Barnes K, Kenny A J, Turner A J
Department of Biochemistry and Molecular Biology, University of Leeds, UK.
Eur J Neurosci. 1994 Apr 1;6(4):531-7. doi: 10.1111/j.1460-9568.1994.tb00297.x.
The subcellular distribution of the plasma membrane ectoenzymes, aminopeptidase N (aminopeptidase M) and dipeptidyl peptidase IV, has been examined by fractionating homogenates of porcine striata by a discontinuous Percoll gradient centrifugation procedure which distinguishes fractions containing pre- and post-synaptic elements. The two enzymes showed different distributions--dipeptidyl peptidase IV did not show a significant pre-synaptic location, whereas aminopeptidase N was present on both pre- and post-synaptic fractions. Immunofluorescent staining on mixed and neuron-enriched primary cultures of pig striatal tissue using affinity purified antibodies to the aminopeptidase and to the dipeptidyl peptidase revealed the ectoenzymes on distinct populations of cells. The astrocytic identity of the aminopeptidase N-staining cells was established by correlation with immunostaining for glial fibrillary acidic protein and for vimentin by confocal microscopy. Ultracryosections of striatum immunostained with gold-labelled immunoglobulins of differing diameters demonstrated aminopeptidase N on pericytes and confirmed its location on endothelial and astrocytic glial cells. Thus, several independent approaches indicated that aminopeptidase N, in addition to being present on endothelial and synaptic membranes, is found on astrocytes and pericytes in the perivascular neuropil, whereas dipeptidyl peptidase IV is less widely distributed on microvessels and appears not to have a synaptosomal location.
通过不连续的Percoll梯度离心法对猪纹状体匀浆进行分级分离，以区分含有突触前和突触后成分的组分，从而研究了质膜外切酶氨基肽酶N（氨基肽酶M）和二肽基肽酶IV的亚细胞分布。这两种酶表现出不同的分布——二肽基肽酶IV在突触前没有明显定位，而氨基肽酶N在突触前和突触后组分中均有存在。使用针对氨基肽酶和二肽基肽酶的亲和纯化抗体，对猪纹状体组织的混合原代培养物和富含神经元的原代培养物进行免疫荧光染色，结果显示外切酶存在于不同的细胞群体中。通过共聚焦显微镜将氨基肽酶N染色细胞的星形胶质细胞特征与胶质纤维酸性蛋白和波形蛋白的免疫染色相关联得以确定。用不同直径的金标免疫球蛋白对纹状体超薄冰冻切片进行免疫染色，结果显示周细胞上存在氨基肽酶N，并证实其在内皮细胞和星形胶质细胞上的定位。因此，几种独立的方法表明，氨基肽酶N除了存在于内皮细胞和突触膜上之外，还存在于血管周围神经纤维网中的星形胶质细胞和周细胞上，而二肽基肽酶IV在微血管上的分布较窄，且似乎不存在于突触体中。
