IMMUNOELECTRON MICROSCOPIC LOCALIZATION OF THE HPC-1 ANTIGEN IN RAT CEREBELLUM
IMMUNOELECTRON MICROSCOPIC LOCALIZATION OF THE HPC-1 ANTIGEN IN RAT CEREBELLUM
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DOI：
10.1007/bf01218356
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发表时间：
1993-11-01
期刊：
影响因子：
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通讯作者：
TASHIRO, Y
中科院分区：
文献类型：
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作者：
KOH, S;YAMAMOTO, A;TASHIRO, Y
HPC-1 抗原是一种神经元特异性 34 kDa 蛋白，与 p35A（突触蛋白）相同，被认为在突触小泡与突触前活性区的对接或融合中发挥重要作用。在本研究中，我们使用针对 β-半乳糖苷酶和 HPC-1 抗原的融合蛋白的抗体，通过冷冻免疫金技术分析了 HPC-1 抗原在大鼠小脑中的分布。在与浦肯野细胞树突形成突触的突触前束的质膜和突触小泡上以及小脑分子层中平行纤维的质膜上检测到高密度的 HPC-1 抗原。在颗粒细胞层，颗粒细胞的内质网、核膜和质膜上也检测到金颗粒。肾小球中的突触前膜和突触小泡也被金颗粒标记。为了确定膜上 HPC-I 抗原的拓扑结构，将从大鼠小脑制备的突触体部分包埋在琼脂糖中，并进行预包埋蛋白 A-金技术处理。完整的突触体没有被金颗粒标记。然而，当用低渗固定剂固定使质膜破裂时，或当用常张固定剂固定后破裂时，突触前膜和突触小泡的细胞质表面被金颗粒标记。这些结果表明HPC-1抗原的大部分表位位于质膜和突触小泡膜的细胞质表面。
HPC-1 antigen is a neuron-specific 34 kDa protein, identical to p35A (syntaxin), and is thought to play important roles in docking or fusion of synaptic vesicles to presynaptic active zones. In the present study we analyze the distribution of HPC-1 antigen in rat cerebellum by a cryoimmunogold technique using an antibody against the fusion protein of beta-galactosidase and the HPC-1 antigen. HPC-1 antigen was detected at high density on the plasma membranes and synaptic vesicles of presynaptic boutons which formed synapses with dendrites of Purkinje cells, and on the plasma membranes of parallel fibres in the cerebellar molecular layer. In the granule cell layer, gold particles were also detected on the endoplasmic reticulum, nuclear membranes and the plasma membranes of granule cells. Presynaptic membranes and synaptic vesicles in glomeruli were also labelled by gold particles. To determine the topology of HPC-I antigen on the membranes, the synaptosome fraction prepared from rat cerebellum was embedded in agarose, and processed for the pre-embedding protein A-gold technique. Intact synaptosomes were not labelled by gold particles. However, when fixed in hypotonic fixative to rupture plasma membranes, or when ruptured after fixation in normotonic fixative, the cytoplasmic surfaces of presynaptic membranes and synaptic vesicles were labelled by gold particles. These results suggest that most of the epitopes of HPC-1 antigen are located on the cytoplasmic surface of plasma membranes and synaptic vesicle membranes.