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Structure and function of proteasome in higher plants

高等植物蛋白酶体的结构和功能

基本信息

批准号：
05454014
负责人：
NAKAGAWA Hiroki
金额：
$ 4.67万
依托单位：
Chiba University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05454014/
关键词：
26S Proteasome Protease spinach leaves

项目摘要

The 26S proteasome complex catalyzing ATP-dependent breakdown of ubiquitin-ligated proteins was purified from spinach leaves to near homogeneity by chromatography on DEAE-cellurose, gel filtration on Biogel A-1.5m and glycerol density gradient cenrifugation. The purified enzyme was shown to degrade multi-ubiquitinated, but not unmodified, lysozyme in an ATP-dependent fashion, coupled with ATPase activity supplying energy for proteolysis, and isopeptidase activity to generate free ubiquitin. By non-denaturing electrophoresis, the puried enzyme was separated into to distinct forms of the 26S complex, named 26Salpha and 26Sbeta proteasomes, with different electorophoretic mobilities. The 26S proteasome was found to consist of multiple polypeptides with molecular masses of 23-35 and 39-115kDa, which were thought to be those of a 20S proteasome with multicatalytic proteinase activity and an associated regulatory part with ATPase and de-ubiquitinating activities, respectively. the subunit multiplicity of the spinach 26S proteasome dosely resembled that of rat liver with minor differences in certain components. No sulfhydryl bond was involved in the assembly of this multi-component polypeptide complex. Electron microscopy showed that the 26S proteasome complex had a "caterpillar" like shape, consisting of four central protein layrs, assumed to be the 20S proteasome, with asymmetric V-shaped layrs of each end. These structural and functional characteristics of the spinach 26S proteasome showed marked similarity to those of the mammalian 26S proteasomes reported recently, suggesting that the 26S proteasome is widely distributed in eukaryotic cells and is of general importance for catalyzing the soluble energy-and ubiquitin-dependent proteolytic pathway.

通过 DEAE-纤维素色谱、Biogel A-1.5m 凝胶过滤和甘油密度梯度离心，从菠菜叶中纯化出催化 ATP 依赖性分解泛素连接蛋白的 26S 蛋白酶体复合物，使其接近均质。纯化的酶显示出以 ATP 依赖性方式降解多泛素化溶菌酶，但不是未修饰的溶菌酶，同时具有为蛋白水解提供能量的 ATP 酶活性和产生游离泛素的异肽酶活性。通过非变性电泳，纯化的酶被分离成不同形式的 26S 复合物，命名为 26Salpha 和 26Sbeta 蛋白酶体，具有不同的电泳迁移率。发现26S蛋白酶体由分子量为23-35和39-115kDa的多个多肽组成，这被认为是具有多催化蛋白酶活性以及分别具有ATP酶和去泛素化活性的相关调节部分的20S蛋白酶体的多肽。菠菜 26S 蛋白酶体的亚基多样性与大鼠肝脏的亚基多样性非常相似，但某些成分存在微小差异。这种多组分多肽复合物的组装不涉及任何巯基键。电子显微镜显示26S蛋白酶体复合物呈“毛毛虫”状，由四个中心蛋白质层组成，推测为20S蛋白酶体，两端具有不对称的V形层。菠菜26S蛋白酶体的这些结构和功能特征与最近报道的哺乳动物26S蛋白酶体的结构和功能特征显示出明显的相似性，表明26S蛋白酶体广泛分布于真核细胞中，并且对于催化可溶性能量和泛素依赖性蛋白水解途径具有普遍重要意义。