Possenti R, Eldridge J D, Paterson B M, Grasso A, Levi A
Dipartimento di Medicina Sperimentale e Scienze Biologiche, Facoltà di Medicina e Chirurgia, Università Tor Vergata, Roma, Italy.
EMBO J. 1989 Aug;8(8):2217-23. doi: 10.1002/j.1460-2075.1989.tb08345.x.
We have previously described the isolation of a cDNA clone corresponding to an mRNA rapidly induced to high levels in PC12 cells by treatment with NGF. We report here the complete amino acid sequence of the protein (named VGF8a) as deduced by nucleotide sequencing of overlapping cDNA clones. VGF8a is particularly rich in proline residues and has a conspicuous number of short stretches of basic amino acid residues which may represent potential targets for proteolytic cleavage. Antibodies directed against recombinant VGF8a-beta-galactosidase fusion proteins were used for immunofluorescent staining of the protein in PC12 cells as well as for its localization, by Western blot analysis, in subfractions of cell homogenates. We demonstrate that in PC12 cells, VGF8a protein is stored in secretory vesicles and is released in response to a variety of stimuli that are known to induce the regulated secretion of neurotransmitters.
我们之前描述过通过用神经生长因子（NGF）处理，在PC12细胞中快速诱导至高水平的一种与mRNA相对应的cDNA克隆的分离。我们在此报告通过重叠cDNA克隆的核苷酸测序推导得出的该蛋白质（命名为VGF8a）的完整氨基酸序列。VGF8a富含脯氨酸残基，并有大量明显的短碱性氨基酸残基片段，这些片段可能代表蛋白水解切割的潜在靶点。针对重组VGF8a - β - 半乳糖苷酶融合蛋白的抗体用于PC12细胞中该蛋白的免疫荧光染色，以及通过蛋白质印迹分析确定其在细胞匀浆亚组分中的定位。我们证明，在PC12细胞中，VGF8a蛋白存储在分泌小泡中，并响应已知可诱导神经递质调节性分泌的多种刺激而释放。
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