Barnes K, Turner A J, Kenny A J
Department of Biochemistry and Molecular Biology, University of Leeds, England.
J Neurochem. 1992 Jun;58(6):2088-96. doi: 10.1111/j.1471-4159.1992.tb10950.x.
Brains from piglets were dissected and a block of tissue including the substantia nigra, globus pallidus, and entopeduncular nucleus was homogenized and then fractionated on discontinuous Percoll gradients. Ligand-binding assays using (-)-[3H]nicotine and [3H]quinuclidinyl benzilate served to delineate fractions containing nicotinic and muscarinic acetylcholine receptors. In this system endopeptidase-24.11 exhibited a biphasic distribution, consistent with its presence on both pre- and postsynaptic membranes. Peptidyl dipeptidase A (angiotensin converting enzyme; ACE) was associated with membrane fractions containing muscarinic receptors. An immunoblot of these fractions with an affinity-purified polyclonal antibody to ACE revealed only the neuronal form of ACE (Mr 170,000), the endothelial form (Mr 180,000) being undetectable. Electron microscopic immunoperoxidase staining of the substantia nigra, with an affinity-purified antibody to endopeptidase-24.11 at the preembedding stage, showed this antigen to be confined to the plasma membranes of boutons, axons, and some dendrites. Both pre- and postsynaptic membranes were stained, and occasionally other regions of the dendritic membrane were positive. No staining of synaptic vesicles within the boutons was observed. Thus, two independent approaches indicate that endopeptidase-24.11 is present on both pre- and postsynaptic membranes in the pig substantia nigra. The subcellular fractionation suggests that neuronal ACE is confined to dendritic membranes.
解剖仔猪的大脑，将包含黑质、苍白球和脚内核的一块组织匀浆，然后在不连续的Percoll梯度上进行分级分离。使用（-）-[3H]尼古丁和[3H]喹核酯进行配体结合测定，以确定含有烟碱型和毒蕈碱型乙酰胆碱受体的分级分离物。在这个系统中，内肽酶-24.11呈现双相分布，这与其在突触前膜和突触后膜上的存在一致。肽基二肽酶A（血管紧张素转换酶；ACE）与含有毒蕈碱受体的膜分级分离物相关。用针对ACE的亲和纯化多克隆抗体对这些分级分离物进行免疫印迹分析，结果仅显示出神经元形式的ACE（分子量170,000），未检测到内皮形式的ACE（分子量180,000）。在包埋前阶段，用针对内肽酶-24.11的亲和纯化抗体对黑质进行电子显微镜免疫过氧化物酶染色，结果显示该抗原局限于终扣、轴突和一些树突的质膜上。突触前膜和突触后膜均被染色，偶尔树突膜的其他区域也呈阳性。在终扣内未观察到突触小泡的染色。因此，两种独立的方法表明，内肽酶-24.11存在于猪黑质的突触前膜和突触后膜上。亚细胞分级分离表明，神经元ACE局限于树突膜。
