. Another neurotensin binding site may be present on neuronal membranes.
为研究（酪氨酸3 - 125I）标记的神经降压素与肠肌的结合，已从犬肠环形平滑肌中纯化出质膜。纯化过程通过差速离心，随后在蔗糖梯度上进行分离。电子显微镜研究表明，用作膜来源的解剖环形肌不含肌间神经丛，且获得的质膜部分不含任何线粒体或突触小体。所用部分是在梯度上14% - 33%蔗糖密度的界面处获得的，与核后上清液相比，其质膜标记酶5'-核苷酸酶活性富集了25倍。该部分线粒体膜标记酶细胞色素c氧化酶的活性可忽略不计，而假定的内质网标记酶NADPH - 细胞色素 - c还原酶的活性较低。该膜部分含有高密度的神经降压素结合位点。通过动力学和饱和方法对这种结合进行了研究。用计算机程序（EBDA和LIGAND）对饱和结合研究的数据进行分析，提示存在双位点模型（Kd1 = 0.118 nM，Kd2 = 3.18 nM，Bmax1 = 9.73 fmol/mg，Bmax2 = 129.8 fmol/mg）。一部分特异性结合的神经降压素迅速解离。未检测到两种受体类型之间的协同作用。结合的动力学分析得出Kd值等于0.107 nM。发现羧基末端氨基酸残基8 - 13对结合活性至关重要，在置换研究中用色氨酸取代酪氨酸11可使肽的亲和力降低10倍。结合受钠离子和鸟嘌呤核苷酸Gpp[NH]p调节。还发现氯化镁、氯化钙和氯化钾会降低特异性结合。有证据表明与另一个质膜含量低而突触小体丰富的膜部分存在高特异性结合。我们得出结论，犬肠环形平滑肌的质膜含有神经降压素受体，其识别特性与先前在鼠组织中神经降压素结合位点的研究结果不同。另一个神经降压素结合位点可能存在于神经元膜上。
