Fischer-Colbrie R, Diez-Guerra J, Emson P C, Winkler H
Neuroscience. 1986 May;18(1):167-74. doi: 10.1016/0306-4522(86)90185-5.
Antisera against neuropeptide Y, [Met]enkephalin and bombesin were used for characterizing the immunoreactive material in subcellular fractions of bovine adrenal medulla. Neuropeptide Y was identified by high performance liquid chromatography and by immunoblotting. Subcellular fractionation established that neuropeptide Y is present in chromaffin granules. During stimulation of the adrenal it is released concomitantly with catecholamines. The soluble proteins of chromaffin granules contain 1.9 micrograms neuropeptide Y/mg protein which gives 429 copies of neuropeptide Y for a single granule. In two-dimensional immunoblots two peptides of the same molecular size, but with differing pI (6.4 and 7.3) react with the antiserum against neuropeptide Y. There was no evidence for the presence of a larger neuropeptide Y precursor in chromaffin granules. On the other hand, larger enkephalin-containing peptides could be detected by immunoblotting. The subcellular distribution of these enkephalin precursors differed. The larger peptides (23.3 and 18.2 kD) were more concentrated in lighter granules when compared to the smaller precursors (12.6 and 8.6 kD) which is consistent with proteolytic processing of these peptides during granule maturation. An antiserum against bombesin reacts in immunoblots with the chromogranin B family. This study further illustrates that chromaffin granules contain a complex mixture of neuropeptide-immunoreactive material. The combination of immunoblotting with subcellular fractionation appears as a useful tool to characterize these peptides and their precursors.
用抗神经肽Y、[甲硫氨酸]脑啡肽和蛙皮素的抗血清来鉴定牛肾上腺髓质亚细胞组分中的免疫反应性物质。通过高效液相色谱法和免疫印迹法鉴定神经肽Y。亚细胞分级分离表明神经肽Y存在于嗜铬颗粒中。在肾上腺受到刺激时，它与儿茶酚胺同时释放。嗜铬颗粒的可溶性蛋白含有1.9微克神经肽Y/毫克蛋白，单个颗粒中有429个神经肽Y拷贝。在二维免疫印迹中，两种分子大小相同但等电点不同（分别为6.4和7.3）的肽与抗神经肽Y抗血清发生反应。没有证据表明嗜铬颗粒中存在更大的神经肽Y前体。另一方面，通过免疫印迹法可检测到更大的含脑啡肽肽段。这些脑啡肽前体的亚细胞分布不同。与较小的前体（12.6和8.6 kD）相比，较大的肽段（23.3和18.2 kD）更集中在较轻的颗粒中，这与这些肽段在颗粒成熟过程中的蛋白水解加工过程一致。抗蛙皮素抗血清在免疫印迹中与嗜铬粒蛋白B家族发生反应。这项研究进一步表明嗜铬颗粒含有神经肽免疫反应性物质的复杂混合物。免疫印迹法与亚细胞分级分离相结合似乎是鉴定这些肽及其前体的有用工具。
