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Investigation of molecular structures and subcellular localization of 49 kDa apyrasefrom plants

植物 49 kDa 腺苷三磷酸双磷酸酶的分子结构和亚细胞定位研究

基本信息

批准号：
12660296
负责人：
ABE Shunnosuke
金额：
$ 2.18万
依托单位：
Ehime University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12660296/
关键词：
apyrase cytoskeleton Mass spectroscopy heparin affinity immune electron microscopy subcellular localization isotype isoeleciric point 細胞骨格アイソタイプ局在質量分析 Cytoskeleton 2D-PAGE Isotype Localizatin Isoelectric point NTPase /

项目摘要

We isolated a 49 kDa protein from the cytoskeleton fraction from pea (Pisum sativum L.var.Alaska) stems using heparin affinity and cation exchange column chromatography, and identified the protein as apyrase (EC 3.6.1.5). Using an antibody raised against apyrase, we studied its sub-cellular distribution in isolated fractions and found significant amounts in the cell wall (50 %), the nuclei (3 %), the cytoskeleton (14 %), and the supernatant fractions (33 %). Immuno electron microscopy using gold-labeled antibody confirmed that apyrase was present in cell walls, nuclei, and in filamentous structures associated with ribosomes. Immuno fluorescence microscopy and expression of GFP fusion protein also revealed their multi-localization. Even though there is only one gene (with 2 alleles), 2D gels indicated there were at least five isotypes, three being major ones, and the relative abundance of these isotypes differed in different fractions. These isotypes were successfully separated by an anion column chromatography with a pH and KOAc gradients. Mass spectroscopy and C-terminal amino acid sequencing the pea apyrase was cleaved at both the N- and C- termini, and clearly distinguished 4 apyrase isotypes of 46,491, 47,055, 47,260, and 47,450 kDa and that it may be modified in various ways to furnish different forms with different functions and locations.

我们使用肝素亲和和阳离子交换柱层析从豌豆（PisumsativumL.var.Alaska）茎的细胞骨架级分中分离出49 kDa蛋白质，并将该蛋白质鉴定为腺苷三磷酸双磷酸酶（EC 3.6.1.5）。使用针对腺苷三磷酸双磷酸酶产生的抗体，我们研究了其在分离组分中的亚细胞分布，并发现在细胞壁（50%）、细胞核（3%）、细胞骨架（14%）和上清液组分（33%）中有显著量。免疫电子显微镜使用金标记的抗体证实，腺苷三磷酸双磷酸酶存在于细胞壁，细胞核，并在与核糖体相关的丝状结构。免疫荧光显微镜和GFP融合蛋白的表达也显示了它们的多定位。即使只有一个基因（2个等位基因），二维凝胶显示至少有五个同种型，三个是主要的，这些同种型的相对丰度在不同的馏分不同。这些同种型成功地分离的阴离子柱色谱与pH和KOAc梯度。质谱和C-末端氨基酸测序豌豆腺苷三磷酸双磷酸酶在N-和C-末端都被切割，并且清楚地区分46，491、47，055、47，260和47，450 kDa的4种腺苷三磷酸双磷酸酶同种型，并且它可以以各种方式被修饰以提供具有不同功能和位置的不同形式。